secY Gene Research Articles

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.

Multigene characterization of a ‘Candidatus Phytoplasma australasia’ related strain associated with phyllody disease of coriander in India

Coriander is one of the important seed spices cultivated throughout India. The yield of the crop is very low due to the presence of many pests and diseases that are major constraints in the production. Among them phytoplasmas are one of the most economically important pathogens associated with significant yield losses. During a survey, coriander plants exhibiting phyllody symptoms were collected from Chikkabalpur dist, Karnataka (India) and the phytoplasma presence was detected through PCR using 16Sr RNA, secY, ribosomal protein (rp) and secA genes specific primers. All symptomatic coriander samples gave amplification using primers for conserved and less conserved phytoplasma genetic regions. In particular, the virtual RFLP pattern of the 16S rRNA gene, similarity coefficient 0.97 to ‘Ca. P. australasia’ enclosed it the subgroup 16SrII-D with some restriction enzyme differences. Based on the threshold similarity coefficient, this phytoplasma is related to ‘Candidatus Phytoplasma australasia’, however it shows some 16S ribosomal differences that could allow its differentiation from it. This is the first report of phytoplasmas associated with the phyllody disease of coriander from India.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.

Molecular characterization of pathogenic Leptospira sp. in small mammals captured from the human leptospirosis suspected areas of Selangor state, Malaysia

Leptospirosis is caused by the spirochetal bacterium Leptospira of which rodents are considered the most important reservoir. This study aims to determine and characterize virulent Leptospira species among rodents and small mammals found in human settlements and recreational spots within the Hulu Langat and Gombak districts of Selangor, Malaysia; regions that frequently report probable human leptospirosis cases. Molecular analysis revealed an overall Leptospira detection rate of 14.3% among the 266 small mammals captured, and the human settlements were found to have the highest number of isolates (15.1%), followed by recreational sites (14.5%). The molecular characterization conducted based on the lipL32, secY genes and MLST revealed that the strains belonged to four different species, including; Leptospira interrogans (29; 76.3%; ST50, ST238, ST243), L. kirschneri (5; 13.15%; ST110), L. borgpetersenii (3; 8%; ST143) and L. weilii (1; 2.63%; ST242). The study revealed genotypes of circulating strains among small mammals in Malaysia, which include Leptospira locus ST110 L. kirschneri, ST 50 L. interrogans, ST143 L. borgpetersenii and ST242 L. weilii. Among the small mammals studied, 17/105 (16.2%) Rattus norvegicus, 7/59 (11.9%) of Rattus rattus, 5/24 (20.8%) of Maxomys whiteheadi, 4/18 (22.2%) of Sundamys muelleri, 2/22 (9%), Tupaia gliss, 2/16 (12.5%) Rattus tiomanicus and 1/4 (25%) of Suncus murinus carried pathogenic leptospires. The data from the present study may imply that, in addition to rodents, other small mammals also serve as maintenance hosts for Leptospira. Hence, much remains unknown about Leptospira maintenance hosts, and there is need for further investigation to ascertain the prevailing serovars of pathogenic Leptospira in Malaysia. This will assist in the development of efficient diagnostic assays with improved microscopic agglutination test (MAT) panels, and in the implementation of suitable prevention and control measures.

First Report of 'Candidatus Phytoplasma asteris' Associated with Witches' Brooms on Sharp-Flowered Rush (Juncus acutiflorus) in Poland.

Juncus acutiflorus L. (Juncaceae) is a species of rush widespread in Europe, North Africa, and Southwest Asia. The health of sharp-flowered rush is threatened by witches’ brooms occurrence. Typical symptoms are dense masses of shoots, usually growing from a single point, with the resulting structure resembling a broom. Witches’ brooms are typically formed in response to Livia junci (Liviinae, Hemiptera) feeding, oligofag, which typically feeds on Juncus sp. These symptoms are similar to those observed in Juncus articulatus L. (Juncaceae) related to phytoplasma infection in Silesia, Poland (Jarzembowski et al. 2015). In August 2017, samples were collected from nine symptomatic and four asymptomatic plants growing in Jagniątkow, Poland. DNA from 100 mg of inflorescence and leaf samples was extracted using a DNeasy Plant Mini Kit (Qiagen, Syngen Biotech, Wroclaw, Poland) according to the manufacturer’s protocol. Additionally, 10 L. junci specimens (adult and the last larva stage) were collected from symptomatic plants and preserved in ethanol (75%). DNA from the insects (treated as one sample) was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Syngen Biotech). The extracts were used as the template in polymerase chain reaction (PCR) assays with the universal phytoplasma primers P1/P7 followed by R16F2/R16R2 (Lee et al. 1998). Additionally, primers rp1/rp2 followed by rp3/rp4, allowing amplification of fragments of rpl22 and rps3 genes (Nakamura et al. 1996), and primers AYsecYF1/AYsec for amplification of secY gene were applied. Water blank samples were included as negative controls. PCR products of expected size for each primer set were amplified from the insects and six symptomatic plants that were assayed. No amplification was observed in symptomless J. acutiflorus samples or in water blanks. Amplicons representing three genetic loci were sequenced at Genomed S.A., Warsaw, Poland. DNA samples were sequenced at least twice in both directions. Sequencing results of the PCR products confirmed that six symptomatic plants and the insects were infected by a phytoplasma. The obtained sequences were nearly identical, and representative sequences of 16S rDNA fragments (accession nos. MG976242 to MG976246), secY gene (MG983765 to MG983769), and ribosomal protein gene (MG983760 to MG983764) from four plants and one insect sample were deposited in GenBank. Determination of the phytoplasma classification group was based on the nucleotide sequence of the F2n/R2 PCR fragment within the 16S gene. Using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier_legacy.cgi), the sequence of J. acutiflorus phytoplasma shared 99.9% identity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (GenBank accession no. M30790). BLAST analysis performed for ribosomal proteins and secY genes confirmed the highest identity (99%) of analyzed sequences with those of ‘Ca. P. asteris’ (CP015149 and KJ394491, respectively). To our knowledge, this is the first report of a phytoplasma of group 16SrI affecting J. acutiflorus in Poland and in the world. The impact of the detected phytoplasma in the regional ecosystem is being assessed. It is highly probable that L. junci is a possible vector for ‘Ca. P. asteris’.

Prospective study of canine leptospirosis in shelter and stray dog populations: Identification of chronic carriers and different Leptospira species infecting dogs

Dogs are highly susceptible to the leptospiral infection, notably stray and sheltered dogs. Unsanitary conditions often observed in dog shelters may predispose the introduction and spread of leptospires among sheltered populations, potentially increasing the chances for the inadvertent adoption of asymptomatically infected animals. The present work describes a longitudinal study using a multidisciplinary approach for the identification of chronically infected dogs and the characterization of potentially pathogenic strains circulating among stray and sheltered dog populations in São Paulo, Brazil. A total of 123 dogs from three populations were included. The initial evaluation consisted of blood and urine quantitative PCR testing (qPCR), the detection of specific antibodies by microscopic agglutination test (MAT), physical examination and hematological and serum biochemistry analyses. The qPCR-positive dogs were prospectively examined, and reevaluations also included culture from urine samples. Positive qPCR samples were subjected to 16S rRNA and secY gene phylogenetic analysis. The recovered strains were characterized by Multilocus Sequence Typing, polyclonal serogroup identification and virulence determination. Leptospiruria was detected in all populations studied (13/123), and phylogenetic analysis revealed that 10 dogs had L. interrogans infection. Three dogs (3/13) had L. santarosai infection. The secY phylogenetic analysis revealed that the L. santarosai sequences clustered separately from those obtained from other hosts. Ten leptospiruric dogs were reevaluated, and three dogs presented persistent leptospiruria, allowing culturing from two dogs. The strains were characterized as L. interrogans serogroup Canicola (virulent) and L. santarosai serogroup Sejroe (not virulent). Serum samples were retested by MAT using the DU92 and DU114 strains as antigens, and no increased seroreactivity was detected. Asymptomatic L. santarosai infection was observed in all populations studied, suggesting a possible role of dogs in the chain of transmission of this leptospiral species. The results suggest a genetic distinction between lineages of Brazilian L. santarosai maintained by dogs and other animal hosts. Our findings revealed that dogs could act as maintenance hosts for distinct pathogenic Leptospira, highlighting also that asymptomatically infected dogs can be inadvertently admitted and adopted in dog shelters, potentially increasing the risks of zoonotic transmission.

Genotyping Points to Divergent Evolution of 'Candidatus Phytoplasma asteris' Strains Causing North American Grapevine Yellows and Strains Causing Aster Yellows.

Grapevine yellows diseases occur in cultivated grapevine (Vitis vinifera L.) on several continents, where the diseases are known by different names depending upon the identities of the causal phytoplasmas. In this study, phytoplasma strains associated with grapevine yellows disease (North American grapevine yellows [NAGY]) in vineyards of Pennsylvania were characterized as belonging to 16S ribosomal RNA (rRNA) gene restriction fragment length polymorphism group 16SrI (aster yellows phytoplasma group), subgroup 16SrI-B (I-B), and variant subgroup I-B*. The strains (NAGYI strains) were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico three-dimensional modeling of the SecY protein. Although the NAGYI strains are closely related to aster yellows (AY) phytoplasma strains and are classified like AY strains in subgroup I-B or in variant subgroup I-B*, the results from genotyping and protein modeling may signal ongoing evolutionary divergence of NAGYI strains from related strains in subgroup 16SrI-B.

First report of ‘Candidatus Phytoplasma pruni’ infecting cassava in Japan

Phytoplasmas are a threat to cassava production, but have not been found from cassava in Japan. Here, we present the discovery of phytoplasma in asymptomatic plants propagated in Japan. A 16S rRNA gene sequence analysis showed that the phytoplasma was related to ‘Candidatus Phytoplasma pruni’. Analyses based on 16S rRNA, secY, and ribosomal protein gene sequences revealed that the phytoplasma was closely related to phytoplasmas found in cassava in Latin America. Infected plants remained asymptomatic during cultivation for more than 11 months. This is the first molecular evidence of phytoplasma infection in cassava in Japan.

Serological diagnosis and molecular characterization of Leptospira spp. in the blood and urine of bovine females from refrigerated slaughterhouses

Leptospirosis is an important socioeconomic disease in humans, as well as in domestic and wild animals, being caused by Leptospira spp. Bovine animals are considered reservoirs of this disease, because they intermittently disseminate the bacteria into the environment through their urine. In this way, the cattle an important source of Leptospira infection. The objective of this study was to detect Leptospira spp. antibodies and DNA in bovine females from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil. In particular, blood and urine samples from 52 crossbred bovine females older than 36 months from the two slaughterhouses were used. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies, and the polymerase chain reaction (PCR) and subsequent sequencing were used to detect Leptospira DNA. The MAT yielded 22 (42.3%) serum samples considered reagent, while the nested PCR test resulted in one amplified sample (1.9%) of 289 bp. This single sample was then amplified again using primers for the SecY gene (549 bp). Sequencing of this gene characterized the bacteria as L. borgpetersenii that were similar to the serovar Hardjo of the genotype Hardjobovis. This is the first molecular confirmation of Hardjobovis-like L. borgpetersenii in the urine of crossbred bovine females older than 36 months from slaughterhouses in the microregion of Umuarama. This study’s results show that it is important to combine serological and molecular diagnosis in the detection of Leptospira spp. Therefore, both methods were used to improve our understanding of the epidemiology of this disease in bovine animals from the microregion of Umuarama. In addition, the analysis informed the subsequent adoption of preventive measures and educational One Health actions to prevent economic losses related to the herd, as well as social losses related to workers and the environment.

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

AbstractDuring 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.

Important genetic diversity of ‘Candidatus Phytoplasma solani’ related strains associated with bois noir grapevine yellows and planthoppers in Azerbaijan

Bois noir (BN) is an important grapevine yellows endemic to the Euro-Mediterranean basin caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), a non culturable plant pathogenic Mollicute. Bois noir symptoms could be associated with ‘Ca. P. solani’ in two Azerbaijanian vineyards where disease incidence and severity were recorded for five local Vitis vinifera cultivars. In order to gain insight into the epidemiology of Bois noir in Azerbaijan, ‘Ca. P. solani’ isolates infecting plants were characterized by multi-locus sequence analysis and their secY and stamp gene sequences compared to that of the strains detected in other plants and in local Cixiidae planthoppers. Genotypes were determined for two non-ribosomal house-keeping genes, namely tuf and secY, as well as two variable markers namely Stamp and mleP1 genes, that respectively encode the antigenic membrane protein AMP and a 2-Hydroxycarboxylate transporter. The Azerbaijanian BN phytoplasma isolates corresponded to three tufB and secY genotypes. A finer differentiation of Azerbaijanian ‘Ca. P. solani’ isolates was obtained with mleP1 as five different mleP1 genetic variants were found. Finally, Stamp gene allowed differentiating four new genotypes in grapevine among the 10 new Stamp genotypes detected in various plants in Azerbaijan. The preliminary survey for infected insects conducted in northern Azerbaijan, led to the identification of Hyalesthes obsoletus and Reptalus noahi as potential vectors for two ‘Ca. P. solani’ new genotypes phylogenetically distant from the known genetic clusters. Altogether these results indicate an important genetic diversity of BN phytoplasmas in Azerbaijan that certainly result from spread through local insect vectors.

Detection, Characterization and In-Silico Analysis of Candidatus Phytoplasma australasiae Associated with Big Bud Disease of Tomato in India

Tomato plants showing witches broom symptoms were collected from different states of India. The presence of phytoplasma infection was confirmed by PCR using phytoplasma-specific primer of 16S rRNA and SecY gene. The sequence analysis of 16S rRNA and SecY gene of eight tomato big bud phytoplasmas showed maximum nucleotide (nt) identity of 95–100% with Peanut WB group (16SrII). Further in-silico RFLP analysis of 16S rRNA gene of TBB-Pun1, TBB-Ban, TBB-mal, TBB-Guj and TBB-Vns showed similarity coefficient of 0.68–0.95. Since threshold similarity coefficient for classifying the phytoplasma into new subgroup is set at 0.97, the strain under study significantly distinct from the representative strains in the subgroups of pea nut witches broom. Further, the phylogenetic analysis of tomato big bud phytoplasmas revealed that, they are closely clustered with peanut witches’-broom strains (16Sr II), specifically within the 16Sr II-D and 16Sr II-A subgroups. A comprehensive recombination analysis showed the evidence of both intra and inter-species recombination in seven tomato big bud isolates with most part of their 16Sr RNA F2nR2 fragments descending from Ca.P.brasiliense (16Sr XV) as major parent, except isolate TBB-Vns which had an intra species recombination with Cactus witches-broom-16Sr II-L as major parent. Similarly, in case of SecY gene, all the seven isolates have intra-species recombination with major portion descending from Vinca virescence-[16Sr VI-A] and Potato purple top wilt-[16Sr XVIII-B]. The genetic similarities and the potential threat of this new phytoplasma belonging to 16Sr II group of Peanut witches’ broom’ group infecting tomato in India are discussed.

Multigene detection of eggplant little leaf phytoplasma strainsbelonging to 16SrII and 16SrVI groups

Brinjal little leaf (BLL) is a phytoplasma-associated disease widespread in different parts of the world that induces severe economic losses. Recently, it was associated with 16SrII and 16SrVI groups of phytoplasmas in eight states of India. In the present study, thirteen BLL strains were analyzed on three genes secA, secY and tuf using specific primers to verify their efficacy in the detection of 16SrII and 16SrVI phytoplasmas. While the secA and secY gene sequences confirmed the presence of the 16SrVI and 16SrII groups of phytoplasmas the tuf gene was successfully detected only in the 16SrII group infected samples from Uttar Pradesh. The results confirmed the validity and reliability of the secA and secY gene specific primers to detect both 16SrVI and 16SrII group phytoplasmas associated with BLL, while the tuf gene primers resulted in the detection of only the 16SrII phytoplasmas associated with the same disease.

‘Candidatus Phytoplasma noviguineense’, a novel taxon associated with Bogia coconut syndrome and banana wilt disease on the island of New Guinea

Bogia coconut syndrome (BCS) is one of the lethal yellowing (LY)-type diseases associated with phytoplasma presence that are seriously threatening coconut cultivation worldwide. It has recently emerged, and is rapidly spreading in northern parts of the island of New Guinea. BCS-associated phytoplasmas collected in different regions were compared in terms of 16S rRNA gene sequences, revealing high identity among them represented by strain BCS-BoR. Comparative analysis of the 16S rRNA gene sequences revealed that BCS-BoR shared less than a 97.5 % similarity with other species of 'Candidatus Phytoplasma', with a maximum value of 96.08 % (with strain LY; GenBank accession no. U18747). This result indicates the necessity and propriety of a novel taxon for BCS phytoplasmas according to the recommendations of the IRPCM. Phylogenetic analysis was also conducted on 16S rRNA gene sequences, resulting in a monophyletic cluster composed of BCS-BoR and other LY-associated phytoplasmas. Other phytoplasmas on the island of New Guinea associated with banana wilt and arecanut yellow leaf diseases showed high similarities to BCS-BoR and were closely related to BCS phytoplasmas. Based on the uniqueness of their 16S rRNA gene sequences, a novel taxon 'Ca.Phytoplasma noviguineense' is proposed for these phytoplasmas found on the island of New Guinea, with strain BCS-BoR (GenBank accession no. LC228755) as the reference strain. The novel taxon is described in detail, including information on the symptoms of associated diseases and additional genetic features of the secY gene and rp operon.

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

SummaryDuring summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”

Molecular characterization of a phytoplasma associated with Euonymus bungeanus witches’ broom in China

Summary Euonymus bungeanus plants exhibiting symptoms of abnormal branches, small leaves and phyllody, which is indicative of E. bungeanus witches’ broom (EbWB) disease, have recently been found in Beijing, China. A phytoplasma from symptomatic E. bungeanus plants was identified by 16S rRNA polymerase chain reaction (PCR) using the phytoplasma-specific universal primer pair R16mF2/R16mR1. Inoculation of healthy E. bungeanus plants by grafting with diseased scions was also performed. The rp and secY genes of the EbWB phytoplasma were cloned and sequenced as was the 16S rRNA gene. Sequence and phylogenetic analyses of 16S rRNA, rp and secY genes indicated that the phytoplasma associated with E. bungeanus belongs to the 16SrV-B, rpV-C and secY-C subgroup, the same subgroup as the jujube witches’ broom (JWB) phytoplasma that is widely distributed among jujube trees in China. Comparative analyses based on virtual restriction fragment length polymorphism (RFLP) showed that the EbWB phytoplasma is more closely related to another 16SrV-B subgroup strain: RPWB (Robinia pseudoacacia witches’ broom). To the best of our knowledge, this is the first report of a witches’ broom phytoplasma in E. bungeanus in China, and the findings add a new cultivated plant species to the already broad natural host range of 16SrV-B subgroup phytoplasmas.

Identification and characterization of a phytoplasma associated with black locust yellow disease in two provinces of Iran

A phytoplasma disease was associated with black locust trees showing yellows, a devastating disease found in different geographical areas of Iran. Infected trees showed yellowing, reduced leaf size, short internodes and proliferation of buds, dieback and decline. The disease was transmitted to healthy black locust and periwinkle plants in greenhouse. DNA extractions were made from midrib of leaf tissues. Nested PCR assays with R16mF2/R16mR1 universal phytoplasma primers detected a 1.4 kb product and an 850 bp product was obtained using Fu5/Ru3 primers in double nested PCR. Phytoplasmas were detected from all symptomatic black locust trees as well as inoculated black locust and periwinkle plants. Analysis of 16S rRNA gene sequences showed that phytoplasmas associated with black locust yellows (BLYp) were related to ‘Candidatus Phytoplasma phoenicium’ (group 16SrIX). Moreover, four 16S rDNA genetic isolates (BLYp-a to -d), distinguished by 1–10 single nucleotide polymorphisms (SNPs), were identified in BLYp population. Virtual RFLP analysis determined that BLYp-a represents a new tentative subgroup within the 16SrIX group; BLYp-b, BLYp-c and BLYp-d were assigned to subgroup 16SrIX-C. Actual RFLP profiles derived from EcoRI, RsaI, HinfI and TaqI enzymes confirmed virtual RFLP subgroup characterization. Phylogenetic analysis showed that BLYp isolates shared a recent common ancestor with some 16SrIX-C phytoplasma strains. Moreover, comparisons of 16S rRNA and SecY gene nucleotide sequence raised the question as to whether BLYp and other 16SrIX-C phytoplasmas identified in Iran are identical or closely related strains. To the best of our knowledge, this is the first report of 16SrIX phytoplasmas infecting black locust.

Leptospira spp. in Small Mammals from Areas with Low and High Human Hantavirus Incidences in South-West Germany.

Leptospirosis is caused by Leptospira spp. and is considered the most widespread zoonotic disease worldwide. It mimics nephropathia epidemica in humans, a disease mainly caused by Puumala hantavirus (PUUV). Small mammals are reservoirs for Leptospira spp. and PUUV. Seewis virus (SWSV) is a shrew-borne hantavirus with unknown pathogenicity. The objective of this study was to estimate the prevalence for Leptospira spp. and the frequency of Leptospira-hantavirus co-infections in small mammals collected at locations with high and low incidences in humans. In 2012 and 2013, 736 small mammals belonging to seven species (Apodemus flavicollis, Microtus agrestis, Microtus arvalis, Myodes glareolus, Sorex araneus, S. coronatus, and S. minutus) were collected at four high incidence sites (H1-H4) and four low (L1-L4) incidence sites for PUUV infection in humans. Kidney-derived DNA samples were tested for Leptospira spp. by real-time PCR targeting the lipl 32 gene and further analyzed by duplex PCR targeting the flaB and the secY genes. For the detection of Seewis virus, lung-derived DNA was tested via RT-PCR targeting the nucleocapsid gene. Altogether, 42 of the 736 small mammals including 27 of 660 bank voles and 11 of 66 shrews, were positive for Leptospira spp., while Sorex spp. (14.7%) showed significantly higher prevalences compared to bank voles (4.1%). Detected Leptospira spp. were pathogenic species other than L. kirschneri. Significantly more Leptospira-positive bank voles were found at H sites than at L sites. Altogether 22.2% of positive bank voles were infected with PUUV. Double infection of PUUV and Leptospira spp. occurrence in bank voles is 1.86 times (OR = 1.86; 95% CI: 0.72-4.73) more likely than infections with each pathogen separately. Leptospira- positive bank voles are focally positively associated with PUUV infection in bank voles and with human hantavirus cases. It should be considered that shrews may serve as Leptospira spp. reservoirs.

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red-leaf Disease ofSalvia miltiorrhizain China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma-specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN-1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII-A), respectively. The phylogenetic trees showed that LN-1 phytoplasma clustered with the members of 16SrXII-A group, including Ca. P. solani. Computer-simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.