Elizabeth Alvarez

Characterization of a Phytoplasma Associated with Frogskin Disease in Cassava

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.

Characterizing the Sphaceloma fungus, causal agent of superelongation disease in cassava

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 pathogens 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.

Characteristics of Pseudomonas spp. causing grain discoloration and sheath rot of rice, and associated pseudomonad epiphytes

Ninety-five strains of fluorescent (presumed to be Pseudomonas spp.) and nonfluorescent pathogens of rice causing grain and sheath rot, dirty panicle, and manchado de grano, and 21 strains of nonpathogenic fluorescent pseudomonads isolated from rice grain and sheaths from 22 countries were compared with 26 reference strains (Pseudomonas avenae, P. fuscovaginae, P. glumae, P. marginalis, and P. syringae) with the use of morphology, serology and 77 physological traits. A Wards minimum variance cluster analysis grouped the strains into seven clusters corresponding to six bacterial species: P. fuscovaginae (two clusters), P. avenae, P. fluorescens, P. glumae, P. putida, and P. syringae (...)

Characterization of the Colletotrichum Species Causing Anthracnose in 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.

Phytoplasma diseases on major crops in Vietnam

The nested-PCR technique was applied to detect and identify phytoplasmas associated with major crops in Vietnam including sugarcane, cassava and longan. Phylogenetic analysis confirmed that phytoplasmas associated with the sugarcane grassy shoot and white leaf diseases belong to the rice yellow dwarf group (16SrXI). In cassava showing witches’ broom symptoms a mixed infection of phytoplasmas related to ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Ca. P. aurantifolia’ (group 16SrII) was detected. In samples showing longan witches’ broom disease three phytoplasmas including ‘Ca. P. aurantifolia’ (group 16SrII), elm yellows (group 16SrV) and “stolbur” (group 16SrXII) were identified.

Identifying and Analyzing the Diversity of Resistance Gene Analogs in Colombian Rubus Genotypes

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.

Assessing the resistance of Brachiaria hybrids to pathogenic Rhizoctonia

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.

Diversity of Neopestalotiopsis and Pestalotiopsis spp., Causal Agents of Guava Scab in Colombia

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).

Phenotyping Brachiaria Genotypes to Assess Rhizoctonia Resistance by Comparing Three Inoculum Types

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.