Publications

4481

Western X (WX) disease, caused by ‘Candidatus Phytoplasma pruni’, is a devastating disease of sweet cherry resulting in the production of small, bitter-flavored fruits that are unmarketable. Escalation of WX disease in Washington State prompted the development of a rapid detection assay based on recombinase polymerase amplification (RPA) to facilitate timely removal and replacement of diseased trees. Here, we report on a reliable RPA assay targeting putative immunodominant protein coding regions that showed comparable sensitivity to polymerase chain reaction (PCR) in detecting ‘Ca. Phytoplasma pruni’ from crude sap of sweet cherry tissues. Apart from the predominant strain of ‘Ca. Phytoplasma pruni’, the RPA assay also detected a novel strain of phytoplasma from several WX-affected trees. Multilocus sequence analyses using the immunodominant protein A (idpA), imp, rpoE, secY, and 16S ribosomal RNA regions from several ‘Ca. Phytoplasma pruni’ isolates from WX-affected trees showed that this novel phytoplasma strain represents a new subgroup within the 16SrIII group. Examination of high-throughput sequencing data from total RNA of WX-affected trees revealed that the imp coding region is highly expressed, and as supported by quantitative reverse transcription PCR data, it showed higher RNA transcript levels than the previously proposed idpA coding region of ‘Ca. Phytoplasma pruni’.

‘Candidatus Liberibacter asiaticus’ is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of ‘Ca. L. asiaticus’ genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 ‘Ca. L. asiaticus’ strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that ‘Ca. L. asiaticus’ populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ‘Candidatus Liberibacter asiaticus’ and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ‘Ca. L. asiaticus’ inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ‘Ca. L. asiaticus’. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ‘Ca. L. asiaticus’ transmission by ACP. In this study, we report a new method for targeted early detection of ‘Ca. L. asiaticus’ in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that ‘Ca. L. asiaticus’ remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ‘Ca. L. asiaticus’ in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ‘Ca. L. asiaticus’ inoculum removal and preventing HLB from spreading.

Abstract We used phenotypic, genomic and phylogenetic information following the taxono-genomics approach to demonstrate that strain Marseille–P3254, isolated from an ileal sample of a 76-year old woman who underwent upper and lower digestive tract endoscopy for esophagitis and colonic polyp, is representative of a novel bacterial genus within the family Erysipelotrichaceae in the phylum Firmicutes. It is an anaerobic Gram-negative bacterium without catalase and oxidase activities. The genome of strain Marseille–P3254 is 2,468,496-bp long with a 40.1% G + C content. This new bacterium is most closely related to Eubacterium dolichum, with which it shares 90.7% 16S rRNA sequence similarity. In addition, genomic comparison using the digital DNA–DNA hybridization and OrthoANI analyses between the novel organism and the E. dolichum type strain revealed identities of 25.2 and 68.91%, respectively. The major fatty acids were C16: 0, C18: 1n9 and C18: 0. Based on these data, we propose the creation of the new genus Merdibacter gen. nov., with strain Marseille-P3254T (=CSUR P3254 = DSM 103534) being the type strain of the new species Merdibacter massiliensis gen. nov., sp. nov.