Decipher Endophytic Bacteria In Citrus HLB In Guangxi And Developing Novel Nano-silver Ant Imicrobial Compounds

Huanglongbing(HLB)is a devastating phloem-limited bacterial(‘Candidatus Liberibacter asiaticus’,CLas)disease that generates various symptoms in leaves and fruits that threaten the global citrus industry.Several therapeutic strategies have been applied for the control of citrus HLB disease in different regions of Guangxi Zhuang Autonomous Region in the past several years.Nevertheless,there is no effective therapeutic strategy to eliminate/suppress the Candidatus Liberibacter asiaticus in HLB affected trees.This study aims to investigate the HLB affected citrus trees in different orchards in Guangxi province.Further,we analyzed the endophytic microbial communities in the different symptomatic parts of HLB affected citrus trees using 16 s RNA gene high-throughput sequencing technology.We also aim to use the nanobiotechnology route to synthesize biogenic silver nanoparticles using aqueous rhizome extract of Dysosma versipellis as a reducing agent for an antimicrobial agent against citrus HLB pathogen.The results were presented as follows:1.HLB incidence in citrus orchards: A total of 722 HLB affected samples were collected from 5 different citrus orchards in Guangxi.Orchard #1and #2 were completely investigated for CLas infection,while the remaining orchards were analyzed for the HLB symptomatic samples for HLB incidence.Q-PCR was performed to detect the CLas bacteria in the collected citrus samples.Q-PCR analysis showed that 14.4 % of fertile oranges were affected by HLB disease,whereas 6.2 % of the other varieties were affected.This discrepancy can be due to the different prevention and control measures used in five orchards to control the occurrence and spread of HLB disease.The infection rate of 4.9 %and 18.26 % were detected in 610 samples collected from Orchard #1 and #2,respectively.The disease index(DI)for orchard #1 and #2 was 2.4 % and10.98 %,respectively.The HLB detection rate of orchards #3,#4,and #5 were87.5 %,25 %,and 30.3 %,and the disease index was 84.3%,14.37 % and14.72 %,respectively.Different therapeutic strategies were applied to control the occurrence and spread of HLB in five tested orchards.Planting healthy citrus seedlings is one of the most effective management that reduces the possibility of HLB occurrence.The authorized seedlings and other preventive measures to control Asian citrus psyllid(Diaphorina citri)were not taken in Orchard #3 and#4,which resulted in a higher incidence of citrus HLB.2.Endophytic microbial community in HLB affected trees: The endophytic microbial community was carried out in both symptomatic and asymptomatic leaves affected by citrus HLB disease.This discrepancy may bedue to the difference between the microbial community and the bacterial population of CLas,which is an essential factor in the determination of HLB disease.The results indicated that there was a significant difference in the endophytic bacterial community of both HLB-affected symptomatic and asymptomatic leaves.However,the diversity of the microbiome was reduced in severely HLB affected leaves.A total of 18 endophytic bacterial genera were significantly higher in the mild HLB-affected leaves than those in the severe ones,of which Bacillus species was negatively correlated with Ca.L.asiaticus.In addition,Bacillus cohnii,Bacillus velezensis,and a large number of unknown Bacillus species were also identified.Conclusively,our study finding revealed that there was no uniformity in the microbiome communities in a high and medium level of HLB infected plants over five years.Bacillus was negatively correlated with CLas bacteria.It might be useful to isolate and identify the endogenous Bacillus species in the CLas-infected citrus plants in determining the role of these endophytic bacteria in association with CLas pathogenicity.3.Biogenic synthesis of silver nanoparticles(AgNPs): The present work described green biosynthetic procedures for the production of silver nanoparticles by using an aqueous rhizome extract of the Dysosma versipellis as a novel reducing agent.The UV–visible spectrum analysis of the synthesized AgNPs showed a broad and robust surface plasmon resonance(SPR)peak at418 nm after 60 min of reaction time.The biosynthesized AgNPs were confirmed and characterized by transmission electron microscopy(TEM),Zeta-potential and Fourier transform infrared spectroscopy(FT-IR).From TEM analysis,the synthesized AgNPs were predominately spherical with an average size of 14 nm.Zeta potential study showed that the biosynthesized AgNPs was stable.The green biosynthesized AgNPs from D.versipellis rhizome can be used in the development of antibacterial nano-emulsion formulation and effective delivery of chemical compounds into citrus phloem via a foliar spray or tree trunk injection for controlling citrus CLas bacteria.