CA2772106C - Fusarium and fusarium mycotoxin biocontrol - Google Patents
Fusarium and fusarium mycotoxin biocontrol Download PDFInfo
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- CA2772106C CA2772106C CA2772106A CA2772106A CA2772106C CA 2772106 C CA2772106 C CA 2772106C CA 2772106 A CA2772106 A CA 2772106A CA 2772106 A CA2772106 A CA 2772106A CA 2772106 C CA2772106 C CA 2772106C
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Abstract
The present invention relates to a novel ascomyceteous fungus, Sphaerodes mycoparasitia strain IDAC 301008-01, for controlling Fusarium plant pathogens, disease symptoms, and mycotoxins in planta and ex planta. Uses, methods, compositions, sequences, and products are also disclosed herein.
Description
FUSARIUM AND FUSARIUM MYCOTOXIN BIOCONTROL
FIELD OF THE INVENTION
The present invention relates to novel biocontrol agents, related compositions with mycocidal effect and uses thereof. In particular, the present agent has a mycocidal effect on fungi of the genus Fusarium. The present invention further relates to the isolated fungal inoculant, genes, proteins and/or organisms as well as uses, methods, compositions, involving the same.
BACKGROUND TO THE INVENTION
Fusarium is a filamentous fungus widely distributed on plants and in the soil.
Certain Fusarium species are plant pathogens. For example, Fusarium oxysporum, causes Fusarium wilt disease in more than a hundred species of plants. It does so by colonizing the plant xylem which can result in blockage and breakdown. When this occurs symptoms such as leaf wilting and yellowing appear in the plant eventually leading to the plant's death. This condition was the primary cause of the decline and disappearance of the Gros Michel banana cultivar from markets around the world. Recently a new strain has begun attacking plants of the dominant Cavendish cultivar leading to fears that, in the absence of a solution, this cultivar will too disappear from world markets.
Fusarium root rot is a major cause of seedling mortality in forest nurseries and also causes reduced survival after outplanting during the first growing season. The disease is caused by several Fusarium species and is common in many parts of the world. The disease is a particular problem in Western Canada and the United States and also in the North Central and Southern States. In addition, Pine pitch canker - caused by the fungus Fusarium circinatum - is a serious disease of pine trees and a threat to the forest industry in particularly in the US and New Zealand. Radiata or Monterey pine is highly susceptible to the disease with mortality rates in mature trees reaching 80% in some areas of California.
Fusarium Head Blight (FHB), also known as 'ear blight' or 'scab', is a disease of wheat, barley, oats and other small cereal grains caused by Fusarium. Corn and maize can be affected by a similar condition known as 'ear rot'. The aforementioned condition can reduce the yield and grade of the crop, and can potentially contaminate the grain with mycotoxins.
It is estimated that FHB costs the cereal industry almost $5 billion annually. In recent years, FHB
has proved a significant and growing problem for the commercially important wheat and barley crops in Western Canada. In the past Fusarium oxysporum and Fusarium avenaceum have been identified as the two species primarily associated with FHB in Canada wherein yields in some affected fields have been reduced by 30% or more. FHB and ear rot harvested grain is often contaminated with mycotoxins such as deoxinivalenol (DON) trichothecenes associated with feed refusal, general digestive disorders, diarrhea, hemorrhages. The emergence of the toxigenic 3-acetyldeoxynivalenol (3-ADON) Fusarium graminearum population in North America is a fairly recent phenomenon. It is replacing 15-ADON chemotype. Moreover, the mycotoxin profiles of F. culmorum - similar to F. graminearum under both laboratory and field conditions-represents an additional threat for corn, maize and wheat production worldwide.
Currently, there are no effective control measures for FHB and associated mycotoxins.
Additionally, there are also no resistant varieties of corn, wheat, barley, oats, or other small grain cereals. Fungicides can be effective but only temporarily suppress the disease.
In addition to being a common plant pathogen, Fusarium spp. can opportunistically infect animals and can be the causative agents of superficial and/or systemic infections in humans.
Fusaria are one of the most drug-resistant fungi making Fusaria infections difficult to treat and invasive infections can prove fatal.
SUMMARY OF THE INVENTION
The present invention relates to a novel ascomyceteous fungus, Sphaerodes mycoparasitia identified as strain IDAC 301008-01 and alternatively as strain SMCD2220-01, that has a mycoparasitic and antimycotoxin effect. In addition, mycotoxic proteins have been isolated from S. mycoparasitia strain IDAC 301008-01. Uses, methods, compositions, sequences, and products are also disclosed herein.
FIELD OF THE INVENTION
The present invention relates to novel biocontrol agents, related compositions with mycocidal effect and uses thereof. In particular, the present agent has a mycocidal effect on fungi of the genus Fusarium. The present invention further relates to the isolated fungal inoculant, genes, proteins and/or organisms as well as uses, methods, compositions, involving the same.
BACKGROUND TO THE INVENTION
Fusarium is a filamentous fungus widely distributed on plants and in the soil.
Certain Fusarium species are plant pathogens. For example, Fusarium oxysporum, causes Fusarium wilt disease in more than a hundred species of plants. It does so by colonizing the plant xylem which can result in blockage and breakdown. When this occurs symptoms such as leaf wilting and yellowing appear in the plant eventually leading to the plant's death. This condition was the primary cause of the decline and disappearance of the Gros Michel banana cultivar from markets around the world. Recently a new strain has begun attacking plants of the dominant Cavendish cultivar leading to fears that, in the absence of a solution, this cultivar will too disappear from world markets.
Fusarium root rot is a major cause of seedling mortality in forest nurseries and also causes reduced survival after outplanting during the first growing season. The disease is caused by several Fusarium species and is common in many parts of the world. The disease is a particular problem in Western Canada and the United States and also in the North Central and Southern States. In addition, Pine pitch canker - caused by the fungus Fusarium circinatum - is a serious disease of pine trees and a threat to the forest industry in particularly in the US and New Zealand. Radiata or Monterey pine is highly susceptible to the disease with mortality rates in mature trees reaching 80% in some areas of California.
Fusarium Head Blight (FHB), also known as 'ear blight' or 'scab', is a disease of wheat, barley, oats and other small cereal grains caused by Fusarium. Corn and maize can be affected by a similar condition known as 'ear rot'. The aforementioned condition can reduce the yield and grade of the crop, and can potentially contaminate the grain with mycotoxins.
It is estimated that FHB costs the cereal industry almost $5 billion annually. In recent years, FHB
has proved a significant and growing problem for the commercially important wheat and barley crops in Western Canada. In the past Fusarium oxysporum and Fusarium avenaceum have been identified as the two species primarily associated with FHB in Canada wherein yields in some affected fields have been reduced by 30% or more. FHB and ear rot harvested grain is often contaminated with mycotoxins such as deoxinivalenol (DON) trichothecenes associated with feed refusal, general digestive disorders, diarrhea, hemorrhages. The emergence of the toxigenic 3-acetyldeoxynivalenol (3-ADON) Fusarium graminearum population in North America is a fairly recent phenomenon. It is replacing 15-ADON chemotype. Moreover, the mycotoxin profiles of F. culmorum - similar to F. graminearum under both laboratory and field conditions-represents an additional threat for corn, maize and wheat production worldwide.
Currently, there are no effective control measures for FHB and associated mycotoxins.
Additionally, there are also no resistant varieties of corn, wheat, barley, oats, or other small grain cereals. Fungicides can be effective but only temporarily suppress the disease.
In addition to being a common plant pathogen, Fusarium spp. can opportunistically infect animals and can be the causative agents of superficial and/or systemic infections in humans.
Fusaria are one of the most drug-resistant fungi making Fusaria infections difficult to treat and invasive infections can prove fatal.
SUMMARY OF THE INVENTION
The present invention relates to a novel ascomyceteous fungus, Sphaerodes mycoparasitia identified as strain IDAC 301008-01 and alternatively as strain SMCD2220-01, that has a mycoparasitic and antimycotoxin effect. In addition, mycotoxic proteins have been isolated from S. mycoparasitia strain IDAC 301008-01. Uses, methods, compositions, sequences, and products are also disclosed herein.
- 2 -BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a micrograph showing Sphaerodes mycoparasitica cultures after two weeks of incubation on (A) Modified Leonian's agar and (B) Potato dextrose agar¨upper sides, (C) and (D) - down sides;
Figure 2 is a micrograph showing Sphaerodes mycoparasitica ascospore germination, showing single-polar and double-polar germination patterns as well as hyphal anastomosis (arrow) formation pattern;
Figure 3 is a micrograph showing Sphaerodes mycoparasitica: (A) Ascoma, (B) Hyaline seta arising from the neck (arrow), (C) Reticulate ascospores (arrows), (D) Smooth ascospore (arrow), (E) Triangular ascospore (arrow), (F) Phialides produced on ascoma surrounding hyphae, (G) Ampulliform phialide arising from the surface of ascoma peridial wall, (H) Formation of mature and starting ascomata, (I) Formation of hook-like structures by S.
mycoparasitica parasitising on living hypha of Fusarium oxysporum (arrows) and (J) Large view of hook-like structure on living hypha of F. avenaceum. Bar scales for (A) and (H) are 50 pm;
for (B), (C), (D), (E), (F), (G) and (J) are 10 pm; for (I) 25 pm;
Figure 4 (A) is a micrograph showing Sphaerodes mycoparasitica ascospores showing the conspicuous wall ornamentation and prominent irregular longitudinal ribs (arrows); (B) shows Sphaerodes quadrangularis ascospores. Bar scales 5 pm;
Figure 5(a) is a micrograph showing inhibition of mycelial growth of (A) Fusarium oxysporum and (B) Fusarium graminearum by extracellular proteins recovered from a Sphaerodes mycoparasitica culture; 5(b) is a chart showing the percent inhibition of mycelial growth of A) Fusarium oxysporum and (B) Fusarium graminearum by S.
mycoparasitica extracellular proteins;
Figure 6 is a FPLC chromatogram showing the fl and 12 extracellular proteins recovered from a Sphaerodes mycoparasitica culture;
Figure 1 is a micrograph showing Sphaerodes mycoparasitica cultures after two weeks of incubation on (A) Modified Leonian's agar and (B) Potato dextrose agar¨upper sides, (C) and (D) - down sides;
Figure 2 is a micrograph showing Sphaerodes mycoparasitica ascospore germination, showing single-polar and double-polar germination patterns as well as hyphal anastomosis (arrow) formation pattern;
Figure 3 is a micrograph showing Sphaerodes mycoparasitica: (A) Ascoma, (B) Hyaline seta arising from the neck (arrow), (C) Reticulate ascospores (arrows), (D) Smooth ascospore (arrow), (E) Triangular ascospore (arrow), (F) Phialides produced on ascoma surrounding hyphae, (G) Ampulliform phialide arising from the surface of ascoma peridial wall, (H) Formation of mature and starting ascomata, (I) Formation of hook-like structures by S.
mycoparasitica parasitising on living hypha of Fusarium oxysporum (arrows) and (J) Large view of hook-like structure on living hypha of F. avenaceum. Bar scales for (A) and (H) are 50 pm;
for (B), (C), (D), (E), (F), (G) and (J) are 10 pm; for (I) 25 pm;
Figure 4 (A) is a micrograph showing Sphaerodes mycoparasitica ascospores showing the conspicuous wall ornamentation and prominent irregular longitudinal ribs (arrows); (B) shows Sphaerodes quadrangularis ascospores. Bar scales 5 pm;
Figure 5(a) is a micrograph showing inhibition of mycelial growth of (A) Fusarium oxysporum and (B) Fusarium graminearum by extracellular proteins recovered from a Sphaerodes mycoparasitica culture; 5(b) is a chart showing the percent inhibition of mycelial growth of A) Fusarium oxysporum and (B) Fusarium graminearum by S.
mycoparasitica extracellular proteins;
Figure 6 is a FPLC chromatogram showing the fl and 12 extracellular proteins recovered from a Sphaerodes mycoparasitica culture;
- 3 -Figure 7 is micrograph of SDS-PAGE gels of purified extracellular proteins recovered from a Sphaerodes mycoparasitica culture. Lane M contains marker proteins.
Lane 1 contains the purified protein from peak fl . Lane 2 contains the purified protein from peak f2;
Figure 8 are micrographs showing the inhibition of F. oxysporum spore germination (A-a-b) and F. graminearum spore germination (B-1-b) by purified proteins fl and f2 compared to controls (A-c and B-c);
Figure 9 is a chart showing the level of 3-ADON degradation by Sphaerodes mycoparasitica in potato-dextrose broth analysed by HPLC;
Figure 10 is a schematic illustration of a system for growing wheat plants in a container (4 x 4 x 16 cm) with different layers of soil-less growing mixes;
Figure 11 is a micrograph of a gel showing SmyITSF/R primers amplified PCR
products for S. mycoparasitica (SM), five Fusarium strains (Fa = F. avenaceum, Fo = F.
oxysporum, Fs =
F. sporotrichioides, Fg3 = F. graminearum chemotype 3, and Fg15 = F.
graminearum chemotype 15), two Trichoderma species (T22 = T. harzianum T22 and Tv = T
viride), two Cladosporium species ACC = C. cladosporioides and CM = C. minourae), and Penicillium aurantiogriseurn (PA) were electrophoresed on 1% agarose gel at 100 V for 20 minutes. The size of the band is around 300 to 400 bp;
Figure 12 are charts showing standard linear curves for (A) Sphaerodes mycoparasitica (in the range of 3.8 x 102 to 3.8 x 10-2 ng in ten-hold decreasing manner);
(B) Fusarium graminearum 3-ADCIN (in the range of 2.7 x 103 to 2.7 x 10-1 ng in ten-hold decreasing manner); (C) Trichoderma harzianum T-22 (in the range of 7.0 x 102 to 7.0 x 10-2 ng in ten-hold increasing manner);
Figure 13 is a chart showing RT-PCR sigmoidal coloured curves for Sphaerodes mycoparasitica (SMCD 2220-01), with 0.025 fluorescence line, in the ranges of 3.8 x 102 to 3.8 x 10-2 ng in a ten-hold decreasing manner;
Figure 14 are charts showing quantities of genomic DNAs for (A) F. graminearum (Fgra); (b) S. mycoparasitica (SM); and (C) Trichoderma harzanium (T-22) monitored in spring
Lane 1 contains the purified protein from peak fl . Lane 2 contains the purified protein from peak f2;
Figure 8 are micrographs showing the inhibition of F. oxysporum spore germination (A-a-b) and F. graminearum spore germination (B-1-b) by purified proteins fl and f2 compared to controls (A-c and B-c);
Figure 9 is a chart showing the level of 3-ADON degradation by Sphaerodes mycoparasitica in potato-dextrose broth analysed by HPLC;
Figure 10 is a schematic illustration of a system for growing wheat plants in a container (4 x 4 x 16 cm) with different layers of soil-less growing mixes;
Figure 11 is a micrograph of a gel showing SmyITSF/R primers amplified PCR
products for S. mycoparasitica (SM), five Fusarium strains (Fa = F. avenaceum, Fo = F.
oxysporum, Fs =
F. sporotrichioides, Fg3 = F. graminearum chemotype 3, and Fg15 = F.
graminearum chemotype 15), two Trichoderma species (T22 = T. harzianum T22 and Tv = T
viride), two Cladosporium species ACC = C. cladosporioides and CM = C. minourae), and Penicillium aurantiogriseurn (PA) were electrophoresed on 1% agarose gel at 100 V for 20 minutes. The size of the band is around 300 to 400 bp;
Figure 12 are charts showing standard linear curves for (A) Sphaerodes mycoparasitica (in the range of 3.8 x 102 to 3.8 x 10-2 ng in ten-hold decreasing manner);
(B) Fusarium graminearum 3-ADCIN (in the range of 2.7 x 103 to 2.7 x 10-1 ng in ten-hold decreasing manner); (C) Trichoderma harzianum T-22 (in the range of 7.0 x 102 to 7.0 x 10-2 ng in ten-hold increasing manner);
Figure 13 is a chart showing RT-PCR sigmoidal coloured curves for Sphaerodes mycoparasitica (SMCD 2220-01), with 0.025 fluorescence line, in the ranges of 3.8 x 102 to 3.8 x 10-2 ng in a ten-hold decreasing manner;
Figure 14 are charts showing quantities of genomic DNAs for (A) F. graminearum (Fgra); (b) S. mycoparasitica (SM); and (C) Trichoderma harzanium (T-22) monitored in spring
- 4 -wheat roots using genus-specific quantification real-time PCR. All values were means of 6 replicates. Error bars indicate SD;
Figure 15 is a chart showing real-time fluorescence curves of tri5 gene sequences amplified by using Tox5- 1/2 primer set from total DNA extracted from dual-culture assays of Fusarium graminearum strains and pre-inoculated Sphaerodes mycoparasitica (SNI) or singly grown F. graminearum 3-ADON and 15-ADON chemotypes;
Figure 16 are micrographs of Sphaerodes mycoparasitica ascomata and ascospores: (A) formation of S. mycoparasitica ascomata on a colony of F. avenaceum (Arrows indicate that ascomata were produced near or surrounding the Fusarium culture); (B) production of numerous S. mycoparasitica ostiolated perithecia being produced on a F. oxysporum colony (Arrows indicate pericthecia were formed on the Fusarium isolate); (C) ungerminated dark-brownish reticulated S. mycoparasitica ascospore; (D) germinating ascospore of S.
mycoparasitica in F.
oxysporum-filtrate suspension showing one polar germ pore: (E) single polar germinating spore of S. mycoparasitica after 3d suspension in a F. oxysporum-filtrate; (F) ascospore of S.
mycoparasitica illustrating two polar germination in F. oxysporum-filtrate suspension after 3d with an additional 1 d on PDA; (G) pattern of S. mycoparasitica two polar germination in F.
avenaceum-filtrate suspension with additional id on PDA: (H) single polar germination in S.
mycoparasitica spore suspended 3d in F. avenaceum-filtrate with id incubation on PDA; (I) single and double polar germinations demonstrated by S. mycoparasitica ascospores after 3d incubation in a F. avenaceum-filtrate suspension plus 1 additional day incubation on PDA. Scale bars for (C) to (H) are 10um and for (I) is 20 m. SM, Fa, and Fo represent S.
mycoparasitica, F.
avenaceum, and F. oxysporum, respectively;
Figure 17 is a chart showing spore germination patterns of Sphaerodes mycoparasitica biotrophic mycoparasitic fungus with spores isolated from a F. oxysporum colony. Single (N) and two (0) polar germination in a F. avenaceum-filtrate suspension; and Single (0) and two (0) polar germination in a F. oxysporum-filtrate suspension. 1 = 1d suspension, 2 = 1 d suspension plus id on PDA incubation, 3 = 3d suspension, and 4 = 3d suspension plus id on PDA
incubation. Each incubation day for S. mycoparasitica was analyzed separately;
Figure 15 is a chart showing real-time fluorescence curves of tri5 gene sequences amplified by using Tox5- 1/2 primer set from total DNA extracted from dual-culture assays of Fusarium graminearum strains and pre-inoculated Sphaerodes mycoparasitica (SNI) or singly grown F. graminearum 3-ADON and 15-ADON chemotypes;
Figure 16 are micrographs of Sphaerodes mycoparasitica ascomata and ascospores: (A) formation of S. mycoparasitica ascomata on a colony of F. avenaceum (Arrows indicate that ascomata were produced near or surrounding the Fusarium culture); (B) production of numerous S. mycoparasitica ostiolated perithecia being produced on a F. oxysporum colony (Arrows indicate pericthecia were formed on the Fusarium isolate); (C) ungerminated dark-brownish reticulated S. mycoparasitica ascospore; (D) germinating ascospore of S.
mycoparasitica in F.
oxysporum-filtrate suspension showing one polar germ pore: (E) single polar germinating spore of S. mycoparasitica after 3d suspension in a F. oxysporum-filtrate; (F) ascospore of S.
mycoparasitica illustrating two polar germination in F. oxysporum-filtrate suspension after 3d with an additional 1 d on PDA; (G) pattern of S. mycoparasitica two polar germination in F.
avenaceum-filtrate suspension with additional id on PDA: (H) single polar germination in S.
mycoparasitica spore suspended 3d in F. avenaceum-filtrate with id incubation on PDA; (I) single and double polar germinations demonstrated by S. mycoparasitica ascospores after 3d incubation in a F. avenaceum-filtrate suspension plus 1 additional day incubation on PDA. Scale bars for (C) to (H) are 10um and for (I) is 20 m. SM, Fa, and Fo represent S.
mycoparasitica, F.
avenaceum, and F. oxysporum, respectively;
Figure 17 is a chart showing spore germination patterns of Sphaerodes mycoparasitica biotrophic mycoparasitic fungus with spores isolated from a F. oxysporum colony. Single (N) and two (0) polar germination in a F. avenaceum-filtrate suspension; and Single (0) and two (0) polar germination in a F. oxysporum-filtrate suspension. 1 = 1d suspension, 2 = 1 d suspension plus id on PDA incubation, 3 = 3d suspension, and 4 = 3d suspension plus id on PDA
incubation. Each incubation day for S. mycoparasitica was analyzed separately;
- 5 -Figure 18 is a chart showing germination of Sphaerodes mycoparasitica ascospores in filtrates of six Fusarium strains and water suspension treatments at four different incubation days. Suspension in different treatments were: Fave-filtrate = F. avenaceum-filtrate; Foxy-filtrate = F. oxysporum-filtrate; Fgra3-filtrate = F. graminearum chemotype 3 filtrate;
Fgra15-filtrate =
F. graminearum chemotype 15 filtrate; Fpro-filtrate = F. proliferatum filtrate; Fspo-filtrate = F.
sporotrichioides filtrate; and water = control. Day of incubations were: id suspension = spores suspended for 1 d in different suspension treatments; ld sus+PDA = spores suspended for 1 d in suspension treatment and then inoculated onto PDA medium for an additional day; 3d suspension = spores suspended for 3d in different suspension treatments; and 3d sus+PDA =
spores suspended for 3d in suspension treatment and then inoculated onto PDA
medium for an additional day;
Figure 19 are charts showing linear mycelial growths of (A) Fusarium graminearum chemotype 3, and (B) Fusarium graminearum chemotype 15 in dual-culture assays challenged with Sphaerodes mycoparasitica in co-inoculation (same day) (¨A- -) and pre-inoculation (1 day prior to Fusarium inoculation) (- = -I.-=-) treatments as well as control (without mycoparasite) for 5 days;
Figure 20 are micrographs of interactions between F. graminearum 3-ADON
(Fgra3) and 15-ADON (Fgral 5) chemotypes on slide culture assays with S. mycoparasitica (S) biotrophic mycoparasitic fungus: (A) Single mycelium of S. mycoparasitica, (B) F.
graminearum mycelium with red complex, (C) Absorption of red complex from F. graminearum by S.
mycoparasitica (arrow), (D) Excretion of red complex in crystal-forms (arrows) by S.
mycoparasitica from mycelium interacting with F. graminearum chemotype 3 only, (E) Formation of series of hook-like structures by S. mycoparasitica, (F) Parasitism of F. graminearum mycelium by S.
mycoparasitica with formation of hook-shaped structures, excretion of crystal-like compounds (arrows), and internal haustorium, (G) Initiation of penetration-peg formation by S.
mycoparasitica on F. graminearum, (H) Infected or penetrated and non-infected myclial cells, (I) Branching of haustorium inside Fusarium host, (J) Formation of extensive short branching structures by F. graminearum chemotype 15 only at the contact zone with S.
mycoparasitica. Bar scales: (A) to (I) in 5 m, and (J) in 20p,m;
Fgra15-filtrate =
F. graminearum chemotype 15 filtrate; Fpro-filtrate = F. proliferatum filtrate; Fspo-filtrate = F.
sporotrichioides filtrate; and water = control. Day of incubations were: id suspension = spores suspended for 1 d in different suspension treatments; ld sus+PDA = spores suspended for 1 d in suspension treatment and then inoculated onto PDA medium for an additional day; 3d suspension = spores suspended for 3d in different suspension treatments; and 3d sus+PDA =
spores suspended for 3d in suspension treatment and then inoculated onto PDA
medium for an additional day;
Figure 19 are charts showing linear mycelial growths of (A) Fusarium graminearum chemotype 3, and (B) Fusarium graminearum chemotype 15 in dual-culture assays challenged with Sphaerodes mycoparasitica in co-inoculation (same day) (¨A- -) and pre-inoculation (1 day prior to Fusarium inoculation) (- = -I.-=-) treatments as well as control (without mycoparasite) for 5 days;
Figure 20 are micrographs of interactions between F. graminearum 3-ADON
(Fgra3) and 15-ADON (Fgral 5) chemotypes on slide culture assays with S. mycoparasitica (S) biotrophic mycoparasitic fungus: (A) Single mycelium of S. mycoparasitica, (B) F.
graminearum mycelium with red complex, (C) Absorption of red complex from F. graminearum by S.
mycoparasitica (arrow), (D) Excretion of red complex in crystal-forms (arrows) by S.
mycoparasitica from mycelium interacting with F. graminearum chemotype 3 only, (E) Formation of series of hook-like structures by S. mycoparasitica, (F) Parasitism of F. graminearum mycelium by S.
mycoparasitica with formation of hook-shaped structures, excretion of crystal-like compounds (arrows), and internal haustorium, (G) Initiation of penetration-peg formation by S.
mycoparasitica on F. graminearum, (H) Infected or penetrated and non-infected myclial cells, (I) Branching of haustorium inside Fusarium host, (J) Formation of extensive short branching structures by F. graminearum chemotype 15 only at the contact zone with S.
mycoparasitica. Bar scales: (A) to (I) in 5 m, and (J) in 20p,m;
- 6 -Figure 21 is a chart showing the differences in diameters between infected and non-infected F. graminearum chemotype 3 (Fgra3) and 15 (Fgral5) host cells in presence of Sphaerodes mycoparasitica on slide culture assays. Basr demarked by different lowercase letters represent significant difference in size of infected vs. non-infected hyphae for the two different F. graminearum chemotypes at P = 0.05 using a T-test;
Figure 22 are charts of the standard curves of Fusarium graminearum chemotype 3 and 15 genomic DNA concentration standards versus cycle threshold (Ct) with PCR
reactions performed in triplicate using primer sets; (A) Tox5-I/2, with genomic DNA
ranging from 270 ng (Logio = 2.90) to 0.27 ng (Logic, = -0.60), readings at 0.005 fluorescence line; (B) Tox5-1/2, with genomic DNA ranging from 30 ng (Logio = 1.48) to 0.03 ng (Logio = -1.52), readings at 0.005 fluorescence line; and (C) Fg16NF/R, with DNA template ranging from 270 ng (Logio = 2.43) to 0.027 ng (Logio = -1.57); in 10-fold dilution series, readings at 0.025 fluorescence line. Error bars indicate standard deviation for the mean of F. graminearum chemotype 3 and 15 standard curves derived from tri5 gene and F. graminearum specific primer set;
Figure 23 is a schematic illustration of the experimental set-up for dual-culture assays used to acquire F. graminearum chemotype 3-ADON or 15-ADON mycelial plugs for DNAs extraction. The sampling zone (S-zone) indicates the 0.5 x 1.5 cm2 sample area situated approximately 0.2 cm behind the interaction zone (I-zone). The I-zone represents the interaction or contact zone between F. graminearum (Fgra) and S. mycoparasitica (SM);
Figure 24 is a chart showing real-time fluorescence curves of F. graminearum sequences amplified using Fg16NF/R primer set from total DNA extracted from dual-culture assays of F.
graminearum strains and pre-inoculated Sphaerodes mycoparasitica (SM), or singly grown F.
graminearum cultures of chemotype 3 and 15;
Figure 25 is a chart showing comparisons between different concentrations of DNA from F. graminearum chemotype 3 and 15 amplified with Tox5-1/2 (tri5 gene specific) and Fg16NF/R
(F. graminearum-specific) primer sets. Fungal DNAs were extracted from 5 d dual-culture assays pre-inoculated with S. mycoparasitica for 1 d. With T-test at P = 0.05, for the comparison between S. mycoparasitica treated and non-treated F. graminearum chemotype 3 and 15 for
Figure 22 are charts of the standard curves of Fusarium graminearum chemotype 3 and 15 genomic DNA concentration standards versus cycle threshold (Ct) with PCR
reactions performed in triplicate using primer sets; (A) Tox5-I/2, with genomic DNA
ranging from 270 ng (Logio = 2.90) to 0.27 ng (Logic, = -0.60), readings at 0.005 fluorescence line; (B) Tox5-1/2, with genomic DNA ranging from 30 ng (Logio = 1.48) to 0.03 ng (Logio = -1.52), readings at 0.005 fluorescence line; and (C) Fg16NF/R, with DNA template ranging from 270 ng (Logio = 2.43) to 0.027 ng (Logio = -1.57); in 10-fold dilution series, readings at 0.025 fluorescence line. Error bars indicate standard deviation for the mean of F. graminearum chemotype 3 and 15 standard curves derived from tri5 gene and F. graminearum specific primer set;
Figure 23 is a schematic illustration of the experimental set-up for dual-culture assays used to acquire F. graminearum chemotype 3-ADON or 15-ADON mycelial plugs for DNAs extraction. The sampling zone (S-zone) indicates the 0.5 x 1.5 cm2 sample area situated approximately 0.2 cm behind the interaction zone (I-zone). The I-zone represents the interaction or contact zone between F. graminearum (Fgra) and S. mycoparasitica (SM);
Figure 24 is a chart showing real-time fluorescence curves of F. graminearum sequences amplified using Fg16NF/R primer set from total DNA extracted from dual-culture assays of F.
graminearum strains and pre-inoculated Sphaerodes mycoparasitica (SM), or singly grown F.
graminearum cultures of chemotype 3 and 15;
Figure 25 is a chart showing comparisons between different concentrations of DNA from F. graminearum chemotype 3 and 15 amplified with Tox5-1/2 (tri5 gene specific) and Fg16NF/R
(F. graminearum-specific) primer sets. Fungal DNAs were extracted from 5 d dual-culture assays pre-inoculated with S. mycoparasitica for 1 d. With T-test at P = 0.05, for the comparison between S. mycoparasitica treated and non-treated F. graminearum chemotype 3 and 15 for
- 7 -Tox5-1/2 and Fg16NF/R primer sets, respectively. (Log10 transformed for DNA
amplified with Tox5-1/2 primers);
Figure 26 are micrographs of Fusarium graminearum mycelia at the contact zone with biological and chemical agents: (A) No visible cell changes with Sphaerodes mycoparasitica biotrophic mycoparasite; (B) Cell abruption with Trichoderma harzianum necrotrophic mycoparasite; (C) 3-ADON chlamydospores formation in chains when challenged with fungicide; (D) 15-ADON chlamydospores formation in clusters when challenged with fungicide.
Scale bars indicate: (A) = 5 [im and (B) ¨ (D) = 10 [an;
Figure 27 are charts showing gene expression of different Tri genes for F.
graminearum chemotype 3 (3-ADON producer) and F. graminearum chemotype 15 (15-ADON
producer) in in vitro assays with three separate treatments: (A) Tri4 gene; (B) Tri5 gene; (C) Tri6 gene; and (D) Tri 10 gene. Legends: B3 = S. mycoparasitica + 3-ADON producing F.
graminearum; F3 =
Folicur + 3-ADON producing F. graminearum; T3 = T harzianum + 3-ADON producing F.
graminearum; B15 = S. mycoparasitica + 15-ADON producing F. graminearum; F15 =
Folicur + 15-ADON producing F. graminearum; and T15 = T. harzianum + 15-ADON producing F.
graminearum. Means for three different treatments in F. graminearum chemotype 3 and 15 were analyzed separately with LSD test at P = 0.05. Values are means SE of three samples;
Figure 28 are charts showing gene expression of PKS genes for F. graminearum chemotype 3 (3-ADON producer) and F. graminearum chemotype 15 (15-ADON
producer) in in vitro assays with three separate treatments: (A) PKS4 and (B) PKS13. Legends:
B3 = S.
mycoparasitica + 3-ADON producing F. graminearum; F3 = Folicur + 3-ADON
producing F.
graminearum; T3 = T harzianum + 3-ADON producing F. graminearum; B15 = S.
mycoparasitica + 15-ADON producing F. graminearum; F15 = Folicur + 15-ADON
producing F. graminearum; and T15 = T harzianum + 15-ADON producing F. graminearum.
Means for three different treatments in F. graminearum chemotype 3 and 15 were analyzed separately with LSD test at P = 0.05. Values are means SE of three samples;
Figure 29 is a micrograph of thin liquid chromatography (TLC) analysis for zearalenone (ZEA) extracted from six separate treatments. Legends: B3 = S. mycoparasitica + 3-ADON
producing F. graminearum; F3 = Folicur + 3-ADON producing F. graminearum; T3 =
T
amplified with Tox5-1/2 primers);
Figure 26 are micrographs of Fusarium graminearum mycelia at the contact zone with biological and chemical agents: (A) No visible cell changes with Sphaerodes mycoparasitica biotrophic mycoparasite; (B) Cell abruption with Trichoderma harzianum necrotrophic mycoparasite; (C) 3-ADON chlamydospores formation in chains when challenged with fungicide; (D) 15-ADON chlamydospores formation in clusters when challenged with fungicide.
Scale bars indicate: (A) = 5 [im and (B) ¨ (D) = 10 [an;
Figure 27 are charts showing gene expression of different Tri genes for F.
graminearum chemotype 3 (3-ADON producer) and F. graminearum chemotype 15 (15-ADON
producer) in in vitro assays with three separate treatments: (A) Tri4 gene; (B) Tri5 gene; (C) Tri6 gene; and (D) Tri 10 gene. Legends: B3 = S. mycoparasitica + 3-ADON producing F.
graminearum; F3 =
Folicur + 3-ADON producing F. graminearum; T3 = T harzianum + 3-ADON producing F.
graminearum; B15 = S. mycoparasitica + 15-ADON producing F. graminearum; F15 =
Folicur + 15-ADON producing F. graminearum; and T15 = T. harzianum + 15-ADON producing F.
graminearum. Means for three different treatments in F. graminearum chemotype 3 and 15 were analyzed separately with LSD test at P = 0.05. Values are means SE of three samples;
Figure 28 are charts showing gene expression of PKS genes for F. graminearum chemotype 3 (3-ADON producer) and F. graminearum chemotype 15 (15-ADON
producer) in in vitro assays with three separate treatments: (A) PKS4 and (B) PKS13. Legends:
B3 = S.
mycoparasitica + 3-ADON producing F. graminearum; F3 = Folicur + 3-ADON
producing F.
graminearum; T3 = T harzianum + 3-ADON producing F. graminearum; B15 = S.
mycoparasitica + 15-ADON producing F. graminearum; F15 = Folicur + 15-ADON
producing F. graminearum; and T15 = T harzianum + 15-ADON producing F. graminearum.
Means for three different treatments in F. graminearum chemotype 3 and 15 were analyzed separately with LSD test at P = 0.05. Values are means SE of three samples;
Figure 29 is a micrograph of thin liquid chromatography (TLC) analysis for zearalenone (ZEA) extracted from six separate treatments. Legends: B3 = S. mycoparasitica + 3-ADON
producing F. graminearum; F3 = Folicur + 3-ADON producing F. graminearum; T3 =
T
- 8 -
9 PCT/CA2010/001253 harzianum + 3-ADON producing F. graminearum; F15 = Folicur + 15-ADON producing F.
graminearum; T15 = T. harzianum + 15-ADON producing F. graminearum, B15 = S.
mycoparasitica + 15-ADON producing F. graminearum. and ZEA = Standard of zearalenone;
Figure 30 is a chart showing the ratio of F. graminearum chemotype challenged with Folicur fungicide to F. graminearum alone control produced for all four different mycotoxins ¨
ZEA, DON, 3ADON and 15ADON. Legends indicate F. graminearum 3-ADON chemotype with Folicur (I) and F. graminearum 15-ADON chemotype with Folicur (0);
Figure 31 is a chart showing relative AUR gene expression in Fusarium strains after co-culturing with biological and chemical agents. Legend: 15ADON - F.
graminearum15-acety/-deoxynivalenol chemotype; 3ADON - F. graminearum15-acetyl-deoxynivalenol chemotype;
F.cul. - F. culmorum; F.ave. - F. avenaceum ; B - Sphaerodes mycoparasitica (biotrophic mycoparasite); Tricho - Trichoderma harzianum (necrotrophic mycoparasite); Fol - Folicur (tebuconazole) fungicide;
Figure 32 is a chart showing changes in AUR gene expression evidenced by the color of fungal hyphae. Legend: Ds #71232B: Red and highly virulent, tolerant at 80 C
for 4 hours; Es #4E040B: Moderately red and moderately virulent, tolerant 40 C for 4 hours; Bs #A86608:
White and non virulent, susceptible 40 C for 4 hours;
Figure 33 are charts showing the effects of inoculating a Fusarium-susceptible barley cultivar with F. graminearum, S. mycoparasitica, and treating F. graminearum-infected barley with different concentrations of S. mycoparasitica on: (A) height of the plants; (b) average number of spikes per plant; and (C) the average weight of 5 spikes;
Figure 34 are charts showing the effects of inoculating a Fusarium-susceptible wheat cultivar with F. graminearum, S. mycoparasitica, and treating F. graminearum-infected wheat with different concentrations of S. mycoparasitica on: (A) height of the plants; (B) average number of spikes per plant; and (C) the average weight of 5 spikes;
Figure 35 is a chart comparing the biocontrol effects of S. mycoparasitica on the severity of Fusarium head blight symptoms with the protection provided by a commercial fungicide;
Figure 36 are charts showing standard curves of Fusarium graminearum chemotype genomic DNA concentration standards versus cycle threshold (Ct) with PCR
reactions performed in triplicate using primer sets: (A) Tox5-1/2, with genomic DNA
ranging from 270 ng (Logi = 2.90) to 0.27 ng (Logi = -0.60), readings at 0.005 fluorescence line; and (B) Fg16NF/R, with DNA template ranging from 270 ng (Logio = 2.43) to 0.027 ng (Logi = -1.57);
in 10-fold dilution series, readings at 0.025 fluorescence line;
Figure 37 is a chart showing the effects of S. mycoparasitica (B) and Folicur fungicide (Fol) treatments on F. graminearum chemotype 3-ADON genomic DNA detected in barley spikes employing RT-PCR. Treatments were: Fus ¨ F. graminearum; B-Fus ¨ S.
mycoparasitica with F. graminearum; Fol-Fus ¨ Folicur fungicide with F. graminearum;
Figure 38 are micrographs showing Sphaerodes mycoparasitica¨Fusarium spp.
mycoparasitism assays: (A¨a). Hook-shaped contact structures (arrows); (B¨b).
Clamp-like clasping cells (arrows). "a" and "b" are diagrammatic drawings for (A) and (B) respectively.
Scale bars = 5 m;
Figure 39 are micrographs showing intracellular parasitism, hyphal inhibition response, and anamorphic stages during the Sphaerodes mycoparasitica¨Fusarium spp.
interactions: (A) Intracellular parasitism by S. mycoparasitica in F. equiseti (arrow); (B) Fusarium hyphal inhibition response when challenged with S. mycoparasitica; deformation of hyphae into rosette-like shapes (arrow); (C) Hyaline S. mycoparasitica anamorphic stages; (D) Sphaerodes mycoparasitica anamorphic stages with adsorption of red pigments from F.
culmorum. Scale bars A, C, D = 5tim; B = 2011m;
Figures 40A and 40B are micrographs of intracellular parasitism by Sphaerodes inside F.
equiseti (arrows), and 40C and 40D are micrographs of intracellular hyphae produced by Sphaerodes inside F. equiseti with hook-shaped contact structure (arrows). 40A
and 40B were captured under light microscopy; whereas in 40C and 40D hyphae were stained with lactofuchsin and images were captured under fluorescent and confocal laser microscopy, respectively. Scale bars = 5 m; and
graminearum; T15 = T. harzianum + 15-ADON producing F. graminearum, B15 = S.
mycoparasitica + 15-ADON producing F. graminearum. and ZEA = Standard of zearalenone;
Figure 30 is a chart showing the ratio of F. graminearum chemotype challenged with Folicur fungicide to F. graminearum alone control produced for all four different mycotoxins ¨
ZEA, DON, 3ADON and 15ADON. Legends indicate F. graminearum 3-ADON chemotype with Folicur (I) and F. graminearum 15-ADON chemotype with Folicur (0);
Figure 31 is a chart showing relative AUR gene expression in Fusarium strains after co-culturing with biological and chemical agents. Legend: 15ADON - F.
graminearum15-acety/-deoxynivalenol chemotype; 3ADON - F. graminearum15-acetyl-deoxynivalenol chemotype;
F.cul. - F. culmorum; F.ave. - F. avenaceum ; B - Sphaerodes mycoparasitica (biotrophic mycoparasite); Tricho - Trichoderma harzianum (necrotrophic mycoparasite); Fol - Folicur (tebuconazole) fungicide;
Figure 32 is a chart showing changes in AUR gene expression evidenced by the color of fungal hyphae. Legend: Ds #71232B: Red and highly virulent, tolerant at 80 C
for 4 hours; Es #4E040B: Moderately red and moderately virulent, tolerant 40 C for 4 hours; Bs #A86608:
White and non virulent, susceptible 40 C for 4 hours;
Figure 33 are charts showing the effects of inoculating a Fusarium-susceptible barley cultivar with F. graminearum, S. mycoparasitica, and treating F. graminearum-infected barley with different concentrations of S. mycoparasitica on: (A) height of the plants; (b) average number of spikes per plant; and (C) the average weight of 5 spikes;
Figure 34 are charts showing the effects of inoculating a Fusarium-susceptible wheat cultivar with F. graminearum, S. mycoparasitica, and treating F. graminearum-infected wheat with different concentrations of S. mycoparasitica on: (A) height of the plants; (B) average number of spikes per plant; and (C) the average weight of 5 spikes;
Figure 35 is a chart comparing the biocontrol effects of S. mycoparasitica on the severity of Fusarium head blight symptoms with the protection provided by a commercial fungicide;
Figure 36 are charts showing standard curves of Fusarium graminearum chemotype genomic DNA concentration standards versus cycle threshold (Ct) with PCR
reactions performed in triplicate using primer sets: (A) Tox5-1/2, with genomic DNA
ranging from 270 ng (Logi = 2.90) to 0.27 ng (Logi = -0.60), readings at 0.005 fluorescence line; and (B) Fg16NF/R, with DNA template ranging from 270 ng (Logio = 2.43) to 0.027 ng (Logi = -1.57);
in 10-fold dilution series, readings at 0.025 fluorescence line;
Figure 37 is a chart showing the effects of S. mycoparasitica (B) and Folicur fungicide (Fol) treatments on F. graminearum chemotype 3-ADON genomic DNA detected in barley spikes employing RT-PCR. Treatments were: Fus ¨ F. graminearum; B-Fus ¨ S.
mycoparasitica with F. graminearum; Fol-Fus ¨ Folicur fungicide with F. graminearum;
Figure 38 are micrographs showing Sphaerodes mycoparasitica¨Fusarium spp.
mycoparasitism assays: (A¨a). Hook-shaped contact structures (arrows); (B¨b).
Clamp-like clasping cells (arrows). "a" and "b" are diagrammatic drawings for (A) and (B) respectively.
Scale bars = 5 m;
Figure 39 are micrographs showing intracellular parasitism, hyphal inhibition response, and anamorphic stages during the Sphaerodes mycoparasitica¨Fusarium spp.
interactions: (A) Intracellular parasitism by S. mycoparasitica in F. equiseti (arrow); (B) Fusarium hyphal inhibition response when challenged with S. mycoparasitica; deformation of hyphae into rosette-like shapes (arrow); (C) Hyaline S. mycoparasitica anamorphic stages; (D) Sphaerodes mycoparasitica anamorphic stages with adsorption of red pigments from F.
culmorum. Scale bars A, C, D = 5tim; B = 2011m;
Figures 40A and 40B are micrographs of intracellular parasitism by Sphaerodes inside F.
equiseti (arrows), and 40C and 40D are micrographs of intracellular hyphae produced by Sphaerodes inside F. equiseti with hook-shaped contact structure (arrows). 40A
and 40B were captured under light microscopy; whereas in 40C and 40D hyphae were stained with lactofuchsin and images were captured under fluorescent and confocal laser microscopy, respectively. Scale bars = 5 m; and
- 10-Figure 41 is a chart showing average hyphal diameters of parasitized and non-parasitized F. equiseti cells (0 ) and F. culmorum (o) on 1-week slide-cultures with Sphaerodes mycoparasitica biotrophic mycoparasite. Data are means and standard deviations. Same lowercase letters indicate no significant difference between parasitized and non-parasitized hyphae at P = 0.05, with T-test.
DETAILED DESCRIPTION OF THE INVENTION
Sphaerodes mycoparasitica (Ascomycetes, Melanosporales), has been isolated from isolates of Fusarium avenaceum, Fusarium graminearum and Fusarium oxysporum originating from wheat or asparagus fields. The species is characterized by a unique combination of ascospore size, shape (fusiform and triangular) and wall ornamentation (reticulate and smooth).
Also, conidia are produced from simple phialides on the surface of ascoma peridial wall, on ascoma surrounding hyphae, and on irregularly branched conidiophores arising from hyphae. S.
mycoparasitica has a phialidic anamorph and produces simple phialides on the surface of ascoma peridial wall or scattered irregularly on ascoma surrounding the hyphae, and on conidiosphores.
S. mycoparasitica forms hook-like structures parasitizing living hyphae of Fusarium.
The 1266bp DNA sequence from the large subunit ribosomal RNA gene (LSU) of S.
mycoparasitica is given in SEQ ID NO: 1.
DETAILED DESCRIPTION OF THE INVENTION
Sphaerodes mycoparasitica (Ascomycetes, Melanosporales), has been isolated from isolates of Fusarium avenaceum, Fusarium graminearum and Fusarium oxysporum originating from wheat or asparagus fields. The species is characterized by a unique combination of ascospore size, shape (fusiform and triangular) and wall ornamentation (reticulate and smooth).
Also, conidia are produced from simple phialides on the surface of ascoma peridial wall, on ascoma surrounding hyphae, and on irregularly branched conidiophores arising from hyphae. S.
mycoparasitica has a phialidic anamorph and produces simple phialides on the surface of ascoma peridial wall or scattered irregularly on ascoma surrounding the hyphae, and on conidiosphores.
S. mycoparasitica forms hook-like structures parasitizing living hyphae of Fusarium.
The 1266bp DNA sequence from the large subunit ribosomal RNA gene (LSU) of S.
mycoparasitica is given in SEQ ID NO: 1.
-11-SEQ ID NO:!.
1 atagggagaa gaagcactgc gattgcccta gtaacggcga gtgaagcggc agcagcccag 61 atttggaatc tggtcctttt ggggcccgag ttgtaatctg cagaggaagc gtctggtgcg 121 gtgccggcct agttccctgg aacgggacgc cgtagagggt gacagccccg tacggtcggc 181 caccaaacct gtgtgtcgct ccttcgaaga gtcgcgtagt ttgggaatgc tgcgtaaagt 241 gggaggtatg ctcctcctaa ggctaaatac cggccagaga ccgatagcgc acaagtagag 301 tgatcgaaag atgaaaagca ccttgaaaat ggggttaaaa agtacgtgaa attgccaaag 361 gggaagcgct cgtggccaga ctcgtgcctt atggatcatc cggctatttc gccggtgcac 421 tccattaggc tcgggccagc gtcggtcggc gccggtacta aaagacagcg cgaacgtggc 481 tctetteggg gagtgttata gcgcgctgtg taatgtgctg gcgccgtccg aggaccgcgc 541 atttatgcaa ggacgctggc gtaatggcca ctagcgaccc gtcttgaaac acggaccaag 601 gagtcgccca gagacgcgag tgtgcgggtg acaaacccct gcgcgaagtg aaagcgaacg 661 ctggtgggaa ccctcacggg tgcaccaccg accgatcctg atgtcttcgg atggatttga 721 gtatgagcgt ttctggtcgg acccgaaaga gggtgaacta tgcttgggta gggtgaagcc 781 agaggaaact ctggtggagg ccccgtttgg gttctgacgt gcaaatcgat ccataaacct 841 gggcatagcg gcgaaagact aatcgaacct tctagtagct ggttcgcatt ctctctctcg 901 cacgagagag agaaaacctc tgtgatatca cgattatcag tgaaaaccac accgagaccc 961 aacggagttc ttctggattt cctcatgctt caattaccac gcctagtgga cctacctgga 1021 gcgctacaat aaagtcatac gaaaatctcg aagatcgggg tgacggtgag ggatcctaag 1081 gttctctcgt tgagtgcgtt ggacgggcat ggccgtcagc gatctggggc gaccgttgcc 1141 ggatcataag ggctttagtg cttaggctat tggtattgag gggtctgaag acggtaatct 1201 gaaaccaaag gctttattct aaacccgcgc agcatgggcg tagtaggaag agacagcgaa 1261 gtctag
1 atagggagaa gaagcactgc gattgcccta gtaacggcga gtgaagcggc agcagcccag 61 atttggaatc tggtcctttt ggggcccgag ttgtaatctg cagaggaagc gtctggtgcg 121 gtgccggcct agttccctgg aacgggacgc cgtagagggt gacagccccg tacggtcggc 181 caccaaacct gtgtgtcgct ccttcgaaga gtcgcgtagt ttgggaatgc tgcgtaaagt 241 gggaggtatg ctcctcctaa ggctaaatac cggccagaga ccgatagcgc acaagtagag 301 tgatcgaaag atgaaaagca ccttgaaaat ggggttaaaa agtacgtgaa attgccaaag 361 gggaagcgct cgtggccaga ctcgtgcctt atggatcatc cggctatttc gccggtgcac 421 tccattaggc tcgggccagc gtcggtcggc gccggtacta aaagacagcg cgaacgtggc 481 tctetteggg gagtgttata gcgcgctgtg taatgtgctg gcgccgtccg aggaccgcgc 541 atttatgcaa ggacgctggc gtaatggcca ctagcgaccc gtcttgaaac acggaccaag 601 gagtcgccca gagacgcgag tgtgcgggtg acaaacccct gcgcgaagtg aaagcgaacg 661 ctggtgggaa ccctcacggg tgcaccaccg accgatcctg atgtcttcgg atggatttga 721 gtatgagcgt ttctggtcgg acccgaaaga gggtgaacta tgcttgggta gggtgaagcc 781 agaggaaact ctggtggagg ccccgtttgg gttctgacgt gcaaatcgat ccataaacct 841 gggcatagcg gcgaaagact aatcgaacct tctagtagct ggttcgcatt ctctctctcg 901 cacgagagag agaaaacctc tgtgatatca cgattatcag tgaaaaccac accgagaccc 961 aacggagttc ttctggattt cctcatgctt caattaccac gcctagtgga cctacctgga 1021 gcgctacaat aaagtcatac gaaaatctcg aagatcgggg tgacggtgag ggatcctaag 1081 gttctctcgt tgagtgcgtt ggacgggcat ggccgtcagc gatctggggc gaccgttgcc 1141 ggatcataag ggctttagtg cttaggctat tggtattgag gggtctgaag acggtaatct 1201 gaaaccaaag gctttattct aaacccgcgc agcatgggcg tagtaggaag agacagcgaa 1261 gtctag
- 12 -The 1233bp DNA sequence from the small subunit ribosomal RNA gene (SSU) of S.
mycoparasitica is given in SEQ ID NO: 2.
SEQ ID NO: 2 1 agtgcggcat gttgtagcct aagcaattat acagcgaaac tgcgaatggc tcattatata 61 agttatcgtt tatttgatag tgccttacta cttggataac cgtggtaatt ctagagctaa 121 tacatgctga aagccccgac ttacggaggg gcgtatttat tagattaaaa accaatgccc 181 ttcggggctc tttggtgatt catgataact tctcgaatcg cacggccttg cgccggcgat 241 ggttcattca aatttcttcc ctatcaactt tcgatgtttg ggtagtggcc aaacatggtg 301 gcaacgggta acggagggtt agggctcgac cccggagaag gagcctgaga aacggctcct 361 acatccaagg aaggcagcag gcgcgcaaat tacccaatct caactcgagg aggtagtgac 421 aataaatacc gatgcagggc tctttagggt cttgcaattg gaatgagtac aatttaaatc 481 ccttaacgag gaacaattgg agggcaagtc tggtgccagc agccgcggta actccagctc 541 caatagcgta tattaaagtt gttgtggtta aaaagctcgt agttgaacct tgggcctggc 601 cggctggtcc gcctaacagc gtgcactggt gcggccgggt cttcccaccg cggagccgca 661 tgtccttcac tgggcgtgtc ggggaagcgg tacttttact gtgaaaaaat tagagtgctc 721 taagcaggcc tatgctcgaa tacattagca tggaataata gaataggaca gtcgttctat 781 tttgttggtt tctaggacgt ctgtaatgat taacagaaac aatcgggggc gtcagtattg 841 catcgtcaga ggtgaaattc ttagatcgat gcaagactaa ctactgcgaa agcattcgcc 901 aagggtgttt tcattaatca ggaacgaaag ttaggggatc gaagacgatc agataccgtc 961 gtagtcttaa ccataaacta tgccgactag ggatcgggcg gtgtaatttt gacccgctcg 1021 gcacttacga gaaatcttaa gtgcttgggc tccaggggag tatggtcgca aggctgaaac 1081 ttaaagaaat gacgaagggc accaccaagg gtgaacctgc ggcctagttg actcacacgg 1141 gaaactcacg aggtaggaat atgtagatga cggatggagc cttcagaata catatgggca 1201 tgcgctctta ataccggtat tgaaaatggg cag Sample cultures of Sphaerodes mycoparasitica have been deposited with Saskatchewan Microbial Collection and Database under the accession number SMCD2220-01, and in the
mycoparasitica is given in SEQ ID NO: 2.
SEQ ID NO: 2 1 agtgcggcat gttgtagcct aagcaattat acagcgaaac tgcgaatggc tcattatata 61 agttatcgtt tatttgatag tgccttacta cttggataac cgtggtaatt ctagagctaa 121 tacatgctga aagccccgac ttacggaggg gcgtatttat tagattaaaa accaatgccc 181 ttcggggctc tttggtgatt catgataact tctcgaatcg cacggccttg cgccggcgat 241 ggttcattca aatttcttcc ctatcaactt tcgatgtttg ggtagtggcc aaacatggtg 301 gcaacgggta acggagggtt agggctcgac cccggagaag gagcctgaga aacggctcct 361 acatccaagg aaggcagcag gcgcgcaaat tacccaatct caactcgagg aggtagtgac 421 aataaatacc gatgcagggc tctttagggt cttgcaattg gaatgagtac aatttaaatc 481 ccttaacgag gaacaattgg agggcaagtc tggtgccagc agccgcggta actccagctc 541 caatagcgta tattaaagtt gttgtggtta aaaagctcgt agttgaacct tgggcctggc 601 cggctggtcc gcctaacagc gtgcactggt gcggccgggt cttcccaccg cggagccgca 661 tgtccttcac tgggcgtgtc ggggaagcgg tacttttact gtgaaaaaat tagagtgctc 721 taagcaggcc tatgctcgaa tacattagca tggaataata gaataggaca gtcgttctat 781 tttgttggtt tctaggacgt ctgtaatgat taacagaaac aatcgggggc gtcagtattg 841 catcgtcaga ggtgaaattc ttagatcgat gcaagactaa ctactgcgaa agcattcgcc 901 aagggtgttt tcattaatca ggaacgaaag ttaggggatc gaagacgatc agataccgtc 961 gtagtcttaa ccataaacta tgccgactag ggatcgggcg gtgtaatttt gacccgctcg 1021 gcacttacga gaaatcttaa gtgcttgggc tccaggggag tatggtcgca aggctgaaac 1081 ttaaagaaat gacgaagggc accaccaagg gtgaacctgc ggcctagttg actcacacgg 1141 gaaactcacg aggtaggaat atgtagatga cggatggagc cttcagaata catatgggca 1201 tgcgctctta ataccggtat tgaaaatggg cag Sample cultures of Sphaerodes mycoparasitica have been deposited with Saskatchewan Microbial Collection and Database under the accession number SMCD2220-01, and in the
- 13 -International Depositary Authority of Canada Collection (1015 Arlington Street, Winnipeg, Canada, R3E 3R2) under the accession number IDAC301008-01.
Sphaerodes mycoparasitica may be useful as an anti-fungal agent. S.
mycoparasitica may be used to treat, ameliorate, or otherwise control infections of Fusarium fungi. S. mycoparasitica seems particularly useful for treating, ameliorating or otherwise control infections of Fusarium avenaceum, Fusarium graminearum, and/or Fusarium oxysporum and improving plant health or growth.
S. mycoparasitica may be used as a prophylactic agent to diminish the chance of a fungal infection, particularly a Fusarium infection, from occurring.
S. mycoparasitica may be used for treating plants affected by Fusarium Wilt Disease or Fusarium Head Blight. S. mycoparasitica may be used for hindering such conditions from spreading or as a prophylactic measure to diminish the risk of such conditions from occurring.
In the present invention, S. mycoparasitica may be isolated from suitable sources. For example, S. mycoparasitica may be isolated from F. graminearum or F. avenaceum isolates originating from wheat fields by the method described in Vujanovic V, et. al.
Can. J. Microbiol.
48(9): 841-847 (2002). S. mycoparasitica is also available deposited with International Depositary Authority of Canada (IDAC301008-01), originally deposited October 30, 2008.
S. mycoparasitica may be used to treat, ameliorate, or otherwise control infections of Fusarium fungi. S. mycoparasitica may be applied in any suitable manner to the organism in need of treatment. For example, S. mycoparasitica isolates may be used directly or they may be further processed into soil-applied granular-based, peat-based, seed-applied, or anthesis-applied liquid-based inoculants.
S. mycoparasitica may be produced via fermentation and formulated into a pesticide composition. A suitable fermentation process includes, selection of fermentation medium (solid state or submerged), concentration of fermentation constituents, oxygen transfer, incubation temperature, time of harvest and post harvest treatments. The aim is to establish the optimum conditions to ensure an abundant, stable, and efficacious mycoparasitic, microbial population.
When formulating S. mycoparasitica into a pesticide composition, care should be taken to
Sphaerodes mycoparasitica may be useful as an anti-fungal agent. S.
mycoparasitica may be used to treat, ameliorate, or otherwise control infections of Fusarium fungi. S. mycoparasitica seems particularly useful for treating, ameliorating or otherwise control infections of Fusarium avenaceum, Fusarium graminearum, and/or Fusarium oxysporum and improving plant health or growth.
S. mycoparasitica may be used as a prophylactic agent to diminish the chance of a fungal infection, particularly a Fusarium infection, from occurring.
S. mycoparasitica may be used for treating plants affected by Fusarium Wilt Disease or Fusarium Head Blight. S. mycoparasitica may be used for hindering such conditions from spreading or as a prophylactic measure to diminish the risk of such conditions from occurring.
In the present invention, S. mycoparasitica may be isolated from suitable sources. For example, S. mycoparasitica may be isolated from F. graminearum or F. avenaceum isolates originating from wheat fields by the method described in Vujanovic V, et. al.
Can. J. Microbiol.
48(9): 841-847 (2002). S. mycoparasitica is also available deposited with International Depositary Authority of Canada (IDAC301008-01), originally deposited October 30, 2008.
S. mycoparasitica may be used to treat, ameliorate, or otherwise control infections of Fusarium fungi. S. mycoparasitica may be applied in any suitable manner to the organism in need of treatment. For example, S. mycoparasitica isolates may be used directly or they may be further processed into soil-applied granular-based, peat-based, seed-applied, or anthesis-applied liquid-based inoculants.
S. mycoparasitica may be produced via fermentation and formulated into a pesticide composition. A suitable fermentation process includes, selection of fermentation medium (solid state or submerged), concentration of fermentation constituents, oxygen transfer, incubation temperature, time of harvest and post harvest treatments. The aim is to establish the optimum conditions to ensure an abundant, stable, and efficacious mycoparasitic, microbial population.
When formulating S. mycoparasitica into a pesticide composition, care should be taken to
- 14 -selected ingredients that: (i) ensure stability during production, processing and storage, (ii) assist application, (iii) protect the pesticide from unfavourable environmental conditions, and (iv) promote pesticidal activity at the target. Exposure to inactivated host/pathogen or its compounds may improve the activity of S. mycoparasitica.
The formulations disclosed herein will usually comprise (i) an active ingredient, (ii) carriers - often an inert material used to support and deliver the densely populated active ingredient to the target, and optionally (iii) adjuvants - compounds that:
promote and sustain the function of the active ingredient by protection from UV radiation, that ensure rain fastness on the target, and that retain moisture or protect against desiccation, or promote the spread and dispersal of the biopesticide using standard agriculture equipments such as those disclosed by Hynes and Boyetchko (2006, Soil Biology & Biochemistry 38: 845-84).
Internal Transcribed Spacer ribosomal DNA from S. mycoparasitica was sequenced. A
560bp DNA sequence from the ITS region is given in SEQ ID NO: 3.
SEQ ID NO: 3 1 ttcgtttctt atcgcattgg tgaccagcgg agggtcatta cgaatcggac catttatgtc 61 atggctctgc caaccctgtg aactttatac ttgtacgttg cctcggcgga acctgccttt 121 tcggcaggcc gccggccggc atatacgcaa acgctctgaa aaagctccgc gctctatctg 181 aataataaaa ctttaacgag taaaaacttt tggcaacgga tctcttggct ctggcatcga 241 tgaaaaacgc agcgaaatgc gatacgtaat gtgaattgca gaattcagtg aaccatcgaa 301 tctttgaacg caccttgcgg ccgccggtaa tccggcggcc atgcccgtcc gagcgtcgtt 361 tccaccctcg ggagttetcc tcctaagaaa atttctcccg gccttgggcc agcgcgttgc 421 gcggctgccc gaccaacggc ggcaggaccg gcgatgtcct ctgtgccctg catttatata 481 aaactcgcat tggtccccgg taaggcttgc cttgcaacca acttctttag gtcgacctca 541 gatcggatag ggatacccgc
The formulations disclosed herein will usually comprise (i) an active ingredient, (ii) carriers - often an inert material used to support and deliver the densely populated active ingredient to the target, and optionally (iii) adjuvants - compounds that:
promote and sustain the function of the active ingredient by protection from UV radiation, that ensure rain fastness on the target, and that retain moisture or protect against desiccation, or promote the spread and dispersal of the biopesticide using standard agriculture equipments such as those disclosed by Hynes and Boyetchko (2006, Soil Biology & Biochemistry 38: 845-84).
Internal Transcribed Spacer ribosomal DNA from S. mycoparasitica was sequenced. A
560bp DNA sequence from the ITS region is given in SEQ ID NO: 3.
SEQ ID NO: 3 1 ttcgtttctt atcgcattgg tgaccagcgg agggtcatta cgaatcggac catttatgtc 61 atggctctgc caaccctgtg aactttatac ttgtacgttg cctcggcgga acctgccttt 121 tcggcaggcc gccggccggc atatacgcaa acgctctgaa aaagctccgc gctctatctg 181 aataataaaa ctttaacgag taaaaacttt tggcaacgga tctcttggct ctggcatcga 241 tgaaaaacgc agcgaaatgc gatacgtaat gtgaattgca gaattcagtg aaccatcgaa 301 tctttgaacg caccttgcgg ccgccggtaa tccggcggcc atgcccgtcc gagcgtcgtt 361 tccaccctcg ggagttetcc tcctaagaaa atttctcccg gccttgggcc agcgcgttgc 421 gcggctgccc gaccaacggc ggcaggaccg gcgatgtcct ctgtgccctg catttatata 481 aaactcgcat tggtccccgg taaggcttgc cttgcaacca acttctttag gtcgacctca 541 gatcggatag ggatacccgc
- 15 -The present invention provides a primer set, SmyITSF/R (SmyITSF is SEQ ID NO:
4 and SmyITSR is SEQ ID NO: 5), useful for identifying S. mycoparasitica.:
SEQ ID NO:4 5'-TCATGGCTCTGCCAACCCTGTAA-3' SEQ ID NO: 5 5'-AATGCAGGGCACAGAGGACATCG-3' The present primer is selective for S. mycoparasitica and can be used to assess and quantify the fungus in industrial products, plant materials, seed samples, and environmental samples by using PCR and RT-PCR technologies.
The SmyITSF/R primer set can be used for quantitative real-time PCR technology for analyzing gene expression, in fungal pathogens detection, and in quantification of fungi in living plants.
The SmyITSF/R primer set was tested with SMCD2220-01, seven Fusarium species, nine different ascomycetous fungal isolates, two zygomycete fungi, and three basidiomycetous fungal strains.
In PCR, this primer set only amplified SMCD2220-01, not the other fungi. Root biomass, total biomass, root length, total length, and seed germination of F.
graminearum infected spring wheat were significantly increased with the treatments of S. mycoparasitica, as compared to inoculation with F. graminearum. In further RT-PCR studies, SmyITSF/R specific primer was used for S. mycoparasitica in combination with F. graminearum-Fg12NFIR and Trichoderma-TGP4-FIR as a control, within which the primer showed high accuracy in assessing b i ocontrol-patho gen-pl ant interactions.
Total extracellular protein extracts from Sphaerodes mycoparasitica demonstrated significant inhibition of Fusarium spore germination. The present invention provides an antifungal agent comprising extracellular protein extracts from S.
mycoparasitica. Two proteins were isolated from the total protein extract, one having a molecular weight of 13 kDa and one having a molecular weight of 50 kDa. The present invention provides an antifungal agent comprising one or both of these proteins.
4 and SmyITSR is SEQ ID NO: 5), useful for identifying S. mycoparasitica.:
SEQ ID NO:4 5'-TCATGGCTCTGCCAACCCTGTAA-3' SEQ ID NO: 5 5'-AATGCAGGGCACAGAGGACATCG-3' The present primer is selective for S. mycoparasitica and can be used to assess and quantify the fungus in industrial products, plant materials, seed samples, and environmental samples by using PCR and RT-PCR technologies.
The SmyITSF/R primer set can be used for quantitative real-time PCR technology for analyzing gene expression, in fungal pathogens detection, and in quantification of fungi in living plants.
The SmyITSF/R primer set was tested with SMCD2220-01, seven Fusarium species, nine different ascomycetous fungal isolates, two zygomycete fungi, and three basidiomycetous fungal strains.
In PCR, this primer set only amplified SMCD2220-01, not the other fungi. Root biomass, total biomass, root length, total length, and seed germination of F.
graminearum infected spring wheat were significantly increased with the treatments of S. mycoparasitica, as compared to inoculation with F. graminearum. In further RT-PCR studies, SmyITSF/R specific primer was used for S. mycoparasitica in combination with F. graminearum-Fg12NFIR and Trichoderma-TGP4-FIR as a control, within which the primer showed high accuracy in assessing b i ocontrol-patho gen-pl ant interactions.
Total extracellular protein extracts from Sphaerodes mycoparasitica demonstrated significant inhibition of Fusarium spore germination. The present invention provides an antifungal agent comprising extracellular protein extracts from S.
mycoparasitica. Two proteins were isolated from the total protein extract, one having a molecular weight of 13 kDa and one having a molecular weight of 50 kDa. The present invention provides an antifungal agent comprising one or both of these proteins.
- 16 -Conventional molecular biological techniques may be used to isolate, characterize and produce the present proteins. For example, in order to identify the gene(s) for the present proteins, S. mycoparasitica could be challenged with F. oxysporum filtrates and upregulated mRNA isolated by standard Northern Blot. cDNA can be produced from the mRNA by Reverse Transcriptase PCR (RT-PCR). The cDNA can be amplified, purified and inserted into an appropriate vector. This vector may be inserted into an appropriate host cell.
Cloning and expression may focus on isolation of genes coding for antimicrobial proteins by designing primers for identified proteins. Isolated genes may be cloned in suitable expression vectors (yeast or bacteria) with suitable/efficient promoters in order to generate industrial strains for large-scale production of the concerned proteins. These vectors can be purchased from Promega, Invitrogen, Clontech, or other companies. The cloned genes may be tested for their protein expression, and the expressed proteins will be purified (using His-tag or other columns).
Purified proteins may be tested against plant-pathogenic fungi for their antifungal activity by disc-plate assay and/or microtitre plate assay methods (e.g., Drummond and Waigh, 2000, Recent Research Developments in Phytochemistry.4:143-152). Then, rDNA
technologies, involving gene mutation or addition of enhancers, introns or protein-specific promoters (GA
inducible), or addition deletion may be used to enhance the production of proteins.
Selected genes may be transformed into wheat and barley to create a transgenic lines with antifungal activity against pathogenic fungi using techniques known to those skilled in these arts (e.g., Sanghyun et.al., 200,8 J. Expt. Botany, 59, 2371-2378; Dennis et. al., 2007. Plant Cell Reports. 26: 631-639).
S. mycoparasitica has demonstrated an ability to degrade the deoxynivalenol (DON) mycotoxin. The present invention provides a method of degrading acetyldeoxynivalenol, especially 3-acetyldeoxynivalenol.
S. mycoparasitica could be challenged with pure DON and upregulated mRNA
isolated by standard Northern Blot. Reverse Transcriptase PCR could be used to identify the gene(s) for the upregulated proteins, an appropriate cDNA construct could then be inserted into an appropriate vector for protein production. Further, recombinant DNA Technology could be applied to create transgenetic plants with antimycotoxin properties.
Cloning and expression may focus on isolation of genes coding for antimicrobial proteins by designing primers for identified proteins. Isolated genes may be cloned in suitable expression vectors (yeast or bacteria) with suitable/efficient promoters in order to generate industrial strains for large-scale production of the concerned proteins. These vectors can be purchased from Promega, Invitrogen, Clontech, or other companies. The cloned genes may be tested for their protein expression, and the expressed proteins will be purified (using His-tag or other columns).
Purified proteins may be tested against plant-pathogenic fungi for their antifungal activity by disc-plate assay and/or microtitre plate assay methods (e.g., Drummond and Waigh, 2000, Recent Research Developments in Phytochemistry.4:143-152). Then, rDNA
technologies, involving gene mutation or addition of enhancers, introns or protein-specific promoters (GA
inducible), or addition deletion may be used to enhance the production of proteins.
Selected genes may be transformed into wheat and barley to create a transgenic lines with antifungal activity against pathogenic fungi using techniques known to those skilled in these arts (e.g., Sanghyun et.al., 200,8 J. Expt. Botany, 59, 2371-2378; Dennis et. al., 2007. Plant Cell Reports. 26: 631-639).
S. mycoparasitica has demonstrated an ability to degrade the deoxynivalenol (DON) mycotoxin. The present invention provides a method of degrading acetyldeoxynivalenol, especially 3-acetyldeoxynivalenol.
S. mycoparasitica could be challenged with pure DON and upregulated mRNA
isolated by standard Northern Blot. Reverse Transcriptase PCR could be used to identify the gene(s) for the upregulated proteins, an appropriate cDNA construct could then be inserted into an appropriate vector for protein production. Further, recombinant DNA Technology could be applied to create transgenetic plants with antimycotoxin properties.
-17-The present invention provides antifungal compositions comprising S.
mycoparasitica, isolates, cultures, or proteins thereof. The present invention also provides a method of controlling a fungal disease of a plant, and a method for mycotoxin detoxification, which methods comprise applying to the locus of the plant S. mycoparasitica, isolates, cultures, or proteins thereof, collectively hereinafter referred to as the `antifungal agent'.
The compositions of the invention may, for example, be applied to the seeds of the plants, to the growth medium (e.g. soil or water), or to the foliage of the plants.
The present invention provides a composition comprising the antifungal agent and an agriculturally acceptable carrier or diluent, which will ensure stability and performance of the final product. Carrier or diluent should compatible with the active ingredient, agriculturally acceptable, have a good absorptive capacity and a suitable bulk density, allowing easy particle dispersion and attachment.
The compositions herein may be applied, as in aqueous sprays, granules and dust formulations in accordance with established practice in the art. An aqueous spray is usually prepared by mixing a wettable powder or emulsifiable concentrate formulation of a compound of the invention with a relatively large amount of water to form a dispersion.
Wettable powders may comprise a finely divided mixture of the antifungal agent, a solid carrier, and a surface-active agent. The solid carrier is usually chosen from among attapulgite clays, kaolin clays, montmorillonite clays, diatomaceous earths, finely divided silica, purified silicates, or combinations thereof. Surfactants which may be useful herein have wetting, penetrating, and/or dispersing ability. They are typically present in an amount of from about 0.5% to about 10% by weight. Surfactants herein may be chosen from, for example, alkylbenzenesulfonates, alkyl sulfates, naphthalenesulfonates and condensed naphthalenesulfonates, sulfonated lignins, and non-ionic surfactants.
Emulsifiable concentrates may comprise the antifungal agent of the invention in a liquid carrier, the carrier being a mixture of a water-immiscible solvent and a surfactant. Solvents that may be useful herein include aromatic hydrocarbon solvents such as the xylenes,
mycoparasitica, isolates, cultures, or proteins thereof. The present invention also provides a method of controlling a fungal disease of a plant, and a method for mycotoxin detoxification, which methods comprise applying to the locus of the plant S. mycoparasitica, isolates, cultures, or proteins thereof, collectively hereinafter referred to as the `antifungal agent'.
The compositions of the invention may, for example, be applied to the seeds of the plants, to the growth medium (e.g. soil or water), or to the foliage of the plants.
The present invention provides a composition comprising the antifungal agent and an agriculturally acceptable carrier or diluent, which will ensure stability and performance of the final product. Carrier or diluent should compatible with the active ingredient, agriculturally acceptable, have a good absorptive capacity and a suitable bulk density, allowing easy particle dispersion and attachment.
The compositions herein may be applied, as in aqueous sprays, granules and dust formulations in accordance with established practice in the art. An aqueous spray is usually prepared by mixing a wettable powder or emulsifiable concentrate formulation of a compound of the invention with a relatively large amount of water to form a dispersion.
Wettable powders may comprise a finely divided mixture of the antifungal agent, a solid carrier, and a surface-active agent. The solid carrier is usually chosen from among attapulgite clays, kaolin clays, montmorillonite clays, diatomaceous earths, finely divided silica, purified silicates, or combinations thereof. Surfactants which may be useful herein have wetting, penetrating, and/or dispersing ability. They are typically present in an amount of from about 0.5% to about 10% by weight. Surfactants herein may be chosen from, for example, alkylbenzenesulfonates, alkyl sulfates, naphthalenesulfonates and condensed naphthalenesulfonates, sulfonated lignins, and non-ionic surfactants.
Emulsifiable concentrates may comprise the antifungal agent of the invention in a liquid carrier, the carrier being a mixture of a water-immiscible solvent and a surfactant. Solvents that may be useful herein include aromatic hydrocarbon solvents such as the xylenes,
- 18-alkylnaphthalenes, petroleum distillates, terpene solvents, ether-alcohols, organic ester solvents or suitable combinations thereof.
When a composition of the invention is to be applied to plant debris or litter, in order to control of the source of contamination and inoculant dispersion, or to the soil, as for pre-emergence protection, granular formulations or dusts are sometimes more convenient than sprays.
In one embodiment the antifungal agents herein are encapsulated into alginate pellets.
The pellets may be prepared in any suitable manner. For example, one useful method is described in Harveson et. al., (2002, Plant Disease; Vol. 86, No. 9 1025-1030).
The composition may comprise other active substances useful in an antifungal agent. For example, the compositions herein may comprise other antifungal agents such as Trichoderma, sulfur, neem oil, rosemary oil, jojoba oil, Bacillus subtilis, allylamines (e.g. terbinafine, antimetabolites (e.g. flucytosine), azoles (e.g. ketoconazole, itraconazole), echinocandins (e.g.
caspofungin), polyenes (e.g. amphotericin B), systemic agents (e.g.
griseofluvin), or combinations thereof.
The compositions herein may contain from about 0.1% to about 95%, by weight, of the antifungal agent and from about 0.1% to about 95%, by weight, of the carrier and/or surfactant.
The direct application to plant seeds prior to planting may be accomplished in some instances by mixing either a powdered solid compound of the invention or a dust formulation with seed to obtain a substantially uniform coating which is very thin and represents only one or two percent by weight or less, based on the weight of the seed. In some instances, however, a non-phytotoxic solvent such as methanol is conveniently employed as a carrier to facilitate the uniform distribution of the compound of the invention on the surface of the seed.
The compositions herein may be useful in the treatment of fungal infections in plants.
Consequently, the compositions herein may comprise S. mycoparasitica, isolates, cultures, or proteins thereof and a pharmaceutically acceptable carrier.
S. mycoparasitica sporulates in the presence of Fusarium. S. mycoparasitica ascospores proved resistant to germination under different standard laboratory conditions (sterile distilled
When a composition of the invention is to be applied to plant debris or litter, in order to control of the source of contamination and inoculant dispersion, or to the soil, as for pre-emergence protection, granular formulations or dusts are sometimes more convenient than sprays.
In one embodiment the antifungal agents herein are encapsulated into alginate pellets.
The pellets may be prepared in any suitable manner. For example, one useful method is described in Harveson et. al., (2002, Plant Disease; Vol. 86, No. 9 1025-1030).
The composition may comprise other active substances useful in an antifungal agent. For example, the compositions herein may comprise other antifungal agents such as Trichoderma, sulfur, neem oil, rosemary oil, jojoba oil, Bacillus subtilis, allylamines (e.g. terbinafine, antimetabolites (e.g. flucytosine), azoles (e.g. ketoconazole, itraconazole), echinocandins (e.g.
caspofungin), polyenes (e.g. amphotericin B), systemic agents (e.g.
griseofluvin), or combinations thereof.
The compositions herein may contain from about 0.1% to about 95%, by weight, of the antifungal agent and from about 0.1% to about 95%, by weight, of the carrier and/or surfactant.
The direct application to plant seeds prior to planting may be accomplished in some instances by mixing either a powdered solid compound of the invention or a dust formulation with seed to obtain a substantially uniform coating which is very thin and represents only one or two percent by weight or less, based on the weight of the seed. In some instances, however, a non-phytotoxic solvent such as methanol is conveniently employed as a carrier to facilitate the uniform distribution of the compound of the invention on the surface of the seed.
The compositions herein may be useful in the treatment of fungal infections in plants.
Consequently, the compositions herein may comprise S. mycoparasitica, isolates, cultures, or proteins thereof and a pharmaceutically acceptable carrier.
S. mycoparasitica sporulates in the presence of Fusarium. S. mycoparasitica ascospores proved resistant to germination under different standard laboratory conditions (sterile distilled
- 19 -water, on water agar and commercially available media) and heat or cold-shock treatments. In contrast, spore germination was obtained on general potato dextrose agar medium amended with Fusarium-filtrates. Significant improvement in percentage of spore germinations were obtained for the spores suspended in Fusarium-filtrates. F. avenaceum and E oxysporum filtrates induced the highest germination, whereas E. sporotrichioides and F. proliferatum triggered lower germination frequency. Filtrates of beneficial fungal inoculants: Trichoderma harzianum (RootShield available from BioWorks Inc., Victor, NY, USA; RootShield is a registered trademark of BioWorks Inc.), Penicillium bilaii (JumpStart available from Novozymes Biologicals Ltd., Saskatoon, SK, CA; JumpStart is a registered trademark of Philom Bios Inc.), and Chaetomium globosum had no impact on germination. Ascospores suspended in F. avenaceum-filtrate, showed double-polar germination pattern. On the other hand, when suspended in F. oxysporum-filtrate, significant amount of spores demonstrated a single-polar germination pattern. S. mycoparasitia grown on F oxysporum kept the same mycoparasitic germination patterns in three offspring generations when transferred on E
avenaceum which indicate a stable genome-regulated expression.
The present invention provides a method of sporulating S. mycoparasitica by exposing them to F. avenaceum or E oxysporum, or filtrates, extracts, or compositions thereof.
The present invention provides a method for producing an antifungal composition comprising S. mycoparasitica or isolates, cultures, genes or proteins thereof.
The method comprises:
(a) inducing S. mycoparasitica sporulation;
(b) culturing S. mycoparasitica;
(c) harvesting S. mycoparasitica.
The method may further comprise any of the following optional steps:
(d) storing/preserving S. mycoparasitica;
(e) applying S. mycoparasitica;
(0 assessing S. mycoparasitica;
(g) producing S. mycoparasitica proteins;
(h) harvesting S. mycoparasitica proteins;
avenaceum which indicate a stable genome-regulated expression.
The present invention provides a method of sporulating S. mycoparasitica by exposing them to F. avenaceum or E oxysporum, or filtrates, extracts, or compositions thereof.
The present invention provides a method for producing an antifungal composition comprising S. mycoparasitica or isolates, cultures, genes or proteins thereof.
The method comprises:
(a) inducing S. mycoparasitica sporulation;
(b) culturing S. mycoparasitica;
(c) harvesting S. mycoparasitica.
The method may further comprise any of the following optional steps:
(d) storing/preserving S. mycoparasitica;
(e) applying S. mycoparasitica;
(0 assessing S. mycoparasitica;
(g) producing S. mycoparasitica proteins;
(h) harvesting S. mycoparasitica proteins;
- 20 -(i) fractionating S. mycoparasitica proteins;
(j) separating S. mycoparasitica proteins;
(k) storing/preserving S. mycoparasitica proteins;
(1) applying S. mycoparasitica proteins;
(m) assessing S. mycoparasitica proteins.
EXAMPLES
Example 1 Sampling, fungal growth and microscopy Myclobutanil-agar (MBA) medium was used for selective isolation of various Fusarium taxa and associated biotrophic mycoparasites from Canadian agriculture fields using the method described in Vujanovic V, et. al. (2002, Can. J. Microbiol. 48(9): 841-847).
Sphaerodes was recovered occasionally from F. graminearum and abundantly from F. avenaceum isolates originating from wheat fields in Saskatchewan; it was also isolated from Fusarium axysporum from asparagus fields in Quebec, Canada. A monosporal, single culture of the mycoparasite was obtained from each Fusarium species according to the method proposed by Harveson &
Kimbrough (2001, Int. J Plant Sci. 162(2):403-410). Single ascospore isolates were maintained on Potato dextrose agar (PDA) (Difco, BD Biosciences, Mississauga, ON, CA) supplemented with antibiotics (100 ug 11 streptomycin sulphate and 13 1,1g 1-1 neomycin sulphate;
Sigma-Aldrich Canada Ltd., Oakville, ON, CA) and stored at -80 C in Saskatchewan Microbial Collection and Database (SMCD2220-01) and in the International Depositary Authority of Canada (IDAC301008-01) collections. Fungal growth was assessed on modified Leonian's agar (MLA) and Potato dextrose agar (PDA) media. Biotrophic interactions between Spha erodes and Fusarium strains were examined with the slide culture method proposed by Jordan & Barnett (1978, Mycologia 70(2):300-312). Morphological studies of ascomata, ascospores, mycelia, and anamorphic structures were performed after two weeks of incubation (21 C - 22 C) under a Carl Zeiss Axioskop2TM with a Carl Zeiss AxioCam ICc1TM camera. Fungal materials for microscopic observation were mounted in lactofuchsin and lactophenol cotton blue dyes.
(j) separating S. mycoparasitica proteins;
(k) storing/preserving S. mycoparasitica proteins;
(1) applying S. mycoparasitica proteins;
(m) assessing S. mycoparasitica proteins.
EXAMPLES
Example 1 Sampling, fungal growth and microscopy Myclobutanil-agar (MBA) medium was used for selective isolation of various Fusarium taxa and associated biotrophic mycoparasites from Canadian agriculture fields using the method described in Vujanovic V, et. al. (2002, Can. J. Microbiol. 48(9): 841-847).
Sphaerodes was recovered occasionally from F. graminearum and abundantly from F. avenaceum isolates originating from wheat fields in Saskatchewan; it was also isolated from Fusarium axysporum from asparagus fields in Quebec, Canada. A monosporal, single culture of the mycoparasite was obtained from each Fusarium species according to the method proposed by Harveson &
Kimbrough (2001, Int. J Plant Sci. 162(2):403-410). Single ascospore isolates were maintained on Potato dextrose agar (PDA) (Difco, BD Biosciences, Mississauga, ON, CA) supplemented with antibiotics (100 ug 11 streptomycin sulphate and 13 1,1g 1-1 neomycin sulphate;
Sigma-Aldrich Canada Ltd., Oakville, ON, CA) and stored at -80 C in Saskatchewan Microbial Collection and Database (SMCD2220-01) and in the International Depositary Authority of Canada (IDAC301008-01) collections. Fungal growth was assessed on modified Leonian's agar (MLA) and Potato dextrose agar (PDA) media. Biotrophic interactions between Spha erodes and Fusarium strains were examined with the slide culture method proposed by Jordan & Barnett (1978, Mycologia 70(2):300-312). Morphological studies of ascomata, ascospores, mycelia, and anamorphic structures were performed after two weeks of incubation (21 C - 22 C) under a Carl Zeiss Axioskop2TM with a Carl Zeiss AxioCam ICc1TM camera. Fungal materials for microscopic observation were mounted in lactofuchsin and lactophenol cotton blue dyes.
-21 -DNA extraction, amplification and sequencing Three Sphaerodes strains: SMCD 2220-01 on F. avenaceum from wheat, SMCD 2220-on F. graminearum from wheat, and SMCD 2220-03 on F. oxysporum from asparagus were cultured on PDA medium at 21 C for a week prior to DNA extraction. Genomic DNA
was extracted with the DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA;
DNEasy is a registered trademark of Qiagen GmbH Corp, Hilden, Fed. Rep. Germany). LSU
(large subunit) rDNA fragments were amplified using primer sets NS1/NS6 using techniques known to those skilled in these arts (e.g., Gardes & Bruns, 1993, Molecular Ecology 2: 113-118; White et al.
1990, PCR Protocols: a guide to methods and application: 315-322. Academic Press, New York) and LS1/LR5 (e.g., Hausner et al. 1993, Canadian Journal of Botany 71:
52-63; Rehner &
Samuels, 1995, Canadian Journal of Botany 73 (Suppl. 1): S816-S823; Zhang &
Blackwell, 2002, Mycological Research 106: 148-155). Target regions of fungal genomic DNA
samples were amplified using polymerase chain reaction (PCR) in a 250 reaction mixture containing 2.50 of 10X buffer, 50 of Q buffer, 0.5 1 10mM dNTPs, ltl of each primer, 0.130 of 0.625 unit of Taq DNA Polymerase, 2 1 of extracted fungal DNA, and 12.871.11 of sterilized ultra-pure MilliporeTM water. The Qiagen Taq PCR core kitsTM were purchased from Qiagen Inc., Mississauga, ON, CA. Purified DNA PCR products were sequenced.
Sequence alignment and phylo genetic analyses Sequences of LSU from this study and sequences retrieved from GenBank were aligned using Clustal X software (version 1.82) (Thompson et al. 1997, Nucleic Acids Research 24:
4876-4882), and edited in Bioedit (Hall, 1999, Nucleic Acids Symposium Series 41: 95-98).
Distance trees were produced with PAUP (Phylogenetic Analysis Using Parsimony) 4.0b10 software (Swofford 2000) using a neighbor-joining approach, and validated using bootstrap analyses with 1,000 repetitions. A fungal distance tree was prepared with sequences showing bootstrap values higher than 50%. Trees were rooted with sequences Xylaria hypoxylon U47841.
Taxonomy: Sphaerodes mycoparasitica Vujanovic, sp. nov. (Figures 1-5) [MycoBank no: MB
515144], in the International Depositary Authority of Canada as Sphaerodes mycoparasitica strain IDAC 301008-01.
was extracted with the DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA;
DNEasy is a registered trademark of Qiagen GmbH Corp, Hilden, Fed. Rep. Germany). LSU
(large subunit) rDNA fragments were amplified using primer sets NS1/NS6 using techniques known to those skilled in these arts (e.g., Gardes & Bruns, 1993, Molecular Ecology 2: 113-118; White et al.
1990, PCR Protocols: a guide to methods and application: 315-322. Academic Press, New York) and LS1/LR5 (e.g., Hausner et al. 1993, Canadian Journal of Botany 71:
52-63; Rehner &
Samuels, 1995, Canadian Journal of Botany 73 (Suppl. 1): S816-S823; Zhang &
Blackwell, 2002, Mycological Research 106: 148-155). Target regions of fungal genomic DNA
samples were amplified using polymerase chain reaction (PCR) in a 250 reaction mixture containing 2.50 of 10X buffer, 50 of Q buffer, 0.5 1 10mM dNTPs, ltl of each primer, 0.130 of 0.625 unit of Taq DNA Polymerase, 2 1 of extracted fungal DNA, and 12.871.11 of sterilized ultra-pure MilliporeTM water. The Qiagen Taq PCR core kitsTM were purchased from Qiagen Inc., Mississauga, ON, CA. Purified DNA PCR products were sequenced.
Sequence alignment and phylo genetic analyses Sequences of LSU from this study and sequences retrieved from GenBank were aligned using Clustal X software (version 1.82) (Thompson et al. 1997, Nucleic Acids Research 24:
4876-4882), and edited in Bioedit (Hall, 1999, Nucleic Acids Symposium Series 41: 95-98).
Distance trees were produced with PAUP (Phylogenetic Analysis Using Parsimony) 4.0b10 software (Swofford 2000) using a neighbor-joining approach, and validated using bootstrap analyses with 1,000 repetitions. A fungal distance tree was prepared with sequences showing bootstrap values higher than 50%. Trees were rooted with sequences Xylaria hypoxylon U47841.
Taxonomy: Sphaerodes mycoparasitica Vujanovic, sp. nov. (Figures 1-5) [MycoBank no: MB
515144], in the International Depositary Authority of Canada as Sphaerodes mycoparasitica strain IDAC 301008-01.
- 22 -Coloniae in agaro potato dextrosum lentior crescents, 4.0 cm ad 7d, floccose, pallido-brunneis. Hyphis septatis, ramosis, anastomosantibus, laevibus, palide fulvis, 2.5-5.0 pm diam compositum. Ascomata superficialia vel immersa, pyriformia vet globosa, ostiolata, flavo-brunnea, 250-300 pm longa, 200-280pm diam. Co//um nul, conicum vet cylindricum, 30-75p.m longum, (0-) 50-70 pm latum ad basim. Peridiurn membranaceum, cellulis 8-15 pm, e 3-6 stratis, 8-15 pm crassum, textura angulari compositum. Setae rectae vet parum curvae, hyalinae vel dilute flavae, crassitunicatae, 10-40 pm longae, septatae. Asci 8-spori, ovoidei vet clavati, 50-75 X 17-25 pm, superne late rotundati, brevistipitati, tenuitunicati, evanescentes.
Paraphysis nullis. Ascosporae unicellulares, irregulariter biseriatae, primum hyalinae, deinde brunneae vet atrobrunneae, crassitunicatae, fusiformes, 18-24 X 9-12 m, reticulatae, costis protrudentibus, e polo visae polygonales, utrinque umbonatae, foramine germinali praeditae.
Phialidis hyalinis, status conidialis.
Culture characteristics: Colonies of Sphaerodes rnycoparasitica strain IDAC
cultured on MLA grew more rapidly than on PDA, 1.1 cm versus 0.6 cm per day (21-22 C), consisting of slightly submerged mycelium and aerial hyphae, granulose due to production of ample number of ascomata. On MLA (Figures 1A and 1C) and PDA Figures 1B and 1D), the cultures produced a woolly mycelium, yellowish to pinky-brownish on both sides. At 37 C, no growth was registered. Hyphae were white to pale yellow, 2.5-5.0 pm diam., septate, anastomosis occurred soon after ascospore germination (Figure 2). Colonies on Potato dextrose agar (PDA) spread with abundant, white to pale yellow aerial mycelium and low number of ascomata. Ascomata, perithecial or cleistotecial, scattered or aggregated in small groups, superficial, pyriform to globose, ostiolate (when mature), light to dark yellowish brown, translucent, appearing black due to mass of mature ascospores, 250-300 !Am high, 200-280 m diam. Neck absent to short conical or cylindrical 25-75 pm long, 20-70 gm wide at the base, sometimes surrounded with a crown of short, upright setae, 10-40 pm long.
Peridium membranaceous, 3-6-layered, 8-10 pm thick, translucent, pale yellow to light brown, composed by cells of 8-15 p.m diam. disposed in textura angularis.. Asci 8-spored, clavate, 50-75 X 17-25 pm, rounded at apex, without apical structures, thin-walled and evanescent when mature.
Paraphyses absent. Ascospores irregularly arranged inside the asci, at first hyaline but becoming brown to dark brown, thick-walled, single-celled, fusiform to rarely triangular, 18-24 X 9-12 pm,
Paraphysis nullis. Ascosporae unicellulares, irregulariter biseriatae, primum hyalinae, deinde brunneae vet atrobrunneae, crassitunicatae, fusiformes, 18-24 X 9-12 m, reticulatae, costis protrudentibus, e polo visae polygonales, utrinque umbonatae, foramine germinali praeditae.
Phialidis hyalinis, status conidialis.
Culture characteristics: Colonies of Sphaerodes rnycoparasitica strain IDAC
cultured on MLA grew more rapidly than on PDA, 1.1 cm versus 0.6 cm per day (21-22 C), consisting of slightly submerged mycelium and aerial hyphae, granulose due to production of ample number of ascomata. On MLA (Figures 1A and 1C) and PDA Figures 1B and 1D), the cultures produced a woolly mycelium, yellowish to pinky-brownish on both sides. At 37 C, no growth was registered. Hyphae were white to pale yellow, 2.5-5.0 pm diam., septate, anastomosis occurred soon after ascospore germination (Figure 2). Colonies on Potato dextrose agar (PDA) spread with abundant, white to pale yellow aerial mycelium and low number of ascomata. Ascomata, perithecial or cleistotecial, scattered or aggregated in small groups, superficial, pyriform to globose, ostiolate (when mature), light to dark yellowish brown, translucent, appearing black due to mass of mature ascospores, 250-300 !Am high, 200-280 m diam. Neck absent to short conical or cylindrical 25-75 pm long, 20-70 gm wide at the base, sometimes surrounded with a crown of short, upright setae, 10-40 pm long.
Peridium membranaceous, 3-6-layered, 8-10 pm thick, translucent, pale yellow to light brown, composed by cells of 8-15 p.m diam. disposed in textura angularis.. Asci 8-spored, clavate, 50-75 X 17-25 pm, rounded at apex, without apical structures, thin-walled and evanescent when mature.
Paraphyses absent. Ascospores irregularly arranged inside the asci, at first hyaline but becoming brown to dark brown, thick-walled, single-celled, fusiform to rarely triangular, 18-24 X 9-12 pm,
- 23 -reticulate to rarely smooth, with irregular transverse sections, and with a strongly umbonate germ pore at each end. Phialides hyaline, ampulliform produced directly on ascomata or on hyphae surrounding ascomata, and on irregularly branched conidiophores.
S. mycoparasitica strain IDAC 301008-01 has a unique combination of features shown in Figure 3. The ascomata height is generally less than 250 11M with a conical to cylindrical neck (Figure 3A). The setae length is generally less than 40 1.tm (Figure 3B) and the spore length is generally less than 23 [tm (Figure 3C). The spores show a conspicuous wall ornamentation and prominent irregular longitudinal ribs indicated by the arrows in Figure 4A.
The spores of Sphaerodes quadrangularis are shown in Figure 4B for comparison. S.
mycoparasitica strain IDAC 301008-01 spores occaisonally show a triangular shape (Figures 3D and 3E). The formation of starting and mature ascoma of S mycoparasitica strain IDAC 301008-01 are shown in Figure 3H. This strain produces simple phialides on the surface of ascoma peridial walls or alternatively, the phialides may be scattered irregularly on ascoma surrounding the hyphae, and on conidiosphores with a distinctive branching pattern (Figures 3F
and 3G). S.
mycoparasitica strain IDAC 301008-01 forms hook-like structures for parasitizing living hyphae of Fusarium (Figures 31 and 3J).
Example 2 Sphaerodes mycoparasitica (SMCD 2220-01) 21 C isolates were cultured in potato dextrose broth (PDB) culture media. About 3 ml of culture were transferred to 250 ml Ehrlenmeyer flasks containing 50 ml PDB growth medium. The flasks were incubated for 7 days on a rotary shaker (150 rpm) at room temperature.
Extracellular protein extraction: Young mycelia were filtered through Whatman No.1 filter paper (Whatman is a registered trademark of Whatman International Ltd., Kent, UK). Filtered culture medium, containing extracellular proteins, and were concentrated by Amicon ultrafiltration centrifuge tube with a 3000 Dalton cut-off membrane by centrifugation at 4000 rpm at 4 C.
Disc diffusion assay: Antifungal activities of extracellular protein extracts were tested under sterile conditions by radial disc plate diffusion assay as described by Roberts & Selitrennikoff
S. mycoparasitica strain IDAC 301008-01 has a unique combination of features shown in Figure 3. The ascomata height is generally less than 250 11M with a conical to cylindrical neck (Figure 3A). The setae length is generally less than 40 1.tm (Figure 3B) and the spore length is generally less than 23 [tm (Figure 3C). The spores show a conspicuous wall ornamentation and prominent irregular longitudinal ribs indicated by the arrows in Figure 4A.
The spores of Sphaerodes quadrangularis are shown in Figure 4B for comparison. S.
mycoparasitica strain IDAC 301008-01 spores occaisonally show a triangular shape (Figures 3D and 3E). The formation of starting and mature ascoma of S mycoparasitica strain IDAC 301008-01 are shown in Figure 3H. This strain produces simple phialides on the surface of ascoma peridial walls or alternatively, the phialides may be scattered irregularly on ascoma surrounding the hyphae, and on conidiosphores with a distinctive branching pattern (Figures 3F
and 3G). S.
mycoparasitica strain IDAC 301008-01 forms hook-like structures for parasitizing living hyphae of Fusarium (Figures 31 and 3J).
Example 2 Sphaerodes mycoparasitica (SMCD 2220-01) 21 C isolates were cultured in potato dextrose broth (PDB) culture media. About 3 ml of culture were transferred to 250 ml Ehrlenmeyer flasks containing 50 ml PDB growth medium. The flasks were incubated for 7 days on a rotary shaker (150 rpm) at room temperature.
Extracellular protein extraction: Young mycelia were filtered through Whatman No.1 filter paper (Whatman is a registered trademark of Whatman International Ltd., Kent, UK). Filtered culture medium, containing extracellular proteins, and were concentrated by Amicon ultrafiltration centrifuge tube with a 3000 Dalton cut-off membrane by centrifugation at 4000 rpm at 4 C.
Disc diffusion assay: Antifungal activities of extracellular protein extracts were tested under sterile conditions by radial disc plate diffusion assay as described by Roberts & Selitrennikoff
- 24 -(1986, Biochim. Biophys. Acta, 880: 161-170). The assay of the isolated protein for antifungal activity toward F. oxysporum and F. graminearurn was carried out in petri plates containing potato dextrose agar. Mycelial plugs from actively growing fungal plates were placed in the center of the petri plates and sterile filter paper discs (5-mm diameter of Whatman filter paper no. 1) were placed on the agar surface at a distance of 0.5 cm away from the rim of the mycelial colony. An aliquot (60 !AL) containing 2.5 fig of extracellular protein was added to a disk. Sterile distilled water and buffer served as controls. The plates were then incubated at room temperature for 4 days and examined for inhibition. The area of the mycelial colony was measured and the inhibition of fungal growth was determined by calculating the % reduction in area of mycelial colony with the controls (Figure 5A). After 4th day of germination, about 30%
inhibition of the hyphal extension of F. oxysporum and 35% inhibition of hyphal extension of F.
graminearum were observed (Figure 5B) Fast protein liquid chromatography (FPLC) of extracellular proteins: Proteins were fractionated through Superdex 75 GL 10/30 column using FPLC AKTA purifier system (GE
Healthcare, Biosciences AB, CA: AKTA is a registered trademark of GE Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden) according to the manufacturer's instructions. The column was previously equilibrated with sterile water and with 50 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaC1, followed by protein injection (about 500 IAL) and elution of proteins with the same buffer with flow rate of 1.0 ml/min. Fractions of 0.8 ml were collected in each tube. Upon gel filtration on Superdex 75 (Superdex is a registered trademark of GE
Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden), proteins were resolved into two distinct peaks (fl and 12) and few smaller peaks Figure 6). Samples from all the peak fractions were pooled, precipitated and tested for their antifungal activity.
Sodium dodecyl sulfate-polyactylamide gel electrophoresis (SDS-PAGE): SDS-PAGE
(12%) of proteins recovered from FPLC fractions was performed according to the method of Laertunli (1970, Nature 227: 680-685). All peaks giving FPLC fractions were pooled and recovered by precipitation in 1:4 volume of chilled acetone and kept at -20 C overnight.
After centrifugation at 12,000 g for 10 min, precipitated proteins (pellets) were dissolved in minimum amount (30 IAL) of assay buffer. Proteins were analyzed by SDS-PAGE having 5% stacking gel (pH 6.8) and 12% separating acrylamide gel (pH 8.8) in Tris-glycine buffer (pH 8.3) and appropriate markers.
inhibition of the hyphal extension of F. oxysporum and 35% inhibition of hyphal extension of F.
graminearum were observed (Figure 5B) Fast protein liquid chromatography (FPLC) of extracellular proteins: Proteins were fractionated through Superdex 75 GL 10/30 column using FPLC AKTA purifier system (GE
Healthcare, Biosciences AB, CA: AKTA is a registered trademark of GE Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden) according to the manufacturer's instructions. The column was previously equilibrated with sterile water and with 50 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaC1, followed by protein injection (about 500 IAL) and elution of proteins with the same buffer with flow rate of 1.0 ml/min. Fractions of 0.8 ml were collected in each tube. Upon gel filtration on Superdex 75 (Superdex is a registered trademark of GE
Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden), proteins were resolved into two distinct peaks (fl and 12) and few smaller peaks Figure 6). Samples from all the peak fractions were pooled, precipitated and tested for their antifungal activity.
Sodium dodecyl sulfate-polyactylamide gel electrophoresis (SDS-PAGE): SDS-PAGE
(12%) of proteins recovered from FPLC fractions was performed according to the method of Laertunli (1970, Nature 227: 680-685). All peaks giving FPLC fractions were pooled and recovered by precipitation in 1:4 volume of chilled acetone and kept at -20 C overnight.
After centrifugation at 12,000 g for 10 min, precipitated proteins (pellets) were dissolved in minimum amount (30 IAL) of assay buffer. Proteins were analyzed by SDS-PAGE having 5% stacking gel (pH 6.8) and 12% separating acrylamide gel (pH 8.8) in Tris-glycine buffer (pH 8.3) and appropriate markers.
-25-Prior to SDS-electrophoresis, the protein was mixed with an equal volume of sample buffer (60 mM Tris¨HC1 buffer, 4% SDS, pH 6.8) containing 5% p-mercaptoethanol. A mixture of standard marker proteins (Bio-Rad protein markers, Bio-Rad Laboratories Inc., Mississauga, ON, CA) was used. V All samples were heated for 5 min at 95 C and cooled to room temperature before loading on gel. Proteins were visualized by silver staining method (Bio-Rad Silver staining kit, Bio-Rad Laboratories Inc., Mississauga, ON, CA). Proteins molecular masses were estimated by comparison with the mobilities of standard molecular mass markers. Protein bands of molecular weight 13 kDa and 50 kDa were detected in fl and f2 peaks respectively (Figure 7).
Microtitre plate assay: Percentage inhibition of spore germination was performed by microtitre plate assay method (Ghosh, 2006, Ann. Bot 98: 1145-1153; Yadav et al. 2007, J.
Med.
Microbiol. 56: 637-644) to test the antifungal activity of proteins recovered from FPLC
fractions. The possible toxicity of the fractionated proteins was tested by a percentage growth inhibition assay using the F. oxysporum and F. graminearum. The in vitro antifungal activities of fractionated proteins were determined in 96-well microtiter plates. In microplate wells, 10 1.11 of potato dextrose broth (PDB; BD Biosciences, Mississauga, ON, CA) was mixed with 3 pl of spore suspensions of F. oxysporum and F. graminearum. An aliquot of 7 IAL of different peak containing proteins fractions were added to suspensions in microtitre plates (12-8 wells). Water 25 and buffer were used as negative controls. The microtitre plate was then incubated at room temperature in dark. Observations were made for inhibition in spore germination in both untreated and treated wells after 24 h using inverted and fluorescent microscope. The number of germinated and non-germinated spores and percentage of area covered by mycelia in microscope were used to determine the percentage of growth inhibition. The fl and f2 protein-containing peaks had inhibitory effects on spore germination after 24 hrs compared to the control treatment.
The discharge of spores from sporangia was inhibited by these protein fractions. Secondary branching (mycelia formation) was observed in control treated spores of F.
oxysporum (Figure 8A-c) and F. graminearum (Figure 8B-c). No branching was observed in fl and f2 protein-treated spores of F. oxysporum (Figures 8A-a-b) and F. graminearum (Figure 8B-a-b). The size of germ tubes (hyphae) from control-treated spores was more than 100 pm compared to 10-15 pm for protein-treated spores (Figures 8A-a-b; 8B-a-b).
Microtitre plate assay: Percentage inhibition of spore germination was performed by microtitre plate assay method (Ghosh, 2006, Ann. Bot 98: 1145-1153; Yadav et al. 2007, J.
Med.
Microbiol. 56: 637-644) to test the antifungal activity of proteins recovered from FPLC
fractions. The possible toxicity of the fractionated proteins was tested by a percentage growth inhibition assay using the F. oxysporum and F. graminearum. The in vitro antifungal activities of fractionated proteins were determined in 96-well microtiter plates. In microplate wells, 10 1.11 of potato dextrose broth (PDB; BD Biosciences, Mississauga, ON, CA) was mixed with 3 pl of spore suspensions of F. oxysporum and F. graminearum. An aliquot of 7 IAL of different peak containing proteins fractions were added to suspensions in microtitre plates (12-8 wells). Water 25 and buffer were used as negative controls. The microtitre plate was then incubated at room temperature in dark. Observations were made for inhibition in spore germination in both untreated and treated wells after 24 h using inverted and fluorescent microscope. The number of germinated and non-germinated spores and percentage of area covered by mycelia in microscope were used to determine the percentage of growth inhibition. The fl and f2 protein-containing peaks had inhibitory effects on spore germination after 24 hrs compared to the control treatment.
The discharge of spores from sporangia was inhibited by these protein fractions. Secondary branching (mycelia formation) was observed in control treated spores of F.
oxysporum (Figure 8A-c) and F. graminearum (Figure 8B-c). No branching was observed in fl and f2 protein-treated spores of F. oxysporum (Figures 8A-a-b) and F. graminearum (Figure 8B-a-b). The size of germ tubes (hyphae) from control-treated spores was more than 100 pm compared to 10-15 pm for protein-treated spores (Figures 8A-a-b; 8B-a-b).
- 26 -Example 3 Active growing mycelia of S. mycoparasitica (SMCD 2220-01) were cultivated on Potato Dextrose Broth (PDB) in a shaking flask at 21 C for 3 days and then washed.
Wet mycelium (0.1 g) were resuspended in 1 ml of PDA medium supplemented 100 lig/mL 3-ADON
(Sigma-Aldrich Canada Ltd., Oakville, ON, CA) and incubated for 10 days.
A 0.2cm2 plug S. mycoparasitica was incubated in lml PDB containing 3-ADON at a 15 concentration of 100 g at RT for 7 days. The sample was analysed for DON
degradation by TLC and HPLC assays.
Extraction of 3-ADON:
3-ADON was extracted by the method disclosed by Vasavada and Hsieh (1987, Appl.
Micro. Biotechnol. 26: 517-521). The spent medium in sampled flasks was filtered through the Whatman filter paper avoiding the mycelia. 3-ADON was extracted from the medium by three 10 ml volumes of ethyl acetate. The mixture of the medium and solvent was vigorously shaken and allowed to stand for 5 min for separation of phases. The organic phase was siphoned off and passed through sodium sulfate to remove residual water. The solvent was allowed to evaporate at room temperature. The residue was redissolved in acetonitrile for analysis.
Analysis of DON by TLC:
Thin layer chromatography (TLC) was used to analyze the DON. TLC was performed by the method disclosed by Andrea etal. (2004, J. Basic Microbiol. 44: 147-156).
Dried residues were dissolved in 75 I and loaded onto silica gel TLC plates coated with fluorescent indicator (60 F254, 0.2 mm layer; Merck Frosst Canada Ltd, Kirkland, PQ, CA). The spots were focused in the solvent system with ethyl acetate-toluene (3:1) for 40 5 mm. The gel plates were sprayed with 20% aluminum chloride in 95% ethanol. Trichothecenes were visualized as dark spots under short wavelength UV light (254 nm). Chromatograms were photographed using gel documentation system. A known standard control of pure 3-ADON (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) was employed to compare with other treatments. The sample containing S.
mycoparasitica showed significantly less 3-ADON than the control sample.
Wet mycelium (0.1 g) were resuspended in 1 ml of PDA medium supplemented 100 lig/mL 3-ADON
(Sigma-Aldrich Canada Ltd., Oakville, ON, CA) and incubated for 10 days.
A 0.2cm2 plug S. mycoparasitica was incubated in lml PDB containing 3-ADON at a 15 concentration of 100 g at RT for 7 days. The sample was analysed for DON
degradation by TLC and HPLC assays.
Extraction of 3-ADON:
3-ADON was extracted by the method disclosed by Vasavada and Hsieh (1987, Appl.
Micro. Biotechnol. 26: 517-521). The spent medium in sampled flasks was filtered through the Whatman filter paper avoiding the mycelia. 3-ADON was extracted from the medium by three 10 ml volumes of ethyl acetate. The mixture of the medium and solvent was vigorously shaken and allowed to stand for 5 min for separation of phases. The organic phase was siphoned off and passed through sodium sulfate to remove residual water. The solvent was allowed to evaporate at room temperature. The residue was redissolved in acetonitrile for analysis.
Analysis of DON by TLC:
Thin layer chromatography (TLC) was used to analyze the DON. TLC was performed by the method disclosed by Andrea etal. (2004, J. Basic Microbiol. 44: 147-156).
Dried residues were dissolved in 75 I and loaded onto silica gel TLC plates coated with fluorescent indicator (60 F254, 0.2 mm layer; Merck Frosst Canada Ltd, Kirkland, PQ, CA). The spots were focused in the solvent system with ethyl acetate-toluene (3:1) for 40 5 mm. The gel plates were sprayed with 20% aluminum chloride in 95% ethanol. Trichothecenes were visualized as dark spots under short wavelength UV light (254 nm). Chromatograms were photographed using gel documentation system. A known standard control of pure 3-ADON (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) was employed to compare with other treatments. The sample containing S.
mycoparasitica showed significantly less 3-ADON than the control sample.
- 27 -High Performance liquid chromatography (HPLC) A Water's HPLC with : 250 x 4.60 mm id Prodigy 5 ODS (3) 100A, 5 micron C18 column (Phenomenex Inc., Torrance, CA USA) and a photodiode-array (PDA) detector was used with a gradient solvent system (water-acetonitrile containing 0.005% (v/v) trifluoracetic acid).
The PDA detector measured the UV spectrum (200-600 nm). Samples were dissolved in acetonitrile and 20 ill was loaded onto the column using an automatic injector. The DON was eluted with solvent as a mobile phase at a rate of 1 ml min't. 3-ADON obtained from Sigma-Aldrich (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) was used as a standard. The results are shown in Figure 9. The sample with S. mycoparasitica contained a much reduced level of 3-ADON.
Example 4 Potato dextrose agar (PDA), Potato dextrose broth (PDB), Yeast extract, Malt extract agar (MEA), Agar, and Peptone were purchased from BD Biosciences (Mississauga, ON, CA) .
Streptomycin sulphate, Neomycin sulphate, and other reagents of analytic grade were from Sigma-Aldrich Canada Ltd. (Oakville, ON, CA) .
Fungal strains and growth conditions Four phytopathogenic Fusarium strains (F. avenaceum, F. sporotrichioides, F.
oxysporum, and F. proliferatum); three beneficial fungal inoculants (Trichoderma harzianum (RootShield), Penicillium bilaii (JumpStart8), and Chaetomium globosum; and one mycoparasitic fungal strain (Sphaerodes mycoparasitica SMCD 2220-01) isolated from F.
oxysporum host, were maintained on PDA amended with antibiotics (100 mg/L
streptomycin sulphate and 12 mg/L neomycin sulphate) and used throughout this study. S.
mycoparasitica mycoparasitic fungal isolate was separated from their F. oxysporum host, and monosporium cultures were achieved according to methods described by Harveson RM, Kimbrough JW, (2001, Int. J. Plant Sci. 162: 403-410) with few modifications. Mature perithecia were picked up, suspended, and shaked in 2-ml sterilized water blanks. The spores-suspension was then spread on PDA plates. After few minutes, with the assistance of a Carl Zeiss Stemi 1000TM dissecting microscope, individual spores were removed and transferred to PDA supplemented with 100
The PDA detector measured the UV spectrum (200-600 nm). Samples were dissolved in acetonitrile and 20 ill was loaded onto the column using an automatic injector. The DON was eluted with solvent as a mobile phase at a rate of 1 ml min't. 3-ADON obtained from Sigma-Aldrich (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) was used as a standard. The results are shown in Figure 9. The sample with S. mycoparasitica contained a much reduced level of 3-ADON.
Example 4 Potato dextrose agar (PDA), Potato dextrose broth (PDB), Yeast extract, Malt extract agar (MEA), Agar, and Peptone were purchased from BD Biosciences (Mississauga, ON, CA) .
Streptomycin sulphate, Neomycin sulphate, and other reagents of analytic grade were from Sigma-Aldrich Canada Ltd. (Oakville, ON, CA) .
Fungal strains and growth conditions Four phytopathogenic Fusarium strains (F. avenaceum, F. sporotrichioides, F.
oxysporum, and F. proliferatum); three beneficial fungal inoculants (Trichoderma harzianum (RootShield), Penicillium bilaii (JumpStart8), and Chaetomium globosum; and one mycoparasitic fungal strain (Sphaerodes mycoparasitica SMCD 2220-01) isolated from F.
oxysporum host, were maintained on PDA amended with antibiotics (100 mg/L
streptomycin sulphate and 12 mg/L neomycin sulphate) and used throughout this study. S.
mycoparasitica mycoparasitic fungal isolate was separated from their F. oxysporum host, and monosporium cultures were achieved according to methods described by Harveson RM, Kimbrough JW, (2001, Int. J. Plant Sci. 162: 403-410) with few modifications. Mature perithecia were picked up, suspended, and shaked in 2-ml sterilized water blanks. The spores-suspension was then spread on PDA plates. After few minutes, with the assistance of a Carl Zeiss Stemi 1000TM dissecting microscope, individual spores were removed and transferred to PDA supplemented with 100
- 28 -Min Of Fusarium filtrate. All fungal isolates were maintained in the culture collection of the Saskatchewan Microbial Collection and Database, Canada (SMCD).
Spores production Mycelium plugs from the margin of the active growing monosporial Sphaerodes culture were cut and placed on Modified Leonian's Agar (MLA: Maltose, 6.25g; Malt extract, 6.25g;
KH2PO4, 1.25g; Yeast extract, 1.0g; MgSO4.7H20, 0.625g; Peptone, 0.625g; Agar, 20g, and 1L
of dH20. Inoculated MLA plates were incubated at room temperature (23 C) in dark condition for a month before collecting the spores for germination assays. With the assistance of dissecting microscope, mature spores exuded from the ascomata and located on the ostiolar opening were harvested by picking up carefully with sterile needles and added to 2 ml tubes in 1 ml sterilized distillated water. The suspension was then filtered through four thin layers of cheesecloth to remove the remaining vegetative cells or mycelia. The density of the spore suspension was counted with a haemocytometer and adjusted to approximately 5-6x105 spores per ml in sterilized water. Freshly prepared aqueous spore suspensions were used.
Preparation of the fungal filtrates Four pathogenic Fusarium strains and three beneficial fungal strains were grown in shake cultures for 7 days at room temperature (23 C) in 500 ml flasks, each with 100 ml PDB medium.
After incubation of 7 days in PDB medium, mycelia were removed by filtering through filter paper and the filtrates were then filter-sterilized. Freshly preparations fungal filtrates were employed for the spore germination tests.
Sphaerodes mycoparasitica strain SMCD 2220-01 was found to sporulate on or when inoculated together with Fusarium species, such as F. avenaceum and F.
oxysporum. S.
mycoparasitica was observed to produce approximately the same amount of ascomata on both F.
oxysporum and F. avenaceum. S. mycoparasitica was not found to produce fruiting bodies on other Fusarium or fungal strains such as F. proliferatum, F. sporotrichioides, P. bilaai, T
harzianum, and C. globosum. In the contact zone between these biotrophic mycoparasites and Fusarium species, hook-shaped contact structures were formed.
Spores production Mycelium plugs from the margin of the active growing monosporial Sphaerodes culture were cut and placed on Modified Leonian's Agar (MLA: Maltose, 6.25g; Malt extract, 6.25g;
KH2PO4, 1.25g; Yeast extract, 1.0g; MgSO4.7H20, 0.625g; Peptone, 0.625g; Agar, 20g, and 1L
of dH20. Inoculated MLA plates were incubated at room temperature (23 C) in dark condition for a month before collecting the spores for germination assays. With the assistance of dissecting microscope, mature spores exuded from the ascomata and located on the ostiolar opening were harvested by picking up carefully with sterile needles and added to 2 ml tubes in 1 ml sterilized distillated water. The suspension was then filtered through four thin layers of cheesecloth to remove the remaining vegetative cells or mycelia. The density of the spore suspension was counted with a haemocytometer and adjusted to approximately 5-6x105 spores per ml in sterilized water. Freshly prepared aqueous spore suspensions were used.
Preparation of the fungal filtrates Four pathogenic Fusarium strains and three beneficial fungal strains were grown in shake cultures for 7 days at room temperature (23 C) in 500 ml flasks, each with 100 ml PDB medium.
After incubation of 7 days in PDB medium, mycelia were removed by filtering through filter paper and the filtrates were then filter-sterilized. Freshly preparations fungal filtrates were employed for the spore germination tests.
Sphaerodes mycoparasitica strain SMCD 2220-01 was found to sporulate on or when inoculated together with Fusarium species, such as F. avenaceum and F.
oxysporum. S.
mycoparasitica was observed to produce approximately the same amount of ascomata on both F.
oxysporum and F. avenaceum. S. mycoparasitica was not found to produce fruiting bodies on other Fusarium or fungal strains such as F. proliferatum, F. sporotrichioides, P. bilaai, T
harzianum, and C. globosum. In the contact zone between these biotrophic mycoparasites and Fusarium species, hook-shaped contact structures were formed.
- 29 -Effects of heat and cold treatments on Sphaerodes spore germination Aqueous ascospore suspensions were heat-shocked at 60 C and 65 C for 20 min, and cold-treated at 4 C, -20 C, and -70 C for 5 min and 20 min. The heat- and cold-shocked spores were then transferred and inoculated onto WA and PDA for 1 day and 3 days. The readings of spore germination were checked daily. Spores not subjected to heat and cold treatments were used as control.
There was no germination observed in any of the heat and cold treatment groups for 3 days. Additional observations were continued up to 7 days and no spore germination was observed.
Germination on various media Aqueous spore suspensions were transferred and inoculated onto the surface of the following media:
1.5% water agar (WA), PDA, MLA, MEA, Carnation leaves agar (CLA);
- 1.5% water agar plus 100 m1/1 of Fusarium filtrate;
- PDA with 100 m1/1 of Fusarium filtrate.
Fusarium strains utilized were F. avenaceum, F. oxysporurn, F. proliferatum, and F.
sporotrichioides. Ascospore-inoculated plates were then incubated at room temperature (23 C) for 3 days. Spore germination was examined daily.
The results are summarized in Table 1. Each incubation day for Sphaerodes was analyzed separately. Numbers in each column represent the mean of ascospore germination (in %) standard deviation. Means within each column for each medium treatment followed by the same letter are not significantly different at P <0.05 after Mann-Whitney U test.
There was no germination observed in any of the heat and cold treatment groups for 3 days. Additional observations were continued up to 7 days and no spore germination was observed.
Germination on various media Aqueous spore suspensions were transferred and inoculated onto the surface of the following media:
1.5% water agar (WA), PDA, MLA, MEA, Carnation leaves agar (CLA);
- 1.5% water agar plus 100 m1/1 of Fusarium filtrate;
- PDA with 100 m1/1 of Fusarium filtrate.
Fusarium strains utilized were F. avenaceum, F. oxysporurn, F. proliferatum, and F.
sporotrichioides. Ascospore-inoculated plates were then incubated at room temperature (23 C) for 3 days. Spore germination was examined daily.
The results are summarized in Table 1. Each incubation day for Sphaerodes was analyzed separately. Numbers in each column represent the mean of ascospore germination (in %) standard deviation. Means within each column for each medium treatment followed by the same letter are not significantly different at P <0.05 after Mann-Whitney U test.
-30-Table 1:: Sporulation of S. mycoparasitica on various media Medium S. mycoparasitica Day 1 Day 3 Water agar (WA) 0 d 0 d F. avenaceum-W A 11.1 1.7 b 35.2 2.6 b F. oxysporum-WA 1.6 1.3 c 18 1.4 c F. proliferatum-W A 0 d 0 d F. sporotrichioides-W A 0 d 0 d Potato dextrose agar (PDA) 0 d 0 d F. avenaceum-PDA 21.5 + 1.4 a 63.2 + 2.4 a F. oxysporum-PDA 2.5 2 c 37.5 2 b F. proliferatum-PDA 0 d 0 d F. sporotrichioides-PDA 0 d 0 d Effects offungal filtrates on spore germination The aqueous spore suspensions were suspended in the filtrates of four separate pathogenic Fusarium and three beneficial fungal strains for 1 day and 3 day in the ratio of 1:2 (1 part of aqueous spore suspension: 2 parts of fungal filtrate), and the filtrate-suspended spores were then transferred and inoculated onto PDA for additional 1 day. Control treatments were suspended with sterilized distilled water or PDB.
Spore germination Microscopic assessments of ascospore germination were conducted after incubation for 1 day and 3 days. Percentage of germinated spores was obtained by scoring the spores on the Petri dish through utilizing the 200x and 400x objectives of the Carl Zeiss Axioskop2 microscope and systematically choosing 50 spores, starting at the top right corner and continuing to count till 50. Each drop of ascospore suspensions on a medium plate was considered as a subunit, and there were three subunits per plate. Each medium plate for each treatment was replicated, there were three replicas per treatments. The experiments were repeated twice. An ascospore was only considered as germinated spore when the germ tube was visibly noticeable.
Germinated ascospores were counted and recorded as a percentage of the total ascospore number.
Spore germination Microscopic assessments of ascospore germination were conducted after incubation for 1 day and 3 days. Percentage of germinated spores was obtained by scoring the spores on the Petri dish through utilizing the 200x and 400x objectives of the Carl Zeiss Axioskop2 microscope and systematically choosing 50 spores, starting at the top right corner and continuing to count till 50. Each drop of ascospore suspensions on a medium plate was considered as a subunit, and there were three subunits per plate. Each medium plate for each treatment was replicated, there were three replicas per treatments. The experiments were repeated twice. An ascospore was only considered as germinated spore when the germ tube was visibly noticeable.
Germinated ascospores were counted and recorded as a percentage of the total ascospore number.
-31 -The results are summarized in Table 2. Numbers in each column represented mean of ascospore germination (in %) + standard deviation. Each incubation day for Sphaerodes was analyzed separately. Means within each column of Sphaerodes for each filtrate-suspension treatment followed by the same letter are not significantly different at P <
0.05 after Mann-Whitney U test.
Table 2: Effects of fungal filtrates on S. mycoparasitica spore germination Shpaerodes sp. Spore germination (in 'Yo) Treatments Id suspension Id sus + Id PDA 3d suspension 3d sus + Id PDA
Water 0 c 0 e 0 e 2 1.2 c PDB 0 c 0 e 0 e 2.8 1.9 c F. avenaceum-filtrate 89.2 6.2 a 91 8.4 a 93.2 6.7 a 94.2 7.9 a F. oxysporum-filtrate 0 c 81.8 11 b 85 7.9 b 91 10 a F. proliferatum-filtrate 2.6 3.6 b 3.2 4.6 d 10 7.4 c 11.2 8 b F. sporotrichioides-filtrate 0 c 3 2.8 d 3.4 3.3d 10 5.6 b P. bilaii-filtrate 0 c 0 e 0 e 0 c T harzianum-filtrate 0 c 9 1.1 c 1.6 1.6 d 10.4 1.5 b C. globosum-filtrate 0 c 0 e 0 e 0 c Example 5 Since F. graminearum 3-ADON is the most pathogenic and mycotoxigenic in wheat, 3-ADON was used to quantifying mycoparasite-Fusarium-wheat root interactions under Phytotron controlled conditions.
Fungal strains and growth Fusarium strains: F. graminearum 3-ADON strain SMCD2243, biotrophic mycoparasite Sphaerodes mycoparasitica SMCD2220-01, and Trichoderma harzianum T-22 (RootShield¨commercial product) as a control strain with mycoparasitic properties were maintained on PDA amended with antibiotics.
0.05 after Mann-Whitney U test.
Table 2: Effects of fungal filtrates on S. mycoparasitica spore germination Shpaerodes sp. Spore germination (in 'Yo) Treatments Id suspension Id sus + Id PDA 3d suspension 3d sus + Id PDA
Water 0 c 0 e 0 e 2 1.2 c PDB 0 c 0 e 0 e 2.8 1.9 c F. avenaceum-filtrate 89.2 6.2 a 91 8.4 a 93.2 6.7 a 94.2 7.9 a F. oxysporum-filtrate 0 c 81.8 11 b 85 7.9 b 91 10 a F. proliferatum-filtrate 2.6 3.6 b 3.2 4.6 d 10 7.4 c 11.2 8 b F. sporotrichioides-filtrate 0 c 3 2.8 d 3.4 3.3d 10 5.6 b P. bilaii-filtrate 0 c 0 e 0 e 0 c T harzianum-filtrate 0 c 9 1.1 c 1.6 1.6 d 10.4 1.5 b C. globosum-filtrate 0 c 0 e 0 e 0 c Example 5 Since F. graminearum 3-ADON is the most pathogenic and mycotoxigenic in wheat, 3-ADON was used to quantifying mycoparasite-Fusarium-wheat root interactions under Phytotron controlled conditions.
Fungal strains and growth Fusarium strains: F. graminearum 3-ADON strain SMCD2243, biotrophic mycoparasite Sphaerodes mycoparasitica SMCD2220-01, and Trichoderma harzianum T-22 (RootShield¨commercial product) as a control strain with mycoparasitic properties were maintained on PDA amended with antibiotics.
- 32 -Taxon specific primers Specific primer sets for: F. graminearum-F gl 2NFiR (Nicholson et al. 1998.
Plant Pathology 53: 17-37.); Trichoderma-TGP4-F IR (Kim and Knudsen 2008. Applied Soil Ecology 40: 100-108); and S. mycoparasitica SMCD2220-01-SmyITSF/R were used in this study.
Mycoparasite/Fusarium/plant interactions Wheat CDC-TEAL 2001 plants were inoculated with mycotoxigenic and pathogenic Fusarium as well as Trichoderma (control mycoparasite) or Sphaerodes SMCD2220-01, and were subjected to RT-PCR quantification to assess the amount of fungal DNAs in roots of wheat under controlled conditions after 14 days of incubation in Phytotron conditions.
Growth conditions and fungal inoculation Five mycelial plugs from S. mycoparasitica, T. harzianum, and F graminearum were cut, transferred and grown in three separate shake cultures for 14 d at room temperature (23 C) in 500 mL tlasks each with 100 mL of PDB (potato dextrose broth). After incubation of 14 d in PDB medium, the fungal cultures were filtered through a Whatman No. 1 filter paper to remove the liquid medium. The mycelia were then transferred to 50 mL sterile Falcon tubes with 20 sterile glass beads and 40 mL of autoclaved distillated water. The Falcon tubes filled with mycelia materials were then vortex vigorously for 1 min to separate the mycelia into smaller pieces. Mycelial suspension was filtered through 2 layers of cheesecloth to remove the glassbeads and bigger mycelial clumps. The flow-through was then used as mycelial suspension stock (10 ) for serial dilution. S-tock of mycelial suspension was further diluted into a series spanning from 10-2, 10-3, and 10-4. This dilution series were plated on PDA
using the pour plate method. The number of CFU (colony forming units) was counted and recorded.
Mycelial suspensions were adjusted with sterile water to about 10-5 ¨ 10-6 CFU/mL for S. mycoparasitica and T harzianum, and to about 10-4 - 10-5 CFU/mL for F. graminearum.
Quantification of interactions between mycoparasite-pathogen-wheat roots was conducted on the spring wheat CDC-TEAL 2001. Wheat plants were grown in containers (4 x 4 x 16 cm) with 10 g of different layers of soil-less growing mix (Figure 10).
All the seeds were surface-sterilized prior to sowing. The containers 10 were lined with filter paper and packed with
Plant Pathology 53: 17-37.); Trichoderma-TGP4-F IR (Kim and Knudsen 2008. Applied Soil Ecology 40: 100-108); and S. mycoparasitica SMCD2220-01-SmyITSF/R were used in this study.
Mycoparasite/Fusarium/plant interactions Wheat CDC-TEAL 2001 plants were inoculated with mycotoxigenic and pathogenic Fusarium as well as Trichoderma (control mycoparasite) or Sphaerodes SMCD2220-01, and were subjected to RT-PCR quantification to assess the amount of fungal DNAs in roots of wheat under controlled conditions after 14 days of incubation in Phytotron conditions.
Growth conditions and fungal inoculation Five mycelial plugs from S. mycoparasitica, T. harzianum, and F graminearum were cut, transferred and grown in three separate shake cultures for 14 d at room temperature (23 C) in 500 mL tlasks each with 100 mL of PDB (potato dextrose broth). After incubation of 14 d in PDB medium, the fungal cultures were filtered through a Whatman No. 1 filter paper to remove the liquid medium. The mycelia were then transferred to 50 mL sterile Falcon tubes with 20 sterile glass beads and 40 mL of autoclaved distillated water. The Falcon tubes filled with mycelia materials were then vortex vigorously for 1 min to separate the mycelia into smaller pieces. Mycelial suspension was filtered through 2 layers of cheesecloth to remove the glassbeads and bigger mycelial clumps. The flow-through was then used as mycelial suspension stock (10 ) for serial dilution. S-tock of mycelial suspension was further diluted into a series spanning from 10-2, 10-3, and 10-4. This dilution series were plated on PDA
using the pour plate method. The number of CFU (colony forming units) was counted and recorded.
Mycelial suspensions were adjusted with sterile water to about 10-5 ¨ 10-6 CFU/mL for S. mycoparasitica and T harzianum, and to about 10-4 - 10-5 CFU/mL for F. graminearum.
Quantification of interactions between mycoparasite-pathogen-wheat roots was conducted on the spring wheat CDC-TEAL 2001. Wheat plants were grown in containers (4 x 4 x 16 cm) with 10 g of different layers of soil-less growing mix (Figure 10).
All the seeds were surface-sterilized prior to sowing. The containers 10 were lined with filter paper and packed with
- 33 -6 g of Pro-Mix soil-less mix (Sun Gro Horticulture Canada Ltd., Delta, BC, CA; Pro-Mix is a registered trademark of Premier Horticulture Ltd., Riviere-du-Loop, PQ, CA) which comprised the first layer 20. This layer 20 was then overlayed with a second layer 30 (1g) of either Pro-Mix* mix amended and homogenized with ¨5 to 6 x 104 CFU of F. graminearum mycelial suspension or alternatively with ProMix mixwith water only. The second layer 30 was followed by a third layer 40 (1g) of either Sunshine peat moss (Sunshine is a registered trademark of Sun Gro Horticulture Canada Ltd., Seba Beach, AB, CA) supplemented and homogenized with ¨ 5 to 6 x 105 CFU of S. mycoparasite or alternatively with T
harzianum mycelial suspension or alternatively with water only. Six spring wheat seeds were then sowed on top of the third layer 40 and topped with a fifth layer 60 of 2 g of Sunshine peat moss (Figure 10). Spring wheat seeds were germinated and grown under a 16-h photoperiod (22 C day/15 C
night) with light intensity of 250 limo' m-2 s-1, watered every 2 d, and fertilized every 14 d using 1300 ppm of NPK (20-20-20) fertilizer. All treatments in the experiment were in three replicates and the experiment was repeated twice.
At the mid-seedling growth, corresponding to Zadok's growth stage 13 (Zadoks et al.
1974, Weed Research 14:415-421), wheat plants with their roots were removed from the pots and washed under running tap water to remove all the soil particles. Washed roots were dried with filter papers. Number of germinated seeds, total biomass, root biomass, total length, and root length were counted and measured. Percentage of seed germination was calculated with the following formula: (Number of germinated seeds in particular treatment/number of germinated seeds in control treatment) x 100%. The roots were then subjected to total DNA
extraction with DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA). Extracted total DNAs from roots of different treatments were employed in real-time PCR quantification.
Statistical analyses Root biomass (g), total biomass (g), root length (cm), total length (cm), seed germination (%); and S. mycoparasitica, T harzianum, and F. graminearum genomic DNA
quantification from the roots of Spring wheat plants were analyzed by using analysis of variance (ANOVA).
Logl transformations were carried out whenever required to meet the ANOVA
requirements.
harzianum mycelial suspension or alternatively with water only. Six spring wheat seeds were then sowed on top of the third layer 40 and topped with a fifth layer 60 of 2 g of Sunshine peat moss (Figure 10). Spring wheat seeds were germinated and grown under a 16-h photoperiod (22 C day/15 C
night) with light intensity of 250 limo' m-2 s-1, watered every 2 d, and fertilized every 14 d using 1300 ppm of NPK (20-20-20) fertilizer. All treatments in the experiment were in three replicates and the experiment was repeated twice.
At the mid-seedling growth, corresponding to Zadok's growth stage 13 (Zadoks et al.
1974, Weed Research 14:415-421), wheat plants with their roots were removed from the pots and washed under running tap water to remove all the soil particles. Washed roots were dried with filter papers. Number of germinated seeds, total biomass, root biomass, total length, and root length were counted and measured. Percentage of seed germination was calculated with the following formula: (Number of germinated seeds in particular treatment/number of germinated seeds in control treatment) x 100%. The roots were then subjected to total DNA
extraction with DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA). Extracted total DNAs from roots of different treatments were employed in real-time PCR quantification.
Statistical analyses Root biomass (g), total biomass (g), root length (cm), total length (cm), seed germination (%); and S. mycoparasitica, T harzianum, and F. graminearum genomic DNA
quantification from the roots of Spring wheat plants were analyzed by using analysis of variance (ANOVA).
Logl transformations were carried out whenever required to meet the ANOVA
requirements.
- 34 -Multiple comparisons for more than two samples were analyzed by utilizing Tukey's studentized range test at P = 0.05 (SPSS 1990).
Wheat growth and fungal inoculation Root biomass, total biomass, root length, total length, and seed germination of F.
graminearum infected spring wheat were significantly increased with the treatments of S.
mycoparasitica compared to inoculation with F. graminearum alone (Table 3).
Table 3: Effects of SMCD2220-01 (SM) and F. graminearum 3-ADON (Fgra) inoculation treatments on root biomass (g), total biomass (g), root length (cm), total length (cm) and seed germination (%) of spring wheat plants Treatment Root biomass Total biomass Root length Total length (g) (g) (cm) (cm) Seed germination (%) Control 0.27 0.06 b 0.56 0.56 b 9.63 0.74 b 31.88 2.39 c NA*
SM 0.32 0.03 ab 0.69 0.69 a 13.88 1.11 a 38.13 2.76 ab 101 2 a Fgra 0.18 0.03 c 0.38 0.48 c 7.88 1.89 c 23.75 1.98d 31.25 1.4c SM-Fgra 0.27 + 0.03 b 0.54 0.54 b 12.13 1.35 ab 32.13 3.91 be 87.25 2 b * Values expressed are means of six replicates standard deviation of the mean. Values followed by same letters within each column are not significantly different using Tukey's range test (P
<0.05).
Table 3, the mycoparasite S. mycoparasitica demonstrates both biomass stimulation and bioprotection or biocontrol.
Conformation of the Sphaerodes-specific primer set The SmyITSF/R primer set was tested with S. mycoparasitica, seven Fusarium species, night different ascomycetous fungal isolates two zygomycete fungi, and three basidiomycetous fungal strains. This primer set only amplified S. mycoparasitica.
Figure 11 shows SmyITSF/R primers amplified PCR products for S. mycoparasitica (SM), five Fusarium strains (Fa = F. avenaceum, Fo = F. oxysporum, Fs = F.
sporotrichioides, Fg3 = F. graminearum chemotype 3, and Fg15 = F. graminearum chemotype 15), two Trichoderma species (T22 = T harzianum T22 and Tv = T viride), two Cladosporium species
Wheat growth and fungal inoculation Root biomass, total biomass, root length, total length, and seed germination of F.
graminearum infected spring wheat were significantly increased with the treatments of S.
mycoparasitica compared to inoculation with F. graminearum alone (Table 3).
Table 3: Effects of SMCD2220-01 (SM) and F. graminearum 3-ADON (Fgra) inoculation treatments on root biomass (g), total biomass (g), root length (cm), total length (cm) and seed germination (%) of spring wheat plants Treatment Root biomass Total biomass Root length Total length (g) (g) (cm) (cm) Seed germination (%) Control 0.27 0.06 b 0.56 0.56 b 9.63 0.74 b 31.88 2.39 c NA*
SM 0.32 0.03 ab 0.69 0.69 a 13.88 1.11 a 38.13 2.76 ab 101 2 a Fgra 0.18 0.03 c 0.38 0.48 c 7.88 1.89 c 23.75 1.98d 31.25 1.4c SM-Fgra 0.27 + 0.03 b 0.54 0.54 b 12.13 1.35 ab 32.13 3.91 be 87.25 2 b * Values expressed are means of six replicates standard deviation of the mean. Values followed by same letters within each column are not significantly different using Tukey's range test (P
<0.05).
Table 3, the mycoparasite S. mycoparasitica demonstrates both biomass stimulation and bioprotection or biocontrol.
Conformation of the Sphaerodes-specific primer set The SmyITSF/R primer set was tested with S. mycoparasitica, seven Fusarium species, night different ascomycetous fungal isolates two zygomycete fungi, and three basidiomycetous fungal strains. This primer set only amplified S. mycoparasitica.
Figure 11 shows SmyITSF/R primers amplified PCR products for S. mycoparasitica (SM), five Fusarium strains (Fa = F. avenaceum, Fo = F. oxysporum, Fs = F.
sporotrichioides, Fg3 = F. graminearum chemotype 3, and Fg15 = F. graminearum chemotype 15), two Trichoderma species (T22 = T harzianum T22 and Tv = T viride), two Cladosporium species
- 35 -(CC = C. cladosporioides and CM = C. minourae), and Penicillium aurantiogriseum (PA) were electrophoresed on 1% agarose gel at 100 V for 20 minutes. The size of the band is around 300 to 400 bp.
Standard curves The standard curves based on known diluted concentrations of DNAs from S.
mycoparasitica, F. graminearurn, and T. harzianum were constructed. Standard curves were achieved using a series of 10-fold diluted DNA spanning from 3.8 x 102 to 3.8 x 10-2 ng for S.
mycoparasitica, 2.7 x 103 to 2.7 x 10-1 ng for F. graminearum chemotype 3, and 7.0 x 102 to 7.0 x 10-2 ng for T harzianum. Quantification demonstrated linear relation (r2 =
0.999 for S.
mycoparasiticaõ r2 = 0.998 for F. graminearum, and r2 = 0.996 for T harzianum) between log! o of fungal genomic DNA (in ng/i_t 1) and real-time PCR threshold cycles (Ct) (threshold fluorescence signal of 0.025 was used for all three fungal isolates) (Figures 12A, 12B, and 12C).
Figure 13 shows RT-PCR sigmoidal coloured curves for Sphaerodes mycoparasitica (SMCD 2220-01), with 0.025 fluorescence line, showing the range of 3.8 x 102 to 3.8 x 10-2 ng in a ten-hold decreasing manner.
RT-PCR confirmation of the Sphaerodes-biocontrol effects Real-time PCR, which evaluated quantity of S. mycoparasitica SMCD2220-01 and F.
graminearum, confirmed that amounts of F. graminearum DNA detected in treatments with S.
mycoparasitica SMCD2220-01 and T harzianum were significantly reduced (Figure 14A).
Previously, treatments with mycoparasitic Sphaerodes retispora had been observed to show significant suppression of F. oxysporum in watermelon plants (Harveson et al.
2002, Plant Dis.
86: 1025-1030). The amount of S. mycoparasitica DNA detected was not significantly different between wheat inoculated with F. graminearum or without Fusarium (Fig.14B).
The amount of T harzianum DNA detected in the treatment inoculated with E graminearum was observed to be significantly reduced, as compared to non-Fusarium treatment (Fig.14C).
In conclusion, during in vitro culture-based studies, only S. mycoparasitica SMCD2220-01 was observed to enhance wheat seed germination and formation of secondary roots, whereas T-22 induced post-emergence damping-off symptoms. Under controlled
Standard curves The standard curves based on known diluted concentrations of DNAs from S.
mycoparasitica, F. graminearurn, and T. harzianum were constructed. Standard curves were achieved using a series of 10-fold diluted DNA spanning from 3.8 x 102 to 3.8 x 10-2 ng for S.
mycoparasitica, 2.7 x 103 to 2.7 x 10-1 ng for F. graminearum chemotype 3, and 7.0 x 102 to 7.0 x 10-2 ng for T harzianum. Quantification demonstrated linear relation (r2 =
0.999 for S.
mycoparasiticaõ r2 = 0.998 for F. graminearum, and r2 = 0.996 for T harzianum) between log! o of fungal genomic DNA (in ng/i_t 1) and real-time PCR threshold cycles (Ct) (threshold fluorescence signal of 0.025 was used for all three fungal isolates) (Figures 12A, 12B, and 12C).
Figure 13 shows RT-PCR sigmoidal coloured curves for Sphaerodes mycoparasitica (SMCD 2220-01), with 0.025 fluorescence line, showing the range of 3.8 x 102 to 3.8 x 10-2 ng in a ten-hold decreasing manner.
RT-PCR confirmation of the Sphaerodes-biocontrol effects Real-time PCR, which evaluated quantity of S. mycoparasitica SMCD2220-01 and F.
graminearum, confirmed that amounts of F. graminearum DNA detected in treatments with S.
mycoparasitica SMCD2220-01 and T harzianum were significantly reduced (Figure 14A).
Previously, treatments with mycoparasitic Sphaerodes retispora had been observed to show significant suppression of F. oxysporum in watermelon plants (Harveson et al.
2002, Plant Dis.
86: 1025-1030). The amount of S. mycoparasitica DNA detected was not significantly different between wheat inoculated with F. graminearum or without Fusarium (Fig.14B).
The amount of T harzianum DNA detected in the treatment inoculated with E graminearum was observed to be significantly reduced, as compared to non-Fusarium treatment (Fig.14C).
In conclusion, during in vitro culture-based studies, only S. mycoparasitica SMCD2220-01 was observed to enhance wheat seed germination and formation of secondary roots, whereas T-22 induced post-emergence damping-off symptoms. Under controlled
-36-conditions in pytotron, S. mycoparasitica SMCD2220-01 was able to reduce the quantity of F.
graminearum in spring wheat root, as well as improving the survival and growth of the spring wheat seedlings. In contrast to T harzianum, the amount of S. mycoparasitica DNA detected was not significantly different between wheat inoculated with F.
graminearum and without Fusarium. Hence, S. mycoparasitica SMCD2220-01 could be a better biocontrol candidate for the F. graminearum pathogen in wheat.
Example 6 Quantitative RT-PCR results from testing Mycoparasite-Fusarium interaction on Wheat host using growing conditions disclosed herein also confirmed the efficiency of S.
mycoparasitica SMCD2220-01 for significantly decreasing an accumulation of genes implicated in Fusarium DON-mycotoxin production.
Strain used: F. graminearum 3-ADON SMCD2243, F. graminearum 15-ADON
SMCD2244 and S. mycoparasitica SMCD2220-01 strains.
Primersets used: Tox5-1/2 for Fusarium (Wu et al. 2002. J. Environ Monit.
4:377-382) and SmyITSF/R (disclosed herein) for S. mycoparasitica.
Results are summarized in the Figure 15. Real-time fluorescence curves of tri5 gene sequences amplified by using Tox5-1/2 primer set from total DNA extracted from dual-culture assays of Fusarium graminearum strains and pre-inoculated Sphaerodes mycoparasitica SMCD2220-01 (SM) or singly grown F. graminearum 3-ADON and 15-ADON chemotypes.
Example 7 The effects of filtrates collected from different Fusarium sp. on the germination of Sphaerodes ascospores were assessed in this study. Also assessed were the ascospore germination patterns.
Media, Reagents, and chemicals: Potato dextrose agar (PDA), potato dextrose broth (PDB), yeast extract, malt extract agar (MEA), agar, and peptone were purchased from Difco (Becton
graminearum in spring wheat root, as well as improving the survival and growth of the spring wheat seedlings. In contrast to T harzianum, the amount of S. mycoparasitica DNA detected was not significantly different between wheat inoculated with F.
graminearum and without Fusarium. Hence, S. mycoparasitica SMCD2220-01 could be a better biocontrol candidate for the F. graminearum pathogen in wheat.
Example 6 Quantitative RT-PCR results from testing Mycoparasite-Fusarium interaction on Wheat host using growing conditions disclosed herein also confirmed the efficiency of S.
mycoparasitica SMCD2220-01 for significantly decreasing an accumulation of genes implicated in Fusarium DON-mycotoxin production.
Strain used: F. graminearum 3-ADON SMCD2243, F. graminearum 15-ADON
SMCD2244 and S. mycoparasitica SMCD2220-01 strains.
Primersets used: Tox5-1/2 for Fusarium (Wu et al. 2002. J. Environ Monit.
4:377-382) and SmyITSF/R (disclosed herein) for S. mycoparasitica.
Results are summarized in the Figure 15. Real-time fluorescence curves of tri5 gene sequences amplified by using Tox5-1/2 primer set from total DNA extracted from dual-culture assays of Fusarium graminearum strains and pre-inoculated Sphaerodes mycoparasitica SMCD2220-01 (SM) or singly grown F. graminearum 3-ADON and 15-ADON chemotypes.
Example 7 The effects of filtrates collected from different Fusarium sp. on the germination of Sphaerodes ascospores were assessed in this study. Also assessed were the ascospore germination patterns.
Media, Reagents, and chemicals: Potato dextrose agar (PDA), potato dextrose broth (PDB), yeast extract, malt extract agar (MEA), agar, and peptone were purchased from Difco (Becton
-37-Dickinson Diagnostics, Sparks, Maryland). Streptomycin sulphate, Neomycin sulphate, and other reagents of analytic grade were from Sigma-Aldrich (Oakville, ON, CA).
Fungal strains and growth conditions: Four phytopathogenic Fusarium strains (F. avenaceum SMCD 2241, F. oxysporum SMCD 2242, F. proliferatum (Matsush.) Nirenberg SMCD
2244, and F.
sporotrichioides Sherb SMCD 2243), three beneficial fungal inoculants: T
harzianum (RootShield8), P. bilaii (PumpStare), and Chaetomium globosum Kunze; and one mycoparasitic fungal strain, S.
mycoparasitica were maintained on PDA amended with antibiotics (100 mg/L
streptomycin sulphate and 12 mg/L neomycin sulphate) and used throughout this study. An isolate of the mycoparasitic fungus, S. mycoparasitica, was separated from its F. oxysporum host on myclobutanil agar (MBA) selective medium as previously described and a monosporic culture was achieved by picking up mature perithecia which were then suspended and shaken in 2-mL sterile distilled water blanks to encourage release of ascospores. The ascospore-suspension was then spread on PDA plates. After a few minutes, a Carl Zeiss Stemi 1000 dissecting microscope was used to identify individual ascospores, which were removed and transferred to PDA supplemented with 100 mL/L of Fusarium filtrate (F. avenaceum and F. oxysporum). All fungal isolates were maintained in the culture collection of the Saskatchewan Microbial Collection and Database, Canada (SMCD).
Ascospore production: Mycelium plugs from the margin of the actively growing monosporic-derived S. mycoparasitica culture were cut and placed on Modified Leonian's Agar (MLA:
maltose, 6.25g;
malt extract, 6.25g; KH2PO4, 1.25g; yeast extract, 1.0g; MgSO4. 7H20, 0.625g;
peptone, 0.625g;
agar, 20g, and 1L of dH20) (Malloch, D., and Cain, R.F. (1971) New genera of Onygenaceae.
Canadian Journal of Botany 49: 839-846). Inoculated Modified Leonian's Agar (MLA) plates were incubated at room temperature (23 C) under darkness conditions for a month before collecting spores for germination assays. Mature ascospores were collected according Goh, Y.K.
and Vujanovic, V.
((2010) Sphaerodes quadrangularis biotrophic mycoparasitism on Fusarium avenaceum. Mycologia 102(4): 757-62).
Preparation of the fungal filtrates: Four pathogenic Fusarium strains and three beneficial fungal strains were grown in shake cultures for 7 d at room temperature (23 C) (with 250 rpm) in 500 mL
flasks, each with 100 mL PDB medium prior to fungal filtrates extraction.
After incubation, the mycelia were removed by filtering through filter paper (Whatman Grade No. 1) and the filtrates were then filter-sterilized with 0.02-nn-pore-size nitrocellulose filter (Fisher Scientific
Fungal strains and growth conditions: Four phytopathogenic Fusarium strains (F. avenaceum SMCD 2241, F. oxysporum SMCD 2242, F. proliferatum (Matsush.) Nirenberg SMCD
2244, and F.
sporotrichioides Sherb SMCD 2243), three beneficial fungal inoculants: T
harzianum (RootShield8), P. bilaii (PumpStare), and Chaetomium globosum Kunze; and one mycoparasitic fungal strain, S.
mycoparasitica were maintained on PDA amended with antibiotics (100 mg/L
streptomycin sulphate and 12 mg/L neomycin sulphate) and used throughout this study. An isolate of the mycoparasitic fungus, S. mycoparasitica, was separated from its F. oxysporum host on myclobutanil agar (MBA) selective medium as previously described and a monosporic culture was achieved by picking up mature perithecia which were then suspended and shaken in 2-mL sterile distilled water blanks to encourage release of ascospores. The ascospore-suspension was then spread on PDA plates. After a few minutes, a Carl Zeiss Stemi 1000 dissecting microscope was used to identify individual ascospores, which were removed and transferred to PDA supplemented with 100 mL/L of Fusarium filtrate (F. avenaceum and F. oxysporum). All fungal isolates were maintained in the culture collection of the Saskatchewan Microbial Collection and Database, Canada (SMCD).
Ascospore production: Mycelium plugs from the margin of the actively growing monosporic-derived S. mycoparasitica culture were cut and placed on Modified Leonian's Agar (MLA:
maltose, 6.25g;
malt extract, 6.25g; KH2PO4, 1.25g; yeast extract, 1.0g; MgSO4. 7H20, 0.625g;
peptone, 0.625g;
agar, 20g, and 1L of dH20) (Malloch, D., and Cain, R.F. (1971) New genera of Onygenaceae.
Canadian Journal of Botany 49: 839-846). Inoculated Modified Leonian's Agar (MLA) plates were incubated at room temperature (23 C) under darkness conditions for a month before collecting spores for germination assays. Mature ascospores were collected according Goh, Y.K.
and Vujanovic, V.
((2010) Sphaerodes quadrangularis biotrophic mycoparasitism on Fusarium avenaceum. Mycologia 102(4): 757-62).
Preparation of the fungal filtrates: Four pathogenic Fusarium strains and three beneficial fungal strains were grown in shake cultures for 7 d at room temperature (23 C) (with 250 rpm) in 500 mL
flasks, each with 100 mL PDB medium prior to fungal filtrates extraction.
After incubation, the mycelia were removed by filtering through filter paper (Whatman Grade No. 1) and the filtrates were then filter-sterilized with 0.02-nn-pore-size nitrocellulose filter (Fisher Scientific
- 38 -Ltd., Nepean, ON, CA). Only fresh preparations of fungal filtrates were employed for the spore germination test in this study.
Effect of heat and cold treatments on S. mycoparasitica spore germination:
Heat activation and cold-shock treatments were performed to investigate ascospore germination in S. mycoparasitica.
Aqueous ascospore suspensions were heat-shocked at 60 and 65 C for 20 min, and cold-treated at 4, -20, and -70 C for 5 min and 20 min. The heat- and cold-shocked spores (10 [IL) were then transferred and inoculated onto water agar (WA) and PDA for 1 d and 3 d. Spore germination was checked daily. Spores not subjected to heat and cold treatments were used as controls. All treatments were in three replicates and experiment was repeated twice.
No germination was observed in all the heat and cold treatments over a 7 d period.
Germination on various media: Aqueous spore suspensions (10 L) of S.
mycoparasitica were transferred and inoculated onto the surface of the following media: 1.5% water agar (WA), PDA, MLA, MEA, Carnation leaf agar (CLA) (Tschanz et al. 1976), 1.5% water agar plus 100 mL/L
of Fusarium filtrate, and PDA with 100 mL/L of the Fusarium filtrate. Fusarium filtrates were created using F. avenaceum, F. oxysporum, F. proliferatum, and F. sporotrichio ides. S.
mycoparasitica ascospore-inoculated plates were then incubated at room temperature (23 C) for 3 d, and spore germination was examined daily. All treatments were in three replicates and experiment was repeated twice.
There was no spore germination on 1.5% WA, PDA, MLA, MEA, and CLA. The effects of 1.5% WA and PDA media amended with 100 mL/L of Fusarium-filtrates on spore germination were examined and are summarized in Table 4.
No germination was recorded on day 3 initially and observations were continued up to 7 d on WA or PDA alone and WA or PDA with either F. proliferatum or F.
sporotrichioides.
However, when either F. avenaceum or F. oxysporum filtrates were added to WA
and PDA, the percentage of ascospore germination in S. mycoparasitica drastically increased after 3 d incubation. On day 3, germination increased on the PDA amended with Fusarium-filtrate, compared to growth on WA supplemented with Fusarium-filtrate, for S.
mycoparasitica
Effect of heat and cold treatments on S. mycoparasitica spore germination:
Heat activation and cold-shock treatments were performed to investigate ascospore germination in S. mycoparasitica.
Aqueous ascospore suspensions were heat-shocked at 60 and 65 C for 20 min, and cold-treated at 4, -20, and -70 C for 5 min and 20 min. The heat- and cold-shocked spores (10 [IL) were then transferred and inoculated onto water agar (WA) and PDA for 1 d and 3 d. Spore germination was checked daily. Spores not subjected to heat and cold treatments were used as controls. All treatments were in three replicates and experiment was repeated twice.
No germination was observed in all the heat and cold treatments over a 7 d period.
Germination on various media: Aqueous spore suspensions (10 L) of S.
mycoparasitica were transferred and inoculated onto the surface of the following media: 1.5% water agar (WA), PDA, MLA, MEA, Carnation leaf agar (CLA) (Tschanz et al. 1976), 1.5% water agar plus 100 mL/L
of Fusarium filtrate, and PDA with 100 mL/L of the Fusarium filtrate. Fusarium filtrates were created using F. avenaceum, F. oxysporum, F. proliferatum, and F. sporotrichio ides. S.
mycoparasitica ascospore-inoculated plates were then incubated at room temperature (23 C) for 3 d, and spore germination was examined daily. All treatments were in three replicates and experiment was repeated twice.
There was no spore germination on 1.5% WA, PDA, MLA, MEA, and CLA. The effects of 1.5% WA and PDA media amended with 100 mL/L of Fusarium-filtrates on spore germination were examined and are summarized in Table 4.
No germination was recorded on day 3 initially and observations were continued up to 7 d on WA or PDA alone and WA or PDA with either F. proliferatum or F.
sporotrichioides.
However, when either F. avenaceum or F. oxysporum filtrates were added to WA
and PDA, the percentage of ascospore germination in S. mycoparasitica drastically increased after 3 d incubation. On day 3, germination increased on the PDA amended with Fusarium-filtrate, compared to growth on WA supplemented with Fusarium-filtrate, for S.
mycoparasitica
- 39 -Table 4: Percentage germination of Sphaerodes mycoparasitica ascospore on various types of Fusarium-filtrate supplemented media, including water agar and potato dextrose agar checks S. mycoparasitica Spore germination (%)*
Medium Day 1** Day 3**
Water agar (WA) 0 d 0 d F. avenaceum-W A 11.1 1.7 b 35.2 2.6 b F. oxysporum-W A 1.6 1.3 C 18 1.4 F. proliferatum-W A 0 d 0 d F. sporotrichioides-W A 0 d 0 d Potato dextrose agar (PDA) 0 d 0 d F. avenaceum-PDA 21.5 1.4 a 63.2 2.4 a F. oxysporum-PDA 2.5 2 C 37.5 2 b F. proliferatum-PDA 0 d 0 d F. sporotrichioides-PDA 0 d 0 d * Numbers in each column represented mean of ascospore germination (in %) standard deviation.
** Each incubation day for Sphaerodes was analyzed separately. Means within each column for each medium treatment followed by the same letter are not significantly different at P < 0.05 after Kruskal-Wallis test.
Effects of fungal filtrates on spore germination: Fungal filtrates from four different phytopathogenic Fusarium species and three beneficial fungal isolates were employed to study spore germination of S. mycoparasitica, and host specificity response. Aqueous spore suspensions of S. mycoparasitica were suspended in the filtrates of four separate pathogenic Fusarium spp. and three beneficial fungal strains for 1 d and 3 d in the ratio of 1:2 (1 part of aqueous spore suspension: 2 parts of fungal filtrate). Filtrate-suspended spores (10 L) were then transferred and inoculated onto PDA for an additional day (spore germination was counted on PDA plate). Spore germination was count according to Goh and Vujanovic (2010) after an additional day of inoculation on PDA medium. Control treatments were suspended with sterilized distilled water or PDB. Abbreviations for different treatments at four separate chronosequences throughout the experiment: id Sus, id Sus + id PDA, 3d Sus, and 3d Sus + id PDA represent 1 day Fusarium-filtrate suspension, 1 day filtrate suspension with an additional day incubation on PDA medium, 3 day suspension, and 3 day filtrate suspension with an additional day incubation on PDA, respectively.
Medium Day 1** Day 3**
Water agar (WA) 0 d 0 d F. avenaceum-W A 11.1 1.7 b 35.2 2.6 b F. oxysporum-W A 1.6 1.3 C 18 1.4 F. proliferatum-W A 0 d 0 d F. sporotrichioides-W A 0 d 0 d Potato dextrose agar (PDA) 0 d 0 d F. avenaceum-PDA 21.5 1.4 a 63.2 2.4 a F. oxysporum-PDA 2.5 2 C 37.5 2 b F. proliferatum-PDA 0 d 0 d F. sporotrichioides-PDA 0 d 0 d * Numbers in each column represented mean of ascospore germination (in %) standard deviation.
** Each incubation day for Sphaerodes was analyzed separately. Means within each column for each medium treatment followed by the same letter are not significantly different at P < 0.05 after Kruskal-Wallis test.
Effects of fungal filtrates on spore germination: Fungal filtrates from four different phytopathogenic Fusarium species and three beneficial fungal isolates were employed to study spore germination of S. mycoparasitica, and host specificity response. Aqueous spore suspensions of S. mycoparasitica were suspended in the filtrates of four separate pathogenic Fusarium spp. and three beneficial fungal strains for 1 d and 3 d in the ratio of 1:2 (1 part of aqueous spore suspension: 2 parts of fungal filtrate). Filtrate-suspended spores (10 L) were then transferred and inoculated onto PDA for an additional day (spore germination was counted on PDA plate). Spore germination was count according to Goh and Vujanovic (2010) after an additional day of inoculation on PDA medium. Control treatments were suspended with sterilized distilled water or PDB. Abbreviations for different treatments at four separate chronosequences throughout the experiment: id Sus, id Sus + id PDA, 3d Sus, and 3d Sus + id PDA represent 1 day Fusarium-filtrate suspension, 1 day filtrate suspension with an additional day incubation on PDA medium, 3 day suspension, and 3 day filtrate suspension with an additional day incubation on PDA, respectively.
-40 -No spore germination was observed in the treatments with P. bilaii and C.
globosum fungal filtrates. Both water and PDB suspension controls appeared to trigger approximately 1.8-3.8% spore germination for S. mycoparasitica, on day 3 with an additional day on PDA only (Table 5). Ascospores of S. mycoparasitica suspended in filtrates of pathogenic F.
sporotrichioides and beneficial T. harzianum showed no germination on day 1 in suspension, but low number of ascospore germination was observed after 1 day in suspension followed by an additional day on PDA, and in the other incubation treatments. Ascospores suspended in F.
proliferatum-filtrate were observed to germinate in low abundance for 1 d, 1 d with an additional day on PDA, 3 d, and 3 d plus an additional day on PDA. Sphaerodes ascospores showed the highest number of germinated ascospores in the F. avenaceum-filtrate suspension. In the day 1 suspension treatments, ascospore germination of S. mycoparasitica in F.
avenaceum-filtrate was significantly higher (89.2%) than in other treatments. When ascospores of the S. mycoparasitica were suspended in F. oxysporum-filtrate, the amount of spore germination was increased compared to control. There was no germination recorded for the 1 d suspension in the F.
oxysporum-filtrate treatment. However, a low number of ascospores were stimulated in F.
oxysporum-filtrate suspension on the second day. Based on the ascospore germination rate and response, S. mycoparasitica showed high specificity to F. avenaceum and F.
oxysporum.
globosum fungal filtrates. Both water and PDB suspension controls appeared to trigger approximately 1.8-3.8% spore germination for S. mycoparasitica, on day 3 with an additional day on PDA only (Table 5). Ascospores of S. mycoparasitica suspended in filtrates of pathogenic F.
sporotrichioides and beneficial T. harzianum showed no germination on day 1 in suspension, but low number of ascospore germination was observed after 1 day in suspension followed by an additional day on PDA, and in the other incubation treatments. Ascospores suspended in F.
proliferatum-filtrate were observed to germinate in low abundance for 1 d, 1 d with an additional day on PDA, 3 d, and 3 d plus an additional day on PDA. Sphaerodes ascospores showed the highest number of germinated ascospores in the F. avenaceum-filtrate suspension. In the day 1 suspension treatments, ascospore germination of S. mycoparasitica in F.
avenaceum-filtrate was significantly higher (89.2%) than in other treatments. When ascospores of the S. mycoparasitica were suspended in F. oxysporum-filtrate, the amount of spore germination was increased compared to control. There was no germination recorded for the 1 d suspension in the F.
oxysporum-filtrate treatment. However, a low number of ascospores were stimulated in F.
oxysporum-filtrate suspension on the second day. Based on the ascospore germination rate and response, S. mycoparasitica showed high specificity to F. avenaceum and F.
oxysporum.
-41-Table 5: Percentage spore germination of Sphaerodes mycoparasitica ascospores suspended in different Fusarium and biocontrol fungi filtrates assesed throughout four chronosequences. Control treatments were suspended with sterilized distilled water 0 (SDW) or potato dextrose broth (PDB).
cio S. mycoparasitica Spore germination (%)*
Treatment**
id Suspension id Sus + id PDA 3d Suspension 3d Sus + id PDA
SDW 0 c 0 e 0 e 2 1.2 PDB 0 c 0 e 0e 2.8 1.9 c E avenaceum-filtrate 89.2 6.2 a 91 + 8.4 a 93.2 6.7 a 94.2 7.9 a F. oxysporum-filtrate 0 c 81.8 11 b 85 7.9 b 91 10 a F. proliferatum-filtrate 2.6 3.6 b 3.2 4.6 d 10 7.4 c 11.2 8b F. sporotrichioides-filtrate 0 c 3 2.8 d 3.3d 10 5.6 b P. bilaii-filtrate 0 c 0 c 0 c 0 c T. harzianum-filtrate 0 c 9 1.1 c 1.6 1.6 d 10.4 1.5 b C'. globosum-filtrate 0 c 0 c 0 c 0 c * Numbers in each column represented mean of ascospore germination (in %) standard deviation.
** Each incubation day for S. mycoparasitica was analyzed separately. Means within each column of S.
mycoparasitica for each filtrate-suspension treatment followed by the same letter (in superscript) are not significantly different at P < 0.05 after Kruskal-Wallis test.
1-d Spore germination assessments: Microscopic assessments of ascospore germination were conducted after incubation for 1 d and 3 d (spore germination was counted on suspensions).
Percentage of germinated spores was obtained by scoring the spores on a Petri dish while observing them through the 200x and 400x objectives of the Carl Zeiss Axioskop2 microscope and systematically choosing 50 spores, starting at the top right corner and continuing to count until 50. Each drop of the ascospore suspension on a growth medium plate was considered as a subunit, and there were three subunits or replicates per plate. The experiments were repeated twice. An ascospore was only considered germinated when the germ tubes exceeded the width of the ascospore (approximately 1211m). Germinated ascospores were counted and recorded as a percentage of the total ascospore number that was counted.
Ascospores from S. mycoparasitica showed two kinds of germination patterns.
Single polar germination was more prevalent in the F. oxysporum-filtrate suspension (Figures 16A, 16B, 16D, 16E). A small number of spores from S. mycoparasitica found to produce shorter double-polar germination in the treatment with F. oxyporum-filtrate at 3 d suspension with additional 1 d on PDA (Figure 16F). In the F. avenaceum-filtrate suspension, ascospores of S.
mycoparasitica showed higher preference for two-polar germination (Figures 16G). Single polar germination was also found in F. avenaceum-filtrate suspension; however it was lower than in F.
oxysporum-filtrate treatment. Commonly, these single-polar germinated spores produced larger web-like organizations and longer hyphal formation (in F. avenaceum-filtrate suspension), which was rarely found in F. oxysporum-filtrate suspended spores (Figures 16H, 161).
The majority of the germinated ascospores in S. mycoparasitica, which was activated through suspension in F.
oxysporum-filtrate were detected to be either at an angle of 90 (Figures 16E, 16F) or between 90 and 180 (Figure 16F) at the tip of the polar germ pores. However, very few spores germinated at an angle of 180 (Figure 16D). Most activated spores (with F.
avenaceum-filtrate treatment) showed germination at an angle of 180 (Figure 16G).
Example 8 The host specificity of S. mycoparasitica to F. graminaerum 3-ADON and 15-ADON
strains were assessed in this study.
Media, reagents and chemicals: Potato dextrose agar (PDA), potato dextrose broth (PDB), yeast extract, malt extract agar (MEA), agar, and peptone were purchased from Difco (Becton Dickinson Diagnostics). Streptomycin sulphate, Neomycin sulphate, and other reagents of analytical grade were from Sigma-Aldrich.(0akville, ON, CA) IQ SYBR Green SupermixTM for real-time PCR reactions was acquired from Bio-Rad Laboratories (Mississauga, ON, CA).
Fungal strains and growth: All phytopathogenic Fusarium strains: Fusarium graminearum 3-ADON (Fgra3) SMCD 2243, and 15-ADON (Fgral5) SMCD 2244 chemotypes, F.
avenaceum (Fave) SMCD 2241, F. oxysporum (Foxy) SMCD 2242, F. proliferatum (Fpro) SMCD
2244, F.
sporotrichioides (Fspo) SMCD 224; and one mycoparasitic Sphaerodes mycoparasitica SMCD
2220 strain were retrieved from Saskatchewan Collection and Database (SMCD), maintained on PDA amended with antibiotics (100 mg/L streptomycin sulphate and 12 mg/L
neomycin sulphate) and used throughout this study.
Spore production and germination assays: Ascospores of S. mycoparasitica were produced on Modified Leonian's agar (MLA), harvested and prepared as previously described.
Also, Fusarium spp. filtrates were prepared, S. mycoparasitica spore germination assays in six different Fusarium filtrates were carried out as previously described.
Sphaerodes mycoparasitica spore germination suspended in both F. graminearum chemotype 3-ADON and 15-ADON filtrates was lower compared to F. avenaceum for the first incubation day, and compared to both F. avenaceum and F. oxysporum for the remaining incubation days (P <0.05; with Mann-Whitney Test) (Figure 17). No significant differences in germination between F. graminearum, F. proliferatum, and F. sporotrichioides filtrate treatments were observed for the first two incubation days. However, treatments with F.
graminearum filtrates showed significantly higher germination rate of S.
mycoparasitica compared to F. sporotrichioides filtrate treatment during later incubation time points (Figure 18).
Dual-culture assays: Dual-culture assays for examining the degree of hyphal reduction/inhibition or damage to F. graminearum chemotypes were assessed as disclosed in Goh and Vujanovic (2009, Mycologia DOT: 10.3852/69-171). S. mycoparasitica is slow-growing fungus as compared to F. graminearumus 3 and 15 strains. Therefore, S. mycoparasitica was pre-inoculated onto the PDA plates for 1 d, at 21 C in darkness, prior to inoculating Fusarium mycelial plugs. Linear mycelial growth of Fusarium strains for both treatments indicated above was measured and recorded daily for 5 days. Around 0.5 x 1.5 cm2 (sampling zones) located approximately 0.2 cm behind the contact zone between F. graminearum and S.
mycoparasitica was excised and subjected to DNA extraction. Each treatment was with three replicates and the experiment was repeated twice. The PDA plate inoculated with F. graminearum only was the positive control. Total genomic DNA was extracted using a DNeasy Plant Mini Kit. The DNA
was eluted once in 50 I of buffer AE and stored at -20 C until real-time PCR
quantification assays (as described below).
Since S. mycoparasitica demonstrated slower mycelial growth (0.56 cm per day;
n = 9) compared to F. graminearum 3-ADON (0.74 cm per day; n=6) and 15-ADON (0.68 cm per day;
n=6) chemotypes, the linear growth of F. graminearum mycelia in dual-culture was assessed using the pre-inoculation method. S. mycoparasitica was pre-inoculated on PDA
for 1 d followed by F. graminearum inoculation. The pre-inoculation approach demonstrated significant differences (starting day 3) in linear growth suppression of F. graminearum chemotype 3 and 15 compared to the co-inoculation approach (Figures 19A and 19B).
Establishment of mycoparasitism: Fusion biotrophic mycoparasitic interactions between S.
mycoparasitica and both F. graminearum chemotype strains, and intracellular parasitism interactions were examined and assessed on slide cultures according the methods described in Goh & Vujanovic (2009).
On day 3 of inoculation on PDA with F. graminearum 3-ADON and 15-ADON, no clamp-like or hook-like structures were formed by Sphaerodes mycoparasitica on Fusarium strains. On day 5 of inoculation, clamp-like and hook-like contact structures as well as Fusarium hyphal cells penetration (with haustoria) were observed (Figures 20E to 201).
Furthermore, on day 3, S. mycoparasitica removed red pigment from the mycelia of F.
graminearum 3-ADON on the slide culture (Figures 20A to 20D). As a result, S. mycoparasitica mycelia adopted reddish color (Figure 20C). Between day 4 and 5, formation of red crystal-like pellets was detected on the surface of mycoparasite hyphae (Figure 20D). The mechanism behind the color changes observed remains unknown. For F. graminearum chemotype 15-ADON, no uptake of red complex or release of red crystal-like structures by S. mycoparasitica hyphae were noted.
Nevertheless, flower-like hyphal structures appeared which could indicate possible growth inhibition of 15-ADON F. graminearum (Figure 20J). Significant differences in diameters of infected and non-infected hyphae were seen for both F. graminearum chemotypes (Figure 21).
Primers and standard curves: F. graminearum-specific (Fg16NF/R) and trichothecene Tri5 gene-specific (Tox5-1/2) primer sets were used in this study. Standard curves for F.
graminearum- and Tri5 gene-primer sets were generated, based on threshold cycles (Ct), by using a series of 10-fold diluted genomic DNAs from F. graminearum (spanning from 2.7 x 102 to 2.7 x 10-2 ng/ 1 for F. graminearum-specific primer set, from 2.7 x 102 to 2.7 x 10-1 ng/ 1 of 3-ADON strain and 3.0 x 101 to 3.0 x 10-2 ng/ l of 15-ADON strain DNAs for Tox5-1/2 primer set). Ct values were recorded and obtained by the Opticon MonitorTM software version 3.1 (Bio-Rad Laboratories Inc., Mississauga, ON, CA). Standard curves for different primer sets were constructed by plotting the threshold cycles (Ct) value versus the logarithm (logio) of the concentration of 10-fold serial diluted F. graminearum DNAs as described above.
Amplifications with different primer sets on the genomic DNAs of two F.
graminearum chemotypes were run in triplicates to obtain the mean and standard deviation of each 10-fold serial dilution.
Real-time PCR quantification: Real-time PCR amplifications on total genomic DNA extracted from the sampling zones (as described above) were performed using MiniOpticonTM (Bio-Rad Laboratories Inc., Mississauga, ON, CA). All the real-time PCR reactions were performed by utilizing the real-time PCR MJ white tubes (Bio-Rad Laboratories Inc., Mississauga, ON, CA) in a total volume of 25 1. The reaction mixture for all real-time PCR assays were: 12.5 1 of IQ
Supermix (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 1 IA of each 10 M
forward/reverse primers (Invitrogen), 9.5 I of sterilized UltraPure Millipore water, and 1 1 of DNA template. Real-time PCR conditions for Fg16NF/R primer set used were outlined in Nicholson et al. (1998, Physiol. Mol. Plant Pathol. 53: 17-37) with melting curve analysis at 60 to 95 C. Parameters for Tox5-1/2 primer set as described in Schnerr et al.
(2001, Int. J. Food microbiol. 71: 53-61).
Standard curves for different primer sets with different F. graminearum DNA
sources were constructed (Figure 22). Growth suppression or inhibition at the sampling zones (Figure 23) for F. graminearum chemotype 3 and 15 was further confirmed with real-time PCR
amplifications with F. graminearum- and tri5 gene-specific primer sets.
Sigmoidal curves for the four different treatments (F. graminearum chemotype 3 or 15 only and F.
graminearum chemotype 3 or 15 pre-inoculated with S. mycoparasitica) with Fg16NF/R primer set were generated using Opticon Monitor software version 3.1 and illustrated in Figure 24.
Using Fg16NF/R primer set, quantity of F. graminearum chemotype 3 DNA in the sampling zones significantly decreased when pre-inoculated with S.
mycoparasitica compared to un-inoculated treatment (P = 0.01) (Figure 25). However, DNA of F. graminearum chemotype 15 did considerably but not significantly reduced (P = 0.085 using T-test).
Using Tox5-1/2 primer set, amount of tri5 gene fragments diminished appreciably in both F.
graminearum chemotype 3 and 15 challenged with S. mycoparasitica (P < 0.05).
Example 9 The effects of S. mycoparasitica (biotrophic mycoparasite) and T. harzianum (necrotrophic mycoparasite) on expression of Tri and PKS genes by F.
graminaerum 3-ADON
and 15-ADON strains were assessed in this study.
Fungal strains and growth: Two Fusarium graminearum 3- ADON (SMCD 2243) and 15-ADON (SMCD 2244) chemotypes, Trichoderma harzianum necrotrophic (SMCD 2166) and Sphaerodes mycoparasitica biotrophic (SMCD 2220) mycoparasites were obtained from the Saskatchewan Microbial Collection and Database (SMCD). All strains were maintained on Potato dextrose agar (PDA, BD Biosciences, Mississauga, ON, CA) amended with antibiotics (100 mg/L streptomycin sulphate and 12 mg/L neomycin sulphate; Sigma-Aldrich Canada Ltd., Oakville, ON, CA) prior to study initiation.
Chemical fungicide control: A concentration of 100 mon tebuconazole was prepared from Folicur 432F (43.2% tebuconazole, Bayer CropScience Inc., Saskatoon, SK, CA;
Folicur is a registered trademark of Bayer Aktiengesellschaft, Leverkusen, Fed. Rep.
Germany). This fungicide preparation was used throughout the study.
In vitro assay, sampling and RNA extraction: Dual-culture assay was carried out between F.
graminearum and Folicur (100 mon tebuconazole) or biological (T harzianum or S.
mycoparasitica) agents on Minimal medium as disclosed by Xue et al. (2009, Can. J. Plant Pathol. 31: 169-179). Inoculated dual-culture plates were incubated at room temperature (23C) in dark conditions for a week. Mycelia of F. graminearum were harvested after a week of inoculation with either chemical or biological agents. A 0.5-cm2 mycelial plug was cut out approximately 0.5 cm away from the border of interaction and fungal cells were disrupted with liquid nitrogen. Total RNA from the samples were extracted using Aurum Total RNA mini kitTM
and extracted RNA was treated with DNAse (Bio-Rad Laboratories Inc., Mississauga, ON, CA) according the manufacturer's recommendations. Samples were then stored at -70C
till gene expression analysis.
During the In vitro studies, both F. graminearum 3-ADON and 15-ADON chemotypes changed mycelia morphology corresponding to each treatment (Figures 26A -26B). However, the main diagnostic distinction was the tendency of F. graminearum chemotypes to abundantly produce chlamidospores in clusters or chains when exposed to tebuconasole fungicide compared to mycoparasites (Figures 26C - 26D). In the case of F. graminearum chemotype 15 (15-ADON
producer), all four trichothecene genes ¨ Tri4, Tri5, Tri6, and Tril0 in this Fusarium strain were found to be induced in high amounts when treated with chemical tebuconazole compared to treatments that co-inoculated with biological agents (either S. mycoparasitica or T harzianum) (Figures 27A - 27D). F. graminearum chemotype 3 (3-ADON producer) was observed to demonstrate induction of Tri4, Tri5 and Tril0 genes when co-inoculated with necrotrophic mycoparasitic Trichoderma harzianum (Figures 27A, 27B, 27D). When F.
graminearum chemotype 3 was treated with Folicur fungicide, only Tril0 gene was being induced (Figure 27D). Generally, all four Tri genes were repressed significantly when challenged with biotrophic mycoparasitic Sphaerodes mycoparasitica (Figure 27A - 27D).
Real-time Reverse-transcription PCR: Real-time RT-PCR was performed by using an IScript One-Step RT-PCR kitTM with SYBR Green on a MiniOpticon Cycler System (Bio-Rad Laboratories Inc., Mississauga, ON, CA), according to the manufacturer's instruction. Primer sets used for amplification and gene expression were summarized in Table 6.
Table 6: Primer sets used to amplify Tri4, Tri5, Tri6, Tri10, PKS4, PKS13, and fl-tubulin by Real-time PCR.
SEQ ID Gene Primer sequences Reference Tri4-F: TAAACGCCCGCGAAGTTCACA Jiao et al. 2008, FEMS Lett.
SEQ ID NO: 6/7 Tri4 Tri4-R: TGGTGATGGTTCGCTTCGAG 285: 212-219 Tr5F: AGCGACTACAGGCTTCCCTC Doohan et al. 1999, Appl.
SEQ ID NO: 8/9 Tri5 Environ. Microbiol.
65:
Tr5R: AAACCATCCAGTTCTCCATCTG
SEQ ID NO:
Tri6 Tri6-1 TCTCTACCAACGGTGGATTCAACC Pinson-Gadais et al. 2008, 10/11 Tri6-2 AGCCTTTGGTGCCGACTTCTTG Mycopathol. 165: 51-59.
SEQ ID NO:Tri1O-F: TCTGAACAGGCGATGGTATGGA
Tri 1 0 Jiao et al. 2008 12/13 Tri1O-R: CTGCGGCGAGTGAGTTTGACA
SE ID NO: PKS4-P S .1 Lysoe et al. 2006, Appl.
Q
14/15 PKS4 GTGGGCTTCGCTAGACCGTGAGTT Environ.
Microbiol.
PKS4-PS .2 ATGCCCTGATGAAGAGTTTGAT 72:3924-3932 PKS13-PS.1 SEQ ID NO: CCCCCAACTCGACGTCAAATCTAT
PKS1 3 PKS13-PS .2 Lysoe et al. 20 TTCTTCCCGCCGACTTCAAAACA
SEQ ID NO: fi- FGtubf GGTCTCGACAGCAATGGTGTT
18/19 tubulin FGtubr GCTTGTGTTTTTCGTGGCAGT Lyscie et al. 2006 RT-PCR sample (-25 'IL) contained 3 mt of RNA template, 8.85 [IL of nucleae-free water, 12.5 1,11_, of RT-PCR reaction mixture (2x), 0.5 iL of IScript RT enzyme mix (50x) (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 0.1 [IL of 50 M solutions of both forward and reverse targeted gene-specific primers (Invitrogen Corp., Carslbad, CA, USA). Real-time PCR
conditions were performed as outlined by manufacturer's recommendations: 50C for 10min, 95C for 5min, followed by 40 cycles of denaturing at 95C for 1 Os and annealing at 55C for 30s, 95C for lmin and 55C for 1 min.
PCR reactions were checked for absence of any primer-dimer formation or non-specific PCR
amplification by performing melting curve analysis. Contamination of RNA
template with residual genomic DNA was eliminated because there was no amplification detected using reverse transcriptase free real-time RT-PCR reaction as template. Fold change in gene expression for each treatment was normalized to 13-tubulin internal reference gene and relative to the expression for the control treatment (Fusariwn alone on minimal medium), using the 2-AAcT method proposed by Livak, K.J., and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-AACT Method. Methods 25(4):402-408. AACT = (CT, target-gene CT, Fgtub)treatment ¨ (CT, target-gene CT, Fgtub)control, where treatment and control indicate F.
graminearum challenged with chemical or biological agent and F. graminearum alone, respectively.
For gene expression analyses based on targeted genes (PKS4 and PKS13) that responsible for zearalenone biosynthesis, these two polyketide synthase genes from both F.
graminearum chemotypes were detected to be repressed in all chemical or biological treatments (Figures 28A -28B). Repression of F. graminearum chemotype 15 PKS4 and PKS13 genes in the treatment with S. rnycoparasitica biotrophic mycoparasite was illustrated to be significantly higher compared to treatments with T harzianum and tebuconazole fungicide (Figures 28A - 28B).
Tri genes were observed to be more sensitive in F. graminearum chemotype 3 when co-inoculated with Trichoderma necrotrophic mycoparasite, however, in E
graminearum chemotype 15, Tri genes appeared to be more responsive towards treatment with chemical fungicide (Figures 27A-27D).
PKS4 gene for both E graminearum chemotypes was monitored to be more sensitive towards treatments with Trichoderma necrotrophic mycoparasitic fungus compared to chemical and biotrophic mycoparasitic agents (Figure 28A). However, PKS13 gene in F.
graminearum chemotype 15 was found to demonstrate higher sensitivity towards chemical stimulus (Figure 28B).
Mycotoxins extraction and analyses: DON, ZEA, 3-ADON and 15-ADON mycotoxins were extracted from agar mycelial plugs (0.5cm2) cut from the sampling zone located approximately 0.5 cm behind the contact zone between E graminearum and S. mycoparasitica.
The extraction was performed by three 10-ml volumes of ethyl acetate. The samples were sonicated on ice and shaken vigorously in ethyl acetate, and then they were allowed to stand for 5 min for separation of phases. The organic phase was siphoned off and passed through sodium sulfate to remove water. The solvent was allowed to evaporate at room temperature (23 C) for 3 days. The residue was then re-dissolved in 2 ml of acetonitrile for thin liquid chromatography (TLC) (only ZEA) and high performance liquid chromatography (HPLC) (all mycotoxins) analyses.
TLC was performed on Silica gel 60 plates (Merck Frosst Canada Ltd, Kirkland, PQ, CA) in the solvent system with ethyl acetate-toluene (3:1) for 40 min. 3-ADON on the gel plates was sprayed with 20% aluminum chloride in 95% ethanol, detected and analyzed following the protocols disclosed by Vasavada and Hsieh (1987, Appl. Microbiol. Biotech. 26: 517-521). Known standard controls of pure DON, ZEA, 3-ADON and 15-ADON (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) were employed to compare with other treatments. All four mycotoxins were quantified using a Water's 2695 HPLC system with: 250 x 4.60 mm, Luna 5 micron C18 (2) 100A
column (Phenomenex Inc., Torrance, CA USA) and a photodiode-array (PDA) detector was used with an isocratic solvent system (methanol: water-methano containing 5% (v/v) (90:10) ratio). The PDA detector measured the UV spectrum (190-500 nm). Samples were dissolved in acetonitrile and 10 I loaded onto the column using automatic injector and mycotoxins were eluted with solvent as a mobile phase at a rate of 0.75 ml min-1 for 25 mins. Peak height method was incorporated to determine the exact amount of DON, ZEA, 3-ADON and 15-ADON
against a standard curves. Ratio between mycotoxins extracted from treatments of F.
graminearum treated with Folicur and F. graminearum only was calculated for HPLC.
The amount of DON produced was between 70 to 90 ug/L. Consequently, it was too low to be detected in TLC analysis. ZEA was high enough to be analyzed with TLC
and both F.
graminearum chemotypes were observed to produce higher amounts of ZEA toxin under the treatment with Folicur compared to other treatments (Figure 29). With HPLC, ZEA was found to be reduced when challenged with Folicur compared to F. graminearum colony only (Figure 30). DON and 3-ADON was detected to increase for both F. graminearum when inoculated together with Folicur (Figure 30). Folicur was monitored to trigger highest amount of 15-ADON under in vitro assay (Figure 30).
Example 10 The effects of S. mycoparasitica (biotrophic mycoparasite) and T harzianum (necrotrophic mycoparasite) on expression of aurofusarin gene by F.
graminaerum 3-ADON and 15-ADON strains were assessed in this study.
Fungi, media, and culture conditions: Fungal strains were obtained from culture collections at University of Saskatchewan (Food and Bioproduct Sciences fungal collection).
Strains of the following fungal species were used, plant pathogenic Fusarium graminearum , F.
graminearum (3ADON), F. graminearum (15ADON), F. avenaceum, F. culmorurnõ F. proliferatum, F.
oxysporum, F. arthrosporoides and mycoparasitic Spaeherodes mycoparasitica and Trichoderma harzianum. Fungi were maintained on potato dextrose agar for 2 wks at 21 C in darkness.
Folicur was also used in this study.
Experiments for the impact of red color nuances on aurofusarin gene expression was carried out using previously classified F. avenaceum isolates, based on VCGs and colony colours. F. avenaceum isolates color identification numbers (CIN) were generated with Hex Color Code Chart. CINs are: Ds #71232B: Red and highly virulent, tolerant at 80 C for 4 hours;
Es #4E040B: Moderately red and moderately virulent, tolerant 40 C for 4 hours;
Bs #A86608:
White and non virulent, susceptible 40 C for 4 hours. Three 0.1 g mycelium samples from three replicates of each isolate was mixed and used for DNA extraction and RT-PCR
analyses.
Fungal DNA Extraction and PCR: Fungal cultures were grown on 1.5 ml PD broth and centrifuged at 10,000 rpm for 5 min. Supernatant was discarded and, subsequently a total DNA
of 2-week old cultures representative of each phenotype/VCG was extracted from the pellet with an Ultra Clean microbial DNA Isolation KitTM (Qiagen Inc., Mississauga, ON, CA) following manufacture's instructions. The purified DNA was resuspended in 50 111 of elution buffer and stored at -200C until further analyses. Full length genomic sequences of PKS
region (7.2Kb) from Fusarium graminearum were obtained from NCBI Genbank. Six pairs of primers were designed (Table 7).
Table 7: PCR Primers designed from fall-length genomic sequences of the PKS
region of F.
graminearum SEQ ID Primer Sequence SEQ ID NO: 20 PKSF1 TCGAGTTTCGTGTTGCGTGT
SEQ IS NO. 21 PKSR1 AGGTAGTTCGCCATACCCGT
SEQ ID NO: 22 PKSF2 AATTGTGCCCGAGGCAGTAC
SEQ ID NO: 23 PKSR2 CATTGGTTCCGCCCGCAATAG
SEQ ID NO: 24 PKSF3 TGACAACTTCGCTGGTTTGGA
SEQ ID NO: 25 PKSR3 CATAGCTTGGCCAGTGCCATC
SEQ ID NO: 26 PKSF4 GAAGTCATTCGGTGTTGAGC
SEQ ID NO: 27 PKSR4 GCTCTGGATTGGGTATCGCAC
SEQ ID NO: 28 PKSF5 ACTCGAGCATCCGTCGCAATG
SEQ ID NO: 29 PKSR5 AGCAACATCTCCGTCTGGAG
SEQ ID NO: 30 PKSF6 GTTGAACTGTCCATGGCTGA
SEQ ID NO: 31 PKSR6 GAATGAAGGCAATCTGCTGC
All the reactions were carried out in 25-0 volumes containing 2.5 ill of 10X
PCR buffer, 1 of Q solution, 1 ill of each primer (10 M), 0.5 111 of 10mM dNTPs mix, and 1.25 U of Taq polymerase (Qiagen Inc., Mississauga, ON, CA). Reaction mixtures were mixed gently and were given flash spin prior to PCR on a Eppendorf Master CyclerTM (ep gradient S).
PCR amplicons were purified using the QIAquick PCR purification kit (Qiagen Inc., Mississauga, ON, CA;
QIAquick is a registered trademark of Qiagen GmbH Corp., Hilden, Fed. Rep.
Germany) and commercially sequenced (Plant Biotechnology Institute, Saskatoon, SK).
Development of a real time RT-PCR assay for detection and quantification of the aur gene in Fusarium species: All sequences were sequenced, sequences were aligned, several primer sets were designed and tested. One set, named Auro RT (SEQ ID NO: 32) and Auro RTR
(SEQ ID
NO: 33) was selected based on efficiency to amplify aurofusarin gene.
SEQ ID NO: 32 ACCTCACTGGAATCAGAGCGCAGC
SEQ ID NO: 33 ATGACRACTTCCCGTGGRCC
The specificity of this set of primers was tested by conventional PCR assay using genomic DNA
from Fusarium and mycoparasites using the amplification reactions volumes indicated above.
The amplification protocol was 1 cycle of 120 s at 94 C, 35 cycles of 30 s at (denaturalization), 30 s at 56 C (annealing), 45 s at 72 C (extension), and 1 cycle of 10 mm at 72 C.
The pair of primers used to amplify the 13-tubulin (tub) gene (the endogenous control gene used to normalize the results) were FGtubf (SEQ ID NO: 34) and Fgtubr (SEQ ID
NO: 35).
SEQ ID NO: 34 GGTCTCGACAGCAATGGTGTT
SEQ ID NO: 35 GCTTGTGTTTTTCGTGGCAGT
The PCR efficiencies of the real time RT-PCR for both genes were checked by performing a 10-fold serial dilution of positive control template to generate a standard curve, and by plotting the Ct as a function of log [10] of template.
Presence of aurofusarin was tested and quantified by RT-PCR in pathogenic Fusarium graminearum , F. graminearum (3ADON), F. graminearum (15ADON), F. avenaceum, F.
culmorum, Mycoparasite, F. proliferatum, F. oxysporum, F. arthrosporoides and mycoparasitic, Sphaerodes mycoparasitica and Trichoderma harzianum. Relative gene expression was studied when Fusarium isolates were co-cultured with Folicur , Sphaerodes and Trichoderma.
The designed RT-PCR primer set was checked for its specificity by conventional PCR on tested fungal species. A single band 157 bp long was amplified in F.
graminearum (3ADON), F.
graminearum (15ADON), F. avenaceum, F. culmorum but not in other strains. One week old grown cultures tested for their aurofusarin relative gene expression and I3-tublin gene was used as an internal control. F. graminearum which produces 15ADON showed highest level gene expression followed by F .graminearum (3ADON) and similar kind of expression results were observed with F. avenacum and F. culmorum. Aurofusarin relative gene expression was quantified when each Fusarium species was co-cultured for a week with Sphaerodes, Trichoderma and Folicur (Figure 31). Among all tested, S. mycoparasitica was most effective in reducing the aurofusarin relative gene expression followed by Trichoderma and Folicur (Figure 31). S. mycoparasitica was most affective on F. graminearum producing 15ADON, but also efficiently reduced the expression level in F. culmorum and F. avenaceum and F.graminearum producing 3ADON (Figure 31). Similarly, Trichoderma was able to somewhat affect F. graminearum producing 15ADON and F. culmorum, but showed no impact on F.
avenaceum, whereas enhanced the expression in F.graminearum producing 3ADON
(Figure 31).
Finally, Folicur could be able to provoke minor reduction in F. graminearum producing 15ADON aur gene expression, but with less impact on F. culmorum and F.
avenaceum.
Inversely, Folicur increased expression almost 2-fold when compared to F.
graminearum producing 3ADON (Figure 31).
RNA isolation, reverse transcription and real time RT-PCR: All the cultures were grown for a week under dark and used for expression studies. Fungal total RNA was isolated using the "Total Quick RNA Cells and Tissues" KitTM (Bio-Rad Laboratories Inc., Mississauga, ON, CA), according to the manufacturer's instructions, and stored at ¨80 C. DNAse I
treatment to remove the chromosomal DNA contamination from the samples was performed using the "Deoxyribonuclease I, Amplification Grade" (Invitrogen Corp., Carlsbad, CA, USA). First strand cDNA was synthesized using the "Iscript RNA PCR Reagent Kit" (Bio-Rad Laboratories Inc., Mississauga, ON, CA). Relative Quantification of aur gene expression was performed in a MiniOpticon Sequence Detection System using the SYBR Green PCR Master Mix (Bio-Rad Laboratories Inc., Mississauga, ON, CA) and the primer pairs indicated above.
The PCR thermal cycling conditions for both genes were as follows: an initial step at 95 C
for 10 min and 40 cycles at 95 C for 15 s and at 60 C for 1 min. SYBR green PCR master mix 12.5 ul (Bio-Rad Laboratories Inc., Mississauga, ON, CA) was used as the reaction mixture, with the addition of 6.5 ul of sterile milli-Q water, 1.0 ul of each primer (5 uM), and 5 ul of template cDNA, in a final volume of 25 iii. In all the experiments, appropriate negative controls containing no template were subjected to the same procedure to exclude or detect any possible contamination or carryover. Each sample (triplicate) was amplified twice in every experiment. The results were normalized using the all fungi cDNA amplifications run on the same plate. The tub2 gene is an endogenous control that was used to normalize quantitation of mRNA target for differences in the amount of total cDNA added to each reaction. Real Time RT-PCR analysis is based on the threshold cycle (CT), which is defined as the first amplification cycle at which the fluorescence signal is greater than the minimal detection level, indicating that PCR
products become detectable. Relative quantitation was the analytical method used in this study. A comparison within a sample is made with the gene of interest (aur) to that of the endogenous control gene (tub2). Quantitation is relative to the control gene by subtracting the CT of the control gene (tub2) from the CT of the gene of interest (aur) (ACT). Each ACT value (corresponding to each sample) was subtracted by the calibrator value (FpMM6-1C) to obtain the corresponding AACT
values. AACT values were transformed to log2 (due to the doubling function of PCR) to generate the relative expression levels.
In total 9 isolates were subjected to RT-PCR analyses for quantitative gene expression analyses. Three kinds of colored samples were used in this study composed of 3 white colored, 3 moderately red colored and 3 dark red colored isolates. White colored isolate was used as control and their expression was normalized and taken as one. As reported moderately red colored isolates were able to tolerate 40 C whereas; dark red isolates were able to tolerate 80 C. When RT-PCR results observed dark colored isolates expressed almost more than 9 folds enhanced relative gene expression whereas, moderately red colored isolates expressed 4 folds enhanced expression (Figure 32).
Example 11 Greenhouse studies were performed to assess the effects of S. mycoparasitica inoculants on development of Fusarium head blight symptoms and the accumulation of tricothecene mycotoxin gene in wheat and barley spikes.
Fungal isolates and growth: S. mycoparasitica SMCD 2220, and F. graminearum 3-ADON
chemotype SMCD 2243 were obtained from the Saskatchewan Microbial Collection and Database (SMCD). These fungal cultures were grown on potato dextrose agar (PDA, BD
Biosciences, Mississauga, ON, CA) supplemented with antibiotics prior to the study. Mycelial suspensions of these fungal strains (104 for F. graminearum and 106 for both biotrophic mycoparasite and antagonistic fungus) were produced for greenhouse experiment, as follows.
F. graminearum 3-ADON chemotype strain was inoculated in potato dextrose broth (PDA, BD Biosciences, Mississauga, ON, CA), incubated at room temperature (-21 C) in darkness using shaker (100rpm), for a week prior to harvesting the mycelia for greenhouse application. Mycelia of F. graminearum were transferred into sterilized commercial blender for cutting the mycelial inoculants into small debris and these mycelia were adjusted to concentration of 104 CFU/mL.
S. mycoparasitica was inoculated in the best preferment yeast peptone dextrose (YPD) (yeast, 5g; peptone, 10g, dextrose, 10g per 1 L of sterile-double distilled water) broth, incubated at room temp in darkness, using shaker at 100 rpm for a week prior to harvesting the mycelia.
Mycelia of S. mycoparasitica were transferred into sterilized commercial blender for cutting the mycelial inoculants into small debris and the mycelia were adjusted to concentration of 104 CFU/mL for greenhouse application.
Greenhouse trials: The Fusarium-susceptible wheat CDC-TEAL and barley BOLD
cultivars were used to test the efficacy of biotrophic mycoparasite S. mycoparasitica and one potential Fusarium antagonistic fungal isolate as potential biocontrol agent for managing Fusarium head blight diseases. Surface-sterilized wheat and barley seeds were planted in 15-cm diameter pots containing Pro-Mix soil (Sun Gro Horticulture, Delta, BC, CA) and maintained at 23-25 C
during the day time and 18-20 C during the night time in a greenhouse with light intensity of 360 mot 111-2 s-I, watered every 2 d, and fertilized every 14 d using 1300 ppm of NPK (20-20-20) fertilizer. All treatments in the experiment were with three replicates and repeated twice.
At the anthesis stage, wheat and barley spikes were sprayed with mycelial suspension of S. mycoparasitica at a concentration of 106 CFU/mL, antagonistic fungal isolate at a concentration of 106 CFU/mL, 65 mon tebuconazole, or sterile distilled water.
The treatments were applied as outlined in Xue et al. (2009, Can. J. Plant Pathol. 31: 169-179) with slight modifications. Following the inoculation of potential biocontrol agents, plants were kept at room temperature and covered with sterile Whirl-Pak bags (Whirl-Pak is a registered trademark of Aristotle Corp., Stamford, CT, USA) for overnight to allow fungal growth prior to F.
graminearum inoculation. Plants were then inoculated with mycelial suspension of F.
graminearum at a concentration of 104 CFU/mL. Fusarium graminearum inoculum was sprayed on the spikes and covered with sterile Whirl-Pak bags for overnight. The inoculated plants were maintained for an additional 21 days prior to sampling. Percentage of infected spikelets (IS) per spike, FHB index, FDK, and weight of 100 seeds were rated as outlined in Xue, A.G., Voldeng, H.D., Savard, M.E., Fedak, G., Tian, X., and Hsiang T. (2009) Biological control of fusarium head blight of wheat with Clonostachys rosea strain ACM941, Canadian Journal of Plant Pathology 31: 169-179. The FHB visual severity scale was determined.
The biocontrol effects of different concentrations of S. mycoparasitica of Fusarium head blight symptoms in barley are shown in Figure 33. The data in Figures 33A, 33B, and 33C show that inoculation of a susceptible barley cultivar with F. graminearum significantly reduced that height of the plants, average numbers of spikes form per plant, and the average weight of 5 spikes. However, inoculation of the barley cultivar with S. mycoparasitica did not affect growth and development. Treatment of F. graminearum-infected barley with S.
mycoparasitica at an inoculation level of 106 CFU/mL prevented the onset of any Fusarium head blight symptoms.
Treatment of F. graminearum-infected barley with lower concentrations of S.
mycoparasitica, i.e., 104 CFU/mL and 105 CFU/mL, did not protect against the occurrence of the disease symptoms (Figures 33A - 33C).
The biocontrol effects of different concentrations of S. mycoparasitica of Fusarium head blight symptoms in wheat are shown in Figure 34. The data in Figures 34A, 34B, and 34C show that inoculation of a susceptible wheat cultivar with F. graminearum significantly reduced that height of the plants, average numbers of spikes form per plant, and the average weight of 5 spikes. However, inoculation of the wheat cultivar with S. mycoparasitica did not affect growth and development. Treatment of F. graminearum-infected wheat with S.
mycoparasitica at an inoculation level of 106 CFU/mL prevented the onset of any Fusarium head blight symptoms.
Treatment of F. graminearum-infected wheat with lower concentrations of S.
mycoparasitica, i.e., 104 CFU/mL and 105 CFU/mL, did not protect against the occurrence of the disease symptoms (Figures 34A - 34C).
The data in Figure 35 and Table 8 show that treatment of F. graminearum-infected barley with S. mycoparasitica at an inoculation level of 106 CFU/mL provided a comparable level of protection against the occurrence of Fusarium head blight symptoms, as was provided by the commercial fungicide Folicur .
t..) o ;o--H--t..) t..) cio o o Table 8: Effect of S. mycoparasitica, Folicur fungicide and F. graminearum on percentage of infected spikelets, Fusarium head blight index, percentage of Fusarium damaged kernels, and weight of 100 seeds of the barley cultivar BOLD in greenhouse trials.
Infected spikelets Fusarium head blight Fusarium damaged Weight of 100 seeds (g) n Treatment (%) index (%) kernels (%) 0 I.) 1 F. graminearum (Fg) 62.6 8.0 a 56.7 11.9 a 75 9.4 a 4.1 0.4 c -.1 I \ ) cri 15.4 3.1 bc 4.1 1.0 b 19 6.5 b 5.4 0.6 ab H
l9 S. mycoparasitica ¨ Fg 0, 1 Folicur ¨ E g 19.6 6.9 b 7.2 4.8 b 16 6.5 b 5.7 0.5 a I.) H
"
I
I \ ) I
I \ ) FP
.0 n ,-i n =
-::--, =
t..) u, c,., Real-time PCR quantification: Total DNA was extracted from the wheat and barly spices with DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA). Extracted total DNAs from spikes of different treatments were employed in real-time PCR quantification. Two different primer sets and PCR conditions used in this study were described in Nicholson et al.
(1998) for Fg16NF/R
primer set and Schnerr et al. (2001) for Tox5-1/2 primer set. Total DNA
extracted from spring wheat and barley spikes harvested from greenhouse trials were carried out in a MiniOpticon (Bio-Rad Laboratories Inc., Mississauga, ON, CA). All real-time PCR reactions were performed using real-time PCR MJ white tubes (Bio-Rad Laboratories Inc., Mississauga, ON, CA) with a total volume of 25 I of IQ supermix (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 1 1 of each 10 M forward/reverse primers (Invitrogen), 3.4 1 of BSA (Bovine Serum Albumin) (1.47 g/ 1) (Ishii, S. and Loynachan, T.E. (2004) Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods. Mycorrhiza 14:271-275), 5.1 I of sterilized UltraPure Millipore water, and 2 1 of DNA template.
Figure 36 shows standard curves of F. graminearum chemotype 3 genomic DNA
concentration standards versus cycle threshold (Ct) with PCR reactions performed in triplicate using primer sets (A) Tox5-1/2, with genomic DNA ranging from 270 ng (Logi =
2.90) to 0.27 ng (Logi() = -0.60), readings at 0.005 fluorescence line and (B) Fg16NF/R, with DNA template ranging from 270 ng (Logi() = 2.43) to 0.027 ng (Logic) = -1.57); in 10-fold dilution series, readings at 0.025 fluorescence line. Error bars indicate standard deviation for the mean of F.
graminearum chemotype 3 standard curves derived from tri5 gene and F.
graminearum specific primer sets. Figure 37 shows effects of S. mycoparasitica (B) and Folicur fungicide (Fol) treatments on F. graminearum chemotype 3-ADON genomic DNA detected in barley spikes employing RT-PCR. Treatments were: Fus ¨ F. graminearum; B-Fus ¨ S.
mycoparasitica with F. graminearum; Fol-Fus ¨ Folicur fungicide with F. graminearum. All values obtained were the means of six replicates. Error bars indicate standard deviation of the mean.
Means of F.
graminearum DNA with Tox 5 primer were log10 transformed prior to LSD test.
Both primer sets were analyzed separately. Values followed by the same letters within each primer set are not significantly different using LSD test at P < 0.05.
Example 12 In addition to the known Fusarium spp. hosts for S. mycoparasitica (i.e., F.
avenaceum, F. graminearum and F. oxysporum), we surprisingly discovered that F. culmorum and F.
equiseti are also mycoparasitised by S. mycoparasitica as disclosed in the following study.
Fungal isolates and growth: Biotrophic mycoparasites S. mycoparasitica SMCD2220, Sphaerodes quadrangularis strain CBS112764, Sphaerodes retispora var.
retispora strain CBS
994.72, and pathogenic Fusarium strains (F. arthrosporioides SMCD2247, F.
culmorum SMCD2248, F. equiseti SMCD2134, F. flocciferum 5MCD2135, F. poae 5MCD2136, and F.
torulosum SMCD2139) were obtained from the Saskatchewan Microbial Collection and Database (SMCD), Saskatchewan, Canada. All fungal isolates were grown and maintained on potato dextrose agar (PDA, BD Biosciences, Mississauga, ON, CA) prior to the study.
Fungal-fungal interactions: For examination of the interaction between isolates of Sphaerodes and Fusarium species, both biotrophic mycoparasite and Fusarium isolates were inoculated and assessed using slide culture assays. Slides were maintained in a sterile humidity chamber and daily observations on the hyphal interactions at the meeting place (contact zone) were performed under a Carl Zeiss Axioskop2 equipped with Carl Zeiss AxioCam ICc 1 camera with 20x, 40x and 100x objectives. Formation of biotrophic mycoparasitic contact structures attaching Sphaerodes species to Fusarium hyphae were examined, recorded and compared to drawings from the literature (Jordan & Barnett, 1978, Mycologia 70: 300-312;
Rakvidhyasastra & Butler, 1973, Mycologia 65: 580-593; Whaley & Barnett, 1963, Mycologia 55: 199-210).
Diameters of both parasitized and non-parasitized Fusarium hyphal cells were measured under light microscopy with a 100x objective lens. Each treatment used six replications consisted of Sphaerodes or Fusarium alone, and Sphaerodes¨Fusarium co-inoculated. The experiment was repeated twice. In the slide-culture assay, Fusarium mycelia infected with Sphaerodes haustoria were stained with lactofuchsin. Stained hyphae of both Fusarium and Sphaerodes in slide-culture were then examined with a Carl Zeiss Axioskop2 fluorescent microscope attached to Carl Zeiss AxioCam ICcl with 40x and 100x objectives. Slide-culture assays were also subjected to Zeiss META 510 confocal laser scanning microscopy (CLSM) analysis to observe intracellular mycoparasitism under a C-Apochromat 63x N.A.1.2 phase-contrast water immersion objective through Z-stacking mode to scan through the Fusarium hyphae with intracellular infection (CLSM with 514nm excitation ¨ argon and LP585 emission filters).
Hyphae-hyphae interactions and contact structures in the contact zone were examined for seven days. On day three, Sphaerodes mycoparasitica was found to produce hook-shaped contact structures on F. equiseti and F. culmorum (Figure 39). On day five, more hook-shaped contact structures and intracellular penetration of F. equiseti were observed (Figures 39A, 39A-39D).
The combination of lactofuchsin dye and fluorescent or confocal laser scanning microscopy revealed that the parasitized or penetrated Fusarium cells became empty (loss of cytoplasm = no fluorescence) or fluoresced with low intensity (very pale) (Figures 39A-39D) as compared to healthy Fusarium cells. During the seven days of observation, no S.
mycoparasitica hyphae were observed within F. culmorum cells. S. mycoparasitica produced hook-shaped contact structures (Figure 38A, a) more frequently than clamp-like contact structures (Figure 38B, b) on both F.
equiseti and F. culmorum. Diameters of F. equiseti, but not F. culmorum, hyphae parasitized by S. mycoparasitica were observed to be significantly reduced compared to non-parasitized Fusarium hyphae (with T-test, P = 0.001 and P> 0.05, respectively) (Figure 41).
None of the Fusarium taxa tested appeared to be suitable hosts for mycoparasitic S.
quadrangularis and S. retispora even after 10 days of co-inoculation on slide cultures. No contact biotrophic parasitic structures or intracellular parasitism by S.
quadrangularis and S.
retispora on the tested Fusarium strains were observed at the interaction or contact zone. Also, F. arthrosporioides, F. flocciferum, F. poae, and F. torulosum did not appear to be suitable hosts for S. mycoparasitica. Around five days after inoculation on slide culture assays, mycelia of F.
art hrosporioides were inhibited by S. mycoparasitica. F. arthrosporioides started to form rosette-like mycelia at the contact zone with S. mycoparasitica (Figure 39B).
On the fifth and seventh days after inoculation, anamorphic structures were produced by S. mycoparasitica more abundantly in the zone of contact with F. culmorum (Figures 40C and 40D). Anamorphic structures or asexual organs in close proximity to F.
culmorum mycelia were red-colored (Figure 40D), whereas the organs at a distance were not (Figure 40C).
cio S. mycoparasitica Spore germination (%)*
Treatment**
id Suspension id Sus + id PDA 3d Suspension 3d Sus + id PDA
SDW 0 c 0 e 0 e 2 1.2 PDB 0 c 0 e 0e 2.8 1.9 c E avenaceum-filtrate 89.2 6.2 a 91 + 8.4 a 93.2 6.7 a 94.2 7.9 a F. oxysporum-filtrate 0 c 81.8 11 b 85 7.9 b 91 10 a F. proliferatum-filtrate 2.6 3.6 b 3.2 4.6 d 10 7.4 c 11.2 8b F. sporotrichioides-filtrate 0 c 3 2.8 d 3.3d 10 5.6 b P. bilaii-filtrate 0 c 0 c 0 c 0 c T. harzianum-filtrate 0 c 9 1.1 c 1.6 1.6 d 10.4 1.5 b C'. globosum-filtrate 0 c 0 c 0 c 0 c * Numbers in each column represented mean of ascospore germination (in %) standard deviation.
** Each incubation day for S. mycoparasitica was analyzed separately. Means within each column of S.
mycoparasitica for each filtrate-suspension treatment followed by the same letter (in superscript) are not significantly different at P < 0.05 after Kruskal-Wallis test.
1-d Spore germination assessments: Microscopic assessments of ascospore germination were conducted after incubation for 1 d and 3 d (spore germination was counted on suspensions).
Percentage of germinated spores was obtained by scoring the spores on a Petri dish while observing them through the 200x and 400x objectives of the Carl Zeiss Axioskop2 microscope and systematically choosing 50 spores, starting at the top right corner and continuing to count until 50. Each drop of the ascospore suspension on a growth medium plate was considered as a subunit, and there were three subunits or replicates per plate. The experiments were repeated twice. An ascospore was only considered germinated when the germ tubes exceeded the width of the ascospore (approximately 1211m). Germinated ascospores were counted and recorded as a percentage of the total ascospore number that was counted.
Ascospores from S. mycoparasitica showed two kinds of germination patterns.
Single polar germination was more prevalent in the F. oxysporum-filtrate suspension (Figures 16A, 16B, 16D, 16E). A small number of spores from S. mycoparasitica found to produce shorter double-polar germination in the treatment with F. oxyporum-filtrate at 3 d suspension with additional 1 d on PDA (Figure 16F). In the F. avenaceum-filtrate suspension, ascospores of S.
mycoparasitica showed higher preference for two-polar germination (Figures 16G). Single polar germination was also found in F. avenaceum-filtrate suspension; however it was lower than in F.
oxysporum-filtrate treatment. Commonly, these single-polar germinated spores produced larger web-like organizations and longer hyphal formation (in F. avenaceum-filtrate suspension), which was rarely found in F. oxysporum-filtrate suspended spores (Figures 16H, 161).
The majority of the germinated ascospores in S. mycoparasitica, which was activated through suspension in F.
oxysporum-filtrate were detected to be either at an angle of 90 (Figures 16E, 16F) or between 90 and 180 (Figure 16F) at the tip of the polar germ pores. However, very few spores germinated at an angle of 180 (Figure 16D). Most activated spores (with F.
avenaceum-filtrate treatment) showed germination at an angle of 180 (Figure 16G).
Example 8 The host specificity of S. mycoparasitica to F. graminaerum 3-ADON and 15-ADON
strains were assessed in this study.
Media, reagents and chemicals: Potato dextrose agar (PDA), potato dextrose broth (PDB), yeast extract, malt extract agar (MEA), agar, and peptone were purchased from Difco (Becton Dickinson Diagnostics). Streptomycin sulphate, Neomycin sulphate, and other reagents of analytical grade were from Sigma-Aldrich.(0akville, ON, CA) IQ SYBR Green SupermixTM for real-time PCR reactions was acquired from Bio-Rad Laboratories (Mississauga, ON, CA).
Fungal strains and growth: All phytopathogenic Fusarium strains: Fusarium graminearum 3-ADON (Fgra3) SMCD 2243, and 15-ADON (Fgral5) SMCD 2244 chemotypes, F.
avenaceum (Fave) SMCD 2241, F. oxysporum (Foxy) SMCD 2242, F. proliferatum (Fpro) SMCD
2244, F.
sporotrichioides (Fspo) SMCD 224; and one mycoparasitic Sphaerodes mycoparasitica SMCD
2220 strain were retrieved from Saskatchewan Collection and Database (SMCD), maintained on PDA amended with antibiotics (100 mg/L streptomycin sulphate and 12 mg/L
neomycin sulphate) and used throughout this study.
Spore production and germination assays: Ascospores of S. mycoparasitica were produced on Modified Leonian's agar (MLA), harvested and prepared as previously described.
Also, Fusarium spp. filtrates were prepared, S. mycoparasitica spore germination assays in six different Fusarium filtrates were carried out as previously described.
Sphaerodes mycoparasitica spore germination suspended in both F. graminearum chemotype 3-ADON and 15-ADON filtrates was lower compared to F. avenaceum for the first incubation day, and compared to both F. avenaceum and F. oxysporum for the remaining incubation days (P <0.05; with Mann-Whitney Test) (Figure 17). No significant differences in germination between F. graminearum, F. proliferatum, and F. sporotrichioides filtrate treatments were observed for the first two incubation days. However, treatments with F.
graminearum filtrates showed significantly higher germination rate of S.
mycoparasitica compared to F. sporotrichioides filtrate treatment during later incubation time points (Figure 18).
Dual-culture assays: Dual-culture assays for examining the degree of hyphal reduction/inhibition or damage to F. graminearum chemotypes were assessed as disclosed in Goh and Vujanovic (2009, Mycologia DOT: 10.3852/69-171). S. mycoparasitica is slow-growing fungus as compared to F. graminearumus 3 and 15 strains. Therefore, S. mycoparasitica was pre-inoculated onto the PDA plates for 1 d, at 21 C in darkness, prior to inoculating Fusarium mycelial plugs. Linear mycelial growth of Fusarium strains for both treatments indicated above was measured and recorded daily for 5 days. Around 0.5 x 1.5 cm2 (sampling zones) located approximately 0.2 cm behind the contact zone between F. graminearum and S.
mycoparasitica was excised and subjected to DNA extraction. Each treatment was with three replicates and the experiment was repeated twice. The PDA plate inoculated with F. graminearum only was the positive control. Total genomic DNA was extracted using a DNeasy Plant Mini Kit. The DNA
was eluted once in 50 I of buffer AE and stored at -20 C until real-time PCR
quantification assays (as described below).
Since S. mycoparasitica demonstrated slower mycelial growth (0.56 cm per day;
n = 9) compared to F. graminearum 3-ADON (0.74 cm per day; n=6) and 15-ADON (0.68 cm per day;
n=6) chemotypes, the linear growth of F. graminearum mycelia in dual-culture was assessed using the pre-inoculation method. S. mycoparasitica was pre-inoculated on PDA
for 1 d followed by F. graminearum inoculation. The pre-inoculation approach demonstrated significant differences (starting day 3) in linear growth suppression of F. graminearum chemotype 3 and 15 compared to the co-inoculation approach (Figures 19A and 19B).
Establishment of mycoparasitism: Fusion biotrophic mycoparasitic interactions between S.
mycoparasitica and both F. graminearum chemotype strains, and intracellular parasitism interactions were examined and assessed on slide cultures according the methods described in Goh & Vujanovic (2009).
On day 3 of inoculation on PDA with F. graminearum 3-ADON and 15-ADON, no clamp-like or hook-like structures were formed by Sphaerodes mycoparasitica on Fusarium strains. On day 5 of inoculation, clamp-like and hook-like contact structures as well as Fusarium hyphal cells penetration (with haustoria) were observed (Figures 20E to 201).
Furthermore, on day 3, S. mycoparasitica removed red pigment from the mycelia of F.
graminearum 3-ADON on the slide culture (Figures 20A to 20D). As a result, S. mycoparasitica mycelia adopted reddish color (Figure 20C). Between day 4 and 5, formation of red crystal-like pellets was detected on the surface of mycoparasite hyphae (Figure 20D). The mechanism behind the color changes observed remains unknown. For F. graminearum chemotype 15-ADON, no uptake of red complex or release of red crystal-like structures by S. mycoparasitica hyphae were noted.
Nevertheless, flower-like hyphal structures appeared which could indicate possible growth inhibition of 15-ADON F. graminearum (Figure 20J). Significant differences in diameters of infected and non-infected hyphae were seen for both F. graminearum chemotypes (Figure 21).
Primers and standard curves: F. graminearum-specific (Fg16NF/R) and trichothecene Tri5 gene-specific (Tox5-1/2) primer sets were used in this study. Standard curves for F.
graminearum- and Tri5 gene-primer sets were generated, based on threshold cycles (Ct), by using a series of 10-fold diluted genomic DNAs from F. graminearum (spanning from 2.7 x 102 to 2.7 x 10-2 ng/ 1 for F. graminearum-specific primer set, from 2.7 x 102 to 2.7 x 10-1 ng/ 1 of 3-ADON strain and 3.0 x 101 to 3.0 x 10-2 ng/ l of 15-ADON strain DNAs for Tox5-1/2 primer set). Ct values were recorded and obtained by the Opticon MonitorTM software version 3.1 (Bio-Rad Laboratories Inc., Mississauga, ON, CA). Standard curves for different primer sets were constructed by plotting the threshold cycles (Ct) value versus the logarithm (logio) of the concentration of 10-fold serial diluted F. graminearum DNAs as described above.
Amplifications with different primer sets on the genomic DNAs of two F.
graminearum chemotypes were run in triplicates to obtain the mean and standard deviation of each 10-fold serial dilution.
Real-time PCR quantification: Real-time PCR amplifications on total genomic DNA extracted from the sampling zones (as described above) were performed using MiniOpticonTM (Bio-Rad Laboratories Inc., Mississauga, ON, CA). All the real-time PCR reactions were performed by utilizing the real-time PCR MJ white tubes (Bio-Rad Laboratories Inc., Mississauga, ON, CA) in a total volume of 25 1. The reaction mixture for all real-time PCR assays were: 12.5 1 of IQ
Supermix (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 1 IA of each 10 M
forward/reverse primers (Invitrogen), 9.5 I of sterilized UltraPure Millipore water, and 1 1 of DNA template. Real-time PCR conditions for Fg16NF/R primer set used were outlined in Nicholson et al. (1998, Physiol. Mol. Plant Pathol. 53: 17-37) with melting curve analysis at 60 to 95 C. Parameters for Tox5-1/2 primer set as described in Schnerr et al.
(2001, Int. J. Food microbiol. 71: 53-61).
Standard curves for different primer sets with different F. graminearum DNA
sources were constructed (Figure 22). Growth suppression or inhibition at the sampling zones (Figure 23) for F. graminearum chemotype 3 and 15 was further confirmed with real-time PCR
amplifications with F. graminearum- and tri5 gene-specific primer sets.
Sigmoidal curves for the four different treatments (F. graminearum chemotype 3 or 15 only and F.
graminearum chemotype 3 or 15 pre-inoculated with S. mycoparasitica) with Fg16NF/R primer set were generated using Opticon Monitor software version 3.1 and illustrated in Figure 24.
Using Fg16NF/R primer set, quantity of F. graminearum chemotype 3 DNA in the sampling zones significantly decreased when pre-inoculated with S.
mycoparasitica compared to un-inoculated treatment (P = 0.01) (Figure 25). However, DNA of F. graminearum chemotype 15 did considerably but not significantly reduced (P = 0.085 using T-test).
Using Tox5-1/2 primer set, amount of tri5 gene fragments diminished appreciably in both F.
graminearum chemotype 3 and 15 challenged with S. mycoparasitica (P < 0.05).
Example 9 The effects of S. mycoparasitica (biotrophic mycoparasite) and T. harzianum (necrotrophic mycoparasite) on expression of Tri and PKS genes by F.
graminaerum 3-ADON
and 15-ADON strains were assessed in this study.
Fungal strains and growth: Two Fusarium graminearum 3- ADON (SMCD 2243) and 15-ADON (SMCD 2244) chemotypes, Trichoderma harzianum necrotrophic (SMCD 2166) and Sphaerodes mycoparasitica biotrophic (SMCD 2220) mycoparasites were obtained from the Saskatchewan Microbial Collection and Database (SMCD). All strains were maintained on Potato dextrose agar (PDA, BD Biosciences, Mississauga, ON, CA) amended with antibiotics (100 mg/L streptomycin sulphate and 12 mg/L neomycin sulphate; Sigma-Aldrich Canada Ltd., Oakville, ON, CA) prior to study initiation.
Chemical fungicide control: A concentration of 100 mon tebuconazole was prepared from Folicur 432F (43.2% tebuconazole, Bayer CropScience Inc., Saskatoon, SK, CA;
Folicur is a registered trademark of Bayer Aktiengesellschaft, Leverkusen, Fed. Rep.
Germany). This fungicide preparation was used throughout the study.
In vitro assay, sampling and RNA extraction: Dual-culture assay was carried out between F.
graminearum and Folicur (100 mon tebuconazole) or biological (T harzianum or S.
mycoparasitica) agents on Minimal medium as disclosed by Xue et al. (2009, Can. J. Plant Pathol. 31: 169-179). Inoculated dual-culture plates were incubated at room temperature (23C) in dark conditions for a week. Mycelia of F. graminearum were harvested after a week of inoculation with either chemical or biological agents. A 0.5-cm2 mycelial plug was cut out approximately 0.5 cm away from the border of interaction and fungal cells were disrupted with liquid nitrogen. Total RNA from the samples were extracted using Aurum Total RNA mini kitTM
and extracted RNA was treated with DNAse (Bio-Rad Laboratories Inc., Mississauga, ON, CA) according the manufacturer's recommendations. Samples were then stored at -70C
till gene expression analysis.
During the In vitro studies, both F. graminearum 3-ADON and 15-ADON chemotypes changed mycelia morphology corresponding to each treatment (Figures 26A -26B). However, the main diagnostic distinction was the tendency of F. graminearum chemotypes to abundantly produce chlamidospores in clusters or chains when exposed to tebuconasole fungicide compared to mycoparasites (Figures 26C - 26D). In the case of F. graminearum chemotype 15 (15-ADON
producer), all four trichothecene genes ¨ Tri4, Tri5, Tri6, and Tril0 in this Fusarium strain were found to be induced in high amounts when treated with chemical tebuconazole compared to treatments that co-inoculated with biological agents (either S. mycoparasitica or T harzianum) (Figures 27A - 27D). F. graminearum chemotype 3 (3-ADON producer) was observed to demonstrate induction of Tri4, Tri5 and Tril0 genes when co-inoculated with necrotrophic mycoparasitic Trichoderma harzianum (Figures 27A, 27B, 27D). When F.
graminearum chemotype 3 was treated with Folicur fungicide, only Tril0 gene was being induced (Figure 27D). Generally, all four Tri genes were repressed significantly when challenged with biotrophic mycoparasitic Sphaerodes mycoparasitica (Figure 27A - 27D).
Real-time Reverse-transcription PCR: Real-time RT-PCR was performed by using an IScript One-Step RT-PCR kitTM with SYBR Green on a MiniOpticon Cycler System (Bio-Rad Laboratories Inc., Mississauga, ON, CA), according to the manufacturer's instruction. Primer sets used for amplification and gene expression were summarized in Table 6.
Table 6: Primer sets used to amplify Tri4, Tri5, Tri6, Tri10, PKS4, PKS13, and fl-tubulin by Real-time PCR.
SEQ ID Gene Primer sequences Reference Tri4-F: TAAACGCCCGCGAAGTTCACA Jiao et al. 2008, FEMS Lett.
SEQ ID NO: 6/7 Tri4 Tri4-R: TGGTGATGGTTCGCTTCGAG 285: 212-219 Tr5F: AGCGACTACAGGCTTCCCTC Doohan et al. 1999, Appl.
SEQ ID NO: 8/9 Tri5 Environ. Microbiol.
65:
Tr5R: AAACCATCCAGTTCTCCATCTG
SEQ ID NO:
Tri6 Tri6-1 TCTCTACCAACGGTGGATTCAACC Pinson-Gadais et al. 2008, 10/11 Tri6-2 AGCCTTTGGTGCCGACTTCTTG Mycopathol. 165: 51-59.
SEQ ID NO:Tri1O-F: TCTGAACAGGCGATGGTATGGA
Tri 1 0 Jiao et al. 2008 12/13 Tri1O-R: CTGCGGCGAGTGAGTTTGACA
SE ID NO: PKS4-P S .1 Lysoe et al. 2006, Appl.
Q
14/15 PKS4 GTGGGCTTCGCTAGACCGTGAGTT Environ.
Microbiol.
PKS4-PS .2 ATGCCCTGATGAAGAGTTTGAT 72:3924-3932 PKS13-PS.1 SEQ ID NO: CCCCCAACTCGACGTCAAATCTAT
PKS1 3 PKS13-PS .2 Lysoe et al. 20 TTCTTCCCGCCGACTTCAAAACA
SEQ ID NO: fi- FGtubf GGTCTCGACAGCAATGGTGTT
18/19 tubulin FGtubr GCTTGTGTTTTTCGTGGCAGT Lyscie et al. 2006 RT-PCR sample (-25 'IL) contained 3 mt of RNA template, 8.85 [IL of nucleae-free water, 12.5 1,11_, of RT-PCR reaction mixture (2x), 0.5 iL of IScript RT enzyme mix (50x) (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 0.1 [IL of 50 M solutions of both forward and reverse targeted gene-specific primers (Invitrogen Corp., Carslbad, CA, USA). Real-time PCR
conditions were performed as outlined by manufacturer's recommendations: 50C for 10min, 95C for 5min, followed by 40 cycles of denaturing at 95C for 1 Os and annealing at 55C for 30s, 95C for lmin and 55C for 1 min.
PCR reactions were checked for absence of any primer-dimer formation or non-specific PCR
amplification by performing melting curve analysis. Contamination of RNA
template with residual genomic DNA was eliminated because there was no amplification detected using reverse transcriptase free real-time RT-PCR reaction as template. Fold change in gene expression for each treatment was normalized to 13-tubulin internal reference gene and relative to the expression for the control treatment (Fusariwn alone on minimal medium), using the 2-AAcT method proposed by Livak, K.J., and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-AACT Method. Methods 25(4):402-408. AACT = (CT, target-gene CT, Fgtub)treatment ¨ (CT, target-gene CT, Fgtub)control, where treatment and control indicate F.
graminearum challenged with chemical or biological agent and F. graminearum alone, respectively.
For gene expression analyses based on targeted genes (PKS4 and PKS13) that responsible for zearalenone biosynthesis, these two polyketide synthase genes from both F.
graminearum chemotypes were detected to be repressed in all chemical or biological treatments (Figures 28A -28B). Repression of F. graminearum chemotype 15 PKS4 and PKS13 genes in the treatment with S. rnycoparasitica biotrophic mycoparasite was illustrated to be significantly higher compared to treatments with T harzianum and tebuconazole fungicide (Figures 28A - 28B).
Tri genes were observed to be more sensitive in F. graminearum chemotype 3 when co-inoculated with Trichoderma necrotrophic mycoparasite, however, in E
graminearum chemotype 15, Tri genes appeared to be more responsive towards treatment with chemical fungicide (Figures 27A-27D).
PKS4 gene for both E graminearum chemotypes was monitored to be more sensitive towards treatments with Trichoderma necrotrophic mycoparasitic fungus compared to chemical and biotrophic mycoparasitic agents (Figure 28A). However, PKS13 gene in F.
graminearum chemotype 15 was found to demonstrate higher sensitivity towards chemical stimulus (Figure 28B).
Mycotoxins extraction and analyses: DON, ZEA, 3-ADON and 15-ADON mycotoxins were extracted from agar mycelial plugs (0.5cm2) cut from the sampling zone located approximately 0.5 cm behind the contact zone between E graminearum and S. mycoparasitica.
The extraction was performed by three 10-ml volumes of ethyl acetate. The samples were sonicated on ice and shaken vigorously in ethyl acetate, and then they were allowed to stand for 5 min for separation of phases. The organic phase was siphoned off and passed through sodium sulfate to remove water. The solvent was allowed to evaporate at room temperature (23 C) for 3 days. The residue was then re-dissolved in 2 ml of acetonitrile for thin liquid chromatography (TLC) (only ZEA) and high performance liquid chromatography (HPLC) (all mycotoxins) analyses.
TLC was performed on Silica gel 60 plates (Merck Frosst Canada Ltd, Kirkland, PQ, CA) in the solvent system with ethyl acetate-toluene (3:1) for 40 min. 3-ADON on the gel plates was sprayed with 20% aluminum chloride in 95% ethanol, detected and analyzed following the protocols disclosed by Vasavada and Hsieh (1987, Appl. Microbiol. Biotech. 26: 517-521). Known standard controls of pure DON, ZEA, 3-ADON and 15-ADON (Sigma-Aldrich Canada Ltd., Oakville, ON, CA) were employed to compare with other treatments. All four mycotoxins were quantified using a Water's 2695 HPLC system with: 250 x 4.60 mm, Luna 5 micron C18 (2) 100A
column (Phenomenex Inc., Torrance, CA USA) and a photodiode-array (PDA) detector was used with an isocratic solvent system (methanol: water-methano containing 5% (v/v) (90:10) ratio). The PDA detector measured the UV spectrum (190-500 nm). Samples were dissolved in acetonitrile and 10 I loaded onto the column using automatic injector and mycotoxins were eluted with solvent as a mobile phase at a rate of 0.75 ml min-1 for 25 mins. Peak height method was incorporated to determine the exact amount of DON, ZEA, 3-ADON and 15-ADON
against a standard curves. Ratio between mycotoxins extracted from treatments of F.
graminearum treated with Folicur and F. graminearum only was calculated for HPLC.
The amount of DON produced was between 70 to 90 ug/L. Consequently, it was too low to be detected in TLC analysis. ZEA was high enough to be analyzed with TLC
and both F.
graminearum chemotypes were observed to produce higher amounts of ZEA toxin under the treatment with Folicur compared to other treatments (Figure 29). With HPLC, ZEA was found to be reduced when challenged with Folicur compared to F. graminearum colony only (Figure 30). DON and 3-ADON was detected to increase for both F. graminearum when inoculated together with Folicur (Figure 30). Folicur was monitored to trigger highest amount of 15-ADON under in vitro assay (Figure 30).
Example 10 The effects of S. mycoparasitica (biotrophic mycoparasite) and T harzianum (necrotrophic mycoparasite) on expression of aurofusarin gene by F.
graminaerum 3-ADON and 15-ADON strains were assessed in this study.
Fungi, media, and culture conditions: Fungal strains were obtained from culture collections at University of Saskatchewan (Food and Bioproduct Sciences fungal collection).
Strains of the following fungal species were used, plant pathogenic Fusarium graminearum , F.
graminearum (3ADON), F. graminearum (15ADON), F. avenaceum, F. culmorurnõ F. proliferatum, F.
oxysporum, F. arthrosporoides and mycoparasitic Spaeherodes mycoparasitica and Trichoderma harzianum. Fungi were maintained on potato dextrose agar for 2 wks at 21 C in darkness.
Folicur was also used in this study.
Experiments for the impact of red color nuances on aurofusarin gene expression was carried out using previously classified F. avenaceum isolates, based on VCGs and colony colours. F. avenaceum isolates color identification numbers (CIN) were generated with Hex Color Code Chart. CINs are: Ds #71232B: Red and highly virulent, tolerant at 80 C for 4 hours;
Es #4E040B: Moderately red and moderately virulent, tolerant 40 C for 4 hours;
Bs #A86608:
White and non virulent, susceptible 40 C for 4 hours. Three 0.1 g mycelium samples from three replicates of each isolate was mixed and used for DNA extraction and RT-PCR
analyses.
Fungal DNA Extraction and PCR: Fungal cultures were grown on 1.5 ml PD broth and centrifuged at 10,000 rpm for 5 min. Supernatant was discarded and, subsequently a total DNA
of 2-week old cultures representative of each phenotype/VCG was extracted from the pellet with an Ultra Clean microbial DNA Isolation KitTM (Qiagen Inc., Mississauga, ON, CA) following manufacture's instructions. The purified DNA was resuspended in 50 111 of elution buffer and stored at -200C until further analyses. Full length genomic sequences of PKS
region (7.2Kb) from Fusarium graminearum were obtained from NCBI Genbank. Six pairs of primers were designed (Table 7).
Table 7: PCR Primers designed from fall-length genomic sequences of the PKS
region of F.
graminearum SEQ ID Primer Sequence SEQ ID NO: 20 PKSF1 TCGAGTTTCGTGTTGCGTGT
SEQ IS NO. 21 PKSR1 AGGTAGTTCGCCATACCCGT
SEQ ID NO: 22 PKSF2 AATTGTGCCCGAGGCAGTAC
SEQ ID NO: 23 PKSR2 CATTGGTTCCGCCCGCAATAG
SEQ ID NO: 24 PKSF3 TGACAACTTCGCTGGTTTGGA
SEQ ID NO: 25 PKSR3 CATAGCTTGGCCAGTGCCATC
SEQ ID NO: 26 PKSF4 GAAGTCATTCGGTGTTGAGC
SEQ ID NO: 27 PKSR4 GCTCTGGATTGGGTATCGCAC
SEQ ID NO: 28 PKSF5 ACTCGAGCATCCGTCGCAATG
SEQ ID NO: 29 PKSR5 AGCAACATCTCCGTCTGGAG
SEQ ID NO: 30 PKSF6 GTTGAACTGTCCATGGCTGA
SEQ ID NO: 31 PKSR6 GAATGAAGGCAATCTGCTGC
All the reactions were carried out in 25-0 volumes containing 2.5 ill of 10X
PCR buffer, 1 of Q solution, 1 ill of each primer (10 M), 0.5 111 of 10mM dNTPs mix, and 1.25 U of Taq polymerase (Qiagen Inc., Mississauga, ON, CA). Reaction mixtures were mixed gently and were given flash spin prior to PCR on a Eppendorf Master CyclerTM (ep gradient S).
PCR amplicons were purified using the QIAquick PCR purification kit (Qiagen Inc., Mississauga, ON, CA;
QIAquick is a registered trademark of Qiagen GmbH Corp., Hilden, Fed. Rep.
Germany) and commercially sequenced (Plant Biotechnology Institute, Saskatoon, SK).
Development of a real time RT-PCR assay for detection and quantification of the aur gene in Fusarium species: All sequences were sequenced, sequences were aligned, several primer sets were designed and tested. One set, named Auro RT (SEQ ID NO: 32) and Auro RTR
(SEQ ID
NO: 33) was selected based on efficiency to amplify aurofusarin gene.
SEQ ID NO: 32 ACCTCACTGGAATCAGAGCGCAGC
SEQ ID NO: 33 ATGACRACTTCCCGTGGRCC
The specificity of this set of primers was tested by conventional PCR assay using genomic DNA
from Fusarium and mycoparasites using the amplification reactions volumes indicated above.
The amplification protocol was 1 cycle of 120 s at 94 C, 35 cycles of 30 s at (denaturalization), 30 s at 56 C (annealing), 45 s at 72 C (extension), and 1 cycle of 10 mm at 72 C.
The pair of primers used to amplify the 13-tubulin (tub) gene (the endogenous control gene used to normalize the results) were FGtubf (SEQ ID NO: 34) and Fgtubr (SEQ ID
NO: 35).
SEQ ID NO: 34 GGTCTCGACAGCAATGGTGTT
SEQ ID NO: 35 GCTTGTGTTTTTCGTGGCAGT
The PCR efficiencies of the real time RT-PCR for both genes were checked by performing a 10-fold serial dilution of positive control template to generate a standard curve, and by plotting the Ct as a function of log [10] of template.
Presence of aurofusarin was tested and quantified by RT-PCR in pathogenic Fusarium graminearum , F. graminearum (3ADON), F. graminearum (15ADON), F. avenaceum, F.
culmorum, Mycoparasite, F. proliferatum, F. oxysporum, F. arthrosporoides and mycoparasitic, Sphaerodes mycoparasitica and Trichoderma harzianum. Relative gene expression was studied when Fusarium isolates were co-cultured with Folicur , Sphaerodes and Trichoderma.
The designed RT-PCR primer set was checked for its specificity by conventional PCR on tested fungal species. A single band 157 bp long was amplified in F.
graminearum (3ADON), F.
graminearum (15ADON), F. avenaceum, F. culmorum but not in other strains. One week old grown cultures tested for their aurofusarin relative gene expression and I3-tublin gene was used as an internal control. F. graminearum which produces 15ADON showed highest level gene expression followed by F .graminearum (3ADON) and similar kind of expression results were observed with F. avenacum and F. culmorum. Aurofusarin relative gene expression was quantified when each Fusarium species was co-cultured for a week with Sphaerodes, Trichoderma and Folicur (Figure 31). Among all tested, S. mycoparasitica was most effective in reducing the aurofusarin relative gene expression followed by Trichoderma and Folicur (Figure 31). S. mycoparasitica was most affective on F. graminearum producing 15ADON, but also efficiently reduced the expression level in F. culmorum and F. avenaceum and F.graminearum producing 3ADON (Figure 31). Similarly, Trichoderma was able to somewhat affect F. graminearum producing 15ADON and F. culmorum, but showed no impact on F.
avenaceum, whereas enhanced the expression in F.graminearum producing 3ADON
(Figure 31).
Finally, Folicur could be able to provoke minor reduction in F. graminearum producing 15ADON aur gene expression, but with less impact on F. culmorum and F.
avenaceum.
Inversely, Folicur increased expression almost 2-fold when compared to F.
graminearum producing 3ADON (Figure 31).
RNA isolation, reverse transcription and real time RT-PCR: All the cultures were grown for a week under dark and used for expression studies. Fungal total RNA was isolated using the "Total Quick RNA Cells and Tissues" KitTM (Bio-Rad Laboratories Inc., Mississauga, ON, CA), according to the manufacturer's instructions, and stored at ¨80 C. DNAse I
treatment to remove the chromosomal DNA contamination from the samples was performed using the "Deoxyribonuclease I, Amplification Grade" (Invitrogen Corp., Carlsbad, CA, USA). First strand cDNA was synthesized using the "Iscript RNA PCR Reagent Kit" (Bio-Rad Laboratories Inc., Mississauga, ON, CA). Relative Quantification of aur gene expression was performed in a MiniOpticon Sequence Detection System using the SYBR Green PCR Master Mix (Bio-Rad Laboratories Inc., Mississauga, ON, CA) and the primer pairs indicated above.
The PCR thermal cycling conditions for both genes were as follows: an initial step at 95 C
for 10 min and 40 cycles at 95 C for 15 s and at 60 C for 1 min. SYBR green PCR master mix 12.5 ul (Bio-Rad Laboratories Inc., Mississauga, ON, CA) was used as the reaction mixture, with the addition of 6.5 ul of sterile milli-Q water, 1.0 ul of each primer (5 uM), and 5 ul of template cDNA, in a final volume of 25 iii. In all the experiments, appropriate negative controls containing no template were subjected to the same procedure to exclude or detect any possible contamination or carryover. Each sample (triplicate) was amplified twice in every experiment. The results were normalized using the all fungi cDNA amplifications run on the same plate. The tub2 gene is an endogenous control that was used to normalize quantitation of mRNA target for differences in the amount of total cDNA added to each reaction. Real Time RT-PCR analysis is based on the threshold cycle (CT), which is defined as the first amplification cycle at which the fluorescence signal is greater than the minimal detection level, indicating that PCR
products become detectable. Relative quantitation was the analytical method used in this study. A comparison within a sample is made with the gene of interest (aur) to that of the endogenous control gene (tub2). Quantitation is relative to the control gene by subtracting the CT of the control gene (tub2) from the CT of the gene of interest (aur) (ACT). Each ACT value (corresponding to each sample) was subtracted by the calibrator value (FpMM6-1C) to obtain the corresponding AACT
values. AACT values were transformed to log2 (due to the doubling function of PCR) to generate the relative expression levels.
In total 9 isolates were subjected to RT-PCR analyses for quantitative gene expression analyses. Three kinds of colored samples were used in this study composed of 3 white colored, 3 moderately red colored and 3 dark red colored isolates. White colored isolate was used as control and their expression was normalized and taken as one. As reported moderately red colored isolates were able to tolerate 40 C whereas; dark red isolates were able to tolerate 80 C. When RT-PCR results observed dark colored isolates expressed almost more than 9 folds enhanced relative gene expression whereas, moderately red colored isolates expressed 4 folds enhanced expression (Figure 32).
Example 11 Greenhouse studies were performed to assess the effects of S. mycoparasitica inoculants on development of Fusarium head blight symptoms and the accumulation of tricothecene mycotoxin gene in wheat and barley spikes.
Fungal isolates and growth: S. mycoparasitica SMCD 2220, and F. graminearum 3-ADON
chemotype SMCD 2243 were obtained from the Saskatchewan Microbial Collection and Database (SMCD). These fungal cultures were grown on potato dextrose agar (PDA, BD
Biosciences, Mississauga, ON, CA) supplemented with antibiotics prior to the study. Mycelial suspensions of these fungal strains (104 for F. graminearum and 106 for both biotrophic mycoparasite and antagonistic fungus) were produced for greenhouse experiment, as follows.
F. graminearum 3-ADON chemotype strain was inoculated in potato dextrose broth (PDA, BD Biosciences, Mississauga, ON, CA), incubated at room temperature (-21 C) in darkness using shaker (100rpm), for a week prior to harvesting the mycelia for greenhouse application. Mycelia of F. graminearum were transferred into sterilized commercial blender for cutting the mycelial inoculants into small debris and these mycelia were adjusted to concentration of 104 CFU/mL.
S. mycoparasitica was inoculated in the best preferment yeast peptone dextrose (YPD) (yeast, 5g; peptone, 10g, dextrose, 10g per 1 L of sterile-double distilled water) broth, incubated at room temp in darkness, using shaker at 100 rpm for a week prior to harvesting the mycelia.
Mycelia of S. mycoparasitica were transferred into sterilized commercial blender for cutting the mycelial inoculants into small debris and the mycelia were adjusted to concentration of 104 CFU/mL for greenhouse application.
Greenhouse trials: The Fusarium-susceptible wheat CDC-TEAL and barley BOLD
cultivars were used to test the efficacy of biotrophic mycoparasite S. mycoparasitica and one potential Fusarium antagonistic fungal isolate as potential biocontrol agent for managing Fusarium head blight diseases. Surface-sterilized wheat and barley seeds were planted in 15-cm diameter pots containing Pro-Mix soil (Sun Gro Horticulture, Delta, BC, CA) and maintained at 23-25 C
during the day time and 18-20 C during the night time in a greenhouse with light intensity of 360 mot 111-2 s-I, watered every 2 d, and fertilized every 14 d using 1300 ppm of NPK (20-20-20) fertilizer. All treatments in the experiment were with three replicates and repeated twice.
At the anthesis stage, wheat and barley spikes were sprayed with mycelial suspension of S. mycoparasitica at a concentration of 106 CFU/mL, antagonistic fungal isolate at a concentration of 106 CFU/mL, 65 mon tebuconazole, or sterile distilled water.
The treatments were applied as outlined in Xue et al. (2009, Can. J. Plant Pathol. 31: 169-179) with slight modifications. Following the inoculation of potential biocontrol agents, plants were kept at room temperature and covered with sterile Whirl-Pak bags (Whirl-Pak is a registered trademark of Aristotle Corp., Stamford, CT, USA) for overnight to allow fungal growth prior to F.
graminearum inoculation. Plants were then inoculated with mycelial suspension of F.
graminearum at a concentration of 104 CFU/mL. Fusarium graminearum inoculum was sprayed on the spikes and covered with sterile Whirl-Pak bags for overnight. The inoculated plants were maintained for an additional 21 days prior to sampling. Percentage of infected spikelets (IS) per spike, FHB index, FDK, and weight of 100 seeds were rated as outlined in Xue, A.G., Voldeng, H.D., Savard, M.E., Fedak, G., Tian, X., and Hsiang T. (2009) Biological control of fusarium head blight of wheat with Clonostachys rosea strain ACM941, Canadian Journal of Plant Pathology 31: 169-179. The FHB visual severity scale was determined.
The biocontrol effects of different concentrations of S. mycoparasitica of Fusarium head blight symptoms in barley are shown in Figure 33. The data in Figures 33A, 33B, and 33C show that inoculation of a susceptible barley cultivar with F. graminearum significantly reduced that height of the plants, average numbers of spikes form per plant, and the average weight of 5 spikes. However, inoculation of the barley cultivar with S. mycoparasitica did not affect growth and development. Treatment of F. graminearum-infected barley with S.
mycoparasitica at an inoculation level of 106 CFU/mL prevented the onset of any Fusarium head blight symptoms.
Treatment of F. graminearum-infected barley with lower concentrations of S.
mycoparasitica, i.e., 104 CFU/mL and 105 CFU/mL, did not protect against the occurrence of the disease symptoms (Figures 33A - 33C).
The biocontrol effects of different concentrations of S. mycoparasitica of Fusarium head blight symptoms in wheat are shown in Figure 34. The data in Figures 34A, 34B, and 34C show that inoculation of a susceptible wheat cultivar with F. graminearum significantly reduced that height of the plants, average numbers of spikes form per plant, and the average weight of 5 spikes. However, inoculation of the wheat cultivar with S. mycoparasitica did not affect growth and development. Treatment of F. graminearum-infected wheat with S.
mycoparasitica at an inoculation level of 106 CFU/mL prevented the onset of any Fusarium head blight symptoms.
Treatment of F. graminearum-infected wheat with lower concentrations of S.
mycoparasitica, i.e., 104 CFU/mL and 105 CFU/mL, did not protect against the occurrence of the disease symptoms (Figures 34A - 34C).
The data in Figure 35 and Table 8 show that treatment of F. graminearum-infected barley with S. mycoparasitica at an inoculation level of 106 CFU/mL provided a comparable level of protection against the occurrence of Fusarium head blight symptoms, as was provided by the commercial fungicide Folicur .
t..) o ;o--H--t..) t..) cio o o Table 8: Effect of S. mycoparasitica, Folicur fungicide and F. graminearum on percentage of infected spikelets, Fusarium head blight index, percentage of Fusarium damaged kernels, and weight of 100 seeds of the barley cultivar BOLD in greenhouse trials.
Infected spikelets Fusarium head blight Fusarium damaged Weight of 100 seeds (g) n Treatment (%) index (%) kernels (%) 0 I.) 1 F. graminearum (Fg) 62.6 8.0 a 56.7 11.9 a 75 9.4 a 4.1 0.4 c -.1 I \ ) cri 15.4 3.1 bc 4.1 1.0 b 19 6.5 b 5.4 0.6 ab H
l9 S. mycoparasitica ¨ Fg 0, 1 Folicur ¨ E g 19.6 6.9 b 7.2 4.8 b 16 6.5 b 5.7 0.5 a I.) H
"
I
I \ ) I
I \ ) FP
.0 n ,-i n =
-::--, =
t..) u, c,., Real-time PCR quantification: Total DNA was extracted from the wheat and barly spices with DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, CA). Extracted total DNAs from spikes of different treatments were employed in real-time PCR quantification. Two different primer sets and PCR conditions used in this study were described in Nicholson et al.
(1998) for Fg16NF/R
primer set and Schnerr et al. (2001) for Tox5-1/2 primer set. Total DNA
extracted from spring wheat and barley spikes harvested from greenhouse trials were carried out in a MiniOpticon (Bio-Rad Laboratories Inc., Mississauga, ON, CA). All real-time PCR reactions were performed using real-time PCR MJ white tubes (Bio-Rad Laboratories Inc., Mississauga, ON, CA) with a total volume of 25 I of IQ supermix (Bio-Rad Laboratories Inc., Mississauga, ON, CA), 1 1 of each 10 M forward/reverse primers (Invitrogen), 3.4 1 of BSA (Bovine Serum Albumin) (1.47 g/ 1) (Ishii, S. and Loynachan, T.E. (2004) Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods. Mycorrhiza 14:271-275), 5.1 I of sterilized UltraPure Millipore water, and 2 1 of DNA template.
Figure 36 shows standard curves of F. graminearum chemotype 3 genomic DNA
concentration standards versus cycle threshold (Ct) with PCR reactions performed in triplicate using primer sets (A) Tox5-1/2, with genomic DNA ranging from 270 ng (Logi =
2.90) to 0.27 ng (Logi() = -0.60), readings at 0.005 fluorescence line and (B) Fg16NF/R, with DNA template ranging from 270 ng (Logi() = 2.43) to 0.027 ng (Logic) = -1.57); in 10-fold dilution series, readings at 0.025 fluorescence line. Error bars indicate standard deviation for the mean of F.
graminearum chemotype 3 standard curves derived from tri5 gene and F.
graminearum specific primer sets. Figure 37 shows effects of S. mycoparasitica (B) and Folicur fungicide (Fol) treatments on F. graminearum chemotype 3-ADON genomic DNA detected in barley spikes employing RT-PCR. Treatments were: Fus ¨ F. graminearum; B-Fus ¨ S.
mycoparasitica with F. graminearum; Fol-Fus ¨ Folicur fungicide with F. graminearum. All values obtained were the means of six replicates. Error bars indicate standard deviation of the mean.
Means of F.
graminearum DNA with Tox 5 primer were log10 transformed prior to LSD test.
Both primer sets were analyzed separately. Values followed by the same letters within each primer set are not significantly different using LSD test at P < 0.05.
Example 12 In addition to the known Fusarium spp. hosts for S. mycoparasitica (i.e., F.
avenaceum, F. graminearum and F. oxysporum), we surprisingly discovered that F. culmorum and F.
equiseti are also mycoparasitised by S. mycoparasitica as disclosed in the following study.
Fungal isolates and growth: Biotrophic mycoparasites S. mycoparasitica SMCD2220, Sphaerodes quadrangularis strain CBS112764, Sphaerodes retispora var.
retispora strain CBS
994.72, and pathogenic Fusarium strains (F. arthrosporioides SMCD2247, F.
culmorum SMCD2248, F. equiseti SMCD2134, F. flocciferum 5MCD2135, F. poae 5MCD2136, and F.
torulosum SMCD2139) were obtained from the Saskatchewan Microbial Collection and Database (SMCD), Saskatchewan, Canada. All fungal isolates were grown and maintained on potato dextrose agar (PDA, BD Biosciences, Mississauga, ON, CA) prior to the study.
Fungal-fungal interactions: For examination of the interaction between isolates of Sphaerodes and Fusarium species, both biotrophic mycoparasite and Fusarium isolates were inoculated and assessed using slide culture assays. Slides were maintained in a sterile humidity chamber and daily observations on the hyphal interactions at the meeting place (contact zone) were performed under a Carl Zeiss Axioskop2 equipped with Carl Zeiss AxioCam ICc 1 camera with 20x, 40x and 100x objectives. Formation of biotrophic mycoparasitic contact structures attaching Sphaerodes species to Fusarium hyphae were examined, recorded and compared to drawings from the literature (Jordan & Barnett, 1978, Mycologia 70: 300-312;
Rakvidhyasastra & Butler, 1973, Mycologia 65: 580-593; Whaley & Barnett, 1963, Mycologia 55: 199-210).
Diameters of both parasitized and non-parasitized Fusarium hyphal cells were measured under light microscopy with a 100x objective lens. Each treatment used six replications consisted of Sphaerodes or Fusarium alone, and Sphaerodes¨Fusarium co-inoculated. The experiment was repeated twice. In the slide-culture assay, Fusarium mycelia infected with Sphaerodes haustoria were stained with lactofuchsin. Stained hyphae of both Fusarium and Sphaerodes in slide-culture were then examined with a Carl Zeiss Axioskop2 fluorescent microscope attached to Carl Zeiss AxioCam ICcl with 40x and 100x objectives. Slide-culture assays were also subjected to Zeiss META 510 confocal laser scanning microscopy (CLSM) analysis to observe intracellular mycoparasitism under a C-Apochromat 63x N.A.1.2 phase-contrast water immersion objective through Z-stacking mode to scan through the Fusarium hyphae with intracellular infection (CLSM with 514nm excitation ¨ argon and LP585 emission filters).
Hyphae-hyphae interactions and contact structures in the contact zone were examined for seven days. On day three, Sphaerodes mycoparasitica was found to produce hook-shaped contact structures on F. equiseti and F. culmorum (Figure 39). On day five, more hook-shaped contact structures and intracellular penetration of F. equiseti were observed (Figures 39A, 39A-39D).
The combination of lactofuchsin dye and fluorescent or confocal laser scanning microscopy revealed that the parasitized or penetrated Fusarium cells became empty (loss of cytoplasm = no fluorescence) or fluoresced with low intensity (very pale) (Figures 39A-39D) as compared to healthy Fusarium cells. During the seven days of observation, no S.
mycoparasitica hyphae were observed within F. culmorum cells. S. mycoparasitica produced hook-shaped contact structures (Figure 38A, a) more frequently than clamp-like contact structures (Figure 38B, b) on both F.
equiseti and F. culmorum. Diameters of F. equiseti, but not F. culmorum, hyphae parasitized by S. mycoparasitica were observed to be significantly reduced compared to non-parasitized Fusarium hyphae (with T-test, P = 0.001 and P> 0.05, respectively) (Figure 41).
None of the Fusarium taxa tested appeared to be suitable hosts for mycoparasitic S.
quadrangularis and S. retispora even after 10 days of co-inoculation on slide cultures. No contact biotrophic parasitic structures or intracellular parasitism by S.
quadrangularis and S.
retispora on the tested Fusarium strains were observed at the interaction or contact zone. Also, F. arthrosporioides, F. flocciferum, F. poae, and F. torulosum did not appear to be suitable hosts for S. mycoparasitica. Around five days after inoculation on slide culture assays, mycelia of F.
art hrosporioides were inhibited by S. mycoparasitica. F. arthrosporioides started to form rosette-like mycelia at the contact zone with S. mycoparasitica (Figure 39B).
On the fifth and seventh days after inoculation, anamorphic structures were produced by S. mycoparasitica more abundantly in the zone of contact with F. culmorum (Figures 40C and 40D). Anamorphic structures or asexual organs in close proximity to F.
culmorum mycelia were red-colored (Figure 40D), whereas the organs at a distance were not (Figure 40C).
Claims (10)
1. An isolated culture of Sphaerodes mycoparasitica, wherein the species is characterized by a combination of:
(a) fusiform and triangular ascopore shape, and reticulate and smooth wall ornamentation;
(b) conidia produced from simple phialides on the surface of ascoma perdial wall on ascoma surrounding hyphae, and on irregularly branched conidiosphores arising from hyphae; and (c) forming hook-like structures parasitizing living hyphae of Fusarium;
wherein the isolated culture of Sphaerodes mycoparasitica comprises a gene encoding a large subunit of ribosomal RNA gene as shown in SEQ ID NO:1; a gene encoding a small subunit of ribosomal RNA as shown in SEQ ID NO:2; and a gene encoding an internal transcribed spacer ribosomal DNA as shown in SEQ ID NO:3.
(a) fusiform and triangular ascopore shape, and reticulate and smooth wall ornamentation;
(b) conidia produced from simple phialides on the surface of ascoma perdial wall on ascoma surrounding hyphae, and on irregularly branched conidiosphores arising from hyphae; and (c) forming hook-like structures parasitizing living hyphae of Fusarium;
wherein the isolated culture of Sphaerodes mycoparasitica comprises a gene encoding a large subunit of ribosomal RNA gene as shown in SEQ ID NO:1; a gene encoding a small subunit of ribosomal RNA as shown in SEQ ID NO:2; and a gene encoding an internal transcribed spacer ribosomal DNA as shown in SEQ ID NO:3.
2. The isolated culture of Sphaerodes mycoparasitica according to claim 1, wherein the isolated culture is Sphaerodes mycoparasitica strain deposited as IDAC 301008-01.
3. A use of the isolated culture of Sphaerodes mycoparasitica of claim 1 or 2 for controlling Fusarium spp.
4. The use of claim 3, for controlling Fusarium spp. in plants.
5. A use of the isolated culture of claim 1 or 2, for modulating synthesis of or degrading one of a Fusarium trichothecene mycotoxin deoxynivalenol (DON), mycotoxin 3-acetyldeoxynivalenol, mycotoxin 15-acetyldeoxynivalenol, mycotoxin zerelanone, and mycotoxin aurofusarin.
6. A composition comprising the isolated culture of Sphaerodes mycoparasitica according to claim 1 or 2 and a carrier.
7. The composition of claim 6, further comprising an additional antifungal agent.
8. A method for controlling Fusarium spp. in plants, the method comprising treating a batch of seeds with the culture of claim 1 or 2 or the composition of claim 6 or 7 and then culturing the treated seeds into plants.
9. A method for controlling Fusarium spp. in plants, the method comprising treating growth medium contaminated with the Fusarium spp. with the culture of claim 1 or 2 or the composition of claim 6 or 7.
10. The method of claim 9, wherein the growth medium is soil.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23790609P | 2009-08-28 | 2009-08-28 | |
| US61/237,906 | 2009-08-28 | ||
| PCT/CA2010/001253 WO2011022809A1 (en) | 2009-08-28 | 2010-08-30 | Fusarium and fusarium mycotoxin biocontrol |
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| BR112015014303A2 (en) | 2012-12-19 | 2017-07-11 | Basf Se | compounds of formula I, process for the preparation of compounds of formula I, agrochemical compositions, use of a compound of formula I, method for combating fungi, seed and intermediate compounds xx3 |
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| UA121106C2 (en) | 2013-10-18 | 2020-04-10 | Басф Агрокемікал Продактс Б.В. | Use of pesticidal active carboxamide derivative in soil and seed application and treatment methods |
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| EP2949649A1 (en) | 2014-05-30 | 2015-12-02 | Basf Se | Fungicide substituted [1,2,4]triazole and imidazole compounds |
| EP2949216A1 (en) | 2014-05-30 | 2015-12-02 | Basf Se | Fungicidal substituted alkynyl [1,2,4]triazole and imidazole compounds |
| EP2952506A1 (en) | 2014-06-06 | 2015-12-09 | Basf Se | Substituted [1,2,4]triazole and imidazole compounds |
| EP2952512A1 (en) | 2014-06-06 | 2015-12-09 | Basf Se | Substituted [1,2,4]triazole compounds |
| EP2952507A1 (en) | 2014-06-06 | 2015-12-09 | Basf Se | Substituted [1,2,4]triazole compounds |
| EP3209818B1 (en) | 2014-10-24 | 2019-12-11 | Basf Se | Organic pesticid particles |
| EP3111763A1 (en) | 2015-07-02 | 2017-01-04 | BASF Agro B.V. | Pesticidal compositions comprising a triazole compound |
| CN107916266B (en) * | 2017-12-05 | 2020-08-11 | 华中农业大学 | Fusarium toxin detoxification pathway related genes ADH, AKR6D1 and AKR13B2 and application thereof |
| EP3628158A1 (en) | 2018-09-28 | 2020-04-01 | Basf Se | Pesticidal mixture comprising a mesoionic compound and a biopesticide |
| CN110286190B (en) * | 2019-06-26 | 2024-03-22 | 山东农业大学 | Method for simultaneously measuring DON and multiple DON bioconversion products |
| CN110724643B (en) * | 2019-11-18 | 2022-07-15 | 河南省农业科学院植物保护研究所 | Penicillium citrinum with disease prevention and growth promotion functions, microbial inoculum and application thereof |
| CN113832038B (en) * | 2021-09-27 | 2023-05-23 | 中国农业科学院植物保护研究所 | Fusarium equisetum (Fusarium equiseti) K2017-696 and application thereof |
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| US20120156173A1 (en) * | 2009-08-28 | 2012-06-21 | Vladimir Vujanovic | Fusarium and other pathogenic fungi and mycotoxin biocontrol |
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| EP2470659A1 (en) | 2012-07-04 |
| IN2012DN02475A (en) | 2015-08-21 |
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| CA2772106A1 (en) | 2011-03-03 |
| UA104050C2 (en) | 2013-12-25 |
| JP2013502902A (en) | 2013-01-31 |
| EP2470659A4 (en) | 2013-07-10 |
| EA201270327A1 (en) | 2013-03-29 |
| WO2011022809A1 (en) | 2011-03-03 |
| AU2010286276B2 (en) | 2013-12-12 |
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