Juan Fernando Mejía

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.

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.

Detection and multigene characterisation of a ‘Candidatus Phytoplasma prunorum’-related phytoplasma associated with witches’ broom symptoms in pineapple fruit

Pineapple samples showing witches’ broom symptoms in the leaves at the top of the fruit were analysed to verify phytoplasma presence. 16SrX group phytoplasmas were detected after nested PCR amplification with specific primers and RFLP analyses. Sequencing of 16Sr DNA allows verification that phytoplasmas from pineapple show a 99% shared identity with ‘Candidatus Phytoplasma prunorum’. Further characterisation of the phytoplasma on ace, imp and secY genes by RFLP analyses on amplicons obtained in nested PCR assays enable the differentiation of the strain infecting pineapple from those detected in fruit trees in Europe and used as reference strains. This is the first report of phytoplasmas belonging to the 16SrX-B group from a species different from pome and stone fruit worldwide.

Lagenaria siceraria yellows associated with phytoplasma presence in Brazil

Lagenaria siceraria, the bottle gourd or calabash gourd, is distributed in Brazil and locally known as “porongo”. It is used as a green vegetable and some cultivars are marketed as handicraft. In the State of Rio de Janeiro, naturally diseased plants were observed with reduced leaf size, leaf malformation and yellowing. Molecular analysis revealed the presence of a phytoplasma related to ‘Candidatus Phytoplasma pruni’ that was classified as a new subgroup -Y into the 16SrIII ribosomal group. This is the first report of the genus Lagenaria harbouring a phytoplasma worldwide.