Broom Symptoms Research Articles

Identification, expression and miRNA targeting of auxin response factor genes related to phyllody in the witches' broom disease of jujube.

Jujube witches' broom (JWB) disease, associated with the presence of phytoplasmas, induces huge crop losses in the woody perennial fruit tree Ziziphus jujuba. An imbalance in the phytohormone auxin is thought to be a key factor in the development of the witches' broom symptoms, and in the alteration of floral development into leafy structures, termed phyllody. The Auxin Response Factor (ARF) gene family controls auxin-responsive gene expression during plant growth and development. However, it remains unknown if the ARF genes are involved in the formation of leaf-like flowers. In the present study, sixteen jujube ARF genes were identified bioinformatically and annotated based on the Z. jujuba cv. Dongzao genome. The ZjARFs were homologous to 12 out of the 23 Arabidopsis ARFs and were distributed in 8 jujube chromosomes and 3 unmapped scaffolds. Phylogenetic analysis grouped the ZjARFs into three classes. Spatio-temporal expression analysis revealed that the ZjARF genes were differentially expressed among different tissues during normal development. The expression of seven ZjARF genes was significantly decreased from flower buds to flowering. JWB-infected jujube plants developed the typical phyllody symptoms and showed lower auxin accumulation during floral development. ZjARF1, ZjARF2, ZjARF3, ZjARF4 and ZjARF8 resulted differentially regulated after phytoplasma infection. ZjARF4 was down-regulated before and during floral development in phytoplasma-infected plants, but it was significantly up-regulated before flowering and down-regulated during flowering in the healthy plants. Target site analysis showed that miRNA167, miRNA529 and miRNA2950 could directly target ZjARF4. Together, the data showed that the auxin-controlled ARF4 gene is likely involved in the disruption of floral development in phytoplasma-infected jujube plants.

Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

First Report of a ‘Candidatus Phytoplasma aurantifolia’ Strain Associated with Virescence, Floral Proliferation, and Dwarf Symptoms on Indigofera suffruticosa in Taiwan

Indigofera suffruticosa Mill., generally known as anil or Guatemalan indigo, is a perennial shrub planting in tropical and subtropical regions, including Taiwan; it was commonly used to produce the indigo dye, which is still being used in many personal workshops in Taiwan. There were a few reported cases of the association of phytoplasma with Indigofera spp. In 1983, I. suffruticosa showing phyllody and small leaf symptoms was first described, but no molecular identification was conducted (Albanese et al. 1995). Indigofera linifolia with little leaf, phyllody, and proliferation symptoms and Indigofera colutea showing little leaf symptoms in Australia caused by phytoplasma strains were reportedly associated with papaya yellow crinkle disease (Padovan and Gibb 2001). Affected Indigofera juncea and Indigofera hirsuta exhibiting witches’-broom symptoms were reported to be the same as the sesame phyllody phytoplasma isolate in Thailand (Nakashima et al. 1995). In February 2016, I. suffruticosa plants showing virescence, floral proliferation, and dwarf symptoms were collected from commercial fields of Mingjian Township, Nantou County, Taiwan. Two each of symptomatic and asymptomatic samples were collected from the field for further study. Transmission electron microscopic observation revealed the presence of the pleomorphic phytoplasma cells only in the phloem sieve cells of the two symptomatic I. suffruticosa plants. The two asymptomatic and two symptomatic samples were used for total DNA extraction with the modified CTAB method, and the amplicons of the nested PCR with universal primer pairs for nearly full-length 16S rDNA amplification (P1/P7 followed by R16F2n/R16R2) were electrophoresed in 0.8% agarose gel (Fulton et al. 1995; Lee et al. 1998). A 1.2-kb fragment (GenBank accession no. KX268346) was identified only from the two symptomatic I. suffruticosa plants. Analysis of 16S rDNA sequences by iPhyClassifier indicated that the phytoplasma associated with virescence symptoms in I. suffruticosa shared a 98.2% identity with that of the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession no. U15442). Virtual restriction fragment length polymorphism patterns were identical to those of the 16SrII-V subgroup (Zhao et al. 2009). Taken together, the results indicated that the phytoplasma associated with I. suffruticosa in Taiwan is a ‘Ca. P. aurantifolia’-related strain. The occurrence of phytoplasma in I. suffruticosa could have direct implication on economic profit for growers and the handicraft industry in Taiwan. To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma causing virescence, floral proliferation, and dwarf symptoms on I. suffruticosa in Taiwan.

Cannabis sativa L.: A Potential Natural Reservoir of Sugarcane Grassy Shoot Phytoplasmas in India

Cannabis sativa plants growing nearby sugarcane fields affected with grassy shoot disease at Muzaffarnagar, Uttar Pradesh, India, showed severe witches’ broom symptoms. The DNA from symptomatic Cannabis and sugarcane samples was isolated and subjected to nested PCR assays with universal phytoplasma-specific primer pairs (P1/P7 and R16F2n/R16R2). A DNA fragment of ~ 1.2 kbp was amplified in all the symptomatic samples of sugarcane and Cannabis with R16F2n/R16R2 primer pair but was absent in non-symptomatic samples. Pairwise sequence comparison, phylogeny and in silico RFLP analysis of 16S rDNA sequences confirmed the identity of a rice yellow dwarf phytoplasma group-related strain (16SrXI-B) associated with Cannabis which suggested its role as potential natural reservoir of SCGS phytoplasma in nature.

Mixed infection of phytoplasma and begomovirus associated with leaf curling and witches’ broom disease of Zinnia elegans in India

Zinnia elegans belonging to the asteraceae family is a flowering annual summer plant and is native to Mexico. Symptoms of excessive leaf curling, little leaf, stunting and witches’ broom were observed on Z. elegans in Tripura state of India during 2016–2017. PCR assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 and tomato leaf curl virus coat protein specific primers yielded specific amplifications of ~ 1.25 kbp and ~ 800 bp, respectively from the symptomatic zinnia leaf samples. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene sequences of zinnia isolate confirmed the presence of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup). Further coat protein sequence comparison and phylogenetic tree analysis of coat protein gene confirmed association of begomovirus with zinnia symptomatic samples and identified it as a strain of tomato leaf curl virus. Our results confirmed a mixed infection of ‘Ca. P. asteris’ and begomovirus with zinnia leaf curl and witches’ broom symptoms.

First report of association of a 16SrII group phytoplasma with a witches'broom disease of Croton bonplandianum

The diseases associated with peanut witches’ broom phytoplasmas are observed in several legume crops and weed species with typical phyllody and witches’ broom symptoms. The presence of a phytoplasma strains belonging to 16SrII-D subgroup is reported in the common Indian weed Croton bonplandianum showing witches’ broom and phyllody. C. bonplandianum is producing widespread infestations in agriculture fields and barren lands and then pose a serious threat as possible secondary host of this phytoplasma. This is the first identification of a ‘Candidatus Phytoplasma australasia’-related strains in C. bonplandianum showing witches’ broom symptoms.

First report of a ‘Candidatus Phytoplasma asteris‘‐related strain (16SrI‐B) associated with Morus alba (white mulberry) witches' broom in Iran

White mulberry (Morus alba), belongs to the family Moraceae and is grown domestically and commercially in Iran for use as fresh fruit or dried nuts. During 2018-19, white mulberry plants showing witches' broom (WMWB) symptoms (Fig. 1) were observed sporadically in the Marvast district of Yazd province, Iran. The maximum incidence of disease was 4%. In affected trees, the witches' broom was restricted to some branches and did not affect the whole canopy. Total DNA was extracted from 0.2g of midrib tissue of fresh leaves from a total of 14 diseased plants from five different orchards, and three asymptomatic plants. DNA samples were tested for the presence of phytoplasmas by nested PCR, using primers P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) in the first round, and primers R16F2n/R16R2 (Gundersen & Lee, 3) in the nested round. PCR amplicons of 1.8 and 1.25 kb (respectively) were obtained from all diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using RsaI, KpnI, HpaII, HaeIII, AluI, MseI, HhaI, TaqI, BfaI and ThaI restriction endonucleases showed RFLP patterns similar to the 16SrI phytoplasma group. The PCR products from three randomly selected WMWB plants were sequenced and showed 100% sequence identity with each other and 99.6% identity with ‘Ca. P. asteris’ (M30790). One R16F2n/R16R2 16S rDNA sequence from the Marvast WMWB strain was submitted to GenBank (Accession No. MT075612). BLAST searches showed that the WMWB phytoplasma shared more than 99% sequence identity with those of ‘Ca. P. asteris’ and related strains. Phylogenetic analysis using Mega7 software confirmed that the phytoplasma strain associated with WMWB clustered within the 16SrI phytoplasma clades closest to ‘Ca. P. asteris’ (M30790) (Fig. 2). Many phytoplasma strains related to ‘Candidatus Phytoplasma aurantifolia’ (from carrot and sunflower), ‘Ca. Phytoplasma australasia’ (from alfalfa, almond, beetroot, marigold, sesame, squash and tamarisk), ‘Ca. Phytoplasma trifolii’ (from alfalfa, aubergine, cucumber and tomato), ‘Ca. Phytoplasma solani’ (from bindweed and grapevine) and ‘Ca. Phytoplasma omanensis’ (from bindweed and peach) have been reported previously in Yazd province. A 16SrI-B related phytoplasma has been reported associated with Eruca sativa (rocket) phyllody in Yazd province (Esmailzadeh Hosseini et al., 2). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’ (16SrI-B) strain associated with witches' broom of Morus alba in Iran.

First Report on ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) Strain Associated with Pineapple Shoot Proliferation and Witches’ Broom Symptoms in Tripura, India

First Report on ‘<i>Candidatus</i> Phytoplasma asteris’ (16SrI-B subgroup) Strain Associated with Pineapple Shoot Proliferation and Witches’ Broom Symptoms in Tripura, India

Identification of a phytoplasma associated with witches&amp;#8217; broom symptoms in calafate (Berberis microphylla G. Forst.)

Identification of a phytoplasma associated with witches&amp;#8217; broom symptoms in calafate (Berberis microphylla G. Forst.)

First Report of ‘Candidatus Phytoplasma trifolii’ Associated with Witches’ Broom Disease of Rauwolfia serpentina from Northeast Region of India

HomePlant DiseaseVol. 103, No. 7First Report of ‘Candidatus Phytoplasma trifolii’ Associated with Witches’ Broom Disease of Rauwolfia serpentina from Northeast Region of India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of ‘Candidatus Phytoplasma trifolii’ Associated with Witches’ Broom Disease of Rauwolfia serpentina from Northeast Region of IndiaM. K. Kalita, G. P. Rao, Madhupriya, and A. K. GogoiM. K. KalitaBiswanath College of Agriculture, Assam Agricultural University, Biswanath Chariali, Assam 784176, IndiaSearch for more papers by this author, G. P. Rao†Corresponding author: G. P. Rao; E-mail Address: gprao_gor@rediffmail.comhttp://orcid.org/0000-0003-1057-7826Division of Plant Pathology, ICAR–Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, IndiaSearch for more papers by this author, MadhupriyaDivision of Plant Pathology, ICAR–Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, IndiaSearch for more papers by this author, and A. K. GogoiBiswanath College of Agriculture, Assam Agricultural University, Biswanath Chariali, Assam 784176, IndiaSearch for more papers by this authorAffiliationsAuthors and Affiliations M. K. Kalita1 G. P. Rao2 † Madhupriya2 A. K. Gogoi1 1Biswanath College of Agriculture, Assam Agricultural University, Biswanath Chariali, Assam 784176, India 2Division of Plant Pathology, ICAR–Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, India Published Online:18 May 2019https://doi.org/10.1094/PDIS-01-19-0150-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Rauwolfia serpentina Benth ex. Kutz (sarpagandha) also known as snakeroot or serpentine root, belongs to the family Apocynaceae, well known for its medicinal properties to treat and cure many human diseases. During November 2016, phytoplasma-suspected typical symptoms of little leaf, dwarfing, and profuse auxiliary proliferation and ultimately severe witches’ broom symptoms were noticed on R. serpentina plants in the medicinal and herbal garden of Biswanath College of Agriculture, Assam Agricultural University, Biswanath Chariali, Assam (26.7233′N, 93.1364′E), India. The disease incidence was recorded as 12% under field conditions by visual observation of symptomatic versus nonsymptomatic plants. Fresh samples (both symptomatic and asymptomatic in triplicate) were collected and DNA was extracted, which was used as a template for direct and nested polymerase chain reaction assays primed by phytoplasma universal primer pairs of 16S rDNA region (P1/P7 followed by R16F2n/R16R2n), using previously described conditions (Schneider et al. 1997) and secA gene primer pair (SecAfor1/SecArev3 followed by SecAfor1/SecArev2) (Hodgetts et al. 2008). Approximately 1.2-kb and 480-bp amplified products were obtained, which were sequenced bidirectionally to obtain maximum length of sequence, and the consensus sequences were deposited in the GenBank with accession numbers MG721532 for 16S rRNA gene and MG721533 for secA gene. Pairwise sequence comparison of 16S rDNA and secA gene sequences of R. serpentina phytoplasma isolate revealed 99 to 100% sequence identities with strains of ‘Candidatus Phytoplasma trifolii’. The MEGA6-constructed neighbor-joining phylogenetic analysis and virtual restriction fragment length polymorphism analysis using 17 restriction enzymes of 16S rRNA gene sequences allowed affiliating the R. serpentina phytoplasma-India strain with 16SrVI-D subgroup strains with a similarity coefficient of 1.0 (Hiruki and Wang 2004). The 16S rRNA and secA gene sequences comparison confirmed the close association of phytoplasma strain associated with R. serpentina with 16SrVI-D subgroup phytoplasmas. Earlier, 16SrVI-D subgroup phytoplasma has been reported associated with various vegetables and horticultural crops in India (Rao et al. 2017). Phytoplasma diseases have been reported in over 200 medicinal plants worldwide (Marcone 2016). To the best of our knowledge, this is the first report of the association of 16SrVI-D subgroup phytoplasma strain causing little leaf, witches’ broom, and stunting symptoms on R. serpentina plant, which is an important medicinal plant species in India and has a large area under cultivation in different parts of India. Because the 16SrVI-D group phytoplasma has been reported on a new host, R. serpentina, in addition to many crop plants and weeds, the studies on the disease epidemiology and common insect vectors would be helpful in developing management strategies.The author(s) declare no conflict of interest.

First report of a 16SrII phytoplasma (subgroup D) associated with Robinia pseudoacacia witches' broom and dieback in Iran

Black locust (Robinia pseudoacacia) is an important shade tree in urban green spaces in Iran. During 2017-18, R. pseudoacacia trees showing witches' broom and dieback symptoms (Fig. 1) were observed in Safayieh, Yazd province, Iran. …

Spread and colonization pattern of ‘Candidatus Phytoplasma aurantifolia’ in lime plants [Citrus aurantifolia (Christm.) Swingle] as revealed by real-time PCR assay

Lime witches’-broom (WBDL) is a devastating disease of Mexican lime trees in southern Iran. ‘Candidatus Phytoplasma aurantifolia’ is associated with the disease. Understanding the pattern of colonization and spread of ‘Ca. P. aurantifolia’ within lime trees is critical for its accurate detection during the latent period of the disease. In the present study, the distribution pattern of the WBDL phytoplasma in graft inoculated lime seedlings was investigated under greenhouse conditions and its seasonal variation of concentration in symptomless lime trees was evaluated in an infected orchard using qPCR assay. Repeated sampling from various tissues of graft-inoculated lime seedlings was carried out at various times post-inoculation. In the field, leaf samples were taken from symptomless trees in an infected orchard in March, August and December. Our results showed that after entrance into the lime seedlings, ‘Ca. P. aurantifolia’ moved downward into the roots as well as upward into the apical leaves. The concentration of phytoplasma in apical leaves showed an initial high followed by a decrease before establishment of infection and subsequent symptom appearance. The spread of the phytoplasma from root to the aerial parts was rather slow. Severe witches’ broom symptoms appeared on the grafted branches 10 months after grafting. At this time, the highest phytoplasma DNA concentration was found in descending order in stems and leaves of witches’ brooms, crown, various parts of the root, and stem bark and apical leaves of the symptomless branches. Results of the field experiment revealed that concentration of ‘Ca. P. aurantifolia’ cells in infected trees was highest in August compared to that in December and March. Our result would be useful for rapid detection of WBDL phytoplasma during the latent period of the disease.

A new phytoplasma strain in group 16SrV associated with witches'broom disease of Ziziphus oenoplia in Karnataka, India

Ziziphus oenoplia, also known as jackal jujube or small fruited jujube is a spiny, evergreen small tropical fruit tree. During a survey in October-November 2019, the jujube trees exhibited severe witches’ broom symptoms, therefore, an investigation was carried out, to verify the presence of phytoplasmas in these symptomatic plants. Total DNA was extracted from leaves of jujube plants using a CTAB method and a PCR assay was performed using 16S rRNA gene specific primer pairs P1/P7 and 3Far/3Rev. Amplicons of about 1.3 kb were consistently amplified from all the symptomatic plant samples in nested PCR assays, while, no amplification was observed in non-symptomatic samples. The phylogenetic analysis of 16Sr DNA sequence of the detected phytoplasma strain indicates its clustering with ‘Candidatus Phytoplasma balanitae’ strains and also its close relatedness to 16SrV-F subgroup phytoplasmas. The virtual RFLP analysis of the 16Sr DNA sequence revealed that it could tentatively be classified into a novel phytoplasma subgroup since its AluI restriction profile was unique to earlier reported subgroup strains in 16SrV group.

First report of ‘Candidatus Phytoplasma trifolii’-related strain presence in Juniperus procubens witches’ broom in Iran

During 2018 in the Kordkouy (Golestan province) of Iran, Japanese juniper trees with witches’ broom symptoms similar to those associated with phytoplasma presence were observed and sampling was carried out to verify the presence of these bacteria. Phylogeny of PCR amplified 16S rRNA gene sequence revealed that individual trees were infected by a ‘Candidatus Phytoplasma trifolii’-related strain. This study reveals that this phytoplasma is more widespread geographically than previously reported in Iran. This is the first report of these phytoplasmas presence in juniper witches’ broom disease worldwide.

Diversity of Phytoplasmas Associated with Several Plants in Western Java-Indonesia

Diseases caused by phytoplasmas have been reported in field crops, ornamentals, and weeds in Indonesia. However, most of phytoplasmas have not been subjected to further identification and thus, they remain unaffiliated with proper classification scheme. More reliable identification of phytoplasmas mostly rely on molecular methods. The aim of this study was to characterize the phytoplasma as the causal agent of naturally infected plants in western Java-Indonesia based on their 16S rRNA nucleotide sequences. Plant exhibiting phytoplasmal symptoms were observed and taken for further molecular examination. Eight plant species from three families in Bogor, Tangerang, and Bekasi, i.e. peanut, soybean, snakebean, Opuntia sp., betung bamboo, apus bamboo, Bermuda grass, and digitaria grass (Digitaria fuscescens) have been observed and taken as samples for further molecular examination. Nested-PCR with primer pairs P1/P7 followed by R16F2n/R16R2 resulted in amplification of products of approximately 1.2 kb from all symptomatic plant samples tested. BLAST analysis of the nucleotide sequences, phylogenetic analyses, and similarity coefficients derived from RFLP in silico revealed that there were association of a phytoplasma of 16SrII-A subgroup with phytoplasmas identified in peanut, soybean, and snakebean. Such an association showed witches’ broom symptoms; 16SrII-C with Opuntia sp. causing proliferation and mosaics; and phytoplasmas displaying yellowing and little leaf of two kinds of bamboos and white leaf of Bermuda grass and digitaria grass that were closely related to 16SrXIV-A subgroup. To our knowledge, this is the first report molecular identification of 16SrXIV-A associated with apus bamboo and digitaria grass in Indonesia.

Phytoplasma effector SWP1 induces witches’ broom symptom by destabilizing the TCP transcription factor BRANCHED1

Phytoplasmas are insect-transmitted phytopathogenic bacteria, which secrete effector proteins that are often responsible for altering the plant morphology and behaviours of their vectors. Phytoplasma multifunctional effector proteins TENGU and SAP11 induce typical witches' broom symptoms, but their mode of action remains unknown. Previously, we have identified a SAP11-like effector from wheat blue dwarf phytoplasma, SWP1, which induces witches' broom symptoms in Nicotiana benthamiana. In this study, we observed that SWP1-expressing transgenic Arabidopsis thaliana plants showed typical witches' broom symptoms. On overexpression of SWP1 truncation mutants in N.benthamiana, we identified that the coiled-coil domain and nuclear localization were responsible for the induction of witches' broom symptoms. In addition, using yeast two-hybrid and bimolecular fluorescence complementation assays, we demonstrated that SWP1 interacts with A.thaliana transcription factor TCP18 (BRC1), the key negative regulator of branching signals in various plant species. Moreover, inplanta co-expression analysis showed that SWP1 promotes the degradation of BRC1 via a proteasome system. These findings suggest that the phytoplasma effector SWP1 induces witches' broom symptoms through targeting of BRC1 and promoting its degradation.

Detection and characterisation of phytoplasma strains associated with field bindweed witches’ broom disease in Iran

During 2013–2015 surveys in Fars, Lorestan and Yazd provinces (Iran), a field bindweed witches’ broom (FBWB) disease was observed. The main symptoms were reduction of leaves size, yellowing, internode shortening, witches’ broom and stunting. The agent of FBWB was dodder transmitted to periwinkle plants inducing phytoplasma-type symptoms. Amplifications of nearly 1.8 and 1.2 kbp were, respectively, obtained from 15 symptomatic bindweed plants and 28 symptomatic dodder-inoculated periwinkles. Virtual RFLP analyses showed that the phytoplasma detected belonged to 16SrXII-A subgroup, and it was the same in all the samples examined; phylogenetic analyses confirmed it as a ‘Candidatus Phytoplasma solani’-related strain. This is the first report of 16SrXII-A phytoplasmas presence in bindweed plants showing witches’ broom symptoms in Fars, Lorestan and Yazd provinces. As a perennial widespread weed, it may act as a 16SrXII-A phytoplasma source for alfalfa, grapevine, Sophora alopecuroides, tomato, hemp and Japanese spindle reported diseases in these Iranian provinces.

Combination of iTRAQ proteomics and RNA-seq transcriptomics reveals jasmonate-related-metabolisms central regulation during the process of Jujube witches’ broom recovery by tetracycline treatment

Witches’ broom disease, caused by phytoplasma, is currently the most destructive disease of jujube. Tetracycline derivatives have been used to treat witches’ broom, and can allow recovery from the phytoplasma infection. Genes related to jasmonic acid (JA) biosynthesis and JA-induced protein-like play roles in phytoplasma infection. Jujube shoots (Ziziphus jujuba Mill. ‘Huizao’) infected by phytoplasma were excised and grown in vitro before treatment with tetracycline. RNA-Seq and iTRAQ analyses of samples treated for different lengths of time were performed during recovery from jujube witches’ broom (JWB) symptoms and diminishing presence of phytoplasma. Phytoplasma was not detected by PCR in the shoots after 6 months of tetracycline treatment (MTT). RNA-Seq and iTRAQ analyses identified 26,790 genes and 6184 proteins from jujube shoot samples. There were 272 differentially expressed genes (DEGs) and 20 differentially expressed proteins (DEPs) between the 3 and 6 MTT samples, respectively. The largest number and the greatest changes of DEGs and DEPs were for those related to alpha-linolenic acid metabolism, jasmonate biosynthesis, and jasmonate induced protein-like (JIPs). JA content slightly decreased at the sixth month compared with the third month. The research avenues explored here showed that genes in the JA biosynthesis pathway, proteins that respond to JA (JIPs), and JA content itself were concurrently regulated during JWB recovery. Alpha-linolenic acid metabolism pathway had higher RichFactor in comparisons between the 3 and 6 MTT samples during JWB recovery, which suggesting JA played vital roles during JWB recovery. The results in this study will help us to understand the roles JA plays in host-phytoplasma interactions during recovery and infection of JWB.

‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group, is associated with witches broom disease of Azadirachta indica in India

Five samples from neem trees exhibiting witches broom (NeWB) symptoms were collected from the Raichur district, Karnataka State, India. The identity of the phytoplasma associated with all five neem samples was confirmed through PCR using phytoplasma 16Sr RNA gene specific universal primers. The amplified products were cloned, sequenced and nucleotide (nt) sequence comparisons were made with published phytoplasmas 16S rRNA gene nt sequences available at NCBI database. The 16Sr RNA gene nt sequence of NeWB phytoplasma had 99 to 99.8% identity with ‘Candidatus Phytoplasma’ group (16SrVI) isolates reported from different parts of the world. This was supported by the close clustering of NeWB phytoplasma in the current study with members of clover proliferation group-16SrVI in the phylogenetic analysis. The virtual RFLP pattern generated for the phytoplasma from neem was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI and subgroup D (Brinjal little leaf-16VI-D, NCBI Ac.No.: X83431). The analysis further confirmed that the phytoplasma associated with NeWB disease of neem belongs to 16Sr group VI and subgroup 16Sr IV-D. This is the first report of ‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group associated with witches broom disease of Azadirachta indica from India.

Characterization of a 16SrII subgroup D phytoplasma strain associated with Calendula officinalis phyllody in Iran.

Calendula officinalis plants with phyllody symptoms (CaoP) were observed in Yazd and Ashkezar (Yazd province, Iran) during 2013-2016. Twenty-one symptomatic and four asymptomatic plants were transferred individually to the greenhouse and potted for the biological and molecular characterization of associated phytoplasma. The dodder transmission from symptomatic potted marigold plants, induced virescence, phyllody and witches' broom symptoms in periwinkle. Total DNAs extracted from symptomatic and symptomless plants and dodder-inoculated periwinkles were subjected to nested PCR assay using primer pairs amplifying phytoplasma ribosomal DNA. Expected PCR amplification was detected in all CaoP plant and dodder-inoculated periwinkle samples. RFLP analysis of the amplicons obtained in direct PCR with P1/P7 primers using RsaI, AluI, MseI, HinfI and HaeIII restriction enzymes showed profiles identical to each other and related to phytoplasmas in all the 21 positive samples. R16mF2/R16mR2 amplicons from six CaoP plant samples were sequenced where consensus sequences had 100% of identity among each other. R16F2n/R16R2-trimmed sequences (1248bp) of representative samples from Yazd and Ashkezar were deposited in GenBank under accession numbers KU297202 and MH065715, respectively. BLAST search and phylogenetic analysis showed that the CaoP phytoplasma had 99% homology and clusters with phytoplasmas in group 16SrII. Computer-simulated analysis using iPhyClassifier suggests that the CaoP RFLP 16S rRNA gene pattern was identical to 16SrII-D phytoplasmas subgroup. Phytoplasma strains (16SrII-D) were reported as alfalfa witches' broom disease agentpreviously in the same geographic areas, and it is possible that alfalfa plays a role in the epidemiology of CaoP disease or vice-versa.