Phytoplasma Infection Research Articles

Tomato and Jute Mallow are Two New Hosts of Papaya Bunchy Top Phytoplasma, a 16SrXII-O Subgroup Strain in Nigeria.

Nigerian papaya bunchy top (NGPBT) phytoplasma was first identified in diseased papaya plants growing in Ibadan, Oyo State, Nigeria (Kazeem et al. 2021). The NGPBT phytoplasma is a 'Candidatus Phytoplasma convolvuli'-related strain and represents a subgroup lineage, 16SrXII-O (the accession number of the reference strain is MW530522, Kazeem et al. 2021). The present communication reports that NGPBT phytoplasma can also infect tomato (Solanum lycopersicum) and jute mallow (Corchorus olitorius). Since May 2020, tomato and jute mallow grwn in Ibadan have been observed to develop yellowing, little leaf, and stunting symptoms (Fig. 1). Because the symptomatic plants occurred in the region approximately 1 km adjacent to where the NGPBT disease was reported, and the symptoms of infected plants resembled those of phytoplasma infection, molecular diagnostic assays for phytoplasma detection were deployed. Total DNAs were extracted from symptomatic plants, including four tomato plants and three jute mallows, as well as from asymptomatic two tomato and two jute mallow plants. The DNA samples were subjected to semi-nested PCR using phytoplasma 16S rRNA gene-specific primers P1A and P7A, followed by P1A and 16S-SR (Lee et al. 2004). An amplicon of 1.5 kb was obtained from each of the symptomatic plants, while no amplicon resulted from DNA samples of asymptomatic plants or negative controls without DNA templates (water and PCR reagents only). PCR products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), and three clones were chosen for each sample for Sanger sequencing (Psomagen Inc., Rockville, MD, USA). The nearly full-length 16S rRNA gene sequences (1.53kb) derived from tomato (OP123558) and jute mallow (OP123559) samples were identical. Based on the iPhyClassifier phytoplasma classification web tool (Zhao et al. 2009) and the BLAST search against the NCBI nucleotide database, these phytoplasma strains showed 100% sequence identity in 16S rRNA gene with the NGPBT phytoplasma (16SrXII-O, MW530522). Moreover, two additional genetic loci including ribosomal protein genes rplV-rpsC, and rplO-secY-adk were also amplified by nested PCR or semi-nested PCR with specific primers rpStolF/rpStolR followed by rpStolF2/rpStolR (Martini et al. 2007), and SecYF1a (Xll)/MapR-703-a, followed by SecYF2a (Xll)/MapR-703-a (Lee et al. 2010). Gene fragments of rplV-rpsC (1238bp) and rplO-secY-adk (2064bp) were amplified from DNAs of diseased papaya, tomato, and jute mallow plants. The obtained sequences were deposited into GenBank, respectively: rplV-rpsC (OP123560, OP123562, and OP123563) and rplO-secY-adk (OP123565, OP123567, and OP123568). Multilocus sequence analysis (MLSA) indicated that the sequences of phytoplasmas amplified from three different plant hosts were also identical in rp, secY, and adk genes. The MLSA results demonstrate that tomato and jute mallow are two new hosts of NGPBT phytoplasmas. This also marks the first time that phytoplasma diseases are associated with tomato and jute mallow in Nigeria, as prior to this study, phytoplasma diseases were only reported in coconut palm and papaya in the country (Osagie et al. 2016; Kazeem et al. 2021). Results from the present study suggest that insect vector(s) for the transmission of the NGPBT phytoplasma are present in the region. Since both tomato and jute mallow are important vegetable crops in Nigeria, timely dissemination of emerging disease information is needed to alert growers and extension personnel in the region. In addition, ongoing incidence, and prevalence surveys of NGPBT disease indicate that more infected papaya and tomato plants have been observed in the region than in previous years. A better understanding of the NGPBT phytoplasma disease epidemiology will help devise strategies to control the diseases associated with the NGPBT phytoplasma.

Evidence of mixed infection of phytoplasma and begomovirus associated with Withania somnifera and Capsicum annum plants from Uttar Pradesh, India

Symptoms of yellow discoloration, curling of leaves, and little leaves with excessive branching were observed on Withania somnifera and Capsicum annum plants, with an average incidence of 5 and 8%, respectively, in Hardoi District of Uttar Pradesh, India during 2016–17 and 2017–18. The little leaf and excessive branching suggested the possibility of phytoplasma association. However, the presence of whiteflies in the vicinity and leaf curling suggested begomovirus infection. Three leaf samples from Withania ­somnifera and Capsicum annum symptomatic plants and one from non-symptomatic leaf were used for DNA isolation and were subjected to PCR using P1/P6 primers and nested PCR R16F2n/R16r2 primers, respectively, for the detection of phytoplasma. The begomovirus coat protein-specific primer AV1F/AV1R was used to detect begomovirus infection. Nested PCR amplified the ∼1.2 kb amplicon in all six symptomatic leaves and no amplification was observed in non-symptomatic leaves. The CP region primer yielded ∼800 bp amplicons in all three symptomatic samples of each plant. Amplified products from both primers were eluted, purified, and sequenced. The phytoplasma sequence obtained from Withania somnifera (MH789552) shared the highest sequence identity (99.92%) with other isolates of a clover proliferation group (16SrVI-D) group of phytoplasmas. However, Capsicum annum plants shared the highest identity (99%) with the Ca. P. asteris-related (16SrI-B) group of phytoplasma. In silico RFLP analysis of the 1.2 kbp product of the 16S rRNA sequence of the W. somnifera and C. annum phytoplasma strains submitted to the pDRAW32 tool (https://www.acaclone.com/) and phylogenetic analysis through the MEGA 6.0 tool confirmed that it as a member of the 16SrVI-D subgroup and 16SrI-B subgroup, respectively. The CP gene sequence of the W. somnifera isolate (MW176071) showed maximum identity (99%) with several isolates of the Tomato leaf curl virus reported from various places in India. However, the C. annum isolate (MW420480) shared maximum identity with the Ageratum enation virus. The investigation confirmed the mixed infection of ToLCV and 16SrVI-D group phytoplasma in the W. somnifera plant and 16SrI-B alongwith AEV in C. annum plants.

Integrated full-length transcriptome and metabolome analysis of the early defense mechanisms in Paulownia witches’ broom disease

ABSTRACT Paulownia witches’ broom (PaWB) is a devastating disease of Paulownia spp. caused by phytoplasma infection. To reveal the reason for the absence of morphological changes at the early stage of phytoplasma infection, in this study, a combination of second-and third-generation sequencing (single-molecule real-time long-read isoform) and liquid chromatography tandem mass spectrometry were performed to analyse the changes of full-length transcripts and metabolites at the early stage of phytoplasma infection. A total of 140,528 non-redundant full-length transcripts, 597 transcription factors, 1,658 long non-coding RNAs, and 645 metabolites were identified. The third-generation sequencing improved the annotation of the reference Paulownia fortunei (Seem.) Hemsl. genome by 8.86%. Combination analysis of the full-length transcriptome and metabolome revealed that phenolics and phenylalanine ammonia-lyase synthesis related genes were significantly up-regulated in the early stage of phytoplasma-infected P. fortunei. Based on the results of full-length transcriptome and metabolome integrative analysis, we constructed an early defence model diagram of PaWB disease. Our results provide powerful insights into the reason for absence of morphological at the early stage of phytoplasma infection.

Comparative Biochemical and Transcriptomic Analyses Provide New Insights into Phytoplasma Infection Responses in Cucumber.

Flat stem and witches’ broom phytoplasma-like symptoms in the cucumber inbred line C17 were observed in a greenhouse at Yangzhou University, China for three consecutive planting seasons; these symptoms resulted in a decreased yield. To better understand the cause of these symptoms, 16S rRNA PCR, plant hormones, mineral elements, and RNA-seq profiling were performed using symptomatic and normal stem samples. The results showed that the causal agent was classified as the Candidatus phytoplasma asteris strain, a plant pathogenic prokaryote that could not be cultured in vitro. Measurement of plant hormones showed that auxin, salicylic acid, and jasmonic acid contents were significantly increased, whereas that of ethylene’s immediate biosynthetic precursor, 1-aminocyclopropane-1-carboxylic acid, was decreased in the phytoplasma-infected stems compared with the healthy stems. Furthermore, measurement of mineral element composition showed that magnesium, calcium, sodium, iron, and zinc concentrations significantly changed in the phytoplasma-infected cucumber stems compared with the uninfected stems. Comparative RNA-seq identified 253 differentially expressed genes, including 179 upregulated and 74 downregulated genes. Further analyses suggested that genes related to phenylpropanoid biosynthesis, phenylalanine metabolism, and plant hormone signal transduction contributed to phytoplasma infection. Taken together, this study presents the first in-depth assessment of disease symptoms and biochemical content of cucumber stems known to be infected with phytoplasma.

Detection and characterization of rice orange leaf phytoplasma infection in rice and Recilia dorsalis

Phytoplasmas are small bacterial parasites that lack cell walls and are transmitted in a persistent-propagative manner by insect vectors. However, detailed multiplication patterns and movements of phytoplasmas within host plant and insect vector remain elusive. In this study, a specific antibody against the immunodominant membrane protein (Imp) of rice orange leaf phytoplasma (ROLP) was generated and subjected to the frozen section immune gold labeling assay, immune gold labeling microscopy, and immune fluorescence labeling assay to investigate how ROLP enters, propagates, and spreads in rice and its leafhopper vector Recilia dorsalis at the ultrastructural level. During acquisition and transmission by insect vectors, ROLPs could squeeze and penetrate the multiple membrane/tissue barriers such as microvilli, apical plasmalemma, and basal lamina in the intestines or salivary glands by endocytosis- and exocytosis-like mechanism. Furthermore, ROLP infection is restricted to the type IV and V cells of salivary glands. In contrast to the classical binary fission used by most bacteria, electron microscopy reveals that ROLP exploits an asymmetrical budding strategy to replicate in plant hosts and insect vectors. In this process, a cellular protrusion of ROLP forms a bud to reproduce the offspring by fission at the junction between the bud and the ROLP main body. These results clarify the infection characteristics of ROLP in rice and R. dorsalis, which will help guide the formulation of ROLP prevention and control strategies.

Acquisition and Transmission of the Lethal Bronzing Phytoplasma by Haplaxius crudus Using Infected Palm Spear Leaves and Artificial Feeding Media.

Lethal bronzing (LB) is a fatal palm disease caused by the phytoplasma 'Candidatus Phytoplasma aculeata'. This disease causes significant economic losses in palm industries and landscapes. The American palm cixiid, Haplaxius crudus, recently was identified as the vector of the phytoplasma. However, knowledge about LB phytoplasma transmission is limited due to the lack of a method to generate phytoplasma-infected insects in the laboratory. In this study, the acquisition and transmission of the LB phytoplasma by H. crudus were investigated. Successful acquisitions of the phytoplasma by H. crudus were observed at 2 days acquisition access period on LB-infected palm spear leaves. Analyses revealed increased phytoplasma infection rates of H. crudus with longer acquisition access periods and latent periods. A significantly higher phytoplasma infection rate was shown after various acquisition access periods and latent periods than the infection rate of the field-collected H. crudus population. Transmission of the phytoplasma from LB-infected spear leaves to sucrose media by H. crudus also was observed using digital PCR assays. These results further support the vector status of H. crudus and offer valuable information to understand LB phytoplasma transmission. Additionally, these results generate a critical baseline for future LB phytoplasma-vector research by providing a way to generate vectors with high phytoplasma infection rates in the laboratory setting.

First Report of 'Candidatus Phytoplasma fragariae' Infecting Hazelnut in Italy.

Italy is the second largest producer of hazelnuts in the world after Turkey. The Viterbo province (Latium region) is one out of the four main productive districts, accounting for approximately 30% of the total Italian production. Several pathogens can severely affect hazelnut trees (Corylus avellana), mainly fungi and bacteria that are commonly spread in all the growing areas. However, other pathogens such as phytoplasmas, are known to infect this crop worldwide. Among these, 'Candidatus Phytoplasma fragariae' (16SrXII-E) has been recently associated with the decline and death of hazelnut trees in UK where it was identified for the first time on this plant species (Hodgetts et al., 2015), and in Slovenia (Mehle et al., 2018; Mehle et al., 2019). In July 2020, a survey was carried out in two orchards located in the Viterbo province, where symptoms resembling those induced by phytoplasmas and consisting in yellowing and rolling of leaves and tree decline (Supplementary figure 1) were recorded by growers. To ascertain the possible presence of phytoplasma infections, leaf samples were collected from five symptomatic plants: one plant from orchard 1, cv. Tonda Gentile Romana and four plants from orchard 2, cv. Tonda di Giffoni. Samples from four non-symptomatic plants (two per each orchard) were also collected and used as negative controls in the detection assays. DNA was extracted from 0.6 g of leaf ribs homogenised in CTAB buffer according to Marzachì et al. (1999). DNA extracts were amplified by direct and nested PCR employing the universal primer pairs P1/P7 and R16F2n/R2 according to the EPPO Standard PM7/133(1) - Appendix 2 (EPPO, 2018). Nested amplicons of the expected size (1,250 bp) were obtained from the five symptomatic samples whereas no bands were observed for the symptomless ones. The R16F2n/R2 fragments were submitted to both Sanger sequencing and RFLP analysis for the phytoplasma identification. Nucleotide BLAST analysis (NCBI) of the obtained sequences showed an identity percentage ranging from 99.65% and 99.91% with numerous strains of 'Ca. Phytoplasma fragariae' (16SrXII-E). The RFLP analysis was performed with the MseI restriction enzyme, and the obtained profiles were compared in the Codon Code Aligner software to those virtually generated from reference sequences of phytoplasmas belonging to the 16SrXII subgroups identified so far (Kazeem et al., 2021). All the five samples showed a common RFLP profile which was ascribable to that virtually generated from the 16SrXII-E reference sequence DQ086423 (Valiunas et al., 2006) (Supplementary figure 2). To our knowledge, this is the first finding of 'Ca. P. fragariae' on hazelnut in Italy since the previous records on woody plant hosts regarded Cornus sanguinea and Sambucus nigra (Filippin et al. 2008). Further surveys are ongoing to assess the spread of 'Ca. P. fragariae' in the Italian hazelnut orchards and to identify the putative insect vectors. The nucleotide sequences obtained in this study were deposited in GenBank NCBI database (accession numbers: MW767127, MW767128, MW767129, MW767130, MW767131).

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters

Main conclusionLoss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport.Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant’s response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Phytochemical composition of essential oils from aerial parts of sweet basil infected by a mixed infection of tobacco mosaic virus and phytoplasma

Ocimum basilicum, commonly known as sweet basil, was found naturally infected showing mosaic mottling and witches’ broom symptoms. Semi-nested PCR using specific primers revealed the presence of a mixed infection of TMV and Candidatus phytoplasma. This study aims to evaluate and compare the phytochemical composition of essential oils from aerial parts produced in sweet basil. The comparison covered uninfected, mixed infected, TMV infected, and phytoplasma infected sweet basil. Thirty different essential oils were isolated and evaluated using gas chromatography-mass spectrometry (GC-MS) analysis. The essential oils produced were grouped into 5 main classes: monoterpenes hydrocarbons, oxygenated monoterpenes, sesquiterpenes hydrocarbons, oxygenated sesquiterpenes and ketones. All classes were severely altered due to TMV and Candidatus phytoplasma infection compared to the control. The pathological stress caused by the mixed infection was significantly higher than each pathogen separately. The monoterpenes hydrocarbons, oxygenated monoterpenes and ketones showed a significant increase in the essential oil percentages induced in the mixed infection than that of single infections. Yet, a significant decrease in sesquiterpenes hydrocarbons and oxygenated sesquiterpenes was reported in the infected plants. Furthermore, α-bergamotene, (+)spathulenol, α bergamotene, δ-guaiene, cubenol, bornyl acetate and 1,3-bis(cinnamoyloxymethyl)adamantane, were found only in healthy plants, while α-pinene, camphene, sabinene, β-pinene, β-myrcene, trans-sabinene hydrate, eugenol and trans-caryophyllene were detected only in infected plants. This study reports the possible metabolic consequences and defense mechanisms that induced in sweet basil due to TMV and phytoplasma infection.

Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.).

Simple SummarySesame (Sesamum indicum L.) is an important oilseed crop that is well known for its highly nutritious oil content. Due to its high oil content and nutritional properties, sesame is also known as the ‘queen of oilseeds’. Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. Sesame phyllody is the leading biotic constraint drastically affecting sesame productivity and resulting in yield losses of up to 80% in major sesame-producing countries in the world. In this study, we explored the role of DNA methylation in phytoplasma infection. Whole Genome Bisulfite Sequencing and a Quantitative analysis of DNA methylation using real-time PCR (qAMP) revealed an alteration of methylation pattern upon phytoplasma infection. Few selected development and defense-related loci were either hypo- or hypermethylated. We hereby report the first methylome study in healthy and phytoplasma-infected sesame. Our study provides fundamental insights into the role of DNA methylation in the symptom development of phytoplasma infection in sesame plants.Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

Phytoplasmas, the Fast Spreading Vector Borne Pathogens of Flower Crops: Indian Scenario

Floriculture trade in India is one of the most lucrative fields, earning valuable forex to the national exchequer. Exchange of planting material via different modes plays a major role in spreading of the pathogen. Floriculture being an international industry, the transmission of pests or pathogens through live plants, seeds, cuttings and flowers etc., is very high. In recent years, the occurrence of phytoplasmal diseases in flower crops is increasing due to extensive exchange amongst many groups. The prominent symptoms of phytoplasma infection are the flowers turning into leafy structures called phyllody and the formation of sterile green flowers called virescence. In floricultural crops, these two symptoms are of major disadvantages as the flowers are the main economic product. The phyllody symptoms are most often mistaken for novel plant type and are multiplied unwittingly. Phytoplasmas are switch between plant and animal kingdoms. Phytoplasma spread in the field through leaf hoppers by inhabiting in their gut, haemolymph, salivary glands etc. Among the ornamental plants and flower crops, various groups of phytoplasma have been reported from India. More than 40 per cent of the infections are reported from the members of the Asteraceae family with 16SrI-B group, ‘Candidatus Phytoplasma asteris’.

Detection, Identification, and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Digera muricata in Taiwan.

Digera muricata (L.) Mart. is a pantropical annual herb belonging to the Amaranthaceae family. In August 2021, D. muricata with indicative phytoplasma symptoms of phyllody, witches'-broom, and virescence was discovered adjacent to a peanut field in Mailiao, Yunlin, Taiwan. The causal agent of the observed symptoms was detected and identified by a series of molecular characterizations. Sieve elements of the phloem tissue were perused under the transmission electron microscope and revealed the presence of pleomorphic phytoplasma-like organisms. Nested PCR using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 was able to amplify a 1.2-kb DNA fragment for the 16S rRNA gene only from the symptomatic D. muricata. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP further affirmed that the phytoplasma associated with the diseased D. muricata can be classified into the 16SrII-V subgroup. Moreover, displayed evident symptoms were explained by the concomitant detection of PHYL1 and SAP11, the virulence genes responsible for the development of leaf-like flowers and shoot proliferation, respectively. Although phytoplasma infection on the noncrop species does not have a direct economic impact, its role in disease spread and perpetuation is indubitable.

Micromorphological analysis indicates reversion of sesame extrafloral nectary to flower-like structure with the onset of phytoplasma infection reflecting its evolutionary path

Plant pathogens are known to cause diverse symptoms in plants. Candidatus Phytoplasma (Class: Mollicutes), a phloem inhabiting bacterial pathogen is transmitted by sap sucking insects and is becoming a serious concern in sesame crops grown for oilseed, especially in the Asiatic region. Sesame (Sesamum indicum L.), a member of family Pedaliaceae, bears prominent extrafloral nectaries (EFN) at each node. Our aim was to investigate the morphological and micromorphological changes in EFN development in phytoplasma infected sesame plants with phyllody disease. Scanning electron microscopy was used to study the micromorphological changes from freshly collected EFNs from healthy and phyllody diseased sesame plants. Observations revealed that the extrafloral nectary in sesame gets converted into flower (green in color) on phytoplasma infection. In diseased plants, this reversion was also seen in early stages of developing EFN primordia. Apart from the reversion, different variants were observed in EFN such as developed EFN showing indeterminacy but retaining the EFN structure or instead occasional morphological alterations were observed such as elongation or splitting of EFN tissue into halves, that often showed conversion to spatula shaped structures with leafy characters. In a few instances, inflated gynoecium like-structure emerged from the center of a split EFN. Rarely, deformed stalked flowers were also observed in place of EFN, with positional and structural abnormalities in stamens, gynoecium defects, leafiness of reduced petals and sepal narrowing cum elongation. Phytoplasma infection in sesame caused development of each EFN into a flower indicating that the EFN gland is a suppressed flower in sesame, the suppression of which is released on infection of the plant pathogen. Instead of solitary nature seen in most Sesamum indicum varieties, the cymose nature of inflorescence is thereby resumed. Phytoplasma-induced EFN conversion is a unique symptom among plant pathogens. In addition, EFNs from affected plant also showed indeterminacy and transition organs show leaf-like features indicating a shift of reproductive to vegetative developmental program on phytoplasma infection. Bushiness on affected sesame is mainly due to conversion of EFN to phylloid flowers, especially those with indeterminate nature. In addition, loss of EFN is suggestive of increased susceptibility of phyllody affected sesame to insect infestation. Further studies on anatomy of symptomatic parts will provide a deeper understanding of the conclusions derived from micromorphological data of phytoplasma associated sesame EFN reversion, presented in the current study.

Hatay İli Bazı Kabak (Cucurbitaceae) ve Tarlasarmaşığı'nda (Convolvuluceae) Fitoplazmaların Belirlenmesi ve Karakterizasyonu

Aims: During a disease survey conducted in 2019, typical symptoms including yellowing of the leaves, stunting, shortening of internodes, proliferation of auxiliary shoots (witches'-broom), the bunchy appearance of growth at the ends of stems (rosetting), virescence of flowers or sterility, phyllody, small and deformed leaves and fasciation were observed on suspected cucurbit plants growing in Hatay province. Therefore, this study was carried out to determine the phytoplasma infection(s) on cucurbit crops and bindweed in Hatay province of Turkey.Methods and Results: In total; 4 pumpkins (winter squash: Cucurbita moschata Duchesne), 12 summer squash (Cucurbita pepo L.) and 2 bindweed (Convolvulus arvensis L.) samples were collected from suspected plants in cucurbit fields in Hatay province. Some of the plants (2 pumpkins, 10 summer squash and 2 bindweeds) exhibited symptoms related to phytoplasmas. Total nucleic acid was extracted by CTAB method. PCR amplification of 16S rDNA with phytoplasma specific primer pairs; F1/R0 followed by R16F2n/R2 confirmed the phytoplasma presence. Expected size amplicons of ~1.2 kb were obtained from infected samples from symptomatic cucurbits (12/12) and bindweed (1/2), but not from asymptomatic cucurbit plants (0/4). The sequences of the 4 symptomatic samples were deposited in NCBI GenBank (MT163353, MT163393, MT163396, and MT163469).Conclusions: After sequencing and phylogenetic analyses it was revealed that the squash isolates had 99% sequence identity with “Candidatus phytoplasma trifolii” (16SrVI) and the bindweed isolate had 99% sequence identity with “Candidatus phytoplasma solani” (16SrXII).Significance and Impact of the Study: There is a lack of knowledge on the status of phytoplasma infections and natural hosts in cucurbits in Turkey. According to our knowledge, this is the first report of a member of 16SrVI group, Candidatus phytoplasma trifolii associated with phytoplasma infection in winter squash-pumpkin (C. moschata) and summer squash (C. pepo) in Turkey.

First report of a 16SrII‐V phytoplasma associated with peanut phyllody in India

Peanut (Arachis hypogaea, Fabaceae), also known as groundnut, is an important oilseed and food legume crop cultivated in a wide range of climatic conditions across the globe. India is the second largest producer of peanut in the world after China (Food and Agriculture Organization of the United Nations, 2022). Peanut plants exhibiting phyllody, little leaf, excessive shoot proliferation, negative geotropism of pegs with absence of pod formation were observed in February 2021 in Pune, Maharashtra, India (Figures 1, 2). The diseased plants could be easily identified in the field due to their distinct appearance, suggestive of the presence of phytoplasma. Eight peanut fields were surveyed and the overall average disease incidence was four percent. Ten diseased and four asymptomatic plants were sampled to identify and characterise the associated phytoplasma. DNA was extracted from leaf petioles (100 mg) using DNeasy® Plant Mini Kit (Qiagen, Germany). The extracted DNA was subjected to PCR using P1/P7 universal primers (Deng & Hiruki, 1991; Schneider et al., 1995). Products from the first round PCR were used in nested PCR with R16F2n/R2 primers (Gundersen & Lee, 1996). Amplicons of the expected size, c. 1.8 kb and 1.2 kb were obtained in all diseased samples in PCR and nested PCR assay, respectively. No amplification was recorded in asymptomatic samples. The sequences of the nested PCR products were identical to each other and the sequence was deposited in GenBank (Accession No. ON139214). A BLASTn search showed more than 99% sequence identity with phytoplasma sequences including peanut witches’-broom (JN681274; L33765), Desmodium ovalifolium witches’-broom (MK956144), soybean witches’-broom (MW115943) and cowpea phyllody (MZ831318). The peanut phyllody phytoplasma sequence clustered with selected sequences belonging to the 16SrII phytoplasma group following phylogenetic analysis using the neighbour-joining method (MEGA X program) (Figure 3). Virtual RFLP using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/) demonstrated that the peanut phyllody phytoplasma is affiliated to group 16SrII, subgroup V. Phytoplasmas belonging to subgroup 16SrII-V have been recently reported to infect legumes such as mungbean and soybean in Taiwan (Chen et al., 2021; Wang et al., 2021). To our knowledge, this the first report of a 16SrII-V phytoplasma associated with phyllody in peanut as well as the first report of the occurrence of a 16SrII-V phytoplasma in India. The finding is of epidemiological importance as the phytoplasma infection reduces pod formation leading to yield and economic losses for farmers. Moreover, infected peanut plants could serve as natural reservoirs of the phytoplasma aiding its spread to other legume, vegetable and flower crops cultivated in the vicinity. The authors thank the Director, ICAR-IARI, New Delhi and Head, ICAR-IARI, RS, Pune for providing lab facilities.

‘Candidatus Phytoplasma asteris’ (16SrI-B alt grup) ile infekteli marul (Lactuca sativa L.)’da biyokimyasal değişimler

Phytoplasma infections are able to limit the lettuce growth around the world. The alterations of biochemical contents in the host physiology following phytoplasma infection in lettuce remain to be elucidated. In this study, changes in total protein and chlorophyll content, proline, malondialdehyde (MDA) accumulation, peroxidase (POD) and catalase (CAT) enzyme levels were investigated in leaves of lettuce plant after Candidatus Phytoplasma asteris infection. Symptoms observed in plants infected with phytoplasma were yellowing, little leaf, stunting, and a general decline. Phytoplasma agent detected in all infected lettuce by PCR-RFLP studies. Total protein and chlorophyll contents of phytoplasma-infected plants were lower than those of healthy control. Proline, MDA accumulation, POX and CAT enzyme activities were increased in infected plants as compared to those of control. The results show that phytoplasma infection can modify the host physiology of lettuce. In conclusion, this study indicated that the previously identified Ca. P. asteris was still pathogen with no changes in its DNA sequence and it was able to reduce the quality parameters of the lettuce plant and possess potential danger to the lettuce growing areas.

The Crosstalk of the Salicylic Acid and Jasmonic Acid Signaling Pathways Contributed to Different Resistance to Phytoplasma Infection Between the Two Genotypes in Chinese Jujube

Jujube witches’ broom disease (JWB), one of the most serious phytoplasma diseases, usually results in the destruction of Chinese jujube (Ziziphus jujuba Mill.). Although most jujube cultivars are sensitive to JWB, we found a few genotypes that are highly resistant to JWB. However, the molecular mechanism of phytoplasma resistance has seldom been studied. Here, we used Chinese jujube “T13,” which has strong resistance to JWB, and a typical susceptible cultivar, “Pozao” (“PZ”), as materials to perform comparative transcriptome, hormone, and regulation analyses. After phytoplasma infection, the differential expression genes (DEGs) were detected at all three growth phases (S1, S2, and S3) in “PZ,” but DEGs were detected only at the first growth phase in “T13.” Meanwhile, no phytoplasma was detected, and the symptoms especially witches’ broom caused by JWB were not observed at the last two growth phases (S2 and S3) in “T13.” Protein–protein interaction analysis also showed that the key genes were mainly involved in hormone and reactive oxygen species (ROS) signaling. In addition, during the recovered growth phase in “T13” from S1 to S2, the level of hydrogen peroxide (H2O2) was significantly increased and then decreased from S2 to S3. Moreover, jasmonic acid (JA) was significantly accumulated in “PZ” diseased plants, especially at the S2 phase and at the S2 phase in “T13,” while the content of salicylic acid (SA) decreased significantly at the S2 phase of “T13” compared to that in “PZ.” The changes in H2O2 and JA or SA were consistent with the changes in their key synthesis genes in the transcriptome data. Finally, exogenous application of an SA inhibitor [1-aminobenzotriazole (ABT)] rescued witches’ broom symptoms, while the contents of both JA and MeJA increased after ABT treatment compared to the control, demonstrating that exogenous application of an SA inhibitor rescued the symptoms of jujube after phytoplasma infection by decreasing the contents of SA and increasing the contents of JA and MeJA. Collectively, our study provides a new perspective on the transcriptional changes of Chinese jujube in response to JWB and novel insights that the crosstalk of JA and SA signaling communicated together to contribute to “T13” JWB resistance.

Fitoplazma: Taşınması, Hastalık Gelişimi ve Fonksiyonel Genomları

In recent years the number of phytoplasma diseases that negatively affect yield and quality of many plants has been increasing around the world. As is known grafting, plant propagation materials (seed, bulb, tuber), parasitic plants and insect vectors play active role in the spread of phytoplasmas causing these diseases. These organasim which get through plate in the sieve tube element and systemically colonize the plant, it invade and multiply salivary glands, midgut, heomocel, brain and fat tissue after sucking up insect body with stylet. Subsequently they continue the cycle by moving to a new plant with a diseased insect feeding on a healthy plant. Although the existence of functional genomics in this cycle is known, there is little information about transmission of phytoplasma from vector to host plant or from host plant to vector, the mechanism of disease caused by phytoplasma in the plant and genomics involved in different reactions in the host plant against phytoplasmal infection.

Detection, Identification, and Molecular Characterization of a 16SrII-V Subgroup Phytoplasma Associated with Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Viviani, commonly known as curl-leaved tobacco, is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani, found in Yunlin County, Taiwan, was observed to have phyllody, virescence, and witches'-broom, which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S ribosomal RNA (rRNA)-based phylogenetic analysis and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches'-broom phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain. Further identification of SAP54/PHYL1 and SAP11 homologs in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches'-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease, which poses a threat to the agricultural sector and food security in Taiwan.

Infection by phloem-limited phytoplasma affects mineral nutrient homeostasis in tomato leaf tissues

Phytoplasmas are sieve-elements restricted wall-less, pleomorphic pathogenic microorganisms causing devastating damage to over 700 plant species worldwide. The invasion of sieve elements by phytoplasmas has several consequences on nutrient transport and metabolism, anyway studies about changes of the mineral-nutrient profile following phytoplasma infections are scarce and offer contrasting results. Here, we examined changes in macro- and micronutrient concentration in tomato plant upon ‘Candidatus Phytoplasma solani’ infection. To investigate possible effects of ‘Ca. P. solani’ infection on mineral element allocation, the mineral elements were separately analysed in leaf midrib, leaf lamina and root. Moreover, we focused our analysis on the transcriptional regulation of genes encoding trans-membrane transporters of mineral nutrients. To this aim, a manually curated inventory of differentially expressed genes encoding transporters in tomato leaf midribs was mined from the transcriptional profile of healthy and infected tomato leaf midribs. Results highlighted changes in ion homeostasis in the host plant, and significant modulations at transcriptional level of genes encoding ion transporters and channels.