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.