Detection, characterisation and transmission by Macrosteles leafhoppers of watercress yellows phytoplasma in Hawaii

A new yellows disease of watercress (Nasturtium officinale) in Hawaii has symptoms of reduced leaf size, leaf yellowing and crinkling, and occasionally witches’ brooms. This disease is found on all watercress farms on Oahu but has not yet been found on other Hawaiian islands. Watercress plants were tested for phytoplasma infection by polymerase chain reaction assays using phytoplasma‐specific primers. Amplicons of the expected sizes were produced from all symptomatic plants but not from healthy plants raised from seed. Phylogenetic analysis of the 16S rRNA gene indicated that watercress yellows was caused by a phytoplasma in the aster yellows group, with sequence similarity to onion yellows from Japan. Six weed species collected from the vicinity of affected watercress farms, Amaranth sp., Eclipta prostrata, Emilia sonchifolia, Plantago major, Myriophyllum aquaticum and Sonchus oleraceus, were also determined to be hosts of this phytoplasma. Leafhoppers, identified as Macrosteles sp. (Hemiptera, Cicadellidae), collected from symptomatic watercress transmitted this phytoplasma to watercress, plantain and lettuce (Lactuca sativa) in greenhouse experiments.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Vector-pathogen-host plant relationships of chrysanthemum yellows (CY) phytoplasma and the vector leafhoppers Macrosteles quadripunctulatus and Euscelidius variegatus

Chrysanthemum yellows (CY) phytoplasma is a plant-pathogenic mollicutes belonging to the 16Sr-IB genetic group which infects a variety of dicotyledonous plants and is transmitted in nature by some species of Cicadellidae Deltocephalinae. The transmission characteristics of CY and the factors influencing the vector efficiencies of the leafhoppers Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum are described in the present study using transmission experiments and phytoplasma-specific polymerase chain reaction (PCR) assays. Vector insects were allowed to acquire CY under different experimental conditions and then transferred to healthy test plants for inoculation and/or sampled for DNA extraction and amplification. The transmission efficiency of CY was very high and almost all the leafhoppers became infective following acquisition on CY-infected daisies. The latent period in the vector ranged from 16 to 20 days after the start of the acquisition and infectivity lasted, in general, for life. The PCR assay was successful in detecting CY phytoplasmas in the insects well before they became infective (5 versus 16–18 days) and was used to estimate the proportion of infective insects. When analysed for CY presence by PCR, all the leafhoppers fed for 7–18 days on source daisy reacted positively while, following one day of acquisition, some insects failed to provide amplification. Host-plant species influenced CY acquisition, and daisy appeared a more efficient source for both leafhoppers compared to periwinkle. Life stage did not appear to be critical for CY acquisition, although newly-hatched nymphs of E. variegatus acquired CY less efficiently than fifth instar nymphs.     

Molecular data reveals California as the potential source of an invasive leafhopper species, Macrosteles sp. nr. severini, transmitting the aster yellows phytoplasma in Hawaii

A species of aster leafhopper ( Macrosteles sp.) became established in 2001 on Oahu, Hawaii, and through the transmission of the aster yellows phytoplasma, caused devastating losses to the island's watercress industry. DNA sequence data were analysed from two mitochondrial genes [cytochrome oxidase subunit 1( CO1 ) and nicotinamide adenine dinucleotide 1 ( NADH1 )] and one nuclear gene (wingless, Wg ) (combined total of 1874 bp) to reconstruct phylogenetic relationships between putative US mainland source populations of aster leafhoppers and those introduced to Hawaii. These data were applied to elucidate the origin(s) and identity of Hawaiian infestations and the amount of genetic diversity within introduced invasive populations. Both phylogenetic search criteria (Bayesian and maximum likelihood models) converged onto similar tree topologies for all three gene regions and suggested that Hawaii infestations represent a single undescribed leafhopper species unrelated to the common aster leafhopper, Macrosteles quadrilineatus . An exact haplotype match was found from a specimen intercepted from watercress shipped to Hawaii from Los Angeles, California, suggesting this region as the potential source for Hawaiian infestations. Two mitochondrial haplotypes were identified in Hawaii suggesting two or perhaps just a single introduction of more than one female.     

Ability of Reptalus quinquecostatus (Hemiptera: Cixiidae) to inoculate stolbur phytoplasma to artificial feeding medium

Laboratory trials were carried out on wild individuals of Reptalus quinquecostatus (Cixiidae), a potential vector of stolbur phytoplasma to grapevine, to assess its ability to inoculate the phytoplasma in artificial feeding medium. Seventy‐seven specimens of the cixiid were tested on a sucrose–TE (Tris–ethylenediaminetetraacetic acid) diet and 62 of them survived less than 24 h. Polymerase chain reaction (PCR) assays performed on the insect bodies detected the presence of stolbur phytoplasma, with an infection rate of 32.5%. Restriction fragment length polymorphism analysis of the tuf gene, amplified by PCR, revealed Vergilbungskrankheit type I (VK‐I) in 20 specimens, VK‐II in 4 specimens and both types in 1 specimen. Ten of the 25 infected R. quinquecostatus specimens successfully inoculated VK‐I in the sucrose solution, that is, a 40% inoculation efficiency despite the brief survival. The results indicate that R. quinquecostatus is a competent species to transmit the stolbur phytoplasma in artificial conditions. The repeated observation of adults feeding on grapevine strengthens the hypothesis that the species is a vector of stolbur phytoplasma to this plant.     

Variability of phytoplasma associated with few ornamental crop species in Uttar Pradesh,India

Abstract

The economic importance of ornamentals has been advancing significantly in many countries due to continuous international demand. Like other crops, ornamental plants are also affected by a number of pathogens and the phytoplasmas diseases are equally important group of pathogens which is responsible for damaging the different ornamental plant species and thereby affecting their market value. In the present investigation, total 24 symptomatic leaf samples of 8 ornamental plant species (C. roseus, R. rubiginosa, C. variegatum, Z. elegans, J. gendurusa, T. erecta, H. rosa sinensis, J. sambrac) were analyzed which were found positive in nested PCR assays with R16F2n/R16R2 primers. The phytoplasma associated with ornamental plant species collected from different districts of UP in the present investigation are belongs to 16Sr I, II, VI and XIV group.     

Sugarcane white leaf phytoplasma in tissue culture: long-term maintenance,transmission, and oxytetracycline remission

Sugarcane white leaf (SCWL)-diseased sugarcane plants collected from Udornthani Province, in north-eastern Thailand, were the source for tissue culture experiments. Explants from axillary buds, meristem tips, and leaves grew optimally in Murashige-Skoog medium containing 0.5 mg/l -naphthaleneacetic acid, 0.5 mg/l 6-benzylaminopurine, and 15% coconut water. Callus development and shoot/root proliferation were more rapid in cultures from diseased than from healthy plants. Disease symptoms continued for 6 years after culture initiation, and SCWL phytoplasma persisted, as confirmed by polymerase chain reaction using both 16S rDNA and 16S-23S rDNA primers. Phytoplasmas in the cultured plantlets were transmissible by grafting to sugarcane and periwinkle, and by feeding of the leafhopper vector Matsumuratettix hiroglyphicus to sugarcane. Although 50% of the plantlets were killed by oxytetracycline at 500 mg/ml, 70–100% of plantlets grown with 200–500 mg/ml oxytetracycline showed symptom remission through 5–8 subcultures. Typical phytoplasma-like bodies, visible by electron microscopy in sieve tubes of untreated diseased plantlets, were absent in antibiotic-treated plantlets. Thus, tissue culture provides a convenient and reliable in vivo system for investigation of SCWL phytoplasma. A preliminary report of this study was presented at the Eighth International Congress of Plant Pathology, Christchurch, New Zealand, 2–7 February 2003     

Detection and seed transmission of Bermudagrass phytoplasma in maize in Turkey

During 2017, maize cultivation areas in the provinces of Adana and Kahramanmara? (Turkey) were surveyed to inspect maize plants with symptoms similar to those associated with of phytoplasma disease, that is, yellowing, short internodes and small corncobs. Thirty fields were inspected and two hundred samples from symptomatic and asymptomatic plants were collected, together with insects considered as potential vectors of phytoplasmas. All samples were assayed by polymerase chain reaction (PCR) and subsequently analysed by restriction fragment length polymorphism and sequencing to identify the phytoplasmas detected in the plant material and insects. Results of laboratory assays and phylogenetic analyses showed that the Bermudagrass white leaf phytoplasma ('Candidatus Phytoplasma cynodontis') was present in both maize plants and seeds, showing 99% sequence identity with other reported phytoplasma strains from GenBank, whereas no PCR amplifications were obtained from tested insects. The seeds of infected plants, sown in an insect‐proof screenhouse, produced plantlets that were found PCR‐positive for the Bermudagrass white leaf phytoplasma, indicating its seed transmission.     

Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

Graft and dodder transmission of phytoplasma affecting lily to experimental hosts

Experimental infection of Alstroemeria seedlings with naturally infected lily ‘Casablanca’ with stunting and flower bud deficiency phytoplasma resulted 3–4 weeks after top grafting in chlorotic and/or necrotic stripes, whitening of the leaves, shoot necrosis and die back. Flower discoloration or malformation were not observed. Attempts to transmit phytoplasma from naturally infected lily and experimentally infected Alstroemeria to Catharanthus roseus by top grafting resulted in stunted growth, dull yellowing and malformation of the leaves in 4–6 weeks. Some plants were temporary entirely vegetative and did not produce flowers. The periwinkle plants that were bridged by Cuscuta odorata from the diseased lilies and Alstroemerias showed similar symptoms as top-grafted ones. With the universal primer pairs rU3/fU5 specific PCR product with expected length ∼900 was amplified from samples collected from lilies with severe symptoms and top grafted test plants. All PCR products used for RFLP analysis after digestion with Alu I showed the same restriction profiles. Position of three obtained bands corresponded to the lengths of the DNA fragments of American aster yellows (AAY) phytoplasma group.     

Population structure and incidence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) among geographic regions in Switzerland

The dissemination of stolbur phytoplasma (16Sr‐XIIA group)‐induced yellows diseases depends on the dispersal biology and host plant fidelity of the planthopper vector Hyalesthes obsoletus (Hemiptera: Cixiidae). We analysed the degree of these two properties in H. obsoletus by studying its population genetic structure and stolbur infection rates relative to the two major host plants, Convolvolus arvensis and Urtica dioica, in order to infer relevant divisions for stolbur epidemiology in Swiss viticultural regions. Three regional populations with the potential to determine stolbur epidemiology in distinct ways were identified. First, populations associated with U. dioica in northern Switzerland were most related to genetically distinct U. dioica host race populations identified previously in Germany. Second, populations in central and southwest Switzerland were undifferentiated relative to host plant and likely have wider stolbur transmission breadths than the northern specialized populations. Third, populations in south of the Alps (Ticino) were undifferentiated relative to host plant but geographically isolated from other Swiss regions, thus implying separate population dynamics in this area. The knowledge of these three distinct epidemiological cycles will help to adapt management programmes against stolbur diseases in Swiss vineyards.     

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

Chrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers-exposed plants were analyzed by dot-blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.     

Molecular detection and characterization of a stolbur phytoplasma associated with Narcissus tazetta phyllody in Iran

During field surveys in 2015, a phytoplasma‐associated disease was identified in Narcissus tazetta plants in Behbahan, Iran. The characteristic symptoms were phyllody and virescence. The presence of phytoplasma in symptomatic plants was confirmed using PCR amplification and sequencing of 16S rRNA, tuf, secY and vmp1 genes. Based on the blastn results, the sequences of 16S rRNA, tuf, secY and vmp1 genes shared, respectively, 99%, 100%, 99% and 99% sequence identity with phytoplasma strains in 16SrXII‐A subgroup. RFLP and phylogenetic analyses using the sequences of 16S rRNA, tuf and secY genes confirmed the assortment of studied strains to 16SrXII‐A phytoplasma subgroup. Sequence comparison of these four genes revealed that all the sequences of 28 strains studied were identical. To the best of our knowledge, the association of “Candidatus Phytoplasma solani” with N. tazetta was demonstrated for the first time in the world.     

Occurrence,identification and transmission of the phytoplasma associated with tomato big bud disease and identification of its vector and weed host in Pakistan

Abstract

Tomato (Solanum lycopersicum L.) plants showing stunting, big bud, leaves yellowing or reddening and witches’-broom symptoms were observed since 2009 in Pakistan. A weed Parthenium hysterophorus grown in and around tomato fields also exhibited witches’-broom like symptoms. Fluorescence light microscopy of hand-cut stem stalk sections treated with Dienes’ stain showed blue areas in the phloem region of both tomato and P. hysterophorus symptomatic plants that indicated the association of phytoplasma with the complex. Amplification of 1.2?kb 16S rDNA fragment in nested PCR confirmed that the symptomatic tomato and P. hysterophorus plants are infected by a phytoplasma. Partial sequencing of 16S rRNA (GenBank accession: LT671581 and LT671583) and virtual restriction fragment length polymorphism confirmed that the phytoplasma associated with both plant species had the greatest homology to 16SrII-D subgroup. Disease was successfully transmitted by grafting and leafhopper Orosius albicinctus in tomato plants. This is the first report of natural occurrence of 16SrII-D phytoplasma in tomatoes and a weed P. hysterophorus in Pakistan.     

Survival relative to new and ancestral host plants,phytoplasma infection,and genetic constitution in host races of a polyphagous insect disease vector

Dissemination of vectorborne diseases depends strongly on the vector's host range and the pathogen's reservoir range. Because vectors interact with pathogens, the direction and strength of a vector's host shift is vital for understanding epidemiology and is embedded in the framework of ecological specialization. This study investigates survival in host‐race evolution of a polyphagous insect disease vector, Hyalesthes obsoletus, whether survival is related to the direction of the host shift (from field bindweed to stinging nettle), the interaction with plant‐specific strains of obligate vectored pathogens/symbionts (stolbur phytoplasma), and whether survival is related to genetic differentiation between the host races. We used a twice repeated, identical nested experimental design to study survival of the vector on alternative hosts and relative to infection status. Survival was tested with Kaplan–Meier analyses, while genetic differentiation between vector populations was quantified with microsatellite allele frequencies. We found significant direct effects of host plant (reduced survival on wrong hosts) and sex (males survive longer than females) in both host races and relative effects of host (nettle animals more affected than bindweed animals) and sex (males more affected than females). Survival of bindweed animals was significantly higher on symptomatic than nonsymptomatic field bindweed, but in the second experiment only. Infection potentially had a positive effect on survival in nettle animals but due to low infection rates the results remain suggestive. Genetic differentiation was not related to survival. Greater negative plant‐transfer effect but no negative effect of stolbur in the derived host race suggests preadaptation to the new pathogen/symbiont strain before strong diversifying selection during the specialization process. Physiological maladaptation or failure to accept the ancestral plant will have similar consequences, namely positive assortative mating within host races and a reduction in the likelihood of oviposition on the alternative plant and thus the acquisition of alternative stolbur strains.     

Infection of Bois‐Noir tuf‐type‐I stolbur phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) larvae and influence on larval size

Recent dramatic spread of the grapevine yellows disease Bois Noir (BN) in Germany is above all explained by highly increased abundances of the vector Hyalesthes obsoletus (Hemiptera: Cixiidae) associated to the plant Urtica dioica, the reservoir of the BN pathogen stolbur tuf‐type‐I. The vector acquires BN‐phytoplasma as larvae whilst feeding on the roots of infected U. dioica. To understand the dynamics of the Urtica‐cycle, we tested at what instar larvae become infected and whether infection affects larvae size (i.e. growth) at two sites in the Mosel Valley, Germany. Larvae were tested from infected plants and collected at instar‐stages 3, 4 and 5. Larvae at stage 3 were already infected but infection rates increased significantly between stage 3 and 5, mean infection rates: 0.12–0.62. There was no effect of infection on larval size at any instar stage.     

Vector activity of Hyalesthes obsoletus living on nettles and transmitting a stolbur phytoplasma to grapevines: a case study

We report a case study on the vector activity of a Hyalesthes obsoletus (Hemiptera: Cixiidae) population living on nettle plants (Urtica dioica) and transmitting a stolbur phytoplasma (Sp) to grapevines (Vitis vinifera). The research was conducted in a site that included a vineyard bordered with a large fallow area where nettles were the predominant plant species together with sparse old grapevines. Nettles hosted a high population of H. obsoletus. By using transparent sticky traps to sample adults, we observed that the daily flight activity of males and females to grapevines in the fallow was unimodal peaking between 15 and 21 h in the day. Adults were unable of great dispersion into the vineyard and the pattern of insect captures inside the planting reflected the pattern of Sp‐infected grapevines in the late autumn. When insects were forced to feed on grapevine cuttings for transmission assays, survival of H. obsoletus decreased after 24–48 h. The scarce propensity of the vector to move into the vineyard and feed on grapevines was counterbalanced by the rapidity of H. obsoletus to inoculate Sp to grapevines (estimated minimum inoculation access period ranged from 3 to 6 h) and a relative high incidence of Sp in the population of H. obsoletus that ranged between 20% and 30% of sampled insects as shown by a polymerase chain reaction–based procedure. Characterisation of Sp by restriction fragment length polymorphism analysis of nonribosomal phytoplasma DNA showed the occurrence of an Sp strain known to infect H. obsoletus associated to nettles and grapevines in Germany.     

Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines,bindweeds and vegetables

Aim: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results: Phytoplasma isolates belonging to stolbur subgroup 16SrXII‐A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf‐a type and 68% by tuf‐b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf‐b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.     

ATP6蛋白影响异沙叶蝉对小麦蓝矮植 原体的传播效率

【目的】探索异沙叶蝉Psammotettix alienus ATP6蛋白对小麦蓝矮植原体(wheat blue dwarf phytoplasma, WBDp)传播效率的影响。【方法】将异沙叶蝉mRNA反转录合成cDNA,构建分离泛素酵母双杂交膜系统cDNA文库;用WBDp的虫传相关蛋白P2-4为诱饵,从异沙叶蝉cDNA文库杂交筛选互作蛋白;通过RNAi沉默ATP6基因,验证ATP6蛋白对异沙叶蝉3龄若虫传播WBDp的影响。【结果】构建了异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库,用P2-4从cDNA文库中杂交筛选获得了ATP6等多种互作蛋白。RNAi结果发现沉默ATP6基因后异沙叶蝉3龄若虫的WBDp传播率比对照组显著下降了23.33%±3.33%,表明ATP6是异沙叶蝉参与WBDp传播的关键蛋白。【结论】明确了异沙叶蝉ATP6是参与WBDp传播的一个关键蛋白。为进一步探明异沙叶蝉传播WBDp的分子机制和植原体病害的田间防治提供了理论基础。     

Identification,occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

A petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.