CN102382776B - Small spore phoma microspora for controlling conyza sumatrensis - Google Patents
Small spore phoma microspora for controlling conyza sumatrensis Download PDFInfo
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Abstract
The invention discloses small spore phoma microspora SMBC022 with a biological activity for controlling conyza sumatrensis, which belongs to the field of biological control over agricultural weeds. The small spore phoma microspora is separated from a host in a natural environment and is identified to be small spore phoma microspora according to morphological characteristics and molecular biology; the optimal culture medium for growing the small spore phoma microspora is a PDA (potato dextrose agar); the optimal temperature is 28 DEG C; and the optimal pH is 7.2. Pathogenic crude toxins can be obtained from a fungal culture liquid through ethyl acetate extraction. The suspension liquid of the microspora and toxin solution thereof do not have adverse effect on 13 important crops such as rice; the SMBC022 strain preparation and the toxin solution have very strong pathogenic and weed-killing effects on malignant weed conyza sumatrensis, and can be produced into an environment-friendly fungal weedicide by a fermented bacterium or toxin biomimetic synthesizing technology. The small spore phoma microspora SMBC022 has important commercial developmental value and application value.
Description
Technical field
The present invention relates to the biological control technical field of agricultural weed; Particularly relate to and use the pathogenic micro-organism that agricultural weed is had very strong pathogenic effects and control effect as environmentally friendly campelyco, it is mould specifically to be that a strain is used to prevent and treat the little spore stem point of root of Sumatra Conyza.
Background technology
Root of Sumatra Conyza originates in South America, introduces China mid-term in 19th century, and root of Sumatra Conyza growth in recent years is rapid, and reproduction speed is fast; Produce a large amount of seeds, distribute extensively (particularly regional along the Yangtze River) is around their growths; Almost can't see the eciophyte growth, the place of particularly " breaking ground " is because original vegetation is damaged; This kind of plant is taken advantage of a weak point, and the growth of trying to be the first forms growth vigor; Not only cause a large amount of underproduction of land for growing field crops food crop, influence economic structure and social stability, and since its raised growth in the Yangtze valley one the band; Being easy to cause ecological security, causing serious consequence, is the typical grass of disliking; Present stage, domestic research to root of Sumatra Conyza was less, mainly concentrated on biology and ecological characteristics such as its morphological specificity, occurrence characteristic and harm and prevented and kill off aspect such as countermeasure, bred characteristic and aspects such as photosynthetic physiological characteristics and genetics and the research of root of Sumatra Conyza is focused mostly in phyletic evolution, the reproduction of yellow Chrysanthemum plant abroad.The measure that root of Sumatra Conyza is prevented and kill off by China mainly is that chemical herbicides such as Glyphosate 62 IPA Salt are pulled out and used in manual work; Artificial weeding is time-consuming and be difficult to reach the ideal effect; And too much use chemical herbicide can cause environmental pollution and residue problem (Lou Yuan comes etc., 2002).
Root of Sumatra Conyza in the generation area of China still in continuous expansion, endanger and the financial loss that causes more and more serious.Therefore; Seeking has pathogenic micro-organism pathogenic by force and the biological and ecological methods to prevent plant disease, pests, and erosion control action kou to this malignant weed; Further develop noresidue, campelyco free from environmental pollution thus; Can not only control causing harm of root of Sumatra Conyza safely and effectively,, and have the important commercial development and application values with the tremendous economic loss of avoiding causing thus.
Summary of the invention
The purpose of this invention is to provide a strain, to be used to prevent and treat the little spore stem point of root of Sumatra Conyza mould, and this fungal bacterial strain has very strong pathogenic effects and control effect to root of Sumatra Conyza.According to morphological feature and based on the molecular biotechnology of " internal transcribed spacer district " gene (ITS) with its be accredited as little spore stem point mould (
Phoma microspora); Confirmed its growth product spore and infected morbific optimum condition through the biological characteristics test determination; And it has been made up system affinity evolutionary tree, discover that it makes the dead pathogenesis of weeds plant morbidity through producing toxin, has analyzed host's specialization of germ and toxin thereof.
1. little spore stem is put mould SMBC22 strains separation purifying and screening: Various Seasonal is repeatedly gathered root of Sumatra Conyza natural occurrence plant in Chongqing; Adopt potato dextrose agar (PDA); Obtain dissimilar fungies through indoor tissue culture and purifying; Demonstrate,prove the disease method with Ke He Shi and confirm pathogenic bacterium wherein, relatively screen through pathogenic test at last and obtain thisly to kill careless pathogenic fungi (SMBC022 bacterial strain) what root of Sumatra Conyza had a very strong pathogenic effects.
2.SMBC022 the morphological specificity of pathogenic strains (Fig. 1) is identified: the strong pathogenic smbc022 that separation is obtained is inoculated on the PDA flat board and cultivates; Rounded or the subcircular of the bacterium colony that grows up to; Neat in edge, the growth of wheel line shape, later stage concentric wheel stripe protuberance is more obvious.The surface white mycelium, bacterium colony is thinner, and aerial hyphae is undeveloped, and is velvet-like, and the bacterium colony base portion is a white in 3 days, and the rear section was transformed into chocolate gradually in 3 days, and final base portion forms the alternate synthetic fibre line shape of grey black.The bacterium colony suitable growth temperature is 15-30 ℃, and optimum growth temperature is 28 ℃.Hyphae colorless, branch, tool is separated, wide 3.1-4.6 μ m, bacterium colony is producing spore about cultivation 12d under the dark 12h/12h of PDA substratum glazing.Pycnidium black, scattered, bury and give birth to or partly bury and be born in the substratum, ball-type or subsphaeroidal, diameter 72～140 μ m, the top is porose, and producing the spore mode be a bottle stalk formula.Conidium is oval or avette, colourless, unit cell, and the part middle part of cell has excessive contracting, and size is (4.5～5.8) μ m * (1.6～2.1) μ m, and 1-2 oily ball arranged.According to the categorizing system of Sutton (1980) and Boerema et al. (2004), be that little spore stem point is mould with this pathogenic bacteria preliminary evaluation with reference to relevant data.
3.SMBC022 the pathogenic strains molecular engineering is identified: through chloroform/primary isoamyl alcohol method extracting obtain SMBC022 because of group DNA; The sample gene group dna solution that is extracted diluted respectively as template carry out pcr amplification, pcr amplification adopts rDNA ITS section universal primer ITS4 and ITS5.ITS4:5 '-CCTCCGCTTATTGATATGC-3 '; ITS5:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' after purified recovery of PCR product and order-checking, obtains the rDNA ITS section gene order of this fungi; Its length is 524bp, is HQ645974 in the accession number of Genbank.The bacterial strain sequence that obtains is landed the NCBI website; Compare through BLAST (Basic Local Alignment Search Tool); Comparison result shows that listed DQ474092 sequence similarity is 99 % among smbc-022ITS sector sequence and the NCBI GenBank, and has uniqueness.Remove 18s and 28s the ITS sequence of choosing the higher similar kind of homology among pathogenic bacteria SMBC022 and the Genbank is accurately compared with analysis software, use sequence analysis software to adopt the Neighbor-joining method in addition institute's calling sequence and close sequence construct systematic evolution tree.Identify finally that in conjunction with the morphological feature of pathogenic bacteria this bacterium is that little spore stem point is mould.Its latin name is:
Phoma microspora, it belongs to mycota
Mycota, the imperfect fungi door
Deuteromycota, chamber born of the same parents' guiding principle
Coelomycetes, Sphaeropsidales
SphaeropsidalesIn Phoma
Phoma, Chinese is that little spore stem point is mould.
The live body pure culture of this bacterial classification has been preserved in ' China Committee for Culture Collection of Microorganisms common micro-organisms center ', preserving number: CGMCC No. 4417 on December 08th, 2010.
4. the mould the best of root of Sumatra Conyza stem point is cultivated and the inoculation infection condition: this fungal colony is grown and produced conidial optimum medium is potato dextrose agar (PDA); 28 ℃ of optimum temperutures; Optimum pH is pH7.2; Under these conditions, colony growth is fast, produces pycnidium and a large amount of conidiums; Illumination is produced spore to fungal growth does not have obvious influence.The morbific top condition of infection process is that inoculum density is 10
5Individual/the mL order of magnitude or higher, preserve moisture after the inoculation and half-light 24h, and keep 23～28 ℃ of temperature in the whole phase of infecting.
5. the mould pathogenic and specialization of root of Sumatra Conyza stem point: root of Sumatra Conyza stem point is mould to have good pathogenic effects to root of Sumatra Conyza.Mainly infect plant leaf and stem stalk.Behind the direct spray inoculation of the conidial suspension of indoor germ, can fall ill in 3 days, sick plant rate reaches 100%, blade dehydration blackening; It is withered to inoculate 7 days rear blades, and its control effect and Glyphosate 62 IPA Salt to root of Sumatra Conyza is suitable.Inoculation back effect manifests delay slightly in the field, the whole yellows in plant top in the 5th day, and it is dead to wilt after 2 weeks.
Use high density conidial suspension (10 under optimum conditions
8Individual/mL) inoculation test result shows; This fungi is all not pathogenic at interior 13 kinds of water of 6 sections, Dry crop to important crops such as paddy rice, wheat, corn, pea, rape, capsicum, tomato, cottons; Do not influence their seed germination and plant strain growth yet; This explanation root of Sumatra Conyza stem point is mould to be the obligate pathogenic bacterium of root of Sumatra Conyza, if be used to develop the root of Sumatra Conyza weedicide, can guarantee the safety of other plant in the habitat.
6. the thick toxin of root of Sumatra Conyza:, obtain the thick toxin that causes a disease through ethyl acetate extraction, rotatory evaporator vacuum-evaporation with the nutrient solution of fungi.Flood respectively behind root of Sumatra Conyza blade and the stem stalk with the water liquid of this toxin and promptly to show signs of toxicity in the 24h, the 72h intra vane becomes to flood the shape blackening.Do not observe signs of toxicity handle seed and the plant of aforementioned 13 kind of plant with this thick toxin soiutions after, explain that this toxin also is highly specialized.
Advantage of the present invention is:
Separating the little spore stem that obtains from the root of Sumatra Conyza of self-sow and put mould SMBC022 bacterial strain, is a new bacterial strain of finding first and identifying, its conidium inoculum is to the important exotic invasive weed of China---root of Sumatra Conyza (
C. sumatrensis) having very strong pathogenic control action kou, the toxin that it produces also has the deadly effect of very strong toxicity to this weeds.Microbial inoculum of germ and toxin all do not have detrimentally affect to other plant in important crop and the environment; Show to the height of root of Sumatra Conyza and specially change removing activity; Can in biological control practice, have potential business development and using value through the environment-friendly type fungoid weedicide of fermentation thalline or bionical synthetic technology production noresidue to root of Sumatra Conyza.
Description of drawings
The little spore stem of Fig. 1 is put the morphological specificity of mould bacterium colony: A is a bacterium colony; B is a mycelia; C is a pycnidium; D is a conidium
The little spore stem point of Fig. 2 mould to the outdoor inoculation SMBC022 of the pathogenic effect of the indoor and outdoors of root of Sumatra Conyza: A after root of Sumatra Conyza leaf incidence map (a1=1d, a2=3d, a3=7d); B. the incidence map of root of Sumatra Conyza leaf behind the indoor inoculation smbc022 (b1=1d, b2=3d, b3=8d); C. indoor in vitro inoculation and CK map (c1=1d, c2=3d, c3=6d)
The little spore stem of Fig. 3 is ordered the pathogenic effect of mycotoxin to root of Sumatra Conyza: A and B are contrast, and to be the blade B. that handles of sterilized water be the blade with the thick toxin dilution processing of ethyl acetate extraction to A
The little spore stem point of Fig. 4 mould (
P.microspora) phylogenetic tree set up according to homology analysis
The internal transcribed spacer gene ITS sequence of the little spore stem point of Fig. 5 mould.
Embodiment
Below in conjunction with embodiment the present invention is further described
Mould separation and purification and the screening of embodiment 1. little spore stem points
1.1 the fungi separation and Culture is with potato sucrose nutrient agar (PDA), its prescription is the fresh potato 200g of peeling, and each 20g of glucose and agar adds tap water to 1000mL.It is well-done with yam to add water earlier, and double gauze filters the back and adds sucrose and agar, replenishes an amount of water to 1000mL, in the triangular flask of packing into after fully mixing evenly, puts into sterilizer sterilization back and places.Heating and melting before using is poured in sterilization petridish or the test tube and is processed flat board or slant medium, supplies fungi separation and Culture or bacterial classification to preserve and uses.
1.2 separation and Culture and purifying: under natural condition, gather root of Sumatra Conyza plant sample with typical disease symptom; The clean back of rinsing clip leaf spot lesion and healthy part are had a common boundary organizes little (2 mm); Put into 5% chlorine bleach liquor's surface sterilization and use the aqua sterilisa rinsing later on 3 times; Place the PDA flat board, 25
oCultivate in the C incubator and observe every day, treat that different bacterium colonies grow after, transfer to and continue to cultivate into pure bacterium colony on the new PDA flat board, transfer to again on the PDA test tube slant substratum, and numbering indicates bacterial strain, preserve subsequent use.
1.3 strong pathogenic bacterium screening: confirm pathogenic bacteria according to the sick rule of Ke He Shi card step.Smear or the inoculation of acupuncture drop for healthy root of Sumatra Conyza plant leaf with obtaining various fungies after the separation and purification of sick leaf texture, guarantee the lucifuge 24h that preserves moisture, 28
oC is growth down, observes incidence.Cause the morbidity of inoculation blade, the fungi of performance same symptoms can be confirmed as pathogenic bacteria.Above-mentioned test is confirmed that all pathogenic bacterias inoculate to the root of Sumatra Conyza plant with identical method (smearing mist or acupuncture drop); And identical and cultivate to observe the severity of inoculation plant blade disease under the suitable condition; Relatively filter out and cause the serious bacterial strain of root of Sumatra Conyza morbidity; Thus, screening has obtained root of Sumatra Conyza stem point mould among the present invention.
1.4 the field of strong pathogenic strains SMBC022 control effect: mainly grow (not the spraying any agricultural chemicals, root of Sumatra Conyza growing way basically identical) of root of Sumatra Conyza plant of field experiment mountain a slice behind the Southwestern University South Area experimental field carried out.With random division experimental field is different sub-districts, and root of Sumatra Conyza 10 strains are chosen in each sub-district.The smbc022 for preparing being loaded on respectively in the hand sprayer with other pathogenic bacterium bacterium colony spore suspensions, being sprayed on the plant leaf surface, is controlled trial with the sterilized water, three sub-districts of each bacterial strain and control treatment, quantitative check root of Sumatra Conyza plant extent of injury.Bacterium colony sprays back temperature and continues do not have rainfall at 25-35 ℃ in the week.From spraying bacterium colony day, investigate once every day, and each sub-district is arbitrarily chosen 5 strains Soviet Union door and understood the wine grass, writes down every strain root of Sumatra Conyza plant extent of injury, calculates control effect, continuous observation 10 d according to state of an illness grade scale.
1.5 the evaluation of pathogenic bacteria: (1) morphological observation and evaluation. on the PDA flat board, cultivate pathogenic bacteria, observe colonial morphology, pycnidium and conidial microscopic features in good time; (2) molecular biology identification. according to the technology that Geiser sets up, extract the genomic dna of bacterial strain, with fungal transcriptional transcribed spacer universal primer ITS4:5 '-CCTCCGCTTATTGATATGC-3 '; ITS5:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' does the transcribed spacer gene order that pcr amplification obtains this fungi, checks order, and sequence is signed in among the Genbank; This sequence is Blast analyzes, the highest sequence title of contrast similarity.Use sequence analysis software Mega4.1 in addition, the Clustal supervisor adopts the Neighbor-joining method to do confidence level analysis (repeating 1000 times) with institute's calling sequence and close sequence construct systematic evolution tree and with bootstrap (bootstrap).Confirm the classification position of this germ.
Cultivation of embodiment 2. little spore stem point moulds and inoculation condition test
2.1 humid test: on potato dextrose agar (PDA) culture medium flat plate of cultivating 5d, use the mycelia piece of punch tool cut-off footpath as the SMBC22 bacterial strain of 0.5cm; Be inoculated on the PDA substratum; Establish 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ of 8 Temperature Treatment respectively, each handles repetition 3 times.Place 7 pre-set temperature required illumination boxs to cultivate, and measure the bacterium colony size day by day with vertical cross method, every 24h measures 1 time, continuously measured 5 times, record data.The potential of hydrogen of PDA substratum is about pH7.0.Confirm the mould optimum growth temperature of little spore stem point thus.
2.2 the potential of hydrogen experiment: the PDA substratum is adopted in experiment, 11 potential of hydrogen is set altogether handles.With the NaOH of 1.0 mol/L and HCl solution the pH value of substratum is adjusted to 4.0,5.0,6.0,6.5,7.0,7.5,8.0,9.0,10.0,11.0 and 12.0 (measuring with the electronics pH meter) respectively respectively, each handles repetition 5 times; Germ inoculation and growth measuring method are with 2.1.Connect the bacterium processing and be placed on 25 ℃ of cultivations down, every 24h measures a colony diameter, continuously measured 6 times.Confirm the optimal ph of little spore stem point mould-growth thus.
2.3 the screening of kinds of culture medium: 6 kinds of influences that substratum is grown to little spore stem point mold colony have been compared in experiment altogether; Except potato glucose substratum (PDA), other five kinds of substratum are respectively: 1) potato sucrose nutrient agar (PSA): water 1000mL, yam 200g, glucose 20g and agar powder 20g; 2) clear water agar medium (WA): water 1000 mL and agar powder 20g; 3) Radix Dauci Sativae substratum (CAA): water 1000mL, Radix Dauci Sativae 200g, agar powder 20g; 4) the root of Sumatra Conyza rhizome is fried in shallow oil juice substratum (ARS): water 1000 mL, root of Sumatra Conyza rhizome 200g and agar powder 20g; 5) the root of Sumatra Conyza blade is fried in shallow oil juice substratum (ALA): water 1000 mL, root of Sumatra Conyza blade 200g and agar powder 20g.When preparation PDA, CAA, ARS and ALA; Yam, Radix Dauci Sativae, weeds rhizome or blade with aequum shreds and puts into water well-done (boiling approximately 30 minutes) earlier; And after-filtration is removed residue and be settled to 1000 mL; The last chemical substance composition that adds aequum again stirs, and contains in the triangular flask subsequent use behind the moist heat sterilization respectively.The pH value of various substratum is about 6.8-7.0,25 ℃ of culture temperature.Confirm the optimal medium that germ is cultivated thus.
2.4 the influence of exposure experiments to light: uses the punch tool cut-off directly as the mycelia piece of the SMBC022 bacterial strain of 0.5cm on the PDA flat board of 5d cultivating, be inoculated on the PDA substratum, the illumination gradient is set under 1500Lux, shine respectively 0,6,12,18 and 24h.Each illumination gradient is done 5 repetitions, measures the bacterium colony size day by day with vertical cross method, and every 24h measures 1 time, continuously measured 5 times.Analyze the upgrowth situation under different light of pathogenic bacterium thus.
The thick toxin of embodiment 3. pathogenic bacterias separates
3.1 the preparation of germ culturing filtrate: in the 500mL triangular flask, pour 250 mL potato glucose (PD) nutrient solutions respectively into, the sterilization back is for use.Choose and cultivate the bacterium colony that 6-7d grows fine, get the bacterium cake of Φ 6 mm size, be inoculated in the triangular flask that 250mL PS nutrient solution is housed, 8 of every bottle graft kinds, static cultivation 15 days under 12h illumination/sky condition in 25 ℃ of incubators with punch tool.On Bechtop, filter with qualitative filter paper (middling speed), can obtain culturing filtrate.Prepare the usefulness that enough germ culturing filtrates supply the toxin separation and Extraction thus.
3.2 the extraction of toxin and activity test: organic solvent extraction: culturing filtrate is extracted with ETHYLE ACETATE; The culturing filtrate of each volume is with isopyknic ethyl acetate extraction 3 times; Each ethyl acetate with 1/3 volume; Merge organic phase then,, can obtain enriched material behind the recovery organic solvent through rotary evaporation.
Ethyl acetate extraction is obtained thick toxin to be done leaves of hyacinth sheet dip treating is measured its activity with using after the water dissolution respectively.Extract all shows very strong toxicity to the root of Sumatra Conyza blade as a result.
Safety (specialization) the property mensuration of embodiment 4. pathogenic bacterias and toxin thereof
4.1 supply the crop of test: 13 kinds of important floods and droughts crops having selected 6 sections; Comprise paddy rice gramineous, wheat, corn; The tomato of Solanaceae, capsicum and tobacco, cucumber cucurbitaceous and watermelon, sponge gourd, wax gourd, the radish of Cruciferae, wild cabbage, rape and Chinese cabbage; The peanut of pulse family, pea, broad bean, soybean and mung bean, and the cotton of Malvaceae.
4.2 seed germination test: the healthy seed of selecting aforementioned 13 kinds of crops; (the small-sized seed treatment time is short to use mycelia and spore suspension, the thick toxin soiutions of germ and clear water (contrast) to soak 12～36h respectively; The large seed treatment time is long), be placed on the wet filter paper, 25
oSprout record seed germination rate in back in the C growth.The result shows that the crop seed germination rate of being tested does not all have significant difference with contrast.
4.3 crop plant growth test:, be placed on the wet filter paper, 25 with warm water soaking 12～36h (deciding) according to seed size
oVernalization in the C growth.Treat to be seeded in the potted plant alms bowl after plumule exposes.Add sandy soil in the alms bowl, add a certain amount of nutritive medium (adding amount of urea, ammonium sulfate and potassiumphosphate in the water) then through washing and hyperthermia drying.Growth under greenhouse experiment is after planting treated that plant is long when high, to use mycelia and spore suspension, the thick toxin soiutions of germ and clear water (contrast) spraying to handle to 20 centimetres respectively, serves as to fall ill to contrast to handle root of Sumatra Conyza simultaneously.Handle the pathology situation of back each kind of plant of observed and recorded every day, results plant during to 15d, the increment of mensuration different treatment.The result shows; Handle the back root of Sumatra Conyza with little spore stem point mould bacterium liquid and toxin solution and after processing, promptly showed tangible disease and toxin symptom respectively in the 3rd day and the 2nd day; And all 13 kinds of crop plants also have no pathology after handling 15 days, do not have significant difference between growth increment after handling with bacterium liquid and toxin solution and the increment of clear water adjoining tree.This explanation germ and toxin thereof do not have any detrimentally affect to supplying to study thing.
Claims (1)
- One strain be used to prevent and treat the little spore stem point of root of Sumatra Conyza mould ( Phoma microspora), be the root of Sumatra Conyza of from physical environment, growing ( Conyza sumatrensis) on the incidence of leaf separation and purification obtain, the toxin that its conidium and thalline extract all has very strong pathogenic effects and control effect to root of Sumatra Conyza, it is characterized in that bacterial strain is SMBC022, the preserving number of this bacterial strain is CGMCC NO.4417.
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