‘Candidatus Liberibacter asiaticus’ is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of ‘Ca. L. asiaticus’ infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus—the pomelo Citrus maxima and the mandarin C. reticulata ‘Tankan’—relative to their measured ‘Ca. L. asiaticus’ infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased ‘Ca. L. asiaticus’ infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. ‘Ca. L. asiaticus’ infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with ‘Ca. L. asiaticus’ scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of ‘Ca. L. asiaticus’ replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against ‘Ca. L. asiaticus’ infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of ‘Ca. L. asiaticus’ infection.
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.
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.
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.
Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to ‘Candidatus Phytoplasma rubi’ strain RS (GenBank Accession No. CP114006). To further characterize the ‘Ca. P. rubi’ strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with ‘Ca. P. rubi’ RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % ‘Ca. Phytoplasma’ strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference ‘Ca. P. rubi’ RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of ‘Ca. P. rubi’ from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.
Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I (‘Ca. P. asteris’) and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a ‘Ca. P. asteris’, 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.
Stone fruits are a multi-billion-dollar industry for the U.S. and Canada, one that has repeatedly suffered significant economic losses to outbreaks of the X-disease phytoplasma (Candidatus Phytoplasma pruni) over the last century. Orchards and entire production areas have been abandoned, with corresponding losses to growers, fruit packers, and consumers. The most recent outbreak, in the U.S. Pacific Northwest, caused an estimated $65 million (USD) in lost revenue between 2015 and 2020 and is only increasing in incidence. Already present across much of the continental U.S. and Canada, the phytoplasma has a broad host range beyond stone fruit and is transmitted by at least eight leafhopper species therefore stone fruit production in any state is at significant risk. This recovery plan was produced as part of the National Plant Disease Recovery System (NPDRS) and is intended to provide a review of pathogen biology, assess the status of critical recovery components, and identify disease management research, extension, and education needs.