Study On Establishment And Application Of A Semi-in Vitro Culture System For "Candidatus Liberibacter Asiaticus" In Citrus

Citrus Huanglongbing（HLB）is the most devastating disease in citrus worldwide.At present,HLB has occurred in more than 50 countries in the world,which has caused serious economic losses to citrus industry in many countries including China.HLB is caused by a phloem-limitedα-Proteobacterium“Candidatus Liberibacter”.According to the sequence characteristics of 16S r DNA,they can be divided into three species:Candidatus Liberibacter asiaticus（CLas）,Ca L.africanus（CLaf）and Ca L.americanus（CLam）.Among them,CLas is the most widely distributed and most harmful in the world and is also the pathogen of HLB in China.There are no resistant commercial citrus varieties and no effective cures so far.Once a citrus plant is infected with CLas,it has to be cut down.CLas has not been successfully cultured in vitro to date.The pathogenesis of HLB is still unclear,which brings great challenges to the prevention and control of the disease.Furthermore,the titer of CLas in citrus trees was low and its distribution in trees was uneven,which has greatly limited the study of pathogen biology and pathogen-plant host interactions.In this study,an efficient semi-in vitro culture system for CLas was established through in vitro regeneration of axillary buds from CLas-infected citrus plants.Using the semi-in vitro culture system,the multiplication of CLas and the community structure of endophytic bacteria in the regenerated shoots were investigated by quantitative polymerase chain reaction（q PCR）and high-throughput sequencing,respectively.To understand the response mechanisms of citrus to CLas infection under semi-in vitro culture condition,transcriptomic profiles of the regenerated shoots was constructed by RNA-seq.In addition,two nanocarriers（carbon dots,CDs）that can both excite fluorescence and bind tetracycline molecules were synthesized.The transport and distribution of CDs and drug-carrying nanoparticles in citrus seedlings were analyzed by in vivo fluorescence imaging and fluorescence detection in tissue sections.The effect of CDs based drug carrier against CLas was evaluated using the the semi-in vitro culture system.The main results are as follows:1.Establishment of an efficient semi-in vitro culture system for CLasFirstly,rapid multiplication of CLas in the regenerated shoots of axillary bud was detected through in vitro culturing stem segments derived from the HLB-affected‘Changyecheng’（Citrus sinensis）plant.A high CLas titer was detected in the regenerated shoots on an optimized medium at30 days post bud initiation（dpi）.This titer was 28.2-fold higher than in the midribs corresponding to the axillary buds of CLas-infected trees under the terms of greenhouse.To minimize culture contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’and cultured in a liquid medium.Then efficient semi-in vitro culture system for CLas was established through in vitro culture of citrus plantlets.In the semi-in vitro culture system,CLas could be detected in 10%,55%and 70%regenerated shoots at 10,30 and 40dpi,respectively,and the bacterial population rapidly increased by more than 1,600 times from 10 to30 dpi（10⁴CLas cells/μg citrus DNA at 10 dpi and approximately 10⁸CLas cells/μg citrus DNA at30 dpi）,and also the percentages of shoots with>10⁸ CLas cells/μg citrus DNA were very high（30%at 30 dpi and 40%at 40 dpi）.Direct tissue blot immune assay（DTBIA）showed that the distribution of CLas was much more evenly in regenerated plantlets than that in CLas-infected trees under the terms of greenhouse.Defoliation symptoms were observed in the CLas-infected citrus plantlet in vitro at the early stage of 10 dpi.In the semi-in vitro culture of CLas,the main symptoms in plantlets were defoliation,stunted growth,leaf necrosis/yellowing,and die-back,and the mortality rates of the plantlets increased rapidly from 19.9%to 82.0%during 40 to 60 dpi.2.Characteristics of the endophytic bacterial community in the host of CLas in semi-in vitro cultureTo investigate the composition and structure of the endophytic bacterial community in semi-in vitro culture,the bacterial species in CLas-infected citrus plantlets were characterized by high-throughout sequencing.Firstly,citrus seedlings were infected with CLas by micrografting.When the CLas concentration enriched to high level above 10⁷ CLas cells/μg citrus DNA in plantlets,the endophytic bacterial communities in plant tissue were analyzed by Illumina Mi Seq sequencing for bacterial 16S r DNA.A total of 535 operational taxonomic units（OTUs）were obtained and 116OTUs were annotated to species level.Species annotation showed that those endophytic bacteria belonged to 21 phyla,49 classes,106 orders,156 families,242 genera.There were significant differences in richness and diversity between the CLas-infected and healthy citrus plantlets.In healthy citrus plantlets,Cyanobacteria were the most abundant bacterial phylum;however,the most abundant phyla were Proteobacteria,Firmicutes and Actinobacteriota in the citrus plantlets for semi vitro culture of CLas.The dominant genera of endophytic bacteria in CLas-infected citrus plantlets in vitro were Candidatus Liberibacter,Bacillus,Alcaligenes,Brevundimonas,Burkholderia,Stenotrophomonas,Cutibacterium,Serratia,Methylobacterium,and Staphylococcus.In addition to CLas,B.marcescens,A.faecalis,Burkholderia,and Cutibacterium can be used as biomarker between the CLas-infected and healthy group.Correlation analysis showed that there was a significantly positive correlation between the richness of B.marcescen,A.faecalis and CLas,suggesting that B.marcescens and A.faecalis have symbiotic relationship with CLas.Our data showed that the diversity of endophytic bacteria was abundant in the host of CLas in semi-in vitro culture.3.Transcriptomic profiles of citrus in response to CLas infection in semi-in vitro cultureTo explore molecular mechanisms of HLB-susceptible‘Changyecheng’sweet orange in response to CLas infection in semi-in vitro culture,transcriptomic comparision of the regenerated shoots derived CLas-infected and healthy‘Changyecheng’plant at 10 and 20 dpi were performed using RNA-seq.Significant changes were detected in transcription profiles in response to CLas during the early stage of germination.At 10 dpi,a total of 1256 differentially expressed genes（DEGs）were obtained.Among them,836 DEGs were up-regulated and 420 DEGs were down-regulated in CLas-infected shoots.A total of 4365 DEGs were identified at 20 dpi,but 56%of DEGs were down-regulated expression（2474 DEGs down-regulated and 1891 DEGs up-regulated）.GO functional enrichment analysis showed the significantly enriched GO terms of DEGs at 10 dpi were mainly involved in molecular function,including signal recognition,transcriptional regulation,and redox,etc.,while the GO terms significantly enriched by DEGs at 20 dpi were related to the components of molecular functional,cellular component,and biological processes.KEGG enrichment analysis revealed that the up-regulated DEGs at 10 dpi involved in plant-pathogen interaction,plant MAPK signal and hormone signal transduction pathways.DEGs functional classification was further performed using Map Man.The results showed that 407 DEGs were involved in plant response to biotic stress,such as signal transduction,transcription regulation,hormone metabolism,resistance proteins,cell wall metabolism,protein degradation,redox reaction and secondary metabolism.Overall,genes related to signal transduction and transcriptional regulation in response to biotic stress was significantly induced by CLas infection.Salicylic acid and jasmonic acid and ethylene signal transduction pathways were positively affected by CLas infection while auxin and gibberellin synthesis and signaling were negatively affected.Notably,receptor-like protein kinase gene（RLP15,RLP56）,disease-resistant genes（EDS1,TIR-NBS-LRR gene,NPR1,NDR1,PR1,PR4 and PR5a）,lipoxygenase gene（LOX2）and phloem protein gene（PP2-B10）were significantly up-regulated in the CLas-infected regenerated shoots at 10 and 20 dpi.Many genes such as RIN4（a negative regulator of resistance to pathogens）,ethylene response factor gene（ERF1B and ERF2）,ACC synthase gene（ACS1）and leaf senescence gene SAG12 that regulates programmed cell death,were also up-regulated.Totally,our RNA-seq showed that many pathways including defense related signalings and functions were significantly regulated by CLas infection in semi-in vitro culture.4.Nanocarrier can effectively enhance the bactericidal activity of tetracycline hydrochloride（TC）to CLas in citrus plantlets in semi-in vitro cultureTo evaluated effects of nanocarriers on the bactericidal activity of tetracycline hydrochloride（TC）to CLas,two kinds of nanocarriers were prepared:green fluorescent carbon dots（CDs^a）and blue fluorescent carbon dots（CDs^b）.The optimal emission wavelength of the CDs^a was 502 nm with green fluorescence and the optimal emission wavelength of the CDs^b was 443 nm with blue fluorescence.The optimal excitation wavelength of the two CDs is 360 nm.The average particle size of the two CDs is less than 10 nm.Both CDs particles have–OH,–NH₂,–C=O active functional groups on the surface,which can be combined with TC molecules and can be used as its nanodrug carrier.In vivo fluorescence imaging and fluorescence detection of CDs in tissue section showed that the two types of CDs and TC loaded nanoparticles（CDs^a-TC and CDs^b-TC）could be absorbed through roots of citrus seedling and transported to stems and leaves.CDs can also be absorbed through leaf surfaces and transported to stems and roots.Unique fluorescence spectra was also observed in tissue sections of citrus seedlings uptake of TC by fluorescent microscope,but the fluorescence distribution showed that the concentration of TC in phloem tissues was low.However,the distribution of CDs^a-TC and CDs^b-TC in the phloem of root,stem and leaf of citrus seedlings was significantly enhanced,indicating that both the nanocarriers could effectively improve the penetration of TC into the phloem.The phytotoxicity of the two CDs was low,but TC is toxic to citrus plants.When TC concentration was higher than 500 mg/L,seed germination rate and root elongation of citrus were seriously suppressed as well as the antioxidant enzyme systems in citrus seedlings were damaged.The bactericidal activity of CDs^a-TC,CDs^b-TC and TC with concentrations of 150,300 and 450 mg/L to CLas were further determined by soaking the root of CLas-infected citrus plantlets in semi-in vitro culture.The results showed that the survival rate of infected citrus plantlets in the treatment group was significantly higher than that of the control group,and the positive detection rate of CLas was significantly lower than that in the control group.The positive rate of CLas in the CDs^a-TC and CDs^b-TC treatment group was also lower than that of the TC treatment group.In addition,PCR detection results of CLas in the materials of the two treatment groups（300 mg/L CDs^a-TC and 450 mg/L CDs^b-TC）were all negative.The results indicated that nanocarrier could effectively enhance the disinfection effectiveness of tetracycline on CLas.