A strain of Paecilomyces lilacinus JBC033 and its bacterial agent and application

Abstract

The invention discloses a strain of Paecilomyces lilacinus JBC033 and its application. The Paecilomyces lilacinus JBC033 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on July 31, 2023, and the deposit number is CGMCC No.40809. The lethality rate of the strain to southern root-knot nematode J2 is 86.3%, the growth inhibition rate to Fusarium oxysporum is 63.2%, and the inhibition rate to Pseudomonas syringae is 72.9%. The microbial agent prepared by Paecilomyces lilacinus JBC033 can be used for the prevention and treatment of root-knot nematode diseases of crops, and has the characteristics of high efficiency, safety, environmental friendliness, etc.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and in particular relates to a strain of Paecilomyces lilacinus JBC033 and its application. Background Art

[0002] Root-knot nematode complex diseases of crops refer to diseases caused by root-knot nematodes and soil-borne pathogens that inhabit the same ecological niche and jointly infect the same crop, which has a great impact on modern agricultural production. The wounds caused by the second-instar larvae of root-knot nematodes infecting the root system of crops are conducive to the infection of a variety of soil-borne pathogenic fungi and bacteria. Complex diseases caused by the coordinated infection of root-knot nematodes and a variety of plant pathogenic fungi and bacteria, such as complex diseases caused by the combined infection of pathogenic bacteria Pseudomonas, Chryseobacterium and pathogenic fungi Fusarium oxysporum, Phytophthora spp., and Phytophthora spp., are extremely common and cause serious economic losses. Root-knot nematodes have been widely reported in my country's economic crops such as tomatoes, Chinese medicinal materials, tobacco, fruits, cotton, bananas, and beans, causing serious damage and huge losses.

[0003] At present, it has become urgent to prevent crop diseases. The main ways to prevent and control diseases are chemical control, physical control, biological control, etc. There are few studies on the control of root-knot nematodes, and the control effect needs to be improved. Biological control refers to the use of one organism to fight against another organism, so as to achieve the purpose of preventing and controlling crop diseases. Paecilomyces lilacinus belongs to endoparasitic fungi and is an important natural enemy of some plant parasitic nematodes. It can parasitize eggs and infect larvae and females. It can significantly reduce the damage of plant nematode diseases such as root-knot nematodes, cyst nematodes, and stem nematodes in various crops. Paecilomyces lilacinus has the characteristics of high efficiency, safety, and environmental friendliness, and has attracted the attention of researchers at home and abroad. There are many reports on the use of biocontrol bacteria to control crop diseases, which have achieved remarkable results in the prevention and control of crop diseases. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a strain of Paecilomyces lilacinus JBC033, aiming to provide a microorganism Paecilomyces lilacinus JBC033 that can effectively prevent and control soil-borne root-knot nematode diseases of crops.

[0005] The invention of the present application is achieved as follows: a Paecilomyces lilacinus JBC033, wherein the Paecilomyces lilacinus JBC033 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40809 and a deposit date of July 31, 2023.

[0006] Another object of the present invention is a microbial agent comprising the above-mentioned Paecilomyces lilacinus JBC033, wherein the spore content is 0.2-10 billion CFU / g.

[0007] Preferably, the bacterial agent is used for the prevention and treatment of root-knot nematode diseases of crops.

[0008] Preferably, the bacterial agent is applied by seed mixing, root dipping, furrow application, or hole application, and the application amount is 1.0-5.0 kg / mu.

[0009] The present invention has the following advantages:

[0010] The Paecilomyces lilacinus JBC033 of the present invention is separated from the roots of crops with root-knot nematode diseases, can resist root-knot nematodes, and inhibit pathogenic bacteria and pathogenic fungi. The microbial agent prepared by the JBC033 strain can effectively prevent and control root-knot nematode diseases of crops, and has the characteristics of low use cost, no residue, and safety for humans and animals. It is a very safe biological control product, showing that the bacteria has great potential to be developed into a commercialized biological pesticide. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the colony morphology and hyphae characteristics of Paecilomyces lilacinus JBC033.

[0012] Figure 2 Schematic diagram of the mycelium of Paecilomyces lilacinus JBC033 infecting the oocysts of root-knot nematodes. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solution and advantages of the present application more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0014] Example 1

[0015] Isolation, Screening and Identification of Paecilomyces lilacinus Strain JBC033

[0016] 1. Strain isolation and screening

[0017] The roots of tomatoes infected with root-knot nematodes were collected from vegetable planting areas in Dali County, Weinan City, Shaanxi Province. After the surface of the nodules was cleaned and disinfected, the egg capsules and female worms of the root-knot nematodes were separated and picked, mixed with physiological saline and inoculated on a Bengal red culture medium plate. After culturing at 25°C for 7 days, a total of 37 strains of nematode epiphytic fungi were obtained by the traditional streak separation method. These 37 strains were cultured in Czapek liquid medium at 28°C and 150rpm for 7 days to obtain bacterial fermentation liquid. The lethality of the fermentation liquid of each strain to southern root-knot nematode J2 was determined. The results showed that the lethality of 18 strains to J2 was above 60%. Then, the pathogenic fungus Fusarium oxysporum and the pathogenic bacterium Pseudomonas syringae of the root-knot nematode complex disease were used as targets to determine the antagonism of the fermentation liquid of these 18 fungi to the two pathogens. The results showed that among the 18 strains, 4 strains (JBC014, JBC027, JBC033, and JBC035) could simultaneously resist three target pathogens, among which JBC033 strain had the best activity, with a lethality rate of 86.3% against southern root-knot nematode J2, a growth inhibition rate of 63.2% against Fusarium oxysporum, and a inhibition rate of 72.9% against Pseudomonas syringae (Table 1). This showed that the JBC033 strain of Paecilomyces lilacinus had good potential for biological control applications.

[0018] Table 1 Determination of the inhibitory effect of fungi on the pathogens of root-knot nematode complex disease

[0019]

[0020] 2. Identification of strain JBC033

[0021] The screened strain JBC033 was inoculated into PDA medium and cultured at 25°C. Observation showed that the mycelium of the strain was white after 3 days of culture, and spores were produced after 7 days of culture, which were light pink, and the conidia were oval or spindle-shaped.

[0022] The strain was inoculated in liquid PDA medium for fermentation. After culturing at 28°C for 5 days, the bacteria were collected and ground with liquid nitrogen. The fungal strain DNA was extracted using a new plant genomic DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd.). The extraction steps were referred to the kit instructions. The strain DNA was PCR amplified using the universal primers ITS1 / ITS4 for the fungal rDNA-ITS segment. The amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence was Blast compared using NCBI data, and phylogenetic analysis was performed using MEGA7.0 software. After phylogenetic analysis and combined with bacterial morphological observation, the strain JBC033 was determined to be Paecilomyces lilacinus, and the strain was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40809 and a deposit date of July 31, 2023.

[0023] Example 2

[0024] 1. Cultivation of strain JBC033 and preparation of microbial agents

[0025] Test tube slant seed culture of strain JBC033: The strain was inoculated onto a conventional PDA medium slant and cultured at 28°C for 5 days to obtain slant seeds.

[0026] 2. Liquid seed culture of strain JBC033: Inoculate the slant seeds into a Erlenmeyer flask filled with PDA liquid culture medium and culture in a shaking incubator at 28°C and 135 rpm for 3 days to obtain liquid seeds.

[0027] 3. Strain JBC033 was cultured in a large number of fermentation tanks: In a 1000-liter solid fermentation tank, liquid seeds were cultured in a sterilized rice bran composite medium at a ratio of 10% (V / V). The fermentation conditions were a stirring speed of 30 rpm, a temperature of 28°C, sterile air with a humidity of 55%, an air volume of 340 L / min, and a fermentation time of 7 days. The fermentation ended when the spore production was more than 10 billion CFU / gram.

[0028] 4. Preparation of strain JBC033 inoculant: The bacteria and its metabolites obtained by fermentation tank culture were evenly mixed with appropriate amounts of diatomaceous earth, Tween 80 and starch, and then vacuum dried at 45°C to a moisture content of 5%. The fineness of the obtained powder was 60 mesh using a cyclone mill to obtain the spore content of 2 billion CFU / g of the Paecilomyces lilacinus JBC033 inoculant.

[0029] Application Example 3

[0030] Experiment on the efficacy of the fungicide Paecilomyces lilacinus JBC033 against tomato root-knot nematodes and their combined diseases

[0031] Test location: Tomato planting area in Yunyang Town, Jingyang County, Xianyang City, Shaanxi Province. The test site is a greenhouse where southern root-knot nematode disease and its complex diseases are severely incurred.

[0032] Experimental crop: Zhongza No. 9 tomato.

[0033] Test agents: JBC033 microbial agent, spore content of 2 billion CFU / g, prepared according to the method of the present invention; the control pesticide for controlling root-knot nematodes is 10% thiazolyl granules (Syngenta Crop Protection Co., Ltd., Switzerland), the control pesticide for controlling soil-borne fungal diseases is 30% carbendazim aqueous solution (Weifang Huanuo Biotechnology Co., Ltd.), and the control pesticide for controlling soil-borne bacterial diseases is 20% thiophanate-copper suspension (Yancheng Limin Agrochemical Co., Ltd., Jiangsu Province). Tomato seedlings were raised according to conventional methods and the drugs were used when transplanting.

[0034] Experimental method: There are three treatments as follows, each with 3 replications and each replication covering 0.3 mu.

[0035] Treatment 1: JBC033 microbial agent, 2.0 kg / mu. Take 1.0 kg of the agent when transplanting, dilute it with 200 kg of water and apply it to the roots; 60 days after transplanting, take 1.0 kg of the agent, dilute it with 800 kg of water and irrigate the roots.

[0036] Treatment 2: During land preparation, spread 10% thiamethoxam granules (1500 g / mu) on the soil and mix it evenly into the ridges. Seven days after transplanting, dilute 30% carbendazim aqueous solution (500 ml / mu) and 20% thiophanate-methyl suspension (500 ml / mu) with water according to the instructions and then irrigate the roots.

[0037] Treatment 3: blank control without any pesticide application.

[0038] Investigation and statistical methods: Six months after transplanting, 100 tomato plants were randomly selected from each treatment, and the plant disease levels for root-knot nematode disease and combined diseases were divided, and the disease index and prevention effect were calculated.

[0039] Calculation of root-knot nematode index and control effectiveness survey

[0040] The root knot grading standards are as follows: Level 0, no root knots are found on the roots; Level 1, a small number of root knots can be found through careful identification; Level 2, no root knots on the main root, and a small number of root knots on the fibrous roots can be clearly identified; Level 3, no root knots on the main root, and slightly larger root knots can be found on the fibrous roots; Level 4, no root knots on the main root, and mainly larger root knots on the fibrous roots; Level 5, 50% of the roots are infected, and the main root is very slightly infected; Level 6, root knots are found on the main root; Level 7, most of the main roots are infected by root knots; Level 8, all main roots are infected or very few roots are not infected; Level 9, all roots can be severely infected or the plant usually dies; Level 10, all roots are severely infected and there are no roots.

[0041] The root knot index and relative control effect are calculated as follows:

[0042] Root knot index = [∑(number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value)] × 100.

[0043] Relative control effect = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100%.

[0044] Calculation of soil-borne disease classification and control effectiveness investigation method

[0045] Grading standard: Level 0: no disease symptoms on the whole plant; Level 1: mild vascular discoloration, no external symptoms; Level 2: moderate vascular discoloration, no external symptoms; Level 3: the lower leaves turn yellow and gradually wilt; Level 4: most of the leaves wilt; Level 5: the plant dies.

[0046] Disease index = [∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100.

[0047] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0048] Table 2 Determination of the efficacy of JBC033 microbial agent against pathogens of tomato root-knot nematode complex disease

[0049]

[0050] Test results: Table 2 shows that in treatment 1, 3.0 kg of JBC033 microbial agent was applied per mu, and the JBC033 spore content was 2 billion CFU / g, and the prevention effects on tomato root-knot nematode disease and combined diseases were 81.89% and 69.47% respectively; in treatment 2, three pesticides for combined diseases of root-knot nematodes, fungi and bacteria were applied, and the prevention effects on tomato root-knot nematode disease and combined diseases of root-knot nematodes were 81.12% and 72.03% respectively. The two treatments of microbial agent and chemical agent had equivalent prevention effects on root-knot nematode disease and combined diseases of greenhouse tomatoes.

[0051] Application Example 4

[0052] Experiment on the efficacy of the fungicide Paecilomyces lilacinus JBC033 against yellow knot nematodes and their combined diseases

[0053] Test location: Coptis chinensis planting base of the cooperative in Tuanjie Village, Huaping Town, Zhenping County, Ankang City, Shaanxi Province. The test site is a field with severe occurrence of root-knot nematodes and their complex diseases.

[0054] Experimental crop: Coptis chinensis.

[0055] Test agent: JBC033 microbial agent, spore content of 5 billion CFU / g, the control pesticide prepared according to the method of the present invention for controlling root-knot nematodes is 10% thiazolyl granules (Syngenta Crop Protection Co., Ltd., Switzerland), the control pesticide for controlling soil-borne fungal diseases is 30% carbendazim aqueous solution (Weifang Huanuo Biotechnology Co., Ltd.), and the control pesticide for controlling soil-borne bacterial diseases is 20% thiophanate-methyl suspension (Jiangsu Yancheng Limin Agrochemical Co., Ltd.). Coptis chinensis was raised in a conventional manner and the drug was used during transplanting.

[0056] Experimental method: There are three treatments as follows, each with 3 replications and each replication covering 0.5 mu.

[0057] Treatment 1: JBC033 microbial agent 6.0kg / mu. Take 2.0kg of the agent when transplanting, dilute it with 500kg of water and apply it to holes; 3 months after transplanting the seedlings, take 2.0kg of the agent, dilute it with 500 times of water and irrigate the roots once; 6 months after transplanting, take 2.0kg of the agent, dilute it with 500 times of water and irrigate the roots once, and manage water and fertilizer normally.

[0058] Treatment 2: During land preparation, spread 10% thiamethoxam granules (2000 g / mu) on the ground and mix it evenly into the tillage layer. Ten days after transplanting, dilute 30% carbendazim aqueous solution (800 ml / mu) and 20% thiophanate-methyl suspension (800 ml / mu) with water according to the instructions and then irrigate the roots.

[0059] Treatment 3: blank control with no treatment.

[0060] Investigation and statistical methods: 9 months after transplanting, statistics were conducted after the disease was fully developed. A five-point sampling method was used for each plot, with 5-10 plants checked at each point. The root-knot nematode disease and combined disease were classified into plant disease levels, and the disease index and prevention effect were calculated according to the method in Example 3.

[0061] Table 3 Determination of the efficacy of JBC033 microbial agent against the pathogens of the complex disease of Coptis chinensis root knot nematodes

[0062]

[0063] Test results: Table 3 shows that in treatment 1, 6.0 kg of 10 billion CFU / g of the microbial agent was applied per mu, and the spore content of JBC033 was 5 billion CFU / g, and the control effects on Coptis root knot nematode disease and its composite disease were 73.83% and 69.10%, respectively. In treatment 2, chemical pesticides targeting root knot nematodes, pathogenic fungi and pathogenic bacteria were applied simultaneously, and the control effects on Coptis root knot nematode disease and composite diseases were 77.57% and 54.32%, respectively. The control effect of JBC033 microbial agent on root knot nematode disease was lower than that of chemical pesticides, and the control effect of JBC033 microbial agent on composite diseases was significantly higher than that of chemical pesticides.

[0064] In summary, the microbial agent prepared by the Paecilomyces lilacinus JBC033 strain in the present invention has a prevention effect of more than 73% on root-knot nematode diseases of tomato and Coptis chinensis crops, and a prevention effect of more than 69% on root-knot nematode complex diseases. It can be seen that the microbial agent can effectively prevent root-knot nematode diseases of various crops, and also has a certain prevention effect on soil-borne fungal diseases and bacterial diseases, and has the characteristics of low use cost, no residue, and safety for humans and animals.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Paecilomyces lilacinus JBC033, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.40809 and the deposit date of July 31, 2023.

2. A microbial agent comprising the Paecilomyces lilacinus JBC033 according to claim 1, wherein the spore content is 0.2-10 billion CFU / g.

3. A use of the bacterial agent as claimed in claim 2, characterized in that: Used for the prevention and control of root-knot nematode diseases of crops.

4. The use according to claim 3, characterized in that: The application method is seed mixing, root dipping, furrow application, and hole application. The application amount is 1.0-5.0 kg / mu.

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