Luis A. Ramos

Handheld Raman spectroscopy for the early detection of plant diseases: Abutilon mosaic virus infecting Abutilon sp.

Plant diseases have a direct impact on the productivity of crops, and therefore the early detection of diseases is crucial. Abutilon mosaic virus (AbMV) (family Geminiviridae; genus Begomovirus) is an important virus infecting ornamental crops throughout the world. Abutilon hybridum showing bright yellow mosaic symptoms were observed in gardens in Tumbaco, Ecuador. The infection was confirmed by Polymerase Chain Reaction (PCR) using degenerate begomovirus primers. The amplicon (∼500 bp) was sequenced, submitted to NCBI (KP877621) and showed 68.7–100% and 72.7–100% sequence identity with other begomoviruses at nucleotide and amino acid levels, respectively. In order to evaluate Raman spectroscopy as a diagnostic tool for AbMV infection, spectra of leaves from healthy and infected plants were recorded. Raman signals of carotenoids are the most important features and there is a significant decrease in the intensity of these bands when the plant is infected. The difference in the intensity of the bands, particularly the one at 1526 cm−1, is proposed as a basis for the early detection of viral infection in plants.

Distinction of Ecuadorian varieties of fermented cocoa beans using Raman spectroscopy

Cocoa (Theobroma cacao) is a crop of economic importance. In Ecuador, there are two predominant cocoa varieties: National and CCN-51. The National variety is the most demanded, since its cocoa beans are used to produce the finest chocolates. Raman measurements of fermented, dried and unpeeled cocoa beans were performed using a handheld spectrometer. Samples of the National and CCN-51 varieties were collected from different provinces and studied in this work. For each sample, 25 cocoa beans were considered and each bean was measured at 4 different spots. The most important Raman features of the spectra were assigned and discussed. The spectroscopic data were processed using chemometrics, resulting in a distinction of varieties with 91.8% of total accuracy. Differences in the average Raman spectra of cocoa beans from different sites but within the same variety can be attributed to environmental factors affecting the cocoa beans during the fermentation and drying processes.

Alcohol-based solutions for bovine testicular tissue fixation:

Tissue fixation, a central element in histotechnology, is currently performed with chemical compounds potentially harmful for human health and the environment. Therefore, alternative fixatives are being developed, including alcohol-based solutions. We evaluated several ethanol-based mixtures with additives to study fixative penetration rate, tissue volume changes, and morphologic effects in the bovine testis. Fixatives used were Bouin solution, 4% formaldehyde (F4), 70% ethanol (E70), E70 with 1.5% glycerol (E70G), E70 with 5% acetic acid (E70A), E70 with 1.5% glycerol and 5% acetic acid (E70AG), and E70 with 1.5% glycerol, 5% acetic acid, and 1% dimethyl sulfoxide (DMSO; E70AGD). Five-millimeter bovine testicular tissue cubes could be completely penetrated by ethanol-based fixatives and Bouin solution in 2–3 h, whereas F4 required 21 h. Bouin solution produced general tissue shrinkage, whereas the other fixatives (alcohol-based and F4) caused tissue volume expansion. Although Bouin solution is an excellent fixative for testicular tissue, ethanol-based fixatives showed good penetration rates, low tissue shrinkage, and preserved sufficient morphology to allow identification of the stages of the seminiferous epithelium cycle, therefore representing a valid alternative for histotechnology laboratories. Common additives such as acetic acid, glycerol, and DMSO offered marginal benefits for the process of fixation; E70AG showed the best preservation of morphology with excellent nuclear detail, close to that of Bouin solution.

FIRST REPORT OF TOMATO SPOTTED WILT VIRUS INFECTING PEPPER IN ECUADOR

Pepper (Capsicum annum L.), a member of the family Solanaceae, is native of South and Central America. In April 2015 mosaic and necrotic symptoms were observed on leaves and stems of pepper plants in the Imbabura province of Ecuador. Based on the type of symptoms, infection by a tospovirus was suspected. Symptomatic pepper samples were screened by TAS-ELISA using specific antibodies to Tomato spotted wilt virus (TSWV) (Agdia, USA), a member of the genus Tospovirus, family Bunyaviridae transmitted by thrips in a persistent and circulative manner. The presence of TSWV was ascertained by RT-PCR using total RNA isolated from ELISA-positive samples with the RNeasy Plant Mini kit (Qiagen, USA). DNA fragments of ca. 800 bp were amplified using degenerate universal tospovirus primers (Chu et al., 2001). A PCR product was custom-sequenced (Macrogen, South Korea) and the sequence was deposited in GenBank as accession No. KT590401. Sequence analysis (BioEdit v. 7.05) of the TSWV isolate from Ecuador with other TSWV isolates showed 97.3% and 95.3% maximum identity at the nucleotide and amino acid levels, respectively. A phylogenetic tree based on nucleotide sequences constructed using MEGA version 4.1 revealed a clustering of TSWV-Pepper from Ecuador with TSWV-Chrysanthemum from South Korea (KC261974), TSWV-Pepper from South Korea (HM581940), TSWV-Leonurus sibiricus from South Korea (KM076651) and TSWV-Tree tomato from Ecuador (KP772268). To the best of our knowledge this is the first report of the occurrence of TSWV on capsicum in Ecuador.

FIRST REPORT OF POTATO YELLOWING VIRUS INFECTING PEPPER IN ECUADOR

Pepper (Capsicum annum L.) is a solanaceous species originating from South and Central America. Potato yel- lowing virus (PYV), an unclassified putative member of the genus Ilarvirus, was first found in potato in Peru (Fuentes et al., 1993), later in wild potato (Solanum fernandezianum) in Chile (Valkonen et al., 1992) and more recently in a native potato (Solanum phureja) in Ecuador (Silvestre et al., 2011). In December 2014, foliar mosaic and necrotic spotting of leaves and stems accompanied by bud and fruit necrosis were observed in pepper fields of Puembo, in the Pichincha prov- ince of Ecuador. Based on the symptomatology, the occur- rence of PYV was suspected and confirmed in symptomatic leaves by DAS-ELISA with a specific antiserum provided by G. Muller, International Potato Center, Lima, Peru, and by RT-PCR using universal primers for members of the family Bromoviridae (Untiveros et al., 2010). The 300 bp amplicon obtained from a symptomatic pepper was purified, custom- sequenced (Macrogen, South Korea) and the nucleotide se- quence was deposited in GenBank (KP772263). Sequence analysis (BioEdit v. 7.05) showed 93.1-97% and 78-90% iden- tity with other PYV isolates at the nucleotide and amino acid levels, respectively. A phylogenetic tree constructed at the nucleotide level using MEGA version 4.1 showed that the Ecuadorian PYV isolate from capsicum was closely related to isolate Loja 2 from S. phureja in Ecuador (HQ141056). Solanum isolates Canete (HQ141057), Russia (KM244740), Loja 1 (HQ141053) and Azuay (HQ141054) formed a sepa- rate cluster. To the best of our knowledge, this is the first report of PYV on Capsicum in Ecuador.