| Root-knot nematodes are pathogens that seriously damage plant growth.Because of their many species,rapid reproduction,and destructiveness,they cause heavy losses in the output and quality of protected vegetables,and have become a key factor restricting the development of protected vegetable planting in China.The control of root-knot nematode diseases has mainly depended on pesticides for a long time.However,chemical nematicides are highly toxic and pose potential safety hazards to the environment and human health.Therefore,it is urgent to develop new pesticides that are safe,efficient,and low-toxic.In this study,root-knot nematode disease samples and rhizosphere soil were collected from vegetable greenhouses in eleven regions(counties)of Shanxi Province.Female,male and second-stage juveniles(J2)of root-knot nematodes collected from different samples were extracted.Molecular identification of primers using sequence characterized amplified-PCR(SCAR),phylogenetic analysis based on the r DNA gene sequence of the 28 S D2/D3 segments of the root-knot nematode,the species and dominant population of the root-knot nematode of vegetable greenhouse pathogens in Shanxi Province were determined.A high-efficiency biocontrol fungus B22 was isolated and screened from the J2,further taxonomic status of the strain,biological control mechanism,plant growth promotion,liquid-solid fermentation culture conditions,preparation of wettable powder and field control effect conducted systematic research,and mainly achieved the following research results:1.Four speices was identified from greenhouse vegetable in Shanxi province: Meloidogyne incognita,M.arenaria,M.javanica and M.hapla.Based on statistical analysis of experimental samples,the isolation frequency of M.incognita is 81.6%,M.arenaria 7.1%,M.javanica 5.1%,and M.hapla 6.2%.Meloidogyne incognita is the dominant species extracted from greenhouse vegetables in Shanxi Province.2.A strain B22,which isolated from the infected nematodes,had significant lethal effects on root-knot nematodes and was identified as Phanerochaete chrysosporium Burdsall.By in vitro toxicity test,the lethal rate of strain B22 to the J2 of root-knot nematodes was as high as 78.1%,and the hatching rate of eggs was only 30.1%;Pot test,the strain B22(spore concentration 1×10~7cfu/ml)had been applied as soil drenching was of an inhibitory effect on J2,that can effectively reduce root gall number and the occurrence of root-knot nematode diseases.3.Phanerochaete chrysosporium strain B22 has parasitic lethal effect on eggs,J2 and females of root-knot nematodes.Strain B22 parasites on eggs and causes the shrinkage and deformation of the egg shell,shrinkage and distortion,and abnormal embryonic development;Parasitic on J2,the conidia of strain B22 adhere to the nematode wall,germinate hyphae,penetrate and kill J2,causes the body wall of the J2 to sag,being uneven,and the outer surface of the body to ablate;Parasitic to females,the spores of strain B-22 surrounded adult females and stuck to their surfaces.The contents of the female's bodies leaked out,and short hyphae grew out from bodies.The adult female body looked severely atrophied and partly dissolved.4.The parasitic rate of P.chrysosporium strain B22 on root-knot nematode increased significantly with the increase of treatment time.After 11 days of inoculation,the parasitic rate of strain B22 on eggs,J2 and female were 78%,77% and 49% respectively.The parasitism of strain B22 on J2 was observed only 1day;It needed 3 day and 4 day on eggs and female.Moreover,the strain B22 has a significant lethal effect on the J2 which can only infect the host.And the lethal effect was enhanced with the increase of the spore concentration and the treatment time.Especially at the treatment in 72 h with spore suspension concentration of 3×10~8cfu/ml,the mortality of the J2 was 76.6%,the corrected mortality was 71.9%,and the LC50 was 9.6×10~7cfu/ml.5.Metabolites produced by P.chrysosporium strain B22 have a strong lethal effect on root-knot nematodes.The strain B22 have protease-producing activity,which can kill the root-knot nematodes.At the same time,HILIC UHPLC-Q-TOF MS was used to analyze the major metabolites on the fermentation broth of the strain B22 and the toxicity of Meloidogyne incognita was determined at 500?g/ml,the corrected mortality rate of Mono-2-ethylhexyl phthalate against Meloidogyne incognita was 52.56%,and1-palmitoyl-2-hydroxy-SN-glycerol-3-phosphate ethanolamine was 62.91%.6.Phanerochaete chrysosporium strain B22 was of promoting plant growth and enhancing the host plant's ability to resist root-knot nematodes.The conidia suspension(1.0×10~7cfu/m L)of strain B22 significantly promotes the germination of tomato seeds,enhances the growth potential of tomato seedlings,and stimulates plant height,leaf area,stem thickness,root length,fresh(dry)weight to enhance the resistance of host plants.The main mechanism is that strain B22 can promote the significant increase of tomato root protective enzymes(phenylalanine ammonia lyase,polyphenol oxidase and peroxidase),which participate in the defense response of plant pathogens.And the strain B22 has moderate drought resistance and salt tolerance.7.The best optimization conditions for liquid-solid two-phase fermentation of P.chrysosporium strain B22 are: in the liquid fermentation stage,6 m L inoculation amount(spore concentration 10~7cfu/ml),and100 ml culture medium in 250 ml triangle flask,30 ? culture temperature,160 rpm rotate speed,the optimal fermentation time is 90 hours after cultivation;in solid fermentation stage,the oat medium is best,16% inoculation amount,30 ? culture temperature,6% water content of the culture medium,the termination time of the solid fermentation is 19 days after inoculation,and the drying condition is drying at40°C for 24 hours.8.Phanerochaete chrysosporium strain B22 spore powder was produced through liquid-solid two-phase fermentation.The powder contains 3.5×10~9 conidia per gram,and has 93.7% average natural germination rate.Diatomaceous earth was used as the carrier for Strain B22 wettable powder preparation,Nekal BX as the wetting aiding agent,and sodium carboxymethyl cellulose as a dispersing aid.The fungicide B22 was used for the biocontrol experiment on the root-knot nematodes in greenhouse.The results showed that the corrected control effect was 51.91%,plant growth was promoted and tomato yield increased by 32.8%.In summary,P.chrysosporium strain B22 can control the root-knot nematodes,reduce the root gall index,inhibite the occurrence of root-knot nematode diseases,and promote tomato yield increase in field. |
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