Elm Yellows Research Articles

First Report of a Phytoplasma Strain in the Elm Yellows Group (16SrV) associated with Virginia Creeper in Maryland, USA.

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) is a deciduous flowering vine in the Vitaceae family. Native to eastern North America, it is often used ornamentally as a climbing vine or as ground cover due to its rapid growth and foliage color in the fall. In July of 2022, along exterior walls of a private property in Lanham, MD, two Virginia creeper (VC) vines were observed displaying symptoms of yellow mottling and premature reddening of leaves. To investigate the cause of these symptoms, two symptomatic leaf samples and one asymptomatic leaf samples from a third vine in the same vicinity were collected for further analysis. A Qiagen DNeasy Plant Mini Kit was used to extract total DNA from leaf tissues according to the manufacturer's instructions. A phytoplasma-specific real-time PCR (Hodgetts et al. 2009) was used to test the DNA extracts, which detected the presence of phytoplasmas in the two DNA samples derived from symptomatic vines. The near full-length of the 16S ribosomal RNA gene was then amplified by seminested PCR from these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004) and Sanger sequenced using primers P1A and 16S-SR. Analysis of the obtained 16S rDNA sequences revealed no variation between the two plant samples, and one sequence was deposited in GenBank representing the phytoplasma strain named VC-MD1 (GenBank PP746981). A BLASTn search of the 16S rRNA gene sequence in the NCBI nucleotide database, showed 99.93% sequence identity with the phytoplasma strain AldY-WA1 (GenBank MZ557341) from red alder in Washington, a phytoplasma associated with VC plants in southern Florida (GenBank AF305198) (Harrison et al. 2001), and other strains detected in grapevines in Europe described as "flavescence dorée" phytoplasma (GenBank AF176319) (Davis, and Dally 2001). The virtual restriction fragment length polymorphism pattern derived from iPhyClassifier (Zhao et al. 2009), indicated that VC-MD1 is indeed a member of the 16SrV-C phytoplasma subgroup. To confirm the identification, the partial spc operon and the partial tuf gene were amplified as previously described (Lee et al. 2010; Makarova et al. 2012). Specifically, the spc operon region was amplified using a nested PCR approach with primer set L15F1A-a/MapR1 followed by L15F1A-b/MapR1A-b. Sequence data obtained from the two loci were deposited to GenBank with accession numbers PP746982 (spc) and PP746983 (tuf). BLAST searches querying the nucleotide sequences of the spc operon and tuf gene showed 95.39% and 99.05% identity, respectively, to the corresponding loci of 'Candidatus Phytoplasma rubi' strain RS (GenBank CP114006) and hemp dogbane yellows phytoplasma strain HD1 (GenBank FR686506). Phylogenetic analysis based on secY and tuf gene sequences suggest that the VC-MD1 strain is evolutionary closest to 16SrV-C phytoplasma strains detected in various hosts in the United States, including HD1 and AldY-WA1. These North American strains cluster together on a distinct branch within the elm yellows group phytoplasmas. For the State of Maryland, this detection represents the first report of a phytoplasma strain member of the16SrV-C subgroup infecting VC plants. A phytoplasma of the same subgroup was previously detected in Florida in asymptomatic VC vines (Harrison et al. 2001). The 16S rRNA gene sequences of the two VC phytoplasma strains are nearly identical, differing by just a single nucleotide. The disease transmission vectors of the VC-MD1 strain and the prevalence of the disease in the region remains undetermined.

Multigene characterization of 'Candidatus Phytoplasma ulmi'‐related isolates associated with elm yellows disease of Ulmus minor Mill. in Poland

Abstract‘Candidatus Phytoplasma ulmi’, associated with elm yellows, shoot proliferation and dieback of elm (Ulmus) species trees was reported in United States of America and in many European countries. Until now its presence in elm trees in Poland has not been detected. In 2017–2018, during visual inspection of elm trees grown in four areas of southern Silesia Province leaf yellowing, shoot proliferation, phloem necrosis and dieback of branches were observed on European field elm trees grown in nature reserve close to the border with the Czech Republic. Phloem tissue from shoots collected from six symptomatic and twenty asymptomatic trees was used for extraction nucleic acids. Nested PCR with primer pairs amplifying 16Sr RNA sequences of phytoplasmas resulted in specific products for samples from six symptomatic trees however the restriction fragment length polymorphism (RFLP) and phylogenetic analyses of the sequenced products did not allow to clearly classify newly detected isolates within the elm yellows group (16SrV). The isolates found in Poland clustered on the boundary of the 16SrV‐A and 16SrV‐C ribosomal subgroups. Sequence analyses of less conservative genes rpl22‐ rps3 and secY confirmed the affiliation of the phytoplasma isolates found in Poland to ‘Candidatus Phytoplasma ulmi’; however, the significant nucleotide changes in signature sequences for all three genes were shown compared with EY1T reference strain. Surprisingly, the newly detected isolates shared a common origin and were phylogenetically closer to EY1‐SRB and EY10‐SRB strains from Serbia as well as NK16 strain from Croatia strains than to the EYCZ1 strain from the Czech Republic. This is the first report on detection and molecular characterization of ‘Candidatus Phytoplasma ulmi’ in Poland based on sequence analysis of three genetic locus.

Strains related to 'Candidatus Phytoplasma ulmi'- are associated with Chinese Elm yellows in Korea

Strains related to 'Candidatus Phytoplasma ulmi'- are associated with Chinese Elm yellows in Korea

First report of a Jujube witches’‐broom phytoplasma (16SrV) strain associated with witches’‐broom and little leaf disease of Solanum melongena in India

Solanum melongena (aubergine or brinjal; family Solanaceae) is a popular vegetable grown extensively in India, Bangladesh, Pakistan, China, Japan, Philippines, Egypt, France, Italy, and the United States (Chadha et al., 1993). Brinjal's cultivation is hampered by the Brinjal little leaf (BLL) phytoplasma. A phytoplasma survey was conducted in various brinjal production areas of Hoshangabad in Central India (Figure 1) during May 2019. Phytoplasma-like symptoms such as proliferation of axillary shoots, witches’- broom, excessive branching, little leaf and the absence of flowers and fruits when compared to symptomless brinjal plants were observed (Figure 2). For molecular detection of phytoplasma, total DNA was isolated from diseased and symptomless leaf samples (100 mg). The phytoplasma 16S rRNA gene was amplified by direct PCR using P1/P6 primers (Deng & Hiruki, 1991) and nested PCR with universal primers R16F2n/R16R2 (Gundersen & Lee, 1996), producing amplicons of the expected size (c. 1.2 kb) for the diseased (4/4) samples but not from the symptomless plant tested. Two nested PCR amplicons were purified (Wizard SV gel extraction kit; Promega, USA) and sequenced bidirectionally (Bioinovations Pvt. Ltd., India). The consensus partial 16S rRNA gene sequences were identical and the sequence data was submitted to GenBank, Accession Nos. MW025971 (BR01) and MW025257 (BR02). BLASTn analysis showed that the sequence shared 99% sequence identity with the 16S rRNA gene sequence of Jujube witches'-broom phytoplasma (MH972556, MH972553, MH972548) and ‘Candidatus Phytoplama balanitae’ (HG937644, LT558785, MH819290), Periwinkle yellows phytoplasma (EU375835) and the Elm yellows group (16SrV). Phylogenetic analysis (MEGA v 7.1) showed the closest relationships with strains of Jujube witches'-broom phytoplasma (MH972556, H744152 and MH972548) and ‘Ca. P. balanitae’ (LT558785), a member the group 16SrV, ‘Ca. P. ulmi’ (Figure 3). Six phytoplasma ribosomal groups (16SrI, 16SrII, 16SrIII, 16SrVI, 16SrIX, 16SrXII) have been associated with S. melongena globally. BLL phytoplasmas were first reported in India in 1995 (Schneider et al., 1995). In India and Japan, a BLL phytoplasma was identified as a member of the group 16SrI, ‘Ca. P. asteris’ (Kumar et al., 2012). The group 16SrVI, ‘Ca. Phytoplasma trifolii’ was also associated with BLL symptoms in India (Yadav et al., 2015). However, this is the first report of a ‘Ca. P. ulmi’ strain (group 16SrV), associated with a witches'-broom disease of brinjal in India. This finding has a significant impact for future epidemiological studies since brinjal could be an alternative host for 16SrV phytoplasma strain which affect several other important vegetable crops cultivated in Hoshangabad, Central India. The authors would like to thank to the Head of the Department of Microbiology and Vice Chancellor, Barkatullah University, Bhopal for support and encouragement.

A chrysanthemum decline associated with phytoplasma presence in Italy

The chrysanthemum is a popular ornamental flower all around the world for both cut flowers and potted plants. In recent days, bacterial infections are reducing in several cases its market value. Irregular flowering, yellowing and decline symptoms were observed at a commercial enterprise located in North Italy. To verify the presence of phytoplasmas, DNA was extracted from symptomatic and asymptomatic potted plants and subjected to specific polymerase chain reaction followed by restriction fragment length polymorphism analysis on phytoplasma 16S ribosomal gene. The tests were carried out three times in 7 months to verify the persistence of detected phytoplasmas over time. Phytoplasma classified in group 16SrV (elm yellows) were detected in all the examined samples independently from the presence of decline symptoms, while group 16SrXII (“stolbur”) phytoplasmas were only detected in the most severely declining plants. This latter phytoplasma strain was also characterized on tuf and leu genes by RFLP analyses and sequencing, respectively, to cross confirm its presence and identity. The detection of this phytoplasma in chrysanthemum plants is the first report in Italy, while it has already been identified in plants with malformed inflorescence in Europe, but not in other parts of the world.

Widespread occurrence of \u2018Candidatus Phytoplasma ulmi\u2019 in elm species in Germany

Background‘Candidatus Phytoplasma ulmi’ is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany.ResultsAbout 6500 samples from Ulmus minor, Ulmus laevis and Ulmus glabra, were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel ‘Ca. P. ulmi’ specific qPCR assay targeting the 16S–23S spacer region. Both assays revealed that 28% of the samples were infected by ‘Ca. P. ulmi’, but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of ‘Ca. P. ulmi’ in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year.Conclusions‘Ca. P. ulmi’ is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.

Molecular identification of Jujube witches'-broom phytoplasma (16SrV) associated with witches'-broom disease of Ziziphus oenoplia in India

Severe witches’-broom disease of Ziziphus oenoplia was observed with significant disease incidence in Bhopal, India, during 2019. Phytoplasma was detected from symptomatic leaf samples by polymerase chain reaction (PCR) using phytoplasma 16S rRNA gene specific primers which revealed positive amplification of expected size ~1.2 kb DNA band. The positive amplicons of the phytoplasma 16S rRNA (1.2 kb) were sequence and sequenced data was submitted in GenBank database (Accession no. MK975463 and MK975462). On the basis of highest 99% sequence identities, closest phylogenetic relationships and In silico of the under study both the phytoplasma isolates associated with witches'-broom disease of Ziziphus oenoplia identified as a species of Jujube witches'-broom phytoplasma as a member of Elm yellows group (16SrV ). To the best of our knowledge, this is the first report on the association of Jujube witches'-broom phytoplasma species of Elm yellows group (16SrV) with witches'-broom disease of Z. oenoplia in India.

First Report of an Association of Multispecies Phytoplasmas with Mango Phyllody and Little Leaf Disease from Western India

HomePlant DiseaseVol. 103, No. 10First Report of an Association of Multispecies Phytoplasmas with Mango Phyllody and Little Leaf Disease from Western India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of an Association of Multispecies Phytoplasmas with Mango Phyllody and Little Leaf Disease from Western IndiaKiran Kirdat, Vipool Thorat, Tushar Ghole, Pramod Takawale, and Amit YadavKiran KirdatNational Centre for Microbial Resource, National Centre for Cell Science, Pashan, Pune 411 021, IndiaSearch for more papers by this author, Vipool ThoratNational Centre for Microbial Resource, National Centre for Cell Science, Pashan, Pune 411 021, IndiaSearch for more papers by this author, Tushar GholeNational Centre for Microbial Resource, National Centre for Cell Science, Pashan, Pune 411 021, IndiaSearch for more papers by this author, Pramod TakawaleBAIF-Development Research Foundation, Urulikanchan, Pune 412 202, IndiaSearch for more papers by this author, and Amit Yadav†Corresponding author: A. Yadav; E-mail Address: [email protected]http://orcid.org/0000-0002-4882-9075National Centre for Microbial Resource, National Centre for Cell Science, Pashan, Pune 411 021, IndiaSearch for more papers by this authorAffiliationsAuthors and Affiliations Kiran Kirdat1 Vipool Thorat1 Tushar Ghole1 Pramod Takawale2 Amit Yadav1 † 1National Centre for Microbial Resource, National Centre for Cell Science, Pashan, Pune 411 021, India 2BAIF-Development Research Foundation, Urulikanchan, Pune 412 202, India Published Online:7 Aug 2019https://doi.org/10.1094/PDIS-03-19-0599-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Mango (Mangifera indica L., family Anacardiaceae) trees with malformed vegetative tissues at branch nodes and phyllody in panicles with thicker and stunted peduncle were observed in Pune region, Western India, in February and March 2018. Although the etiology of mango malformation disease is established by performing Koch’s postulate, confirming species of Fusarium are the causative agents (Chakrabarti 2011; Crespo et al. 2014), the observed symptoms, namely, excessive vegetative growth resulting in “bunchy top” appearance with shootlets bearing small, scaly leaves and leafy girdles, however, were also suggestive of phytoplasma infection. The growing symptomatic tissues were collected from one seedling, two panicles, and four branch nodal tissues from separate plants. Eight other asymptomatic samples were also collected. To confirm the presence of phytoplasmas, 2 g of malformed tissue from symptomatic plants was used for total DNA extraction using the CTAB method. The DNA was also extracted from asymptomatic plants. The phytoplasma 16S rRNA gene was amplified from 50 ng of DNA using primers P1/P7 followed by nested primers R16F2n/R16R2. The 16S rRNA gene of phytoplasma was detected in a symptomatic seedling, one nodal tissue, and one panicle sample, except in one symptomatic nodal tissue and a panicle. This may be owing to unevenly spread low phytoplasma titer, as known from previous studies (Abeysinghe et al. 2016; Caicedo et al. 2015). No amplification was observed in asymptomatic samples. All symptomatic samples were also tested for the presence of Fusarium DNA using universal PCR primers ITS1/ITS4; however, none of the samples tested positive, nor was fungal mycelium observed when sections of symptomatic tissue stained with cotton blue were observed under the microscope. The obtained 16S rRNA gene sequences from a symptomatic panicle (MH744151), nodal tissue (MH744152), and seedling (MH744153) samples showed 99.30% sequence similarity with ‘Candidatus Phytoplasma asteris’ AYWB (CP000061), 16SrI group (aster yellows); 99.53% with ‘Ca. P. balanitae’ Balanites triflora (AB689678), 16SrV group (elm yellows); and 98.57% with ‘Ca. P. aurantifolia’ WBDL (U15442), 16SrII group (peanut witches’ broom), respectively, when analyzed using EzTaxon database and iPhyClassifier. So far, unknown phytoplasma associated with malformed mango inflorescence was reported from Egypt using electron microscopy (El-Banna et al. 2007), and ‘Ca. P. asteris’ (group 16SrI) was PCR detected from Pakistan (Fahmeed et al. 2009). These results suggest that polyphagous sap-sucking insect vectors must be involved in phytoplasma transmission from other alternate plant hosts to mango, and further studies are required. India produced over 19 million tons of mangoes, which is over 40% of the world’s production, and exported over U.S.$60 million worth of mangos in 2016 to 2017. To our knowledge, this is the first formal evidence of the association of multispecies phytoplasma strains with a mango malformation disease in India. The malformation is a severe disease of mango that causes serious damage to the seedlings and flowering tissue, resulting in severe economic losses. It is therefore essential to understand the etiology of mango malformation disease associated with phytoplasmas beyond the existing knowledge by collecting additional samples from different agro-climatic regions of India, ultimately for the better management of the disease.The author(s) declare no conflict of interest.

The Distribution of Phytoplasmas in South and East Asia: An Emerging Threat to Grapevine Cultivation.

Grapevine is largely cultivated in several parts of the world, and a spurt in its cultivation has occurred in the last two decades in grapevine cultivated areas of South and East Asia, mainly in China, India, Japan, Korea, Thailand, and Indonesia. Grapevine yellows (GY) represent one of the most important diseases in viticultural areas of the world, and they have been assigned to five different groups: aster yellows [AY (16SrI)], peanut witches’ broom [PnWB (16SrII)], X-disease (16SrIII), elm yellows [EY (16SrV)], and Stolbur (16SrXII). This study provides a comprehensive overview of the presence of phytoplasma strains and their vectors associated with GY complex, and their potential impact on viticulture of the South and East Asia. In general, both AY and EY were reported on several herbaceous plants and/or cultivated plants in South and East Asia, along with its vectors that were largely reported in China and sporadically in Japan. Interestingly, AY and EY are yet not found in South and East Asia grapevine regions; however, their presence on different plant species suggests the potential spread of the pathogens that may occur in grapevine regions in the near future. Additionally, a few reports also suggest the presence of Stolbur group in Asian countries, along with one study that found a Stolbur-related strain in China on Vitis vinifera. Similarly, PnWB was also frequently reported in India and China on several plant species, but not in grapes. Conversely, sporadic detections of phytoplasma strains related to X-disease in Thailand, South Korea, and China indicate that their potential influence in viticulture is rather negligible. Our review suggests that monitoring and control strategies against GY are essential in order to prevent epidemic phytoplasma spread, especially in vine-allocated areas in Asia.

First Report of ‘Candidatus Phytoplasma trifolii’ Associated with a Witches’ Broom Disease in Cannabis sativa in Nevada, U.S.A.

In September 2018, symptoms including leaf curling, mottling, chlorosis, witches’ broom, stunting, and node shortening were detected in Cannabis sativa L. plants at two growing sites in Central and Southern Nevada, U.S.A., respectively. Incidence of the disease varied between 5 and 20% at the growing site in Central Nevada, and 30% of plants were affected at the growing site in Southern Nevada. Symptomatic leaves showed “green islands” that were constrained by the veins on the upper leaf surface, and interveinal discoloration on the lower surface, forming a pale “pustule-like” appearance. One infected plant with 80% of leaves exhibiting symptoms was sampled from the Central Nevada site. One infected plant with 50% of leaves exhibiting symptoms was sampled from the Southern Nevada site. Two nonsymptomatic plants were provided by a local grower in California. Total DNA was extracted from the petioles of the most symptomatic leaves. DNA extracted from nonsymptomatic plants was used as the negative control. A diagnostic TaqMan real-time PCR test was conducted using the universal primer pair JH-F/JH-R (Hodgetts et al. 2009) targeting the 23S rRNA gene. Positive amplification signals were generated only from the symptomatic plants indicating the presence of Phytoplasma species. To identify the species, the 16S ribosomal RNA gene was amplified with the universal primer pairs P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), P1/Tint (Smart et al. 1996), followed by nested PCR primer pair R16F2/R2 (Lee et al. 1993). The anticipated amplicons (1.8, 1.6, and 1.2 kb, respectively) were gel purified and subjected to Sanger sequencing (UC Davis DNA Sequencing Facility). The sequence data were deposited in GenBank (accession no. MK377248). BLASTn results suggested a 99% sequence similarity with ‘Candidatus Phytoplasma trifolii’ (accession no. KF178706.1), a member of 16SrVI group associated with alfalfa witches’ broom disease. iPhyClassifier analysis (Zhao et al. 2013) was conducted to generate a restriction fragment length polymorphism profile using the amplified 16S rRNA gene sequence, revealing the most similarity (98.8%) to ‘C. P. trifolii’. Phytoplasmas of elm yellows group (16SrV) (Zhao et al. 2007) and aster yellows group (16SrI) (Raj et al. 2008), both associated with witches’ broom disease on Cannabis sp., have been reported in China and India, respectively. To our knowledge, this is the first report of 16SrVI group ‘Ca. P. trifolii’ infecting C. sativa in the United States. The identified pathogen may pose a significant threat to the production of C. sativa in the United States.

First report of ‘Candidatus Phytoplasma rubi’ on blackberry in Belgium

In May 2018 stunted blackberry plants were observed on a small-scale organic farm producing several small fruit types in West Flanders, Belgium. Besides severe stunting, leaf yellowing and distortions could also be observed (Figs. 1-2). Only Rubus fruticosus plants of the cultivar Obsidian showed symptoms but disease incidence was estimated to be almost 100%, of which almost half the plants were stunted. Other cultivars did not seem affected. Seven symptom-bearing plants, of which four had severe stunting, and three only leaf symptoms, were sampled and tested. Additionally, three symptomless plants from neighbouring rows and belonging to different cultivars, were tested. All samples consisted of leaves and roots, and tests were conducted on a pooled leaf/root sample. Total genomic DNA was extracted by a CTAB protocol. The 16S rRNA gene was partially amplified using the phytoplasma universal primer pair P1/P7 (Deng & Hiruki, 2; Schneider et al., 5) followed by a nested PCR with R16F2/R2 primers (Lee et al., 3) as a diagnostic test, and P1/Tint primers (Smart et al., 6) on positive samples for sequencing. All of the diseased plants tested positive for phytoplasma in nested PCR tests. Additionally, specific phytoplasma group V primers targeting the tuf (FDTUF-F1/R1 & FDTUF-F2/R2; Malembic-Maher et al., 4), and secY (primer sets FD9f2L/FD9r & FD9f3L/FD9r2L; Arnaud et al., 1) genes were used in nested PCRs and yielded fragments of 998 and 1174 bp respectively. The obtained 16SrRNA (P1/Tint), tuf and secY fragments were gel purified (SmartPure, Eurogentec), and sequenced directly (Genewiz, Leipzig, Germany). BLAST analysis of the 16SrRNA sequence, revealed the highest identity (99.85%) with a ‘Rubus stunt phytoplasma’ strain from blackberry (R. fruticosus) in Italy (isolate RuS400; GenBank Accession No. AY197649). Partial sequence of the 16S ribosomal RNA (1607 bp), and tuf (835 bp) and secY (1053 bp) genes were submitted to GenBank (MH801133, MH809672 and MH809673, respectively). Phylogenetic analysis was undertaken on all three fragments, and the phytoplasma was identified as ‘Candidatus Phytoplasma rubi’, ‘elm yellows’ group 16SrV-E (Figs. 3-5). To our knowledge, this is the first report of ‘Ca. P. rubi’ on blackberry in Belgium. An additional nationwide survey is needed to assess the phytopathological impact of this outbreak. Although a growing number of reports indicate the increased importance of Rubus stunt, associated with ‘Ca. P. rubi’, unlike the related ‘Ca. P. ulmi’, associated with elm trees, no regulatory actions are currently taken against this pathogen.

A new elm yellows phytoplasma strain associated with leaf yellowing disease of Tamarindus indica in India

Tamarind is an economically important tree species; its fruit pulp is widely used in kitchen in India. A symptomatic tamarind tree with yellow leaf symptoms found in the western part of India was assayed for phytoplasma presence. The 16S rDNA sequence of the detected phytoplasma (TM01) was found clustering with sequences in the elm yellows (16SrV) phytoplasma group. The virtual RFLP analysis of obtained 16S rDNA sequence TM01 revealed that it could be tentatively classified into a novel phytoplasma subgroup, 16SrV-L since its AluI, HaeIII and MseI restriction profiles of R16F2n/R16R2 sequenced amplicons generated were unique as compared to the earlier reported profiles of this phytoplasma subgroup. This is the first phytoplasma report in Tamarindus indica.

Occurrence of ‘Candidatus Phytoplasma ulmi’ in native elm trees in Germany

A countrywide survey has been performed to examine the occurrence of the elm yellows phytoplasma in three elm species native to Germany. The DNA of more than 5, 000 trees was extracted and the presence of phytoplasmas verified by universal and pathogen-specific quantitative PCR assays. In total, 33% of all plants resulted phytoplasma-positive. The highest presence was found in the European white elm, followed by the wych elm and the field elm. Regional clusters of high incidence were found in east and in south-west Germany, while in the western states the phytoplasma presence was low or absent. Disease symptoms were only occasionally visible as witches’ broom for wych elm and yellowing and small leaves for field elm. The survey revealed that ‘Candidatus Phytoplasma ulmi’ is widespread in Germany questioning the current quarantine status of this pathogen.

Multilocus genotyping identifies a highly homogeneous phytoplasma lineage associated with sweet cherry virescence disease in China and its carriage by an erythroneurine leafhopper

Phytoplasmas are a diverse group of insect-transmitted, cell wall-less bacteria that colonize plant phloem sieve elements and cause numerous diseases in economically important crops. Living a trans-kingdom parasitic life, phytoplasmas possess dynamic genomes and evolve rapidly toward formation of distinct ecological lineages in their adaptation to specific ecological niches. In an effort aimed at identification of the etiological agent responsible for a repeatedly-occurring sweet cherry virescence (SCV) disease in China, we found that the SCV disease was consistently associated with infection by a phytoplasma belonging to subgroup B of the elm yellows phytoplasma group (16SrV-B). Further analysis of genetic loci that encode important phytoplasma cellular components, including an array of ribosomal proteins and preprotein translocase subunit SecY, revealed that the SCV phytoplasma was essentially indistinguishable from the subgroup 16SrV-B phytoplasma strains responsible for jujube witches’-broom (JWB) disease and diseases of many other plants. Evidence gathered in the present study indicated that SCV-JWB phytoplasma strains formed a highly homogenous ecological lineage. The study also revealed that a polyphagous leafhopper, Táoyīdiǎnyèchán (桃一点叶蝉 in Chinese and often being cited as Erythroneura sudra), was able to carry the SCV phytoplasma, emphasizing a need to investigate whether this erythroneurine leafhopper plays a role in spreading the SCV phytoplasma among sweet cherry plants.

First Report of ‘Candidatus Phytoplasma trifolii’-Related Strain of 16SrVI-A Phytoplasma Subgroup, Associated with Elm Yellows Disease in American Elm (Ulmus americana L.) in Ohio, U.S.A

During the investigation of the sudden and early onset of yellowing, followed by mortality of American elm (Ulmus americana L.) trees at the USDA Forest Service Northern Research Station in Delaware, Ohio, a phytoplasma of the clover proliferation group (16SrVI) was detected as the putative causal agent of the disease outbreak. Onset of symptoms was rapid and widespread, occurring in late July 2016 and affecting ∼60 trees across two elm research plantations. Symptoms included a general yellowing of individual tree canopies, epinasty of foliage throughout the canopy, phloem discoloration, and on a subset of trees, a strong odor of methyl salicylate (observed in phloem tissue extracted from the lower stem). Similar symptoms in elms have been attributed to the classic elm yellows ‘Candidatus Phytoplasma ulmi’ (16SrV-A) (Lee et al. 2004) and the Illinois elm yellows phytoplasma (16SrVI-C) (Jacobs et al. 2003). In July 2016, samples were collected from 12 symptomatic and 8 asymptomatic American elm trees. DNA from the leaf midrib and branch phloem was isolated and analyzed for phytoplasma via seminested polymerase chain reactions (PCR). PCRs were first primed by phytoplasma universal primer pair P5/P7 (Jomantiene et al. 1998), followed by P7 and the reverse complement of the universal phytoplasma primer R16R2 for amplification of the phytoplasma 16S-23 ribosomal (r) DNA (16S-23 rRNA gene) sequences as per Gundersen and Lee (1996). The predicted band size of the second PCR product is 487 base pair (bp). The product bands were isolated, purified, and sequenced using primer P7. Sequencing results of the PCR products indicated that nine of the symptomatic and one of the asymptomatic American elm trees tested were infected by a phytoplasma. A BLAST search of the DNA sequences indicated high similarities to members of the ‘Ca. P. trifolii’ group 16SrVI-A. The sequences of all 10 phytoplasma-infected trees were identical to each other. To further confirm that a strain of ‘Ca. P. trifolii’ was infecting the elms, a PCR product was cloned and sequenced. The 1,557 bp band was the product of primers P1a/P7, followed by PCR primers designed from the sequence of ‘Ca. P. trifolii’. This band was cloned into the pMiniT 2.0 vector. Plasmid sequencing used standard sequencing primers (SP6 and T7 promoters), the primers included with the vector, and finally, custom designed phytoplasma primers that eliminate other bacterial DNA from getting amplified. The entire plasmid clone was sequenced in both directions with four to six times of coverage per base. Determination of the phytoplasma classification group was based on the nucleotide sequence within the phytoplasma universal primers F2n/R2 PCR fragment within the 16Sr gene. Using iPhyClassifier, the online tool for phytoplasma classification and taxonomic assignment (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), the sequence similarity between the Delaware elm phytoplasma and ‘Ca. P. trifolii’ (GenBank accession no. AB279597.1) is 99.9% in the 16Sr region, which places the Delaware elm phytoplasma in the 16SrVI-A group. The sequence of the plasmid was deposited in GenBank under accession number MF385584. Elm decline and yellows diseases in North America have been associated with the Illinois elm yellows phytoplasma (16SrVI-C, GenBank accession no. AF268893.1) (Jacobs et al. 2003), ‘Ca. Phytoplasma ulmi’ (16SrV-A) (Lee et al. 2004), and phytoplasma in the aster yellows group (16SrI-C) (Lee et al. 1995).

Molecular Diversity of Phytoplasmas Associated with Grapevine Yellows Disease in North-Eastern Italy.

A 3-year survey was conducted in Northern Italy to verify the presence and diversity of phytoplasmas in selected vineyards showing symptoms of severe yellows. Symptomatic and asymptomatic grapevines were sampled, and insects were collected using yellow sticky traps. The phytoplasmas detected in grapevine samples were different according to the years: "flavescence dorée" (16SrV-C/D) was detected together with other phytoplasmas such as 16SrXII-A ('Candidatus Phytoplasma solani'-related, bois noir), 16SrI-B ('Ca. P. asteris'-related, aster yellows), 16SrX-B ('Ca. P. prunorum'-related, European stone fruit yellows), and 16SrV-A ('Ca. P. ulmi'-related, elm yellows). Moreover, phytoplasmas belonging to 16SrVII-A ('Ca. P. fraxini'-related) and 16SrVI ('Ca. P. trifolii'-related) subgroups were also identified. Identification of phytoplasmas was also carried out from insects and showed the presence of some of these phytoplasmas in Scaphoideus titanus and Orientus ishidae: 16SrXII-A, 16SrVII, and 16SrVI phytoplasmas were detected in specimens of both species, while 16SrXII-A and 16SrI-B phytoplasma strains were identified in Orientus ishidae and Hyalesthes obsoletus, and 16SrX-B in S. titanus. Direct sequencing of selected amplicons obtained from 16S rRNA, rp, and tuf genes from grapevine and insect samples confirmed the phytoplasma identification. The 16SrVII-A and 16SrVI phytoplasmas were never detected before in grapevine, S. titanus and Orientus ishidae in Europe and their epidemiological importance is being monitored.

Elms Revisited

Until Dutch elm disease (DED) was accidentally introduced into the United States around 1930, the streets in many states were lined with American elms (Ulmus americana). This review highlights the aftermath of DED, and updates readers on the advances in our knowledge of the pathosystem, which consists of a tree, a fungal pathogen, and an insect vector. Conventional breeding has produced new cultivars of American elm that are more disease-tolerant, although still not resistant. Suitable DED-resistant hybrid elms have been bred using species from Europe and Asia. The discovery of diploid populations of American elm may open new opportunities in elm hybridization and genome analysis. Growing knowledge of resistance mechanisms reveals a complex interaction of anatomy, physiology, environmental factors, and tree age. The beetle’s role is largely understood but appears not to be a viable point of attack in the war on DED. The genome of the fungal pathogen has been sequenced, and gene expression studies are well under way. There is a renewed interest in understanding the evolution, genetics, and physiology of the DED pathogen. The genetic engineering of elms has been demonstrated but not with the specificity and vigor as has been reported for genetically engineered American chestnut. Elm yellows, caused by a phytoplasma, are still a deadly problem for elms, although outbreaks are more regional than for DED. Germplasm resources are critical to elm improvement, and the first comprehensive survey of living elm species, hybrids, and cultivars growing in America is presented in tabular form.

Grapevine flavescence dorée phytoplasma. [Distribution map

Abstract A new distribution map is provided for Grapevine flavescence dorée phytoplasma [ Phytoplasma vitis ]. Acholeplasmatales: Acholeplasmataceae: Candidatus Phytoplasma: 16SrV (Elm yellows group). Host: grapevine ( Vitis spp.). Information is given on the geographical distribution in Europe (Austria, Croatia, France, Corsica, Mainland France, Hungary, Italy, Portugal, Serbia, Slovenia, Spain, Switzerland).

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

SummaryElm yellows (EY) is a lethal or decline phytoplasma disease that affects several Ulmus (elm) species and hybrids, which is widespread in North America and Europe. The symptoms vary among the elm species. In those native to North America, main symptoms include epinasty, chlorosis, premature casting of the leaves, yellow to brown discoloration of the phloem in the roots and stem and tree death that usually occurs within 1 or 2 years from the appearance of foliar symptoms. In contrast, affected trees of European and Asian species are primarily characterized by witches’ broom as a specific symptom, do not show phloem discoloration and are less prone to decline. The disease is caused by a relatively genetically homogeneous phytoplasma, the EY agent “Candidatus Phytoplasma ulmi,” a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV‐A. In nature, this pathogen exhibits a high plant host specificity. The elm leafhopper Scaphoideus luteolus is the only confirmed vector of EY phytoplasma in North America, whereas Macropsis mendax has been reported as a natural vector in Northern Italy. However, other insect vectors are likely to be involved in its natural spread. Phytoplasmas of other taxonomic groups or 16SrV subgroups, which are known to infect a wide range of plant hosts, have been identified in naturally infected elm trees. However, the pathological relevance of these “non‐elm” phytoplasmas needs to be confirmed in many cases. Their detection is mainly based on the highly sensitive nested PCR assays, while pathological data are lacking. This study summarizes, within the framework of a single comprehensive review, the current knowledge of EY. Gaps in knowledge of this disease and prospects for future research are also critically discussed.

Molecular characterization of elm yellows phytoplasmas in Croatia and their impact on Ulmus spp.

‘Candidatus (Ca.) Phytoplasma ulmi’, the causal agent of elm yellows, was found widely distributed across elm populations in Croatia, infecting Ulmus laevis and Ulmus minor. Especially high prevalence of the infection, approximately 75%, was detected in U. laevis, but more than half of the trees were symptomless. ‘Candidatus Phytoplasma solani’ and ‘Ca. Phytoplasma asteris’ were also detected. The latter could possibly represent a new 16SrI subgroup, most closely related to 16SrI‐B. Diversity of ‘Ca. Phytoplasma ulmi’ in Croatia was determined by sequencing of the 16S rRNA gene, ribosomal protein genes rpl22 and rps3, secY and secY‐map genes, in 62 phytoplasma isolates. Phylogenetic analysis indicated that Croatian isolates share a common origin and are closely related to strains of ‘Ca. Phytoplasma ulmi’ from southeastern Europe. However, comparative sequence analysis revealed mutations at positions where variability has never been detected before, including positions within sequences unique to ‘Ca. Phytoplasma ulmi’ in the 16S rRNA gene, rpl22, rps3 and secY. New genotypes were identified based on the sequenced genes. This study points to a significantly higher genetic diversity than previously reported, and a necessity to revise the formal description of this phytoplasma species and to include newly discovered characteristics.