First Report of Cymbidium mosaic virus and Odontoglossum ringspot virus in Orchids in Mexico.

In 2010, a survey for viral diseases in commercial, orchid-producing greenhouses was carried out in Morelos, Mexico. Many symptomatic plants were observed. The most common leaf symptoms were yellow mottle, yellow streaks, and chlorotic and necrotic ringspots. Leaf samples were collected from eight symptomatic plants from the following genera: Encyclia, Oncidium, Shomburghia, Brassia, Guarianthe, Cattleya, Epidendrum, Vanilla, Xilobium, Laelia, and Brassocattleya. Samples were tested using double-antibody sandwich (DAS)-ELISA (Agdia, Elkhart, IN) with antiserum for Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cymbidium ringspot mosaic virus, and Tobacco mosaic virus (TMV) and a general antiserum for potyviruses. At least one plant from each genus was positive to CymMV and ORSV as individual or mixed infections. Encyclia and Laelia plants were the most frequently found with mixed infections by both viruses. All genera were negative for TMV and potyviruses. Total RNA extracts were obtained from all ELISA-positive samples by a modified silica capture protocol (2). Reverse transcription (RT)-PCR was carried out with general polymerase (RdRp) gene primers corresponding to the Potexvirus group (3) and specific primers for the coat protein gene (CP) of CymMV and ORSV (1). The PCR amplification from a positive sample of each genus was resolved in agarose gels. Amplification products of the expected size were obtained for CymMV and ORSV. Five CymMV RdRp gene clones from five different plants of Laelia (GenBank Accession Nos. HQ393958, HQ393959, HQ393960, HQ393961, and HQ393962), two CP gene clones of CP gene of CymMV from two different plants of Oncidium (GenBank Accession Nos. HQ393956 and HQ393957), and three CP clones of CP of ORSV from three different plants of Encyclia (GenBank Accession Nos. HQ393953, HQ393954, and HQ393955) were sequenced. The nucleotide sequences of the Mexican orchid CymMV isolates were 96 to 97% identical to CymMV sequences in the GenBank, while those of ORSV were 99 to 100% identical to deposited ORSV sequences. To our knowledge, this is the first report of CymMV and ORSV in orchids in Mexico, which are two of the most important quarantine virus in orchids in Mexico. References: (1) P. Ajjikuttira et al. J. Gen. Virol. 86:1543, 2005. (2) J. R. Thompson et al. J. Virol. Methods 111:85, 2003. (3) R. A. A. van der Vlugt and M. Berendsen. Eur. J. Plant Pathol. 108:367, 2002.

Leaf Stripe and Stem Rot Caused by Burkholderia gladioli, a New Disease of Maize in México.

A new maize disease appeared in the State of Veracruz, Mexico during 2003-2004. Initial symptoms in the leaves were small, white-yellow, watery spots, which coalesced into dry necrotic stripes that were 0.3 wide and 8 cm long. Reddening sometimes developed on these leaves. Stems developed a rot in the crown. The flag leaf showed a rot and necrosis at the base, rolled inward, and dried out. Necrosis developed at the base of the corn ears and their growth was halted. A bacterium characterized by white colonies was consistently isolated from lesions on casamino acid peptone and glucose (CPG), King's medium B, and nutrient agar media. Ten isolates were chosen for further characterization. Pathogenicity was confirmed in the greenhouse (25 to 30°C) on 45-day-old ASGROW 7573 maize plants by injection of bacterial suspensions (107 CFU/ml) at the base of the stem. Control maize seedlings were injected with phosphate buffer. Symptoms similar to those observed in the field were observed after 3 days on all inoculated plants but were not observed on control plants. Koch's postulates were fulfilled with reisolation of the inoculated strain from the inoculated tissues and confirmation was fulfilled by phenotypic characters and 16S rDNA sequences. The white colonies on CPG were slightly convex, shiny, circular with entire margins, gram negative, lacked arginine dihydrolase, did not produce fluorescent pigment on Pseudomonas F medium, and grew aerobically. The strains were able to utilize l-arabinose, d-mannitol, and cellobiose, but unable to utilize d-maltose and l-rhamnose. Gel hydrolysis was positive but starch hydrolysis was not positive. Colonies grew at 40°C. These characteristics are the same as those described previously for Burkholderia gladioli (3). The identity of these isolates was confirmed by 16S analysis with two universal primers, FD1 and RD1, for Eubacteria (2), which generate a 1,600-bp fragment. Two primers specific for the genus Burhkolderia, RHG-F and RHG-R that generate a 500-bp fragment (1) were also used for amplification. Comparison of sequences using Clustal W and Megaline, DNAstar software showed 100% similarity among strains isolated from the three counties (GenBank Accession Nos. EU161873 to EU161878) and 99% similarity of field isolates with B. gladioli in the GenBank database. To our knowledge, this is the first report of B. gladioli causing leaf stripe and stem rot of maize in Mexico. References: (1) J. J. Lipuma et al. J. Clin. Microbiol. 37:3167, 1999. (2) L. M. Rodrigues et al. Appl. Environ. Microbiol. 69:4249, 2003. 3) N. W. Shaad et al., eds. 3rd ed. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001.

First Report of Prunus necrotic ringspot virus in Peach in Mexico.

Peach (Prunus persica (L.) Batsch) is one of the most important fruit crops in the temperate regions of Mexico. In 2006, during a survey conducted in commercial peach orchards in Puebla, Mexico for viral diseases, many trees were observed with foliar symptoms that included yellow mottle, ringspot, line patterns, and mosaic. Samples (flowers, young shoot tips, and leaves) were collected from 120 symptomatic trees in three locations (San Martin Texmelucan, Domingo Arenas, and Tepetzala). All samples were tested using double-antibody sandwich (DAS)-ELISA kits (Agdia, Inc., Elkhart, IN) for the presence of the following viruses: Apple mosaic virus, Plum pox virus, Prune dwarf virus, and Prunus necrotic ringspot virus (PNRSV). Sap extracts from young symptomatic leaves and shoots were used to mechanically inoculate Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana tabacum cv. Xanthi, N. glutinosa, N. clevelandii, N. benthamiana, Datura stramonium, Capsicum annuum, and Solanum lycopersicum. Plants were kept in a greenhouse with approximate temperatures of 25 to 35°C, humidity of 70%, and 12 h of light. Sap extracts were also used for dsRNA extraction and analyses (2) and RNA extraction for use in reverse transcription (RT)-PCR with the Access RT-PCR system (Promega, Madison, WI) and primers that annealed to a conserved region in the PNRSV coat protein gene (1). The expected size amplicons of approximately 450 bp were generated from all field-collected samples. The PCR products from three geographically distinct PNRSV isolates (Domingo Arenas [Accession No. DQ979004], Tepetzala [Accession No. DQ979005], and San Martin Texmelucan [Accession No. EF456771]) were directly sequenced with a Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA) and their nucleotide and deduced amino acids sequences were more than 93% identical to corresponding sequences of PNRSV available in the NCBI/GenBank database. PNRSV was the only virus detected by DAS-ELISA in flowers and young shoots from 60 of the symptomatic field samples tested from the three locations. DsRNA banding patterns were obtained from 40 field-collected symptomatic samples; all showed three bands of approximately 3.6, 2.5, and 1.8 kb, the expected sizes for RNAs 1, 2, and 3 of PNRSV, respectively. DsRNAs were not detected in asymptomatic plants. PNRSV transmission by mechanical inoculation induced mosaic symptoms in N. tabacum cv. Xanthi and necrotic local lesions in G. globosa. Although G. globosa is reported to be a systemic host of PNRSV and N. tabacum is not reported to be a host, symptomatic plants were positive for PNRSV in DAS-ELISA tests. It is possible that there was an additional virus not detected in our assays that was responsible for the unexpected reactions in the host range studies. To our knowledge, this is the first report of PNRSV in peach in Mexico. References: (1) D. J. MacKenzie et al. Plant Dis. 81:222, 1997. (2) R. A. Valverde et al. Plant Dis. 74:255,1990.

First Report of Tomato severe leaf curl virus in México.

San Luis Potosí and Morelos are states situated in the north-central and south-central regions of Mexico, respectively, where a considerable area of agricultural land is occupied by tomato crops. In the summer of 2005, stunting and leaf curling/crumpling symptoms were observed in several tomato (Lycopersicon esculentum L.) fields in Rioverde, San Luis Potosí (Rioverde-SLP). These symptoms and the existence of large populations of whiteflies (Bemisia tabaci Gennadius) in the affected fields suggested a viral etiology. Symptomatic tomato leaves collected during July and September of 2005 from several locations throughout the Rioverde area were assessed for begomovirus presence using polymerase chain reaction (PCR) with three sets of degenerate primers: PAL1v1978/PAR1c496 (3), pCP70for/pCP70rev (1), and two new primers that specifically amplify DNA from viruses of the Squash leaf curl virus (SLCV) lineage, prSL060-for (CGGCGTTRTRRTARACGTCGTC) and prSL150-rev (GCWGCC-AAAGACACCAAYGCCGT). These primers amplify overlapping DNA segments encompassing the complete begomovirus genome A. Amplicons were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. The complete sequence for component A of isolates from two different fields in the Rioverde Valley were assembled and compared with sequences available in the GenBank database using BlastN and the Clustal alignment method (MegAlign, DNASTAR, Madison, WI). The 2588-bp sequence of the Rioverde-SLP1 isolate (Accession No. DQ347946) and the 2594-bp sequence of Rioverde-SLP2 isolate (Accession No. DQ347947) were 97.2% identical. Both field isolates displayed the highest similarity (97.1 and 97.3% nt identity, respectively) with Tomato severe leaf curl virus from Guatemala (ToSLCV-GT96; Accession No. AF130415). Similarity of SLP isolates with Tomato severe leaf curl virus from Nicaragua (Accession Nos. AJ508784 and AJ508785) was significantly lower, 89.9 and 89.7%, respectively. A parallel survey of tomato fields in Xochitepec, Morelos, located 550 km southeast of Rioverde-SLP, was performed during September, 2005. Leaf samples from six plants displaying leaf curling/crumpling symptoms were collected and assessed for begomovirus presence using PCR with the degenerate primers, prC889 (4) and prSL060-for. The 1.4-kb PCR fragments obtained were subsequently analyzed by restriction fragment length polymorphism using MspI and HhaI. Restriction fragment patterns were the same for all amplicons. The 1435-bp DNA A sequence of one isolate from Morelos was determined (Accession No. DQ267157) and compared with sequences available for other begomoviruses using Clustal alignment method. The highest identity (98%) was with ToSLCV-SLP and ToSLCV-GT96 isolates. These data confirm that ToSLCV is infecting tomato in different horticultural regions of Mexico. The presence of this begomovirus has been previously reported in Honduras, Guatemala, and Nicaragua (2). To our knowledge, this is the first report of ToSLCV in Mexico. References: (1) R. De La Torre-Almaraz et al. Plant Dis. 90:378, 2006. (2) M. K. Nakhla et al. Acta Hort. (ISHS) 695:277. Proc. First Int. Symp. on Tomato Diseases. M. T. Momol et al., eds., 2005. (3) M. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

A New Begomovirus Inducing Yellow Mottle in Okra Crops in Mexico is Related to Sida yellow vein virus.

Okra (Abelmoschus esculentus L. Moench), an annual vegetable of African origin, has been cultivated in Mexico for 3 decades. Since 2000, the most important okra-producing areas in the states of Guerrero and Morelos have been affected by a disease causing yellow streak and severe distortion of fruits, a bright yellow mottle, and curling and distortion of leaves. These symptoms and the presence of whiteflies (Bemisia tabaci Gennadius) suggest a viral etiology. Samples of symptomatic plants from three localities, Iguala (Guerrero), Mazatepec, and Xochitepec (Morelos) were collected in November 2004 and tested for the presence of viruses. Single whitefly transmissions, grafting experiments, and experimental inoculation of healthy plants by biolistic delivery of DNA extracts from symptomatic plants consistently induced yellow mottle in okra plants and suggest the presence of a DNA virus. Total DNA extracts from symptomatic plants from field and greenhouse conditions were analyzed by Southern blot hybridization using the coat protein gene of Pepper yellow vein huasteco virus as a probe at low stringency. More than 20 positive samples were subsequently used as templates for polymerase chain reaction (PCR) amplification with the degenerate primers pRepMot and pCPMot (1). PCR products of approximately 600 bp were obtained and directly sequenced. Eight isolates from the three localities (GenBank Accession Nos. AY624016 to AY624023) shared 97 to 100% nucleotide identity but were significantly different from other known begomoviruses. The complete genome A sequence of one isolate from Mazatepec (Ok-M3) was determined using PCR amplification of viral DNA with the degenerate primers PAL1v1978 and PAL1c1960 (3) and four new universal primers, pRepQGR (5'-TCCCTGWATGTTYGGATGGAAATG-3'), pRepQGR-rev (5'-CATTTCCATCCRAACATWCAGGGA-3'), pCp70-MAC (5'-GTC TAGACCTTRCANGGNCCTTCACA-3'), and pCp70-MAC-rev (5'-GAA GGSCCNTGYAAGGTNCAGTC-3'). Partially overlapping PCR products of 0.9, 1.3, and 1.7 kb were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. The 2612-bp DNA-A sequence of Ok-M3 (GenBank Accession No. DQ022611) was compared with sequences available from GenBank using the Clustal alignment method (MegAlign, DNASTAR software, London). The highest sequence identity was obtained with Sida yellow vein virus (SiYVV; Accession No. Y11099), Sida golden mosaic Honduras virus (SiGMHV; Accession No. Y11097), and Chino del tomate virus (CdTV; Accession No. AF101478) that had 85.4, 85.4, and 84.4% nucleotide sequence identity with the Ok-M3 isolate, respectively. Comparative analysis of the intergenic region of the Ok-M3 isolate and its closest relatives revealed that these viruses display different putative Rep-binding sites (iterons): Ok-M3 (GGTACACA), SiYVV (GGAGTA), and SiGMHV (GGKGTA). Current taxonomic criteria for the classification of begomoviruses establishes that less than 89% DNA-A nucleotide sequence identity with the closest relative of a virus is indicative of a separate species (2). Our results indicate that the okra-infecting virus identified in this study is a new begomovirus species, and the provisional name of Okra yellow mottle Mexico virus is proposed. References: (1) J. T. Ascencio-Ibañez et al. Plant Dis. 86:692, 2002. (2) C. Fauquet et al. Arch. Virol. 148:405, 2003. (3) M. Rojas et al. Plant Dis. 77:340, 1993.

First Report of a Geminivirus Inducing Yellow Mottle in Okra (Abelmoschus esculentus) in Mexico.

Okra is an annual vegetable species native to Africa. In Mexico, the states of Tamaulipas, Guerrero, and Morelos contain the most important okra-producing areas. Viral-like diseases have recently affected the fruit production. In the field, the most common symptoms encountered include yellow streak, distortion of fruits, and foliar yellow mottle. Total DNA extracts from symptomatic okra plants were used as a template for polymerase chain reaction (PCR)-based detection using begomovirus-specific primers. RepMot and CPMot primers (1) were used for the amplification of DNA fragments that included the Rep and coat protein (CP) genes of begomoviruses. PCR results suggested the presence of a begomovirus in symptomatic plants. Southern and dot blot hybridization analysis were performed using a DNA fragment containing the CP gene of Pepper huasteco virus as a probe. Hybridization conducted under low stringency conditions confirmed the presence of a geminivirus. Additionally, transmission by grafting and biolistic (total DNA extracts from symptomatic plants) inoculations induced consistently severe streak fruits and yellow mottle symptoms in okra plants. Cloning of the PCR products (approximately 632-bp fragment) was performed in the PCRTopo vector (Invitrogen, San Diego, CA). Cloned viral inserts were sequenced (Genbank Accession No. AF349113). Nucleotide sequence comparisons were performed using the Clustal Method (MegAlign, DNAStar software, Madison, WI) with the GenBank database. Analysis of the PCR products confirmed the begomovirus nature of the sequence. The first 64 amino acids of the CP had 89% identity with Squash leaf curl virus while the intergenic region had 85% identity with Sida golden mosaic virus. To our knowledge, this is the first report of a begomovirus infecting okra in Mexico. Reference: 1) J. T. Ascencio et al. Plant Dis. 86:692, 2002.