WO2015114552A1 - Plant growth promoting rhizobacterial strains and their uses - Google Patents

Plant growth promoting rhizobacterial strains and their uses Download PDF

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WO2015114552A1
WO2015114552A1 PCT/IB2015/050672 IB2015050672W WO2015114552A1 WO 2015114552 A1 WO2015114552 A1 WO 2015114552A1 IB 2015050672 W IB2015050672 W IB 2015050672W WO 2015114552 A1 WO2015114552 A1 WO 2015114552A1
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strain
plant
plants
strains
tubers
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French (fr)
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Nico LABUSCHAGNE
Ahmed Idris HASSEN
Theresa PRETORIUS
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University Of Pretoria
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Priority to AP2016009398A priority Critical patent/AP2016009398A0/en
Publication of WO2015114552A1 publication Critical patent/WO2015114552A1/en
Priority to ZA2016/05308A priority patent/ZA201605308B/en

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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/25—Paenibacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/11—Bacillus megaterium

Definitions

  • This invention relates to specific methods for selectively isolating free-living rhizosphere inhabiting bacteria which have the ability to fix atmospheric nitrogen and assessing their efficacy as plant growth enhancers in the greenhouse.
  • the invention further relates to procedures for identifying effective Plant Growth Promoting Rhizobacterial strains, isolating these strains, growing them in pure culture, and screening the isolates for efficacy as plant growth enhancers and biocontrol agents by means of specific assays.
  • the invention also describes the isolation, culturing, characterisation and evaluation of specific strains of Bacillus megaterium (isolate A-07), Paenibacilius barcinonensis (isolate A-10), Pseudomonas fluorescens (isolate N-04), Bacillus cere us (isolate T-11 ), Lysinibaciilus sphaericus (isolate T-19), Paenibacilius alvei (isolate T-22) and Paenibacilius alvei (isolate T29) which have shown to be effective plant growth enhancers and biocontrol agents of plant disease in greenhouse trials and in some instances field trails.
  • These strains have also been shown to have antagonistic activity against plant pathogens, the ability to soiubilize phosphate, fix atmospheric nitrogen asymbiotically and produce phytohormones, siderophores, chitinases and antifungal volatiles.
  • Plant Growth-Promoting Rhizobacteria are naturally occurring bacteria that colonize the rhizosphere (the surrounding soil volume close to the root and the soi! adhering to the roots of plants). These bacteria are able to enhance plant growth directly as biofertilizers and indirectly through biological control of plant pathogens also known as biocontrol agents.
  • Rhizobacteria with biocontrol efficacy often provide long term protection from soilborne pathogens at the root surface due to the fact that they are often rhizosphere competent i.e. they have the capacity to rapidly colonize the rhizosphere and spread down the root from a single seed treatment or drench application into the soil (Rangarajan et al. 2003; Whipps, 2007).
  • Plant Growth-Promoting Rhizobacteria as a group of free-living bacteria occupying the rhizophere and rhizoplane (root surface area) of plants, may be classified into a number of different groupings, including biopesiticides and biofertilizers.
  • Biopesticides have been defined by enn and Hall (1999) as microbials or products derived from microbials, plants, and other biological entities, applied for control of plant pests.
  • Biofertilizers have been defined by Vessey (2003) as substances which contain living microorganisms that, when applied to seed, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply and availability of primary nutrients to the host plant.
  • Phytophthora spp. are known to be an important group of plant pathogenic fungi causing diseases on a large range of crops worldwide.
  • the species Phytophthora capsici in particular, is an aggressive pathogen causing diseases on numerous crops, including aerial blights of the leaves, stems and fruits as well as root, fruit and crown rots on peppers, tomatoes, macadamia, lima bean, water melon, squash and other cucurbit crops (Hwang and Kim, 1995; Lamour and Hausbeck, 2001 ).
  • Rhizoctonia spp. are important plant pathogens infecting a wide range of crops, especially in the solanaceous group of plants that include potatoes.
  • Other examples of hosts infected by Rhizoctonia spp. include amongst others alfalfa, peanut, soybean, lima bean, cucumber, papaya, eggplant, maize and several other hosts (Anees ef a/., 2010).
  • R.solani causes black scurf and stem canker which is of great economic importance in many countries (Banville, and Car!ing, 2004).
  • Fusarium spp include many plant pathogens attacking a wide range of crops globally. Root and crown rot caused by Fusarium is a chronic disease found globally, in all production regions at different severity levels (Liu and Griffiths 2009). Several species of Fusarium cause crown and root rot of wheat. Fusarium graminearum and F. pseudograminearum are generally considered the most pathogenic and widespread species responsible for these diseases. Other species are also known to cause crown and root rot, but their economic importance is related to the area in which they occur. Van Wyk er al. (1987), for instance, showed that Fusarium avenaceum, F. culmorum and F. graminearum are important pathogens of wheat roots in specific areas of the south Western Cape.
  • Rhizobacteria which are able to inhibit plant pathogens and suppress plant diseases (i.e. biocontrol agents), act through various mechanisms including parasttization, production of various types of antifungal and antibacterial metabolites (i.e. antibiosis) (Bloemberg and Lugtenberg, 2001 ; Mazzola et al., 1992), siderophore production (Bultreys, ef al., 2001 ), by inducing systemic resistance of the plant (Jetiyanun and Kloepper, 2002) or by displacing the pathogen through competition (O'Suilivan and O'Gara, 1992).
  • biocontrol PGPR have the capacity to rapidly colonize the rhizosphere and compete with deleterious microorganisms including soiiborne pathogens at the root surface (Rangarajan er al., 2003).
  • Root-knot nematodes are some of the most devastating pests in agriculture and cause huge yield and economic loss to a variety of crops, including vegetables and soybeans (Kiewnick and Sikora, 2006).
  • M. incognita is the most damaging root-knot nematode on soybean in warmer regions such as South Africa. Root-knot nematodes cause losses of between 9% and 100% on soybean in South Africa (Fourie, McDonald & De Waele, 2006).
  • Internationally chemical nematodes are being withdrawn from the market due to toxicity concerns, thereby precipitating a crisis in terms of nematode control measures.
  • Biological control methods including application of Plant Growth Promoting Rhizobacteria (PGPR) is therefore a viable alternative with which to fill this gap.
  • PGPR Plant Growth Promoting Rhizobacteria
  • PGPR which directly enhance the growth of plants (biofertilisers) can do so through various mechanisms including synthesis of phytohormones (Vancura and Jander, 1986), asymbiotic fixation of atmospheric nitrogen ⁇ Malik ef a/., 1997), soiubilisation and mineralisation of organic and inorganic phosphates and improvement of plant nutrition status (Bai ef al, 2002).
  • an isolated Bacillus megaterium strain having NCIMB Accession No. 42317; an isolated Paenibacillus barcinonensis strain (isolate A-10) having NCIMB Accession No. 42318; an isolated Pseudomonas fluorescens (isolate N-04) strain having NCIMB Accession No. 42319; an isolated Paenibacillus alvei strain (isolate T-22) having NCIMB Accession No. 42322 all of which were deposited with the NCIMB on 12 January 2015.
  • an isolated Bacillus cereus strain (isolate T- 11 ) having NCIMB Accession No. 42321 ; an isolated Lysinibacillus sphaericus strain (isolate T-19) having NCIMB Accession No. 42320; and an isolated Paenibacillus alvei strain (isolate T-29) having NCIMB Accession No. 42323 ail of which were deposited with NCIMB on 26 January 2015.
  • a biocontroi and biofertiliser composition comprising of at least one isolated strain selected from the group comprising Bacillus megaterium (isolate A-07), Paenibacillus barcinonensis (isolate A-10), Pseudomonas fluorescens (isolate N-04), Bacillus cereus (isolate T-11 ), Lysinibacillus sphaericus (isolate T- 9), Paenibacillus alvei (isolate T-22) and Paenibacillus alvei (isolate T-29) or any combination thereof.
  • Bacillus megaterium isolate A-07
  • Paenibacillus barcinonensis isolate A-10
  • Pseudomonas fluorescens isolate N-04
  • Bacillus cereus isolate T-11
  • Lysinibacillus sphaericus isolate T- 9
  • Paenibacillus alvei
  • the biocontroi and biofertiliser composition may include an agricultural carrier, such as an organic wheat based carrier containing gluten, a fertiliser, peat, a pelletised compost carrier, an inert carrier such as Per!ite®, fertilizer, insecticide, fungicide, wetting agent, extender, coating agent, abrading agent, clay, talc, polysaccharide, water, or mixtures thereof.
  • an agricultural carrier such as an organic wheat based carrier containing gluten, a fertiliser, peat, a pelletised compost carrier, an inert carrier such as Per!ite®, fertilizer, insecticide, fungicide, wetting agent, extender, coating agent, abrading agent, clay, talc, polysaccharide, water, or mixtures thereof.
  • an agricultural carrier such as an organic wheat based carrier containing gluten, a fertiliser, peat, a pelletised compost carrier, an inert carrier such as Per!ite®, fertilizer, insecticide, fungicide, wetting agent, extender, coating agent
  • Yet a further aspect of the invention provides for a method of protecting plants against plant pathogens wherein the method comprises applying to plants, plant propagation material such as cuttings, tubers and plant seeds, or soil surrounding plants, plant cuttings, tubers or seeds, a biocontro! composition as previously described, under conditions effective to protect the plants or plants produced from the plant seeds against a plant pathogen.
  • plant propagation material such as cuttings, tubers and plant seeds, or soil surrounding plants, plant cuttings, tubers or seeds
  • the plant pathogen is preferably a fungus. More preferably, the fungus is selected from the group comprising Phytophora spp. including P. capsici and P. nicotianae, Rhizoctonia spp. including R. solani, Fusarium spp. including F. oxysporum, F. graminearum and F. pseudograminearum, Pythium spp. including P. ultimum, VerticHHum spp. including V. dahiiae.
  • the plant pathogen can also be a plant parasitic nematode (phyto nematode). More preferably the nematode is selected from the group comprising Meloidogyne spp. including M. incognita.
  • the biocontrol and biofertiliser composition is used to treat plants or plant tubers or seeds by topical application.
  • the method further comprises a step of growing plants from the tubers or seeds which have been topically treated with the biocontrol agent.
  • the biocontrol agent may be used to treat soil around the plants.
  • a method of enhancing plant growth comprising applying to plants, plant tubers, plant seeds, or soil surrounding plants or plant tubers or seeds, the biocontrol and biofertiliser composition as described, under conditions that are effective to enhance growth of the plants or in the plants produced from the plant tubers or seeds.
  • the method further comprises a step of growing plants from tubers or seeds topically treated with the biocontrol and biofertiliser composition.
  • the biocontrol agent is used to treat soil around the plants.
  • Figure 1 Neighbor-Joining (NJ) phylogenetic tree of five rhizobacterial strains and ten reference sequences from the Genebank based on 16S rRNA sequence analysis. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences. Evolutionary analyses were conducted in MEGA5. Isolate A-07 clusters with Bacillus reference groups, T-29 and A-10 to Paenibacillus group, and N-04 to Pseudomonas group.
  • Figure 2 Plant growth enhancement by PGPR strains. Effect of rhizobacterial strains Paenibacillus barcinonensis (A-10) and Bacillus megaterium (A-07) on growth of tomato plants in a seedling-tray based greenhouse assay. Data is expressed as effect on fresh root weights and total plant weights in grams. Statistical analysis indicate that isolate A-10 differs significantly from iso!ate A-07 and the control. Isolate A-07 differs significantly form isolate A- 10 but not from the control.
  • Figure 3 Biological control of the pathogen Phytophthora capsici on tomatoes through application of Pseudomonas fluorescence strain N-04.
  • “Positive control” signifies plants infected with the pathogen and not treated with bacteria.
  • "Negative control” signifies uninfected untreated plants.
  • Figure 4 Biological control of the pathogen Phytophthora capsici on tomatoes by Pseudomonas fluorescens strain N-04.
  • the graphs represent disease progression curves for P. capsici on tomato plants indicating suppression of the pathogen by P. fluorescens strain N-04.
  • "Positive control” signifies plants infected with the pathogen and not treated with bacteria.
  • "Negative control” signifies uninfected untreated plants.
  • BacUp® is a commercial microbial product included for comparison.
  • ME signifies Malt Extract Agar
  • NA signifies Nutrient Agar
  • PDA signifies Potato Dextrose Agar.
  • FIG. 6 Phosphate solubilisation activity of rhizobacterial strains in the laboratory, based on diameter of clear halos formed around each bacterial colony on Pikovskaya medium.
  • the data indicate phosphate soiubiiising activity by Pseudomonas fluorescens strain N-04 and Paenibacillus barcinonensis strain A-10 whereas Bacillus megaterium strain A-07 showed no activity.
  • Figure 7 Effect of rhizobacteria! seed treatments on yield of maize in three different soil types under field conditions. Error bars indicate error of the means.
  • Figure 8 Effect of rhizobacterial seed treatments on yieid of maize in three types of soil under field conditions, expressed as the difference in yield (tons/hectare) compared to the untreated control.
  • LSD Least Significant Difference
  • Figure 11 Example of inhibition of mycelial growth of F. oxysporum on MEA (left) and V. dahliae on PDA (right) by rhizobacteria! strain T29 during a dual culture assay.
  • Figure 12 Effect of rhizobacterial strains on soft rot of potato tubers in a tuber slice assay. Tuber slices inoculated with Pc. brasiliensis (A), Pc. brasi!iensis and strain N04 (B) and sterile water (C) after incubation for 3 days at 28 °C in a humid container.
  • Pc. brasiliensis A
  • Pc. brasi!iensis and strain N04 B
  • C sterile water
  • Figure 13 Disease rating scale used for the rating of ail tubers. 0 being diseased free tubers and 3 being the tuber skin 3 ⁇ 4 covered with scierotia.
  • Media compositions used in PGPR cell culture Table 1 Media compositions used in PGPR cell culture Nitrogen-free semisolid 5 g.L "1 D-L maiic acid, 0.5 g.L “ ' K 2 HPO.,, 0.2 g.L “1 MgS0 4 , 4 g.L “1 KOH, medium (NFb medium) 0.1 g.L “1 NaCl, 0.02 g.L “1 CaCi 2 , 2 ml 5% bromothymol blue (in ethanof),
  • FeEDTA 1 ml vitamin solution (containing: 10 mg biotin; 20 mg pyridoxine in 100m! dH 2 0), 2 ml trace element solution (containing: 200 mg Na 2 o0 4 .2H 2 0; 235 mg MnS0 4 .H 2 0; 280 mg H 3 B0 3 ; 8 mg CuS0 4 .5H 2 0; 24 mg ZnS0 4 .7H 2 0) and 1.75 g.L "1 agar in 1 Litre dH 2 0.
  • Congo red (0.25%) and 15 g.L "1 agar. Adjust pH to 7 using NaOH.
  • NBRIY media 10 g glucose, 5 g Ca 3 (P0 4 ) 2 , 0.5 g (NH 4 ) 2 S0 , 0.2 g NaCl, 0.1 g
  • V8 juice agar 100 ml of V8 juice, 900 ml distilled water, 1 g of CaC0 3 , 15 g of
  • a 0.1 mi aliquot of the serially diluted suspension was spread-plated on King's B medium (Oxoid distributed by Quantum Biotechnologies (Pty) Ltd, Ferndale, South Africa) (King er a/., 1954), Pseudomonas agar (Merck, Longmeadow Business Estate, Modderfontein, South Africa) and tryptone glucose yeast extract medium (Merck, Longmeadow Business Estate, Modderfontein, South Africa) in triplicate.
  • the spread-piate cultures were incubated for 24 hours at 28°C. Colonies, with different morphological appearances, were selected from the countable plates and re-streaked on new plates of the same media to obtain pure colonies.
  • Free living nitrogen fixing bacteria were selectively isolated from the roots and/or rhizosphere soil of the plants sampled using nitrogen free semi solid medium (NFb) and Congo red agar (CRA) medium.
  • NFb nitrogen free semi solid medium
  • CRA Congo red agar
  • NFb medium Ten ml of NFb medium was dispensed into a 20 ml vial and autoclaved. Adhering soil was removed from the roots of the sampled plants and the roots were washed in rapidly running tap water. Subsequently, the roots were rinsed in sterile water and cut into 5- 0 mm long pieces. The root pieces were then macerated with forceps. The macerated roots were introduced into the 10 ml semisolid NFb medium and incubated for 72 hours at 30°C. The formation of a white halo 3-6 mm below the surface of the medium was taken to be an indication of nitrogenase activity.
  • isolates of Paenibacitlus barcinonensis (strain A-10), Bacillus megaterium (strain A- 07) and Pseudomonas fluorescens (strain N-04) all showed surprisingiy high levels of plant growth enhancing and biocontroi activity. More speciftcaily, Paenibacitlus barcinonensis ⁇ strain A-10) and Bacillus megaterium (strain A-07) showed plant growth enhancing activity in tomato plants and Pseudomonas fluorescens (strain N-04) showed effective biocontrol activity by suppressing disease caused by the plant pathogen Phytophthora capsici in tomato plants in greenhouse trials.
  • Bacillus cereus T-1 , Lysinibaciilus sphaericus T-19, Paenibacillus alvei T-22 and Paenibacillus alvei T29 showed effective biocontrol activity in wheat plants.
  • a pure culture of each isolate was sent to Inqaba Biotechnical Industries (Hatfield, South Africa) for sequencing of the 16S rRNA gene region.
  • the DNA was extracted with Zymo Fungal/Bacterial DNA extraction kit (Zymo Research Corp.), the PCR performed using DreamTaq (Fermentas Life Sciences, DreamTacTM Green PCR Master Mix) and the primers 27-F (GAG TTT GAT CCT GGC TCA G) and 1492-R (GGT TAC CTT GTT ACG ACT).
  • the sequencing reactions were performed with AB! BigDye v3.1 and the clean-up performed with the Zymo Sequencing Clean-up kit (ZR-96, DNA Sequencing Clean-up KitT ).
  • the rhizobacterial strains were identified based on 16S rRNA sequence comparison of the GENBANK database sequences using the BLAST function. The results of the molecular identification of the strains is shown in Table 3.
  • Figure 1 shows a neighbor-joining (NJ) phylogenetic tree of five rhizobacterial strains including A-07; A-10 and N-04 and 10 reference sequences from Genebank.
  • Tomato seeds (variety Moneymaker® Starke Ayres) were planted in autoclaved vermiculite in polystyrene seedling trays. Tomato seeds were surface sterilized prior to planting by dipping them in 70% ethanol for 30 seconds, thereafter the seeds were transferred to a 1 % hypochlorite solution for 1 minute after which they are rinsed with sterile distilled water. One seed was planted in each cell of the seedling tray. Seed trays were placed in a greenhouse where the temperatures ranged from 10°C - 30 D C. The plants were watered regularly with heat sterilized water. No fertilizer was applied. The tomato plants were grown under these conditions for 5.5 weeks.
  • the isolated rhizobacterial strains were grown in 100 ml nutrient broth (Biolab, Wadesvi!le, South Africa) on a rotary shaker for 48 hours at room temperature. Cell suspensions where diluted or concentrated to a concentration of 1 x 10 8 cfu/ml by means of centrifugation of each of the isolates in a 50 ml capacity plastic tube, the supernatant was discarded and the cell pellet was re-suspended in sterile 1 ⁇ 4 strength Ringer s solution.
  • Each of the rhizobacterial isolates A-10 and A-07 were tested for promotion of plant growth.
  • the tomato plants were individually treated with 5 ml of the rhizobacterial suspension at a concentration of 1 x10 s cfu/ml of each isolate.
  • Control samples were included in which 5 mi of sterile 1 ⁇ 4 strength Ringer ' s solution was applied to each of the control plants as opposed to the rhizobacterial suspension.
  • the experiment was designed as a cateiy randomized (block-less) design.
  • Each treatment consisted of 6 replications.
  • a replication consisted of one cell of a seedling tray with one plant in it. The plants were allowed to grow for 3 weeks subsequent to the commencement of the treatments before they were harvested and the fresh root and shoot mass were determined by weighing.
  • Figure 2 illustrates the plant growth enhancing effect of each of the bacterial strains Paenibacillus barcinonensis strain A-10 and Bacillus megaterium strain A-07.
  • Paenibacillus barcinonensis strain A-10 showed significant enhancement of plant growth as compared to the untreated control plants.
  • the Bacillus megaterium strain A-07 also showed a trend towards enhanced plant growth although the enhancement was not statistically significant.
  • Tomato seedlings were grown under the conditions specified in example 1. The plants were watered daily with heat sterilised tap water. Replicates comprising two 4 week old seedlings were transferred to 500 ml plastic pots containing steam pasteurised soil and a single application of soluble fertilizer (Multifeed-P® at 90 ppm nitrogen) was applied as a soil drench after transplanting. Pots were watered daily with heat sterilised tap water. Greenhouse temperatures ranged from a minimum of 14°C to a maximum of 33°C. Each pot containing two seedlings was regarded as a replication. Three replications were prepared for each treatment.
  • Test treatment - plants were treated with the rhizobacterial strain Pseudomonas fluorescens N-04 and simultaneously infected with the Phytophthora capsici zoospore suspension.
  • the rhizobacterial cell suspension was prepared as described in example 1 , above. 25 ml of the rhizobacterial cell suspension was applied to each of the relevant pots respectively as a soil drench.
  • a zoospore suspension of P. capsici was prepared according to a modification of the method described by Pau!itz and Loper (1991 ) as follows: The fungus was grown on V8 juice agar plates (100ml of V8 juice, 900ml distilled water, 1g of CaC0 3 , 15g of agar) for 5 - 7 days at 25°C. The plates were then incubated at room temperature under a near-UV light for at least 5 days. The whole culture was then cut into small blocks by means of a sterile scalpel. Fifteen ml of sterile distilled water was then added to each plate. Plates were incubated at 4°C for 30 minutes.
  • the 24 hour period between inoculation and the infection was in order to allow the bacteria! strains to adjust to the new environment and to start colonizing the rhizosphere of the plants in each pot.
  • the rhizobacterial strain and commercial microbial product were therefore predominantly screened for their ability to prevent infection and decrease disease progression.
  • Plants were harvested three weeks after commencement of the trial. Total wet mass, wet root mass and wet shoot mass were determined by weighing. The results are shown in Figure 3(a). Pseudomonas fluorescens strain N-04 produced significantly higher biomass yields based on total wet mass, wet shoot mass and wet root mass as compared to the positive control and the comparative treatment with the commercial microbial product BacUp®. The results indicate that Pseudomonas fluorescens strain N-04 shows significant biocontrol activity.
  • Figure 4 shows disease progression curves for the treatments with Pseudomonas fluorescens strain N-04 in comparison with those of the positive control treatment (i.e. pathogen only) and the commercial microbial product BacUp®. Treatment with Pseudomonas fluorescens strain N-04 slowed disease progression significantly, confirming effective biocontrol activity, whereas the commercial microbial product BacUp® was ineffective.
  • This assay was performed to test for antibiosis activity of the rhizobacterial strains as a mechanism of action for biocontrol of the plant pathogen Phytophthora capsici.
  • the rhizobacterial strains were grown on nutrient agar plates (Biolab, Wadesville, South Africa) for 24 - 48 hours at 25°C. When distinct colonies became visible, bacterial cells from single colonies were transferred to sterile nutrient broth in 250 ml flasks by means of a sterile inoculation loop. Alternatively, cultures stored at -70°C in microbeads (Davies diagnostics) were cultured by adding one microbead from a pure culture of the relevant isolate to the nutrient broth. The cultures were then incubated at room temperature on a shake-incubator for 48 hours. To test for antibiosis activity the dual-culture technique was used (Paulitz el a!., 1992).
  • control plates which were not treated with any rhizobacterial isolates showed no inhibition zone. Further, the surface of the control piates was completely covered by P. capsici growth.
  • Pikovskaya medium (Nautijal, 1999) was prepared. It is a well known fact that on this medium phosphate solubilisation activity is identified by the formation of a clear halo which develops around bacterial colonies having the ability to so!ubilise phosphate. During the assessment, clear halos were easily distinguished from the surrounding medium as Pikovskaya ' s medium has a milky, whitish colour.
  • the rhizobacterial isolates of the invention were revived from storage cultures in microbeads (Davies diagnostics) at -70°C and grown on nutrient agar plates for 24 - 48 hours. An inoculum of the pure colonies was then transferred from the nutrient agar plate to the Pikovskaya piates by means of a sterilised toothpick. The phosphate medium was pierced at three positions equidistant from one another with the inoculated toothpick. Control plates were pierced with sterile toothpicks without bacterial inoculum.
  • Pikovskaya plates were incubated for 14 days at 25°C. Two piates were prepared for each treatment and as each halo around a single bacterial colony was regarded as a replication, 6 replications for each treatment were performed. After 14 days, the size of each of the halos was determined by measuring the total halo diameter and subtracting the bacterial colony diameter to give a final measurement of halo diameter in millimeters. Figure 6 sets out the data from the phosphate solubitisation assay.
  • Pseudomonas fluorescens strain N-04 and Paenibacillus barcinonensis strain A-10 showed significant phosphate soiubilisation activity whereas Bacillus megaterium strain A-07 did not show any phosphate soiubilisation activity.
  • Rhizobacteriai isolates from the University of Pretoria's Plant Growth Promoting Rhizobacteria collection were assessed in the greenhouse for their ability to promote the growth of wheat seedlings.
  • Bacterial inoculum was prepared by inoculating 100 ml Nutrient broth (NB) (Biolab) with one frozen bead containing the bacteria isolate. The cultures were incubated on a rotary shaker for 24 hours at 25°C and 180 rpm. After incubation the cell concentration was determined by spectrophotometer at 550 nm. The cultures were transferred to 50 ml centrifuge tubes and centrifuged at 3000 x g for 10 minutes. The pellet was resuspended in sterile Ringer's solution to a concentration of approximately 10 & cfu/ml.
  • the bacterial isolates were applied to ten day old wheat seedlings that had been transplanted into 500 ml (10 cm diameter) plastic pots, containing steam pasteurized sandy loam soil, at the rate of 5 seedlings per pot.
  • the respective treatments were: 1 ) untreated control, 2) 25 ml bacterial suspension ( 0 8 cfu/ml) of each bacterial strain respectively. Treatments were replicated four times, each treatment unit consisting of a pot containing five seedlings. The pots were arranged in a randomised block design.
  • the trial was harvested four weeks after the plants were transplanted in to the pots. Plants were harvested by carefully removing them from the pots and washing the soil from the roots under running tap water. The fresh mass of the roots and foliage was then determined by weighing. The plants were then dried in an oven at approximately 75°C for three days and the dry root and foliage masses were determined by weighing.
  • the results of assessment of growth promotion of wheat by the rhizobacterial strains are shown in Table 2.
  • the fresh shoot mass of wheat was significantly increased in plants treated with Bacillus cereus T-11 , Paenibacillus alvei T-22 and Paenibacillus alvei T-29, whereas dry shoot mass was significantly increased by all four strains, namely Bacillus cereus T-1 1 , Lysinibacillus sphaericus T-19, Paenibacillus alvei T-22 and Paenibacillus alvei T-29 in comparison with the control.
  • These increases in dry foliage mass represent increases of 19%, 51 %, 53% and 65% respectively.
  • a dual culture assay on solid agar media was performed to test for the ability of the rhizobacteria to inhibit mycelial growth of plant pathogenic fungi by means of antibiosis. Inhibition of fungal pathogens by the bacterial isolates was determined by means of the dual culture method on Water-yeast agar (WYA) and Potato dextrose agar (PDA). WYA was used because it is a minimal medium that mimics the carbon- limiting environment of soil.
  • WYA Water-yeast agar
  • PDA Potato dextrose agar
  • a single isolate of the bacterium to be tested was stab inoculated in three places equidistant from the centre and each other onto a WYA plate. A 5 mm diameter fungal plug was then placed in the centre of the plate between the bacterial spots (De Boer et al.
  • Tabie 4 inhibition of plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Fusarium graminearum myce!ial growth by rhizobacterial strains on potato dextrose agar and water yeast agar
  • Chitinase activity of bacteria is indicative of their ability to produce chitinase as a mechanism of biocontrol activity against plant pathogenic fungi.
  • each strain was stab inoculated in triplicate onto water agar with chitin (WAC) media and chitin minima! media (CMM). Both media are opaque and the formation of a clear halo around the bacterial colony indicates the production of chitinase.
  • Colloidal chitin for the preparation of WAC and C M was prepared by grinding crab shell chitin (Sigma, Moscow South Africa) to a fine powder with a coffee grinder. Forty grams of the powdered chitin was dissolved in 400 ml concentrated hydrochloric acid and left overnight at 4°C to digest. The colloidal chitin was precipitated from the acid by adding the chitin-acid solution to 5 litres cold water and recovered by filtration. To determine the chitin concentration of the filter cake a sample was weighed and dried in the oven overnight (adapted from Hsu and Lockwood 1975)
  • Chitinase production by the bacterial strains was assessed on two different media namely CCM and WAC.
  • the CCM contained nutrients whereas the WCA only consisted of colloidal chitin, agar and water.
  • Bacillus cereus T-11 and Paenibacillus aivei T-22 gave a positive reaction for chitinase activity indicating that these isolates might have chitinoiitic activity as part of their potential biocontrol mode of action.
  • Paenibacillus alvei T-22 produced chitinase on the WCA but not on the CCM media. Results of this assay are presented in Table 5.
  • CAS-agar plates were prepared as described by Hassen 2007. This method is a modification of Schwyn and Nielands (1987) through the substitution of piperazine-1 ,4-bis(2-ethane sulfonic acid) (PIPES) with 3-(N-morpholtno propane sulphonic acid) (MOPS).
  • PPES piperazine-1 ,4-bis(2-ethane sulfonic acid)
  • MOPS 3-(N-morpholtno propane sulphonic acid)
  • the CAS media was prepared by separately making CAS indicator and basal solutions and 20 m! sterile 50% glucose solution.
  • the CAS indicator solution consisted of 60.5 mg chrome azurol S in 50 ml ddH 2 0 mixed with 10 ml Fe 3+ . A solution of 72.0 mg HDTMA in 40 ml ddH 2 0 was slowly added to the above mixture.
  • CAS basal medium was prepared by dissolving 30 g MOPS, 0.1 g NH 4 CI, 0.5 g NaC!, and 0.3 KH 2 P0 4 in 880 ml ddH 2 0 and adjusting the pH to 6.8 with 6 M NaOH. Fifteen grams agar was added to the solution while stirring. The so!utions were autoclaved separately. After autoclaving the solutions were cooled to 50°C and the glucose solution added to the basal medium. The 100 ml CAS indicator solution was then added to the basal medium by carefully pouring it in along the flask wall. The resulting blue-green agar solution was poured into Petri dishes.
  • Bacillus cereus T-11 , Paenibacillus alvei T-22 and Paenibacillus alvei J - 29 strains showed an ability to solubilise phosphate with Bacillus cereus T-11 and Paenibacillus alvei T-29 giving the strongest activity. Results of this assay are presented in Table 5.
  • the ability of bacterial isolates to fix atmospheric nitrogen was determined through an assay that demonstrate the ability of the bacteria to grow in nitrogen free media ⁇ Baldani and Dobereiner 1980).
  • a loop full of a single bacterial isolate from a singie coiony was transferred to vials containing NFb.
  • the vials were incubated at 25°C and observed for the formation of a white halo or pellicle indicating bacterial growth after 4 and 7 days. Vials were incubated for a further three days after the first observation because it was difficult to determine the presence or absence of growth in some vials.
  • Table 5 Specific modes of action exhibited by the rhizobacterial strains.
  • Nitrogen fixation was determined by pellicle formation by bacterial isolates inoculated in N-free media prepared as described by Baidani and Dobereiner (1980) and Caceres
  • a modified paper doll root colonisation assay was used to assess root colonisation by the bacterial strains. Bacterized seeds were placed on seedling germination paper in a single line 2 cm from the upper edge of the paper and then rolled into a paper doll. The paper dolls were then placed upright into an incubator and incubated at 25°C. After 7 days the roots were excised and 0.5 g roots placed in 45 ml steriie Ringer's solution in a beaker. The beakers were then sonicated for 30 seconds and a dilution series prepared from the resultant solution. After counting the number of resultant colonies which developed the number of colony forming units was calculated.
  • the nitrogen and phosphate soil nutrient levels were adjusted with superphosphate and ammonium nitrate mixture to standard levels of 100 kg nitrate per hectare and 75 kg phosphate per hectare.
  • a two row Monosem® planter was adjusted to deliver the correct amount of fertilizer and seed planting settings for each of the different soil types.
  • Each replication consisted of 4 x 90 m lines with an intra-row spacing of 50 cm and an intra-row spacing of 90 cm.
  • Maize seed (cultivar phb16 5) was treated with the fungicide Thiram according to manufacturer specifications.
  • Bo!lworm was controlled with De!tamethrin and weeds with Giyphosate (Round-up®) due to the use of the round-up ready maize cultivar phb1615.
  • the Huttons and Arcadian soil ecotype trials were grown under dryland conditions until maturity at 12% moisture whereas the Shortiand soil ecotype trial received supplementary irrigation when moisture stress was observed.
  • the rhizobacterial strains of the invention namely Lysinibacillus sphaericus T- 19, Paenibacillus alvei T-29 as well as other comparative strains viz. Bacillus cereus S7, Brevundiomonas vesicularis A40 and Chryseobacteriu indologenes (isolate A26), were applied as a dry powder seed treatment at the rate of 200 g / 50 kg seed.
  • a commercial bacterial biofertiliser product B-rus® (Stimupiant, PO Box 2013 Swavelpoort 0036) was also included for comparison. The latter product was applied as a seed treatment at the same rate as the other bacterial strains.
  • Data collection consisted of: germination date, bimonthly growth rate and dry biomass and maize cob yield at 12% moisture.
  • strains Lysinibacilius sphaericus T- 9 and Paenibacillus alvei T-29 rendering the best yield increases, thereby outperforming all other strains including the commercial reference product B-rus.
  • the best performing inoculants, the strains Lysinibacilius sphaericus T-19 and Paenibacillus alvei T-29 showed yield increases of 2.88 tons/ha and 2.50 tons/ha respectively (i.e. increases of 30.86% and 24.43 % respectively, compared to the control).
  • the commercial reference treatment B-rus resulted in a yield of 1.42 tons/ha.
  • Bacillus cereus S7 rendered the best yield (5.11 tons/ha) followed by the Lysinibacilius sphaericus T-19, Chryseobacterium indologenes A-26, Paenibacillus alvei T-29, B-rus, Control and Brevundiomonas vesicularis A-40 with 4.76 tons/ha, 4.5 tons/ha, 4.43 tons/ha, 3.87 tons/ha, 3.82 tons/ha and 3.59 tons/ha respectively.
  • the aim of this work was thus to determine whether the six selected PGPR isolates, namely: Lysinibacillus sphaericus (strain T-19), Paenibacillus alvei (strain T-29), Bacillus cereus (strain S7), Pseudomonas fluorescens (strain N-04), Paenibacillus barcinonensis (strain A-10) and Bacillus megaterium (strain A-07) are able to enhance the yield of potatoes.
  • a greenhouse trial was conducted to evaluate the selected PGPR for their ability to promote the growth of potatoes. The trial was conducted at the University of Pretoria's greenhouse facilities and commenced on the 8th March 2012.
  • Pathogen free mini-tubers obtained from Rascal Seed Research Laboratories, cv. Up-to-Date, size 1 1 - 30 g
  • the tubers were planted approximately 6-7 cm below the soil surface. Pots were watered daily to keep the soil at field water capacity.
  • the bacterial inoculum was prepared by inoculating 200 ml sterile nutrient broth (Biolab), in 500 ml Erlenmeyer flasks, with a loop-full of a single bacterial isolate.
  • the isolates were obtained from the University of Pretoria's PGPR culture collection.
  • the inoculated broth was incubated in a shaking incubator at 25°C and 150 rpm for two days.
  • a single 15 ml drench application of a single PGPR isolate was applied to each pot.
  • Paenibacillus barcinonensis strain A-10 15 ml broth suspension containing 10 8 cfu/ml per pot,
  • Paenibacillus alvei strain T-29 15 ml broth suspension containing 0 8 cfu/ml per pot,
  • Bacillus cereus strain S7 15 ml broth suspension containing 10 8 cfu/ml per pot,
  • Bacillus megaterium strain A-07 15 mi broth suspension containing 10 s cfu/ml per pot,
  • Pseudomonas fluorescens strain N-04 15 ml broth suspension containing 10 8 cfu/ml per pot.
  • the trial was harvested on the 18th June 2012 (102 days after planting). The plants were allowed to die down completely before harvesting.
  • the potato tubers were harvested by transferring the soil from the pots to a wire mesh basket (1 cm 2 openings) allowing the soil to fali through and the tubers to remain on top of the wire mesh. The number of tubers, and total tuber weight was recorded for each pot.
  • the experiment was arranged in a completely randomised design, with five replicates per treatment. Each replicate consisted of 5 pots, each containing one plant per pot.
  • Biocontrol of selected bacterial and fungal pathogens of potato by application of rhizobacterial strains Bacillus me aterium A07; Paenibacillus barcinonensis A10; Lvsinibacillus sphaericus T19; Paenibacillus alvei T29; and Pseudomonas fluorescens N04
  • Plant growth promoting rhizobacteria are known to control diseases by several mechanisms, including induced systemic resistance (ISR), reduction of ethylene levels and production of antibiotics, volatiies, antimicrobial enzymes and siderophores (Zahir ei a/., 2004).
  • ISR induced systemic resistance
  • PGPR could be developed, as a part of integrated plant disease management, to reduce conventional pesticide application (Zahir er a/., 2004).
  • Pectobacterium carotovorum subsp. carotovorum pathogens of potato in South Africa and many other potato producing countries
  • Pectobacterium carotovorum and Dickeya species can infect plants through wounds in stems or from infected planting material.
  • Soft rot bacteria are carried asymptomatically on tuber surfaces until a sufficient concentration is reached to cause post-harvest or pre-emergence tuber rot.
  • S. scabies can infect plants from the soil or from planting material. No chemical controls are currently available against soft rot bacteria, and chemical control of common scab provides inconsistent results (Gouws & Geldenhuys, 2011 ).
  • Rhizoctonia solani anastomosis group 3 The most important route of infection by Rhizoctonia solani anastomosis group 3 is infected planting material in South Africa, although infection from soil is also important. Chemical control of this disease currently provides inconsistent results (Denner, 2011 ).
  • Antagonistic PGPR that control aforementioned soil- and tuber-borne pathogens at the soil-plant interface and protect roots and daughter tubers from infection would be of value to the potato industry.
  • a bacterium antagonistic to A. alternata could be applied as foliar sprays (PGPR have successfully been applied as foliar sprays) (Zahir et a/., 2004).
  • the dual culture assay testing for antibiosis is the most commonly used assay used to identify promising PGPR for biologica! control of bacteria and fungi.
  • Inhibition of growth of cultures may occur due to production of antibiotics, antimicrobial enzymes or volatiles (Fravel, 1988) or transfer of genetic material detrimental to the recipient cells (Schottel, Schimizu & Kinkel, 2001 ).
  • the relative sizes of inhibition zones formed by PGPR in antibiosis tests may correlate with the relative efficacy of these strains in the field as shown by Liu, Anderson and Kinkel (1995).
  • Biosurfactants can lyse pathogens, or can induce resistance against pathogens (Czajkowsi et al., 2012).
  • Tuber slice assays can be used to identify PGPR that reduce damage by pathogens with postharvest aspects such as Fusarium spp. (Gould et al., 2008) and Dickeya spp. (Czajkowsi et al., 2012).
  • the inventors determined a) which of the selected rhizobacterial strains are most promising as biocontrol agents against selected bacterial and funga! pathogens of potato (Pc. carotovorum, Pc. brasiliensis, D. dadantii and S. scabies and A. alternata, C. coccodes, F. oxysporum f. sp. tuberosi, R. solani AG 3 and V. dahliae) and b) by studying the mechanisms of biological control of these bacterial strains.
  • the PGPR isolates were obtained from the long-term storage culture collection at the University of Pretoria. The cultures were streaked on nutrient agar (NA) plates and incubated at 37°C for 2 days before use. The cultures were then stored at 25°C until being used (cultures older than 7 days were discarded). The cultures were stored at -20°C in 80% aqueous glycerol as a source of cultures for the duration of the study.
  • Pathogen cultures were obtained from the long-term storage culture collection of the Potato Pathology Group of the University of Pretoria, with the exception of V. dahliae, which was obtained from the Agricultural Research Council- Roodep!aat.
  • the fungai pathogens were grown on potato dextrose agar (PDA) plates at 25°C before use. The cultures were stored on agar slants for the duration of the study. The soft rot bacteria were grown on NA at 37°C for 2 days and then at 25°C for 4 days before being used. The soft rot bacteria were stored in the same manner as the PGPR for the duration of the experiment. S. scabies was grown for 3 weeks at 25°C on malt extract agar ( EA) before being used. No cultures were plated out more than 3 times from the originally obtained cultures.
  • PDA potato dextrose agar
  • Pathogen suspensions were prepared by suspending 3 colonies of Pc. brasiliensis or D. dadantii (or 15 colonies of Pc. carotovorum, which produces smaller colonies than the other soft rot bacteria) in 15 mL of Ringers solution. The suspensions were then vortexed at maximum speed for 3 minutes.
  • the Pc. brasiliensis suspension contained 4x10 8 coiony-forming units (CFUs)/ml_.
  • the D. dadantii suspension contained 1.4x10 8 CFUs/mL.
  • the Pc. carotovorum suspension contained 1 x10 7 CFUs/mL. Hundred ⁇ !
  • Rhizobacterial strains T29 and N04 expressed antibiosis against the soft rot pathogens Pc. brasiliensis and D. dadantii (Table 8). Strain N04 formed significantly larger inhibition zones in mats of Pc. brasiliensis, and to a lesser extent also inhibited Pc. carotovorum. Ho clear zones were formed on NA (data not shown), and D. dadantii failed to grow on MEA. Around strain T29 the clear zones in mats of Pc. brasiliensis and D. dadantii increased in size after 3, 5 and 1 days at 25' C possibly indicating lysis of the pathogen's cells.
  • Table 8 In vitro antibiosis activity by rhizobacterial strains against soft rot bacterial pathogens of potato. Values are the mean radii (mm) of clear zones around PGPR colonies on mats of soft rot bacteria
  • the antibiosis assay was performed as for the soft rot bacteria, except that a S. scabies spore suspension was prepared by transferring 15 mL of Ringers solution with 0.05% Tween 80 (as discussed by Bashan, Holguin and Lifshitz ,1993 ⁇ to a S. scabies culture. The plate was manually rotated for 3 minutes and the suspension transferred to a glass tube. The spore suspension contained 4 * 10 6 CFUs/mL. The sizes of the clear zones were recorded daily for 1 month after preparing the dual cultures by marking the borders of the clear zones on the bottoms of the plates.
  • Tabie 9 In vitro antibiosis activity of rhizobacterial strains against S. scabies, the causal organism of common scab of potato. Formation of c!ear inhibition zones around PGPR colonies in mats of S. scabies on NA, PDA and MEA are indicated. Consistent formation of clear zones is indicated by a +, inconsistent formation of clear zones is indicated by a -7+ and no formation of clear zones is indicated by a -.
  • Fungal plugs 4 mm in diameter were cut from the edges of fungal cultures grown on PDA.
  • the fungai plugs were piaced in the centre of NA, PDA and MEA plates (the use of the 3 media allows the consistency of inhibition to be estimated) (Walker, Powell & Seddon, 1998).
  • the fungal plugs were then allowed to grow at 25°C until the colonies were 10 mm to 20 mm in diameter.
  • the test strains A07, A10, N04, S7, T19 and T29 were stab-inoculated at two equidistant points 3 cm from the centre of each plate. Control plates were prepared for each medium in triplicate, and were inoculated with fungal plugs without inoculating with PGPR.
  • C mean of measurement c for control plates (where pathogen was grown without the biocontro! agent on the same medium)
  • T mean of measurement t for plate.
  • Light microscopy was used to examine parts of fungal colonies near PGPR with visibly altered morphology.
  • the rhizobacterial strain T29 showed the best overall antifungal activity, inhibiting the most fungal pathogens (Table 10). This strain caused more than 50% inhibition of F. oxysporum on MEA and significantly more inhibition of F. oxysporum than the other strains on all 3 media (Table 10). Strain T29 also inhibited R. solani, V. dahliae and C. coccodes.
  • Table 10 In vitro inhibition of mycelial growth of fungal pathogens of potato by rhizobacterial strains during dual culture assays on three different agar media. Inhibition expressed as %.
  • strain T29 prevented sporulation of nearby C. coccodes hyphae, caused nearby A. alternate hyphae to swell and become filled with vacuoles and caused the nearby F. oxysporum hyphae to become swollen and highly pigmented, with granulation of cellular contents.
  • the rhizobacterial strains A07, A10, N04, S7, T 9 and T29 were assayed for volatile production using the method of Mallesh (2008).
  • a loopful of each test strain was streaked in a zig-zag pattern across NA plates, using a template to ensure consistency.
  • Fungal plugs were placed in the centre of PDA plates.
  • the test strain plates and fungal pathogen plates were sealed together with the test strain plates inverted over the fungal pathogen plates.
  • Positive controls consisted of pathogen- inoculated PDA plates sealed together with sterile NA plates. All treatments were performed in triplicate. The plates were incubated until a pathogen colony had reached the edge of the plate. The radial growth of the pathogen colonies were measured in 4 perpendicular directions.
  • the percentage inhibition due to volatile production on each plate was measured using the formula:
  • Table 11 In vitro inhibition of mycelial growth of fungal pathogens of potato by rhizobacterial strains due to production of volatile substances.
  • brasiliensis suspensions were diluted to a concentration of 10 6 CFUs/mL.
  • Small-medium cv. Music tubers Visser, 201 1 ) were rinsed with tap water, thoroughly sprayed with 70% ethanol, allowed to dry in a laminar flow, rinsed with sterilised, demineralised water, allowed to dry in a laminar flow and cut into 9mm-thick slices.
  • Each replicate consisted of a petri plate containing two slices from a tuber, and each replicate was obtained from a different tuber.
  • Wells, 4 mm deep and 4 mm in diameter were cut from the centre of each slice and inoculated with 100 ⁇ of the pathogen suspension and 100 pi of a rhizobacterium suspension.
  • Strains N04 and T29 showed more than 50% inhibition of Pc. brasiliensis as reflected by a reduction in the diameters of rotted lesions on potato tuber slices (Table 12).
  • Table 12 Suppression of Pc. brasiliensis soft rot of potato by rhizobacterial strains during the
  • Rhizobacterial strains A10, A07.T19 and T29 were grown in nutrient broth for twelve hours at 28 ° C with orbital shaking. The cultures were then centrifuged for 20 minutes at 5000 rpm using an Eppendorf® 5804R centrifuge. The supernatant of each culture was collected and analysed for phytohormone content by means of Liquid Chromatography Mass-spectroscopy (LC-MS/MS), by RPS Mountainheath Ltd Centurion Court, 85 Milton Park, Abingdon, Oxfordshire OX144RY in England.
  • LC-MS/MS Liquid Chromatography Mass-spectroscopy
  • Table 14 Phytohormone concentrations (mg/kg) in rhizobacterial culture broth as determined by means of Liquid Chromatography Mass-spectroscopy.
  • IAA lndole-3- Acetic Acid
  • KN Kinetin
  • ZR Trans-Zeatin
  • BAP 6-Benzyi-aminopurine
  • IPA Isopentyl-adenosine
  • ZR Trans-zeatin Riboside
  • PAA Phenyl Acetic Acid
  • !BA lndole-3 Butyric Acid
  • 2iP Isopentenyf adenine.
  • Strain A07 was the most promising for control of S. scabies, the causal organism of common scab of potato, during the antibiosis assay. This strain produced biosurfactants which are known to play a role in the biocontrol of plant pathogens.
  • Strain N04 was found to be promising for control of soft rot bacteria and R. solani during the antibiosis assays. This strain was demonstrated to produce antifungal volatiies that inhibit R. solani, and also produced biosurfactants.
  • Strain T29 showed the best suppression of the soft rot bacterial pathogen Pc. brasitiensis during the tuber slice assay, and also suppressed the soft rot bacterium D. dadantii and the fungal pathogens R. solani, F. oxysporum and V. dahliae during the dual culture assays.
  • the data indicate rhizobacterial strains N04 and T29 to be potential broad- spectrum biocontrol agents, possibly able to control bacteria and fungi simultaneously.
  • Rhizobacterial strains A10, A07, T19 and T29 all produced high levels of phytohormones in Iiquid culture, thereby demonstrating their inherent potential to promote plant growth via the production of plant growth enhancing hormones.
  • Efficacy of rhizobacterial strains Paenibacillus barcinonensis A10, Lvsinibacillus sphaericus T19 and Paenibacillus alvei T29 for bioloqicai control of black scurf of potatoes caused by the fungal pathogen Rhizoctonia solani Rhizoctonia solani is a soil borne plant pathogen capable of infecting a wide range of hosts.
  • the species is classified into 13 anastomosis groupings (AG), with each AG showing a preference in target host, plant part or host growth stage. (Carling & Leiner 1990 ;Tsror, 2010).
  • the most common anastomosis group infecting potatoes is AG3.
  • Potato tuber infections occur at the end of the growing season when haulms start to die. The dying plant cells release certain voiatiles which stimulate and attract mycelial growth of R. sotani, increasing the probability of tuber infection and sclerotia development (Wharton ei al. 2007). Sclerotia appearing on the tubers are the most prominent symptom of R. solani AG3. These sclerotia appear as dark structures on the tuber skin , thereby lowering the market value of the tubers.
  • inoculum of the rhizobacterial strains was prepared as either a dry powder (seed treatment) formulation or a liquid bacterial cell suspension.
  • the liquid cell suspension was prepared as follows: The rhizobacterial strains were streaked onto nutrient agar (Biolab, Wadevclude South Africa) and incubated for two days at 25°C. Erlen Meyer fiasks (250 ml capacity) each containing 100 ml nutrient broth (Biolab, Wadevclude, South Africa) were prepared according to the suppliers' specifications. Each flask was inoculated asepttcaliy with a bacterial culture by means of an inoculation loop (two flasks per isolate). The flasks were incubated for two days at 25°C on a rotary shaker in an incubator to allow bacteria to reach populations of approximately 10 8 cfu/ml.
  • Inoculum for the seed treatment was prepared by initially growing the selected strains in nutrient broth as explained above.
  • Perlite ® powder obtained from Stimuplant in autociavable plastic bags (200 g/bag). Twenty one mil!ilitres of the selected bacterial isolate broth culture was aseptically injected into a bag with a sterile syringe before the bag was manually shaken and incubated. The contents of each bag was manually mixed every second day to allow adequate growth and spread of the bacterial inoculum within the bag. After 2-3 weeks, the inoculum was ready to be used.
  • a strain of R. solani AG3 isolated from diseased potato tubers was used for the biocontrol trials.
  • Three types of pathogen inoculum was used, a sand-bran inoculum, a vermicuiite inoculum and inoculum comprising a macerated culture of the fungus.
  • the R. solani was cultured on potato dextrose agar (PDA, Bio!ab, Wadeville, South Africa). Three week old PDA cultures were used for the agar plug inoculation.
  • the vermicuiite inoculum was prepared by adapting the method described by Atkinson et al, 2010: Three week old PDA cultures of the R. solani AG3 were macerated and mixed with 20 litres sterilised (autociaved) vermicuiite and 5 litre of sterile distilled water. The vermicuiite mixture was incubated at room temperature for four days and mixing of contents occurred on day two. The inoculum was used on day five.
  • a macerated culture inoculum was prepared by mixing macerated R. solani cultures with sterile distilled water.
  • the inoculum was prepared in Schott bottles by mixing macerated cultures from 8 1 ⁇ 2 x 9cm petri dish cultures per litre sterile distilled water. The bottles were shaken to insure adequately mixing of the inoculum.
  • tuber treatment with bacterial strains T 19, T29and A10, applied as a powder tuber treatment formulation, al! resulted in a significant reduction in disease severity compared to the control, with strain Paenibacillus barcinonensis A10 being the most effective (Figure 14).
  • Planting was done one week after soil preparation. The plots were subdivided into sub-plots (1.8m x 2m) and each plot served as one treatment replicate. Five different treatments were planted in each plot. Tubers (cultivar "Up-to-Date”) were planted approximately 20cm apart and 5cm deep.
  • the treatments were: 1 ) pathogen inoculated control 2) a mixture of rhizobacterial strains T19, T29 and A10 (treatment labelled "GM”) applied as a drench over the tuber.
  • GM rhizobacterial strains
  • tubers were harvested by using a garden fork to dig them from the ground. For each treatment replicate the tubers were weighed and counted. Fifty 50 tubers were randomly selected from each treatment replicate, soil washed off and disease severity on each tuber assessed according to the disease rating scale (Figure 13).
  • GM The treatment labelled "GM” containing a combination of strains T19, T29 and A10 significantly reduced disease severity when applied to the tuber as a drench treatment at a rate of 10 ml/tuber, under field conditions (Figure 18).
  • the primary focus of this study was to select and evaluate rhizobacteria that are able to control black scurf disease of potato tubers caused by the fungal pathogen R. solani.
  • the rhizobacterial strains were first screened under greenhouse conditions and subsequently evaluated in mini field plots.
  • tuber treatment with a powder formulation of strains T29 and A10 showed a significant reduction in disease severity, with strain A10 being the most effective, reducing disease severity by 50%. This finding was substantiated in a second (repeat) greenhouse trial.
  • Biocontroi efficacy of a combination (i.e. mixture) of the rhizobacterial strains Paenibacillus barcinonensis A10, Lysinibacillus sphaericus T19 and Paenibacillus alvei T29 was subsequently evaluated under field conditions.
  • Treatment with the mixture of strains applied as a bacterial eel suspension (i.e. liquid) over the tubers effectively reduced black scurf disease severity of potatoes with 43.3 % and the disease index by 60.6 %.
  • incognita is the most damaging root-knot nematode on soybean in warmer regions such as South Africa. Root-knot nematodes cause losses of between 9% and 100% on soybean in South Africa (Fourie, McDonald & De Waele, 2006).
  • the selected rhizobacterial strains were screened for control of Meloidogyne incognita using a modified soybean seedling bioassay based on that described by Padgham & Sikora (2007).
  • Silica sand (0.95 mm filter sand; Silica Quartz (Pty) Ltd) was mixed with medium grade vermiculite (Hygrotech SA (Pty) Ltd) in a ratio of 1 :1 by volume, transferred to autoc!aveable plastic bags and autoclaved at 121 °C for 1 hour.
  • Plastic pots (425 mL) and saucers were sterilised overnight in 1% sodium hypochlorite. The plastic containers were then rinsed with autoclaved tap water.
  • Stationary phase rhizobacterium cultures were prepared by adding a loopful bacteria growing on nutrient agar to an Erlenmeyer flask containing 100 mL of autoclaved nutrient broth (Biolab, Merck (Pty) Ltd). The Erlenmeyer flasks were sealed with aluminium foil and the cultures incubated at 37 °C for 2 days with shaking (200 rpm). Before application, the bacteria cells were centrifuged from the broth culture at 6000g for 10 minutes. The supernatants were discarded and the bacterial pellets re-suspended in autoclaved quarter-strength Ringer's solution (Merck (Pty) Ltd) to produce the same volume as the stationary phase culture.
  • the plastic pots were placed on saucers and filled with sand-vermicuiite mixture up to 1cm from the top of the pots.
  • a soybean cv. LS 6248 R seed was placed on the surface of the sand-vermiculite in the centre of each pot and 10 mL of bacterial suspension in Ringer's solution was pipetted onto each seed. Controls were treated with 10mL sterile quarter-strength Ringer's solution only.
  • the pots were then filled with sand-vermiculite mixture to the rims of the pots.
  • the pots were placed in a greenhouse maintained at an average ambient temperature of 25 °C. Each pot was watered daily with 60 mL autociaved tap water. At emergence of the first true leaves of the soybean seedlings ⁇ approximately 8 days after planting) each plant was inoculated with 2000 M. incognita juveniles.
  • M. incognita juveniles were obtained using the method of Fourie (2005).
  • Tomato cv. Moneymaker plants infected with a M. incognita race 2 culture obtained from Prof. H. Fourie were uprooted, the root systems washed with tap water and the roots cut into 1 cm-iong pieces. The root pieces were shaken by hand for 4 minutes in a 1% sodium hypochlorite solution. The resulting suspension was washed through a nested set of 20 cm-diameter stainless steel sieves (apertures of 75 pm, 25 pm and 10 pm respectively). The sieves were rinsed with sterilised tap water while the bottom sieve was connected to a vacuum pump.
  • the material collected on the 10 pm sieve was gently washed onto the 25 pm sieve.
  • the 25 pm sieve was piaced in a plastic container with the sieve surface submerged in sterilised tap water and the container incubated in the dark at 25 °C allowing for nematode egg hatching.
  • the water was replaced after 1 day. Every day onward, the water was collected and washed onto a 10 pm sieve.
  • the hatched nematode juveniles were then washed from the sieve into a conical flask.
  • the concentration of M. incognita juveniles in the suspension was determined by counting 6 aliquots of 20 pi each on a counting grid / in a counting chamber under a stereomicroscope at 32X magnification.
  • Inoculation of the soybean seedlings was done by first exposing the uppermost lateral roots around each seedling by opening up the growth medium around the root system with a sterilised plastic rod. Three mL sterile tap water containing 2000 M. incognita juveniles were then pipetted onto the uppermost lateral roots of each seedling. The nematode juveniles were kept in suspension during counting and inoculation by means of a magnetic stirrer. After inoculation, the exposed roots of each seedling were covered up with growth medium.
  • the seedlings were assessed 17 days after nematode inoculation. Plants were removed from the growth medium and their root systems washed with tap water. The remaining vermiculite was removed with tweezers. The roots were blotted dry with a paper towel before determining fresh mass by weighing. After weighing, the root systems were kept in tap water while galls were counted. The number of root galls per plant was recorded with the aid of a magnifying glass (2X magnification). Root and shoots were excised and their fresh mass was determined by weighing.
  • Lysinibacillus sphaericus strain T19, Paenibacillus alvei T22 and Pseudomonas fluorescens strain N04 reduced the number of galls per plant by 67%, 32% and 44%, respectively, and the number of galls per gram root by 67%, 34% and 45%, respectively.
  • Tabie 15 Results of first soybean seediing bioassay indicating the effect of rhizobacterial treatments on root knot nematode (Meloidogyne incognita) infection parameters (galling) and piant growth parameters (biomass). Data expressed as least squares means.
  • the least squares mean differs significantly from that of the corresponding nematode-only control.
  • Table 16 Results of second soybean seediing bioassay indicating the effect of rhizobacterial treatments on root knot nematode (Meloidogyne incognita) infection parameters (galling) and plant growth parameters (biomass). Data expressed as least squares means.

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Abstract

This invention relates to a biocontrol composition comprising at least one isolated strain selected from the group consisting of Bacillus megaterium strain A-07, Paenibacillus barcinonensis strain A-10, Pseudomonas fluoresceins strainN-04, Bacillus cereus strain T-1 1, Lysinibacillus sphaericus strain T-19, Paenibacillus alvei strain T-22 and Paenibacillus alvei strain T29 which have have been shown to be effective plant growth enhancers and biocontrol agents of plant disease in greenhouse and in field trials. The invention further relates to procedures for identifying effective Plant Growth Promoting Rhizobacterial strains, isolating these strains, growing them in pure culture, and screening the isolates for efficacy as plant growth enhancers and biocontrol agents of plant pathogens by means of specific assays.

Description

PLANT GROWTH PROMOTING RHIZOBACTERIAL STRAINS A D THEIR USES
BACKGROUND OF THE INVENTION
This invention relates to specific methods for selectively isolating free-living rhizosphere inhabiting bacteria which have the ability to fix atmospheric nitrogen and assessing their efficacy as plant growth enhancers in the greenhouse.
The invention further relates to procedures for identifying effective Plant Growth Promoting Rhizobacterial strains, isolating these strains, growing them in pure culture, and screening the isolates for efficacy as plant growth enhancers and biocontrol agents by means of specific assays.
The invention also describes the isolation, culturing, characterisation and evaluation of specific strains of Bacillus megaterium (isolate A-07), Paenibacilius barcinonensis (isolate A-10), Pseudomonas fluorescens (isolate N-04), Bacillus cere us (isolate T-11 ), Lysinibaciilus sphaericus (isolate T-19), Paenibacilius alvei (isolate T-22) and Paenibacilius alvei (isolate T29) which have shown to be effective plant growth enhancers and biocontrol agents of plant disease in greenhouse trials and in some instances field trails. These strains have also been shown to have antagonistic activity against plant pathogens, the ability to soiubilize phosphate, fix atmospheric nitrogen asymbiotically and produce phytohormones, siderophores, chitinases and antifungal volatiles.
Plant Growth-Promoting Rhizobacteria (PGPR) are naturally occurring bacteria that colonize the rhizosphere (the surrounding soil volume close to the root and the soi! adhering to the roots of plants). These bacteria are able to enhance plant growth directly as biofertilizers and indirectly through biological control of plant pathogens also known as biocontrol agents.
Rhizobacteria with biocontrol efficacy often provide long term protection from soilborne pathogens at the root surface due to the fact that they are often rhizosphere competent i.e. they have the capacity to rapidly colonize the rhizosphere and spread down the root from a single seed treatment or drench application into the soil (Rangarajan et al. 2003; Whipps, 2007).
Supplying nutrients to crops in order to ensure optimum yields and the combating of plant diseases are two of the major factors influencing any crop production system. Soilborne diseases are responsible for major crop losses worldwide. Chemical pesticides including fungicides used to control plant diseases, and synthetic fertilizers which are used to increase yields are major contributors to increased input costs in agriculture. Alternatives to the use of synthetic agrochemicals are increasingly being sought due to amongst other reasons, the detrimental effects of chemical compounds on the environment, and market forces requiring lower pesticide residues on/in food. The use and application of PGPR is a viable biological alternative to the use of synthetic agrochemicals.
Plant Growth-Promoting Rhizobacteria as a group of free-living bacteria occupying the rhizophere and rhizoplane (root surface area) of plants, may be classified into a number of different groupings, including biopesiticides and biofertilizers. Biopesticides have been defined by enn and Hall (1999) as microbials or products derived from microbials, plants, and other biological entities, applied for control of plant pests. Biofertilizers have been defined by Vessey (2003) as substances which contain living microorganisms that, when applied to seed, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply and availability of primary nutrients to the host plant.
In the context of biocontrol of plant diseases Phytophthora spp. are known to be an important group of plant pathogenic fungi causing diseases on a large range of crops worldwide. The species Phytophthora capsici in particular, is an aggressive pathogen causing diseases on numerous crops, including aerial blights of the leaves, stems and fruits as well as root, fruit and crown rots on peppers, tomatoes, macadamia, lima bean, water melon, squash and other cucurbit crops (Hwang and Kim, 1995; Lamour and Hausbeck, 2001 ). Conventional fungicides, apart from being expensive and often detrimental to the environment, are not very effective against this disease, and repeated applications result in development of pathogen resistance against the fungicides (Hausbeck and Lamour, 2004). As it is a soiiborne pathogen, chemical fumigants such as methyl bromide, metam sodium and chloropicrin are normally applied.These chemicals are toxic and hazardous to the environment. Alternative control methods which are cost-effective, environmentally friendly and effective against soiiborne diseases are needed. One of these alternatives is the use of Plant Growth Promoting Rhizobacteria (PGPR) as biocontrol agents of plant diseases and general plant growth enhancers.
Similarly, Rhizoctonia spp. are important plant pathogens infecting a wide range of crops, especially in the solanaceous group of plants that include potatoes. Other examples of hosts infected by Rhizoctonia spp. include amongst others alfalfa, peanut, soybean, lima bean, cucumber, papaya, eggplant, maize and several other hosts (Anees ef a/., 2010). On potatoes R.solani causes black scurf and stem canker which is of great economic importance in many countries (Banville, and Car!ing, 2004).
Fusarium spp include many plant pathogens attacking a wide range of crops globally. Root and crown rot caused by Fusarium is a chronic disease found globally, in all production regions at different severity levels (Liu and Griffiths 2009). Several species of Fusarium cause crown and root rot of wheat. Fusarium graminearum and F. pseudograminearum are generally considered the most pathogenic and widespread species responsible for these diseases. Other species are also known to cause crown and root rot, but their economic importance is related to the area in which they occur. Van Wyk er al. (1987), for instance, showed that Fusarium avenaceum, F. culmorum and F. graminearum are important pathogens of wheat roots in specific areas of the south Western Cape.
Rhizobacteria which are able to inhibit plant pathogens and suppress plant diseases (i.e. biocontrol agents), act through various mechanisms including parasttization, production of various types of antifungal and antibacterial metabolites (i.e. antibiosis) (Bloemberg and Lugtenberg, 2001 ; Mazzola et al., 1992), siderophore production (Bultreys, ef al., 2001 ), by inducing systemic resistance of the plant (Jetiyanun and Kloepper, 2002) or by displacing the pathogen through competition (O'Suilivan and O'Gara, 1992). Such biocontrol PGPR have the capacity to rapidly colonize the rhizosphere and compete with deleterious microorganisms including soiiborne pathogens at the root surface (Rangarajan er al., 2003).
Root-knot nematodes ( eloidogyne spp.) are some of the most devastating pests in agriculture and cause huge yield and economic loss to a variety of crops, including vegetables and soybeans (Kiewnick and Sikora, 2006). M. incognita is the most damaging root-knot nematode on soybean in warmer regions such as South Africa. Root-knot nematodes cause losses of between 9% and 100% on soybean in South Africa (Fourie, McDonald & De Waele, 2006). Internationally chemical nematodes are being withdrawn from the market due to toxicity concerns, thereby precipitating a crisis in terms of nematode control measures. Biological control methods, including application of Plant Growth Promoting Rhizobacteria (PGPR) is therefore a viable alternative with which to fill this gap.
PGPR which directly enhance the growth of plants (biofertilisers) can do so through various mechanisms including synthesis of phytohormones (Vancura and Jander, 1986), asymbiotic fixation of atmospheric nitrogen {Malik ef a/., 1997), soiubilisation and mineralisation of organic and inorganic phosphates and improvement of plant nutrition status (Bai ef al, 2002).
SUMMARY OF THE INVENTION
According to a first aspect of this invention there is provided for an isolated Bacillus megaterium strain (isolate A-07) having NCIMB Accession No. 42317; an isolated Paenibacillus barcinonensis strain (isolate A-10) having NCIMB Accession No. 42318; an isolated Pseudomonas fluorescens (isolate N-04) strain having NCIMB Accession No. 42319; an isolated Paenibacillus alvei strain (isolate T-22) having NCIMB Accession No. 42322 all of which were deposited with the NCIMB on 12 January 2015. Further according to this aspect of the invention there is provided for an isolated Bacillus cereus strain (isolate T- 11 ) having NCIMB Accession No. 42321 ; an isolated Lysinibacillus sphaericus strain (isolate T-19) having NCIMB Accession No. 42320; and an isolated Paenibacillus alvei strain (isolate T-29) having NCIMB Accession No. 42323 ail of which were deposited with NCIMB on 26 January 2015.
According to a second aspect of this invention there is provided for a biocontroi and biofertiliser composition comprising of at least one isolated strain selected from the group comprising Bacillus megaterium (isolate A-07), Paenibacillus barcinonensis (isolate A-10), Pseudomonas fluorescens (isolate N-04), Bacillus cereus (isolate T-11 ), Lysinibacillus sphaericus (isolate T- 9), Paenibacillus alvei (isolate T-22) and Paenibacillus alvei (isolate T-29) or any combination thereof.
The biocontroi and biofertiliser composition may include an agricultural carrier, such as an organic wheat based carrier containing gluten, a fertiliser, peat, a pelletised compost carrier, an inert carrier such as Per!ite®, fertilizer, insecticide, fungicide, wetting agent, extender, coating agent, abrading agent, clay, talc, polysaccharide, water, or mixtures thereof. Preferably the biocontroi composition of the invention contains about 1x102 to about 1x109 CFU/mi of the carrier.
Yet a further aspect of the invention provides for a method of protecting plants against plant pathogens wherein the method comprises applying to plants, plant propagation material such as cuttings, tubers and plant seeds, or soil surrounding plants, plant cuttings, tubers or seeds, a biocontro! composition as previously described, under conditions effective to protect the plants or plants produced from the plant seeds against a plant pathogen.
The plant pathogen is preferably a fungus. More preferably, the fungus is selected from the group comprising Phytophora spp. including P. capsici and P. nicotianae, Rhizoctonia spp. including R. solani, Fusarium spp. including F. oxysporum, F. graminearum and F. pseudograminearum, Pythium spp. including P. ultimum, VerticHHum spp. including V. dahiiae.
The plant pathogen can also be a plant parasitic nematode (phyto nematode). More preferably the nematode is selected from the group comprising Meloidogyne spp. including M. incognita.
Preferably the biocontrol and biofertiliser composition is used to treat plants or plant tubers or seeds by topical application. In instances where the biocontrol and biofertiliser composition is used to treat plant tubers or seeds the method further comprises a step of growing plants from the tubers or seeds which have been topically treated with the biocontrol agent. It will be appreciated that the biocontrol agent may be used to treat soil around the plants.
In yet a further aspect of the invention there is provided for a method of enhancing plant growth comprising applying to plants, plant tubers, plant seeds, or soil surrounding plants or plant tubers or seeds, the biocontrol and biofertiliser composition as described, under conditions that are effective to enhance growth of the plants or in the plants produced from the plant tubers or seeds. In instances where the biocontrol and biofertiliser composition is used to treat plant tubers or seeds the method further comprises a step of growing plants from tubers or seeds topically treated with the biocontrol and biofertiliser composition.
in one embodiment of the invention the biocontrol agent is used to treat soil around the plants.
BRIEF DESCRIPTION OF THE FIGURES
Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures.
Figure 1 : Neighbor-Joining (NJ) phylogenetic tree of five rhizobacterial strains and ten reference sequences from the Genebank based on 16S rRNA sequence analysis. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences. Evolutionary analyses were conducted in MEGA5. Isolate A-07 clusters with Bacillus reference groups, T-29 and A-10 to Paenibacillus group, and N-04 to Pseudomonas group.
Figure 2: Plant growth enhancement by PGPR strains. Effect of rhizobacterial strains Paenibacillus barcinonensis (A-10) and Bacillus megaterium (A-07) on growth of tomato plants in a seedling-tray based greenhouse assay. Data is expressed as effect on fresh root weights and total plant weights in grams. Statistical analysis indicate that isolate A-10 differs significantly from iso!ate A-07 and the control. Isolate A-07 differs significantly form isolate A- 10 but not from the control.
Figure 3: Biological control of the pathogen Phytophthora capsici on tomatoes through application of Pseudomonas fluorescence strain N-04. Wet mass (Figure 3(a)) and dry mass (Figure 3(b)) of tomato plants indicating biocontrol activity of P.capsici by P.fluorescens strain N-04. "Positive control" signifies plants infected with the pathogen and not treated with bacteria. "Negative control" signifies uninfected untreated plants. BacUp® is a commercial microbial product included for comparison. Dry shoot weight was not included in Figure 3(b) as the F-value did not meet the required standards. *Means with the same letter are not significantly different at p=0.05 according to the LSD t-test using the GLM procedure.
Figure 4: Biological control of the pathogen Phytophthora capsici on tomatoes by Pseudomonas fluorescens strain N-04. The graphs represent disease progression curves for P. capsici on tomato plants indicating suppression of the pathogen by P. fluorescens strain N-04. "Positive control" signifies plants infected with the pathogen and not treated with bacteria. "Negative control" signifies uninfected untreated plants. BacUp® is a commercial microbial product included for comparison.
Figure 5: Antibiosis activity of PGPR isolates indicated by inhibition of mycelial growth of Phytophthora capsici by rhizobacterial isolates during dual culture tests in vitro. Percentage inhibition was calculated based on the size of the inhibition zones formed around the rhizobacterial isolates on the agar plates. * eans with the same letters are not significantly different from one another as determined by the Least Significant Difference LSD t- test (p=0.05) using the GLM procedure on SAS-9.2 software. ME signifies Malt Extract Agar; NA signifies Nutrient Agar; PDA signifies Potato Dextrose Agar.
Figure 6: Phosphate solubilisation activity of rhizobacterial strains in the laboratory, based on diameter of clear halos formed around each bacterial colony on Pikovskaya medium. The data indicate phosphate soiubiiising activity by Pseudomonas fluorescens strain N-04 and Paenibacillus barcinonensis strain A-10 whereas Bacillus megaterium strain A-07 showed no activity. *Means with the same letters are not significantly different from one another as determined by the Least Significant Difference LSD t- test (p=0.05) using the GLM procedure on SAS-9.2 software. Figure 7: Effect of rhizobacteria! seed treatments on yield of maize in three different soil types under field conditions. Error bars indicate error of the means.
Figure 8: Effect of rhizobacterial seed treatments on yieid of maize in three types of soil under field conditions, expressed as the difference in yield (tons/hectare) compared to the untreated control.
Figure 9: Effect of various PGPR isolates on growth (yieid) of potato plants in the greenhouse, expressed in terms of tuber mass. Values are means of five replicates. Values with the same letters indicated above individual bars do not differ significantly (P=0.05) according to the Least Significant Difference (LSD) test using the GLM procedure. LSD = 2.7671 ).
Figure 10: Effect of various PGPR isolates on number of potato tubers produced in the greenhouse. Values are means of five replicates. Values with the same letters indicated above individual bars do not differ significantly (P=0.05) according to the Least Significant Difference (LSD) test using the GLM procedure.
Figure 11 : Example of inhibition of mycelial growth of F. oxysporum on MEA (left) and V. dahliae on PDA (right) by rhizobacteria! strain T29 during a dual culture assay.
Figure 12: Effect of rhizobacterial strains on soft rot of potato tubers in a tuber slice assay. Tuber slices inoculated with Pc. brasiliensis (A), Pc. brasi!iensis and strain N04 (B) and sterile water (C) after incubation for 3 days at 28 °C in a humid container.
Figure 13: Disease rating scale used for the rating of ail tubers. 0 being diseased free tubers and 3 being the tuber skin ¾ covered with scierotia.
Figure 14: Greenhouse trial 1 : Effect of rhizobacterial strains applied as a powder seed treatment to potato tubers on black scurf [Rhizoctonia solani) severity in the greenhouse. Treatments with the same aiphabetica! letters indicated above each bar do not differ significantly from each other according to Duncan's multiple range test (p=0.05).
Figure 15: Greenhouse trial 1 : Efficacy of rhizobacteria applied as a powder tuber treatment on black scurf (R. solani) disease index in the greenhouse, p = 0.5, SE = 0.145, CV = 20.05. Treatments with different alphabetical letters indicated above each bar are significantly different from one another according to Duncan's multiple range test (p=0.05). Error bars indicate standard deviation at 0.148.
Figure 16: Greenhouse trial 2: Effect of rhizobacterial strains applied as a powder treatment to potato tubers, on black scurf (Rhizoctonia solani) severity in the greenhouse.Treatments with the same alphabetical letters indicated above each bar do not differ significantly from each other according to Duncan's multiple range test (p=0.05).
Figure 17: Greenhouse trial 2: Effect of powder seed treatment with rhizobacterial strains on black scurf {Rhizoctonia solani) disease index in the greenhouse. Treatments with the same alphabetical letters indicated above each bar do not differ significantly from each other according to Duncan's multiple range test (p=0.05).
Figure 18: Field trial: Effect of tuber drench treatment with a mixture of three rhizobacteria! strains (GM) on black scurf (Rhizoctonia solani) severity in the field. Treatments with different alphabetical letters indicated above each bar are significantly different from one another according to Duncan's Multiple Range Test (p=0.05).
Figure 19: Field trial: Effect of tuber drench treatment with a mixture of three rhizobacterial strains (GM) on black scurf (Rhizoctonia solani) disease index in the field. Treatments with different alphabetical letters indicated above each bar are significantly different from one another according to Duncan's Multiple Range Test (p=0.05).
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.
The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
Provided herein are specific procedures for the selective isolation of specific groups, species and effective strains of plant growth promoting rhizobacteria. Further, provided herein are procedures for growing these isolates or strains in pure culture and for screening the isolates for btocontro! and plant growth enhancing activities. Further protocols are described for testing the isolates for their mechanisms of action including antibiosis activity, phosphate solubilization ability, nitrogen fixing abilities, production of siderophores, chitinases, antifungal volatiles and phytohormones.
Media compositions used in PGPR cell culture Table 1 : Media compositions used in PGPR cell culture Nitrogen-free semisolid 5 g.L"1 D-L maiic acid, 0.5 g.L"' K2HPO.,, 0.2 g.L"1 MgS04, 4 g.L"1 KOH, medium (NFb medium) 0.1 g.L"1 NaCl, 0.02 g.L"1 CaCi2, 2 ml 5% bromothymol blue (in ethanof),
4 ml FeEDTA, 1 ml vitamin solution (containing: 10 mg biotin; 20 mg pyridoxine in 100m! dH20), 2 ml trace element solution (containing: 200 mg Na2 o04.2H20; 235 mg MnS04.H20; 280 mg H3B03; 8 mg CuS04.5H20; 24 mg ZnS04.7H20) and 1.75 g.L"1 agar in 1 Litre dH20.
Congo Red Agar Medium 5 g.L"1 D-L malic acid, 0.5 g.L"1 K2HP04, 0.2 g.L"1 MgS04, 4 g.L"1 KOH, (CRA) 0.1 g.L"1 NaCl, 0.5 g.L"1 yeast extract, 0.015 g.L'1 FeCI3.6H20, 15 ml
Congo red (0.25%) and 15 g.L"1 agar. Adjust pH to 7 using NaOH.
Pikovskaya medium 10 g glucose, 5 g Ca3(P04)2, 0.5 g (NH )2S04, 0.2 g NaCl, 0.1 g
MgSC\,.7H20, 0.2 g KCi, 0.5 g yeast extract, 0.002 g MnS04,H20, 0.002g FeSO4.7H20, 15 g bacteriological agar in 1 L distilled water, pH adjusted to 7
NBRIY media 10 g glucose, 5 g Ca3(P04)2, 0.5 g (NH4)2S0 , 0.2 g NaCl, 0.1 g
MgSO4.7H20, 0.2 g KCi, 0.002 g nS04.H20, 0.002g FeSO4.7H20, 15 g bacteriological agar in 1 L distilled water, pH adjusted to 7.
V8 juice agar 100 ml of V8 juice, 900 ml distilled water, 1 g of CaC03, 15 g of
bacteriological agar
Water yeast agar (WAY) 20 g agar, 5 g NaCl, 1 g KH2P04, 0.1 g yeast extract in 1 L distilled
water
Water agar with chitin 20 g bacteriological agar (Biolab) with 0.4 % colloidal chitin in 1 L medium (WAC) distilled water, pH adjusted to 7
Chitin minimal medium 1 g (NH4)2S04, 1 g K2HP04, 1 g NaCl, 0.1g MgS04.7H2tO 0.5 g yeast (CMM) extract, 0.4 % colloidal chitin and 20 g bacteriological agar in 1 L of distilled water, pH adjusted to 7
Collection of rhizosphere soil samples
Soil samples were collected from the rhizosphere of indigenous South African grasses. Grass root samples were excavated from the soil, transferred into plastic bags, placed in cooler boxes and transported to the laboratory for further processing. Rhizosphere soil was collected from the grass roots by shaking the adhering soil from each root system and collecting the soil in sterile plastic containers. Each rhizosphere soil sample was sieved to remove plant debris before being further processed for the isolation of bacteria.
Isolation of rhizobacteria from soil samples
Each soil sample was mixed manually by hand in the plastic bag and one gram was transferred to 9 ml quarter strength sterile Ringer's solution (Merck, Halfway House, South Africa). Subsequently, a dilution series was prepared for each sample by adding 1 g of soil to 9 ml of sterile distilled water in a test tube, vortexing the suspension for 30 seconds and subsequently transferring 1 mi of the resulting suspension to another 9 ml of sterile distilled water in a test tube and repeating the procedure until a 104 dilution was obtained. A 0.1 mi aliquot of the serially diluted suspension was spread-plated on King's B medium (Oxoid distributed by Quantum Biotechnologies (Pty) Ltd, Ferndale, South Africa) (King er a/., 1954), Pseudomonas agar (Merck, Longmeadow Business Estate, Modderfontein, South Africa) and tryptone glucose yeast extract medium (Merck, Longmeadow Business Estate, Modderfontein, South Africa) in triplicate. The spread-piate cultures were incubated for 24 hours at 28°C. Colonies, with different morphological appearances, were selected from the countable plates and re-streaked on new plates of the same media to obtain pure colonies.
Selective isolation of free-living nitrogen fixing bacteria
Free living nitrogen fixing bacteria were selectively isolated from the roots and/or rhizosphere soil of the plants sampled using nitrogen free semi solid medium (NFb) and Congo red agar (CRA) medium.
Ten ml of NFb medium was dispensed into a 20 ml vial and autoclaved. Adhering soil was removed from the roots of the sampled plants and the roots were washed in rapidly running tap water. Subsequently, the roots were rinsed in sterile water and cut into 5- 0 mm long pieces. The root pieces were then macerated with forceps. The macerated roots were introduced into the 10 ml semisolid NFb medium and incubated for 72 hours at 30°C. The formation of a white halo 3-6 mm below the surface of the medium was taken to be an indication of nitrogenase activity.
Cultures which showed positive nitrogenase activity were then streaked out on CRA plates, individual colonies where transferred to semisolid NFb medium. Pellicle formation in the NFb medium is indicative of growth of free living nitrogen fixing bacteria. Purified colonies were transferred to nutrient agar slants (Biolab, Wadesviile, South Africa) for further study.
A large number of bacteria! isolates were obtained in this manner and grown in pure culture. The purified isolates were subsequently screened for piant growth enhancing activity, as well as biocontroi activity using tomato plants infected with the plant pathogen Phytophthora capsici. The most effectual isolates were identified based on partial 16S rRNA gene sequences and assessed for MOA including antibiosis in dual culture assay, chitinase production, siderophore production, phosphate solubilisation, growth in nitrogen free media, and root colonisation of tomato and wheat seedlings.
isolates of Paenibacitlus barcinonensis (strain A-10), Bacillus megaterium (strain A- 07) and Pseudomonas fluorescens (strain N-04) all showed surprisingiy high levels of plant growth enhancing and biocontroi activity. More speciftcaily, Paenibacitlus barcinonensis {strain A-10) and Bacillus megaterium (strain A-07) showed plant growth enhancing activity in tomato plants and Pseudomonas fluorescens (strain N-04) showed effective biocontrol activity by suppressing disease caused by the plant pathogen Phytophthora capsici in tomato plants in greenhouse trials.
Bacillus cereus T-1 , Lysinibaciilus sphaericus T-19, Paenibacillus alvei T-22 and Paenibacillus alvei T29 showed effective biocontrol activity in wheat plants.
The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1
Molecular identification of plant growth promoting rhizobacterial strains
A pure culture of each isolate was sent to Inqaba Biotechnical Industries (Hatfield, South Africa) for sequencing of the 16S rRNA gene region. At Inqaba the DNA was extracted with Zymo Fungal/Bacterial DNA extraction kit (Zymo Research Corp.), the PCR performed using DreamTaq (Fermentas Life Sciences, DreamTacTM Green PCR Master Mix) and the primers 27-F (GAG TTT GAT CCT GGC TCA G) and 1492-R (GGT TAC CTT GTT ACG ACT). The sequencing reactions were performed with AB! BigDye v3.1 and the clean-up performed with the Zymo Sequencing Clean-up kit (ZR-96, DNA Sequencing Clean-up KitT ).
The rhizobacterial strains were identified based on 16S rRNA sequence comparison of the GENBANK database sequences using the BLAST function. The results of the molecular identification of the strains is shown in Table 3.
Table 3: Results of molecular identification of four rhizobacterial strains
Isolate code 16S rRNA sequence identification
T-11 Bacillus cereus
T-19 Lysinibaciilus sphaericus
T-22 Paenibacillus alvei
T-29 Paenibacillus alvei
The rhizobacterial strains A-07, A-10 and N-04 were also identified based on 16S rRNA sequence analysis. Figure 1 shows a neighbor-joining (NJ) phylogenetic tree of five rhizobacterial strains including A-07; A-10 and N-04 and 10 reference sequences from Genebank.
EXAMPLE 2
Plant growth promotion assay Tomato seeds (variety Moneymaker® Starke Ayres) were planted in autoclaved vermiculite in polystyrene seedling trays. Tomato seeds were surface sterilized prior to planting by dipping them in 70% ethanol for 30 seconds, thereafter the seeds were transferred to a 1 % hypochlorite solution for 1 minute after which they are rinsed with sterile distilled water. One seed was planted in each cell of the seedling tray. Seed trays were placed in a greenhouse where the temperatures ranged from 10°C - 30DC. The plants were watered regularly with heat sterilized water. No fertilizer was applied. The tomato plants were grown under these conditions for 5.5 weeks.
The isolated rhizobacterial strains were grown in 100 ml nutrient broth (Biolab, Wadesvi!le, South Africa) on a rotary shaker for 48 hours at room temperature. Cell suspensions where diluted or concentrated to a concentration of 1 x 108 cfu/ml by means of centrifugation of each of the isolates in a 50 ml capacity plastic tube, the supernatant was discarded and the cell pellet was re-suspended in sterile ¼ strength Ringer s solution.
Each of the rhizobacterial isolates: A-10 and A-07 were tested for promotion of plant growth. The tomato plants were individually treated with 5 ml of the rhizobacterial suspension at a concentration of 1 x10s cfu/ml of each isolate. Control samples were included in which 5 mi of sterile ¼ strength Ringer's solution was applied to each of the control plants as opposed to the rhizobacterial suspension. The experiment was designed as a compteteiy randomized (block-less) design. Each treatment consisted of 6 replications. A replication consisted of one cell of a seedling tray with one plant in it. The plants were allowed to grow for 3 weeks subsequent to the commencement of the treatments before they were harvested and the fresh root and shoot mass were determined by weighing.
Figure 2 illustrates the plant growth enhancing effect of each of the bacterial strains Paenibacillus barcinonensis strain A-10 and Bacillus megaterium strain A-07. Paenibacillus barcinonensis strain A-10 showed significant enhancement of plant growth as compared to the untreated control plants. The Bacillus megaterium strain A-07 also showed a trend towards enhanced plant growth although the enhancement was not statistically significant.
EXAMPLE 3
Assessment of PGPR isolates for biocontrol of Phytophthora capsici on tomatoes Tomato seedlings were grown under the conditions specified in example 1. The plants were watered daily with heat sterilised tap water. Replicates comprising two 4 week old seedlings were transferred to 500 ml plastic pots containing steam pasteurised soil and a single application of soluble fertilizer (Multifeed-P® at 90 ppm nitrogen) was applied as a soil drench after transplanting. Pots were watered daily with heat sterilised tap water. Greenhouse temperatures ranged from a minimum of 14°C to a maximum of 33°C. Each pot containing two seedlings was regarded as a replication. Three replications were prepared for each treatment.
The treatments were performed as follows: a) Negative control - plants were untreated and uninfected;
b) Positive control - plants were untreated and infected with the Phytophthora capsici zoospore suspension;
c) Test treatment - plants were treated with the rhizobacterial strain Pseudomonas fluorescens N-04 and simultaneously infected with the Phytophthora capsici zoospore suspension.
d) Comparative treatment - plants were treated with a commercial bacteria! biocontrol product, BacUp® and simultaneously infected with the Phytophthora capsici zoospore suspension.
The rhizobacterial cell suspension was prepared as described in example 1 , above. 25 ml of the rhizobacterial cell suspension was applied to each of the relevant pots respectively as a soil drench.
A zoospore suspension of P. capsici was prepared according to a modification of the method described by Pau!itz and Loper (1991 ) as follows: The fungus was grown on V8 juice agar plates (100ml of V8 juice, 900ml distilled water, 1g of CaC03, 15g of agar) for 5 - 7 days at 25°C. The plates were then incubated at room temperature under a near-UV light for at least 5 days. The whole culture was then cut into small blocks by means of a sterile scalpel. Fifteen ml of sterile distilled water was then added to each plate. Plates were incubated at 4°C for 30 minutes. After this period plates were incubated at 25°C for 15 - 30 minutes. The water in each plate is added to a sterile bottle or small container. After a period of 24 hours, a 1 ml zoospore suspension of Phytophtora capsici was applied to the relevant treatments at a concentration of 2,5 x 103 zoospores/mi and each pot was watered to saturation point.
The 24 hour period between inoculation and the infection was in order to allow the bacteria! strains to adjust to the new environment and to start colonizing the rhizosphere of the plants in each pot. The rhizobacterial strain and commercial microbial product were therefore predominantly screened for their ability to prevent infection and decrease disease progression.
During the trial plants were assessed according to a disease rating as follows: 0 = no visible symptoms
1 = appearance of lower stem lesion and plant slightly wilted
2 = stem lesion extending 1 - 5 cm from surface of soil and 25% of plant wilted
3 = stem lesion progressed up to half of the plant and 50% of plant is wilted
4 = stem lesion is continuous and > 50% of plant is wilted
5 = plant is dead
Plants were harvested three weeks after commencement of the trial. Total wet mass, wet root mass and wet shoot mass were determined by weighing. The results are shown in Figure 3(a). Pseudomonas fluorescens strain N-04 produced significantly higher biomass yields based on total wet mass, wet shoot mass and wet root mass as compared to the positive control and the comparative treatment with the commercial microbial product BacUp®. The results indicate that Pseudomonas fluorescens strain N-04 shows significant biocontrol activity.
Figure 4 shows disease progression curves for the treatments with Pseudomonas fluorescens strain N-04 in comparison with those of the positive control treatment (i.e. pathogen only) and the commercial microbial product BacUp®. Treatment with Pseudomonas fluorescens strain N-04 slowed disease progression significantly, confirming effective biocontrol activity, whereas the commercial microbial product BacUp® was ineffective.
EXAMPLE 4
Dual culture assay for screening of rhizobacterial isolates for in vitro antibiosis activity as indicated by inhibition of Phytophthora capsici mycelial growth.
This assay was performed to test for antibiosis activity of the rhizobacterial strains as a mechanism of action for biocontrol of the plant pathogen Phytophthora capsici.
An isolate of Phytophthora capsici was obtained from the Vegetable and Ornamental Plant institute at Roodeplaat, South Africa and grown in pure culture on Potato Dextrose Agar (PDA) plates (Biolab, Wadesville, South Africa).
The rhizobacterial strains were grown on nutrient agar plates (Biolab, Wadesville, South Africa) for 24 - 48 hours at 25°C. When distinct colonies became visible, bacterial cells from single colonies were transferred to sterile nutrient broth in 250 ml flasks by means of a sterile inoculation loop. Alternatively, cultures stored at -70°C in microbeads (Davies diagnostics) were cultured by adding one microbead from a pure culture of the relevant isolate to the nutrient broth. The cultures were then incubated at room temperature on a shake-incubator for 48 hours. To test for antibiosis activity the dual-culture technique was used (Paulitz el a!., 1992). Three drops of bacterial suspension were placed on the margins of a Potato Dextrose Agar (PDA) plate, equidistant from one another using 50 μΙ drops of a 10s cfu/ml bacterial suspension. The piates were then incubated at 28°C.
After 24 hours of incubation, a 4 mm agar disc from a Phytophthora capsici culture grown on PDA for 7 days at 25*C was placed in the centre of the PDA plate, equidistant from the three bacteria! suspension drops. The plate was then incubated at about 27°C for seven days. The percentage inhibition was calculated by means of the following formula: (R - r)/R x 100, where R = Radius of growth in uninhibited region and r = radius of mycelial growth in the inhibited region {Idris et al, 2007).
As shown in Figure 5, Paenibacillus barcinonensis A10, Bacillus megaterium A07, and Pseudomonas fluorescens N04 inhibited mycelial growth of the pathogen P. capsici by 50.01%, 26.48% and 20.46% respectively.
The control plates which were not treated with any rhizobacterial isolates showed no inhibition zone. Further, the surface of the control piates was completely covered by P. capsici growth.
EXAMPLE 5
Screening of rhizobacterial isolates for potential phosphate soiubilisation activity
To test for phosphate solubilisation activity of the rhizobacterial isolates, Pikovskaya medium (Nautijal, 1999) was prepared. It is a well known fact that on this medium phosphate solubilisation activity is identified by the formation of a clear halo which develops around bacterial colonies having the ability to so!ubilise phosphate. During the assessment, clear halos were easily distinguished from the surrounding medium as Pikovskaya's medium has a milky, whitish colour.
The rhizobacterial isolates of the invention were revived from storage cultures in microbeads (Davies diagnostics) at -70°C and grown on nutrient agar plates for 24 - 48 hours. An inoculum of the pure colonies was then transferred from the nutrient agar plate to the Pikovskaya piates by means of a sterilised toothpick. The phosphate medium was pierced at three positions equidistant from one another with the inoculated toothpick. Control plates were pierced with sterile toothpicks without bacterial inoculum.
The Pikovskaya plates were incubated for 14 days at 25°C. Two piates were prepared for each treatment and as each halo around a single bacterial colony was regarded as a replication, 6 replications for each treatment were performed. After 14 days, the size of each of the halos was determined by measuring the total halo diameter and subtracting the bacterial colony diameter to give a final measurement of halo diameter in millimeters. Figure 6 sets out the data from the phosphate solubitisation assay. Pseudomonas fluorescens strain N-04 and Paenibacillus barcinonensis strain A-10 showed significant phosphate soiubilisation activity whereas Bacillus megaterium strain A-07 did not show any phosphate soiubilisation activity.
EXAMPLE 6
Assessment of rhizobacteriai strains for growth promotion of wheat in the greenhouse
Rhizobacteriai isolates from the University of Pretoria's Plant Growth Promoting Rhizobacteria collection were assessed in the greenhouse for their ability to promote the growth of wheat seedlings.
Bacterial inoculum was prepared by inoculating 100 ml Nutrient broth (NB) (Biolab) with one frozen bead containing the bacteria isolate. The cultures were incubated on a rotary shaker for 24 hours at 25°C and 180 rpm. After incubation the cell concentration was determined by spectrophotometer at 550 nm. The cultures were transferred to 50 ml centrifuge tubes and centrifuged at 3000 x g for 10 minutes. The pellet was resuspended in sterile Ringer's solution to a concentration of approximately 10& cfu/ml.
The bacterial isolates were applied to ten day old wheat seedlings that had been transplanted into 500 ml (10 cm diameter) plastic pots, containing steam pasteurized sandy loam soil, at the rate of 5 seedlings per pot. The respective treatments were: 1 ) untreated control, 2) 25 ml bacterial suspension ( 08 cfu/ml) of each bacterial strain respectively. Treatments were replicated four times, each treatment unit consisting of a pot containing five seedlings. The pots were arranged in a randomised block design.
A!l trials were conducted with wheat seeds variety SST822 from Sensako (Randburg, South Africa) Batch number: 20025A07VV. The seeds were commercially treated with the fungicides Vitavax Plus, and K-Orbito! by Sensako. During the current study these fungicides were removed and the seeds surface sterilised before the experiments were conducted by immersion in 70% ethanoi for 5 minutes followed by immersion in 1 % sodium hypochlorite for one minute thereafter the seeds were rinsed five times with sterile water.
All experiments were conducted at the University of Pretoria's Plant Pathology Greenhouses. The seeds were geminated and plants grown in the following manner: The seeds were germinated in sterile vermiculite. One week after sowing in the vermiculite the seedlings were transferred to pots containing sandy loam soil. Greenhouse temperatures were maintained at 16 - 25°C and the seeds and plants received daily watering with municipal tap water.
The trial was harvested four weeks after the plants were transplanted in to the pots. Plants were harvested by carefully removing them from the pots and washing the soil from the roots under running tap water. The fresh mass of the roots and foliage was then determined by weighing. The plants were then dried in an oven at approximately 75°C for three days and the dry root and foliage masses were determined by weighing.
For the pot trials the treatments were arranged in a randomised block design with four replicate pots per treatment. Each experimental unit consisted of one pot with five wheat
Table 2: Efficacy of rhizobacteria! isolates for promoting growth of wheat seedlings in the greenhouse
Figure imgf000019_0001
T29 5.032 abc ll§i¾! abc 0.593 ab illiil: a 28 abc abc 15 ab Hill a
* % change in mass [(treatment - control)/ control x 100] therefore negative values are treatments that are (ess than the untreated control and positive values are treatments with a higher mass than the untreated control.
** Treatment means within the same column followed by the same letter do not differ significantiy, (P=0.05) according to the Least Significant
Difference (LSD) tests using the GL procedure. Significant values are also shaded.
seedlings, and treatments were replicated four times. Data was subjected to analysis of variance (ANOVA) using SAS-9.2 software. Means values in each treatment were compared using the least significant difference (LSD) test at 5% (p = 0.05) level of significance.
The results of assessment of growth promotion of wheat by the rhizobacterial strains are shown in Table 2. The fresh shoot mass of wheat was significantly increased in plants treated with Bacillus cereus T-11 , Paenibacillus alvei T-22 and Paenibacillus alvei T-29, whereas dry shoot mass was significantly increased by all four strains, namely Bacillus cereus T-1 1 , Lysinibacillus sphaericus T-19, Paenibacillus alvei T-22 and Paenibacillus alvei T-29 in comparison with the control. These increases in dry foliage mass represent increases of 19%, 51 %, 53% and 65% respectively.
These results demonstrate that the rhizobacteria! strains of the present invention have the ability of enhancing the growth of wheat in the absence of additional fertiliser applications. This finding indicates the possibility that wheat growth under field conditions may be maintained or even enhanced whilst reducing synthetic fertiliser application rates.
EXAMPLE 7
Dual culture assay for screening of rhizobacterial isolates for antibiosis activity against the plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Fusarium praminearum
A dual culture assay on solid agar media was performed to test for the ability of the rhizobacteria to inhibit mycelial growth of plant pathogenic fungi by means of antibiosis. Inhibition of fungal pathogens by the bacterial isolates was determined by means of the dual culture method on Water-yeast agar (WYA) and Potato dextrose agar (PDA). WYA was used because it is a minimal medium that mimics the carbon- limiting environment of soil. A single isolate of the bacterium to be tested was stab inoculated in three places equidistant from the centre and each other onto a WYA plate. A 5 mm diameter fungal plug was then placed in the centre of the plate between the bacterial spots (De Boer et al. 2007). Three replicate plates were used for each isolate. When the fungal colonies on the control plate without bacteria reached the edge of the plate (after approximately four days), the growth towards and away from the bacterial colonies was recorded and the % inhibition of mycelial growth calculated. The % inhibition of myce!ia! growth was calculated by means of the formula [(R2-R1 )/R2] x 00, with R1 being the distance of mycelial growth towards the bacterial colonies and R2 the maximum mycelial growth on the control plate. During the dual culture assay with Rhizoctonia abnormal mycelial growth was observed and recorded as well.
The dual culture (antibiosis) results are shown in Table 4. In general the largest mycelial inhibition zones were observed with the Fusarium isolates grown on PDA. F. oxysporum UPGH 132 was only inhibited by PaenibacHlus alvei T-29 on PDA where a 24% inhibition was observed. The greatest inhibition against F. graminearum WP4F was observed with PaenibacHlus alvei T-29 (46%) on PDA.
Tabie 4: inhibition of plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Fusarium graminearum myce!ial growth by rhizobacterial strains on potato dextrose agar and water yeast agar
Isolate
l!ii! iYAf ii iiAiiii
T-11 5 5 0 Gc 43 0
T-19 4 0 0 NGC 0 0
T-22 0 0 0 NG 0 0
T-29 0 0 45 NG 46 0
* ycelia! growth inhibition was calculated as [(R - r)/ R] x 100 where R is mycelial growth away from the bacterial colony (the maximum growth of fungal mycelia) and r is the mycelia growth towards the bacteria.
a PDA = Potato dextrose agar
b WYA = Water yeast agar
0 For interactions where no inhibition zone was present, the bacterial isolates were rated by whether the fungal mycelium grew over the bacterial colony or not. G = overgrown; N G= not overgrown.
EXAMPLE 8
Assessment of rhizobacterial strains for chitinase activity
Chitinase activity of bacteria is indicative of their ability to produce chitinase as a mechanism of biocontrol activity against plant pathogenic fungi. In order to determine whether the bacterial strains of the invention produced chitin, each strain was stab inoculated in triplicate onto water agar with chitin (WAC) media and chitin minima! media (CMM). Both media are opaque and the formation of a clear halo around the bacterial colony indicates the production of chitinase.
The plates were incubated at 25°C and after seven days incubation the presence of halos in the media was recorded. Colloidal chitin for the preparation of WAC and C M was prepared by grinding crab shell chitin (Sigma, Johannesburg South Africa) to a fine powder with a coffee grinder. Forty grams of the powdered chitin was dissolved in 400 ml concentrated hydrochloric acid and left overnight at 4°C to digest. The colloidal chitin was precipitated from the acid by adding the chitin-acid solution to 5 litres cold water and recovered by filtration. To determine the chitin concentration of the filter cake a sample was weighed and dried in the oven overnight (adapted from Hsu and Lockwood 1975)
Chitinase production by the bacterial strains was assessed on two different media namely CCM and WAC. The CCM contained nutrients whereas the WCA only consisted of colloidal chitin, agar and water. Bacillus cereus T-11 and Paenibacillus aivei T-22 gave a positive reaction for chitinase activity indicating that these isolates might have chitinoiitic activity as part of their potential biocontrol mode of action. Paenibacillus alvei T-22 produced chitinase on the WCA but not on the CCM media. Results of this assay are presented in Table 5.
EXAMPLE 9
Assessment of rhizobacterial isolates for siderophore production
Siderophore production by bacteria is a known biocontrol mechanism against plant pathogenic fungi. Siderophore production by the strains was determined by using the chrome azurol S (CAS)-agar assay modified from Adesina er al. (2007). Briefly, CAS-agar plates were overlaid with a thin layer (5 mm) of Nutrient agar. These plates were then stab inocuiated with the bacterial isolates from nutrient agar in triplicate and incubated at 25°C. Halo formation was recorded after three days.
CAS-agar plates were prepared as described by Hassen 2007. This method is a modification of Schwyn and Nielands (1987) through the substitution of piperazine-1 ,4-bis(2-ethane sulfonic acid) (PIPES) with 3-(N-morpholtno propane sulphonic acid) (MOPS). The CAS media was prepared by separately making CAS indicator and basal solutions and 20 m! sterile 50% glucose solution. The CAS indicator solution consisted of 60.5 mg chrome azurol S in 50 ml ddH20 mixed with 10 ml Fe3+. A solution of 72.0 mg HDTMA in 40 ml ddH20 was slowly added to the above mixture. The result is a dark blue solution with a total volume of 100 ml. CAS basal medium was prepared by dissolving 30 g MOPS, 0.1 g NH4CI, 0.5 g NaC!, and 0.3 KH2P04 in 880 ml ddH20 and adjusting the pH to 6.8 with 6 M NaOH. Fifteen grams agar was added to the solution while stirring. The so!utions were autoclaved separately. After autoclaving the solutions were cooled to 50°C and the glucose solution added to the basal medium. The 100 ml CAS indicator solution was then added to the basal medium by carefully pouring it in along the flask wall. The resulting blue-green agar solution was poured into Petri dishes.
Siderophore production was assessed using the CAS media overlay method of Adesina et al, 2007. Paenibacillus alvei T-22 and Paenibacillus alvei T-29 caused a change in the colour of the CAS media indicating that they produced siderophores. Results of this assay are presented in Table 5.
EXAMPLE 10
Assessment of rhizobacterial isolates for phosphate solubilisation activity
The ability of bacterial isolates to solubilise phosphate was determined on Pikovskaya medium (PVK) and the National Botanical Research Instituted phosphate growth medium (NBRIY) (Nauttyal 1999). Bacterial isolates were stab inoculated onto the agar using a sterile inoculation needle. Four isolates were inoculated on each plate using three replicate plates. After 14 days incubation at 25°C the colonies and halos1 (if present) diameters were measured. The halo size was calculated by subtracting the colony diameter from the halo diameter.
The Bacillus cereus T-11 , Paenibacillus alvei T-22 and Paenibacillus alvei J - 29 strains showed an ability to solubilise phosphate with Bacillus cereus T-11 and Paenibacillus alvei T-29 giving the strongest activity. Results of this assay are presented in Table 5.
EXAMPLE 11
Assessment of rhizobacterial isolates for nitrogen fixing ability
The ability of bacterial isolates to fix atmospheric nitrogen was determined through an assay that demonstrate the ability of the bacteria to grow in nitrogen free media {Baldani and Dobereiner 1980). A loop full of a single bacterial isolate from a singie coiony was transferred to vials containing NFb. The vials were incubated at 25°C and observed for the formation of a white halo or pellicle indicating bacterial growth after 4 and 7 days. Vials were incubated for a further three days after the first observation because it was difficult to determine the presence or absence of growth in some vials.
The Bacillus cereus T-11 , Lysinibacillus sphaericus T-19 and Paenibacillus alvei T-29 strains were able to grow and produce a pellicle in the NFB-free media, demonstrating their ability to fix atmospheric nitrogen. Results of this assay are presented in Table 5.
Table 5: Specific modes of action exhibited by the rhizobacterial strains.
Siderophore Phosphate Chitinolytic
Isolate production3 solubitisationb activity0 N-fixationd
PVK NBRIYR WAC CMM N-free media
T-11 - +++ - + + +
T-19 ' - - - - +
T-22 + + + + -
T-29 + ++ ++ - +
The presence of a halo on CAS agar plates due to the presence of siderophores, + = the presence of a halo, - = no halo.
b Phosphate solubiiisaiion was assessed on Pikovskaya (PVK) and the National
Botanical Research Institute's phosphate growth medium (NBRIY) agar: - = no clearing zone, + = 0-1 mm zone, ++ = 1-2 mm zone, +++ = 2-3mm zone
c Chitinolytic activity was determined by measuring the diameter of the clearing zone on chitin minima! medium (CMM) and water agar containing chitin (WCA): 0 = no clearing zone; + = 1-2 mm zone, ++ = 2-4 mm zone surrounding the bacterial colony.
d Nitrogen fixation was determined by pellicle formation by bacterial isolates inoculated in N-free media prepared as described by Baidani and Dobereiner (1980) and Caceres
(1982).
- = no pellicle; + = presence of pellicle EXAMPLE 12
Assessment of root colonisation ability
A modified paper doll root colonisation assay was used to assess root colonisation by the bacterial strains. Bacterized seeds were placed on seedling germination paper in a single line 2 cm from the upper edge of the paper and then rolled into a paper doll. The paper dolls were then placed upright into an incubator and incubated at 25°C. After 7 days the roots were excised and 0.5 g roots placed in 45 ml steriie Ringer's solution in a beaker. The beakers were then sonicated for 30 seconds and a dilution series prepared from the resultant solution. After counting the number of resultant colonies which developed the number of colony forming units was calculated.
A modified paper doll assay was used to assess root colonisation. The results are shown in Table 5. The Bacillus cereus T-11 , Paenibacillus alvei T-22 and Paenibacillus alvei T-29 strains showed effective colonization of wheat roots. The strains tested in this study can therefore be considered average (104 cfu/gram root) to good (107cfu/gram root) root colonisers (Table 6). Table 6: Ability of rhizobacteria! strains to colonise the roots of wheat seedlings germinated in paper dolls.
T-11 1.05 x 106 6.02
T-22 2.0 x 106 6.30
T-29 2.0 x 104 4.30
EXAMPLE 13
Assessment of rhizobacterial strains for yield enhancement of maize under field conditions
Field trials were conducted over a two year period namely the 2011/2012 growing season. The 2011/2012 season biofertilizer trial was planted on Huttons soil and Arcadian soil ecotype at Towoomba ADC and the Shortlands ecotype on the farm Turfbult. The 2012/2013 trial was planted on the farm Turfbult under supplementary irrigation but the Arcadian and Huttons soil ecotype could not be replicated due to unfavorable climatic conditions. The trials that extended over the two seasons had the same layout and implementation and can be regarded as replicates over the two seasons.
The nitrogen and phosphate soil nutrient levels were adjusted with superphosphate and ammonium nitrate mixture to standard levels of 100 kg nitrate per hectare and 75 kg phosphate per hectare. A two row Monosem® planter was adjusted to deliver the correct amount of fertilizer and seed planting settings for each of the different soil types.
Each replication consisted of 4 x 90 m lines with an intra-row spacing of 50 cm and an intra-row spacing of 90 cm. Maize seed (cultivar phb16 5) was treated with the fungicide Thiram according to manufacturer specifications. Bo!lworm was controlled with De!tamethrin and weeds with Giyphosate (Round-up®) due to the use of the round-up ready maize cultivar phb1615. The Huttons and Arcadian soil ecotype trials were grown under dryland conditions until maturity at 12% moisture whereas the Shortiand soil ecotype trial received supplementary irrigation when moisture stress was observed.
The rhizobacterial strains of the invention namely Lysinibacillus sphaericus T- 19, Paenibacillus alvei T-29 as well as other comparative strains viz. Bacillus cereus S7, Brevundiomonas vesicularis A40 and Chryseobacteriu indologenes (isolate A26), were applied as a dry powder seed treatment at the rate of 200 g / 50 kg seed. A commercial bacterial biofertiliser product B-rus® (Stimupiant, PO Box 2013 Swavelpoort 0036) was also included for comparison. The latter product was applied as a seed treatment at the same rate as the other bacterial strains.
Data was collected exclusively from the two inner rows and yield data collected when harvest maturity was reached at 12% by hand harvesting 80 plants per replicate. As for the 2012/2013 season the trial was replicated as in the 2011/2012 season but only the Shortlands survived to the point of usable data collection.
Data collection consisted of: germination date, bimonthly growth rate and dry biomass and maize cob yield at 12% moisture.
The results presented in Figure 7 show the yield data for the 2011/2012 season for a combination of all three the different soil types (Muttons, Arcadia and Shortlands). in Figure 8 only the 2012/20 3 data for the Shortlands soil is presented.
As is evident from Figure 7 significant yield increases during the 2011/2012 season were only recorded in the Shortlands soil, with the bacteria! strains Lysinibacilius sphaericus T- 9 and Paenibacillus alvei T-29 rendering the best yield increases, thereby outperforming all other strains including the commercial reference product B-rus. The best performing inoculants, the strains Lysinibacilius sphaericus T-19 and Paenibacillus alvei T-29 showed yield increases of 2.88 tons/ha and 2.50 tons/ha respectively (i.e. increases of 30.86% and 24.43 % respectively, compared to the control). The commercial reference treatment B-rus resulted in a yield of 1.42 tons/ha. During the 2012/2013 season in Shortlands soil, Bacillus cereus S7 rendered the best yield (5.11 tons/ha) followed by the Lysinibacilius sphaericus T-19, Chryseobacterium indologenes A-26, Paenibacillus alvei T-29, B-rus, Control and Brevundiomonas vesicularis A-40 with 4.76 tons/ha, 4.5 tons/ha, 4.43 tons/ha, 3.87 tons/ha, 3.82 tons/ha and 3.59 tons/ha respectively.
When consistency in performance is examined over the two season's it was seen that the most consistent results were obtained with the Lysinibacilius sphaericus T-19 and Paenibacillus alvei T-29 strains, with mean increases in maize yield over the two seasons of 30.86% and 24.43% respectively in comparison with the untreated control.
Regarding performance of the strains in the different soil types it is evident that Lysinibacilius sphaericus T-19 and Paenibacillus alvei T-29 gave the best results in marginal soils. EXAMPLE 14
Evaluation of selected PGPR strains for growth promotion of potato in the greenhouse
Six selected plant growth promoting rhizobacteria (PGPR), Lysinibacillus sphaericus (strain T-19), Paenibacillus alvei (strain T-29), Bacillus cereus (strain S7), Pseudomonas fluorescens (strain N-04), Paenibacillus barcinonensis (strain A-10) and Bacillus megaterium (strain A-07), were screened in the greenhouse to determine whether they are able to enhance the growth and yield of potato, especially increasing the tuber yield. Mini tubers (cv. Up-to-Date) were planted in five litre pots in sandy loam soil. When the potato shoots emerged the PGPR isolates were applied as a drench. Once the potato plants had died down, the tubers were harvested and assessed for weight and tuber number per treatment. Tuber mass indicated that Paenibacillus alvei (strain T-29), Paenibacillus barcinonensis (strain A- 10), Lysinibacillus sphaericus (strain T-19), and Bacillus megaterium (strain A-07) significantly improved tuber mass compared to the untreated control. The greatest increase in tuber mass was obtained with Paenibacillus barcinonensis (strainA-10) which increased tuber mass by 56.95%. Pseudomonas fluorescens (strain -04) significantly increased the number of tubers. The results indicate that the selected PGPR isolates have potential to improve the yield of potato and further screening to determine their commercial application is warranted.
Globally in agriculture there is a trend to adopt more sustainable production practices and reduce the reliance on chemical fertilisers. In the search for alternatives to chemical fertilisers, the role of beneficial soil microbes, and in particular the "plant growth promoting rhizobacteria" (PGPR) has received much attention. The presence of these organisms improves plant growth and the general health of plants. These positive effects can be attributed to a variety of modes of action, broadly categorised as direct growth promotion or biological control of plant pathogens. Direct growth promotion activities of PGPR include production of phytohormones and solubilisation of minerals such as phosphate. The aim of this work was thus to determine whether the six selected PGPR isolates, namely: Lysinibacillus sphaericus (strain T-19), Paenibacillus alvei (strain T-29), Bacillus cereus (strain S7), Pseudomonas fluorescens (strain N-04), Paenibacillus barcinonensis (strain A-10) and Bacillus megaterium (strain A-07) are able to enhance the yield of potatoes. With this objective in mind a greenhouse trial was conducted to evaluate the selected PGPR for their ability to promote the growth of potatoes. The trial was conducted at the University of Pretoria's greenhouse facilities and commenced on the 8th March 2012. Pathogen free mini-tubers (obtained from Rascal Seed Research Laboratories, cv. Up-to-Date, size 1 1 - 30 g) were planted in 25 cm diameter (4 litre capacity) pots (one tuber per pot) in steam pasteurised sandy loam soil. The tubers were planted approximately 6-7 cm below the soil surface. Pots were watered daily to keep the soil at field water capacity.
At emergence of the tuber sprouts from the soil (12th March 2012), the PGPR were inoculated as a drench. The bacterial inoculum was prepared by inoculating 200 ml sterile nutrient broth (Biolab), in 500 ml Erlenmeyer flasks, with a loop-full of a single bacterial isolate. The isolates were obtained from the University of Pretoria's PGPR culture collection. The inoculated broth was incubated in a shaking incubator at 25°C and 150 rpm for two days. A single 15 ml drench application of a single PGPR isolate was applied to each pot.
Treatments:
1. Untreated control
2. Paenibacillus barcinonensis strain A-10: 15 ml broth suspension containing 108 cfu/ml per pot,
3. LysinibaciHus sphaericus strain T- 9: 15 m! broth suspension containing 108 cfu/ml per pot,
4. Paenibacillus alvei strain T-29: 15 ml broth suspension containing 08 cfu/ml per pot,
5. Bacillus cereus strain S7: 15 ml broth suspension containing 108 cfu/ml per pot,
6. Bacillus megaterium strain A-07: 15 mi broth suspension containing 10s cfu/ml per pot,
7. Pseudomonas fluorescens strain N-04: 15 ml broth suspension containing 108 cfu/ml per pot.
The trial was harvested on the 18th June 2012 (102 days after planting). The plants were allowed to die down completely before harvesting. The potato tubers were harvested by transferring the soil from the pots to a wire mesh basket (1 cm2 openings) allowing the soil to fali through and the tubers to remain on top of the wire mesh. The number of tubers, and total tuber weight was recorded for each pot.
The experiment was arranged in a completely randomised design, with five replicates per treatment. Each replicate consisted of 5 pots, each containing one plant per pot.
Paenibacillus alvei T-29, Paenibacillus barcinonensis A-10, Lysinibacillus sphaericus T-19, and Bacillus megaterium A-07 significantly improved tuber mass, compared to the untreated control. Paenibacillus barcinonensis A-10 and Paenibacillus alvei T-29 rendered more than 50% yield increase over the uninoculated control. Pseudomonas fluorescens N-04 significantly increased the number of tubers. The three strains that performed the best were Paenibacillus barcinonensis A-10, Paenibacillus alvei T-29 and Lysinibacillus sphaericus T-19. In comparison to the control these strains resulted in an increase in mass of 59% for Paenibacillus barcinonensis A-10, 57% for Paenibacillus alvei T-29 and 37.5% for Lysinibacillus sphaericus T-19. Results are presented in Figure 9 and 10 and in Table 7.
Table 7: Effect of various rhizobacterial strains on growth (yield) of potato plants in the greenhouse. Values are means of five replicates. Values in columns followed by the same letters do not differ significantly (P=0.05) according to the Least Significant Difference (LSD) test using the GLM procedure (LSD = 2.7671)
Tuber Weight (g) 111®
Control 9.41 c 0.00 2.80 B
T-19 12.94 ab 37.46 2.67 B
A-10 14.77 a 56.95 3.00 Ab
T-29 15.00 a 59.30 2.60 B
S-7 11.62 be 23.40 2.67 B
A-07 12.69 ab 34.78 3.00 Ab
N-04 11.79 be 25.22 3.60 A
EXAMPLE 15
Biocontrol of selected bacterial and fungal pathogens of potato (Solanum tuberosum L.) by application of rhizobacterial strains Bacillus me aterium A07; Paenibacillus barcinonensis A10; Lvsinibacillus sphaericus T19; Paenibacillus alvei T29; and Pseudomonas fluorescens N04
Diseases contribute significantly to yield loss of potato, sufficiently so to warrant costly control measures, and have caused regional crop failures (Hide & Lapwood, 1992). Plant growth promoting rhizobacteria (PGPR) are known to control diseases by several mechanisms, including induced systemic resistance (ISR), reduction of ethylene levels and production of antibiotics, volatiies, antimicrobial enzymes and siderophores (Zahir ei a/., 2004). PGPR could be developed, as a part of integrated plant disease management, to reduce conventional pesticide application (Zahir er a/., 2004).
Important bacteria! pathogens of potato in South Africa and many other potato producing countries include Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliensis, Dickeya dadantii and Streptomyces scabies. Pectobacterium carotovorum and Dickeya species can infect plants through wounds in stems or from infected planting material. Soft rot bacteria are carried asymptomatically on tuber surfaces until a sufficient concentration is reached to cause post-harvest or pre-emergence tuber rot. S. scabies can infect plants from the soil or from planting material. No chemical controls are currently available against soft rot bacteria, and chemical control of common scab provides inconsistent results (Gouws & Geldenhuys, 2011 ).
important fungal pathogens of potato in South Africa and many other potato producing countries include Colietotrichum coccodes (cause of black dot), Fusarium oxysporum f. sp. Tuberosi (cause of dry rot), Rhizoctonia solani (cause of black scurf) and Verticillium dahlia (cause of Verticillium wilt) (Denner, 2011 ). Alternaria alternate is also an important pathogen of potatoes in South Africa, and may cause yield losses of up to 50% (Van der Waals ei a/., 2011 ). Fusarium and Verticillium pathogens of potato are spread both through soil and planting material in South Africa. No conventional fungicides are currently available in South Africa for control of these diseases (Denner, 2011 ).
The most important route of infection by Rhizoctonia solani anastomosis group 3 is infected planting material in South Africa, although infection from soil is also important. Chemical control of this disease currently provides inconsistent results (Denner, 2011 ).
Antagonistic PGPR that control aforementioned soil- and tuber-borne pathogens at the soil-plant interface and protect roots and daughter tubers from infection would be of value to the potato industry. A bacterium antagonistic to A. alternata could be applied as foliar sprays (PGPR have successfully been applied as foliar sprays) (Zahir et a/., 2004). The dual culture assay testing for antibiosis is the most commonly used assay used to identify promising PGPR for biologica! control of bacteria and fungi. Inhibition of growth of cultures may occur due to production of antibiotics, antimicrobial enzymes or volatiles (Fravel, 1988) or transfer of genetic material detrimental to the recipient cells (Schottel, Schimizu & Kinkel, 2001 ).The relative sizes of inhibition zones formed by PGPR in antibiosis tests may correlate with the relative efficacy of these strains in the field as shown by Liu, Anderson and Kinkel (1995). Biosurfactants can lyse pathogens, or can induce resistance against pathogens (Czajkowsi et al., 2012). Tuber slice assays can be used to identify PGPR that reduce damage by pathogens with postharvest aspects such as Fusarium spp. (Gould et al., 2008) and Dickeya spp. (Czajkowsi et al., 2012).
The inventors determined a) which of the selected rhizobacterial strains are most promising as biocontrol agents against selected bacterial and funga! pathogens of potato (Pc. carotovorum, Pc. brasiliensis, D. dadantii and S. scabies and A. alternata, C. coccodes, F. oxysporum f. sp. tuberosi, R. solani AG 3 and V. dahliae) and b) by studying the mechanisms of biological control of these bacterial strains.
Cultures
The PGPR isolates were obtained from the long-term storage culture collection at the University of Pretoria. The cultures were streaked on nutrient agar (NA) plates and incubated at 37°C for 2 days before use. The cultures were then stored at 25°C until being used (cultures older than 7 days were discarded). The cultures were stored at -20°C in 80% aqueous glycerol as a source of cultures for the duration of the study. Pathogen cultures were obtained from the long-term storage culture collection of the Potato Pathology Group of the University of Pretoria, with the exception of V. dahliae, which was obtained from the Agricultural Research Council- Roodep!aat. The fungai pathogens were grown on potato dextrose agar (PDA) plates at 25°C before use. The cultures were stored on agar slants for the duration of the study. The soft rot bacteria were grown on NA at 37°C for 2 days and then at 25°C for 4 days before being used. The soft rot bacteria were stored in the same manner as the PGPR for the duration of the experiment. S. scabies was grown for 3 weeks at 25°C on malt extract agar ( EA) before being used. No cultures were plated out more than 3 times from the originally obtained cultures.
Antibiosis assays against soft rot bacteria
Pathogen suspensions were prepared by suspending 3 colonies of Pc. brasiliensis or D. dadantii (or 15 colonies of Pc. carotovorum, which produces smaller colonies than the other soft rot bacteria) in 15 mL of Ringers solution. The suspensions were then vortexed at maximum speed for 3 minutes. The Pc. brasiliensis suspension contained 4x108 coiony-forming units (CFUs)/ml_. The D. dadantii suspension contained 1.4x108 CFUs/mL. The Pc. carotovorum suspension contained 1 x107 CFUs/mL. Hundred μ! of freshly prepared pathogen suspension was pipetted onto each NA, PDA and MEA piate {the suspension was briefly vortexed before each transfer). The transferred suspensions were immediately spread over the surface of the agar plates using a sterilised cell spreader. After spreading the pathogens, each plate was stab-inocuiated at 3 equidistant points, each point 3 cm from the centre of the plate, with each of the test strains respectively (A07, A10, N04, S7, T19 and T29). Experimental controls consisted of plates inoculated only with the pathogen to determine whether the pathogen was evenly spread on the plates. The concentrations of the pathogen suspensions were determined using plate counts on NA. The inhibition zones were observed after 3 days. Two perpendicular diameters of the partial inhibition zones around each rhizobacterium colony (that were visibly less dense than the surrounding bacterial lawn) and the clear zones around each bacterial colony were measured. The mean radii of the clear zones on each plate on day 3 were recorded. The method is similar to that of Schottel, Schimizu and Kinke! (2001 ), but uses stab inoculation of the PGPR, discussed by Bashan, Holguin and Lifs ttz (1993).
Rhizobacterial strains T29 and N04 expressed antibiosis against the soft rot pathogens Pc. brasiliensis and D. dadantii (Table 8). Strain N04 formed significantly larger inhibition zones in mats of Pc. brasiliensis, and to a lesser extent also inhibited Pc. carotovorum. Ho clear zones were formed on NA (data not shown), and D. dadantii failed to grow on MEA. Around strain T29 the clear zones in mats of Pc. brasiliensis and D. dadantii increased in size after 3, 5 and 1 days at 25' C possibly indicating lysis of the pathogen's cells.
Table 8: In vitro antibiosis activity by rhizobacterial strains against soft rot bacterial pathogens of potato. Values are the mean radii (mm) of clear zones around PGPR colonies on mats of soft rot bacteria
Figure imgf000032_0001
Means followed by the same letters are not significantly different (σ=0.05). Antibiosis assay against S. scabies
The antibiosis assay was performed as for the soft rot bacteria, except that a S. scabies spore suspension was prepared by transferring 15 mL of Ringers solution with 0.05% Tween 80 (as discussed by Bashan, Holguin and Lifshitz ,1993} to a S. scabies culture. The plate was manually rotated for 3 minutes and the suspension transferred to a glass tube. The spore suspension contained 4 * 106 CFUs/mL. The sizes of the clear zones were recorded daily for 1 month after preparing the dual cultures by marking the borders of the clear zones on the bottoms of the plates.
The experiment was repeated three times. The clear zones were marked daily on each plate.
Uneven growth of the pathogen colonies made quantitative measurements difficult, therefore the results were recorded qualitatively and not quantitatively (Table 9), Al the strains except T 9 showed antibiosis on one or more of the media. Strains A07 gave the most consistent inhibition of S. scabies.
Tabie 9: In vitro antibiosis activity of rhizobacterial strains against S. scabies, the causal organism of common scab of potato. Formation of c!ear inhibition zones around PGPR colonies in mats of S. scabies on NA, PDA and MEA are indicated. Consistent formation of clear zones is indicated by a +, inconsistent formation of clear zones is indicated by a -7+ and no formation of clear zones is indicated by a -.
Figure imgf000033_0001
Antibiosis assays against fungal pathogens of potato
Fungal plugs 4 mm in diameter were cut from the edges of fungal cultures grown on PDA. The fungai plugs were piaced in the centre of NA, PDA and MEA plates (the use of the 3 media allows the consistency of inhibition to be estimated) (Walker, Powell & Seddon, 1998). The fungal plugs were then allowed to grow at 25°C until the colonies were 10 mm to 20 mm in diameter. The test strains A07, A10, N04, S7, T19 and T29 were stab-inoculated at two equidistant points 3 cm from the centre of each plate. Control plates were prepared for each medium in triplicate, and were inoculated with fungal plugs without inoculating with PGPR. When pathogen colonies on a plate reached the edge of the plate, the 2 lengths of radial growth nearest to the rhizobacterium colonies (t) and the 2 lengths of radial growth farthest from the rhizobacterium colonies (c') were measured for the medium. The radial growth of the fungal colonies on the control plates (c) were measured in 4 perpendicular directions. The results of antibiosis assays against C. coccodes, F. oxysporum and V. dahliae were obtained after 12, 9 and 21 days of incubation at 25°C, respectively. The plates with R. solani on NA plates were measured after 5 days at 25°C, the PDA plates after 6 days and the MEA plates after 8 days. The NA and PDA plates with A. alternate had been measured after 6 days at 25°C (before the controls had reached the edges of the plates) due to mite contamination, while the MEA plates were measured after 17 days. The percentage inhibition of the treatment plates were measured as follows:
Percentage inhibition of plate = [(C-T)/Cj100, where C = mean of measurement c for control plates (where pathogen was grown without the biocontro! agent on the same medium) and T= mean of measurement t for plate. A similar formula was used by Uppai et al. (2007).
Light microscopy was used to examine parts of fungal colonies near PGPR with visibly altered morphology.
The rhizobacterial strain T29 showed the best overall antifungal activity, inhibiting the most fungal pathogens (Table 10). This strain caused more than 50% inhibition of F. oxysporum on MEA and significantly more inhibition of F. oxysporum than the other strains on all 3 media (Table 10). Strain T29 also inhibited R. solani, V. dahliae and C. coccodes.
Table 10: In vitro inhibition of mycelial growth of fungal pathogens of potato by rhizobacterial strains during dual culture assays on three different agar media. Inhibition expressed as %.
Figure imgf000034_0001
F. oxysporum A07 23.96a 15.65ab 13.646b
A10 21.32a 17.46ab 1.426c
N04 15.65ab 9.573bc
S7 25.28a -2.04bc 1.426c
T19 1.10a -10.66c 16.498b
T29 24.84a 26.98a
R. solani A07 27.93a 35.01a
A10 7.82a 17.65ab
N04 32.96a 36.13a
S7 60.95ab 17.32a 7.09ab
T19 7.82a 7.00b
T29 48.81ab eoisi
V. dahliae A07 23.12ab 20.70bc -2.051b
A10 12.91ab -15.45d 1.538b
N04 1.50b 24.78ba 46.154a
S7 K32i3a¾¾ 20.70bc 9.744b
T19 21.92ab -4.37dc -2.051 b
T29 21.92ab 47.25a 39.487a
Mean percentages of inhibition are indicated. Means followed by the same alphabetical letters are not significantly different (a=0.05). Shaded cells indicate strains showing significant inhibition of 30% or more.
All of the rhizo bacteria I strains showed inhibition of the black scurf pathogen R. solani, but strain N04 showed the greatest activity, causing more than 50% inhibition of the fungus on PDA and significantly more inhibition of R. solani than the other strains on all 3 media.
Only strain A07 was able to significantly inhibit A. alternata.
Light microscopy revealed that strain T29 prevented sporulation of nearby C. coccodes hyphae, caused nearby A. alternate hyphae to swell and become filled with vacuoles and caused the nearby F. oxysporum hyphae to become swollen and highly pigmented, with granulation of cellular contents.
Examples of dual culture assay effects are shown in Figure 11.
Assays for antifungal volatile production
The rhizobacterial strains A07, A10, N04, S7, T 9 and T29 were assayed for volatile production using the method of Mallesh (2008). A loopful of each test strain was streaked in a zig-zag pattern across NA plates, using a template to ensure consistency. Fungal plugs were placed in the centre of PDA plates. The test strain plates and fungal pathogen plates were sealed together with the test strain plates inverted over the fungal pathogen plates. Positive controls consisted of pathogen- inoculated PDA plates sealed together with sterile NA plates. All treatments were performed in triplicate. The plates were incubated until a pathogen colony had reached the edge of the plate. The radial growth of the pathogen colonies were measured in 4 perpendicular directions. The percentage inhibition due to volatile production on each plate was measured using the formula:
Percentage inhibition of plate = [(C"-T')/C"]100, where C" = mean radial growth on control plates and T - mean radial growth of treatment plate.
Ali tested rhizobacterial strains except N04 produced antifungal voiatiies that inhibited radial growth of C. coccodes, and all tested PGPR strains produced antifungal voiatiies that inhibited R. solani (Table 1 1 ). Only strain N04 produced antifungal voiatiies that inhibited V. dahliae. No antifungal volatile activity was observed against A. alternate and F. oxysporum (data not shown).
Table 11 : In vitro inhibition of mycelial growth of fungal pathogens of potato by rhizobacterial strains due to production of volatile substances.
Figure imgf000036_0001
Mean percentages of inhibition are indicated. Means followed by the same letters are not significantly different (a=0.05).
Potato tuber slice assay
An adaption of the technique of Czajkowski et al (2012) was used. A selection of rhizobacterial strains from the long-term storage culture collection were grown by adding a loopful of each strain to 100 m! nutrient broth and incubating the broth in a shaker incubator for 2 days at 36 °C. This is expected to produce a suspension of 108 CFUs/mL (unpublished results). Pc. brasiliensis was grown in nutrient broth and adjusted to a concentration of 108 CFUs/mL (0.1 absorbance at 600nm). The cells were pelleted through centrifugatton at 6000g and washed twice. The rhizobacteria were used at concentrations of 108 CFUs/mL, while the Pc. brasiliensis suspensions were diluted to a concentration of 106 CFUs/mL. Small-medium cv. Mondial tubers (Visser, 201 1 ) were rinsed with tap water, thoroughly sprayed with 70% ethanol, allowed to dry in a laminar flow, rinsed with sterilised, demineralised water, allowed to dry in a laminar flow and cut into 9mm-thick slices. Each replicate consisted of a petri plate containing two slices from a tuber, and each replicate was obtained from a different tuber. Wells, 4 mm deep and 4 mm in diameter, were cut from the centre of each slice and inoculated with 100 μΙ of the pathogen suspension and 100 pi of a rhizobacterium suspension. Positive controls were inoculated with 100 μΙ sterilized, demineralised water and 100 μ! of the pathogen suspension, while water controls were inoculated with 100 μί of sterilised, demineralised water. The plates were incubated at 28 °C for 4 days in humid chambers. Due to the large amount of work, the test strains were divided into 3 groups, and each group was tested independently. Diameters of soft rot lesions on the potato slices were measured in two perpendicular directions. For irregularly shaped lesions, the longest and shortest measurement was obtained. The percentage reduction in the size of the soft rot lesion was calculated using the formula:
Percentage inhibition of plate - [(C'"-T")/C"']100, where C" = mean diameter of rotted lesions in positive control plates and T"= mean diameter of rotted lesions in treatment plate.
Examples of tuber slice assay results are shown in Figure 12.
Strains N04 and T29 showed more than 50% inhibition of Pc. brasiliensis as reflected by a reduction in the diameters of rotted lesions on potato tuber slices (Table 12).
Table 12: Suppression of Pc. brasiliensis soft rot of potato by rhizobacterial strains during the
Figure imgf000037_0001
Means followed by the same letters are not significantly different (a=0.05)
Biosurfactant assay
An adaption of the technique of Czajkowski et al (2012) was used. The six selected rhizobacterial strains A07, A10, N04, S7, T 9 and T29 were grown in nutrient broth for twelve hours at 28 "C with orbital shaking. The cultures were then centrifuged for 20 minutes at 5000 rpm using an Eppendorf® 5804R centrifuge. The supernatant of each culture was collected. 20 μΙ mineral oil (bio erieux® sa, Marcy TEtoile, France) was placed on 30 mL of demineralized water in cleaned petri dishes, after which 2.5 pi of supernatant was placed on each oil drop. Each treatment was repeated in triplicate. The size of each oil drop was measured immediately. The diameters of each oil drop were measured perpendicularly immediately after the supernatant was applied and the oil droplet stabilised. Controls consisted of oil droplets on which 2.5 μΐ of sterilised water was placed. The percentage increase in sizes of oil drops was measured using the formula: Percentage increase = (Y-Z)/Z*100%, where Y = mean diameter of treated oil droplet, and Z= mean diameter of controls.
Strains A07, N04 and T19 produced biosurfactants detectable in the supernatants of the cultures (Table 13).
Table 13: Results of biosurfactant assay with rhizobacterial strains.
Figure imgf000038_0001
Means followed by the same alphabetical letters are not statistically different (a=0.05) Phytohormone analysis
Rhizobacterial strains A10, A07.T19 and T29 were grown in nutrient broth for twelve hours at 28 °C with orbital shaking. The cultures were then centrifuged for 20 minutes at 5000 rpm using an Eppendorf® 5804R centrifuge. The supernatant of each culture was collected and analysed for phytohormone content by means of Liquid Chromatography Mass-spectroscopy (LC-MS/MS), by RPS Mountainheath Ltd Centurion Court, 85 Milton Park, Abingdon, Oxfordshire OX144RY in England.
Results of the plant hormone analysis are shown in Tabie 14. Strains A10, A07, T19 and T29 all produced the auxin lndole-3-acetic acid (IAA), with the highest amount being produced by strain T19. All four strains also produced high amounts of the auxin Phenyl acetic acid (PAA) with the highest amount being produced by strain A07. These results confirm phytohormone production by the rhizobacterial strains as an inherent plant growth promoting mode of action.
Table 14: Phytohormone concentrations (mg/kg) in rhizobacterial culture broth as determined by means of Liquid Chromatography Mass-spectroscopy.
Figure imgf000038_0002
Abbreviations used herein refer to: IAA: lndole-3- Acetic Acid; KN: Kinetin; ZR: Trans-Zeatin;
BAP: 6-Benzyi-aminopurine; IPA: Isopentyl-adenosine; ZR: Trans-zeatin Riboside; PAA: Phenyl Acetic Acid; !BA: lndole-3 Butyric Acid; and 2iP: Isopentenyf adenine. Statistical analysis
The results were analysed using Fisher's least-significant-difference test at a level of 5%, using SAS®9.3 (SAS Institute Inc., Cary, NC, USA).During the analysis of the antibiosis assay results, the results on different media were analysed as independent data sets. Only bacteria that caused 50% or more inhibition of fungal pathogens or that reduced the size of rotted lesions by 50% or more (in the case of the antibiosis assays against fungal pathogens and the tuber slice assays, respectively) were considered promising biocontrol agents.
Discussion
All five of the rhizobacterial strains tested, showed activity against at least one potato pathogen, but most strains showed activity against multiple pathogens.
Strain A07 was the most promising for control of S. scabies, the causal organism of common scab of potato, during the antibiosis assay. This strain produced biosurfactants which are known to play a role in the biocontrol of plant pathogens.
Strain N04 was found to be promising for control of soft rot bacteria and R. solani during the antibiosis assays. This strain was demonstrated to produce antifungal volatiies that inhibit R. solani, and also produced biosurfactants.
Strain T29 showed the best suppression of the soft rot bacterial pathogen Pc. brasitiensis during the tuber slice assay, and also suppressed the soft rot bacterium D. dadantii and the fungal pathogens R. solani, F. oxysporum and V. dahliae during the dual culture assays.
The data indicate rhizobacterial strains N04 and T29 to be potential broad- spectrum biocontrol agents, possibly able to control bacteria and fungi simultaneously.
Rhizobacterial strains A10, A07, T19 and T29 all produced high levels of phytohormones in Iiquid culture, thereby demonstrating their inherent potential to promote plant growth via the production of plant growth enhancing hormones.
EXAMPLE 16
Efficacy of rhizobacterial strains Paenibacillus barcinonensis A10, Lvsinibacillus sphaericus T19 and Paenibacillus alvei T29 for bioloqicai control of black scurf of potatoes caused by the fungal pathogen Rhizoctonia solani Rhizoctonia solani (R. solani) is a soil borne plant pathogen capable of infecting a wide range of hosts. The species is classified into 13 anastomosis groupings (AG), with each AG showing a preference in target host, plant part or host growth stage. (Carling & Leiner 1990 ;Tsror, 2010). The most common anastomosis group infecting potatoes is AG3. Potato tuber infections occur at the end of the growing season when haulms start to die. The dying plant cells release certain voiatiles which stimulate and attract mycelial growth of R. sotani, increasing the probability of tuber infection and sclerotia development (Wharton ei al. 2007). Sclerotia appearing on the tubers are the most prominent symptom of R. solani AG3. These sclerotia appear as dark structures on the tuber skin , thereby lowering the market value of the tubers.
Bacterial inoculum preparation
inoculum of the rhizobacterial strains was prepared as either a dry powder (seed treatment) formulation or a liquid bacterial cell suspension.
The liquid cell suspension was prepared as follows: The rhizobacterial strains were streaked onto nutrient agar (Biolab, Wadeviile South Africa) and incubated for two days at 25°C. Erlen Meyer fiasks (250 ml capacity) each containing 100 ml nutrient broth (Biolab, Wadeviile, South Africa) were prepared according to the suppliers' specifications. Each flask was inoculated asepttcaliy with a bacterial culture by means of an inoculation loop (two flasks per isolate). The flasks were incubated for two days at 25°C on a rotary shaker in an incubator to allow bacteria to reach populations of approximately 108cfu/ml.
Inoculum for the seed treatment was prepared by initially growing the selected strains in nutrient broth as explained above. Perlite® powder obtained from Stimuplant in autociavable plastic bags (200 g/bag). Twenty one mil!ilitres of the selected bacterial isolate broth culture was aseptically injected into a bag with a sterile syringe before the bag was manually shaken and incubated. The contents of each bag was manually mixed every second day to allow adequate growth and spread of the bacterial inoculum within the bag. After 2-3 weeks, the inoculum was ready to be used.
Pathogen inoculum preparation
A strain of R. solani AG3 isolated from diseased potato tubers was used for the biocontrol trials. Three types of pathogen inoculum was used, a sand-bran inoculum, a vermicuiite inoculum and inoculum comprising a macerated culture of the fungus.
The R. solani was cultured on potato dextrose agar (PDA, Bio!ab, Wadeville, South Africa). Three week old PDA cultures were used for the agar plug inoculation.
For the sand-bran inoculum graded filter sand, 0.95mm - 1.1mm effective sizes, Silica quartz, Farm Groenfontein, Delmas) and bran (Snowflake, Premier foods, !sando, South Africa) were mixed in a 1 L Schott bottle at a 20:1 sand-bran ratio. This mixture was moistened by the addition of 60ml distilled water and autociaved once per day for three consecutive days.
Two hundred grams of the autociaved sand-bran mixture was placed in an autoclaveable plastic bag strengthened with an additional autoclaveable plastic bag by placing the one within the other and sealed. To allow steam to escape the bags were punctured with a hypodermic syringe needle. The bags were autociaved twice thereafter. After the complete sterilization process, each bag was inoculated with 10 agar plugs cut from two weeks old R. solani AG3 cultures on PDA using a 1cm diameter cork borer. The bags were re-sealed and the pathogen mycelium was allowed to grow and establish itself within the mixture for approximately two months. The bags were manually mixed once a week to aid myce!ia dispersion within the bag.
The vermicuiite inoculum was prepared by adapting the method described by Atkinson et al, 2010: Three week old PDA cultures of the R. solani AG3 were macerated and mixed with 20 litres sterilised (autociaved) vermicuiite and 5 litre of sterile distilled water. The vermicuiite mixture was incubated at room temperature for four days and mixing of contents occurred on day two. The inoculum was used on day five.
A macerated culture inoculum was prepared by mixing macerated R. solani cultures with sterile distilled water. The inoculum was prepared in Schott bottles by mixing macerated cultures from 8 ½ x 9cm petri dish cultures per litre sterile distilled water. The bottles were shaken to insure adequately mixing of the inoculum.
Disease assessment
At harvest of each trial disease severity was assessed as follows: Tubers were washed clean in tap water and each tuber was inspected and a disease rating was given based on the amount of sclerotia visible on the tuber surface. The ratings ranged from 0 to 3 (0 being no sclerotia present and 3 being tuber surface approximately ¾ covered with sclerotia, (Appendix A) (adapted from Atkinson er al. 2011 ).
Assessment of rhizobacterial strains for biological control of black scurf of potatoes caused by Rhizoctonia solani on potato tissue culture plants in the greenhouse
One month old tissue culture plantiets (obtained from ANSABI MASS tissue culture Laboratory, Johannesburg, South Africa) were hardened off by transplanting the plantiets into seedling trays filled with a sterilized sand/coir (1 :1 ) mixture. The tray was placed in a mist bed for two weeks until the plantiets gained sufficient strength to survive transplanting to pots. Individual plants were planted into 15 cm pots filled with steam pasteurized sandy loam soil amended with 0.15 g of super phosphate per 500 g soil. One ml of NH4N03 was applied per plantlet one day after transplant. One week after transplant, each plant was drenched with 10 mi PGPR broth. One day after the bacterial application, 3 g of the sand/bran pathogen inoculum was placed near the root zone of the plant approximately 1 cm below soil surface. Five replications of each treatment were placed in a completely randomized arrangement. The plants were watered as required until the haulms died. After the haulms had died the tubers were harvested. The tuber number and mass was recorded and each tuber rated for disease development (disease rating scale in Figure 13).
In the first greenhouse trial, tuber treatment with bacterial strains T 19, T29and A10, applied as a powder tuber treatment formulation, al! resulted in a significant reduction in disease severity compared to the control, with strain Paenibacillus barcinonensis A10 being the most effective (Figure 14).
When disease was measured according to disease index strains T29 and A10 showed a significant reduction in the disease compared to the control, with Paenibacillus barcinonensis A10 being the most effective (Figure 15).
In the second greenhouse trial all the bacterial strains again showed a significant reduction in disease severity when compared to the pathogen inoculated control treatment, with strain Paenibacillus barcinonensis strain A10 again being the most effective (Figure 16).
Similarly the disease index data (Figure 17) indicated significant disease suppression by al the bacterial strains, with strain A10 being the most effective. Biological control of black scurf of potatoes caused by R. solani with a mixture of selected rhizobacterial strains under field conditions
Field trials were planted at the experimental farm of the University of Pretoria. Mini field plots (9m x 2m) containing sandy loam soil were fumigated using formaldehyde at a concentration of 15 ml/ L water. Soil was amended with potassium chloride at a dosage of 460 kg/ha and with LAN (28% Nitrogen) at 785 kg/ha as recommended based on the soil analysis. This amounts to a total of 220:0:230kg N:P:K per hectare.
Planting was done one week after soil preparation. The plots were subdivided into sub-plots (1.8m x 2m) and each plot served as one treatment replicate. Five different treatments were planted in each plot. Tubers (cultivar "Up-to-Date") were planted approximately 20cm apart and 5cm deep.
The treatments were: 1 ) pathogen inoculated control 2) a mixture of rhizobacterial strains T19, T29 and A10 (treatment labelled "GM") applied as a drench over the tuber.
For the tuber/soil drench treatment, 10 ml of the bacterial inoculum was applied onto the tuber before covering with soil. The pathogen was inoculated at planting by placing 10ml of R. solani colonised vermiculite on each tuber. Approximately 3 weeks prior to harvest the soil was re-inoculated with R. solani by placing 5ml macerated mycelial suspension in each of four holes made 10 cm away from the base of each plant and equidistant from each other. The plants were watered with borehole water at regular intervals and allowed to grow to maturity. Near the end of the trial the plants were sprayed with Glyphosate (1 %) to speed up haulm death.
After the plants had died completely and the foliage was removed, the tubers were harvested by using a garden fork to dig them from the ground. For each treatment replicate the tubers were weighed and counted. Fifty 50 tubers were randomly selected from each treatment replicate, soil washed off and disease severity on each tuber assessed according to the disease rating scale (Figure 13).
The treatment labelled "GM" containing a combination of strains T19, T29 and A10 significantly reduced disease severity when applied to the tuber as a drench treatment at a rate of 10 ml/tuber, under field conditions (Figure 18).
According to the disease index parameter, the mixture of strains T19, T29 and A10 (treatment labelled GM) significantly reduced the disease when compared to the pathogen inoculated, untreated control (Figure 19). Statistical analysis
All trials in this example were set up according to a randomized block design to eliminate gradients such as sunlight and air circulation (in greenhouse pot trials). Results from each trial were subjected to an analysis of variance (ANOVA) using SAS 9.2 software. Duncan's Multiple Range Test at 5% (p=0.05) was applied. In cases where the data was not normally distributed the data was transformed using the Logarithmic function.
Discussion
The primary focus of this study was to select and evaluate rhizobacteria that are able to control black scurf disease of potato tubers caused by the fungal pathogen R. solani. The rhizobacterial strains were first screened under greenhouse conditions and subsequently evaluated in mini field plots. In the first greenhouse trial, tuber treatment with a powder formulation of strains T29 and A10 showed a significant reduction in disease severity, with strain A10 being the most effective, reducing disease severity by 50%. This finding was substantiated in a second (repeat) greenhouse trial.
Biocontroi efficacy of a combination (i.e. mixture) of the rhizobacterial strains Paenibacillus barcinonensis A10, Lysinibacillus sphaericus T19 and Paenibacillus alvei T29 was subsequently evaluated under field conditions. Treatment with the mixture of strains applied as a bacterial eel suspension (i.e. liquid) over the tubers effectively reduced black scurf disease severity of potatoes with 43.3 % and the disease index by 60.6 %.
These results demonstrate that the rhizobacterial strains Paenibacillus barcinonensis A10, Lysinibacillus sphaericus T19 and Paenibacillus alvei T29 either individually or in combination, are effective biocontroi agents of Rhizoctonia solani, the causa! fungus of black scurf and stem canker of potatoes.
EXAMPLE 17
Biological control of the root-knot nematode MeloidoQvne incognita race 2 on soybean (Glycine max (L.) Merrill) by means of plant growth-promoting rhizobacterial strains Lysinibacillus sphaencus T19; Paenibacillus alvei T22; and Pseudomonas fluorescens N04 Root-knot nematodes {Meloidogyne spp.) are some of the most devastating pests in agriculture and cause huge yield and economic loss to a variety of crops, including vegetables and soybeans (Kiewnick and Sikora, 2006). M, incognita is the most damaging root-knot nematode on soybean in warmer regions such as South Africa. Root-knot nematodes cause losses of between 9% and 100% on soybean in South Africa (Fourie, McDonald & De Waele, 2006).
Internationally chemical nematicides are being withdrawn from the market due to toxicity concerns, thereby precipitating a crisis in terms of nematode control measures. Biological control methods, including application of Plant Growth Promoting Rhizobacteria (PGPR) is therefore a viable alternative with which to fill this gap- Soybean seedling bioassay
The selected rhizobacterial strains were screened for control of Meloidogyne incognita using a modified soybean seedling bioassay based on that described by Padgham & Sikora (2007).
Silica sand (0.95 mm filter sand; Silica Quartz (Pty) Ltd) was mixed with medium grade vermiculite (Hygrotech SA (Pty) Ltd) in a ratio of 1 :1 by volume, transferred to autoc!aveable plastic bags and autoclaved at 121 °C for 1 hour. Plastic pots (425 mL) and saucers were sterilised overnight in 1% sodium hypochlorite. The plastic containers were then rinsed with autoclaved tap water.
Seeds of the South African soybean cultivar LS 6248R which is highly susceptible to root-knot nematodes (Fourie 2005), were surface sterilised by soaking in 3.5% sodium hypochlorite for 5 minutes and rinsed five times with autoclaved tap water.
Stationary phase rhizobacterium cultures were prepared by adding a loopful bacteria growing on nutrient agar to an Erlenmeyer flask containing 100 mL of autoclaved nutrient broth (Biolab, Merck (Pty) Ltd). The Erlenmeyer flasks were sealed with aluminium foil and the cultures incubated at 37 °C for 2 days with shaking (200 rpm). Before application, the bacteria cells were centrifuged from the broth culture at 6000g for 10 minutes. The supernatants were discarded and the bacterial pellets re-suspended in autoclaved quarter-strength Ringer's solution (Merck (Pty) Ltd) to produce the same volume as the stationary phase culture.
The plastic pots were placed on saucers and filled with sand-vermicuiite mixture up to 1cm from the top of the pots. A soybean cv. LS 6248 R seed was placed on the surface of the sand-vermiculite in the centre of each pot and 10 mL of bacterial suspension in Ringer's solution was pipetted onto each seed. Controls were treated with 10mL sterile quarter-strength Ringer's solution only. The pots were then filled with sand-vermiculite mixture to the rims of the pots. The pots were placed in a greenhouse maintained at an average ambient temperature of 25 °C. Each pot was watered daily with 60 mL autociaved tap water. At emergence of the first true leaves of the soybean seedlings {approximately 8 days after planting) each plant was inoculated with 2000 M. incognita juveniles.
M. incognita juveniles were obtained using the method of Fourie (2005). Tomato cv. Moneymaker plants infected with a M. incognita race 2 culture obtained from Prof. H. Fourie (North-West University, South Africa) were uprooted, the root systems washed with tap water and the roots cut into 1 cm-iong pieces. The root pieces were shaken by hand for 4 minutes in a 1% sodium hypochlorite solution. The resulting suspension was washed through a nested set of 20 cm-diameter stainless steel sieves (apertures of 75 pm, 25 pm and 10 pm respectively). The sieves were rinsed with sterilised tap water while the bottom sieve was connected to a vacuum pump. The material collected on the 10 pm sieve was gently washed onto the 25 pm sieve. The 25 pm sieve was piaced in a plastic container with the sieve surface submerged in sterilised tap water and the container incubated in the dark at 25 °C allowing for nematode egg hatching. The water was replaced after 1 day. Every day onward, the water was collected and washed onto a 10 pm sieve. The hatched nematode juveniles were then washed from the sieve into a conical flask. The concentration of M. incognita juveniles in the suspension was determined by counting 6 aliquots of 20 pi each on a counting grid / in a counting chamber under a stereomicroscope at 32X magnification.
Inoculation of the soybean seedlings was done by first exposing the uppermost lateral roots around each seedling by opening up the growth medium around the root system with a sterilised plastic rod. Three mL sterile tap water containing 2000 M. incognita juveniles were then pipetted onto the uppermost lateral roots of each seedling. The nematode juveniles were kept in suspension during counting and inoculation by means of a magnetic stirrer. After inoculation, the exposed roots of each seedling were covered up with growth medium.
The seedlings were assessed 17 days after nematode inoculation. Plants were removed from the growth medium and their root systems washed with tap water. The remaining vermiculite was removed with tweezers. The roots were blotted dry with a paper towel before determining fresh mass by weighing. After weighing, the root systems were kept in tap water while galls were counted. The number of root galls per plant was recorded with the aid of a magnifying glass (2X magnification). Root and shoots were excised and their fresh mass was determined by weighing.
All bacteria could not be assessed at the same time and were therefore assessed in groups. The experiments were laid out as completely randomised designs. Data was analysed using PROC GLM (Statistical Analysis System version 9.3, SAS Institute). The Dunnett test was used to determine whether the rhizobacteria! treatments had caused a significant reduction in nematode infection parameters, or a significant increase or decrease in plant growth parameters (a =0.05). The experiment was repeated. In some instances least squares means were used because of missing data points due to seeds that did not germinate.
Data from the first seedling bioassay experiment is presented in Table 15. Lysinibacillus sphaericus strain T19, Paenibacillus alvei strain T22 and Pseudomonas fluorescens strain N04 reduced the number of galis per plant by 31 %, 38% and 32%, respectively, and the number of galls per gram root by 29%, 44% and 19%, respectively. The difference between the least squares means of the rhizobacterium treatment and the nematode-oniy control was only significant in the case of strain T22 (for both galls per plant and galls per gram root). Strain T22 increased shoot mass by 46% compared to the nematode-oniy control, whiie other bacteria did not significantly affect shoot mass. None of the bacteria affected root mass significantly.
When the experiment was repeated, the number of nematode juveniles inoculated per plant was lowered to 1000 juveniles, which is sufficient to cause maximal M. incognita reproduction on cv. LS 6248 R, according to the results of Fourie (2005). The number of replicates per treatment was also increased to 8. in the repeat experiment, ail 3 bacterial strains caused significant reductions in nematode infection parameters (Table 16). Lysinibacillus sphaericus strain T19, Paenibacillus alvei T22 and Pseudomonas fluorescens strain N04 reduced the number of galls per plant by 67%, 32% and 44%, respectively, and the number of galls per gram root by 67%, 34% and 45%, respectively. Tabie 15: Results of first soybean seediing bioassay indicating the effect of rhizobacterial treatments on root knot nematode (Meloidogyne incognita) infection parameters (galling) and piant growth parameters (biomass). Data expressed as least squares means.
Shoot fresh mass GaMs/plant Galls/gram
N04 1.3 1.5 320 235
T19 1.6 1 .6 327 205
Nematode-onl Control 1.7 1.6 473 289
T22 2.3 2.4* 184* 83*
Nematode-only control 2.1 1.6 297 147
* The least squares mean differs significantly from that of the corresponding nematode-only control. The Dunnett test was used to determine whether the rhizobacterial treatments caused a significant reduction in nematode infection parameters, or a significant increase or decrease in plant root mass and shoot mass (a =0.05).
Table 16: Results of second soybean seediing bioassay indicating the effect of rhizobacterial treatments on root knot nematode (Meloidogyne incognita) infection parameters (galling) and plant growth parameters (biomass). Data expressed as least squares means.
AS hoot fresh m ass Galls/ptant Galls/gram mass (grams)
N04 1.4 1.4 55* 42*
T19 1.3 1.2 33* 25*
T22 1.4 1.4 66* 51 *
Nematode-only control 1.3 1.4 98 76
No nematode control 1.0 1.1 0* 0
* The ieast squares mean differs significantly from the corresponding nematode-only control. The Dunnett test was used to determine whether the rhizobacterial treatments caused a significant reduction in nematode infection parameters, or a significant increase or decrease in plant root mass and shoot mass (a =0.05).
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Claims

1. A biocontroi composition comprising of at least one isolated strain of bacteria selected from the group consisting of an isolated Bacillus megaterium strain (isolate A-07); an isolated Paenibacillus barcinonensis strain (isolate A-10); an isolated Pseudomonas fluorescens strain (isolate N-04), an isolated Bacillus cereus strain (isolate T-11 ), an isolated Lysinibacillus sphaericus strain (isolate T-19), an isolated Paenibacillus alvei strain (isolate T-22) and an isolated Paenibacillus alvei strain (isolate T-29) or any combination thereof.
2. The biocontroi composition of claim 1 , including an agricultural carrier.
3. A biocontroi composition according to claim 2, wherein the agricultural carrier comprises a wheat based carrier containing gluten, pe!letised compost, an inert carrier such as Perlite® , fertilizer, insecticide, fungicide, wetting agent, coating agent, abrading agents, clay, talc, polysaccharides, water, or mixtures thereof.
4. A biocontroi composition according to any one of claims 1 to 3 wherein each of the isolated strains is present in an amount ranging from about 1X10Z to about 1X109 CFU/ml of carrier.
5. A method of protecting plants against plant pathogens comprising applying to plants, plant tubers, plant seeds, or soil surrounding plants or plant cuttings, tubers or seeds, the biocontroi composition according to any one of claims 1 to 4, under conditions effective to protect said plants or the plants produced from said plant cuttings, tubers or seeds against said plant pathogens.
6. The method of claim 5 wherein the plant pathogen is a selected from the group consisting of a fungus, a bacterium or a plant parasitic nematode.
7. The method of claim 5 wherein the plant pathogen is selected from the group consisting of Phytophora spp. including P. capsici and P. nicotianae, Rhizoctonia spp, including R. soiani, Fusarium spp. including F. oxysporum , F. graminearum and F. pseudograminearum, Pythium spp. including P. ultimum, Verticillium spp. including V. dahliae, Alternaria spp. including A. alternata, Colletotrichum spp. including C. coccodes, Dickeya dadantii, Pectobacterium carotovorum carotovorum, Pectobacterium carotovorum brasiliensis Streptomyces scabies and Me!oidogyne incognita.
8. The method according to any one of claims 5 to 7, wherein the biocontrol composition is used to treat plants by topical application.
9. The method according to any one of claims 5 to 7, wherein the biocontrol composition is used to treat plant cuttings, seeds or tubers by topical appiication, said method further comprising growing plants from cuttings, seeds or tubers topically treated with the biocontrol agent.
10. The method according to any one of claims 5 to 7, wherein the biocontrol agent is used to treat soil around the plant's roots.
1 . A method of enhancing plant growth comprising applying to plants, plant cuttings, tubers, seeds, or soil surrounding the plants' roots or plant cuttings, tubers or seeds, a biocontrol composition according to claim 1 , under conditions effective to enhance growth in said plants or in the plants produced from said plant cuttings, tubers or seeds.
12. The method according to claim 11 , wherein the biocontrol composition is used to treat plant cuttings.tubers or seeds by topical application, said method further comprising growing plants from cuttings, tubers or seeds topically treated with the biocontrol composition.
13. The method according to claim 11 , wherein the biocontrol agent is used to treat soil around the plants' roots.
14. The isolated Bacillus megaterium strain of claim 1 having MTCC Accession No. 42317.
15. An isolated Paenibacillus barcinonensis strain of claim 1 having MTCC Accession No. 42318.
16. An isolated Pseudomonas fiuorescens strain of claim 1 having MTCC Accession No. 42319.
17. An isoiated Lysinibacillus sphaericus strain of claim 1 having MTCC Accession No. 42320.
18. An isolated Bacillus cereus strain of claim 1 having MTCC Accession No. 42321.
19. An isolated Paenibacillus alvei strain of claim 1 having MTCC Accession No. 42322.
20. An isolated Paenibacillus alvei strain of claim 1 having MTCC Accession No. 42323.
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