Phytoplasma Research Articles

Mediterranean pine forest decline: A matter of root-associated microbiota and climate change

Forest ecosystems worldwide currently face worrying episodes of forest decline, which have boosted weakening and mortality of the trees. In the Mediterranean region, especially in the southeast Iberian Peninsula, Pinus sylvestris forests are severely affected by this phenomenon, and it has been commonly attributed to drought events. Remarkably, the role of root microbiota on pine decline has been overlooked and remains unclear. We therefore used metabarcoding to identify the belowground microbial communities of decline-affected and unaffected pine trees. Taxonomic composition of bacterial and fungal rhizosphere communities, and fungal populations dwelling in root endosphere showed different profiles depending on the health status of the trees. The root endosphere of asymptomatic trees was as strongly dominated by ‘Candidatus Phytoplasma pini’ as the root of decline-affected pines, accounting for >99 % of the total bacterial sequences in some samples. Notwithstanding, the titer of this phytopathogen was four-fold higher in symptomatic trees than in symptomless ones. Furthermore, the microbiota inhabiting the root endosphere of decline-affected trees assembled into a less complex and more modularized network. Thus, the observed changes in the microbial communities could be a cause or a consequence of forest decline phenomenon. Moreover, ‘Ca. Phytoplasma pini’ is positively correlated to Pinus sylvestris decline events, either as the primary cause of pine decline or as an opportunistic pathogen exacerbating the process once the tree has been weaken by other factors.

The 'Candidatus phytoplasma ziziphi' effectors SJP1 and SJP2 destabilise the bifunctional regulator ZjTCP7 to modulate floral transition and shoot branching.

Phytoplasmic SAP11 effectors alter host plant architecture and flowering time. However, the exact mechanisms have yet to be elucidated. Two SAP11-like effectors, SJP1 and SJP2, from 'Candidatus Phytoplasma ziziphi' induce shoot branching proliferation. Here, the transcription factor ZjTCP7 was identified as a central target of these two effectors to regulate floral transition and shoot branching. Ectopic expression of ZjTCP7 resulted in enhanced bolting and earlier flowering than did the control. Interaction and expression assays demonstrated that ZjTCP7 interacted with the ZjFT-ZjFD module, thereby enhancing the ability of these genes to directly bind to the ZjAP1 promoter. The effectors SJP1 and SJP2 unravelled the florigen activation complex by specifically destabilising ZjTCP7 and ZjFD to delay floral initiation. Moreover, the shoot branching of the ZjTCP7-SRDX transgenic Arabidopsis lines were comparable to those of the SJP1/2 lines, suggesting the involvement of ZjTCP7 in the regulation of shoot branching. ZjTCP7 interacted with the branching repressor ZjBRC1 to enhance suppression of the auxin efflux carrier ZjPIN3 expression. ZjTCP7 also directly bound to and upregulated the auxin biosynthesis gene ZjYUCCA2, thereby promoting auxin accumulation. Our findings confirm that ZjTCP7 serves as a bifunctional regulator destabilised by the effectors SJP1 and SJP2 to modulate plant development.

First report of a ‘Candidatus Phytoplasma trifolii’-related strain (16SrVI-A) associated with watermelon in Türkiye

First report of a ‘Candidatus Phytoplasma trifolii’-related strain (16SrVI-A) associated with watermelon in Türkiye

Putting 'X' into context: the diversity of 'Candidatus Phytoplasma pruni' strains associated with the induction of X-disease.

Recurrent epiphytotics of X-disease, caused by 'Candidatus Phytoplasma pruni', have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes, and more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multi-locus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define 'true' X-disease-inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extra-orchard environment.

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

Vom Chaos zur Klarheit. Die Neuordnung der Apfel TCP Gen-Benennung

Apple Proliferation (AP) is a widespread disease affecting apple orchards throughout Europe, including South Tyrol.The disease is associated with a phytoplasma, 'Candidatus Phytoplasma mali' ('Ca. P. mali'), which manipulates the plant's physiological processes through the secretion of small peptides called effectors. One well-studied phytoplasma effector is SAP11, which targets Teosinte Branched 1/Cycloidea/Proliferating Cell Factor 1 (TCP) genes in multiple plant species, including apple. TCP genes encode plant-specific transcription factors involved in various biological processes, such as growth, development, and responses to stimuli. The identification and naming of TCP genes have been inconsistent, leading to confusion and redundancy in sequence databases. In apple, 52 TCP genes (MdTCP) were identified in 2014 on the 2010 assembly of the apple genome, which is now considered outdated. To address this issue, a comprehensive investigation was conducted to identify and name TCP genes in apple using the high-quality genome assembly, GDDH13v1.1. Existing TCP sequences were aligned with the genome excluding redundant, fragmented, and non-TCP sequences. The revised set comprised 40 unique MdTCPs, including three novel genes. To establish a standardised nomenclature, each MdTCP was BLASTedagainst the Arabidopsis thaliana gene database, in order to identify the best hits for each MdTCP. The MdTCP genes were then renamed, incorporating the letter "a" or "b" to differentiate between MdTCPs showing homology to the same AtTCP, and also between the existing and proposed nomenclatures. The present study highlights the need for clarity and organisation in sequence databases, especially in respect of TCP genes. The presence of redundant and fragmented sequences in databases complicates accurate identification and annotation of genes. By providing a comprehensive and standardised nomenclature system, we aim to enhance the coherence and interoperability of future TCP gene research in plant and crop science. In our view, this work will serve as a valuable resource for researchers studying TCP genes in apple and will provide insights into the evolutionary dynamics and functional roles of TCP genes in plants.

First report of Candidatus Phytoplasma australasia (16SrII- subgroup D) associated with virescence of Chia (Salvia hispanica L.) from India.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

First report of Candidatus Phytoplasma trifolii (16SrVI-D) associated with little leaf disease of Nyctanthes arbor-tristis in the world

First report of Candidatus Phytoplasma trifolii (16SrVI-D) associated with little leaf disease of Nyctanthes arbor-tristis in the world

First Report of 'Candidatus Phytoplasma citri' related strain (16SrII-C subgroup) associated with phyllody disease of cumin in India.

Cumin (Cuminum cyminum L.), is an important export-oriented seed spice crop for India. Cumin is popularly used for flavouring food, including soups, pickles and vegetables, and for herbal medicine. India is the largest producer, consumer and exporter of cumin seed with an annual production of 0.795 million tones over an area of 1.09 million hectares. During 2020-21, India exported about 0.299 million tons of cumin worth of Rs 33280 million (Anonymous, 2021). Recently, phytoplasma suspected symptoms were observed in cumin at Agricultural Research Station, Mandor, Jodhpur, Rajasthan, India from 2019. The symptoms related to phytoplasma infection were first recorded after 70-75 days of sowing in the month of January of the years 2019 to 2022. The major symptoms recorded were yellowing, phyllody, witches-broom, yellowing and deformed elongated seeds. Disease incidence was recorded as 0.25-1.0%, 0.5-1.5%, 0.5-2.5 % and 0.5-10.6% during the years 2019, 2020, 2021 and 2022, respectively using quadrate method. In 2022, among different genotypes assessed GC 4, MCU 73, MCU 105, and MCU 32 exhibited lower disease incidences ranging from 0.5% to 1.5%, while MCU 78 recorded the highest disease incidence at 10.6%. To detect the association of phytoplasma with symptomatic cumin samples, genomic DNA was extracted from four representative cumin genotypes (CuPP-MND-01 to CuPP-MND-04) and one asymptomatic cumin plant 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) (Schneider et al., 1995; Gundersen and Lee, 1996) and secA gene specific primers (SecAfor1/SecArev3 followed by nested PCR primers SecAfor5/ SecArev2) (Hodgetts et al. 2008; Bekele et al. 2011). The amplicons of ∼1.25 kb with 16S rRNA gene and ∼600 bp with secA gene specific primers were amplified in all symptomatic cumin plant samples and positive control of brinjal little leaf. PCR amplified products from the four selected positive samples (CuPP-MND-01 to CuPP-MND-04) of 16S rRNA gene and secA gene, were sequenced from both ends. The 1,245 bp sequences were deposited in GenBank (OQ299007-10), which showed 100% sequence identity with each other and 99.4% identity with 'Candidatus Phytoplasma citri' reference strain (GenBank accession: U15442) (Rodrigues Jardim et al. 2023). The phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16S rRNA gene sequences through iPhyclassifier allowed affiliating the cumin phytoplasma strains with 16SrII-C subgroup strain with a similarity coefficient of 1 to the reference pattern of 16Sr group II, subgroup C (GenBank accession: AJ293216) (Zhao et al. 2009). In addition, the phylogenetic analysis of the secA gene-based sequences (OQ305073-76) further confirmed the close association of 16SrII-C group phytoplasmas with phyllody and witches' broom disease of cumin. Earlier 16SrII-C subgroup phytoplasma has been reported from various crops and weeds in India (Rao et al. 2021). However, no phytoplasma association has been reported earlier with cumin in India and abroad. To the best of our knowledge, this is the first report on the association of 16SrII-C group phytoplasma causing phyllody, witches' broom in cumin genotypes. This report has economic and epidemiological implications and needs immediate attention to reduce export losses due to phytoplasma disease in cumin and to prevent the potential spread to other crops.

First Report of 'Candidatus Phytoplasma australasiaticum' Associated with Phyllody, Virescence, and Witches'-Broom Disease in Chrysanthemum morifolium in Taiwan.

Chrysanthemum morifolium (Asteraceae) is commonly grown as commercial cut flowers or pot mums worldwide. Common diseases of chrysanthemum include bacterial blight, fungal diseases, viruses, and phytoplasmas (Verma et al. 2003; Taloh et al. 2020). In June 2022, C. morifolium plants showing virescence, stunting, witches' broom, and phyllody symptoms were observed in 10 plants representing 10% of the estimated 100 plants in a field in Taichung City, Taiwan (Fig. S1). Three symptomatic samples along with three asymptomatic ones were collected for further study. Nested PCR was performed with two primer sets, P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) to amplify nearly full-length of 16S rDNA from the collected samples. The target 1.2-kb DNA band was only amplified from the symptomatic chrysanthemum plants. The amplicons were sequenced and a representative sequence deposited in GenBank under accession number OR501416. This sequence was used to search GenBank database by the Basic Local Alignment Search Tool (BLAST) program through the web service of National Center for Biotechnology Information (NCBI). In the 16S rDNA analyses, the three randomly picked amplicons from chrysanthemum phyllody phytoplasma (CPP) shared 100% identity with one another, and all shared 99.5% identity with the, 'Candidatus Phytoplasma australasiae' reference phytoplasma strain (Y10097). Further analysis using iPhyClassifier (Wei et al. 2007) revealed that CPP was most similar to the pattern of the peanut witches' broom phytoplasma in the 16SrII-A subgroup (GenBank Acc. No. L33765), with a pattern similarity coefficient of 1.0. For confirmation, the secY gene was amplified by secY-F/R primers (Li et al. 2014), the 1.2-kb band was sequenced and deposit in GenBank (Acc. No. OR508986). BLAST analysis showed that the secY sequence of CPP shared 99.93% of sequence identities to several 'Ca. P. australasiaticum' strains (MN543069, CP097312, CP120449, KC953013, MW085916, MW070030, CP040925). The phylogenetic tree analysis based on the secY gene by MEGA11 employing maximum-likelihood algorithm was performed and the bootstrap value was set as 1000 times for support of the stability for the clades. The result showed that CPP is closely related to other strains in 16SrII group (Fig. S2). Taken together, CPP is a 'Ca. P. australasiaticum' related-strain in 16SrII-A subgroup. This is the first report of chrysanthemum as a host of this phytoplasma in Taiwan, and might have an impact to the horticultural industry and the growers.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Bois noir management in vineyard: a review on effective and promising control strategies.

Among grapevine yellows, Bois noir (BN), associated with 'Candidatus Phytoplasma solani', represents the biggest threat in the main wine-growing areas worldwide, causing significant losses in berry quality and yields. BN epidemiology involves multiple plant hosts and several insect vectors, making considerably complex the development of effective management strategies. Since application of insecticides on the grapevine canopy is not effective to manage vectors, BN management includes an integrated approach based on treatments to the canopy to make the plant more resistant to the pathogen and/or inhibit the vector feeding, and actions on reservoir plants to reduce possibilities that the vector reaches the grapevine and transmit the phytoplasma. Innovative sustainable strategies developed in the last twenty years to improve the BN management are reviewed and discussed.

Is the Removal of Nettles along Ditches Effective in Controlling Bois Noir in Vineyards?

Bois noir (BN) is a grapevine yellows disease associated with ‘Candidatus Phytoplasma (Ca. P.) solani’ that is transmitted to grapevines by the planthopper Hyalesthes obsoletus Signoret which uses herbaceous plants such as Urtica dioica as a pathogen reservoir. Urtica dioica is often widespread along ditches bordering vineyards, and a gradient in decreasing BN symptomatic grapevines is observed from the vineyard edges facing these ditches. In two vineyards in north-eastern Italy, over eight or seven years, the ditch bordering one edge of each vineyard was divided into two sections; in one of these, U. dioica was chemically weeded in April, while the other one remained untreated. The impact of chemical weeding on the spatial distribution of both H. obsoletus captures and newly BN symptomatic grapevines was assessed. The reduction in H. obsoletus captures in the vineyard sector facing the section of the ditch subjected to weeding corresponded to a decrease in newly symptomatic grapevines. These findings demonstrated that nettle removal from areas surrounding vineyards can effectively control BN.

Insects as potential vectors of phytoplasmas in urban trees in a mega-city: a case study in Bogotá, Colombia

AbstractUrban trees offer many ecosystem services which can be affected by pathogens, including phytoplasmas. Phytoplasmas cause regulator growth imbalances in infected plants, which produce deviations from the normal plant architecture, among other symptoms. In Bogotá, Colombia, ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma fraxini’ infect at least 11 species of urban trees, including Andean oak. This tree species is endemic to Colombia and Panama, and it plays an important ecologic role in the Colombian Andes wild forests. In Bogotá, knowledge about the insect vectors associated with the transmission of phytoplasmas to Andean oak is scarce. In this work we sampled the entomofauna of Andean oaks and evaluated the role of Cicadellidae and Psylloidea taxa as potential vectors of phytoplasmas in Bogotá. Insects were captured using direct and indirect sampling methods from Andean oaks at three contrasting sites in Bogotá, and also from neighbouring trees. The insects were identified by morphological and molecular methods and tested for phytoplasmas. In total, 510 Cicadellidae members of 13 taxa and 129 Psylloidea of 8 taxa were captured; richness and abundance depended on the type and sampling site. The most abundant species were Scaphytopius (Convelinus) sp. (Cicadellidae) and Acizzia uncatoides (Psylloidea). Leafhoppers positive for phytoplasmas were Chiasmodolini, Dikrella (Readionia) sp., Empoasca sp. and Scaphytopius sp. taxa. Our results suggest that several potential species of phytoplasmas may be dispersing phytoplasmas to Andean oaks and other urban tree species. It is necessary to conduct transmission tests to evaluate the transmission capacity of the potential vectors.

First report of ‘Candidatus Phytoplasma asteris’ (16SrI) associated with yellows disease of Trachyspermum ammi from India

First report of ‘Candidatus Phytoplasma asteris’ (16SrI) associated with yellows disease of Trachyspermum ammi from India

Seasonal and lifecycle changes in behavior affect the trapping efficiency of an insect vector, Circulifer tenellus (Hemiptera: Cicadellidae)

Abstract Accurate sampling of pests is the foundation of pest management. Choosing the best trap for pest monitoring can be complex, however, because trap performance is affected by pest preferences and behaviors. Moreover, preservation of DNA in traps is a consideration when insect specimens are used in molecular assays, such as the detection of insect-borne pathogens. We assessed the efficiency of 2 trap designs and 2 trap placements on the capture of beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), vector of “Candidatus Phytoplasma trifolii” and Beet curly top virus. Trap designs included standard yellow sticky cards and 3D-printed traps that capture insects directly in a DNA preservative. We found that yellow sticky cards captured more adults than did 3D-printed traps during summer months but captured fewer adults during autumn when leafhoppers move to overwintering sites. 3D-printed traps captured more nymphs than sticky cards, regardless of season, and traps hung at ground level captured more nymphs and adults compared to traps at 1-m height. Contrary to predictions, we did not find differences between trap types in the molecular detection of Ca. P. trifolii or Beet curly top virus, perhaps because 3D-printed traps captured few leafhoppers during summer when the pathogen rates were highest. Our results suggest behavioral differences in C. tenellus trap preference based on seasonality and life stage and underscore the importance of understanding insect behaviors when choosing trap designs for pest monitoring as well as for properly interpreting trap capture data.

Pentastiridius leporinus (Linnaeus, 1761) as a Vector of Phloem-Restricted Pathogens on Potatoes: 'Candidatus Arsenophonus Phytopathogenicus' and 'Candidatus Phytoplasma Solani'.

In Germany, the phloem-sucking planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) currently represents the epidemiological driver for the spread of the syndrome "Basses Richesses" in sugar beets, which results in a reduced sugar content and an economic loss for the farmers. This disease is associated with the γ-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the Stolbur phytoplasma 'Candidatus Phytoplasma solani'. Recently, P. leporinus was found in potato fields in Germany and is associated with Stolbur-like symptoms in this crop. In this study, we confirmed that the vector completes its lifecycle on sugar beets as well as on potatoes when reared under controlled conditions. Transmission experiments with adults of this vector combined with molecular analyses showed, for the first time, that both pathogens are transmitted by this vector to potatoes. For an accurate assessment of the Ca. P. solani and Ca. A. phytopathogenicus titers in the vector and host plants, gBlocks derived from the hsp20- and 16S rRNA genes were employed, respectively. For Ca. P. solani, the limit of detection was determined in potato and sugar beet material. The results of this study will further the research on the epidemiology of the syndrome "Basses Richesses" and "Stolbur" diseases and the response of host plants and vector insects to both pathogens.

First Report of Lingonberry Stunted Yellows Disease of Vaccinium vitis-idaea L. associated with 'Candidatus Phytoplasma trifolii'-Related Phytoplasma Strain in Lithuania.

Lingonberries (Vaccinium vitis-idaea L.) are low-growing, evergreen shrubs of cooler, northern regions of North America and Europe. These plants produce berries that are unique in flavor, bear high economic significance, and play a vital role in maintaining the diversity of the northern ecosystems (Kowalska, 2021). In October 2023 diseased plants of lingonberry were discovered in Labanoras Forest (55°14'N 25°42'E) (Lithuania). The plants expressed symptoms of stunting, yellowing, little leaf, shortened internodes, and stem distortions. Samples (leaves) were collected and tested from ten asymptomatic and ten symptomatic lingonberry plants. Total genomic DNAs of all samples were extracted by a CTAB protocol. Extracted DNAs were used as a template in direct and nested PCRs using the universal primer pairs P1/P7 and R16F2n/R2, respectively, to amplify phytoplasma 16S rRNA gene 1.2 kb fragments (Lee et al. 1998). The primer pairs SecAFor1/SecARev3 and SecAFor2/SecARev3 were used in direct and semi-nested PCRs, respectively, to amplify phytoplasma secA genes 0.5 kb fragment (Dickinson and Hodgetts, 2013). PCR amplicons of the 16S rRNA and secA genes specific for the phytoplasmas were only obtained from all sampled symptomatic plants. Three R16F2n/R2 and three SecAFor2/SecARev3 amplicons were cloned and submitted for Sanger sequencing (Nature Research Centre, Vilnius, Lithuania by 3500 Genetic Analyser). The three 16S rDNAs as well as the three secA gene fragments were identical. The BLAST analysis (NCBI) of the obtained sequences showed a similarity percentage, ranging from 99.75% to 100% (1247-1250 bp from 1250 bp) for 16SrRNA, and 98.13% to 99,15% (473-478 bp from 482 bp) for secA amplicons, with numerous strains of 'Candidatus (Ca.) Phytoplasma (P.) trifolii' (first hit MT674293 and KR906724, respectively). Additionally, 16S rDNA sequences by using iPhyClassifier were used to create virtual RFLP pattern (Zhao et al. 2009). The generated pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A. The phytoplasma strain detected in lingonberries was designated as lingonberry stunted yellows, LingbSY. Furthermore, the enzymatic RFLP analysis was performed with the 14 restriction enzymes (Lee et al., 1998), and obtained profiles were compared with virtually generated using iPhyClassifier. This yielded the same classification of detected phytoplasma to the 16SrVI-A phytoplasma subgroup. The phylogenetic analysis of both marker gene sequences revealed the same LingbSY phytoplasma classification. Selected sequences were deposited in GenBank (NCBI) with Accession No: PP237769 (16S rRNA gene) and No: PP238489 (secA gene). Phytoplasmas of 16SrI phytoplasma group were identified in lingonberries in Canada (Brochu et al. 2022). Strains of 16SrVI phytoplasma group were reported in Vaccinium myrtillus in Austria (Fernandez et al. 2007). This is the first report of 'Ca. P. trifolii' strain belonging to 16SrVI-A phytoplasma subgroup infecting lingonberry worldwide. Also, this is the first report of 16SrVI phytoplasma group in Lithuania. The presence of this phytoplasma poses a threat to the natural ecosystem and could eventually spread into agricultural settings in our country. Therefore, it's crucial to conduct surveillance for insect vectors, and assess effective control methods. Without proactive action, long term sustainability of lingonberries and their ecosystems may be jeopardized.

Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.

Distribution and Frequency of Little Cherry Virus 2 Genotypes in Both Production and Ornamental Fruit Trees in the Pacific Northwest

Little cherry virus 2 (LChV2) is the causal agent of little cherry disease, a recurring problem affecting cherry production the U.S. Pacific Northwest. Previous research has shown that there are two genotypes (LC5 and Rube-74) of LChV2 present in Washington. Here, we used data and samples from 2017 to 2022 to determine the incidence of each genotype in the Pacific Northwest, their distribution in cultivated versus ornamental host systems, and evidence of coinfection of both genotypes in individual plants. We found that there was no geographic pattern in Washington, as both genotypes are distributed throughout the state, whereas in Oregon, LChV2 positives were concentrated on one region. In both states, the LC5 genotype was the most common and widespread of the two genotypes, suggesting that the Rube-74-like isolates found were more recently introduced but are being actively transmitted. There were no host-specific patterns found, as both genotypes were found in cultivated and ornamental cultivars. Coinfection of a single plant by both genotypes was common in both states, with no evidence of superinfection-exclusion, although, interestingly, LChV2 titer and frequency decreased in the presence of ‘ Candidatus Phytoplasma pruni’. Cumulatively, these data will help us better understand the epidemiology of this pathogen in the Pacific Northwest.