CN115044493B - Nematode biocontrol bacterial strain and application thereof in preventing and controlling tobacco root knot nematode disease - Google Patents
Nematode biocontrol bacterial strain and application thereof in preventing and controlling tobacco root knot nematode disease Download PDFInfo
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Abstract
The invention discloses a nematode biocontrol bacterial strain and application thereof in preventing and treating tobacco root knot nematode disease, belonging to the technical field of microorganisms. The biocontrol bacteria are nocardia clock (Nocardioides stalactiti) YC3001 strain, and the YC3001 strain is preserved in China center for type culture collection (China) for 12 months and 17 days in 2021; deposit unit address: eight ways of universities of Wuhan in the mountain area of mountain in Wuhan City of Hubei province; deposit number: cctccc: m20211636. The microbial thallus and the metabolite thereof can be mixed with a conventional pesticide adsorption carrier and an auxiliary agent to prepare the YC3001 microbial inoculum. Application of YC3001 in preparation of microbial inoculum for preventing and treating tobacco root knot nematode disease.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a nematode biocontrol bacterial strain and application thereof in preventing and controlling tobacco root knot nematode disease.
Background
Root knot nematodeMeloidogynespp.) are important pathogens for a variety of crops, an economy that is created annually worldwideLosses are up to 1570 billion dollars (Abad et al, 2008). Tobacco root knot nematode disease is a worldwide tobacco disease, according to the statistics of united nations grain and agricultural organization (FAO), the direct economic loss of tobacco caused by nematode disease in the world reaches 4 hundred million dollars each year, except northeast tobacco areas in China, all large tobacco areas commonly occur, according to the statistics of national tobacco pest measurement and report central stations, only the occurrence area of the national tobacco root knot nematode disease in 2015 is about 310 ten thousand mu, and the yield loss is nearly 15 hundred million yuan; the root knot nematode disease land in main-production core tobacco areas of Pitch, southwest of Guizhou and the like is endangered throughout the year to reduce the yield of tobacco leaves by about 30 percent, and partial plots in serious disaster year are dead-harvested; the annual incidence area of Yunnan tobacco region is more than 2 ten thousand hm 2 The annual yield reduction of the tobacco leaves in the ill plots reaches more than 30%; in the root-knot nematode disease disaster area of the cold mountain and the branch climbing flowers of Sichuan, the incidence rate exceeds 70 percent, the annual tobacco loss rate reaches 10 to 20 percent, and the sustainable development of flue-cured tobacco is seriously influenced (Zhang Zongjin and the like, 2019).
Tobacco root knot nematode disease is one of typical diseases which are difficult to prevent and treat, and is mainly represented by: (1) Root-knot nematode disease is classified as rhizome disease, but the pathogen is animals, and the pathogenic catastrophe mechanism, host interaction mechanism and the like of the root-knot nematode disease are more complex than bacterial, fungal and viral diseases; (2) multiple pathogenic species, wide distribution and strong pathogenicity; (3) The conventional agricultural prevention and control measures are difficult to take effect, the root-knot nematodes have no host with obvious specialization, can infect almost all cultivated plants (more than 3000 plants in 114 families), and most plants in the farmland ecological system can be parasitized, so that the nematodes are very beneficial to avoiding complex environmental changes and agronomic activity interference; (4) The application of disease-resistant varieties is limited, and the resistance of the current main cultivated varieties only aims at a certain physiological race of a certain root-knot nematode and does not have the resistance to the mixed population of the tobacco root-knot nematode; (5) The high-efficiency low-toxicity chemical wire-killing agent has the advantages of rare variety, high cost, outstanding resistance/drug tolerance, ecological safety and other problems, and the chemical prevention and treatment prospect is not optimistic (Ristaino & Thomas, 1998). In contrast, biological wire killers developed by utilizing natural enemy microorganisms of nematodes and natural products of plant sources are expected to be more practical and operable due to the safety of people and livestock and environmental friendliness of the biological wire killers.
Aiming at the root knot nematode disease of crops, the prior utilizationBacillus firmus、B. amyloliquefaciens、 Pasteuria penetrans、Burkholderia cepaciaIsotogenic bacteriaPurpureocillium lilacinum、Pochonia chlamydosporiaBiological nematicides developed by isophytol fungi have been registered at home and abroad and used for preventing and treating root knot nematode disease (Davies& Spiegel，2011）。
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a novel green, efficient and novel nematode biocontrol bacterial strain and application thereof in preventing and controlling tobacco root knot nematode disease.
In order to achieve the above purpose, the present invention adopts the following technical scheme: the invention relates to a nematode biocontrol bacterial strain, which is opal nocardia clock（Nocardioides stalactiti）YC3001 strain with the accession name of nocardia rubra（Nocardioides stalactiti）YC3001 strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation unit address is the university of Wuhan in the mountain area of Wuhan, hubei province, and the preservation date is: 2021, 12, 17; preservation number: cctccc: m20211636.
Further, YC3001 strain is a gram positive aerobic bacterium, and is shown in R 2 Culturing on the culture medium A at 30deg.C for 3 days, wherein the colony is white, round, convex or smooth at the center, and has a diameter of 1.5-3.5 mm; round bar shape with width of 0.55-0.65 μm and length of 0.85-2.2 μm.
Further, said R 2 Medium a: yeast extract powder 0.5 g, peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, potassium dihydrogen phosphate 0.3 g, anhydrous magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, agar 15 g, and sterilized water to 1000 ml with pH 7.2.
Further, the strain grows in a temperature range of 10-40 ℃ and an optimal growth temperature of 30 ℃ and can grow in a pH range of 6.0-9.0.
Further, the strain has an optimum growth pH of 7.0 at R 2 A cultureThe highest concentration of NaCl-tolerant on the medium was 4.5% (W/V).
The invention relates to a nematode biocontrol bacterial agent prepared from a nematode biocontrol bacterial strain.
Further, the active ingredients are at least one of the following (a), (b) and (c):
(a) A fermentation culture of said nematode biocontrol bacteria;
(b) The obtained spore suspension of the nematode biocontrol bacteria;
(c) Ultrasonic lysis precipitation of the obtained nematode biocontrol bacterial cells.
The invention relates to opal nocardia clock（Nocardioides stalactiti）Application of YC3001 strain in preparation of microbial inoculum for preventing and treating tobacco root knot nematode disease.
The beneficial effects are that: the microbial inoculum prepared by the YC3001 can effectively prevent and treat the tobacco root knot nematode disease, and has the characteristics of low use cost, high prevention effect, no residue and safety to people and livestock.
Compared with the prior art, the invention has the following advantages: (1) The microbial inoculum prepared by the YC3001 strain has the effect of preventing and controlling the root knot nematode disease of crops up to more than 68 percent.
(2) The microbial inoculum prepared by the YC3001 strain can effectively prevent and treat tobacco root knot nematode disease, and has the characteristics of low use cost, no residue and safety to human and livestock.
(3) In the research of recently screening root-knot nematode biocontrol bacteria, the invention separates a bacterium with extremely strong lethal activity to tobacco root-knot nematode second-instar larva (J2) from the surface of limestone in Guizhou farmland, the strain number is YC3001, and the strain is identified as Nocardia of stalactites @Nocardioides stalactiti). Nocardia of the stalactites class @ andNocardioides stalactiti) Is a new species of bacteria reported by Zhou et al in 2021 (Zhou et al, 2021), and the biological function of the bacteria has not been reported so far.
Description of the embodiments
The present invention will be described in further detail with reference to examples. The microbial agent in the embodiment is prepared according to a conventional microbial fermentation method and a conventional microbial agent preparation method.
Examples
The invention relates to a nematode biocontrol bacterial strain, which is opal nocardia clock（Nocardioides stalactiti）YC3001 strain with the accession name of nocardia rubra（Nocardioides stalactiti）YC3001 strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation unit address is the university of Wuhan in the mountain area of Wuhan, hubei province, and the preservation date is: 2021, 12, 17; preservation number: cctccc: m20211636.
The YC3001 strain is a gram positive aerobic bacterium, and is shown in R 2 Culturing on the culture medium A at 30deg.C for 3 days, wherein the colony is white, round, convex or smooth at the center, and has a diameter of 3.5 mm; round bar shape of the cells, 0.55 μm wide and 1 μm long.
Said R is 2 Medium a: yeast extract powder 0.5 g, peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, potassium dihydrogen phosphate 0.3 g, anhydrous magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, agar 15 g, and sterilized water to 1000 ml with pH 7.2.
The strain grows in a temperature range of 10 ℃ and an optimal growth temperature of 30 ℃ and can grow in a pH range of 8.0.
At R 2 The highest concentration of NaCl-tolerant on the A medium was 4.5% (W/V).
The nematode of the invention further provides a nematode biocontrol bacterial agent prepared by the biocontrol bacterial strain.
The active ingredients are at least one of the following (a), (b) and (c):
(a) A fermentation culture of said nematode biocontrol bacteria;
(b) The obtained spore suspension of the nematode biocontrol bacteria;
(c) Ultrasonic lysis precipitation of the obtained nematode biocontrol bacterial cells.
The invention relates to application of nocardia rubra (Nocardioides stalactiti) YC3001 strain in preparation of a microbial inoculum for preventing and treating tobacco root knot nematode disease.
Examples
Example 2 differs from example 1 in that: the YC3001 strain is a gram positive aerobic bacterium, and is shown in R 2 Culturing on the culture medium A at 30deg.C for 3 days, wherein the colony is white, round, convex or smooth at the center, and has a diameter of 2.5 mm; round bar shape of the cells, 0.6 μm wide and 2.2 μm long. The strain grows in a temperature range of 30 ℃ and an optimal growth temperature of 30 ℃ and can grow in a pH range of 7.0.
Examples
Example 3 differs from example 1 in that: the YC3001 strain is a gram positive aerobic bacterium, and is shown in R 2 Culturing on the culture medium A at 30deg.C for 3 days, wherein the colony is white, round, convex or smooth at the center, and has a diameter of 1.5 mm; round bar shape of the cells, 0.55 μm wide and 0.85 μm long. The strain grows in a temperature range of 40 ℃ and an optimal growth temperature of 30 ℃ and can grow in a pH range of 6.0.
Slant seed culture of YC3001 strain test tube: inoculating the strain into R 2 And A, culturing on the slant of the culture medium at 30 ℃ for 3 days to obtain slant seeds.
Liquid seed culture of YC3001 strain: inoculating the slant seed to the seed containing R 2 A liquid Medium (R) 2 Agar was not added to the medium A), and liquid seeds were obtained by shaking culture at 30℃and 180 rpm for 3 days.
YC3001 strain fermentor mass culture: the liquid seeds are added into beef extract peptone liquid culture medium in a fermentation tank according to the proportion of 5% (V/V), and the culture condition in the fermentation tank of 1000 liters is controlled as follows: the temperature was 30℃and the stirring speed was 160 rpm, and the fermentation time was 56 hours.
Preparation of YC3001 strain microbial inoculum: the microbial thallus and its metabolite obtained by fermentation tank culture are mixed with proper amount of diatomite, and then dried or dried at 65 deg.C until the water content is less than 5%, and then crushed. The viable count content of YC3001 strain in the microbial inoculum is ensured to be 5 multiplied by 10 9 More than one per gram.
Collecting root system of tobacco plant infected with root knot nematode, picking ovum from root nodule, placing in culture medium with gauze at 25deg.CAnd 7 days after water bath, collecting the hatched J2, and centrifugally concentrating to obtain J2 suspension. Slant seed of YC3001 strain was inoculated into a strain containing R 2 A, shake culturing in a triangular flask of the liquid culture medium at 30 ℃ and 180 rpm for 3 days to obtain fermentation liquor. 2 ml of the fermentation broth was placed in a petri dish, 0.1 ml of J2 suspension (about 500 bars) was added and mixed well. At equal volume R 2 A liquid culture medium is used as a control, after 5 replicates are treated, nematodes are inoculated, 24 h are inoculated, the number of dead nematodes is counted under a dissecting scope, the mortality rate of the nematodes is counted, the mortality rate is corrected, and the number of the nematodes counted per dish is more than 100.
Mortality (%) = (number of dead nematodes-total number of nematodes counted) ×100
Corrected mortality (%) = treated mortality-control mortality
Test results: the corrected mortality of the YC3001 strain fermentation broth on the tobacco root-knot nematode J2 was 95.7%.
Test site: tobacco planting field in the flat dam area of Anshun city in Guizhou province.
Test crop: flue-cured tobacco (variety: yunyan 87).
Test agent: YC3001 microbial inoculum with viable cell count content of 5×10 9 Each gram, prepared according to the above-described method of the invention; 10% fosthiazate granules produced by Shandong province United agricultural chemical industry Co.
The test method comprises the following steps: three treatments were set up, 3 replicates per treatment, 120 cigarettes per replicate.
Treatment 1: the dosage of YC3001 microbial inoculum is 500 g/mu. During transplanting, 500 g of microbial inoculum is mixed in 100 kg of water, and after uniform stirring, tobacco seedling roots are soaked in the liquid medicine for 20-30 minutes, and then transplanting is performed according to a conventional method.
Treatment 2:10% fosthiazate granules 2 kg/mu. When soil preparation is carried out, the granules are uniformly spread on the soil surface, and are mixed into the soil by a tillage machine, and then transplanted according to a conventional method.
Treatment 3: and (3) transplanting the blank control without applying any nematicide according to a conventional method.
Investigation and statistical methods: plant nodule disease grade index (grade 0: no nodule; grade 1: 1-20% root system nodule; grade 2: 21-40% root system nodule; grade 3: 41-60% root system nodule; grade 4: 61-80% root system nodule; grade 5: 81-100% root system nodule) was investigated 180 days after transplanting, and disease index and control were calculated according to the following formula:
disease index= (n) 1 ×1+ n 2 ×2+ n 3 ×3+ n 4 ×4+ n 5 ×5)/(S×5)×100, n 1 - n 5 Respectively representing the total number of plants corresponding to the 1-5 grades of the root nodule disease grade index, and S representing the total number of plants investigated.
Control (%) = 100 (1-x/y), x, y represent disease index of treatment and blank control, respectively.
Test results: table 1 shows that the application of 500 g YC3001 microbial inoculum per mu can achieve 80.1% of the control effect on tobacco root-knot nematodes by adopting the root dipping method, which is slightly lower than the control effect (82.1%) of 2 kg of 10% fosthiazate granules per mu. The control effect of the YC3001 microbial inoculum on tobacco root knot nematode is shown in table 1:
TABLE 1
| Treatment of | Index of disease (%) | Preventing effect (%) |
| Treatment 1 (YC 3001 microbial agent) | 17.4 | 80.1 |
| Treatment 2 (10% fosthiazate granules) | 15.6 | 82.1 |
| Treatment 3 (blank) | 87.3 | 0 |
Test site: black stone town tobacco planting field in Weining county, guizhou province.
Test crop: flue-cured tobacco (variety: yunyan 87).
Test agent: YC3001 microbial inoculum with viable cell count content of 6.5X10 9 Each gram, prepared according to the above-described method of the invention; 10% fosthiazate granules produced by Shandong province United agricultural chemical industry Co.
The test method comprises the following steps: three treatments were set up, 3 replicates per treatment, 120 cigarettes per replicate.
Treatment 1: the dosage of YC3001 microbial inoculum is 500 g/mu. During transplanting, 500 g of microbial inoculum and 200 kg of farmyard manure are uniformly mixed, shi Yao g/plant is planted in holes, and then transplanting is carried out according to a conventional method.
Treatment 2:10% fosthiazate granules 2 kg/mu. When the soil preparation is carried out, the granules and 200 kg of farmyard manure are uniformly mixed and then are applied in holes, and then transplanted according to a conventional method.
Treatment 3: and (5) performing blank control of 200 kg of farmyard manure in holes, and transplanting according to a conventional method.
Investigation and statistical methods: plant nodule disease grade index (grade 0: no nodule; grade 1: 1-20% root system nodule; grade 2: 21-40% root system nodule; grade 3: 41-60% root system nodule; grade 4: 61-80% root system nodule; grade 5: 81-100% root system nodule) was investigated 180 days after transplanting, and disease index and control were calculated according to the following formula:
disease index= (n) 1 ×1+ n 2 ×2+ n 3 ×3+ n 4 ×4+ n 5 ×5)/(S×5)×100, n 1 - n 5 Respectively representing the total number of plants corresponding to the 1-5 grades of the root nodule disease grade index, and S representing the total number of plants investigated.
Control (%) = 100 (1-x/y), x, y represent disease index of treatment and blank control, respectively.
Test results: table 2 shows that 500 g YC3001 microbial inoculum is applied to each mu of holes, the control effect on tobacco root knot nematode reaches 74.6 percent, which is slightly higher than the control effect (72.6 percent) of 2 kg of 10 percent fosthiazate granules applied to each mu. The control effect of YC3001 microbial agent hole application on tobacco root-knot nematodes is shown in Table 2:
TABLE 2
Test site: tobacco planting field in Qian and West county of Guizhou province Jin Po.
Test crop: flue-cured tobacco (variety: yunyan 87).
Test agent: YC3001 microbial inoculum with viable cell count content of 6.5X10 9 Each gram, prepared according to the above-described method of the invention; 10% fosthiazate granules produced by Shandong province United agricultural chemical industry Co.
The test method comprises the following steps: three treatments were set up, 3 replicates per treatment, 120 cigarettes per replicate.
Treatment 1: the dosage of YC3001 microbial inoculum is 800 g/mu. 7 days after transplanting, 800 g of microbial inoculum and 400 water are uniformly mixed and root irrigation is carried out, and the volume of the microbial inoculum is about 320 ml/plant.
Treatment 2:10% fosthiazate granules 2 kg/mu. The granules were mixed with 400 water and root irrigated at about 320 ml/plant.
Treatment 3:400 kg root is irrigated, about 320 ml/plant.
Investigation and statistical methods: plant nodule disease grade index (grade 0: no nodule; grade 1: 1-20% root system nodule; grade 2: 21-40% root system nodule; grade 3: 41-60% root system nodule; grade 4: 61-80% root system nodule; grade 5: 81-100% root system nodule) was investigated 180 days after transplanting, and disease index and control were calculated according to the following formula:
disease index= (n) 1 ×1+ n 2 ×2+ n 3 ×3+ n 4 ×4+ n 5 ×5)/(S×5)×100, n 1 - n 5 Respectively representing the total number of plants corresponding to the 1-5 grades of the root nodule disease grade index, and S representing the total number of plants investigated.
Control (%) = 100 (1-x/y), x, y represent disease index of treatment and blank control, respectively.
Test results: table 3 shows that 800 g YC3001 microbial inoculum is irrigated per mu, the control effect on tobacco root knot nematode reaches 68.9%, and is slightly lower than the control effect (70.2%) of 10% fosthiazate granules applied per mu. The control effect of YC3001 microbial inoculum root irrigation on tobacco root knot nematodes is shown in table 3:
TABLE 3 Table 3
The practical application shows that: the bacterial agent prepared by YC3001 strain has effective viable count of 5×10 9 When the application amount per gram is more than one gram and per mu is 500-800 grams, the root is dipped, planted in holes or irrigated, the prevention effect on tobacco root knot nematode disease is more than 68%, and the application method has the characteristics of low use cost, no residue and safety to people and livestock.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Claims (4)
1. A nematode biocontrol bacterial strain, characterized by: the nematode biocontrol bacterial strain is opal nocardia clock（Nocardioides stalactiti）YC3001 strain with the accession name of nocardia rubra（Nocardioides stalactiti）YC3001 strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation unit address is the university of Wuhan in the mountain area of Wuhan, hubei province, and the preservation date is: 2021, 12, 17; preservation number: cctccc: m20211636.
2. The bacterial agent for controlling tobacco nematodes prepared from the bacterial strain for controlling nematode living in claim 1, wherein the content of viable bacteria in the bacterial agent is 5×10 9 More than one per gram.
3. The nematode biocontrol bacterial agent of claim 2, wherein the active ingredient is at least one of the following (a) (b):
(a) The fermentation culture of nematode biocontrol bacteria of claim 1;
(b) A spore suspension of the nematode biocontrol bacterium of claim 1.
4. Nocardia clock of claim 1（Nocardioides stalactiti）Application of YC3001 strain in preparation of microbial inoculum for preventing and treating tobacco root knot nematode disease.
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