Population Structure Of ’Candidatus Liberibacter Asiaticus’ In China Revealed By SSR Markers And Transcriptome Profiling Of Sweet Orange Infected With HLB

Citrus Huanglongbing (HLB) is the most devastating citrus disease worldwide. The infected trees develop a series of typical symptoms:1) yellow shoots with blotchy mottling of leaves, usually accompanied with microelement deficiency symptoms or uniformly yellowing in late stage.2) lopsided or color inversion fruits, so-called ’red nose fruits’, with a significant increase in bitter and acidic flavor.3) tree vigor declined, causing death in late stage. HLB is temporarily known to be caused by the three species of genus Candidatus Liberibacter, including ’Ca. Liberibacter asiaticus’(CaLas),’Ca. Liberibacter africanus’(CaLaf) and ’Ca. Liberibacter americanus’(CaLam). So far, its Koch’s postulates have not been completely fulfilled due to the difficulty encountered in cultivation of the pathogen in vitro, also becoming a great obstacle for study in depth and little is known on the bacterial biology and epidemiology. Since its first report in China in1913, HLB has occurred in ca.50countries, including many ones in Asian, Africa,North America and South America. To date, no curative chemicals and resistant varieties or cultivars are available, the most efficient three measures to reduce HLB spread are to pull off the infected trees, to control the insect vector psyllids (Diaphorina citri, Trioza erytreae and Cacopsylla citrisuga) in a large scale, and to use HLB-free nursery trees. Since the first occurrence reported in Sao Paulo, Brazil in2004, and in in Florida, USA2005, respectively, HLB has been becoming the biggest issue towards their citrus industries, more and more attentionhas been being attracted to control this disease, including much efforts on researches.In this study, the following three aspects have been focused:1) a few papers on’successful’ in vitro cultivation of CaLas and CaLaf had been published. However, their results could never be independently repeated in many other Labs throughout the world when I started this study. So we tried to repeat and explore the in vitro cultivation methods.2)285CaLas isolates from eight provinces in China were collected and analyzed for evaluating their diversities and population structure, The results would prompt further understanding on the origin and prevalence of HLB in China.3) the transcriptome profiling of sweet orange infected with CaLas was approached.The main results obtained are as follows.1. The qPCR method for detecting CaLas established by Teixeira was experimentally confirmed to be stable and sensitive, with lowest false positive detecting rate among all three reported qPCR methods in this study. Using the Teixeira’s qPCR method, the standard curve was established for estimating the CaLas titers during cultivation in vitro. The cultivation methods addressed in my study included those reported with ’successful’ CaLas cultivation in vitro, as well as the methods reported for other citrus fastidious bacteria, such as Xylella fastidiosa and Spiroplasma citri. Although a lot of attempts have been made in my study, the results were still negative without success. Among the eight cultivation media tested, the2G medium was able to be detected the presence of CaLas for a longer period of10-12days during cultivating, which might be the residual nucleic acid of inoculated CaLas, since no accretion of CaLas was monitored. Further efforts on seeking the key factors for cultivating of CaLas are still very much in demand.2. By selecting the reported23simple sequence repeat (SSR) loci, five SSR loci with Nei’s gene diversity index ranging from0.1542to0.9556, were identified for this study. The population structure of285CaLas isolates collected from various geographical locations in China, was analyzed by the5SSR loci. The result indicated that all isolates could be clustered into two main groups. The isolates collected from the Southeast areas mainly dropped into one group, whereas those from the Southwest areas mainly dropped into another group, but the former possessed a higher level of the population mixture than the later, which might be due to the circulation disorder of nursery trees and vector insects transmission. It was also found that populations of those isolates in Yunnan and Sichuan were constituted nearly by a single group. One exception was found in Fujian, of which the population structure was similar to those in the Southwest areas. All symptoms induced by the above two groups of CaLas isolates were nearly the same according to the observation in the field and ingraft-inocualated plants. We also compared the population structure of CaLas among China and other countries based on three loci of the five, all isolates were also clustered into two groups, those in Brazil, India, Cambodia and China were similar, dropped into one group. Notably Chinese population possessed high mixture level, which might be due to long history of HLB prevalence in China. Populations of CaLas isolates in United States mainly dropped into another group with less mixture level.3. Comparative transcriptome approach was applied to describe the response of Citrus sinensis leaves infected with CaLas using deep sequencing technology.1077differentially expressed genes (DEGs) were identified. These DEGs were assumed to be involved in a variety of different biological processes, including transport, photosynthesis, carbohydrate metabolism and plant hormones. In carbohydrate metabolism related pathways, the DEGs related to sucrose invertase were up-regulated nearly4times, the DEGs related to phosphofructokinase were down-regulated2times, the DEGs related to y-amylase also down-regulated2times. These changes might lead to starch accumulation and photosynthesis reaction repression. Few DEGs related to carbohydrate metabolism was found, which might be due to the early infection stage of C. sinensis by CaLas. The pathways of plant in response to stress were activated, including the up-regulated DEGs of ascorbate synthesis, salicylic acid signal transduction, glutathione S-transferase (GST) and heat shock protein (HSP) pathways. In photosynthesis pathways, the DEGs related to antenna proteins and light-harvesting complex pathways, were down-regulated, which would be directly negative effects on the photosynthesis reactions. Besides, the down-regulated DEGs related to auxin and cytokinin in plant hormones pathways were adverse impact on plant growth and development as well.4.CaLas was graft-inoculated into HLB sensitive cultivar Yanxiwanlu ponkan (C. reticulata) and tolerant cultivar Carrizo citrange (Poncirus trifoliata). CaLas could be detected by qPCR within49-56days post inoculation (dpi) in Yanxiwanlu ponkan, reaching high titers at105dpi, while it could not be detected in Carrizo citrange at the same time. For assuring the effectiveness of the inocula with CaLas used for grafting in this trial, thus, the shoots flushed from the inoculum buds grafted onto Carrizo citrange, were addressed to test if CaLas was there, all showed positive with CaLas. This phenomenon indicated that Carrizo citrange partially restricted the colonization of CaLas. Compared to the symptoms of the two hosts, tree vigor of Yanxiwanlu ponkan declined, with some new leaves getting yellow and smaller, whereas Carrizo citrange showed no symptom. The gene expression levels of the two cultivars on photosynthesis, plant hormones, calcium signal transduction and zinc transport proteins pathways were compared by relative qPCR method. Less DEGs related to photosynthesis pathways were down-regulated, and more DEGs inresponse to stress in plant hormone pathways were up-regulated in Carrizo citrange, and most selected DEGs expressed at higher levels, this might be associated with the tolerance of Carrizo citrange towards CaLas.