CN102191198B - Biocontrol strain JH21 for controlling rice sheath blight and rice blast and biocontrol bacterial agent thereof - Google Patents
Biocontrol strain JH21 for controlling rice sheath blight and rice blast and biocontrol bacterial agent thereof Download PDFInfo
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Abstract
The invention relates to a biocontrol strain JH21 for controlling rice sheath blight and rice blast, belonging to the technical field of plant protection. The bacteria are Pantoea agglomerans. The preparation method of the bacterial agent thereof comprises the following steps: using the bacteria to perform shaking cultivation in potato dextrose agar(PDA) culture solution at 30 DEG C in 180rpm for20-24 hours, then centrifuging in 6000rpm for 10 minutes, and using wet bacteria and the preservative fluid of the laboratory to prepare the bacterial agent according to a ratio of 1:40 (g/ml), wherein the total concentration of the live bacteria in the finished product is 3*10<9>-3*10<12>CFU/ml. The control rate of the JH21 bacterial agent to rice blast is about 65-75%, the yield can be increased by 40-50%; and the control rate to rice sheath blight is more than 60-70% and the yield can be increased by 40-60%. The JH21 bacterial agent can control rice sheath blight and rice blast simultaneously in one field; and the bacterial agent can obviously promote the growth of rice and increase the yield of rice. The bacterial agent can be used through soaking during seeding and after rice is transplanted, the bacterial agent is sprayed to control diseases.
Description
One, technical field
The present invention relates to a kind of biocontrol strain JH21 and biocontrol fungicide thereof of preventing and treating rice sheath blight disease and rice blast, belong to the farm crop technical field of preventing and curing diseases, be exclusively used in of the control of paddy rice main producing region rice blast and rice sheath blight disease.
Two, background technology
Paddy rice is one of China's staple food crop, and cultivated area accounts for 1/4 of national cultivated area at present, and YO is about 1/2 of national total output of grain.Yet the lasting generation of rice disease is having a strong impact on the production of paddy rice always.Prevent and treat under the condition existing, the whole nation reaches 20,000,000,000 kg because of various rice diseases cause the paddy underproduction every year on average, and as not preventing and treating, the underproduction can reach 30,000,000,000 kg.Therefore, it is very urgent to study and prevent and treat rice disease.In rice disease, rice blast and hypochnus generation area are big, and popular strong, harm is serious, and difficulty is bigger in the control, is topmost two big diseases in the Rice Production.
Rice blast is claimed rice blast again, the pest etc. of kowtowing, and is a kind of worldwide fungal disease, and global Rice Production is caused very big harm; Classified as first of the paddy rice three big diseases; Be rice seedling and heading stage most important disease, in all rice districts of China generation is arranged all, generally cause the underproduction 10%～20% in the popular time of disease; The time underproduction 40%～50% that disease is heavy, even No kernels or seeds are gathered, as in a year of scarcity.Since the 90's of 20th century, area takes place all at 3,800,000 hm in the year of China's rice blast
2More than, year loss paddy reaches several hundred million kilograms.Attending China in production mainly is that the applied chemistry agricultural chemicals is prevented and treated rice blast at present, generally when seedling stage, adopts chemical bactericide seed soaking control seedling pest, use foliar spray control leaf pest and panicle blast behind the transplanting Honda.Present stage produce to go up main use kasugamycin, tricyclazole or spray at seedling stage and heading stage with the composite composite bactericide of tricyclazole and sulphur, tricyclazole and iprobenfos prevent and treat rice blast.But rice blast receive kind, weather effect bigger; Occurrence degree, the prediction precision of emergence period to rice blast are not high; Applied chemistry pesticide control rice blast because of medicament itself does not have interior absorption, is difficult in and prevents and treats the optimum period medication; And, make preventive effect constantly descend because germ strengthens medicament commonly used is drug-fast gradually.The use at double of chemical agent in addition, farmland ecosystem is seriously damaged, and the rice pesticide residue that the results post-treatment becomes are serious, bring the very important serious danger side of body to health of people.
By rhizoctonia solani
(Rhizoctonia solani)The rice sheath blight disease that causes is one of rice fungus diseases evil of the refractory reason of paddy rice.Along with the popularizing planting of short-stalked variety and hybridisation rice and the raising of cultivation management level, hypochnus takes place serious day by day.Wherein, be injured the heaviest with the high-yield rice district especially.Hypochnus causes that mainly sheath is withered withered with leaf, and the paddy rice setting percentage is reduced, and rate of immatured grain increases, and grain heavily descends, and serious underproduction 40-50% takes place general underproduction 10-20%.And, do not find high anti-kind both at home and abroad so far as yet to this disease because the saprophytic ability of sheath blight fungus is strong and host range is wide; Main control medicament---jingganmycin has produced tangible resistance to this disease to domestic, and per season medication number of times is increased to present 4～7 times for 2 times before 20 years, dosage also from before 1.5kg/ hm
2Be increased to present 4.5～6.0kg/ hm
2Cause the problem that natural ecological environment pollutes and the rice pesticide residue increases also more and more outstanding because of preventing and treating the long-term a large amount of agricultural chemicals that use of hypochnus.
Chemical pesticide is to the havoc of farmland ecosystem, and pathogenic bacteria is more and more obvious to the resistance of medicament.Simultaneously, chemical pesticide is difficult for degraded, and high residue is absolutely unsafe to people and animals and has all seriously limited the use in agriculture prodn in the future.Therefore, property prophylactico-therapeutic measures has non-chemically become the core mode of preventing and treating rice disease at present, and biological control then is the most promising method wherein.In the prior art according to this breadboard preservative fluid for thallus patent (publication CN1358838, open day: on July 17th, 2002) method, but thalline room temperature preservation 2～3 years.
Three, summary of the invention
Technical problem:
The objective of the invention is to rice blast in the present agriculture prodn and rice sheath blight disease controlling difficulty big; Cost is high, and the chemical pesticide resistance becomes increasingly conspicuous, problems such as pesticide residue height; Screen efficient biocontrol strain and make biocontrol fungicide, rice blast and rice sheath blight disease are all had preventive effect.
Technical scheme
A kind of biocontrol strain JH21 that prevents and treats rice sheath blight disease and rice blast be pantoea agglomerans (
Pantoea agglomerans), on June 18th, 2009 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, and Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.3126.
The method that biocontrol strain JH21 is used to prepare biocontrol fungicide is:
1) this inoculation is cultivated 30 ℃ of 180rpm respectively in the PDA nutrient solution, in Bechtop, take a sample respectively after 16～20 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; To plant daughter bacteria liquid and add in the PDA nutrient solution, 30 ℃ of 180rpm shaking culture 20～24 hours, the centrifugal 10min of 6000rpm then with the 1:100 volume ratio; Every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline is got wet bacterium and preservative fluid for thallus and is made into microbial inoculum by 1 g:40ml, and the viable bacteria total concn is 3 * 10 in the finished product
9～3 * 10
12CFU/ml is used to prevent and treat rice blast and rice sheath blight disease.
Used PDA nutrient solution compound method is: be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze; Filtrating adds 20 gram common sucrose; Be settled to 1000 milliliters, the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then.Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
Beneficial effect
Advantage of the present invention and positively effect show: the present invention is the biological prevention and control agent that is directed against the exploitation of rice blast and rice sheath blight disease specially.Owing to be biotechnological formulation, there are not a series of problems that use brought fully because of chemical pesticide, thereby the ecosystem that can effectively protect the fields, help the nuisanceless production of rice, implement the Sustainable development of agricultural.This biocontrol fungicide preventive effect is good, and price is lower than chemical pesticide, can be the peasant and reduces expenses; This microbial inoculum also has the short raising the output function of giving birth to, and the high-quality pollution-free rice of producing can be the peasant and increases income.
Dull and stereotyped antagonistic effect shows that the JH21 preparation has the obvious suppression effect to rice blast pathogen and rice sheath blight disease pathogenic bacteria.Greenhouse test shows: the JH21 preparation can effectively be controlled Pyricularia oryzae and Rhizoctonia solani Kuhn to the infecting of paddy rice, and suppresses mycelial growth, reaches the effect that disease controlling takes place.Simultaneously, the JH21 preparation can promote the growth of rice plant and improve its resistance.Field experiment result shows: the JH21 microbial inoculum is to being about 65～70% to the rice blast preventive effect, and raising the output can reach 40～45%; To the rice sheath blight disease preventive effect is more than 60～70%, and raising the output can reach 40～60%.
This microbial inoculum (belongs to this laboratory granted patent with wet bacterium and this breadboard preservative fluid for thallus; Patent No. ZL02112559.7; Publication CN1358838, open day: on July 17th, 2002) be made into microbial inoculum by 1:40 (g/ml), but room temperature preservation 2～3 years.
This mix bacterium agent uses through spraying and prevents and treats disease, also can handle through seed soaking.
Four embodiments
A kind of from paddy rice rhizosphere soil the biocontrol strain JH21 that prevents and treats rice sheath blight disease and rice blast that obtains of screening, this bacterium be general Pseudomonas (
Pantoea agglomerans), 8h produces single bacterium colony on the LB substratum, and bacterium colony is yellow behind the cultivation 24h, and the quality homogeneous is circular to irregular, moistening, central uplift, smooth surface, somatic cells is a direct rod shape, G
-
The preparation method of JH21 microbial inoculum adopts conventional bacterial cultivation:
1) this inoculation is cultivated 30 ℃ of 180rpm respectively in the PDA nutrient solution, in Bechtop, take a sample respectively after 16～20 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when the OD value is not between 0.1～1, each bacterium is continued to cultivate; Every surveyed once at a distance from 3 hours, up to each OD value all in this scope. when being cultured to the OD value between 0.5～0.8 the time, will plant daughter bacteria liquid with in the 1:100 volume ratio adding PDA nutrient solution; 30 ℃ of 180rpm shaking culture 20～24 hours; The centrifugal 10min of 6000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline;
Get wet bacterium and preservative fluid for thallus and be made into the JH21 biocontrol fungicide by 1 g:40ml, the viable bacteria total concn is 3 * 10 in the finished product
9～3 * 10
12CFU/ml.Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
Used PDA nutrient solution compound method is: be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze; Filtrating adds 20 gram common sucrose; Be settled to 1000 milliliters, the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then.
(1) dull and stereotyped antagonism bacteriostatic test
Utilize dull and stereotyped face-off culture method, measure the restraining effect of JH21 bacterial strain Rhizoctonia solani Kuhn and Pyricularia oryzae.
(2) greenhouse test
1. seed treatment:
Rice paddy seed carries out surface sterilization to be handled: sterilized water is washed three times after handling 15 s with 75% alcohol, and sterilized water was washed three times after 5% chlorine bleach liquor handled 15min.It is 10 that the rice paddy seed of surface sterilization is soaked in concentration
8Behind the CFU/mlJH21 microbial inoculum 30min, sow seedling raising after the vernalization of preserving moisture.
2. inoculation of pathogenic bacteria and JH21 microbial inoculum uses
With plastics keg dress rice field soil, transplant 3 caves, 5 rice shoots in every cave for every barrel.Inoculation pathogenic bacteria when rice plant grows to tillering phase.With Rhizoctonia solani Kuhn (
Rhizoctonia solani) AG-1 (IA) for supply the examination pathogenic bacteria (Inagaki K. Dispersal of rice sheath blight fungus,
Rhizoctonia solaniAG-1 (IA), and subsequent disease development in paddy fields, from survey of vegetative compatibility groups [J].
Mycoscience1998 (39): 391-397.), adopt toothpick inoculation method (Yi Runhua, Zhu Xiru; Zhou Erxun. the research [J] of Rhizoctonia solani Kuhn artificial inoculation method. Guangzhou University's journal (natural science edition); 2003, (03)) inoculation: it is inboard to sign the leaf sheath that embeds plant through the wooden brachydont in the length 2 cm left and right sides that connects the bacterium cultivation, preserves moisture.
With Pyricularia oryzae (
Magnaporthe grisea) the 91-17-2 physiological strain is pathogenic bacteria (Mu Changqing, Liu Xue, Lu Qingguang; Jiang is carefully good; Zhu Changxiong. subtilis B-332 bacterial strain reaches the effect [J] of growing surely to the control effect of rice blast. plant protection journal, 2007, (02)); Inoculate seedling stage: after germ is induced the product spore, inoculate to the foliage-spray spore suspension.
When treating that the state of an illness appears in clear water control group paddy rice, begin to spray the JH21 microbial inoculum and handle.
3. statistics
Statistics JH21 microbial inoculum is to the preventive effect of rice sheath blight disease and rice blast, and to the growth promoting function of paddy rice.
(3) field test
Tongshan County and Anhui Province's Lujiang County carry out the field test of field control rice blast and rice sheath blight disease in Jiangsu Province in May, 08 to November to use the JH21 microbial inoculum, added up the JH21 microbial inoculum to the control effect of disease with to the short living effect of increasing production of paddy rice.
Experimental result
1. dull and stereotyped bacteriostatic test
According to " planting the disease organon " middle plateform face-off culture experiment method; JH21 is estimated the inhibition effect of Pyricularia oryzae and Rhizoctonia solani Kuhn; Statistics is following when treating that clear water control treatment group germ mycelia is covered with flat board; JH21 is 74.44% to the Pyricularia oryzae growth inhibition ratio, and the Rhizoctonia solani Kuhn growth inhibition ratio is 47.78% (shown in the table 1).
2. the sick greenhouse test of leaf blast in rice
Behind the inoculation magnaporthe grisea spore,, control group sprays the JH21 microbial inoculum when beginning to fall ill for the first time, after this every JH21 microbial inoculum that sprayed a time at a distance from 7-10 days.To rice ear sprouting period, investigation preventive effect and living weight.With 20% tricyclazole WP (Jiangsu bumper harvest farmingization ltd) is the contrast medicament.The JH21 microbial inoculum reaches 73.60% to the rice blast preventive effect in greenhouse test, be higher than the tricyclazole treatment group.Simultaneously, use the JH21 microbial inoculum can obviously promote the growth of rice plant, the living weight rate of increase is 40.83% (shown in the table 2).
3. rice sheath blight disease greenhouse test
Behind the inoculation Rhizoctonia solani Kuhn, spray the JH21 microbial inoculum when control group begins to fall ill for the first time, sprayed once in after this every separated 10-15 days.Paddy rice is investigated preventive effect and living weight to milk stage.With the contrast medicament is the very clear SC (Shanghai Nongle Biological Product Co., Ltd.) of 20% line.The JH21 microbial inoculum reaches 81.07% to the rice sheath blight disease preventive effect in greenhouse test, be higher than the agricultural chemicals control group.Simultaneously, the JH21 microbial inoculum can promote the growth of rice plant, and the living weight rate of increase is 62.41% (shown in the table 3).
4. rice blast is prevented and treated field test
Field test is chosen in rice blast and takes place in heavier Anhui Province's Lujiang County and the Jiangsu Province's Tongshan County rice terrace year after year.Establish 3 processing altogether: A:JH21 microbial inoculum 450ml converts water to 40L/667m
2B:20% tricyclazole (WP) 100g converts water to 40L/667m
2C: clear water contrast.Every processing is established 4 sub-districts and is repeated, and every sub-district area is 330m
2, each handles district's group arrangement at random.Isolate with guard rows between the sub-district, rich water quality management is carried out by routine in the experimental plot.Test is with reference to local plant protection unit state of an illness forecast medication, and the medication for the first time in early stage of tillering whenever combines the rice disease and pest general status control medication of each field at a distance from 15-17d afterwards.The paddy rice milk stage, discontinues medication and adds up the disease severity.Whole rice growing season is prevented and treated 4 times altogether.Application method is: seed soaking is handled before the sowing; Adopt hand sprayer carefully to be sprayed to paddy rice leaf table after transplanting Honda.In order to estimate the ability that the JH21 microbial inoculum promotes paddy growth, carried out living weight mensuration in earlier stage in paddy rice booting, add up output behind the rice harvesting.
Test-results shows; The JH21 microbial inoculum is all obtained effect preferably in the field test of two provinces two places control rice blast; The preventive effect of Lujiang County and the Jiangsu Tongshan County difference 61.99% and 64.53% in Anhui; All be higher than pesticide-treated group JH21 microbial inoculum and can promote the growth of rice plant and increase rice yield that units increased in production is respectively 48.13% and 36.26% (shown in the table 4 and 5).
5. rice sheath blight disease field test
Field test is chosen in rice blast and takes place in heavier Anhui Province's Lujiang County and the Jiangsu Province's Tongshan County rice terrace year after year.Establish 3 processing altogether: A:JH21 microbial inoculum 450ml converts water to 40L/667m
2The very clear SC 75ml of B:20% line converts water to 40L/667m
2C: clear water contrast.Every processing is established 4 sub-districts and is repeated, and every sub-district area is 330m
2, each handles district's group arrangement at random.Isolate with guard rows between the sub-district, rich water quality management is carried out by routine in the experimental plot.Test is with reference to local plant protection unit state of an illness forecast medication, and the medication for the first time in early stage of tillering is afterwards whenever at a distance from the each field of 15-17d rice disease and pest general status control medication.The paddy rice milk stage, discontinues medication and adds up the disease severity.Whole rice growing season is prevented and treated 4 times altogether, and application method adopts hand sprayer slightly to be sprayed to rice clump base portion.In order to estimate the promotes growth ability of JH21 microbial inoculum to paddy rice, living weight mensuration has been carried out in paddy rice booting in earlier stage.Add up output behind the rice harvesting.
Test-results shows that the JH21 microbial inoculum is all obtained effect preferably in two provinces two places field control rice sheath blight disease, and the preventive effect of Lujiang County and Jiangsu Tongshan County is respectively 67.81% and 71.57% in Anhui, all is higher than the pesticide-treated group.The JH21 microbial inoculum can promote the growth of rice plant and increase rice yield that units increased in production is respectively 59.20% and 56.57% (shown in the table 6 and 7).
Claims (2)
1. biocontrol strain JH21 who prevents and treats rice blast and rice sheath blight disease, for pantoea agglomerans (
Pantoea agglomerans), on June 18th, 2009 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, and Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.3126.
2. with the control rice blast of the described biocontrol strain JH21 preparation of claim 1 and the biocontrol fungicide of rice sheath blight disease.
3, prepare the method for the said biocontrol fungicide of claim 2, it is characterized in that:
1) the described inoculation of claim 1 30 ℃ of 180rpm in the PDA nutrient solution is cultivated after 16～20 hours in Bechtop the OD value that the 600nm place is surveyed in sampling respectively, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; To plant daughter bacteria liquid and add in the PDA nutrient solution, 30 ℃ of 180rpm shaking culture 20～24 hours, the centrifugal 10min of 6000rpm then with the 1:100 volume ratio; Every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline;
3) get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1 g:40ml, the viable bacteria total concn is 3 * 10 in the finished product
9～3 * 10
12CFU/ml, preservative fluid for thallus are the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
4, method according to claim 3 is characterized in that, used PDA nutrient solution compound method is:
Be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze, filtrating adds 20 gram common sucrose, is settled to 1000 milliliters, and the pH value transfers to 7.2～7.4, sterilizes 20 minutes for 121 ℃ then; Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
5, the application of the said biocontrol fungicide of claim 2 aspect control rice blast and rice sheath blight disease.
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| CN103952338B (en) * | 2014-04-11 | 2016-08-17 | 山西省农业科学院农产品贮藏保鲜研究所 | Pantoea agglomerans strain X M2 and the preparation method of bacteria suspension thereof and the prevention and controls to Pear black spot |
| CN104694422B (en) * | 2015-02-11 | 2018-04-27 | 齐齐哈尔大学 | Separate the general bacterium HYL01 bacterial strains of pineapple obtained and its application in watermelon root growth is promoted |
| CN108192850B (en) * | 2018-03-13 | 2021-03-19 | 长沙艾格里生物肥料技术开发有限公司 | Rhodococcus sulfidophilus strain, Rhodococcus sulfidophilus microbial inoculum and application thereof |
| CN110643551B (en) * | 2019-11-18 | 2022-03-15 | 吉林大学 | Stenotrophomonas rhizophila S11 for preventing and treating rice blast and application thereof |
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|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| CN1831116A (en) * | 2006-04-03 | 2006-09-13 | 西北农林科技大学 | One strain forming a block type fanjun fungus, fermentation culturing method and application thereof |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| CN1831116A (en) * | 2006-04-03 | 2006-09-13 | 西北农林科技大学 | One strain forming a block type fanjun fungus, fermentation culturing method and application thereof |
Non-Patent Citations (2)
| Title |
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| E. Costa等.Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products.《Appl Microbiol Biotechnol》.2001,第56卷第367-371页. * |
| 薛梦林等.拮抗菌B501的鉴定及其对采后冬枣黑斑病的抑制效果.《中国生物防治》.2008,第24卷(第2期),第122-127页. * |
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