Publications

4457

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

AbstractHere a stable glycogen accumulating organisms (GAOs) system was operated by anaerobic–aerobic mode in the sequencing batch reactor. We focused on the metabolic mechanisms of PHAs storage from GAOs. Our system showed the classic characteristic of glycogen accumulating metabolism (GAM). Glycogen consumption was followed by acetic acid uptake to synthesize poly-β-hydroxyalkanoates (PHAs) during the anaerobic period, and glycogen was synthesized by PHAs degradation in the aerobic stage. Microbial community structure indicated that Candidatus Contendobacter was the most prevalent GAOs. We found that the ethylmalonyl-CoA (EMC) pathway was the crucial pathway supplying the core substance propionyl-CoA for poly-β-hydroxyvalerate (PHV) synthesis in Candidatus Contendobacter. All genes in EMC pathway were mainly located in Candidatus Contendobacter by gene source analysis. The key genes expression of EMC pathway increased with Candidatus Contendobacter enrichment, further validating that propionyl-CoA was synthesized by Candidatus Contendobacter predominantly via EMC pathway. Our work revealed the novel mechanisms underlying PHV synthesis through EMC pathway and further improved the intercellular storage metabolism of GAOs.

SUMMARY‘Candidatus Phytoplasma tritici’ (‘Ca. P. tritici’) is an insect‐borne obligate pathogen that infects wheat (Triticum aestivum) causing wheat blue dwarf disease, and leads to yield losses. SWP12 is a potential effector secreted by ‘Ca. P. tritici’ that manipulates host processes to create an environment conducive to phytoplasma colonization, but the detailed mechanism of action remains to be investigated. In this study, the expression of SWP12 weakened the basal immunity of Nicotiana benthamiana and promoted leaf colonization by Phytophthora parasitica, Sclerotinia sclerotiorum, and tobacco mild green mosaic virus. Moreover, the expression of SWP12 in wheat plants promoted phytoplasma colonization. Triticum aestivum WRKY74 and N. benthamiana WRKY17 were identified as host targets of SWP12. The expression of TaWRKY74 triggered reactive oxygen species bursts, upregulated defense‐related genes, and decreased TaCRR6 transcription, leading to reductions in NADH dehydrogenase complex (NDH) activity. Expression of TaWRKY74 in wheat increased plant resistance to ‘Ca. P. tritici’, and silencing of TaWRKY74 enhanced plant susceptibility, which indicates that TaWRKY74 is a positive regulator of wheat resistance to ‘Ca. P. tritici’. We showed that SWP12 weakens plant resistance and promotes ‘Ca. P. tritici’ colonization by destabilizing TaWRKY74.

AbstractBACKGROUNDAsian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1‐21. However, the pathogen elimination and colonization potential of B. subtilis L1‐21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown.RESULTSInitially, we suggested that endophyte L1‐21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium‐Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1‐21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1‐21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1‐21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively.CONCLUSIONSWe propose that B. subtilis L1‐21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

Abstract Anaerobic ammonium oxidation (Anammox) bacteria are a group of extraordinary bacteria exerting a major impact on the global nitrogen cycle. Their phylogenetic breadth and diversity, however, are not well constrained. Here we describe a new, deep-branching family in the order of Candidatus Brocadiales, Candidatus Bathyanammoxibiaceae, members of which have genes encoding the key enzymes of the anammox metabolism. In marine sediment cores from the Arctic Mid-Ocean Ridge (AMOR), the presence of Ca. Bathyanammoxibiaceae was confined within the nitrate-ammonium transition zones with the counter gradients of nitrate and ammonium, coinciding with the predicted occurrence of the anammox process. Ca. Bathyanammoxibiaceae genomes encode the core genetic machinery for the anammox metabolism, including hydrazine synthase for converting nitric oxide and ammonium to hydrazine, and hydrazine dehydrogenase for hydrazine oxidation to dinitrogen gas, and hydroxylamine oxidoreductase for nitrite reduction to nitric oxide. Their occurrences assessed by genomes and 16S rRNA gene sequencings surveys indicate that they are present in both marine and terrestrial environments. By introducing the anammox potential of Ca. Bathyanammoxibiaceae and charactering their ideal niche in marine sediments, our findings suggest that the diversity and abundance of anammox bacteria may be higher than previously thought, and provide important insights on cultivating them in the future to not only assess their biogeochemical impacts but also constrain the emergence and evolutionary history of this functional guild on Earth.

AbstractEleven haplotypes of the bacterium, ‘Candidatus Liberibacter solanacearum’, have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for ‘Ca. L. solanacearum’ by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the ‘Ca. L. solanacearum’ 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of ‘Ca. L. solanacearum’ haplotype F was not detected in this study.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati’s blood samples in Midwestern Brazil. Twenty-five samples (25/165—15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with ‘Candidatus Anaplasma brasiliensis’, respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely ‘Candidatus Ehrlichia dumleri’, and Anaplasma sp. closely related to the previously described ‘Candidatus Anaplasma brasiliensis’.