Bacterial Stem Rot of Oncidium Orchid Caused by a Dickeya sp. (ex Pectobacterium chrysanthemi) in Mainland China

In December 2007, stem rot symptoms on orchids (Oncidium Gower Ramsey) were observed at a flower nursery in the Zhejiang Province of China. Initial symptoms were water-soaked lesions starting at the base of the stem. As these lesions expanded and elongated, the stem and leaf tissues became soft and watery. When examined with a microscope, cut edges of symptomatic stem and leaf tissues consistently exhibited bacterial streaming. The bacteria were isolated by streaking on nutrient agar (3). All isolates were gram-negative, facultative, anaerobic rods with peritrichous flagella. Infiltration of tobacco leaves (Nicotiana tabacum cv. Samsun) with the bacterial suspension of 108 CFU/ml resulted in typical hypersensitivity reactions within 24 h. Five representative isolates were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) by the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TSBA50). The five isolates were identified as Erwinia chrysanthemi (Pectobacterium chrysanthemi) with a Biolog and FAME similarity index of 0.81 to 0.88 and 0.62 to 0.75, respectively. The transfer of P. chrysamthemi to a novel genus, Dickeya gen. nov., was recently proposed (2). The almost complete 16S rDNA sequence from Oncidium isolate SCH-01 (1,604 bp; EMBL Accession No. FM946179) was determined according to the method of Li et al. (1). A subsequent GenBank search showed that this isolate is 98% identical to that of type strain CFBP 1269T of Dickeya dadantii (EMBL Accession No. AF520707) and CFBP 1200T of Dickeya dianthicola (EMBL Accession No. AF520708). Nevertheless, species identification within genus Dickeya is still difficult since only a limited number of strains of each species have been characterized fully. Koch's postulates were completed with the inoculation of Oncidium seedlings with cell suspensions (108 CFU/ml) by a pinprick at the base of the stem. All five representative isolates induced stem rot similar to that observed in natural infections. No symptoms were noted on the control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of Oncidium plants. To our knowledge, this is the first report of stem rot on Oncidium orchid in Mainland China caused by the bacterium formerly referred to as P. chrysanthemi, now proposed as Dickeya sp. References: (1) B. Li et al. J. Phytopathol. 154:711, 2006. (2) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

First Report of Rice yellow mottle virus in Rice in The Gambia

Rice yellow mottle virus (RYMV) of the genus Sobemovirus is a major biotic constraint to rice (Oryza sativa) production in Africa. First reported in Kenya during 1966, RYMV was later found in most countries in Africa where rice is grown (1). In countries in westernmost Africa (The Gambia, Guinea-Bissau, Mauritania, and Senegal), plants with leaf yellowing and mottling symptoms were observed, but RYMV was never isolated. Rice is the staple food in The Gambia. In 2006, four samples were collected from local rice varieties in the Kuntaur Region in the center of The Gambia. Mechanical inoculation with leaf extracts from all samples caused typical yellow mottle symptoms on the susceptible rice varieties BG90-2, Bouaké 189, and IR64. RYMV was detected in the four samples collected by ELISA with polyclonal antisera (2). The 720-nt coat protein gene was amplified for each isolate by reverse-transcriptase-PCR with primers 5′-CAAAGATGGCCAGGAA-3′ (sense) and 5′-CTCCCCCACCCATCCCGAGAATT-3′ (antisense) (2). The RT-PCR products were directly sequenced (EMBL Accession Nos. AM765810, AM765811, AM765812, and AM765813) and then aligned using ClustalW with a pool of RYMV coat protein sequences from West African isolates (EMBL Accession Nos. AJ279905, AJ279901, AJ885137, AJ885124, and AJ279935). Phylogenetic reconstruction by maximum-likelihood with PAUP indicated that the isolates from The Gambia formed a monophyletic group with over 97% nucleotide identity and are closely related to isolates of other countries in West Africa (Burkina Faso, Côte d'Ivoire, Guinea, Mali, and Sierra-Leone) with 91 to 94% identity. Detection of RYMV in The Gambia indicates that RYMV is present in westernmost Africa, which is referred to as the ‘rice belt’ of Africa, and shows that RYMV is widely distributed from eastern Africa (Tanzania) to the western part of the continent. References: (1) N. K. Kouassi et al. Plant Dis. 89:124, 2005. (2) A. Pinel et al. Arch. Virol. 145:1621, 2000.

First Report of Apple chlorotic leaf spot virus in Quince in Greece

The sanitary status of pome fruit trees was evaluated in central and northern Greece during a survey in the autumn of 2004 and spring of 2005. Twenty-six leaf samples were collected from five symptomless and 21 symptomatic quince trees showing fruit deformation (FD) symptoms and diffuse chlorotic leaf spots. All samples were tested for the presence of Apple chlorotic leaf spot virus (ACLSV), a member of the Trichovirus genus, initially by ELISA and then by a specific reverse transcription (RT)-PCR assay reported previously (1). ACLSV has a broad host range that includes most, if not all, Prunoidae (peach, apricot, plum, and cherry) and Maloidae (apple, pear, and quince) fruit tree species. Although it has been tentatively linked with fruit, leaf, bark, and growth retardation symptoms in quince (2,3), its geographic distribution and association with specific symptoms is still poorly determined. ACLSV was initially detected by serology in two plants, one symptomless and one showing FD symptoms. ACLSV presence in these two samples and in an additional symptomless plant was confirmed by the ACLSV-specific RT-PCR assay. Sequencing of the RT-PCR amplicon from the symptomatic isolate (EMBL Accession No. AM292923), which was positive in both assays, confirmed the identification of ACLSV. The obtained sequence shows 93% nucleotide identity with an apple isolate of ACLSV (EMBL Accession No. AY677103). To our knowledge, these findings represent the first report of the presence of ACLSV in quince in Greece. They further indicate that at least for some host-cultivar/virus isolate combinations, ACLSV may be asymptomatic in quince and that the symptoms observed in the plants sampled are unrelated to ACLSV infection. References: (1) T. Candresse et al. Acta Hortic. 386:136, 1995. (2) J. C. Desvignes. Page 202 in: Virus Diseases of Fruit Trees. CTIFL Publishing, 1999. (3) S. Paunovic and M. Rankovic. Jugosl. Vocarstvo 31:231, 1997.

First Report of Tomato yellow leaf curl virus Infecting Common Bean (Phaseolus vulgaris) in Greece

Tomato yellow leaf curl is one of the most devastating virus diseases of tomato (Lycopersicon esculentum Mill) crops worldwide. Several whitefly-transmitted viruses are associated with the disease and all are assigned to the genus Begomovirus, family Geminiviridae. In Greece, Tomato yellow leaf curl virus (TYLCV) was first reported to infect greenhouse and open-field tomatoes in 2000 (2). During 2006, a survey was conducted in the southwestern part of Peloponnese (mainland) within the areas of Kyparissia and Filiatra (Perfecture of Messinia) to identify the prevalence and natural hosts of the disease. During this survey, yellow mosaic, severe leaf curling, and leaf crumple symptoms were observed in greenhouse bean plants (Phaseolus vulgaris) that were cultivated together with tomatoes showing typical TYLCV symptoms. In all affected greenhouses, the incidence of the disease ranged from 1 to 5% in beans and 90 to 100% in tomato plants. Both bean and tomato plants were highly infested with Bemisia tabaci (Gennadius) populations and produced unmarketable fruits. Twenty-four symptomatic bean plants were collected from four greenhouses that tested positive by triple-antibody sandwich-ELISA using TYLCV-specific antibodies purchased from NEOGEN, EUROPE, Ltd. DNA was extracted from all infected bean plants, and a 580-bp fragment of the coat protein gene was amplified by PCR using the TY(+)/TY(-) primer pair (1). Amplified fragments were then analyzed by restriction fragment length polymorphism with Ava II cutter enzyme. Two DNA fragments of 277 and 302 bp in agarose gels were produced from all isolates and the restriction pattern corresponded to TYLCV. The amplified DNA from four infected bean plants was cloned and sequenced. All four sequences were 100% identical (EMBL Accession No. AM418398) and showed 99% nucleotide identity to a TYLCV isolate from Italy (EMBL Accession No. DQ144621). To our knowledge, this is the first report of TYLCV infection of P. vulgaris, which is an important commercial crop in Messinia, Greece. Within the last decade, TYLCV has emerged as an important pathogen for several cultivated plants in many regions and different TYLCV variants have been reported to infect P. vulgaris (3). Bean is often used as an intercrop between tomato crops, and thus, infected plants may serve as a potential reservoir for virus survival and spread (4). References: (1) G. P. Accotto et al. Eur. J. Plant Pathol. 106:179, 2000. (2) A. D. Avgelis et al. Plant Dis. 85:678, 2001. (3) J. Morris et al. EPPO Bull. 32:41, 2002. (4) J. Navas-Castillo et al. Plant Dis. 83:29, 1999.

Molecular Characterization of a Begomovirus Associated with Tomato Leaf Curl Disease in Uganda

During the summer of 2003, leaf curl symptoms were observed in tomato (Lycopersicon esculentum) plantings in the Iganga District of Uganda. Begomoviral infection was suspected. Twelve symptomatic samples were collected. Begomoviral DNA was extracted and amplified using polymerase chain reaction (PCR) with the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (4). The expected 1.4-kb PCR products were obtained from 11 of 12 samples. The 1.4-kb PCR product of one of the samples was cloned and sequenced. Based on the sequence of the 1.4-kb DNA product, specific primers were designed to complete the DNA-A sequence. The DNA-A consisted of 2,747 nucleotides (GenBank Accession No. DQ127170) and was found to contain seven predicted open reading frames (ORFs V1, V2, C1, C2, C3, C4, and C5). A BLAST analysis was conducted with geminivirus sequences available in the GenBank database at the National Center for Biotechnology Information (Bethesda, MD), and MegAlign (DNASTAR, Inc, Madison, WI) software was used for further comparisons. The DNA-A sequence of the virus associated with leaf curl of tomato from Uganda showed less than 79% sequence identity with cassava mosaic viruses from Uganda (GenBank/EMBL Accession Nos. AF126800, AF126802, AF126804, AF126806, and Z83257), the only begomoviruses from the country so far in the public domain. Highest sequence identity (83%) was with Tomato leaf curl Mayotte virus from Dembeni, Mayotte, Comoros Islands (ToLCYTV-[Dem], EMBL Accession No. AJ865341). Pairwise comparison with ToLCYTV-[Dem] showed 60, 88, 91, 82, 84, 86, and 80% sequence identities in the intergenic region, V2, V1, C1, C2, C3, and C4 ORFs, respectively. Only low sequence identities (ranging from 71 to 82%) were obtained with other tomato bego-moviruses reported from Africa (GenBank/EMBL Accession Nos. AF261885, AJ865337-AJ865340, AY044137-AY044139, AY502934, AY502936, AY594174, AY736854, and U73498). There was no evidence for the presence of DNA-B or DNA-beta using PCR with the DNA-B specific primer pairs DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2) and the DNA-beta primer pair Beta01/Beta02 (1), respectively. Detection of possible recombination was by RDP2 software (3) using DNA-A sequences of begomoviruses from Uganda and tomato begomoviruses from Africa. The DNA-A was found to contain a small recombinant fragment from ToLCYTV-[Dem] in the 411 to 969 nucleotide position with 92% sequence identity. Based on DNA-A sequence comparisons, the tomato leaf curl virus from Uganda most likely constitutes a distinct new begomovirus. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) D. P. Martin et al. Bioinformatics 21:260, 2005. (4) M. R. Rojas et al. Plant Dis.77:340, 1993.

A New Tomato leaf curl virus from Mayotte

In June 2003, symptoms of stunting and leaf curling resembling symptoms of tomato leaf curl disease, as well as reductions in yields, were observed on tomato plants in the western (Combani and Kahani) and eastern (Dembeni, Kaoueni, and Tsararano) regions of Mayotte, a French island in the Comoros Archipelago located in the northern part of the Mozambique Channel. The whitefly, Bemisia tabaci (Gennadius), was observed colonizing tomato plants and other vegetable crops at low levels. Overall, 13 leaf samples with symptoms were collected from tomato plants among the five regions and tested for the presence of begomoviruses using a polymerase chain reaction (PCR) assay with two sets of degenerate primers designed to amplify two regions of the A component of begomoviruses. Primers MP16 and MP82 amplify an approximately 500-bp fragment located between the intergenic conserved nonanucleotide sequence and the first 200 bp of the coat protein (CP) gene (2). Primers AV494 and AC1048 amplify the approximately 550-bp core region of the CP gene (3). Six leaf samples, one from Combani, three from Dembeni, and two from Kahani, gave a PCR product of the expected size with both sets of primers. No PCR products were obtained with degenerate primers designed for begomovirus DNA B or β. The approximately 500- and 550-bp PCR products from one sample each of Combani (EMBL Accession Nos. AJ620912 and AJ620915, respectively), Dembeni (EMBL Accession Nos. AJ620911 and AJ620914, respectively), and Kahani (EMBL Accession Nos. AJ620913 and AJ620916, respectively) were sequenced. For the 489-bp sequences obtained with the MP16/MP82 primer set, the three isolates had 90 to 95% nucleotide identity (DNAMAN; Lynnon BioSoft, Quebec). The most significant sequence alignments (NCBI and BLAST) were with begomoviruses; 80 to 83% nucleotide identity was obtained with the Tomato yellow leaf curl Morondava virus (TYLCMV) isolates from Madagascar (EMBL Accession Nos. AJ422123 and AJ422124), 80 to 82% nucleotide identity was obtained with the South African cassava mosaic virus (SACMV) isolates (GenBank and EMBL Accession Nos. AF155806 and AJ422132), and 79 to 81% nucleotide identity was obtained with the East African cassava mosaic Malawi virus (EMBL Accession No. AJ006460). For the 522-bp sequences obtained with the AV494/AC1048 primer set, 95 to 97% nucleotide identity was shown between the three isolates. The most significant sequence alignments were also with begomoviruses; TYLCMV isolate Morondava (EMBL Accession No. AJ422125) with 86 to 88% nucleotide identity, Tomato yellow leaf curl virus isolates (GenBank and EMBL Accession Nos. AF105975, AJ489258, AB014346, AF024715, AF071228, and X76319) with 86 to 87% nucleotide identity, and SACMV isolate M12 (EMBL Accession No. AJ422132) with 85 to 86% nucleotide identity. According to the current taxonomic criteria for the provisional classification of a new begomovirus species, nucleotide sequence identity in the core region of the CP <90% (1), the tomato begomovirus from Mayotte is a new species and is provisionally named Tomato leaf curl Mayotte virus. References: (1) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (2) P. Umaharan et al. Phytopathology 88:1262, 1998. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

First Report of Tomato yellow leaf curl virus in Tomato in Guadeloupe

In September 2001, symptoms of stunting and chlorotic curled leaves of reduced size were observed on tomato plants in Guadeloupe. These symptoms were different from those described for Potato yellow mosaic virus, which has been present since 1993, but similar to those described for Tomato yellow leaf curl virus (TYLCV). Samples from symptomatic plants were collected and analyzed by polymerase chain reaction (PCR). Primers PC1 (5′-TGACTATGTCGAAGCGACCAGG-3′) and PC2 (5′-CGACATTACAGCCTCAGACTGG-3′) were designed to amplify a 950-bp fragment within the coat protein gene (CP) of TYLCV-IL species (2). Primer pair MP16/MP82 (3) amplified a 550-bp fragment from the conserved nonanucleotide sequence (TAATATTAC) to the 5′ end of the CP gene. Products of expected sizes were obtained with both pairs of primers from all symptomatic samples but not from uninfected samples. A 950-bp and a 550-bp PCR product were cloned into a pGEM-T Easy Vector (Promega, Madison, WI) and sequenced with plasmid specific primers (SP6 and T7). Sequences were compared with those available in the NCBI database using BlastN. Fifteen of the sequences that gave the highest score with BlastN were aligned with the Guadeloupe sequences using Clustal W. The nucleotide sequence of the 950-bp fragment (GenBank Accession No. AY319645) shared at least 97% sequence identity with that of TYLCV from Israel (EMBL Accession No. X15656), Puerto Rico (GenBank Accession No. AY134494), Cuba (EMBL Accession No. AJ223505), and the Dominican Republic (GenBank Accession No. AF024715). Similar percentages of identity were obtained with the 550-bp sequence (GenBank Accession No. AY319646). These results confirm that a begomovirus belonging to the species TYLCV-Israel is infecting tomato in Guadeloupe. To our knowledge, this is the first report of TYLCV in this region of the Caribbean. Puerto Rico is the closest location from which TYLCV was previously reported (1). In May 2002, typical TYLCV symptoms were observed in all tomato production areas at an incidence of 80 to 100%. References: (1) J. Bird et al. Plant Dis. 85:1028, 2001. (2) Y. Martinez et al. Rev. Prot. Veg. 18:168, 2003. (3) P. Umaharan et al. Phytopathology 88:1262, 1998.