A kind of Candida citrinum and its application in preventing and controlling root-knot nematodes

Abstract

The present invention relates to the field of microorganisms and plant protection, and specifically to a Candida citrifolia strain and its application in preventing and controlling root-knot nematodes. The strain is isolated from orchard soil, is easy to survive in the soil, and has good prevention effect on root-knot nematodes through indoor and field prevention and control tests, providing an effective biocontrol strain for preventing and controlling the harm of root-knot nematodes, contributing to the green prevention and control of root-knot nematodes and ensuring safe production of vegetables.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and plant protection, and in particular to Candida citrinum and application thereof in preventing and controlling root-knot nematodes. Background Art

[0002] Meloidogyne incognita belongs to the order Meloidogyne, the suborder Meloidogyne, the family Heterodera, the subfamily Meloidogyne, and the genus Meloidogyne. There are four main species of nematodes that cause root-knot nematodes, namely, southern root-knot nematode (Meloidogyne incognita), Javan root-knot nematode (M.javanica), peanut root-knot nematode (M.arearia), and northern root-knot nematode (M.hapla), of which southern root-knot nematode is the main pathogen. Male and female adults of root-knot nematodes are different in shape, with male adults being linear and female adults being pear-shaped. Female adults are fixed and parasitic in the host tissues, with an average length of 440μm to 1,300μm and a width of 325μm to 700μm. The body of the worm is milky white, with a well-developed esophageal bulb and a well-developed stylet. The unique perineal pattern is an important basis for the classification of nematodes. These four nematodes are root-knot nematodes distributed worldwide. In my country, southern root-knot nematodes account for 52%, Java root-knot nematodes account for 31%, peanut root-knot nematodes account for 8%, and northern root-knot nematodes account for 8%. In the survey of nematode occurrence in 121 crops, 33% occurred in tomatoes, eggplants, and tobacco, while tomatoes accounted for 17%.

[0003] Root-knot nematode disease is one of the important diseases of melon vegetables. Worldwide, plant parasitic nematodes cause losses of approximately $6.5 billion to melons each year (2001). The losses are mainly caused by root-knot nematodes. The typical symptom of the damage is that after infecting the root system, root knots of varying sizes are formed on the root system. After infection, root-knot nematodes affect the root system's absorption and upward transportation of water and nutrients. The symptoms in the aboveground part are mainly slow growth, wilting, yellowing of leaves, organ deformities, etc. The manifestations directly related to agricultural production are reduced yield and quality, resulting in a large amount of economic losses. In my country, root-knot nematodes are one of the main soil-borne pathogens, which can infect more than 3,000 host plants belonging to 114 families, especially Cucurbitaceae, Solanaceae and Cruciferae. In recent years, with the adjustment of agricultural industrial structure and the promotion of greenhouse and other facility cultivation technologies, the area of ​​cucumber, gourd and other vegetable cultivation in facilities has become larger and larger. Due to the difficulty of cropping and the increase in multiple cropping index, the root-knot nematode disease provides suitable living conditions for root-knot nematodes due to continuous cropping or repeated cropping throughout the year, resulting in the disaster of cucumber root-knot nematode disease. At the same time, root-knot nematode disease can aggravate the occurrence of other diseases such as wilt, causing huge economic losses.

[0004] The population density of root-knot nematodes is an important factor in root-knot nematode damage. The damage threshold of root-knot nematodes is about 0.5-2 second-instar larvae per gram of soil, or 100 per cubic centimeter (cm 3 ) 1 egg (Di Vito and Greco, 1988). Different nematodes have different tolerance limits on different crops. For example, the threshold of southern root-knot nematodes on cabbage is 0.5 eggs / cm3 (Sasanelli et al. (1992)), on peppers it is 0.3 eggs (Di Vito et al. 1992), and on tomatoes it is 0.55 eggs / cm3 (Di Vito et al. 1991). Therefore, controlling the number of root-knot nematodes in the soil is one of the main goals of root-knot nematode prevention and control.

[0005] At present, the nematicides on the market are mainly divided into two categories: chemical nematicides and microbial nematicides. Chemical nematicides can be divided into halogenated hydrocarbons, methyl thioisothiocyanates, organophosphorus, carbamates, antibiotics, and biological agents according to their structure. In terms of microbial nematicides, currently registered products include Paecilomyces lilacinus, Verticillium chrysosporum and Bacillus, etc. These microbial products play a huge role in the production of green and organic food. Biological control has the characteristics of green and safety, and is closely related to the green control of agricultural pests and diseases. Biological control is increasingly valued. In the study, the biocontrol bacteria isolated from root-knot nematodes involved fungi, bacteria and other microorganisms. In the study, there are few studies on yeast. Yeast is a single-cell fungus that is easy to culture and preserve, survives for a long time in the soil, and can produce rich secondary metabolites. It has great potential in the development of biocontrol bacteria. Summary of the invention

[0006] The object of the present invention is to provide a Candida citrinum strain for preventing and treating root-knot nematode disease and a preparation method thereof. The difference between the present invention and other inventions is that the Candida citrinum strain comes from the rhizosphere soil of an orchard and has a good effect on root-knot nematodes. Experiments have shown that the isolated Candida citrinum strain has a good repelling effect, lethal effect and control effect on root-knot nematodes both indoors and in the field, which is of great significance for preventing and treating the harm of root-knot nematode disease and ensuring the safe production of vegetables.

[0007] By isolating and purifying in the soil of the Yizhiying orchard in Langfang, Hebei, and through microbiological classification and identification, Candida citrinum was obtained, and the strain number was Cq-C08, which constitutes the first aspect of the present invention. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 11, 2022. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is CGMCC NO.25889.

[0008] The strain was cultured in a yeast fermentation liquid medium by shaking, and the fermentation culture liquid and its supernatant were used to treat the second-instar larvae of the root-knot nematode, respectively, and the lethal effect on the root-knot nematode was detected. The results showed that the lethality of the bacterial liquid and the supernatant to the nematode within 12 hours reached 100%. The strain's avoidance of nematodes was detected by the confrontation method in a culture dish, and it was confirmed that the fermentation liquid and the supernatant of the strain could significantly repel the root-knot nematode; in the indoor pot inoculation test, the control result of the Candida citrifolia Cq-C08 strain on the root-knot nematode reached 81.7%. The fermentation liquid of the strain was used for soil irrigation in the field, and it was confirmed that the control effect of the strain on the root-knot nematode reached 54.1%, and the growth-promoting effect on the plant height reached 18.8%.

[0009] Therefore, the second aspect of the present invention is to provide the use of the Candida citrinum Cq-C08 strain in preventing and controlling root-knot nematodes.

[0010] Furthermore, the third aspect of the present invention provides a method for controlling root-knot nematodes using the Candida citrinum, which comprises fermenting and culturing the Candida citrinum, inoculating the fermentation liquid into a soil seedling hole, and then planting plant seedlings in the seedling hole for normal management and cultivation.

[0011] Preferably, the fermentation culture is to inoculate the Candida citrinum into a liquid culture medium and ferment and culture for 2-4 days at 25-32° C. and 150-200 rpm.

[0012] More preferably, the liquid culture medium is NYDB medium: 10 g / L yeast powder, 20 g / L tryptone, 20 g / L glucose, pH: 7.0-7.2, 121° C., sterilization for 20 min.

[0013] More preferably, the culture condition is fermentation culture at 28° C. and 180 rpm for 3 days.

[0014] In a specific embodiment, the plant seedlings are seedlings of vegetable crops such as cucumber, melon, watermelon, tomato and pepper.

[0015] The fourth aspect of the present invention is to provide a biocontrol agent for controlling root-knot nematodes, which is prepared by using the fermentation liquid or concentrated liquid obtained by fermentation and culture of the Candida citrinum.

[0016] The beneficial effect of the present invention is that the fermentation liquid of the citrus yeast Cq-C08 strain has a mortality rate of 100% on the second-instar larvae of the root-knot nematode, a control effect of 81.7% in the pot experiment, a control effect of 54.1% in the field, and promotes plant growth by 18.8%, and the strain has a significant repellent effect on the second-instar larvae of the root-knot nematode, a high and stable control effect, and is suitable for agricultural production needs. At the same time, the citrus yeast Cq-C08 strain is a yeast isolated from rhizosphere soil, which is safe and harmless to crops, and has a control effect on root-knot nematodes, and has safety characteristics in agricultural applications. The present invention screened the citrus yeast for the first time and applied it to the prevention and control of root-knot nematodes, providing an important basis for its biological control application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Colony morphology of Candida citrinum Cq-C08.

[0018] Figure 2 Schematic diagram of the nematode chemotaxis assay.

[0019] Collection information:

[0020] The Cq-C08 strain of the present invention is classified and named Candida quercitrusa, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC for short) on October 11, 2022, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.25889. DETAILED DESCRIPTION

[0021] The technical scheme of the present invention is further described below in conjunction with specific implementation examples. First, the Candida pseudocitrinum Cq-C08 strain was isolated, purified and cultured, and then fermented and cultured using a yeast liquid medium NYDB, and the nematicidal activity and repellent activity of the fermentation broth and its supernatant were tested. At the same time, the fermentation culture broth of the Candida pseudocitrinum Cq-C08 strain was applied to cucumbers for root-knot nematode control experiments under indoor potted and field conditions.

[0022] Example 1: Isolation, purification and identification of Candida citrinum Cq-C08 strain

[0023] The culture medium and preparation used in this example: lactic acid PDA: 200g potato, 20g glucose, 5mL lactic acid, 15g agar powder, fixed to 1L, pH = 5.0. During preparation, peel the potato, weigh 200g, cut into pieces, boil for 15min, filter out the potato pieces and residues with gauze, take the filtrate and add 20g glucose, 15g agar, 5ml lactic acid, sterilize at 121℃ for 20min, and then spread into a plate in a culture dish for isolating and purifying yeast.

[0024] The specific method is as follows:

[0025] (1) Isolation of strains:

[0026] Soil samples were uniformly taken from the rhizosphere of apples in the orchard of Yizhiying Village, Langfang City, for a total of 3 samples, 200g each, and mixed evenly; 50g was added to 1000mL of distilled water, stirred evenly, and allowed to stand for 3min. 1mL of supernatant was taken, diluted 100 times with sterilized water, and then 50μL of the diluted liquid was spread on the lactic acid PDA plate. After the soil dilution was evenly spread, it was inverted in a 28℃ constant temperature incubator and cultured for 2d. After the colonies grew on the plate, single colony purification was performed, and single colonies were picked and separated and purified on the lactic acid PDA medium plate to obtain single colonies. About 30 single colonies can grow on each plate, which are randomly numbered from 1 (Cq-C01), and then the numbered single colonies are expanded and identified to obtain the eighth single colony strain Cq-C08 strain. The strains were morphologically determined according to the "Fungal Identification Manual".

[0027] (2) Morphological identification of strains:

[0028] The strains were morphologically determined according to the Manual of Fungal Identification. Figure 1 , the Cq-C08 strain was obtained on a lactic acid PDA medium plate at 30°C. After 3 days of culture, the colonies were white to cream, with a smooth surface, spherical or hemispherical, milky white and opaque, with a raised center and neat edges. The bacterial cells were round or oval, and no pigment was produced on the culture medium. The vegetative cells budded and had simple pseudohyphae, but no sexual reproduction. The morphology of the Cq-C08 strain was determined according to the "Handbook of Fungal Identification", and the strain was determined to be Candida quercitrusa.

[0029] (3) Molecular identification of strains:

[0030] Pick a single colony with a sterilized toothpick, gently tap the bottom of the PCR tube, stick the bacteria to the bottom of the tube, and use it as a PCR template for bacterial PCR amplification. Use the universal primers NL1 (forward primer) and NL4 (reverse primer) for fungal identification to amplify the D1 / D2 segment of the 26srRNA sequence. The PCR amplification reaction system is 50μL, containing 25μL ExTaq enzyme, 1μL forward primer, 1μL reverse primer, 3μL DNA template, and 20μL sterile water. Amplification conditions: 94℃5min, 94℃30S, 50℃30S, 72℃1min, 30 cycles; 72℃10min to terminate amplification. The amplified products were separated and identified by 1% agarose gel electrophoresis, and the PCR products were directly sequenced in both directions and compared by Blast on the NCBI website.

[0031] The D1 / D2 region sequence of yeast 26SrDNA was amplified using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'), and the PCR product was subjected to 1% agarose gel electrophoresis. The 26SrDNA sequence amplified by the strain was 609bp long (SEQ ID No.1) after sequencing analysis. After the 26SrDNA sequence of the strain was subjected to BLAST analysis, the results showed that the similarity between the Cq-C08 strain and Candida quercitrusa reached 100%. The strain was identified as Candida quercitrusa.

[0032] Example 2: Analysis of the effect of Candida citrinum Cq-C08 strain on nematodes

[0033] The culture medium and its formula used in this embodiment are as follows: NYDA medium: 10 g / L yeast powder, 20 g / L tryptone, 20 g / L glucose, solid culture medium with 15-20 g / L agar added, pH: 7.0-7.2, divided into Erlenmeyer flasks for sterilization at 121°C for 20 min; NYDB medium: 10 g / L yeast powder, 20 g / L tryptone, 20 g / L glucose, pH: 7.0-7.2, sterilized at 121°C for 20 min.

[0034] The test method is as follows:

[0035] (1) Preparation of Cq-C08 strain fermentation broth

[0036] The Candida citrinum Cq-C08 strain was streaked, purified and activated on a NYDA medium plate, cultured at 28°C for 3 days, and the activated colonies were scraped with an inoculation loop and inoculated into NYDB liquid medium. The culture was shaken (180 rpm) at 28°C for 3 days. When the absorbance value of the fermentation liquid was OD 600 When the pH value reached 1.0, the fermentation liquid was used for the test. At the same time, part of the fermentation liquid was centrifuged at 10,000 rpm for 3 min to precipitate the bacteria, and the upper supernatant was used for the test, respectively, for the analysis of the avoidance of the second-instar larvae of the root-knot nematode (Meloidogyne spp.).

[0037] (2) Preparation of root-knot nematodes for testing

[0038] Root-knot nematodes were collected from the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The roots of peppers with severe root-knot nematode disease were taken out and gently rinsed with water. The egg masses were carefully removed from the surface of the roots and placed in 0.5% sodium hypochlorite for disinfection for 3 minutes. Then, they were rinsed with sterile water for 3 times to rinse off the residual reagent on the surface of the egg masses. The sterilized egg masses were placed in a culture dish containing a small amount of sterile water and incubated at 25°C for 24-48 hours. The hatched second-instar larvae of root-knot nematodes were collected for testing.

[0039] (3) Test on nematode avoidance effect

[0040] A culture dish (9 cm) was used for the root-knot nematode tropism analysis. The culture dish was divided into 6 sections (1-6) from left to right and 6 sections from top to bottom, with a total of 6×6 small grids ( Figure 2 ). Use 0.5% water agar to make plates. Add 20μl of fermentation liquid to the center of the left edge of the culture dish (position B), add about 100 second-instar larvae of root-knot nematodes (20μl) to the center of the culture dish (position A), and observe the movement of nematodes at 25°C. In the study, the movement of nematodes from the center to the bacterial solution (1-3 parts) was determined to have an attractive effect, and the movement from the center to the direction away from the bacterial solution (4-6 parts) was determined to have a repulsive (avoidance) effect. At 12h, the number of nematodes in the two parts of each culture dish and the number of nematodes still staying at the nematode inoculation site were counted for comparative analysis. Ten culture dishes were treated for each treatment, and the experiment was repeated 3 times independently. The experiment also set up 20μl Cq-C08 strain supernatant treatment and 20μl sterile water as a control.

[0041] (4) Test results and analysis

[0042] The results showed that at 12h, the second-instar larvae showed strong repulsion to both the fermentation liquid and the supernatant (Table 1). In the fermentation liquid and supernatant treatments, the nematodes tended to stay away from the fermentation liquid and the supernatant, and the number of nematodes in the repulsion area was significantly greater than that in the attraction area. In the fermentation liquid treatment, the number of nematodes in the attraction area and the repulsion area was 12.2 and 88.7, respectively. The supernatant treatment had a similar situation, with 11.6 and 91.0 nematodes in the attraction area and the repulsion area, respectively. In the clear water control, the number of nematodes in the attraction area and the repulsion area was not much different, 55.3 and 54.6, respectively (Table 1).

[0043] Table 1 Analysis of nematode avoidance

[0044] deal with Attracting nematodes Middle inoculation point nematode Nematodes Fermentation Broth 12.2 4.4 88.7 Supernatant 11.6 3.8 91.0 Clear water control 55.3 4.3 54.6

[0045] Example 3: Analysis of the nematicidal effect of Candida citrinum Cq-C08 strain

[0046] (1) Preparation of Cq-C08 strain fermentation broth

[0047] The Cq-C08 strain was streaked and activated on a NYDA medium plate and cultured at 28°C for 3 days. The activated colonies were scraped with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 100 mL NYDB liquid medium. The culture was shaken (180 rpm) at 28°C for 3 days. When the absorbance value of the fermentation liquid was OD 600 When it reached 1.0, the fermentation liquid and its 10-fold and 100-fold dilutions prepared with sterile water were used for the test. At the same time, part of the fermentation liquid was centrifuged at 10,000 rpm for 3 min to precipitate the bacteria. The upper supernatant and 10-fold and 100-fold dilutions prepared with sterile water were taken for the test to treat the second-instar larvae of the root-knot nematode (Meloidogyne spp.) respectively, and the lethal effect of the fermentation liquid of the Cq-C08 strain on the root-knot nematode was determined.

[0048] (2) Preparation of root-knot nematodes for testing

[0049] Root-knot nematodes were collected from the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The roots of peppers with severe root-knot nematode disease were taken out and gently rinsed with water. The egg masses were carefully removed from the surface of the roots and placed in 0.5% sodium hypochlorite for disinfection for 3 minutes. Then, they were rinsed with sterile water for 3 times to rinse off the residual reagent on the surface of the egg masses. The sterilized egg masses were placed in a culture dish containing a small amount of sterile water and incubated at 25°C for 24-48 hours. The hatched second-instar larvae of root-knot nematodes were collected for testing.

[0050] (3) Nematicidal effect test method

[0051] On a sterile 24-well cell culture plate, add 1 mL of fermentation liquid to each well, then add 10 μL of nematode suspension, containing about 100 nematodes, and make 10 wells (samples) for each treatment, repeat 3 times. In the same way, 10 times diluted fermentation liquid, 100 times diluted fermentation liquid, supernatant, 10 times supernatant, 100 times supernatant were treated, and sterile water was used as a control. They were placed at room temperature for 24 hours, and the death of root-knot nematodes was observed. The corrected mortality rate was calculated as the nematode killing effect. The corrected mortality rate calculation formula is as follows: Corrected mortality rate (%) = 100 × (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate).

[0052] (4) Test results

[0053] Through the above test, the lethal effect of fermentation liquid and supernatant on root-knot nematodes after 24 hours of treatment was determined. The results showed that different fermentation liquid multiples had different effects on nematodes. The corrected mortality of the fermentation liquid and the 10-fold dilution on root-knot nematodes was 100%. The control effect was reduced after the fermentation liquid was diluted 100 times, but the effect still reached 72.5% (Table 2). The nematicidal effect of the supernatant of the fermentation liquid of the Cq-C08 strain was similar to that of the fermentation liquid. The corrected mortality of root-knot nematodes in the supernatant and the 10-fold dilution was 100%, and the nematicidal effect in the 100-fold diluted supernatant was 76.2% (Table 3). The experiment shows that the fermentation liquid of the Cq-C08 strain has a good effect on preventing and controlling root-knot nematodes.

[0054] Table 2 Nematicidal effect of fermentation broth of Cq-C08 strain at different concentrations

[0055] Nematode mortality Original solution (%) 10 times solution (%) 100 times solution (%) Clear water control (%) Repeat 1 100 100 75.1 1.1 Repeat 2 100 100 72.6 1.5 Repeat 3 100 100 70.9 1.6 Adjusted mortality rate 100％ 100％ 72.5％ -

[0056] Table 3 Nematicidal effect of supernatant of different concentrations

[0057] Nematode mortality Original solution(%) 10 times solution (%) 100 times solution (%) Clear water control (%) Repeat 1 100 100 77.5 1.5 Repeat 2 100 100 73.4 1.5 Repeat 3 100 100 78.7 1.3 Adjusted mortality rate 100％ 100％ 76.2％ -

[0058] Example 4: Effect of Candida citrinum Cq-C08 strain in potted plants on the prevention and treatment of root-knot nematode disease

[0059] (1) Test method

[0060] The Candida citrifolia Cq-C08 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL NYDB liquid medium and cultured at 28°C with shaking (180 rpm) for 3 days. 600=1.0 for prevention and control tests. The cucumber variety selected was "Zhongnong 16". After the seeds were disinfected and germinated, they were raised in seedling trays. When the cucumber seedlings grew to one true leaf and unfolded, they were used for the test. The cucumber seedlings in the one true leaf stage were transplanted into seedling pots (10 cm in diameter, 10 cm in height). The planting matrix was disinfected peat vermiculite (v / v=2:1). Seven days after planting, when the second true leaf appeared, the inoculation treatment was carried out. During inoculation, 15 mL of Cq-C08 strain fermentation culture fluid was inoculated in the rhizosphere soil of the cucumber seedlings, and then the second-instar larvae of the root-knot nematode were inoculated one day later, with 500 inoculated per plant. The preparation of the second-instar larvae of the root-knot nematode is the same as in Example 2. Normal management was carried out at room temperature, and the number of root knots was detected after 5 weeks to calculate the control effect of the Cq-C08 strain on the root-knot nematode. The test also set up a water control and a drug treatment. The drug treatment was 10% thiazolyl granules (Ishihara, Japan), and the dosage was converted according to 2kg per mu. The dosage for each seedling pot was about 0.03g / pot. When using, gently draw shallow grooves or poke shallow holes on the surface of the seedling root matrix, evenly sprinkle the granules into the matrix, and then lightly cover the matrix. Normal management was carried out after application. Each treatment had 10 cucumber seedlings, repeated 3 times, and the clear water simulation treatment was used as a control. The formula for calculating the potted control effect is as follows:

[0061] Control effect (%) = (1-number of treated nodes / number of control nodes) × 100.

[0062] (2) Test results

[0063] 35 days after inoculation of root-knot nematodes, the soil at the root of the cucumber seedlings was gently washed off, the number of root knots on the root system was detected, and the control effect was analyzed (Table 4). It can be seen from the experiment that the number of root knots on the cucumber root system was significantly reduced after the treatment of the Candida citrinum Cq-C08 strain, and the average number of root knots was 17.7 per plant. In the control treatment, the average number of root knots was 97.0 per plant, and the control effect of the Cq-C08 strain on root-knot nematodes reached 81.7%. The control effect reached 93.5% in the treatment of thiazole. It shows that the Candida citrinum Cq-C08 strain can effectively control cucumber root-knot nematodes. Although the control effect is lower than that of the chemical agent thiazole, it has application potential and value in the control of root-knot nematode disease.

[0064] Table 4 Number of root knots and control effects of different treatments

[0065] Treatment Effect Repeat 1 Repeat 2 Repeat 3 average Control effect% Clear water control 100.3 95.5 95.2 97.0 - Thiazofosfate 6.4 6.9 5.7 6.3 93.5％ Cq-C08 18.3 16.9 18.0 17.7 81.7％

[0066] Example 5: Effect of Candida citrinum Cq-C08 strain on controlling root-knot nematode disease in the field

[0067] (1) Test method

[0068] The Cq-C08 strain was inoculated into a 1000mL triangular flask containing 500mL NYDB liquid culture medium, and cultured at 28°C with shaking (180rpm) for 3 days. About 10L of citrus Candida fermentation liquid was cultured. When OD=1.5, the fermentation liquid was used for prevention and control experiments. The test site was in the greenhouse of the Yizhiying Vegetable Institute base in Langfang City, Hebei Province. The greenhouse root-knot nematodes in the area were serious and suitable for field prevention evaluation. The experimental cucumber variety was "Zhongnong 16", and the field test was carried out when the two true leaves of the cucumber seedlings were fully expanded. During the test, each plot had a furrow, with a furrow width of 1.1 meters and a length of 6 meters. Double rows were planted, with a plant spacing of 40cm and a row spacing of 50cm. About 30 seedlings were planted in each furrow. The test was repeated in 3 plots (furrows), and the three plots were randomly distributed. The test was carried out in two different greenhouses at the same time.

[0069] Before transplanting, dig a 15cm deep hole at the designated location for planting the seedlings, dilute 50ml of the fermentation liquid of the Cq-C08 strain 10 times with clean water and pour it into the seedling hole. After the bacterial liquid has completely penetrated into the soil and there is no accumulation of liquid, the cucumber seedlings are planted in the seedling hole. The experiment also set up a clean water control treatment and a chemical pesticide treatment. In the chemical pesticide treatment, 10% thiazolyl granules (Fukido, Ishihara, Japan) were used, and the dosage was tested according to the instruction standard (2kg / mu). After the seedlings were transplanted, normal production management was carried out. After 45 days, the occurrence of root-knot nematodes was detected, the disease level and disease index were statistically analyzed, and the prevention and control effect was calculated. At the same time, the plant height of the aboveground part was measured, and the growth-promoting effect of the strain on the height of the cucumber plant was compared and analyzed.

[0070] The calculation of disease level, disease index and prevention effect is as follows (Table 5):

[0071] Table 5 Disease classification of root knot severity of individual plants

[0072] Disease level Root Symptom Description 0 No knots on all roots 1 The proportion of roots with knots is 0% to 10% 2 The proportion of roots with knots is 11% to 25% 3 The proportion of roots with knots is 26% to 50% 4 The proportion of roots with knots is 51% to 75% 5 The proportion of roots with knots is 76% to 100%

[0073] The disease index (DI) was calculated according to the following formula (1):

[0074] DI=(∑(s×n) / (N×S))×100…………(1)

[0075] in:

[0076] ∑ is the sum of the product of the numerical value of each disease level and the number of plants at each disease level; s is the numerical value of each disease level; n is the number of diseased plants at each disease level; N is the total number of plants surveyed; S is the numerical value of the highest disease level.

[0077] The formula for calculating the control effect is:

[0078] Control effect (%) = (1-treatment disease index / control disease index) × 100………(2)

[0079] Growth promotion effect calculation formula:

[0080] Growth promotion rate (%) = (treated plant height / control plant height-1) × 100………(3)

[0081] (2) Test results

[0082] It can be seen from the experiment that after the treatment of the Cq-C08 strain of Candida citrinum, the incidence of cucumber root-knot nematode disease was significantly reduced, and the number of root knots on the root system was reduced (Table 6). The disease index in the clear water control was 70.4, and the disease index in the Cq-C08 strain treatment was 32.4. The control effect of the Cq-C08 strain on root-knot nematodes reached 54.1%. The disease index treated with the thiazolyl phosphine agent was 26.0, and the average control effect was 63.1%. The results show that although the Cq-C08 strain of Candida citrinum has some gaps with the thiazolyl phosphine agent, the strain can effectively prevent and control cucumber root-knot nematode disease. It has important application potential in the prevention and control of root-knot nematode disease in agricultural production.

[0083] At the same time, through the detection of plant height (Table 6), the results showed that the citrus Candida Cq-C08 strain has a growth-promoting effect. After the treatment, the average plant height of the cucumber plants treated with the strain was 211.0 cm, while the plant height of the water control was 177.6 cm, and the growth promotion rate reached 18.8%, while the average plant height of the thiazolyl phosphine treatment was 196.5 cm, and the growth promotion rate was 10.6%. The results show that the citrus Candida Cq-C08 strain is significantly higher than the thiazolyl phosphine treatment in terms of growth promotion, and the growth promotion effect is significant.

[0084] Table 6 Disease index and control effect of different treatments

[0085]

Claims

1. A Candida citrinum ( Candida quercitrusa ), characterized in that Its accession number is CGMCCNO.25889.

2. Use of the Candida citrinum as claimed in claim 1 in controlling root-knot nematodes.

3. The method for controlling root-knot nematodes using the Candida citrinum according to claim 1, characterized in that: After the Candida citrinum is fermented and cultured, the fermentation liquid is inoculated into the soil seedling holes, and then plant seedlings are planted in the seedling holes for normal management and cultivation.

4. The method according to claim 3, characterized in that The fermentation culture is to inoculate the Candida citrinum into a liquid culture medium and ferment and culture for 2-4 days at 25-32° C. and 150-200 rpm.

5. The method according to claim 4, characterized in that The liquid culture medium is NYDB culture medium: 10 g / L yeast powder, 20 g / L tryptone, 20 g / L glucose, pH: 7.0-7.2, 121° C., sterilization for 20 min.

6. The method according to claim 4, characterized in that The culture conditions were 28°C and 180 rpm for 3 days.

7. The method according to any one of claims 4 to 6, characterized in that The plant seedlings are cucumber, melon, watermelon, tomato or pepper seedlings.

8. A biological control agent for controlling root-knot nematodes, characterized in that: The method is prepared by using the fermentation liquid or concentrated liquid obtained by fermenting and culturing the Candida citrinum as claimed in claim 1.

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