A raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA-2.

Sequencing of the complete genome of a raspberry bushy dwarf virus isolate from Rubus glaucus in Ecuador revealed that its RNA-1 and RNA-2 were 5449 and 2231 nucleotides (nt) long, respectively, and phylogenetically closest to isolates from Sweden and Slovenia. In dsRNA analysis of infected plants, an additional band of 3 kbp was observed. Sequencing of this band revealed that it was 3279 nt long. BLAST searches revealed that this band contained a modified version of RNA-2, which consisted of RNA-2 (2231 nt) plus an additional 1048-nt fragment that was concatenated in a reverse-complement fashion to its 5' terminus.

First Report of Bacterial Panicle Blight of Rice Caused by Burkholderia gladioli in Ecuador.

Burkholderia gladioli is one of the causal agents of bacterial panicle blight of rice (BPB). Although B. glumae is considered the main pathogen responsible of BPB, B. gladioli can also cause this disease in rice (3). B. gladioli is also of clinical importance because of the ability of some strains to cause respiratory infections in humans (2). Symptoms in rice plantations of Palestina city, like upright panicles with grayish-straw color, grain rot, and vain grains were observed in July 2013, although similar symptoms were first noticed as early as 2012 in other regions of Ecuador. Since then, similar symptomatology has been reported by farmers in coastal provinces, possibly affecting 75% of the crops. One of the causal agents was recently identified as B. glumae but other bacteria were observed in infected rice (1). Plants showing BPB symptoms were collected from Palestina and bacteria were isolated from panicle twigs using the semi selective SPG agar (KH2PO4 1.3 g, Na2HPO4 1.2 g, (NH4)2SO4 5 g, MgSO4·7H2O 0.25 g, Na2MoO4·2H2O 24 mg, EDTA-Fe 10 mg, L-cystine 10 µg, D-sorbitol 10 g, pheneticillin potassium 50 mg, ampicillin sodium 10 mg, cetrimide 10 mg, methyl violet 1 mg, phenol red 20 mg, agar 15 g/liter distilled water). Colonies were then transferred to PDA. Presumptive B. gladioli colonies were classified into two groups according to their color on PDA. Colonies from group one (six strains) were dull yellow, whereas those from group two (two strains) were olive colored. Both groups produced fluorescent colonies with smooth, shiny surfaces on PDA. All cells were gram-negative rods with the following dimensions: 0.8 to 2.0 × 0.4 to 0.6 µm (group one) or 1.5 to 2.5 × 0.4 to 0.7 µm (group two). All colonies were subjected to biochemical tests (API 20NE) and shared a 99% or higher similarity (APIWEB) with B. gladioli. To confirm identity, genomic DNA was extracted (gDNA extraction kit from Invitrogen) and a portion of the 16s rDNA was amplified by PCR using the primers 536F: 5'-GTGCCAGCMGCCGCGGTAATAC-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' followed by sequencing. Sequences of group one strains shared 100% similarity with B. gladioli strain OM1 (GenBank Accession No. EU678361) while the sequences from group two strains were 100% similar to B. gladioli strain BgHL-01 (JX566503). Sequences of the Ecuadorian strains were deposited into NCBI GenBank (group one: KF669879 to KF669882, KF669884, and KF669885; group two: KF669883 and KF669886). Pathogenicity was confirmed by submerging rice seeds in a cell suspension with 108 CFU of the pathogen for 24 h. Seeds were germinated at 28°C and about 70% RH on autoclaved peat. Inoculated seeds yielded plants with BPB symptoms 6 days after planting. Re-isolated strains shared a 99.9% similarity with B. gladioli by APIWEB. To the best of our knowledge, this is the first report of B. gladioli as a rice pathogen in Ecuador. References: (1) C. Riera-Ruiz et al. Plant Dis. 98:988, 2014. (2) C. Segonds et al. J. Clin. Microbiol. 47:1510, 2009. (3) H. Ura et al. J. Gen. Plant Pathol. 72:98, 2006.

First Report of Burkholderia glumae Causing Bacterial Panicle Blight on Rice in Ecuador.

Rice (Oryza sativa L.) is one of the leading crops and the basis of most diets in Ecuador and other countries. Diseases such as bacterial panicle blight (BPB), also known as seedling rot or grain rot, have the potential to threaten rice production worldwide. Burkholderia glumae, a causal agent of BPB, has severely affected the rice industry in many countries of Africa, Asia, and the Americas (1,2,4), but no report of this bacteria in Ecuador can be found in the literature. Rice plantations showing BPB-like symptoms including upright panicles with stained and vain grains were spotted in Palestina city, one of Ecuador's most extensive rice areas, in July 2013, but similar symptoms have been observed in the region since early 2012. Six symptomatic plants from two different groves were collected. Samples were plated on the semi-selective medium S-PG (KH2PO4 1.3 g, Na2HPO4 1.2 g, (NH4)2SO4 5 g, MgSO4·7H2O 0.25 g, Na2MoO4·2H2O 24 mg, EDTA-Fe 10 mg, L-cystine 10 µg, D-sorbitol 10 g, pheneticillin potassium 50 mg, ampicillin sodium 10 mg, cetrimide 10 mg, methyl violet 1 mg, phenol red 20 mg, agar 15 g/liter distilled water) and axenic colonies were transferred to potato dextrose agar (PDA) to test for fluorescence (3). Colonies of the potential pathogen were 1 mm, circular, entire margin, with a smooth and shiny surface. When cultured in PDA, isolates showed a moist texture, dull yellow color, and displayed fluorescence with exposure to UV light. Cells were bacterial gram-negative rods of 1 to 2 × 0.5 µm. Twelve presumptive isolates were submitted to biochemical tests (API 20NE). The biochemical profile (APIWEB) showed that all the isolates belonged to the Burkholderia genus with a 99.9% similarity. To determine the bacterial species, colonies were submitted to ELISA tests using specific antibodies for B. glumae from Agdia, Inc. The two isolates that were positive for B. glumae were sequenced using a part of the 16s rDNA amplified by the primers 536F: 5'-GTGCCAGCMGCCGCGGTAATAC-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The obtained sequences (deposited into GenBank as KF601202) shared 100% similarity with several B. glumae strains after a BLAST query. Isolates were then diluted to 108 UFC/ml and used to inoculate healthy rice plants. Inoculated plants produced BPB-like symptoms including upright panicles with stained vain grains and the bacterium was re-isolated from symptomatic plants. To the best of our knowledge, this is the first report of B. glumae in Ecuador. Further research is ongoing to identify and determine the pathogenicity of the remaining Burkholderia strains that tested negative for B. glumae. References: (1) J. Luo et al. Plant Dis. 91:1363, 2007. (2) R. Nandakumar et al. Plant Dis. 93:896, 2009. (3) T. Urakami et al. Int. J. Syst. Bacteriol. 44:235, 1994. (4) X.-G. Zhou. Plant Dis. 98:566, 2014.

First Report of Banana bract mosaic virus in 'Cavendish' Banana in Ecuador.

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of bract mosaic disease. The disorder has been considered a serious constraint to banana and plantain production in India and the Philippines, where the virus was first identified (3). To date, the presence of BBrMV has been reported only in a few banana-growing countries in Asia (3). In the Americas, BBrMV has been detected by ELISA tests in Colombia only (1). The efficient spread of BBrMV through aphids and vegetative material increases the quarantine risk and requires strict measures to prevent entrance of the virus to new areas. In Ecuador-the world's number one banana exporter-the banana industry represents the main agricultural income source. Thus, early detection of banana pathogens is a priority. In June of 2012, mosaic symptoms in bracts and bunch distortion of 'Cavendish' banana were observed in a commercial field in the province of Guayas, Ecuador. Leaves from 35 symptomatic plants were tested for Cucumber mosaic virus (CMV), Banana streak virus (BSV), and BBrMV using double antibody sandwich ELISA kits from Adgen (Scotland, UK). Twenty-one plants tested positive for BBrMV but not for CMV or BSV. In order to confirm the ELISA results, fresh or lyophilized leaf extracts were used for immunocapture reverse transcription (IC-RT)-PCR. In addition, total RNA was extracted from the ELISA-positive samples and subjected to RT-PCR. The RT reactions were done using both random and oligo dT primers. Several sets of primers, flanking conserved regions of the virus coat protein (CP), have been used for PCR-detection of BBrMV (2,3,4). The Ecuadorian BBrMV isolate was successfully detected by three primer sets with reported amplification products of 324, 280, and 260 nucleotides long, respectively (3,4). Amplification products of the expected size were purified and sequenced. All the nucleotide sequences obtained from 20 PCR-positive symptomatic plants were 100% identical between each other. However, 99% identity was observed when PCR products from the Ecuadorian isolate were compared with the corresponding fragment of a BBrMV isolate from the Philippines (NCBI Accession No. DQ851496.1). PCR products of the Ecuadorian isolate, amplified by the different CP primers described above, were assembled into a 408-bp fragment and deposited in the NCBI GenBank (KC247746). Further testing confirmed the presence of BBrMV in symptomatic plants from four different provinces. To our knowledge, this is the first report of BBrMV in Ecuador and the first BBrMV partial nucleotide sequence reported from the Americas. It is worth mentioning that primer set Bract 1/Bract 2, which amplifies a 604-bp product (2), was not effective in detecting the Ecuadorian isolate. It is hypothesized that nucleotide variation at the reverse primer site is the cause of the lack of amplification with this primer set, since the forward primer is part of the sequenced product and no variation was found. Sequencing of the entire CP region is underway to conduct phylogenetic analysis and determine genetic relationships across several other BBrMV isolates. References: (1) J. J. Alarcon et al. Agron 14:65, 2006. (2) M. F. Bateson and J. L. Dale. Arch. Virol 140:515, 1995. (3) E. M. Dassanayake. Ann. Sri Lanka Dept. Agric. 3:19, 2001. (4) M. L. Iskra-Caruana et al. J. Virol. Methods 153:223, 2008.

First Report of Raspberry bushy dwarf virus in Andean Blackberry (Rubus glaucus) in Central Ecuador.

During the past two decades, several viruses have been identified from Rubus spp. in wild and commercial plantings around the world (2). In Ecuador, approximately 14 tons of blackberries are produced each year from an estimated area of 5,500 ha. In 2012, a preliminary survey was conducted to determine the presence of RNA viruses in Rubus glaucus, the most prevalent blackberry in Ecuador. Fifteen plants showing leaf mottling and severe mosaic were leaf-sampled from each of five different fields in Azuay Province. A total of 12 pooled samples of 20 g were obtained from the collected symptomatic tissue and used for dsRNA extraction using a cellulose-based protocol for detection of RNA viruses in plants (3). Three dsRNA segments of approximately 5 kbp, 2 kbp, and 900 bp were observed from all 12 dsRNA preparations. The dsRNA was heat-denatured and used as template for the generation of cDNA library using the universal random primer 5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3', for reverse transcription (RT), and the anchor primer 5'-GCCGGAGCTCTGCAGAATTC-3'for PCR as described (1). The PCR products were cloned using a StrataClone Kit (Agilent, CA) and sequenced (Macrogen, Korea). Sequence analysis revealed the presence of Raspberry bushy dwarf virus (RBDV), a pollen-borne Idaeovirus naturally found in several Rubus spp. worldwide. Approximately 120 RBDV sequences obtained from the Ecuadorean isolate were assembled into two contigs belonging to RNA1 and RNA2. Both sequences were re-confirmed by RT-PCR using specific primers. Partial sequences were assigned GenBank Accessions KC315894, KC315893, and KC315892 for the replicase, MP and CP, respectively. Furthermore, BLAST searches showed that the nucleotide sequence corresponding to the replicase was 95% similar to an isolate from the resistance breaking R15 strain (S51557.1), whereas the MP and CP nucleotide sequences were up to 98% similar to a Slovenian isolate (EU796088.1). Primers designed to amplify a 427-bp portion of the CP were used to detect RBDV from four blackberry plantings in two distant production areas: Ambato in Tungurahua Province and Paute in Azuay Province. Leaf mottling and severe mosaic was observed in 90% of blackberry fields in those two locations. Leaf samples (n = 90) were randomly collected from both symptomatic and asymptomatic plants in each location. In Ambato, RBDV was detected in 50% and 40% of symptomatic and asymptomatic plants, respectively. In Paute, RBDV was present in 70% of symptomatic plants and 29% of asymptomatic plants. The presence of RBDV in asymptomatic plants suggests the virus might not be the sole causal agent of the disorder. Further studies are needed to determine the role of RBDV in the observed symptoms, since virus complexes responsible for increased severity of symptoms have been commonly reported in Rubus spp. (4). R. glaucus is native to the tropical highlands (from Ecuador to Mexico) and differs from blackberries commercially grown in the United States and Europe. Therefore, RBDV-induced symptoms reported in blackberry grown in the United States and Europe may not be extrapolated to the Andes berry. To the best of our knowledge, this is the first report of RBDV from blackberry in Ecuador. References: (1) P. Froussard. Nucleic Acids Res. 20:2900, 1992. (2) R. R. Martin et al. Plant Dis. 97:168, 2013. (3). T. J. Morris and J. A. Dodds. Phytopathology 69:854. 1979. (4) D. F. Quito-Avila et al. J. Virol. Methods 179:38, 2012.

Xanthomonas albilineans Haplotype B Responsible for a Recent Sugarcane Leaf Scald Disease Outbreak in Cuba.

Over the past 3 years, the incidence of sugarcane leaf scald disease (LSD) caused by Xanthomonas albilineans has increased at alarming rates in some Caribbean countries. LSD was in latent phase since 1978, when the disease was reported in Cuba, until February 1998 when typical symptoms were observed in germ plasm collections and in some commercial plantings. More than 150 bacterial isolates from different sugarcane varieties and from different localities were isolated on Wilbrink agar medium and characterized. All isolates had shown similar cultural and biochemical patterns. However, serological differences between isolates from the recent outbreak and the ones obtained prior to 1998 were detected by indirect ELISA testing. Differences between Cuban isolates obtained prior to 1998 and those from the recent outbreak were confirmed by analysis of repetitive DNA sequences dispersed throughout the genome. According to the pattern obtained, the newer isolates were similar to reference strains classified as haplotype B by pulsed field gel electrophoresis (1). It is concluded that the recent outbreak of LSD was caused by a strain different than the ones previously detected in Cuba. Reference: (1) M. J. Davis et al. Phytopathology 87:316, 1997.

First Report of Virus and Phytoplasma Pathogens Associated with Yellow Leaf Syndrome of Sugarcane in Cuba.

Yellow leaf syndrome (YLS) has been seen recently in sugarcane (Saccharum sp.) in Cuba. The primary symptom is a yellow discoloration of the midrib that may spread from the midrib to the lamina in cane 6 months and older. In certain cultivars, such as CP 5243, EPC 17-395, and F31-156, a reddish coloration has been observed. In severe cases, plants are stunted and can be pulled easily. YLS was first reported from Hawaii, followed by Brazil, Florida, and Australia, where it is associated with a luteovirus: sugarcane yellow leaf virus (ScYLV). However, in South Africa, YLS is associated with a phytoplasma: sugarcane yellow leaf phytoplasma (ScYLP) (1). A survey performed in Jovellanos, Matanzas, Cuba, for ScYLV, using enzyme-linked immunosorbent assay with antiserum provided by B. E. L. Lockhart, showed that only a small percentage of canes with YLS carried the virus. A nested polymerase chain reaction (PCR) (1) was used to amplify phytoplasma 16S/23S rDNA from sugarcane leaves with YLS symptoms, also collected from Jovellanos. Restriction fragment length polymorphism analysis with HaeIII, RsaI, and AluI produced patterns similar to those of members of the aster yellows group for 260 of 277 samples tested. Sequencing of the 16S/23S intergenic rDNA PCR products, followed by BLAST (basic local alignment search tool) analysis, confirmed the high homology (97%) of these amplimers to the DNA of phytoplasmas belonging to the aster yellows I-A subgroup. This is the first report of ScYLV and ScYLP from Cuba, and it demonstrates the difficulty of determining the identity of the YLS pathogen based on symptoms alone. Reference: (1) C. P. R. Cronjé et al. Ann. Appl. Biol. 133:177, 1998.

Havana tomato virus, a new bipartite geminivirus infecting tomatoes in Cuba.

The cloning and nucleotide sequence of a new bipartite geminivirus found in Cuba is described. DNA A (2620 nt) and DNA B (2586 nt) presented a genomic structure resembling that of other geminiviruses transmitted by Bemisia tabaci. Both components had a common region of 168 nt with a 95% identity. Typical elements involved in replication and transcription were found in this region, though group-characteristic arrangement of iterons was not conserved in this virus. Sequence was compared with geminivirus sequences deposited in the GenBank. Interestingly, when total DNAs or individual ORFs and deduced amino acid sequences were compared, the highest scores were for different viruses. It showed to be most closely related to tomato mottle virus (81.9% and 65.5% similarity with DNAs A and B, respectively) and a member of the abutilon mosaic cluster of New World Begomoviruses. When clones A and B were co-agroinoculated they resulted highly infectious and induced symptoms in Nicotiana benthamiana plants. The A component alone was infectious but induced only mild symptoms, while the B component was not infectious. The presence of viral DNA in N. benthamiana plants was confirmed by dot-blot hybridization using specific probes. These data show that the cuban isolate is a new geminivirus for which the name of Havana tomato virus is proposed.

A Bipartite Geminivirus Infecting Tomatoes in Cuba.

Whitefly-transmitted geminiviruses have increased in their distribution and importance during the last decade, apparently due to a wider distribution of their vector Bemisia tabaci (Gennadius). In Cuba, symptoms of geminivirus disease have been observed since the early 1990s. A survey of geminivirus-caused diseases in tomato plants has been conducted since 1994 in the province of La Habana. The most severe symptoms were observed in the commercial varieties Campbell 28, Criollo Quivican, and HC 3880. In some plants the Israeli strain of tomato yellow leaf curl geminivirus (TYLCV) was detected (1). In other cases, a viral DNA with a genome size smaller than that of TYLCV that could not be amplified with primers specific for the Israeli strain was also detected by hybridization. In these samples a polymerase chain reaction amplification with broad-spectrum, degenerate primers designed for whitefly-transmitted bipartite geminivirus (2) was carried out. Two fragments of about 1.1 and 0.6 kb, corresponding to components A and B, respectively, were amplified, indicating that a bipartite geminivirus was present. The 1.1-kb fragment amplified from a sample showing strong leaf yellowing and slight curling was cloned and sequenced. A sequence of 1,174 nucleotides was obtained and compared with geminivirus sequences deposited in the GenBank, by multiple alignment (CLUSTAL W, European Bioinformatics Institute, Hinxton Hall, UK). Genetic distances were calculated by Kimura's two parameters method. Percentages of similarities obtained with the sequence were as follows: tomato mottle virus 78%, abutilon mosaic virus 73.8%, and tomato golden mosaic 61.8%. Partial sequences of the open reading frames AL1 and AR1 and the intergenic region were present. Percentages of similarities obtained with the intergenic region were as follows: tomato mottle virus 78.1%, sida golden mosaic virus 63%, bean dwarf mosaic virus 62.6%, abutilon mosaic virus 57.1%, and tomato golden mosaic virus 32%. These results suggest that we have detected in Cuban tomatoes a new bipartite geminivirus related to tomato mottle virus, and we propose that it be named Havana tomato virus. Frequency of distribution of viruses in commercial varieties and their relationship with the presence of whiteflies are currently being studied. References: (1) Y. Martinez et al. J. Phytopathol. 144:277, 1996. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.