Biological Activity Of Endophytic Bacteria Isolated From Root Nodules Against Meloidogyne Incognita

Root-knot nematode is one of the most destructive species of various crops in plant parasitic nematodes.It has powerful parasitism,a wide range of hosts,a short life cycle and a high breeding frequency,causing dramatic yield losses to agricultural production.Biocontrol agents,which are eco-friendly and generally non-toxic,provide an effective strategy for root-knot nematode disease.In this study,bacteria strains which showed biocontrol effect against Meloidogyne incognita,were screened and identified from 448 endophytic bacterial isolated from root nodules.The pot and field experiments were used to verify its biocontrol effect,and its colonization distribution in tomato roots was explored.The main results were as follows:1.Screening and control effect of endophytic bacteria isolated from root nodules against Meloidogyne incognita: five strains Sneb1994,Sneb1995,Sneb1997,Sneb2000,and Sneb2001,which showed biocontrol effect against Meloidogyne incognita,were screened and identified from endophytic bacterial isolated from root nodules through the first screening in the field experiments and the second screening in the pot experiments.These five endophytic bacteria strains showed high nematicidal activity against M.incognita second-stage juveniles(J2),which was more than 85% after 24 hours of treatment,Furthermore,it inhibited egg hatching and promoted tomato seed germination and growth.The results of pot experiments indicated that root treatment with fermentation broth of five strains significantly reduced the number of root galls and egg masses,respectively,and promoted the growth of tomato plants.Greenhouse field experiments showed that root treatment with fermentation broth of strains Sneb1997 and Sneb2000 had better control effect on tomato root-knot nematode disease,at the same time,it could promote tomato plant growth and increase fruit yield.2.Identification of effective endophytic bacteria from root nodules: Strains Sneb1994,Sneb1995,Sneb1997,Sneb2000,and Sneb2001 were identified as Microbacterium oxydans,Pseudomonas brassicacearum,P.protegens,Bacillus cereus and Serratia plymuthica based upon morphological,physiological and biochemical characteristics,and 16 S r DNA sequence analysis,respectively.This is the first report on the use of M.oxydans as a biocontrol agent for M.incognita.3.Colonization of biocontrol strains Sneb1997 and Sneb2001 in tomato plants: The plasmid PMP2444,which contain the green fluorescent protein(GFP)gene,was transformed into the biocontrol strains Sneb1997 and Sneb2001 by electric shock.Transformants strains Sneb1997-gfp and Sneb2001-gfp were obtained,which could emit bright green fluorescent.The GFP-labeled strain showed the same characteristics such as growth speed and nematicidal activity as wild strain,and the plasmid PMP2444 could be stably inherited in the GFP-labeled strain.After root treatment with fermentation broth of GFP-labeled strain,strain Sneb1997-gfp and Sneb2001-gfp could colonize in tomato roots in a large number.The colonization population of strain Sneb1997-gfp reached the maximum at 15 d after inoculation,with 2.41×106 cfu per gram root.The colonization population of strain Sneb2001-gfp was lower than that of strain Sneb1997-gfp,and the population reached the maximum at 10 d after inoculation,with 8.79 × 105 cfu per gram root.In addition,strains Sneb1997-gfp and Sneb2001-gfp also were detected in tomato stems.Observation by confocal laser scanning microscopy(CLSM)showed that the strain Sneb1997-gfp colonized preferentially on tomato root hairs and root surfaces,and then accumulated in root epidermal cells,root tips and root vascular columns.Furthermore,after 20 days of tomato roots infected by M.incognita,strain Sneb1997-gfp was observed in root giant cells.However,Strain Sneb2001-gfp was observed only in tomato root hair,root surface,root epidermal cells and vascular columns.In this study,five highly effective biocontrol bacteria from 448 endophytic bacterial isolated from root nodules were screened and identified,and this is the first report on the use of M.oxydans as a biocontrol agent for M.incognita.In addition,strains Sneb1997 and Sneb2001 could colonize in tomato roots.The results provided new microbial resources for the biological control of plant-parasitic nematode disease.