Huanglongbing, a globally devastating citrus disease, is associated with Candidatus Liberibacter asiaticus (CLas) and is mainly transmitted by Diaphorina citri. Verification of the distribution and dynamics of CLas in D. citri is critical to understanding CLas transmitted by vectors in nature. Here, the distribution and titers of CLas in different sexes and tissues of D. citri adults were investigated by fluorescence in-situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR). Results showed that CLas had widespread distribution in the brain, salivary glands, digestive system, and reproductive system of both females and males, indicating a systemic infection of CLas in D. citri. Moreover, CLas fluorescence intensity and titers were significantly increased in both the digestive system and the female reproductive system with development and there was a marked decreased in both the salivary glands and the male brain, but there was no significant change in the female brain or the male reproductive system. Furthermore, the distribution and dynamics of CLas in embryos and nymphs were investigated. CLas was observed in all laid eggs and subsequent first–second-instar nymphs, indicating that a high percentage of embryos and nymphs resulting from infected D. citri mothers were infected with CLas.
The phytoplasma “
Candidatus
Phytoplasma rubi” is associated with
Rubus
stunt disease. The complete genome was determined by assembling Oxford Nanopore Technologies system-derived long reads, with short-read polishing with Illumina reads. The genome of strain RS, from Germany, is organized in one circular chromosome with a length of 762 kb.
We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete “
Candidatus
Borrelia fainii” Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.
Vicia faba L. commonly known as broad bean or faba bean is one of the most widely grown protein rich legume crops. Out of more than 50 faba bean-producing countries, about 90% production is concentrated in the Asian, European Union (EU), and African region (FAO, 2020). Owing to its high nutritional value, both the fresh pods and dry seeds are consumed. During March 2022, some plants with little leaf and phyllody symptoms such as leaf-like floral structures were observed in the experimental fields of Indian Agricultural Research Institute (IARI), New Delhi (Fig. 1 a, b, c). The twig samples were collected from two individual symptomatic and one from asymptomatic plant. DNA was extracted using CTAB (cetyl trimethyl ammonium bromide) method (Ahrens and Seemüller, 1992; Marzachi et al. 1998) and examined for the association of phytoplasma through nested PCR using the universal primers P1/P7 and R16F2n/R16R2 targeting the 16SrRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and the other set of primers secAfor1/secArev3 and secAfor2/secArev3 targeting secA gene (Hodgetts et al. 2008). The DNA from symptomatic plants resulted the amplicons of 1200bp and 840bp specific to 16S rRNA and secA gene respectively. The gel purified PCR products were cloned into pGEM®-T Easy Vector system (Promega) and outsourced for Sanger sequencing at Agri Genome Labs, Kerala, India. The resultant 16S rRNA sequences (GenBank Acc. No. OP978231, OP978232) and secA sequences (ON715392 and ON715393) were examined through NCBI BLASTn analysis. The 16S rRNA sequences of the V. faba strains shared a minimum of 99.85% similarity with the phytoplasma strain causing little leaf and phyllody disease of sesame in India (MW622017) and a maximum of 100% sequence identity with the Vigna radiata phyllody and necrosis phytoplasma strain of Jodhpur (OP935760) India, whereas the secA gene sequences showed 100% identity with Tephrosia purpurea witches'-broom phytoplasma (MW603929) from China and a minimum of 91.14% similarity with ‘Candidatus Phytoplasma aurantifolia’ (MW020541) from India. The pairwise comparison results were completely in support of the corresponding phylogenetic sequence analysis results of 16SrRNA and secA gene sequences of faba bean strains in comparison with other strains retrieved from GenBank database, wherein the faba bean strains got clustered with 16SrII-D subgroup related strains (Fig. 2 a and b). Virtual RFLP analysis through iPhyClassifer tool through in silico digestion of R16F2n/R2 region of 16S rRNA gene of the faba bean strain using 17 restriction endonuclease enzymes resulted in the RFLP profiles similar to that of the profile of phytoplasma subgroup 16SrII-D (Y10097: papaya yellow crinkle) used as reference strain with a similarity coefficient value of 1.0. All the results of this investigation confirmed the association of ‘Candidatus phytoplasma aurantifolia’ (16SrII-D) with the diseased faba bean plants in this study. Previous reports of phytoplasma infecting faba bean include a group 16SrIII strain detected in Spain in 2004 (Castro and Romero), a subgroup 16SrII-D strain detected in Sudan in 2012 (Alfaro-Fernandez et al.), a group 16SrII strain detected in Saudi Arabia in 2014 (Al-Saleh and Amer), and subgroup 16SrIII-J strains detected in Egypt in 2014 (Hamed et al.) and in Peru in 2021 (Torres-Suarez et al.). To the best of our knowledge, these findings, document the first report of the association of ‘Candidatus Phytoplasma aurantifolia’ (subgroup 16SrII-D) with faba bean plants in India. This report necessitates further research on the status of distribution of this phytoplasma strain in other locations and hosts in the country so as to develop possible strategies to contain its further spread and management of the disease.
Two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani', threaten sugar beet production in France, Switzerland and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to 'Ca. P. solani' is found predominant in Saxony-Anhalt, unlike in France, where 'Ca. P. solani' has a minor role compared to 'Ca. A. phytopathogenicus'. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The MLSA of non-ribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported 'Ca. P. solani' strains including strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020, and also in Bavaria in southern Germany. Based on 16S rDNA analysis, 'Ca. A. phytopathogenicus' in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany, suggest that more attention should be directed towards understanding phytoplasma infection in sugar beet in this country.
AbstractPhytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches’ broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete ‘Candidatus Phytoplasma ziziphi’ chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.
Abstract
Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) to manipulate autophagy are unknown. We identified a CLas effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. Transgenic SDE4405 in Wanjincheng orange (Citrus sinensis) promotes CLas proliferation and symptom expression via suppressing host immunity response. SDE4405 interacts with ATG8-family proteins (ATG8s) and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing NbATG8s reduces Pseudomonas syringae pv. tomato strain DC3000ΔhopQ1-1 (Pst DC3000ΔhopQ1-1) proliferation in N. benthamiana, and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting SDE4405-ATG8s interaction negatively regulates plant defense. These data show the role of the CLas effector protein in manipulating autophagy and provide new insights into the molecular interaction between CLas and citrus.