Screening Of Streptomyces Fulvissimus And Evaluation Of Its Nematicidal Activity Against Meloidogyne Incognita And Analysis Of Its Components

Plant parasitic nematodes can harm almost all kinds of plants,including economic important crops,causing losses of about 358.24 billion US dollars worldwide every year.A large number of microorganisms and their metabolites have been developed into biological agents by researchers and applied to the biological control of plant parasitic nematodes.Among the more than 10000 identified natural antibiotics reported publicly to data,about 70～80%are produced by actinomycetes,and more than half of them are from Streptomyces spp.In the screening of biocontrol microognisms and thier active secondery metabolites leads for the control of root knot nematode,a standard evaluation system is reqired and plays very important role.The size of plant growing container,solvent type,inoculation time,inoculation amount and culture time are all important factors that need to be clarified in the evaluation system.For the six organic solvents commonly used for compound solubilization,their contact killing rates to the secondery-juvenile（J2）of Meloidogyne incognita and their toxicities to the cucumber seedlings growing in greenhouse were investigated.Acetone showed as the safest solvent that was selected to dissolve compounds used in this study.When the final concentration of acetone was not higher than 2%,it had no contact killing effect on J2 and no plant toxicity was observed.This study established series of biologic detection systems that included to use pure sand test tube,matrix sand（sand:soil:matrix=2:1:1）test tube and cup to grow cucumber in the laboratory,and Microplot to grow the plant in the field.It was determinted that the optimal numbers of J2 inoculated in test tube was 200,in cup was 700 and in Microplot was 1250.The above methods were used to evaluate the activity of six commercially available chemical nematicides,including abamectin B1a,abamectin B2a,emamectin benzoate,fluensulfone,fluopyram and fosthiazate.The results showed that the the system has the advantages of short experimental cycle,high standardization,easy operation and good reproducibility.The EC₅₀ and EC₉₀ calculated by Microplot experiment in field can be used to guide the dosage in field practice.The system can also quickly evaluate the co-toxicity index of different compounds,so as to quickly obtain the best ratio.407 actinomycetes strains were isolated from the soil near Qinghai Lake.Using the above methods,an actinomycete with good nematicidal activity was screened.Using the fermentation broth optimized by fermentation conditions to carry out field Microplot assay,the control effect on Meloidogyne incognita was 77.6%.The control effect of fermentation broth on root knot nematode of Hami melon was 60.9%and that of Pseudo-ginseng root knot nematode was 63.1%in field test.After genome sequencing and collinearity analysis with the control strain,it was confirmed that the strain is Streptomyces fulvissimus.The killing activity of this strain against root knot nematode was found for the first time in this study.Through the investigation of the polarity of the active components in the fermentation broth,it is clear that they belong to fat soluble compounds.To identify the active chemical composition,the petroleum ether and the ethyl acetate were used as the mobile phase to separate the active chemical composition along with bioassay.The compound EAS-5 at the concentration of 1000 mg/L,the inhibition rate of egg hatching in 7 days was 39.7%,the contact killing rate of J2 after 48 h was 100%,and the relative inhibition rate of root knot nematode in-planta assay was 82.9%.The purified active collection was preliminarily analyzed by NMR and speculated as a polycyclic macromolecular compound containing aromatic ring and aliphatic ring and it may also be a compound containing aromatic ring and aliphatic chain.Benzene acetic acid was another compound isolated from the extract of ethyl acetate.At the concentration of 1000mg/L,the inhibition rate of egg hatching in 7 days was 93.8%,the contact killing rate of J2 after 48 h was97.4%,and the relative inhibition rate of root knot nematode in-planta assay was 78.9%.Valinomycin is one of the important antibiotics synthesized by Streptomyces fulvissimus according to the reports in the literature.In this study,some fragments of valinomycin synthesis related gene clusters were amplified in the genome of the strain by designing specific primers.Valinomycin was detected in the extract of fermentation broth by mass spectrometry.It was preliminarily estimated that the content of valinomycin in fermentation broth was about 5.04 mg/L.Valinomycin also showed certain biological activity against Meloidogyne incognita in the test.