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An active miniature inverted-repeat transposable element (MITE), MCLas-A, was previously identified from ‘CandidatusLiberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus’ strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus’ strains were analyzed by next-generation sequencing (NGS) approach. PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80% (7/389) and the jumping-out type of the MITE was predominant (81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus’ strains recently collected from China. Biological roles of transposition of the active MITE remain to be determined.

Citrus Huanglongbing (HLB, yellow shoot disease) is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR (ddPCR) assay was developed for the rapid detection of ‘CandidatusLiberibacter asiaticus’ (Las), the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that ddPCR was superior to quantitative PCR (qPCR) for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples (46.15%) with high Ctvalue (>35) were positive by ddPCR. This methodology showed great potential for early HLB infection diagnosis.

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-derived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus(CYVCV) isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 (JX040635) and YN (KP313242), both of which belong to the genus Mandarivirusin the family Alphaflexiviridae.Mapping analysis of viral-derived siRNA (vsiRNA) profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

Two miniature inverted-repeat transposable elements (MITEs), MCLas-A and MCLas-B, were recently identified from ‘CandidatusLiberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus’ isolates from China, the United States and Brazil. Out of the 12 sequence types, three (T4, T5-2, T6) were reported for the first time. Recombination events were found in the two unique sequence types (T5-2 and T6) which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, São Paulo isolates and a few Florida isolates.