Hishimonus Phycitis Research Articles

Comprehensive analysis of little leaf disease incidence and resistance in eggplant

BackgroundLittle leaf disease caused by phytoplasma infection is a significant threat to eggplant (also known as brinjal) cultivation in India. This study focused on the molecular characterisation of the phytoplasma strains and insect vectors responsible for its transmission and screening of brinjal germplasm for resistance to little leaf disease.ResultsSurveys conducted across districts in the Tamil Nadu state of India during 2021–2022 showed a higher incidence of phytoplasma during the Zaid (March to June), followed by Kharif (June to November) and Rabi (November to March) seasons with mean incidence ranging from 22 to 27%. As the name indicates, phytoplasma infection results in little leaf (reduction in leaf size), excessive growth of axillary shoots, virescence, phyllody, stunted growth, leaf chlorosis and witches’ broom symptoms. PCR amplification with phytoplasma-specific primers confirmed the presence of this pathogen in all symptomatic brinjal plants and in Hishimonus phycitis (leafhopper), providing valuable insights into the role of leafhoppers in disease transmission. BLAST search and phylogenetic analysis revealed the phytoplasma strain as “Candidatus Phytoplasma trifolii”. Insect population and disease dynamics are highly influenced by environmental factors such as temperature, relative humidity and rainfall. Further, the evaluation of 22 eggplant accessions revealed immune to highly susceptible responses where over 50% of the entries were highly susceptible. Finally, additive main effect and multiplicative interaction (AMMI) and won-where biplot analyses identified G18 as a best-performing accession for little leaf resistance due to its consistent responses across multiple environments.ConclusionsThis research contributes essential information on little leaf incidence, symptoms, transmission and resistance profiles of different brinjal genotypes, which together ensure effective and sustainable management of this important disease of eggplants.

Impact of Abiotic Factors on Occurrence of Capsule Borer and Sucking Pests in Sesamum

A field experiment was conducted at Agricultural college, Naira, Srikakulam, Andhra Pradesh, India during early summer (January–April) of 2021 to know about the incidence of leaf webber and sucking pests viz., leafhopper (Hishimonus phycitis Distant) and whitefly (Bemisia tabaci Gennadius) its relation with main weather factors. Results revealed that a peak incidence of mean capsule borer population per plant were recorded in 9th and 11th standard weeks, respectively. The correlation study revealed that the Antigastra larvae had significantly positive correlation with maximum temperature (r=0.48) and significantly negative correlation with relative humidity (r=- 0.71). The regression analysis revealed that these factors accounted for approximately 59.8% of the variation in the capsule borer population (R2=0.598). The incidence of leafhopper (Hishimonus phycitis Distant) and whitefly (Bemisia tabaci Gennadius) were started during 5th and 4th SMW attained their peaks during 11th an 12th SMW respectively. The correlation study revealed that the leafhopper showed significant positive correlation with maximum temperature (r=0.623) and minimum temperature (r=0.283) and significantly negatively correlated with morning and evening relative humidity (r=-0.165, r=-0.122) respectively. While the correlation study between whitefly population and weather parameters revealed that the it showed positive correlation with maximum (r=0.821) and minimum (r=0.120) temperature and negatively correlated with morning and evening relative humidity (r=-0.097, r=-0.182) respectively whereas, rainfall showed non-significant correlation (r=0.00) with the both sucking pests.

First report of 'Candidatus phytoplasma asteris' (16SrI) from Cassia fistula showing symptoms of flat stem and witches'-broom in India.

Cassia fistula commonly known as 'golden shower tree' is a deciduous tree with a greenish-gray bark and complex leaves with lovely clusters of yellow blossoms that is also utilized for several purposes in traditional medicine offer therapeutic characteristics (Pawar et al., 2017). Random spotting of flat stem symptoms along with unopened flower beds was observed in C. fistula plant during March 2022 in IISER (Indian Institute of Science Education and Research), Thiruvananthapuram, Kerala, India and during May 2022 in SKUAST (Sher-e-Kashmir University of Agricultural Sciences and Technology), Jammu, which were suggestive of phytoplasma infection (Fig. 1 a-e). Surge of leaf hoppers was also observed in and around the tree. The leaf samples were collected from 3 individual C. fistula trees showing suspected symptoms of phytoplasma and one sample from asymptomatic plant of both the states. Leafhopper (LH) species were collected using sweep net method from both the locations. DNA was extracted using CTAB (Cetyl trimethyl ammonium bromide) method and nested universal PCR primers P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/secArev3 and SecAfor2/ SecArev3 for SecA gene (Hodgetts et al. 2008) were employed for the analysis of the phytoplasma strain association. The symptomatic plants and leaf hopper species showed positive bands of 1.2kb and 480bp for 16S rRNA and SecA gene respectively along with. Purified PCR products of both the genes (16Sr RNA and sec A) were ligated into pGEM ®T vector and cloned in Escherichia coli (DH5-α) were sequenced at Agri Genome labs, Kerala, India. The comparative sequence analysis using the BLASTn tool results showed 16S rRNA sequences acquired from plant samples (GenBank Acc. No. OP950857, OP950858) and the leafhoppers Hishimonus phycitis (OP538583) and Orosius albicinctus (OP538584) of Kerala had the minimum of 99.84% of similarity with Bitter gourd little leaf phytoplasma from Myanmar and maximum sequence identity (100%) with the Rapeseed phyllody phytoplasma strain from Taiwan. The sequences of phytoplasma strains from Jammu trees (Genbank Acc. No. OP801671 & OP801672) and H. phycitis (OP801673) shared 100% similarity with each other as well as with North American grapevine yellows and a minimum of 97.65% with Beta vulgaris phytoplasma from Poland. The pairwise comparison results were completely supported by the corresponding phylogenetic sequence analysis of 16S rRNA and SecA gene sequences of all the isolates in the study which clustered with 16SrI-B subgroup related strains. Virtual RFLP analysis through iPhyClassifer results that were derived from in silico digestions of R16F2n/R2 region of 16S rRNA gene using 17 restriction endonucleases enzymes indicated that all the samples produced similar virtual RFLP profiles identical to the reference strain of 16SrI-B phytoplasma subgroup (aster yellows: Acc. No. M30790) with a similarity coefficient value of 1.0. To the best of our knowledge, this is the first report of the phytoplasma association of 'Ca. P. asteris' (16SrI-B) subgroup with Cassia fistula in the world.

Efficacy of Certain Insecticides against Sucking Pests and Phyllody in Sesamum

A field experiment was conducted at the Agricultural college, Naira during rabi, 2020-21 on the control of major insect pests on sesamum viz., leafhopper Hishimonus phycitis (Distant) and whitefly Bemisia tabaci (Gennadius) and phyllody disease. The results revealed that tthiamethoxam 25WG @ 0.25 g/ l was very effective against both the pests with 72.2 and 50.5% reduction in incidence, respectively. Thiamethoxam was also effective against phyllody (PDI of 5.72), and gave a maximum seed yield of 6.50 q/ ha and B:C ratio (1:4.7). Seed treatment with imidacloprid 17.5SL @ 5 ml/ kg seed stood as the next best with B:C ratio of 1:3.7.

Studies on Transmission of Phytoplasma in Sesame

The experiment was conducted to artificially transmit phytoplasma, the causal organism of sesame phyllody by mechanical sap inoculation and with insect vectors (Orosius albicinctus and Hishimonus phycitis). The experimental findings revealed that the phytoplasma could not be transmitted by sap inoculation. The mean transmission rate was found to be 54.67 per cent, with the highest rate of phyllody transmission (93.33%) observed in both treatments T8 and T9 with 3 insects/plant + 7 Days of Acquisition Feeding (DAF) + 5 Days of Inoculation Feeding (DIF) and 3 insects/plant + 7 DAF+ 7 DIF, respectively, while the lowest was observed in the treatment T1 with 3 insects/plant + 3 DAF+ 3 DIF (13.3%) phyllody transmission from infected to healthy sesame plants. All the treatments were found to be statistically significant over control, however, the treatments T8 and T9 with three insects/plant and with acquisition feeding period of 7 days and inoculation feeding of 5 and 7 days, respectively were found to be at par with each other statistically.

Detection, characterization and management of brinjal little leaf disease in Assam

Aim: To record the prevalence, detection, molecular and biological characterization of phytoplasma associated with brinjal little leaf disease in Assam and its management. Methodology: Roving survey was conducted during the 2018-2020 brinjal growing seasons to record the disease incidence and symptoms associated with the disease. Insect and dodder transmission studies were carried out along with tetracycline tests. Further, molecular detection of all the samples collected during the survey and after dodder and insect transmission studies were done through PCR assays by using universal primers. Results: The disease incidence varied from 3.62 to 23.63 per cent in surveyed districts. Transmission studies revealed that Hishimonus phycitis (Dist.) is acting as a vector of brinjal little leaf phytoplasma and found to be transmitted by dodder (Cuscuta campestris). The phylogenetic analysis indicated that the Brinjal little leaf phytoplasma understudy was associated with ‘Candidatus phytoplasma trifolii’ and plants like Datura stramonium play a major role as potential alternate host of the phytoplasma. Further, studies on the effect of the antibiotic tetracycline hydrochloride in managing the disease elucidated that the antibiotic was effective only for temporary remission of symptoms. Interpretation: This study reveals a broad picture about the incidence, phytoplasma associated with the disease, putative leafhopper vector transmitting the phytoplasma in the field, alternate host harbouring the phytoplasma, and effect of antibiotic on the disease suppression, which will be useful to develop effective management strategies.

Pest survey card on `Candidatus Phytoplasma aurantifolia’ and its vector Hishimonus phycitis

This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M-2020-0114) at the request of the European Commission. The full pest survey card for `Candidatus Phytoplasma aurantifolia’ and its vector Hishimonus phycitis is published and available online in the EFSA Plant Pest Survey Cards Gallery at the following link and will be updated whenever new information becomes available: https://arcg.is/19SK8v

“Candidatus Phytoplasma aurantifolia” increased the fitness of Hishimonus phycitis; the vector of lime witches’ broom disease

Hishimonus phycitis is one of the main insect vectors of phytoplasmas, in particular of ‘Candidatus Phytoplasma aurantifolia’-related strains associated with witches' broom disease in many crops. A study was conducted to investigate the relationship between H. phycitis infection by phytoplasmas and some of its biological parameters. H. phycitis harboring phytoplasmas resulted in more fecundity (higher number of offspring produced) and higher nymph developmental rate compared to uninfected insects. The finite rate of population increase (λ) and net reproductive rate (R0) were higher in phytoplasma-infected H. phycitis indicating that the overall fitness of the infected population was improved. The survival of phytoplasma-infected female was lower compared with infected male, which suggest that they may be a fitness trade-off in response to phytoplasma infection. A beneficial effect of a plant pathogen on vector fitness may indicate that the pathogen developed a relationship with the insect first, then moving to the plant secondarily. Taken together, our findings suggest that elimination of the infected trees (as a source of inoculum) should be a relevant part of the lime witches' broom disease management.

Hishimonus phycitis: Possible Vector of 16SrI-B Subgroup Phytoplama

Witches-broom symptoms of Catharanthus roseus are caused by ‘Candidatus phytoplasma is a terrible disease in the Uttar Pradesh State of India. The disease has been transmitted to infected C. roseus seedlings by grafting and cutting of stems. Heavy infestation of Hishimonus phycitis was recorded in ornamental garden during rainy season with witches- broom and phyllody disease of C. roseus at surveyed location of Shahjahanpur, Uttar Pradesh. To confirm the phytoplasma in C. roseus and the identification of phytoplasma causing insect vector, DNA were isolated from both of the infected plants and suspected H. phycitis. Isolated DNA of both insects and plants were performed to PCR assay with the help of phytoplasma specific universal primer P1/P7 and nested PCR with R16F2n/R2 which yielded about 1.2 kb amplicon in both the H. phycitis and witches-broom symptomatic samples. Both suspected amplified DNA samples from C. roseus and H. phycitis were sequenced. BLASTn and RFLP analysis were performed for the sequence alignment and subgroup assignment. BLASTn analyses were produces a result of 100% sequence similarity among themselves and 99% similarity with strain of 16SrI group whereas RFLP produces 16SrI-B subgroup. The study indicated that H. phycitis may play role as a vector in the transmission of these phytoplasma in C. roseus crops.

First report on clover proliferation phytoplasma related strain associated with Matthiola incana floral virescence in Uttar Pradesh, India.

Matthiola incana R. Br. (Fam: Brassicaceae) is an ornamental, commonly known as hoary stock has an extremely fragrant flowers, which blooms in dense clusters in a large variety of colors. During a survey of flower nurseries in March 2019 at Indian Institute of Sugarcane Research campus, Lucknow, floral virescence (MiV) symptoms (Fig. 1 A, B) were observed in M. incana pots with an incidence of over 40%. Leaf yellows symptoms were also observed on a weed Acalypha indica (AiLY) in Matthiola nursery (Fig. 1 C). Nested PCR assays were carried out to detect and identify the possible association of phytoplasmas with MiV and AiLY symptoms. Three each of symptomatic MiV and AiLY samples and two non-symptomatic samples were collected and processed for DNA extraction from the leaf midrib by CTAB method. Hishimonus phycitis (HP) (Hemiptera: Cicadellidae) leafhopper feeding on MiV symptomatic plants was also collected and DNA was extracted. The DNA of 8 symptomatic and 4 non-symptomatic plants and from the 10 leafhopper was used as a template for PCR assays. Phytoplasma specific 16Sr RNA gene specific primers (P1/P7 and 3Far/3Rev; Schneider et al. 1995; Manimekalai et al. 2010) and multilocus genes' specific primer pairs for secA (SecAfor1/SecArev3;SecAfo5r/SecARev2; Bekele et al. 2011), secY (SecYF1(VI)/SecYR1(VI);SecYF2(VI)/SecYR1(VI); Lee et al. 2010) and rp genes (rpFIC/rp(I)R1A; rp(VI)F2/ rp(VI)R2; Martini et al. 2007) were employed as previously described. Amplified products of ~1.3kb, ~600bp, ~1.7kb and ~1.0kb of 16S rRNA, secA, secY and rp genes of phytoplasma were consistently amplified in all the MiV and AiLY samples and in the HP leafhopper. No amplifications were achieved in any of the asymptomatic plant samples. Amplified products of all the four genes of MiV, AiLY and HP isolates were purified, sequenced and submitted in GenBank. Sequence comparison and phylogeny analysis of the sequences of the four genes of MiV, AiLY and HP isolates revealed 99% - 100% sequence identity and clustering with clover proliferation phytoplasma related strains (16SrVI group)(Fig.2 A,B,C and D). The virtual RFLP analysis of 17 restriction endonucleases corresponding to the 16S rDNA sequence of MiV, AiLY and HP phytoplasma strains by pDraw program, assigned them into a novel phytoplasma subgroup strain under 16SrVI group, since its HpaII restriction profile was different to earlier classified 16SrVI subgroups but was very close to16SrVI-E subgroup (GenBank acc. no. AY270156) (Fig 3). Earlier, peanut witches' broom (16SrII-A) phytoplasma was identified associated with M. incana from Italy (Davino et al. 2007). However, the association of clover proliferation phytoplasma (16SrVI) related strain associated with virescence symptom of M. incana is the first report in world. The weed (A. indica) and HP leafhopper were also reported as additional hosts of 16SrVI subgroup related new strain in India, which needs further investigation. The report of a new host and new subgroup of clover proliferation phytoplasma related strain in India is having an epidemiological significance and warrants attention.

Genetic diversity of phytoplasma strains infecting chrysanthemum varieties in India and their possible natural reservoirs

Symptoms typical of phytoplasma infection such as phyllody, virescence, witches’ broom and yellowing were observed in 12 varieties of Chrysanthemum morifolium in floral nurseries and experimental fields at New Delhi, Karnataka, Maharashtra and Andhra Pradesh, India, during surveys made from 2015 to 2017. Disease incidence ranged from 15 to 30%. Phytoplasma presence was confirmed in all symptomatic chrysanthemum varieties by molecular identification assays. Sequence comparison, phylogenetic and in silico RFLP analyses of 16S rDNA sequences allowed the identification of the chrysanthemum infecting phytoplasma strains into different ribosomal groups and subgroups, namely 16SrI, 16SrII-D, 16SrVI-D and 16SrXIV. Detection of phytoplasma strains of 16SrII-D subgroup were also confirmed in symptomatic Chenopodium album and Parthenium hysterophorus plants grown in and around the surveyed chrysanthemum fields at New Delhi, whereas 16SrVI-D phytoplasma strains were detected in symptomatic Cannabis sativa weed and leafhopper Hishimonus phycitis individuals collected from the symptomatic chrysanthemum fields at New Delhi. This is the first report on the presence of 16SrVI and 16SrXIV groups of phytoplasmas in chrysanthemum plants. Studies on genetic diversity of phytoplasmas infecting the major chrysanthemum varieties in India and their epidemiological aspects had previously not been reported. The detection and identification of phytoplasmas in different chrysanthemum varieties could contribute to increase the awareness among farmers in the management of these diseases.

A new genus Vittaliana belonging to the tribe Opsiini (Hemiptera: Cicadellidae) from India and its molecular phylogeny.

The new leafhopper genus Vittalianareticulata gen. nov., sp. nov., is described from India, and placed in the tribe Opsiini based on ocelli close to eyes, without carina on anterior margin of the face and bifurcate aedeagus with two gonopores. Phylogenetic analysis with maximum likelihood (ML) using IQtree v1.4.1 of combined data (Histone H3 and 28S rDNA) reveals that the new genus Vittaliana belongs to a clade consisting of Opsius versicolor (Distant, 1908), Opsiini gen. sp., Libengaia sp., Hishimonus phycitis (Distant, 1908) and Yinfomibus menglaensis Du, Liang & Dai (2019) with good branch support, and that the tribe Opsiini is paraphyletic. This resolves the placement of a new genus in the tribe Opsiini under Deltocephalinae.

Evidence for the association of mastrevirus, cucumovirus and phytoplasma with chickpea stunt disease and their putative insect vectors in India

Symptoms of severe stunting, little leaf, yellowing and axillary shoot proliferation were recorded on chickpea plants in three northern states of India (New Delhi, Uttar Pradesh and Rajasthan) during January–March, 2017–2018 with disease incidences of 6–56%. Nested PCR assays with phytoplasma-specific universal primer pairs (P1/P7 and 3Far/3Rev) and 16S rRNA gene sequence analysis confirmed the association of ‘Candidatus Phytoplasma trifolii’ in the symptomatic chickpea variety Pusa Bheema at New Delhi. The in silico RFLP and phylogeny analyses of 16S rRNA sequence of the chickpea stunt (CpS) affected samples confirmed the association of 16SrVI-D subgroup phytoplasma. Similar phytoplasma strain (16SrVI-D subgroup) was detected in the leafhopper species, Hishimonus phycitis collected from symptomatic chickpea fields at New Delhi. Association of icosahedral virus particles of ~25–30 nm diameters were seen under electron microscope in CpS affected samples as well as in the aphids, Myzus persicae (Sulzer) collected from the symptomatic CpS samples at New Delhi. Both the plant and aphid samples were tested positive with the polyclonal antibodies (pAbs) of cucumber mosaic virus (CMV) in DAS-ELISA and ISEM tests. PCR assays using coat protein (CP) gene specific primers for CMV and the resultant sequence comparison also confirmed the association of CMV with the CpS affected symptomatic chickpea plants and M. persicae. The CpS affected samples collected from all the three states were further tested positive with chickpea chlorotic dwarf virus (chickpea chlorotic dwarf virus, CpCDV) CP gene specific primers in PCR assays. The 2.7 kb DNA sequence resulted from the rolling circle amplification (RCA) from the CpS affected chickpea samples of New Delhi revealed a highest sequence similarity of 99% with the CpCDV isolate from Pakistan. These results suggested a wider occurrence of CpCDV in chickpea crops in all the three states and a mixed infection of phytoplasma [‘Ca. P. trifolii’ (16SrVI-D subgroup)] and two plant viruses (CMV and CpCDV) associated with CpS disease at New Delhi.

Identification of sesame phyllody transmitting insect vectors in Assam, India

Different leafhopper species transmitting sesame phyllody phytoplasma were reported. Leafhoppers were collected from sesame phyllody infected field at different time interval and their population was recorded. Four leafhopper species were predominantly observed in the field conditions: Hishimonus phycitis, Exitianus indicus, Cofana unimaculata and Nephotettix nigropictus. Transmission studies were carried out by giving an acquisition and inoculation feeding period of 7 days each. Among all the leafhoppers, H. phycitis was found to transmit sesame phyllody phytoplasma (83.33%) to healthy sesame plants as confirmed by PCR and nested PCR assays followed by sequencing. The BLAST analysis of 16S rDNA sequences from H. phycitis confirmed the phytoplasma presence indicating that is clusters with the aster yellows phytoplasmas.

Incidence, symptomatology and transmission of sugarcane grassy shootdisease in Assam, India

A roving survey was conducted during 2015 and 2016 to study the incidence, symptomatology and transmission of sugarcane grassy shoot (SCGS) disease in the sugarcane growing areas of four districts of Assam, viz. Biswanath, Golaghat, Sonitpur, and Nagoan. Major symptoms observed in the fields were the production of narrow and chlorotic leaves ranging from pale yellow to completely white color, the development of chlorotic streaks in leaves, unproductive tiller proliferation and the bushy appearance of the infected stool, with thin and non millable canes with short internodes. The average disease incidence ranged from 10-22%. The transmission of the disease was attempted by different methods. Out of five predominant leafhopper species (Cofana unimaculata, Exitianus indicus, Hishimonus phycitis, Nephotettix nigropictus, Orosius albicinctus) collected and identified from the sugarcane fields, in all the four districts, only E. indicus was successful in transmitting the SCGS disease. The disease was also transmitted by the setts collected from sugarcane grassy shoot symptomatic canes and by dodder.

An Identification key to the species of Auchenorrhyncha of Iranian fauna recorded as pests in orchards and a review on the pest status of the species

An illustrated dichotomous identification key for a total of 54 Auchenorrhyncha species of Iran is presented. The studied species have been recorded as pests and vectors of diseases to fruit trees. Twenty nine records were contributed to Iranian orchards and 25 of which were from other parts of the world. Hence, the latter group can be considered as potential pests and vectors in Iran. Reviewing the published information on the former group suggests 12 species as pests (4, 2 and 6 species with significant, minor, and unknown recorded economic damage levels, respectively). The pest status of 14 recorded pest species were quite doubtful and 3 of them could not be present in Iran due to the lack of evidence or their limited distribution in other parts of the world. The 4 species which were recognized as well known and significantly important pests belonged to the families: Tropiduchidae (Ommatissus lybicusBergevin, 1930), Cicadidae (Cicadatra alhageos (Kolenati, 1857)), and Cicadellidae (Hishimonus phycitis (Distant, 1908) and Neoaliturus haematoceps (Mulstant et Rey, 1855)). One species Orosanga japonicus is recording for the first time from Iran and added to the key.

Molecular characterization of 'Clover proliferation' phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India.

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches' broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of~1.3kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit,bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of 'clover proliferation' (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetablesand one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of 'clover proliferation' subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.

Sesame phyllody disease-a potential emerging threat to sesamecultivation in the north east region of India

Sesame phyllody disease associated with the presence of phytoplasmas is becoming a serious problem to many sesame growing countries of the world including India. However no systematic study on the disease has been done to assess the disease incidence, characterization of the associated phytoplasma strains and effect on the yield in north eastern states of India. Therefore an extensive survey was conducted from 2015 to 2017 in seven north eastern states of India covering 52 districts to record the incidence of the disease and to perform phytoplasma associated molecular characterization. The infected plants showed symptoms of virescence, phyllody, floral proliferation, flattened stem, splitting of capsules and witches’ broom. The disease incidence varied from 2% to 29% in different locations. The highest disease incidence (29%) was observed in the Sonitpur district of Assam in the variety ST-1683. Sesame phyllody infection was found to reduce the number of pod and seed yield per plant from 72.82%-100% and 57.20%-100%, respectively. A total of 96 symptomatic sesame samples were collected and analysed on extracted DNA with polymerase chain reaction using phytoplasma specific primers, amplifying 16S rRNA and secA genes. PCR and sequencing analysis confirmed the presence of ‘Candidatus Phytoplasma asteris’-related strains in all the symptomatic samples. Pairwise sequence comparison and phylogenetic analyses of 21 strains on 16S rRNA gene and 9 strains on secA gene sequences confirmed the phytoplasma classification in the aster yellows groups. Among the 7 leafhopper species collected only Hishimonus phycitis tested positive for sesame phyllody phytoplasma presence. Seven species of weeds and wavy leaf basket grass (Oplismenus undulatifolius) and flax (Linum usitatissimum) in and around the infected sesame fields were also positive for phytoplasma presence indicating them as potential reservoir hosts of the phytoplasma associated with sesame phyllody disease in the north east region of India.

Pest categorisation of Hishimonus phycitis.

The Panel on Plant Health performed a pest categorisation of Hishimonus phycitis (Hemiptera: Cicadellidae) for the EU. H. phycitis is a well‐defined species, occurring in tropical and subtropical Asian countries from Iran to Malaysia. H. phycitis is polyphagous. Hosts of particular relevance to the EU include Citrus spp. and Solanum melongena. While harmful in its own right as a leafhopper extracting host nutrients through feeding, it is regarded in the Middle East more significantly as a vector of Witches' broom disease of lime phytoplasma, which limits production of Citrus aurantifolia, and in India as a vector of brinjal little‐leaf phytoplasma impacting S. melongena yields. H. phycitis is currently regulated by Council Directive 2000/29/EC, listed in Annex II/AI as Hishomonus phycitis (sic). Eggs planted on host plants for planting could provide a pathway for entry into the EU. The EU has eco‐climatic conditions that are also found in countries where H. phycitis occurs although it is unknown whether H. phycitis occurs in those areas. There is therefore considerable uncertainty around EU establishment. Any establishment is likely to be limited to the warmest areas around the Mediterranean. As a free‐living organism with adults capable of flight, spread within the EU would be possible but confined to the limited area where establishment could occur. Measures are available to inhibit entry via traded commodities (e.g. prohibition on the introduction of Citrus plants for planting; sourcing other hosts from pest free areas). H. phycitis does satisfy all of the criteria that are within the remit of EFSA to assess to be regarded as a Union quarantine pest. It is uncertain if eggs of H. phycitis would carry phytoplasmas into the EU as transovarial transmission from infected females to eggs has not been demonstrated.

Pest categorisation of Witches' broom disease of lime (Citrus aurantifolia) phytoplasma.

The EFSA Panel on Plant Health performed a pest categorisation for the Witches' broom disease of lime (Citrus aurantifolia) phytoplasma for the EU territory. The pest has been reported in a few countries in the Middle East and is not known to occur in the EU. The disease is caused by a well‐defined phytoplasma strain in the ‘Candidatus Phytoplasma aurantifolia' species, for which efficient molecular detection assays are available. The most important known natural host is Citrus aurantifolia, which is only grown for ornamental purposes in the EU. Sweet limes, rough lemon and trifoliate orange are also naturally infected by that phytoplasma. The latter can be transmitted by grafting also to some citrus species. Other citrus species were reported to be resistant; however, their susceptibility has been assessed only by symptom observations, and the possible presence of phytoplasmas in symptomless plants cannot be ruled out. The phytoplasma is transmitted by the leafhopper Hishimonus phycitis, which is not known to occur in the EU. There is no information on the vector status of other phloem feeding insects of citrus present in the EU. The pest is listed in Annex IIAI of Directive 2000/29/EC. The main pathways for entry, plants for planting and the vector insect, are closed by existing legislation on import of citrus plants. Nevertheless, should the pest enter, it could establish and spread. In countries where Witches' broom disease of lime (WBDL) is present, it has significant impact. The main knowledge gaps concern (1) and vertical transmission of the phytoplasma to H. phycitis eggs (2) lack of information regarding susceptibility of citrus crops grown in the EU (3) status of potential insect vectors in the EU. Therefore, the WBDL phytoplasma meets the criteria assessed by EFSA for consideration as a potential Union quarantine pest.