The Pathogens And Intraspecies Differentiation Of Candidatus Liberibacter Asiaticus Of HLB In P.R. China

Citrus Huanglongbing (HLB) is one of the most destructive diseases in the world, which produced show blotchy mottle, yellowing, nutrition deficiency, red nose symptoms and other atypical symptoms in the citrus plants. HLB could infect almost all the citrus cultivars, and caused substantial economic losses by reducing the fruit production and shortening the lifespan of trees, and even killing trees. During 2004-2007, more than 2 million HLB-infected trees have been killed in Sao Paulo State, Brazil and also about 40 million of citrus trees have been killed for this reason in Guangdong, Guangxi and Fujian, China.The agent of HLB is a phloem-restricted, non-cultured, Gram-negative bacterium. The HLB bacterium belongs to a new genus in the alpha subdivision of the Proteobacteria, Rhizobiales, Rhizobiaceae, and was assigned as the Candidatus status, three species of which are currently known as Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. In present, quarantine measure, disease-free citrus plants, timely removal of HLB-infected trees and control of psyllid vector populations are used as the most effective way to control HLB.In order to investigate the kinds of pathogens which associated with HLB, and the diversity of the Ca. Liberibacter asiaticus in China, PCR or nested PCR were used to detect 359 citrus samples with characteristic or atypical symptoms of HLB which collected from 7 provinces Guangxi, Guangdong, Yunnan, Jiangxi, Fujian, Hunan and Sichuan during 2008-2009, and citrus samples with blotchy mottle, "red nose" yellowing, nutrition deficiency symptoms and other asymptomatic were examined and characterized the pathogens which associated with HLB. Furthermore, the diversity of the Ca. Liberibacter asiaticus in China was detected by sequencing and the polymorphism of omp gene was analyzed by restriction endonuclease enzymes TaqI, BclI, ApoI, MssI, SmiI, SspI and EcoRI.The main results were as follows:1. 237 (60.02%) citrus samples were infected with HLB, and the agent was Ca. Liberibacter asiaticus. Moreover,18(5.01%) samples were positive with phytoplasma, and 7 samples were co-infected with Ca. Liberibacter asiaticus.2. Of the 237 Ca. Liberibacter asiaticus positive samples,173 (73%) showed blotchy mottle,30 (13%) showed "red nose",19 (8%) showed yellowing,10 (4%) showed nutrition deficiency, and 5 (2%) showed asymptomatic. Meanwhile, of the 18 phytoplasma positive samples,11(61%) showed yellowing,7 (39%) were showing nutrition deficiency, and no infected-phytoplasma sample was showing blotchy mottle and "red nose", it suggested that phytoplasma might not have direct relationship to HLB.3. All of the amplified fragments from phytoplasma were sequenced and the result showed that they belonged to the Candidatus Phytoplasma asteri.4. To investigate the diversity of the Ca. Liberibacter asiaticus, the omp genes of 23 Ca. Liberibacter asiaticus isolates with different symptoms from seven provinces in China were analyzed by restriction enzymes TaqI, BclI, ApoI,MssI,SmiI,SspI and EcoRI. The results showed that the omp gene of all the isolates was able to be digested by all of the enzymes except Mssl. ApoI and TaqI provided more polymorphism than others. The PCR-RFLP analysis also suggested that all the 23 Ca. Liberibacter asiaticus isolates be separated into 6 groups and all of them contain the single group. The isolates causing blochy mottle had more polymorphism.5. Comparison of all the omp gene sequences of Ca. Liberibacter asiaticus isolates to the GenBank database showed that all the isolates shared 99.3-99.6% identity with Poona isolate (AY642159) and 72.4%-72.7% identity with Nelspruit isolate (AY642158), respectively. BLAST analysis showed that nucleotide sequences of the 23 Ca. Liberibacter asiaticus isolates had high BLASTn match with Ca. Liberibacter asiaticus DNA sequences in GenBank, and omp gene revealed highly conservation among the 23 Ca. Liberibacter asiaticus isolates (99.3% to 99.9%), although these isolates were collected from different areas, hosts and different symptoms. With phylogenetic tree analysis, all the Ca. Liberibacter isolates were separated into two major groups. The Ca. Liberibacter africanus (AY642158) belongs to one group, the 23 Ca. Liberibacter asiaticus isolates and other Ca. Liberibacter asiaticus isolates belong to the other that was separated into 3 subgroups. The first subgroup contained HN-ay and GX-nfmj and the second subgroup contained the isolates from Indonesian (AB480132), Vietnamese (AB480111), Nepal (AY842430), India (AY642159) and SC-qc, and the rest ones belong to the third subgroup.