Screening And Nematicidal Mechanism Of Bacillus Altitudinis AMCC1040 Against Root-knot Nematode

Root-knot nematodes belonging to genus Meloidogyne are one of the important microscopic obligate biotrophic plant parasites,and cause huge economic damage to agriculture throughout the world.In the past few decades,various nematicides and soil fumigants have been widely used for root-knot nematode disease control,however,these highly toxic pesticides have caused serious problems for the environment and human health,as well as the multi-drug resistance of nematodes,hence,many alternative strategies such as using biological agents for plant-parasitic nematode control have been investigated.As an important natural enemy of nematodes,bacteria have been received more attention on developing into commercially available biological agents to control root-knot nematodes.In this study,the diversity and nematicidal activity of cultivable bacteria from suppressive soil in Shandong province,China was investigated.The obtained bacteria strain was identified based on 16S r RNA gene sequence,morphological characteristics and the physiological and biochemical properties.For an important nematicidal bacteria strain Bacillus altitudinis AMCC1040,the nematicidal factors was analysed,meanwhile,the mechanism of virulence factors was further studied in combination with physiological,morphology and transcriptome of nematodes.Field experiments were conducted to evaluate the biocontrol potential of Bacillus altitudinis AMCC1040 against ginger root-knot nematode disease,and to evaluate its ability to colonize the rhizosphere dected by real time-PCR.Futhermore,we have assessed the ecological safety of Bacillus altitudinis AMCC1040 based on its impact on the rhizosphere microbial communities.The main results are as follows:(1)Diversity and nematicidal activity of culturable bacteria from suppressive soils in Shandong province,China.In this study,a total of 110 isolates were obtained from 13sampling sites.Using phylogenetic analysis the 110 sequenced isolates were clustered into16 genera belonging to four phyla,Actinobacteria,Bacteroidetes,Firmicutes,and Proteobacteria,indicating a high diversity of culturable bacteria in suppressive soil.Among the genera,Bacillus was most abundant,suggesting that Bacillus species are well adapted to the rhizosphere soil micro-environments.(2)Screening and identification of effective nematicidal bacteria strains.A direct-contact bioassay was used to evaluate the mortality of M.incognita J2s exposed to the supernatant of all 110 bacterial strain cultures.Among the isolates,41.44%showed high nematicidal activity(>50%).Twenty-three of these isolates caused>75%mortality of second-stage juveniles of the root-knot nematode Meloidogyne incognita in in vitro assays,particularly Bacillus sp.AMCC1040 which were found to cause 100%J2 mortality.Meanwhile,in pot experiments,Bacillus sp.AMCC1040 can also significantly reduce root galling,soil J2 density and egg-mass production.According to the phylogenetic tree,morphological characteristics,and physiological and biochemical characteristics,the bacteria strain AMCC1040 was identified as Bacillus altitudinis.(3)Biocontrol potential of Bacillus altitudinis AMCC1040 against root-knot nematode disease of ginger and its impact on rhizosphere microbial community.Compared with the negative control,the nematode populations in the soil and roots were reduced by 84.48%and 93.68%in Bacillus altitudinis AMCC1040 treatment,there were even 46.48%and58.59%reductions,compared with the positive control.In addition,the application of the bacterial strain significant reduced the nematode reproduction index because it significantly reduced females in the roots.The lowest single root knot or beaded root knot recorded from the Bacillus altitudinis AMCC1040 control was reduced by 63.61%and 70.48%,respectively compared with negative control.Compared with the negative control,the biocontrol efficacy of Bacillus altitudinis AMCC1040 was 57.14%.The amount of Bacillus altitudinis AMCC1040 per gram of soil varied from 5.08 to 5.49 Log₁₀CFU,indicating that the biocontrol microorganism could successfully survive in the rhizosphere soil of ginger.PCo A was performed using the weighted Bray-Curtis metric to analyze the differences and similarities of microbial communities between treatments.The results showed that there were significant differences in the bacterial microbial community structures between three treatments,especially the comparison of positive control with the other two treatments.The above results showed that the application of fosthiazate can significantly affected the soil microecology,while the effect of Bacillus altitudinis AMCC1040 has a small effect on the structure of the soil microbial community.Linear discriminant analysis of effect size(LEf Se)with LDA scores>3 confirmed significantly(P<0.05)differentiated bacterial taxa in different treatments.It should be noted that the taxa enriched in Bacillus altitudinis AMCC1040 treatment were found in Actinobacteria,Gemmatimonadetes and Firmicutes.(4)The investigation of nematicidal substances produced by Bacillus altitudinis AMCC1040.The results of basic properties of active substances emited by Bacillus altitudinis AMCC1040 showed that the compounds had good thermal stability and passage stability,resistant to acid environment while sensitive to alkali conditions.Further analysis showed that it was a volatile component.HS-SPME-GC/MS analysis showed that eight characteristic volatile components were detected in the fermentation supernatant of AMCC1040.Six of them had strong nematicidal activities,including 2,3-butanedione,acetic acid,2-isopropoxy ethylamine,3-methylbutyric acid,2-methylbutyric acid and octanoic acid.Among them,octanoic acid had the strongest nematicidal activity with0.03?L/m L LC_100/12hvalue.(5)Study on the nematicidal mechanism of octanoic acid produced by Bacillus altitudinis AMCC1040 on Meloidogyne incognita.The mechanism of action of octanoic acid was analyzed by transcriptome analysis,morphological observation,physiological and biochemical analysis.The results showed that octanoic acid had an obvious concentration-time relationship.0.03?L/m L was the lowest nematicidal concentration,which was lower than this concentration and had no nematicidal activity on nematodes even after treatment for 48h.At the same time,0.08?L/m L was the characteristic concentration for killing nematodes rapidly,under this concentration,it only took 15min to kill nematodes completely.Changes in the morphology of root knot nematode,M.incognita were observed by light microscopy after incubation with octanoic acid at concentrations of 0.03?L/m L and0.08?L/m L.In the water control,the J2s were active,healthy with normal shapes.Treatment with 0.03?L/m L killed J2s but did not modify J2s structures significantly even after treatment for 72h.However,at 0.08?L/m L,when treated for 24h,J2s were completely dead which accompanied by the formation of multiple giant vacuoles,subsequently,the J2s body became incomplete,structures of intestine,pharynx and other organs were disorganized after 72h of treatment.The GO and KEGG enrichment analysis of DEGs of J2s treated with the above two concentrations of octanoic acid,combined with physiological,biochemical and RT-PCR verification experiments,it was proved that octanoic acid can interfere with the nematode's energy metabolism and signal transmission,and destroy the nematode cuticle structure,it can also inhibit the nematode's nerve and motor system,which eventually leads to the paralysis and death.In addition,nematodes will activate their own defense system when responding to low concentrations of octanoic acid,and resist the damage of octanoic acid by up-regulating the defense enzyme system and heterologous metabolic pathways.