Complete genome resources for Ralstonia bacterial wilt strains UW763 (phylotype I); Rs5, UW700 (phylotype II); UW386, RUN2474, RUN2279 (phylotype III)

We share whole genome sequences of six strains from the Ralstonia solanacearum species complex, a diverse group of beta-Proteobacteria that cause plant vascular wilt diseases. Using single-molecule real-time (SMRT) technology, we sequenced and assembled full genomes of Rs5 and UW700, two phylotype IA-sequevar 7 (IIA-7) strains from the southeastern US that are closely related to the R. solanacearum species type strain, K60, but were isolated >50 years later. Four sequenced strains from Africa include a soil isolate from Nigeria (UW386, III-23), a tomato isolate from Senegal (UW763, I-14), and two potato isolates from the Madagascar highlands (RUN2474, III-19 and RUN2279, III-60). This resource will support studies of the genetic diversity, ecology, virulence, and microevolution of this globally distributed group of high-impact plant pathogens.

Characteristic of Tobacco mosaic virus isolated from cucumber and tobacco collected from East Java, Indonesia

Listihani, Hidayat SH, Wiyono S, Damayanti TA. 2019. Characteristic of Tobacco mosaic virus isolated from cucumber and tobacco collected from East Java, Indonesia. Biodiversitas 20: 2937-2942. Tobacco mosaic virus (TMV) is a newly emerging virus infecting cucumbers in Java, Indonesia. The basic characters of the TMV isolated from cucumber need further study to investigate its differences with that from tobacco. Thus, the research aimed to study the character of both isolates based on their biological, symptomatology and nucleic acid of coat protein (CP) gene properties. The TMV isolates from both cucumber and tobacco were able to infect similar indicator plants with differing symptom expressions, especially on eggplant. Homology of nucleotide and amino acid of coat protein gene among isolates were about 90.3% and 91.0%, and homology to other isolates was about 87.6 to 93.8% and 89.3 to 96.8%, respectively. There were 17 amino acid differences in the CP gene which is presumed to differentiate those two isolates. Phylogenetic analysis CP gene sequences compared with corresponding isolates in GenBank showed the two isolates separated in different clades. The cucumber isolate from Kediri is closely related to tomato isolate from China in clade I, while tobacco isolate from Jember closely related to tobacco isolate from China in clade IIindicating the existence of two differ TMV variants. It will increase the difficulty to manage TMV in the fields.

Complete Genome Sequence of a Tomato Isolate of Parietaria Mottle Virus from Italy

We report here the complete genome sequence of isolate T32 of parietaria mottle virus (PMoV) infecting tomato plants in Turin, Italy, obtained by Sanger sequencing. T32 shares 90.48 to 96.69% nucleotide identity with other two PoMV isolates, CR8 and Pe1, respectively, whose complete genome sequences are available.

Dieback and Wilting Caused by Tomato spotted wilt virus in Arctotis × hybrida in Italy

In winter 2012, some potted plants of African daisy (Arctotis × hybrida L., family Asteraceae) cv. Hannah, propagated by rooted stem cuttings and cultivated for commercial purposes in a greenhouse located at Albenga (Liguria region, Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. Around 130 plants on a total of 3,000 cultivated plants showed symptoms (4 to 5%). One gram of fresh leaves, each collected from three different symptomatic plants, was ground in 4 ml of cold (∼5°C) sodium phosphate 0.03 M buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of carborundum (600 mesh). The inoculum was rubbed on healthy indicator herbaceous plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24/18°C day/night. Healthy and buffer inoculated plants were also included in the test and used as negative control in the subsequent serological and molecular analysis. Sap-inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa, yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano), necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa, necrotic local lesions in petunia (Petunia × hybrida cv. Pink Beauty). No symptoms were recorded on buffer inoculated plants. Leaf samples from both symptomatic hosts and the three original symptomatic African daisy plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and tospoviruses (Tospovirus broad-spectrum, Serogroups I, II, and III, Bioreba AG, Switzerland). Positive reaction was obtained with Tospo-groups antibodies, but not with the CMV ones. Total RNA was extracted from infected leaves of African daisy with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription (RT)-PCR by using the tospovirus universal primers BR60/BR65 that amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all samples tested. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (HF913777). The sequence showed highest homology (99%) with the Tomato spotted wilt virus (TSWV) tomato isolate NJ-JN from South Korea (HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers partially covering the movement protein gene of TSWV (2). The sequence obtained (HF913776) showed the highest homology (99%) with three TSWV isolates: a tomato isolate from Spain (AY744493), a pepper isolate from South Korea (AB663306), and again the tomato NJ-JN isolate from South Korea (HM581936). To our knowledge, this is the first natural report of TSWV infecting African daisy plants. Moreover, since this ornamental is often cultivated with other flowering plants, it can act as reservoir for the virus that can infect other ornamentals and crops, considering that TSWV has a very broad host range (3). This result also represents the first finding of TSWV in the genus Arctotis, family Asteraceae, the greater botanical family of TSWV hosts (3). References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) M. M. Finetti et al. J. Plant Pathol. 84:145, 2002. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003.

First Report of Tomato yellow ring virus (Tospovirus, Bunyaviridae) Infecting Tomato in Kenya

Tomato (Lycoperscion esculentum) is one of the most popular vegetables and a major source of nutrition and income for smallholders in Africa. Thrips-transmitted tospoviruses are among the economically important pathogens of tomatoes that cause significant crop losses worldwide (3). In surveys for Tomato spotted wilt virus (TSWV) in the major tomato production areas of Kenya between March 2010 and January 2012, tomato fruits with chlorotic ring spots on fruits with stem and leaf necrosis were observed frequently. The symptoms were more evident in the dry seasons and disease incidence ranged from 28 to 42%. The pathogen did not react with antiserum specific to TSWV (Agdia Biofords, Ervy, France) in double-antibody sandwich (DAS)-ELISA. Furthermore, the pathogen did not react with antiserum specific to Capsicum chlorosis virus (CaCV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ring spot virus (GRSV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Watermelon silver mottle virus (WSMoV) (Agdia Biofords and DSMZ, Germany) in DAS-ELISA, but reacted positively to antiserum specific to Tomato yellow fruit ring virus (TYFRV) (DSMZ, AS0526). The nucleocapsid (N) gene specific primers (TFfor: 5′-ACTCATTAAAATGCATCGTTCT-3′ and TFrev: 5′-CTAAGTAAACACCATGGCTACC-3′ as forward and reverse primers, respectively) were designed by choosing six conserved regions of the N gene sequences of known TYFRV and Tomato yellow ring virus (TYRV) sequences available from GenBank. Using these primers, TYRV infection of tomatoes collected from Loitokitok, Kenya (2.73°S, 37.51°E) was confirmed by reverse transcription (RT)-PCR. PCR products of approximately 912-bp were obtained from six out of 11 symptomatic tomato samples tested, but not from healthy and water controls. Amplicons were gel-purified using QuickClean II Gel Extraction Kit (GenScript, UK) and sequenced using TFfor and TFrev primers. A consensus sequence was generated using Geneious Pro 5.5.6 Software (Biomatters Ltd., Auckland, NZ). The BLAST revealed that the N-gene sequence of the Kenyan tomato isolate (GenBank Accession No. JQ955615) had sequence identity with the Cineraria isolate (98.5%) (Accession No. DQ788693.1) and the Anemone isolate (98.1%) (Accession No. DQ788694.1) of TYRV (4) from Fars Province, Iran; an Alstroemeria isolate (98.4%) (Accession No. HQ154130.1) and two tomato isolates (98.3%) (Accession Nos. HQ154131.1 and AY686718.1) of TYRV from northern Khorasan Province, Iran, and a tomato isolate (98.1%) (Accession No. AJ493270.1) of TYFRV from Varamin, Iran. The Kenyan tomato isolate differed from a TYFRV potato isolate (87.5%) from Iran (Accession No. EU126931.1) (1), a TYRV potato isolate (87.5%) from Iran (Accession No. JF836812.1); a soybean isolate of TYRV (87.4%) from Iran (Accession No. DQ462163.1) (2), and showed significant divergence from that of Polygonum ringspot virus from Italy (81%) (Accession No. EF445397.1). To our knowledge, this is the first report of TYRV infecting tomatoes in Kenya. Further surveys and monitoring of TYRV incidence and distribution in the region, vector competence of thrips species, and impact on the crop yield are in progress. References: (1) A. R. Golnaraghi et al. Plant Dis. 92:1280, 2008. (2) A. Hassani-Mehraban et al. Arch. Virol. 152:85, 2007. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) R. Rasoulpour and K. Izadpanah, Austral. Plant Pathol. 36:285, 2007.

Ralstonia solanacearum Virulence Increased Following Large Interstrain Gene Transfers by Natural Transformation

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.

First Report of Tomato chlorotic dwarf viroid in Tomato in France

Tomato chlorotic dwarf viroid (TCDVd) is a pospiviroid found naturally infecting tomato (Solanum lycopersicum L.) (3) and several ornamentals such as Brugmansia, petunia (1), and trailing verbena (4). Initially identified in North America (3), it has been reported from India, Europe (the Netherlands and United Kingdom), and Japan. At the end of 2007, 20 to 25% of tomato plants within a group of greenhouses in the Brittany Region of France were observed with top bunching, leaf curling, and epinasty symptoms. Reverse transcription (RT)-PCR with a primer pair specific for several pospiviroids (5′GGGGAAACCTGGAGCGA3′ and 5′GGGGATCCCTGAAGCGC3′) amplified the correctly sized fragment (approximately 360 bp) from total nucleic acid extracts from three symptomatic plants. The sequence of the uncloned amplification product (GenBank Accession No. EU729744) was determined, together with that of five cloned cDNAs. All sequences were highly related with a total of three mutations in these six sequences and they showed 96.9% (GQ169709 and AY372399) to 99.4% (AF162131) identity with TCDVd sequences present in GenBank. Identification of TCDVd was confirmed from the same plant samples by molecular hybridization with a Potato spindle tuber viroid (PSTVd)-specific probe (which cross-hybridizes with TCDVd to a certain extent) and by PCR with the PSTVd/TCDVd-specific 2A-1S primer pair (3) and sequencing of the amplified fragment. The French isolate is most closely related to the original tomato isolate from Canada (GenBank Accession No. AF162131). In a grow-out test involving 2,500 seeds from the original seed lot from which the symptomatic plants were derived, 2 of the 250 pools of 10 plants tested positive for TCDVd infection with the 3H1-2H1 primer pair (2). The sequence of the amplified product proved identical to the isolate detected in the original greenhouse plants, indicating a low level of seed transmission. As with other pospiviroids, which appear to be more and more frequently reported in greenhouse tomatoes, possible sources of infection include contaminated seeds, as seem to be the case in this first outbreak, and also transfer to tomatoes from infected ornamental hosts. This is, to the best of our knowledge, the first report of TCDVd in tomato in France. References: (1) T. James et al. Plant Pathol. 57:400, 2008. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. 4) R. P. Singh et al. Plant Dis. 90:1457, 2006.

First Report of Pepper veinal mottle virus Associated with Mosaic and Mottle Diseases of Tomato and Pepper in Mali

The aphid-transmitted Pepper veinal mottle virus (PVMV; genus Potyvirus, family Potyviridae) has been reported as causing an epidemic in solanaceous crops, including eggplant, pepper, and tomato in Africa (4). In West Africa, PVMV has been detected in Senegal, Sierra Leone, Ivory Coast, Ghana, Togo, Burkina Faso, and Nigeria (2). In April 2009, leaf yellowing, mosaic, mottle, and curling symptoms indicative of viral infection were common on tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants in home gardens and fields in Mali. Symptomatic leaf samples were collected from two sweet pepper and two tomato plants from Baguineda, four tomato plants and one chili pepper plant in Kati, and three chili pepper plants from Samanko. All samples except two chili pepper from Samanko and two sweet pepper and two tomato from Baguineda tested positive for begomovirus by PCR with primers PAL1v1978/PAR1c715 (3). PVMV was detected by double-antibody sandwich (DAS)-ELISA using PVMV antibody (DSMZ, Braunschweig, Germany) in both Baguineda sweet pepper, one Baguineda tomato, and one Samanko chili pepper sample. Three PVMV ELISA-positive samples, one each of sweet pepper, chili pepper, and tomato, were also confirmed by reverse transcription (RT)-PCR and sequencing. The expected 1.8-kb viral cDNA was amplified from all three samples using the potyvirus general primer Sprimer1 (5′-GGNAAYAAYAGHGGNCARCC-3′), which was modified from the Sprimer (1) as upstream primer, and Oligo(dT) (5′-GCGGGATCCCTTTTTTTTTTTTTTTTTT-3′) as downstream primer. The sequences obtained from chili pepper (GenBank Accession No. GQ918274), sweet pepper (GenBank Accession No. GQ918275), and tomato (GenBank Accession No. GQ918276) isolates, excluding the 3′ poly-A tails, were each 1,831 nucleotides (nt) long, comprising the 3′-terminal of the NIb region (1 to 642 nt), the coat protein region (643 to 1,455 nt), and the 3′-untranslated region (1,456 to 1,831 nt). The sequences shared between 99.3 and 99.5% nucleotide identity with each other. A comparison of these sequences with corresponding sequences of potyviruses in GenBank revealed they had greatest nucleotide identity (96.5 to 96.6%) with a tomato isolate of PVMV from Taiwan (PVMV-TW; GenBank Accession No. EU719647), between 81.4 and 95.9% identity with other PVMV isolates, and only as much as 67.2% identity with other potyvirus isolates. Analysis of coat protein regions alone also revealed high nucleotide (96.6 to 96.8%) and amino acid (99.3 to 99.6%) identity with PVMV-TW. The PVMV Baguineda tomato isolate caused mosaic and mottle symptoms on tomato (line CLN1558A) and pepper (cv. Early Calwonder) plants following mechanical inoculation. To our knowledge, this is the first report of PVMV infecting plants in Mali and reinforces the need to take this virus into consideration when breeding tomato and pepper for this region. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) C. Huguenot et al. J. Phytopathol. 144:29, 1996. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) G. Thottappilly, J. Phytopathol. 134:265, 1992.