Isolation,Screening Of Bacillus Safensis J2 From Pigeon Pea[Cajanus Cajan(L.) Millsp] And Its Plant Growth Promoting Function

Pigeon pea is a woody leguminous plant with various values such as medicinal,edible and ecological values.It is mainly distributed in tropical and subtropical regions,and in my country mainly in Guangxi,Hainan,Yunnan and other provinces.At present,the extensive cultivation and production of pigeon pea,low yield and low biomass have restricted the industrial development of pigeon pea.Chemical fertilizers have played an irreplaceable role in improving soil fertility and ensuring food security,but their excessive application will cause environmental pollution and soil depletion.The current trend in forestry and agriculture to improve quality and increase production is to reduce the use of chemical fertilizers and explore environmentally friendly biological growth promoters.Compared with chemical fertilizers,microbial inoculants have gradually replaced chemical fertilizers and become a research hotspot in this field because of their safety,environmental protection and effectiveness.At present,the microbial resources of pigeon pea have not yet been fully developed.This study intends to screen high-efficiency plant growth promoting bacteria(PGPB)from different tissues of pigeon pea and rhizosphere soil,and conduct in-depth research on its growth-promoting function,in order to develop high-efficiency Environmentally-friendly microbial agents provide a scientific basis for increasing crop yields.The specific research results are as follows:1.Using pigeon pea roots,stems,leaves and rhizosphere soil as materials,the tissues with different gradients were diluted and coated on three media of NA,LB and TSA for isolation and purified,41 purified strains were finally obtained.Among them,4 were isolated from roots,5 were isolated from stems,4 were isolated from leaves,28 were isolated from rhizosphere soil.With reference to the "Bacterial Identification Manual" and molecular biological methods,the 41 Endophytic and rhizosphere bacteria were mainly classified as:Pseudomonas sp.,Bacillus sp.,Stenotrophomonas sp.,and Enterobacter sp.based on morphological characters and 16S rRNA sequences2.Forty-one strains of endophytic bacteria and rhizosphere bacteria isolated from pigeon pea were screened for IAA production,ammonia production,phosphorus dissolving,nitrogen fixation,biofilm formation and hydrolase production(protease,pectinase,amylase)in vitro.14 strains showed one or more plant growth-promoting properties.Five strains produced IAA(the concentration ranged from 1.78 g/mL to 32.28 g/mL).Ten strains had the ability to dissolve phosphorus(the D/d values ranged from 1.25-6).10 strains could fix nitrogen.11 strains could produce protease(the D/d values ranged from 1.4-2).Two strains could produce amylase(the D/d values were 2.2 and 1.7).One strain could produce pectinase(the D/d value was 3).9 strains could form biofilm.Ten strains produced ammonia.3.A comprehensive evaluation of a variety of plant growth promoting activities was carried out,and finally an endophytic bacterial strain J2 with excellent plant growth promoting activity was obtained,which was identified as Bacillus safensis.HPLC and UPLC-MS/MS analysis were used to identify the plant growth promoting material basis of B.safensis J2.The results showed that there are three growth hormones:IAA,KT and 6-BA in the ethyl acetate extract of B.safensis J2 fermentation broth.Its contents in the fermentation broth were 41.29?g/mL,46.36 ?g/mL and 27.06 ?g/mL,respectively.4.Using GC/MS to study the volatile plant growth promoting substances in the fermentation broth of B.safensis J2,the results show that J2 can produce diketopiperazine antibacterial substances.The three diketopiperazine compounds are identified as:hexahydropyrrolo[1,2-a]pyrazine-1,4-dione,3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione,which had resistance to pathogenic microorganisms.By PCR detection,it was found that the strain contained the gene NRPS which encodes the biosynthesis of non-ribosomal peptide antibiotics.5.Three plants of Arabidopsis thaliana,maize and pigeon pea were used as experimental materials to verify the effect of PGPB Bacillus safensis J2 on plant growth and development.The results showed that the root development of Arabidopsis thaliana seedlings treated with J2 was better than that of the control group.Growth parameters such as seedling rate,plant height,fresh weight,dry weight,nodulation and pod number of pigeon pea seeds treated with J2 were significantly improved compared to the control group.The root length,plant height,fresh weight and dry weight of the non-host plant maize seedlings under J2 treatment were increased by 7.94%,44.13%,28.85%and 26.67%,respectively.