CN104818216A - Paecilomyces lilacinus for prevention and control of meloidogyne diseases of tomato and grape - Google Patents

Abstract

The invention discloses a strain of Paecilomyces lilacinus used for preventing and treating tomato and grape root-knot nematode diseases. The Paecilomyces lilacinus provided by the present invention is the Paecilomyces lilacinus YT08, whose registration number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCC No.10026, and the preservation date is November 2014 26th. The Paecilomyces lilacinus YT08 CGMCC No.10026 provided by the invention has a strong control effect on tomato and grape root-knot nematodes, and can be developed as a new biological control agent for agricultural root-knot nematodes.

Description

A Paecilomyces lilacinus used to control tomato and grape root-knot nematode diseases

technical field

The invention relates to a Paecilomyces lilacinus used for preventing and treating root-knot nematode diseases of tomatoes and grapes, and belongs to the technical field of microorganisms.

Background technique

Plant nematode disease is one of the four major pathogenic organisms in the cultivation of crops, vegetables, fruit trees, forest trees, flowers and medicinal materials. Its harm is second only to fungi and surpasses bacteria and viruses. Plant parasitic nematodes cause about 100 billion US dollars of losses in world agricultural production every year, and root-knot nematodes are the most harmful type of plant parasitic nematodes.

The control of plant parasitic nematodes mainly includes chemical nematicides, cultivation techniques, breeding resistant varieties and biological pesticides. Because root-knot nematodes usually live in the soil and in the roots of plants, there are still no specific chemical pesticides, and they can only control the occurrence of nematode diseases in a short period of time. Therefore, it is difficult to control the damage of root-knot nematodes with chemical pesticides. Animal health, the use of chemical nematicides is very restricted. In addition, although cultivation techniques (such as crop rotation) and cultivating resistant varieties can play a certain role in prevention and control, there are relatively large limitations in the actual application process. Therefore, as a safe and effective method for preventing and controlling root-knot nematode diseases, the application of biological control technology and its preparation products in the control of plant nematode diseases is particularly important.

Paecilomyces lilacinus belongs to the subphylum Deuteromycetes, Paecilomyces genus, Paecilomyces family, and the genus Paecilomyces. It can widely parasitize nematode eggs. It has high insecticidal effect, safety, high efficiency, and long-lasting effect. It is considered to be the most promising nematode biocontrol fungus. In addition to its nematicidal effect, Paecilomyces lilacinus can also parasitize insects such as Hemiptera, Homoptera, Isoptera, Coleoptera and Lepidoptera. , cucumber anthracnose, cotton wilt and rice bakanae disease, etc.) have antagonistic effects. The secondary metabolites of Paecilomyces lilacinus also have certain physiological effects, such as producing products similar to indole acetic acid, promoting the growth of plant roots and plants, promoting seed germination and growth, and producing a variety of enzymes with certain Degradation effects of chemical pesticides.

Paecilomyces lilacinus has infectivity to root-knot nematodes, but due to differences in the characteristics of individual organisms, the control effects of Paecilomyces lilacinus on root-knot nematodes from different sources are different, so it is necessary to continuously separate them to have a better effect on root-knot nematodes. highly pathogenic strains.

Contents of the invention

The invention provides a strain of Paecilomyces lilacinus used for preventing and controlling root-knot nematode diseases of tomatoes and grapes, which has strong pathogenicity to root-knot nematodes of tomatoes and grapes, and can be developed as a new type of biological control against root-knot nematodes in agriculture agent.

Paecilomyces lilacinus ( Paecilomyces lilacinus ) YT08 used to prevent and control tomato and grape root-knot nematode diseases in the present invention, its registration number in the General Microbiology Center of China Microbiological Culture Collection Management Committee It is CGMCC No. 10026, and the deposit time is November 26, 2014.

Paecilomyces lilacinus YT08 of the present invention is isolated from the seaweed kelp epidermis of Yantai City, Shandong Province. The bacterial strain is cultivated on the separation medium, and the colony is white at the initial stage and the mycelium is dense. As the cultivation time prolongs, it gradually becomes lavender, and the colony It is closely connected with the culture medium and is not easy to pick. The strain produces conidia, the spores are oval, and the conidiophores are broom-shaped.

The Paecilomyces lilacinus YT08 of the invention is insensitive to chloramphenicol, has low nutritional requirements, grows well on brewer's grain solid medium, and can be normally cultured at 20°C-35°C.

The Paecilomyces lilacinus YT08 of the present invention has a strong control effect on tomato and grape root-knot nematode diseases, and the control effect on tomato root-knot nematode is 89.69% in the pot test, and the control effect on grape root-knot nematode is 93.42%.

The Paecilomyces lilacinus species ( Paecilomyces lilacinus ) of the genus Paecilomyces in the present invention belongs to Paecilomyces lilacinus.

Biomaterial Information :

Taxonomic name: Paecilomyces lilacinus .

Strain number: YT08.

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms.

Depository institution abbreviation: CGMCC.

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Deposit date: November 26, 2014.

Registration number of the collection center: CGMCC No. 10026.

Description of drawings

Fig. 1 is the culture characteristic of Paecilomyces lilacinus YT08 on the separation medium; Fig. 2 is the microscopic observation characteristic of Paecilomyces lilacinus YT08.

Detailed ways

The present invention will be further described below in conjunction with specific examples, and these examples are for understanding rather than limiting the present invention.

A strain of Paecilomyces lilacinus used to prevent and control tomato and grape root-knot nematode diseases. The strain is named YT08. General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: November 26, 2014.

Embodiment 1 : the isolation and screening of bacterial strain YT08

Paecilomyces lilacinus YT08 was isolated from kelp epidermis cultured in Yantai City, Shandong Province, China. Separation and screening are carried out in accordance with the following steps: Dilute the collected kelp samples with sterile water at a ratio of 1:100, shake in a constant temperature shaker at 28°C for 30 minutes, and apply after dilution step by step on an ultra-clean workbench On the screening medium plate, keep a constant temperature shaker at 28°C for 4-6 days until a single colony grows, and pick a single colony on the separation medium plate for multiple times of streaking, isolation and purification. Inoculated with Bengal red solid slant medium, cultured in a constant temperature shaker at 28°C for 4-6 days and stored at 4°C, it was Paecilomyces lilacinus YT08.

The components of the screening medium used are as follows: ammonium sulfate 10 g/L, dipotassium hydrogen phosphate 2 g/L, sodium chloride 15 g/L, magnesium sulfate heptahydrate 1 g/L, colloidal chitosan 100 g/L, Agar 20 g/L, pH 5.8, sterilized at 121°C for 20 minutes.

The components of the separation medium used are as follows: glucose 30 g/L, sodium nitrate 2 g/L, dipotassium hydrogen phosphate 1 g/L, iron sulfate 0.01 g/L, potassium chloride 0.5 g/L, magnesium sulfate heptahydrate 0.5 g/L, colloidal chitosan 6 g/L, agar 20 g/L, pH 6.5, sterilized at 121°C for 20 minutes.

The components of Bengal red medium for strain preservation are as follows: peptone 5 g/L, glucose 10 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate heptahydrate 0.5 g/L, agar powder 20 g/L, Bengal red 0.0133 g/L, chloramphenicol 0.1 g/L, pH 6.5, sterilized at 121°C for 20 minutes.

Embodiment 2 : the genus identification of bacterial strain YT08

1. Morphological characteristics of the strain YT08: Streak inoculation of the strain YT08 isolated and screened above on the isolation medium, cultivate in a constant temperature shaker at 28°C, and observe the growth status of the colony regularly. Inoculate the strain into another separation medium by coating method, and insert the sterilized cover glass (1cm×1cm) obliquely into the medium, and place it in a constant temperature incubator at 28°C for 6 days until the hyphae climb. After being placed on the cover glass, the cover glass was taken out and placed under an optical microscope to observe the hyphae and spore morphology of strain YT08.

The colony color of strain YT08 was white at the initial stage of culture, and the color gradually deepened with the prolongation of culture time, and the colony color was lavender after producing spores. Figure 1 shows the morphological characteristics of the strain YT08 cultured on the isolation medium for 6 days. The colonies are raised and round. The colonies are closely connected with the medium and are not easy to pick. The hyphae are dense and spread outward, and the color is lavender. , slightly white around, no soluble pigment secreted. The morphological characteristics of the strain YT08 observed under the microscope are shown in Figure 2. The conidiophores are broom-shaped, the spores are oval, and the clusters are unbranched chains, and the hyphae in the base are light brown.

2. 18S rDNA sequence analysis of strain YT08

The total DNA of strain YT08 was extracted and amplified by PCR with universal primers for fungal 18S rDNA (NS1-5'-GTAGTCATATGCTTGTCTC-3', NS6-5'-GCATCACAGACCTGTTATTGCCTC-3'). The electrophoresis results of the PCR products cut the required DNA target bands, recover them with the gel extraction kit and send them to the company for sequencing.

The 18S rDNA sequence of strain YT08 obtained by sequencing is shown in the sequence table. After comparison and analysis by NCBI Blast, the sequence is consistent with multiple isolates of Paecilomyces lilacinus in GenBank (such as JF824691.1, AB124670.1, AY526475.2, AB084157.1, AB104884.1, AB023945.1, EF638694.1, AB103380.1, AF548079.1, etc.) have a sequence similarity as high as 100%.

By combining the morphological characteristics and 18S rDNA sequence characteristics of the strain YT08, the strain YT08 can be identified as Paecilomyces lilacinus .

Embodiment 3 : the fermentation culture of Paecilomyces lilacinus YT08

1. Activation culture of bacteria

Add 100 ml of strain activation medium to each 500 ml Erlenmeyer flask, and steam sterilize at 121° C. for 20 minutes. The composition of the strain activation medium used (g/L): glycerol 10, soybean peptone 15, MnSO ₄ 2, powdered chitosan with 85% deacetylation degree 8, NaCl 6, CaCl ₂ 1, MgSO ₄ 3, FeSO ₄ 0.5, initial pH 7.

Pick out a small piece of bacterial lawn from the solid slant strains, directly insert it into the liquid strain activation medium, and shake it on a shaking table at 28° C. and 200 rpm for 48 hours until it grows into a light white sticky mycelium.

2. Solid fermentation culture

Solid fermentation medium: 3kg of dried brewer's grains, 20g of sucrose, 60g of soybean peptone, 10g of powdered chitosan with 85% degree of deacetylation, 30g of NaCl, 6g of MgSO ₄ , 4g of MnSO ₄ , and dilute to 7 liters with tap water. Preparation method: Dissolve NaCl, MgSO ₄ , MnSO ₄ , sucrose and soybean peptone with water first, stir evenly, and then mix with other substances, the pH is natural. The above solid fermentation medium was put into a 10L solid fermenter, and steam sterilized at 118° C. for 20 minutes.

According to the inoculation amount of 10% (volume percentage), the seed culture product of Paecilomyces lilacinus YT08 was poured into the solid fermentation medium, and the mechanical stirring was started to mix the bacteria and the fermentation medium evenly. Incubate statically at 28°C and pass through sterile air once every 3 hours. After cultivating for 3 days until the surface of the medium is covered with hyphae, start the mechanical stirring again to mash and mix all the materials in the fermenter, culture statically at 28°C, and inject sterile air once every 5 hours, and continue culturing for 4 days Fermentation is terminated after the culture medium is covered with lavender spores.

After the fermentation is terminated, start the mechanical stirring device, break up the culture and mix it evenly, take it out from the discharge port, put it in a cool and ventilated place to dry, and obtain the solid preparation of Paecilomyces lilacinus YT08. The number of viable bacteria of Paecilomyces lilacinus YT08 in the solid preparation was found to be 4.7×10 ⁹ cfu/g by the method of serial dilution and coating.

Embodiment 4 : the parasiticity of Paecilomyces lilacinus YT08 to tomato root-knot nematode eggs

A treatment group and a control group were set up, and 3 replicates (petri dishes) were set up for each group. Add 2 mL of egg particle suspension to a sterile petri dish, add 2 mL of Paecilomyces lilacinus YT08 spore suspension to each petri dish of the treatment group, and add 2 mL of sterile water to each petri dish of the control group. After culturing at 28°C for 8 days, the egg hatching rate and egg parasitic rate were calculated according to the following formula:

      

      

The parasitic results of Paecilomyces lilacinus YT08 on tomato root-knot nematode eggs are shown in Table 1.

Table 1 Parasitism of the spore suspension of Paecilomyces lilacinus YT08 on the eggs of tomato root-knot nematode

      

The method for obtaining the eggs of tomato root-knot nematode is as follows: collect the root system of tomato root-knot nematode diseased plants in the greenhouse, rinse it with water first, cut it into small pieces, add it to sodium hypochlorite solution, seal it and shake it for 3 minutes, pass it through a 200-mesh sieve and use Rinse with distilled water, pass through a 500-mesh sieve, and collect in a sterile small beaker after repeated rinsing with distilled water, and adjust the suspension to 50 eggs/mL.

The preparation method of the spore suspension of Paecilomyces lilacinus YT08 used is as follows: Paecilomyces lilacinus YT08 was streak-inoculated on Bengal red culture medium, and after cultivating for 7 days at 28°C, Paecilomyces lilacinus YT08 was inoculated with sterile water. The spores were washed out, the spore concentration was calculated by microscopic examination, and a spore suspension with a concentration of 10 ⁷ cfu/mL was prepared.

Embodiment 5 : the parasiticity of Paecilomyces lilacinus YT08 to root-knot nematode ovum

A treatment group and a control group were set up, and 3 replicates (petri dishes) were set up for each group. Add 2 mL of egg particle suspension to a sterile petri dish, add 2 mL of Paecilomyces lilacinus YT08 spore suspension to each petri dish of the treatment group, and add 2 mL of sterile water to each petri dish of the control group. After culturing at 28°C for 8 days, the egg hatching rate and egg parasitic rate were calculated according to the following formula:

      

      

See Table 2 for the parasitic results of Paecilomyces lilacinus YT08 on root-knot nematode eggs.

Table 2 The parasitism of the spore suspension of Paecilomyces lilacinus YT08 to the eggs of root-knot nematode

      

The method of obtaining the root-knot nematode eggs used is as follows: collect the root system of the root-knot nematode diseased plant Stenis sinensis grape, first rinse it with water, cut it into small pieces, add it to the sodium hypochlorite solution, seal it and shake it for 3 minutes, pass it through a 200-mesh sieve and use it. Rinse with distilled water, pass through a 500-mesh sieve, and collect in a sterile small beaker after repeated rinsing with distilled water, and adjust the suspension to 50 eggs/mL.

The preparation method of the spore suspension of Paecilomyces lilacinus YT08 used is as follows: Paecilomyces lilacinus YT08 was streak-inoculated on Bengal red culture medium, and after cultivating for 7 days at 28°C, Paecilomyces lilacinus YT08 was inoculated with sterile water. The spores were washed out, the spore concentration was calculated by microscopic examination, and a spore suspension with a concentration of 10 ⁷ cfu/mL was prepared.

Embodiment 6 : the pot experiment of Paecilomyces lilacinus YT08 control tomato root-knot nematode

The cultivation medium is prepared by mixing disease-free soil, organic fertilizer and sand according to the ratio of 9:1:3, and the cultivation medium used has been tested to be free of insects. Tomato seedlings with a length of about 12 cm were picked and planted in flower pots of 30×30 cm, with 1 plant per pot, 60 plants in total. 200 2nd instar root-knot nematodes of tomato were all applied to the cultivation medium of the above-mentioned 60 pots of tomato seedlings, and 10 g of Paecilomyces lilacinus YT08 solid preparation (see Example 3 for the production steps) was added to 30 pots of them, and another 30 pots were re-applied. Apply 10g of dried distiller's grains as a blank control. After 60 days of routine management, the control effect was calculated.

The tomato root-knot nematode used is acquired as follows: collect the root system of the tomato root-knot nematode diseased plant in the greenhouse, rinse it with water first, cut it into small pieces, add it to the sodium hypochlorite solution, seal it and shake it for 3 minutes, pass it through a 200-mesh sieve and rinse it with distilled water , then passed through a 500-mesh sieve, washed repeatedly with distilled water, and collected in a small sterile beaker. The collected eggs of root-knot nematode were hatched at 26°C for 3 days to obtain the 2nd instar larvae of root-knot nematode tomato.

According to the number of root knots, the severity of the disease is divided into 5 grades: no root knot is grade 0, root knot accounts for 1%-25% of the whole root system is grade 1, and root knot accounts for 26%-50% of the whole root system % is grade 2, root knots account for 51%-75% of the whole root system is grade 3, and root knots account for 76%-100% of the whole root system is grade 4. The calculation formulas of the disease index and the control effect are as follows:

      

      

The pot test results are shown in Table 3. The control effect of Paecilomyces lilacinus YT08 on tomato root-knot nematode is 89.69%. The root weight growth rate, plant height growth rate and aerial part weight growth rate of tomato in the treatment group were significantly higher than those in the control group.

Table 3 Control effect of tested pesticides on tomato root-knot nematode

      

Embodiment 7 : the pot experiment of Paecilomyces lilacinus YT08 control grape root-knot nematode

The cultivation medium is prepared by mixing disease-free soil, organic fertilizer and sand according to the ratio of 9:2:2, and the cultivation medium used has been tested to be free of insects. Pick about 20cm long Gernischt grape seedlings and plant them in 30×30cm flower pots, with 1 plant per pot, 40 plants in total. 200 2nd instar root-knot nematodes were all applied to the cultivation medium of the above-mentioned 40 pots of grape seedlings, and 10 g of Paecilomyces lilacinus YT08 solid preparation (see Example 3 for the production steps) was added to 20 pots of them, and another 20 pots were re-applied. Apply 10g of dried distiller's grains as a blank control. After 90 days of routine management, the control effect was calculated.

The grape root-knot nematode used is acquired as follows: collect the root system of the root-knot nematode diseased plant Stenis sinensis grape, first rinse it with water, cut it into small pieces, add it to the sodium hypochlorite solution, seal it and shake it for 3 minutes, then pass it through a 200-mesh sieve and rinse it with distilled water , then passed through a 500-mesh sieve, washed repeatedly with distilled water, and collected in a small sterile beaker. The collected root-knot nematode eggs were hatched at 26° C. for 3 days to obtain the 2nd instar larvae of root-knot nematode stenognac.

According to the number of root knots, the severity of the disease is divided into 5 grades: no root knot is grade 0, root knot accounts for 1%-25% of the whole root system is grade 1, and root knot accounts for 26%-50% of the whole root system % is grade 2, root knots account for 51%-75% of the whole root system is grade 3, and root knots account for 76%-100% of the whole root system is grade 4. The calculation formulas of the disease index and the control effect are as follows:

      

      

The pot test results are shown in Table 4. The control effect of Paecilomyces lilacinus YT08 on controlling root-knot nematode of grapevine was 93.42%. The root weight growth rate, plant height growth rate and aerial part weight growth rate of the treatment group were significantly higher than those of the control group.

Table 4 Control effect of the tested pesticides on root-knot nematode of grapevine

           

sequence    List    surface

Ludong University

A Paecilomyces lilacinus used to control tomato and grape root-knot nematode diseases

1338 bp

dna

Paecilomyces lilacinus

CTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGCACCTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTATTAGATTAAAAACCAATGCCCTCTGGGCTCTCTGGTGATTCATGATAACTTCTCGAATCGCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTAGTGGCCAAACATGGTTGCAACGGGTAACGGAGGGTTAGGGCTCGACCCCGGAGAAGGAGCCTGAGAAACGGCTACTACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGCACTGGTCCGGCCGGGCCTTTCCCTCTGTGGAACCCCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAAAAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTTGACTCGTTCGGCACCTTACGAGAAAT CAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCAGTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACAG

Claims (1)

1. A strain of Paecilomyces lilacinus used to prevent and control tomato and grape root-knot nematode diseases, characterized in that the bacterial strain is Paecilomyces lilacinus ( Paecilomyces lilacinus ) YT08, which is obtained from the kelp epidermis of seawater culture in Yantai City, Shandong Province The isolated one has a strong control effect on root-knot nematode of tomato and grape, and has been registered and preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on November 26, 2014, with the preservation number CGMCC No. 10026.