“Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China.
CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using Pacbio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G+C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.
AbstractCandidatusLiberibacter asiaticus (CLas) is the causal agent of the devastating citrus Huanglongbing (HLB) disease. Our understanding of the pathogenicity mechanism and biology of CLas remain limited because CLas has not been cultured in artificial media. CLas encodes 1136 proteins of which 415 have unknown functions. Since genetic studies of CLas genes with unknown functions are impossible, we utilized genome-wide protein-protein interactions (PPIs) yeast-two-hybrid (Y2H) assays to help solve the mystery. PPIs are fundamental to all cellular processes and machinery and instrumental in investigating uncharacterized proteins and inferring biological pathways. In total, 916 bait and 936 prey proteins were included in the three-phase screening, which identified 4245 interactions. The false positive rate of the Y2H assay was estimated to be 3.1%. Pull-down assays confirmed the robustness of our Y2H. The average interactions per node for CLas Y2H interactome were approximately 15.6, significantly higher than free-living bacteria, indicating genome reduction has led to a multi-function of proteins. PPIs provide clues for functions of 371 uncharacterized proteins of CLas. Forty HUB node proteins were identified which might play critical roles in CLas, including a quinone oxidoreductase and LysR that are known to protect bacteria against oxidative stress. This explains why CLas survives well in the phloem even though it triggers immune-mediated disease, systemic and chronic production of reactive oxygen species, and phloem cell death. This PPI database facilitates the investigation of CLas cellular biochemistry and physiology, functions of uncharacterized proteins, and pathogenicity mechanisms of the pathogen.
The beet leafhopper, Circulifer tenellus, is an important pest of agricultural crops in the United States, where it transmits Beet curly top virus, Beet leafhopper-transmitted virescence agent phytoplasma and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects Beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast, accurate results to growers for implementation in their insect pest monitoring programs.
Bamboo is used for making structures, furniture, handicrafts, and ropes, as well as a source of food in the Philippines. One of the emerging diseases of bamboo in the country is the bamboo witches’ broom (BWB), which has been occasionally noted in three genera of bamboo – including Dendrocalamus, Gigantochloa, and Schizostachyum from various provinces in the Philippines (Ilocos Norte, Laguna, Batangas, Quezon, Agusan del Sur, Bukidnon, and South Cotabato) since the 1990s. However, studies and information about BWB in the country remain lacking and largely unexplored. In this study, we report a similar disease affecting Dendrocalamus and Gigantochloa bamboo species from Bohol and Davao and – for the first time – in Dendrocalamus merrillianus ("bayog") and Bambusa spinosa ("kawayan-tinik") from Isabela and Nueva Vizcaya. As a result of the surveys conducted from 1999–2019, the disease is now identified in six species across four genera of bamboo – namely, Gigantochloa spp. (G. levis and G. atter), Dendrocalamus spp. (D. asper and D. merrillianus), Schizostachyum lumampao, and Bambusa spinosa recorded in 11 provinces in the Philippines. The BWB symptoms include clustering of leaves forming a rosette-like structure, leaf proliferation, excessive limb formation from a single node, and shortening of internodes. Nested PCR using the universal primers P1/P7 and R16MF2/R1 targeting the phytoplasma 16S ribosomal RNA gene revealed positive amplification in five symptomatic BWB samples from Isabela, Philippines. Subsequent sequencing (~ 1.3kbp) and phylogenetic analysis using the representative BWB isolates from Isabela revealed > 98.65% genetic similarity and clustering to Candidatus Phytoplasma luffae, which belongs to the 16SrVIII group (Loofah Witches’ Broom Group). This paper determined the distribution of BWB in different species of bamboo in the Philippines, as well as the association of 'Ca. Phytoplasma luffae'-related strain (16SrVIII) to BWB.
AbstractMany insects engage in stable nutritional symbioses with bacteria that supplement limiting essential nutrients to their host. While several plant sap-feeding Hemipteran lineages are known to be simultaneously associated with two or more endosymbionts with complementary biosynthetic pathways to synthesize amino acids or vitamins, such co-obligate symbioses have not been functionally characterized in other insect orders. Here, we report on the characterization of a dual co-obligate, bacteriome-localized symbiosis in a family of xylophagous beetles using comparative genomics, fluorescence microscopy, and phylogenetic analyses. Across the beetle family Bostrichidae, most investigated species harbored the Bacteroidota symbiont Shikimatogenerans bostrichidophilus that encodes the shikimate pathway to produce tyrosine precursors in its severely reduced genome, likely supplementing the beetles’ cuticle biosynthesis, sclerotisation, and melanisation. One clade of Bostrichid beetles additionally housed the co-obligate symbiont Bostrichicola ureolyticus that is inferred to complement the function of Shikimatogenerans by recycling urea and provisioning the essential amino acid lysine, thereby providing additional benefits on nitrogen-poor diets. Both symbionts represent ancient associations within the Bostrichidae that have subsequently experienced genome erosion and co-speciation with their hosts. While Bostrichicola was repeatedly lost, Shikimatogenerans has been retained throughout the family and exhibits a perfect pattern of co-speciation. Our results reveal that co-obligate symbioses with complementary metabolic capabilities occur beyond the well-known sap-feeding Hemiptera and highlight the importance of symbiont-mediated cuticle supplementation and nitrogen recycling for herbivorous beetles.