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Next generation sequencing has been used to identify and characterize the full genome sequence of a cassava-infecting torradovirus, revealing the presence of a Maf/HAM1 domain downstream of the RNA-dependent RNA-polymerase (RdRp) domain in RNA1 in all isolates sequenced. A similar domain is also found in unrelated potyvirids infecting Euphorbiaceae hosts in the Americas and cassava in Africa. Even though cassava torrado-like virus (CsTLV) could not be mechanically transmitted to a series of herbaceous hosts, it can be efficiently transmitted by bud graft-inoculation to different cassava landraces. Our bioassays show that CsTLV has a narrow host range. Crystal-like structures of isometric virus-like particles were observed in cells of plants with single infection by CsTLV, and consistently induced chlorotic leaf spots and affected root yields significantly. Moreover, CsTLV infection induces changes in the accumulation of total sugars in storage roots. Field surveys indicated the presence of CsTLV in the main cassava growing regions of Colombia, and the occurrence of two different cassava-infecting torradovirus species. Profiles of small RNAs of 21 to 24 nucleotides in length, derived from CsTLV RNAs targeted by cassava RNA silencing defense mechanisms, are also reported.

Wheat blast (WB), caused by Magnaporthe oryzae Triticum pathotype, recently emerged as a destructive disease that threatens global wheat production. Because few sources of genetic resistance have been identified in wheat, genetic transformation of wheat with rice blast resistance genes could expand resistance to WB. We evaluated the presence/absence of homologs of rice blast effector genes in Triticum isolates with the aim of identifying avirulence genes in field populations whose cognate rice resistance genes could potentially confer resistance to WB. We also assessed presence of the wheat pathogen AVR-Rmg8 gene and identified new alleles. A total of 102 isolates collected in Brazil, Bolivia, and Paraguay from 1986 to 2018 were evaluated by PCR using 21 pairs of gene-specific primers. Effector gene composition was highly variable, with homologs to AvrPiz-t , AVR-Pi9 , AVR-Pi54 , and ACE1 showing the highest amplification frequencies (>94%). We identified Triticum isolates with a functional AvrPiz-t homolog that triggers Piz-t -mediated resistance in the rice pathosystem and produced transgenic wheat plants expressing the rice Piz-t gene. Seedlings and heads of the transgenic lines were challenged with isolate T25 carrying functional AvrPiz-t. Although slight decreases in the percentage of diseased spikelets and leaf area infected were observed in two transgenic lines, our results indicated that Piz-t did not confer useful WB resistance. Monitoring of avirulence genes in populations is fundamental to identifying effective resistance genes for incorporation into wheat by conventional breeding or transgenesis. Based on avirulence gene distributions, rice resistance genes Pi9 and Pi54 might be candidates for future studies.

Burkholderia glumae is responsible for the panicle blight disease of rice. This disease is present worldwide and can result in significant drop in yields. To estimate the genetic diversity of the bacterial strains present in a rice paddy field in Colombia, we sampled 109 strains from infected panicles. To detect fine genetic relationships among related haplotypes, and to overcome a very low nucleotide diversity detected in previous studies, we designed primers to amplify and sequence several highly variable minisatellite loci, or variable number tandem repeats (VNTRs), as well as part of the Toxoflavin toxA gene in all strains. Results show that the toxA nucleotide diversity defined four lineages and was similar to that detected in several fields in Japan; data suggest that B . glumae has spread from Asia to America without major loss of genetic diversity, and that five VNTR loci discriminated the strains within the field revealing single and multi-infections of the rice panicles with a wide distribution of the haplotypes among the different plots. Even though disease levels vary considerably from year to year, the bacterial genetic diversity is maintained within a field. We do not detect any geographical structuring within the field, nor any effect of the rice cultivar on the observed diversity. The consequences on the origin and evolution of the bacteria are discussed.

Cassava ( Manihot esculenta Crantz) has been traditionally grown as a subsistence crop in Laos, but in recent years cassava cultivation in this country has expanded and is becoming a 'cash crop' for farmers (Malik et al., 2020). This also means that cassava vegetative seed (stakes) is rapidly multiplied and distributed. One of the most important diseases affecting cassava in the world is the Cassava Mosaic Disease (CMD), caused by several species of begomoviruses and disseminated by infected stakes or vectored by the whitefly Bemisia tabaci (Legg et al., 2014). Sri Lankan cassava mosaic virus (SLCMV), a bipartite begomovirus, is the virus species causing CMD in Southeast Asia (SEA) and is widespread in Cambodia, Vietnam, Thailand and south China (Siriwan et al., 2020). During field surveys on July 12 to 14, 2020, the team in south Laos, surveyed 8 fields along the border with Cambodia, in the southern provinces of Attapeu and Champassack and identified CMD symptoms (Supplementary Figure 1A) in only one of the fields, located at Kong District of the Champassack province (GPS coordinates 13.94325, 105.99102). From these 8 fields, samples were collected from every third plant in an X pattern. Photographs from each sampled plant were taken and uploaded into CIAT's PestDisPlace platform (https://pestdisplace.org), for CMD symptom confirmation (Supplementary Figure 1B). Leaf samples were sent to the laboratory for PCR using primers SLCMV-F 5'-ATGTCGAAGCGACCAGCAGATATAAT-3' and SLCMV-R 5'-TTAATTGCTGACCGAATCGTAGAAG-3' targeting the AV1 gene (Dutt et al., 2005), following the protocol described in Siriwan et al. (2020) and primers SLCMV-B-F1 5'-ACCGGATGGCCGCGCCCCCCTCT-3' and SLCMV-B-606R 5'-CACCTACCCTGTTATCGCTAAG-3' targeting part of the BV1 gene. Out of 60 samples collected for the field in Kong district, eleven (18.3%) resulted PCR positive to SLCMV (to DNA-A and DNA-B) but only four plants (6.7%) showed symptoms of CMD (see Supplementary Figure 1B and 1C). None of the samples in the other seven fields had CMD symptoms nor was SLCMV detected in any of these plants. Furthermore, the presence of CMD symptoms in the old leaves of the plants in the affected field suggests that the virus was introduced with contaminated stakes. The complete bipartite genome of one isolate (Champ1), was amplified by Rolling Circle Amplification and sequenced with the nanopore MinION technology as described by Leiva et al. (2020). The sequences were submitted to GenBank under accession nos MT946533 (DNA-A) and MT946534 (DNA-B). A phylogenetic tree for SLCMV and a link to the open SLCMV Nextstrain map (Hadfield et al., 2018) is included in Supplementary Figure 2. The sequences of the DNA-A and DNA-B components of the Champ1 isolate were nearly identical to those of anisolate of SLCMV from Ratanakiri, Cambodia (99.72% for DNA-A and 99.82 for DNA-B; Wang et al., 2016). Phylogenetic analysis (Supplementary Figure 2), grouped isolate Champ1 with those that form the cluster of SEA isolates that contain the shorter version of the rep gene (Siriwan et al., 2020). This short version of rep present a deletion of 7 amino acids at the C-terminus, which is involved in host responses to SLCMV (Wang et al., 2020). The confirmation of CMD and SLCMV in the border between Laos and Cambodia should be followed by disease containment and management strategies, particularly given that the majority cassava varieties grown in Laos are from neighbor countries, most of which have already reported the presence of CMD. Acknowledgements We thank all staff from the CIAT's Cassava Program and the Plant Protection Center of Laos in Vientiane. We acknowledge financial support from the Australian Centre for International Agricultural Research (ACIAR) and the CGIAR Research Program on Roots, Tubers and Bananas (RTB) (https://www.cgiar.org/funders/).

Over the last 5 years, Southern blight caused by Sclerotium rolfsii Sacc. has superseded root rots caused by pathogens such as Fusarium and Pythium spp. as a major constraint of common bean ( Phaseolus vulgaris L.) production in Uganda. Although S. rolfsii is prevalent in all bean-growing regions of Uganda, there is a lack of information about its diversity and pathogenicity to guide the development of effective management strategies. In total, 348 S. rolfsii isolates were obtained from bean fields in seven agroecological zones of Uganda, with the following objectives: to characterize their morphology, based on mycelial growth rate, mycelium texture, and number of sclerotia; and to determine the pathogenicity of 75 selected isolates on five common bean varieties in artificially inoculated soils in a screenhouse. We found that mycelial growth rate and the number of sclerotia produced on artificial media varied among agroecological zones but not within a zone. The five bean varieties tested were found to be susceptible to S. rolfsii , including varieties MLB49-89A and RWR719 that are resistant to Fusarium and Pythium root rots, respectively. Preemergence damping-off ranged between 0 and 100%, and disease severity index ranged between 4.4 and 100%. The widespread and high levels of S. rolfsii virulence on varieties of common bean indicate that management intervention is urgently required to help reduce losses incurred by Ugandan smallholder farmers.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Common guava (Psidium guajava L.) is a fruit tree of global economic importance. It is grown in Asia, South and Central America, and Hawaii for its exquisite aroma and flavor, and nutritional and medical properties. However, guava production is limited by guava scab, caused by fungi in the Pestalotiopsis genus. Characteristic symptoms of guava scab are corky, ovoid or round lesions on fruit surfaces. These lesions may thicken, affecting the flesh below and reducing fruit quality and commercial value. We characterized 81 isolates isolated from guava scab lesions on guava leaves and fruit in different regions of Colombia, and identified them as Pestalotiopsis and Neopestalotiopsis spp. We analyzed the morphology, pathogenicity, and genetic diversity of the isolates based on the sequences of the internal transcribed spacer, β-tubulin, and elongation factor genes. Isolates were morphologically, pathogenically, and genetically diverse but the diversity did not correlate with geographical origin, or guava cultivar or tissue from which the isolates were recovered. Selected monosporic isolates included in the multiple-gene analysis were identified as belonging to two genera: Neopestalotiopsis (65 isolates with versicolorous conidia) and Pestalotiopsis (4 isolates with concolorous conidia).

Dry bean (Phaseolus vulgaris L.) is a globally important leguminous food crop. Yields can be reduced by high incidence of soilborne oomycetes that cause seedling disease. Breeders have attempted to develop Pythium root rot-resistant bean varieties; however, relationships between dry bean and most soilborne oomycete species remain uncharacterized. Oomycete species (n = 28), including Pythium spp. and Phytopythium spp., were tested in a growth chamber seedling assay at 20°C and an in vitro seed assay at 20°C and 26°C to evaluate their pathogenicity and virulence on 'Red Hawk' dark red kidney bean and 'Zorro' black bean. Root size or disease severity was significantly impacted by 14 oomycete species, though results varied by bean variety, temperature, and assay. Of these 14 pathogenic oomycete species, 11 species exhibited significant differences in DSI due to temperature on at least one bean variety. Pythium aphanidermatum, P. myriotylum, P. ultimum, P. ultimum var. sporangiiferium, and P. ultimum var. ultimum were the most virulent species in both assays, causing seed rot and pre-emergence damping-off of dry bean. Oomycete species were clustered into three groups based on symptom development: seed rot pathogens, root rot pathogens, or nonpathogens. Intraspecific variability in virulence was observed for eight of the 14 pathogenic oomycete species. Improved understanding of Pythium and Phytopythium interactions with dry bean may enable breeders and pathologists to more effectively evaluate strategies for oomycete seedling disease management.

Rhizoctonia foliar blight, caused by Rhizoctonia solani, is an important disease of Brachiaria spp. in tropical America. Host-plant resistance is an attractive option for disease management. In this study, we evaluated three inoculum types (mycelium-infected agar disc, microdiscs suspensions, and microencapsulated-mycelium suspensions) in order to identify a rapid and accurate method for mass screening of Brachiaria genotypes for resistance to Rhizoctonia spp. in greenhouse trials. Visual damage score, area under the disease progress curve, and percent chlorophyll loss were estimated to determine the most accurate and precise method for evaluating Rhizoctonia resistance. The microencapsulated-mycelium solution (0.75 g/ml in potato dextrose broth sprayed on plants 30 days after planting) caused greater foliar damage than the other inoculum types and allowed effective discrimination between resistant and susceptible genotypes. The effectiveness of spray-applied, microencapsulated-mycelium was further corroborated by the evaluation of 350 genotypes not previously selected for resistance to Rhizoctonia spp., which varied significantly in their reaction to R. solani. The microencapsulated-mycelium methodology has several advantages over existing methods, including its high-throughput capacity, efficient use of time and space, ease of quantification of inoculum, and consistent results over replicate trials. This methodology could be applied to assess resistance to Rhizoctonia spp. in other crops.

Five Andean blackberry Rubus genotypes, three resistant and two susceptible to anthracnose, were used to identify regions in the Rubus genome with homology to disease-resistance genes found in other plant species. Polymerase chain reaction amplification with 12 pairs of primers and fragment cloning yielded 520 clones, of which 151 showed inserts between 500 and 700 bp long. When sequenced, 47 clones showed homology with two types of resistance genes, non-Toll/interleukin-1 receptor (TIR) nucleotide binding site (NBS) leucine-rich repeat (LRR) and TIR-NBS-LRR, thereby confirming their designation as resistance gene analogs (RGAs). The number of RGAs detected per Rubus genotype ranged from 7 to 11, with the highest in a wild resistant and a cultivated susceptible genotype. Rubus RGAs were also homologous with several non-TIR- and TIR-type RGAs found in other members of the Rosaceae family (Rosa hybrid cultivar, Rosa roxburghii, Malus × domestica, M. prunifolia, M. baccata, M. floribunda, Pyrus communis, Prunus persica, P. kansuensis, P. avium, and Fragaria vesca). Three RGAs shared identity with two Rosaceae RGAs associated with the CRPM1 locus for powdery mildew resistance in R. roxburghii and the Rosa hybrid cultivar. This is the first report on RGAs present in the Andean blackberry in Colombia.

Since 1992, anthracnose of Andean blackberry (Rubus glaucus) has generated losses as high as 40% for farmers in Colombia. In this study, our goal was to characterize 240 Colletotrichum isolates from Andean blackberry in eight areas of Colombia. These isolates were evaluated according to morphological characteristics, sensitivity to benomyl, pathogenicity, and genetic variability. Identification of the genus Colletotrichum was achieved by using species complex-specific polymerase chain reaction primers. A multilocus phylogeny approach was used to identify isolates to the species level with sequences from the ribosomal internal transcribed spacer region and partial sequences of the actin, β-tubulin 2, calmodulin, chitin synthase 1, glutamine synthetase, and glyceraldehyde-3-phosphate dehydrogenase genes. Most of the isolates were identified as Colletotrichum gloeosporioides sensu lato, were associated with the Castilla ecotype, showed high sensitivity to benomyl, and were highly aggressive. Isolates identified as C. acutatum sensu lato were found mainly on the Thornless ecotype, were highly resistant to benomyl, and showed intermediate aggressiveness. Only three isolates were identified as C. boninense sensu lato. The species identified included C. fructicola, C. kahawae subsp. ciggaro, C. godetiae, C. karstii, C. brassicicola, and undetermined Colletotrichum spp. This study is the first report of these species associated with anthracnose in Andean blackberry.

Rhizoctonia foliar blight, caused by Rhizoctonia solani anastomosis group 1, is an economically important fungal disease found throughout the world. The fungus attacks numerous crops, including cereals, roots and tubers, legumes, and cruciferous, horticultural, and ornamental plants. In tropical America, this invasive and destructive disease also attacks most Brachiaria spp. used as forages in the ranching industry, especially in the production of cattle. Research to solve this constraint has been ongoing at the International Center for Tropical Agriculture and has generated new Brachiaria hybrids with excellent agronomic performance, tolerance to poor soils, and, particularly, high resistance to biotic factors such as Rhizoctonia foliar blight. These hybrids belong to lines obtained from Brachiaria humidicola, B. brizantha, and B. decumbens. To identify resistance among Brachiaria hybrid genotypes, the hybrid clones were evaluated for their variability in resistance, and their disease reaction was also determined and characterized. Results led to the identification of hybrids that not only were highly resistant to the blight but also had excellent agronomic characteristics.

The distribution of lethal wilt, a severe disease of oil palm, is spreading throughout South America. An incidence of about 30% was recorded in four commercial fields in Colombia. In this study, phytoplasmas were detected in symptomatic oil palm by using specific primers, based on 16S ribosomal DNA (rDNA) sequences, in nested polymerase chain reaction assays. The phytoplasmas were then identified as 'Candidatus Phytoplasma asteris', ribosomal subgroup 16SrI-B, through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing. Cloning and sequencing of 16S rDNA from selected strains, together with phylogenetic analysis, confirmed the classification. Moreover, collective RFLP characterization of the groEL, amp, and rp genes, together with sequence data, distinguished the aster yellows strain detected in Colombian oil-palm samples from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.

Cassava (Manihot esculenta Crantz) is a major staple crop in developing countries and a large source of raw material for industrial purposes as flour, starch, and ethanol. In July 2012, 24 cassava genotypes (corresponding to 1.85% of the accessions) with typical symptoms of frogskin disease (CFSD) were observed in one of the maintenance areas of the Brazilian Cassava Germplasm (located at Embrapa Cassava & Fruits, Cruz das Almas, Bahia State, Brazil). All diseased plants were asymptomatic on the aboveground parts (leaves and stem). However, for accessions BGM 880, BGM 1094, BGM 1100, BGM 1212, BGM 1218, and BGM 1526, all roots showed a woody appearance, thickened cork-like peel with opaque aspect, and coalescent lip-like slits in a honeycomb pattern. Based on literature description, two pathogens could be associated with CFSD: a dsRNA virus (belonging to family Reoviridae) and a 16SrIII-L phytoplasma (1). To investigate the presence of phytoplasma associated with the CFSD symptoms, total DNA was extracted from 0.5 g of root tissue collected from both symptomatic and asymptomatic roots by scratching the secondary vessel at the center of the cassava root with a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/Tint followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.2 kb in length, for the final reaction (4). No phytoplasma was detected in asymptomatic cassava roots that were sampled from the same field. A posterior extraction of total RNA was made but no dsRNA was noticed on the agarose gel, and reaction of RT-PCR with specific primers (2) had no amplification. In order to characterize the strains, the 1.2-kb amplicon was digested with BamHI, MseI, MspI, KpnI, and TaqI endonucleases. The resulting patterns indicated that the symptomatic accessions were infected with a phytoplasma belonging to the 16SrIII group, sharing similarities with pseudo gel mapping from the reference strain of Peach X-Disease Phytoplasma (GenBank Accession No. L33733). Nested PCR products from accessions BGM 1526 and BGM 1212 were purified and sequenced by Macrogen, (Seoul, South Korea) in both directions, manually edited, and the consensus sequences were deposited in the NCBI database (GenBank Accession Nos. KF019184 and KF019185). Phylogenetic studies were conducted based on maximum parsimony, neighbor-joining, and maximum likelihood analysis for 16S rRNA. The phytoplasma 16S rRNA gene sequences from both strains had 99% identity (P < 0.0001) with the 16SrIII-L CFSD phytoplasma (EU346761 and AY737647), described by Alvarez et al. (1) infecting cassava in Colombia. To our knowledge, this is the first report of a phytoplasma associated with Cassava Frogskin Disease in Brazil, where only the dsRNA virus was recognized as causing this symptom (3). This is not likely to be an isolated case, and possibly more cassava plants are infected with this phytoplasma in Brazil. Due to the difficulties to observe the symptoms at the field level, this could be an emerging disease in that country. References: (1) E. Alvarez et al. Plant. Dis. 93:1139, 2009. (2) L. A. Calvert et al. J. Phytopathol. 156:647, 2008. (3) L. S. Poltroniere et al. Comun. Tec., Belem-PA. 006:2p, 1999. (4) C. D. Smart et al. Appl. Environ. Microb. 62:2988, 1996.

Up to 50% of Brachiaria production in the tropics is affected by foliar blight caused by Rhizoctonia spp. Monothallic isolates of Rhizoctonia (n = 147) were cultured from different Brachiaria genotypes in Colombia and morphologically characterized and evaluated in pathogenicity trials in the greenhouse. Based on restriction fragment length polymorphism of the internal transcribed spacer region, 101 of the isolates were identified as Rhizoctonia solani anastomosis group (AG)-1 IA and were multinucleated, with high growth rate, brown mycelium, and high virulence; and 46 isolates were identified as Rhizoctonia sp. AG-D and were binucleated, with low growth rate, white mycelium, and lower virulence on the Brachiaria genotypes tested. The Rhizoctonia isolates also showed variation according to geographic origin, with R. solani AG-1 IA prevalent in warm lowland areas and Rhizoctonia sp. AG-D occurring in cooler areas. Ten Brachiaria genotypes were challenged with different Rhizoctonia isolates and resistant reactions were seen in three of these genotypes, including Brachiaria hybrid (International Center for Tropical Agriculture [CIAT] 36062), Brachiaria brizantha 'Marandú' (CIAT 6294), and Brachiaria hybrid 'Mulato II' (CIAT 36087). These results will contribute to a greater understanding of the interaction of diverse Rhizoctonia isolates on different Brachiaria genotypes, supporting improvement of Brachiaria spp. for disease resistance.

The causal agent of Ascochyta blight disease of chickpea (Cicer arietinum L.) is highly variable because of the presence of a sexual phase (Didymella rabiei). There is also selection pressure on the pathogen due to wide adoption of improved resistant chickpea cultivars in some countries. The pathogen is able to produce pathotypes with specific virulence on particular cultivars. Three pathotypes, I, II, and III, have been reported (3). In this study, we confirmed the presence of a new and highly virulent pathotype that we designate as pathotype IV. To test the pathogenicity of the isolates collected and maintained at ICARDA, 10 isolates representing a wide spectrum of pathogenic variation, including those classified by S. M. Udupa et al. (3) and a putatively identified more virulent type, which was collected from a chickpea production field in the Kaljebrine area, Syria, were inoculated onto a set of differential chickpea genotypes. The differential genotypes, ILC 1929, ILC 482, ILC 3279, and ICC 12004, were sown in individual 10-cm-diameter pots containing potting mix and arranged in a randomized block design with three replications in a plastic house maintained at 18 to 20°C. Each differential genotype was inoculated individually with the 10 isolates following the methodology of S. M. Udupa et al. (3). DNA was extracted from single-spored isolates to compare the genotypes of the isolates using three simple sequence repeat (SSR) markers (ArA03T, ArH05T, and ArH06T) (2) and to determine the frequency of mating types (MAT) through the use of MAT-specific PCR primers for MAT1-1 and MAT1-2 (1). Host genotype reactions were measured on a 1 to 9 rating scale (1 = resistant and 9 = plant death). On the basis of the pathogenicity tests, the isolates were classified into four pathotypes: I (least virulent, killed ILC 1929 but not ILC 482, ILC 3279, or ICC12004); II (virulent, killed ILC 1929 and ILC 482 but not ILC 3279 or ICC12004); III (more virulent, killed ILC 1929, ILC 482, and ILC 3279 but not ICC12004); and IV (highly virulent, killed all four host differentials). Of 10 single-spore isolates tested, four showed similar disease reactions unique to pathotype I, four revealed pathotype II reactions, and one isolate each behaved like pathotype III or pathotype IV. SSR fingerprinting of these isolates provided evidence for genetic diversity since SSR ArH05T was highly polymorphic and amplified five bands, including pathotypes III- and IV-specific bands, which need further investigation to discern if this locus has any role to play in the virulence. MAT-type analysis showed that seven isolates were MAT1-1 while the remaining three isolates were MAT1-2. Only pathotype I showed the profile of MAT1-2 and the other three pathotypes were MAT1-1. Initially, a number of chickpea wild relatives were screened to identify sources of resistance to pathotype IV, but none of the accessions tested showed resistance. However, efforts are underway to combine minor and major gene(s) available in the breeding program in addition to a further search of the wild gene pools to control pathotype IV. References: (1) M. P. Barve et al. Fungal Genet. Biol. 39:151, 2003. (2) J. Geistlinger et al. Mol. Ecol. 9:1939, 2000. (3) S.M. Udupa et al. Theor. Appl. Genet. 97:299, 1998.

Bemisia tabaci biotype B is considered to be the primary vector of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus). However, Trialeurodes abutiloneus also has been shown to transmit SPCSV in a semipersistent manner. Mixed infection of SPCSV with the aphid-transmitted Sweet potato feathery mottle virus (SPFMV, Potyvirus) causes sweetpotato (Ipomoea batatas) virus disease (SPVD), the major virus disease affecting this crop. High populations of B. afer sensu lato are seasonally associated with sweetpotato in Peru during times of low B. tabaci incidence. The transmission of SPCSV (in single and double infection with SPFMV) by laboratory-reared B. afer sensu lato and B. tabaci biotype B was investigated. For SPCSV transmission efficiency, individual adult insects were allowed 48 h for acquisition and inoculation access periods at both 20 and 25°C. SPCSV was transmitted by both whiteflies, with similar transmission efficiency when the virus was acquired from plants singly infected by SPCSV or doubly infected with SPCSV and SPFMV, at 20 and 25°C. We conclude that B. afer sensu lato is a newly identified vector of SPCSV. This finding may have important epidemiological significance for the spread of SPCSV and SPVD.

Cassava frogskin disease (CFSD) is an economically important root disease of cassava (Manihot esculenta) in Colombia and other South American countries, including Brazil, Venezuela, Peru, Costa Rica, and Panama. The roots of severely affected plants are thin, making them unsuitable for consumption. In Colombia, phytoplasma infections were confirmed in 35 of 39 genotypes exhibiting mild or severe CFSD symptoms either by direct or nested polymerase chain reaction (PCR) assays employing ribosomal (r)RNA operon primer pairs. The CFSD-associated phytoplasmas were identified as group 16SrIII strains by restriction fragment length polymorphism (RFLP) and sequence analyses of amplified rDNA products, and results were corroborated by PCRs employing group 16SrIII-specific rRNA gene or ribosomal protein (rp) gene primers. Collectively, RFLP analyses indicated that CFSD strains differed from all phytoplasmas described previously in group 16SrIII and, on this basis, the strains were tentatively assigned to new ribosomal and ribosomal protein subgroups 16SrIII-L and rpIII-H, respectively. This is the first molecular identification of a phytoplasma associated with CFSD in cassava in Colombia.

An accurate greenhouse screening method has not been developed previously to identify host response to sheath blight disease caused by Rhizoctonia solani Kühn that causes significant economic losses in rice yield worldwide. The unavailability of a robust screening system in the greenhouse has made it difficult to quantify disease reactions to R. solani, and has hampered studies on the genetics of resistance and plant breeding efforts to improve resistance. In an effort to develop a standardized laboratory micro-chamber screening method to quantify resistance to R. solani in rice, five rice cultivars, representing a wide range of observed disease reactions under field conditions, were examined in a blind inoculation test at three locations (Arkansas, Texas, and Colombia). Rice seedlings were inoculated at the three- to four-leaf stage with potato dextrose agar plugs containing mycelium and then covered with a 2- or 3-liter transparent plastic bottle for maintaining high humidity after inoculation. Two cultivars, Jasmine 85 and Lemont, that consistently have shown the highest and lowest levels of resistance, respectively, in previous field and greenhouse studies, were used as standards. Concurrent field experiments in Arkansas and Texas also were performed to compare the greenhouse disease ratings with those observed under field conditions. Overall, the relative disease ratings of the seven test cultivars were consistent between test locations and with field evaluations. Thus, the micro-chamber screening method can be used as an effective approach to accurately quantify resistance to the sheath blight pathogen under controlled greenhouse conditions and should help expedite the selection process to improve resistance to this important pathogen.

Coffee crispiness ("crespera"), a disease of uncertain etiology, has been endemic in coffee (Coffea arabica L.) plantations in Colombia for at least 60 years. Symptoms typically consist of bud proliferation, abundant short and narrow leaves, phyllody, floral abortion, monospermic fruit, and dwarfing of plants. In severe cases, coffee crispiness disease (CCD) can affect production significantly. In this study, association of a phytoplasma with CCD was indicated by the accumulation of Diene's stain, or 4', 6-diamidino-2-phenylindole fluorescence, only in phloem of affected plant tissues. The presence of polymorphic phytoplasma cells in phloem sieve tube elements was confirmed by transmission electron microscopy. The disease was transmitted successfully by grafting symptomatic shoots from CCD-affected C. arabica plants onto young, healthy rootstocks; however, symptoms failed to develop after mechanical inoculation of young plants with extracts derived from diseased plant tissues. A nested polymerase chain reaction (PCR) assay employing primer pairs P1/P7 followed by FU5/rU3 amplified a 16S ribosomal DNA product (941 bp) exclusively from DNA of diseased plants. Sequence and phylogenetic analysis of the nested PCR product identified the CCD phytoplasma as a new strain member of group 16SrIII (X-disease group). This is the first report of a phytoplasma infecting coffee plants.

Plantains (Musa AAB) are important sources of food and income for millions of people in Colombia and other developing countries. Colombia is the largest producer of plantains (2) and the third largest exporter of bananas in the world. In 2001, plants of 'Dominico-Hartón' plantain showing mild chlorotic streak symptoms were observed in northwestern Colombia. Electron microscopy of symptomatic tissue extracts revealed the presence of filamentous virus-like particles approximately 800 nm long. Immunocapture reverse-transcription polymerase chain reaction was performed to test for the presence of Banana mild mosaic virus (BanMMV) as described by J. E. Thomas (unpublished, Queensland Department of Primary Industries, Australia) and Sharman et al. (3). For polymerase chain reaction (PCR), the upstream primer No. 193 (5'-CAC TTA GGT TTG TGT GAT GT-3') (designed in this study by using the computer Program DNAMAN Version 4.13) and the downstream primer Poty1 (5'-GGA TCC CGG GTT TTT TTT TTT TTT TTT V-3') (1,3; J. E. Thomas, unpublished, Queensland Department of Primary Industries, Australia) were used. Amplification products of the expected size (approximately 900 bp) were obtained and sequenced after cloning in a pCR2.1 plasmid vector. Analyses of nucleic acid sequences using the international sequence databases and the BLAST program yielded nucleotide and amino acid sequence similarities of 80 to 83% and 90 to 92%, respectively, with an Australian isolate of BanMMV (GenBank Accession No. AF314662). The coat protein (CP) gene of the Colombian BanMMV isolate consists of 717 nucleotides. When the CP of the Colombian BanMMV isolates (GenBank Accession Nos. AY319331, AY319332, and AY319333) was compared with the CP of the Australian isolate, a highly variable region was observed in the N-terminus region. To our knowledge, this is the first report of BanMMV isolated from plantains in Colombia and the presence of molecular variability in the CP of BanMMV isolates. BanMMV has been found in Colombia associated with Banana streak virus and Cucumber mosaic virus in plantain. References: (1) A. Gibbs and A. Mackenzie. J. Virol.Methods 63:9, 1997 (2) N. S. Price. Infomusa 8(2):26, 1999. (3) M. Sharman et al. J. Virol. Methods 89:75, 2000.

The fungus Sphaceloma manihoticola causes superelongation disease in cassava, a starchy root crop grown widely in the tropics. Isolates were collected from infected plants grown in six localities of Colombia. Morphological analyses of the fungus showed that colony growth and color are not stable characteristics over time. Pathogenicity studies, using the susceptible cassava variety M Col 22 and the resistant M Ven 77, showed that M Col 22 was tolerant of 29% of pathogen isolates studied and had an intermediate reaction to 71%. Variety M Ven 77 also showed tolerance of 16.2% of the isolates, had an intermediate reaction to 80.6%, and was susceptible to 3.2%. Significant cultivar × isolate interactions indicated pathogenic specialization. This study is the first to describe this pathogen's molecular characteristics. A homogeneous and reporducible band of about 545 bp was obtained with polymerase chain reaction which, when digested by restriction enzymes, showed an equal pattern of bands for all isolates. The isolates thus belonged to one species. Random amplified polymorphic DNA analysis revealed intraspecific genetic diversity. By better understanding the pathogen, we can apply more appropriate disease management strategies, such as selection of germ plasm tolerant of superelongation disease.