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Microsatellite markers and the mitochondrial sequence of mtCOI gene were used for the molecular characterization of the MEAM1 species of Bemisia tabaci (Gennadius) (Hemiptera, Aleyrodidae) in Cuba. Whitefly specimens collected from cucumber (Cucumis sativa L.), sugar beet (Beta vulgaris L.), tomato (Solanum lycopersicum L.), squash (Cucurbita pepo L), fruits of economic interest like papaya (Carica papaya L.), and two species of Bemisia weed hosts like American black nightshade (Solanum americanum Mill) and sage (Salvia officinalis L) were selected in non-productive ecosystems The results made evidente the presence of MEAM1 population with low molecular divergence in all hosts examined. DNA sequencing revealed the coexistence of MEAM1 with the New World (NW) species in S. officinalis L in natural habitat. [ABSTRACT FROM AUTHOR]

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Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi, is a devastating plant disease that affects several crops of worldwide economic importance. The pathogen ability for surviving in different environments and causing latent infections becomes an accurate diagnosis as a key point for the disease prevention. The aim of this work was the critical review of these techniques and the analysis of those factors affecting their performance parameters. Traditional methods based on in vitro cultures and pathogenicity tests are laborious, dependent of plant pathogen interactions and slow for obtaining the results. Advanced techniques based on serological principles and nucleic acid detection allow the automated and sensitive pathogen detection, but serology depends on antibody quality and molecular techniques are limited by oligonucleotide specificity and presence of inhibitory substances in the samples. Post-test probability analysis in function of the prevalence provides a higher accuracy in the decision making about the appropriate techniques for diagnosis. Combined use of several techniques based on different biological principles increases diagnosis reliability, and is therefore recommended for quarantine and certification of plant material imported by Cuba. The inclusion of a new technique into a diagnosis scheme requires a previous exhaustive testing of its performance with inter and intra laboratories evaluations. [ABSTRACT FROM AUTHOR]

Rapid detection and accurate identification of seed-borne bacteria are critical steps to prevent pathogen dissemination. Molecular-based methods such as PCR have greatly improved detection. However, plant inhibitors can interfere its detection. Despite, pre-enriching bacteria on agar media (BIO-PCR) can increase the sensitivity and reduce the inhibitor's effects. Three primer sets were designed for detecting Burkholderia glumae (Bg-BIO), Pseudomonas fuscovaginae (Pfus-BIO) and Pseudomonas syringae (Ps-BIO), and specificity and sensitivity of conventional PCR assessed. Detection limits of PCR and BIO-PCR were compared, and so were pathogen detection in artificially-infected seeds and in symptomless plant material. It was found that PCR using the designed primers were specific (detection limits 4-400 pg/µL of target DNA). Pfus-BIO and Ps-BIO were highly sensitive and allowed increasing detection limits significantly compared with conventional PCR. Conversely, Bg-BIO was only 10-times more sensitive than conventional PCR. Bacteria were efficiently detected by PCR and BIO-PCR from artificially inoculated seeds, the same results achieved for both methods with large pathogen amounts and macerated seed extracts. P. fuscovaginae and B. glumae were detected in 26 (87%) and 18 (60%) out of the 30 symptomless plant material samples tested, whereas P. syringae was undetected in any of the samples. Conventional PCR did not allowed target amplification of any sample. In summary, the designed BIO-PCR tests are reliable and efficient tools for diagnosing B. glumae, P. fuscovaginae, and P. syringae, enabling to sow only bacteria-free propagating material and moving it from one place to another. [ABSTRACT FROM AUTHOR]

'Elbita' arises from the need to have tomato varieties adapted to the Cuban tropical climate for open field planting. It is a variety resistant to TYLCV-IL [CU], begomovirus of greater incidence in the country and tolerant to fungal diseases, such as Alternariosis. It has characteristics which allows it to be used as a cultivar for opening (seedbeds in August) and closing of the season (seedbeds in January) of sowing. [ABSTRACT FROM AUTHOR]

Orthotospoviruses are RNA viruses transmitted by thrips (Thysanoptera: Thripidae). They have been considered one of the most economically important pests since the last century and today the losses caused by TCSV keep being an important factor to take into account in the different vegetable and grain producing areas in Cuba. Seventy-two samples of tomato, lettuce, carrot, cow pea, and pepper showing characteristic symptoms of infection by orthotopoviruses were collected in 2018-2020 (November to January) at the "Las Piedras" farm, Guanabacoa. The samples were analyzed by DAS-ELISA to detect TSWV, GRSV and TCSV. Twenty-six samples, corresponding to tomato and cow pea, resulted positive. These results corroborate the possibility of using serological techniques to detect orthotopoviruses in areas of intensive tomato production like "Las Piedras" farm and suggest that the cow pea crop can act as a viral host. The presence of orthotospovirus in tomato constitutes a serious risk for production of other crops of high economic impact because of the wide host range and the incidence of its transmitting vector in the producing areas of potato, tobacco, beans and other vegetables. [ABSTRACT FROM AUTHOR]

The response of 19 foreign genotypes and 21 national genotypes to the African cassava mosaic virus (ACMV) was evaluated, and the disease incidence and severity were analyzed. The total number of the plants planted by genotypes at the Namibe Agricultural Experiment Station, Angola, was sampled in February 2017, June 2017, and February 2018. The whitefly adult density per genotype was averaged to calculate the density index on each date, which was compared using a simple analysis of variance and the Duncan's multiple range test for a confidence level of 95 %. The cluster analysis showed that the genotypes were grouped according to the disease severity. Some genotypes were detected with high severity indexes (0.8-1); however, TMS-84004, TMS-0276, TMS-300027, TMS-40142, TMS-800098, TMS-3021, TMS-300027, TMS-42025, Mz-3, Kassakala, and Canfufu, showed severity indexes of 0.2, the lowest index obtained. This result allowed suggesting genotypes that under high vector density conditions showed lower rates of disease severity, which can be used under adequate pest management conditions. [ABSTRACT FROM AUTHOR]

Climate changes, increasing mobility of humanity, indiscriminate use of pesticides, genetic resistance, crop rotation and cultural practices for reducing pests, have been combined to increase the risk of changes in ecosystems and the evolution of pathogens and their vectors causing the emergence of invasive pests. The aim of the present paper was to carry out a study on begomovirus populations present in tomato 20 years after the first detections of this pathogen. The results confirmed that Tomato yellow leaf curl virus, Israel strain (TYLCVIL) was the predominant begomovirus in the cultivation of tomatoes and peppers in Cuba, with a distribution of 83.1% and 42.8%, respectively. Defective molecules of TYLCV-IL replication were characterized. Defective genomes associated with infection by TYLCV-IL (CU) were first described, with a complex structure including deletions, inversions, translocations and duplications of the viral genome fragments. Also, the appearance of new species of bipartite begomovirus in tomato and in associated weeds has caused high risk epidemiological situations for horticultural productions, increasing density and diversity of the vector populations. Mixed infection symptoms of more than one viral genus have been observed, coinciding with high vector populations. These studies will strengthen the phytosanitary surveillance and management programs being carried out in the country to control vector-borne diseases in crops of economic interest, mainly in tomato. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented on the topic of genetic aspects of whitefly species.

The Cuban tomato line LD3 , in which the Ty-1 gene from Solanum chilense LA 1969 was detected, was crossed with an F1 hybrid carrying the genes Ty-3 from S. chilense LA 2779 and Sw-5 from S. peruvianum. A hundred F2 plants were transplanted to a ferralitic soil in the field and the F2 plants individually evaluated in the field with high natural TYLCV-IL (Cu) incidence by using a scale from 0 (no symptom) to 4 (severe stunting). The total DNA extracted from the F2 plants were analyzed per individual plant by using PCR markers linked to the genes Ty-3 (resistance to TYLCV) and Sw-5 (resistance to TSWV) so that both genes could be detected in a same genotype. Eighty two F2 plants showed no symptoms under natural TYLCV incidence (0 symptoms) with a productivity ranging from 1.6 to 9.7 kilograms per plant. The Principal Component Analysis (PCA) permitted grouping the different genotypes. Six F2 plants showed the Ty-3 gene in homozygosis and 12 in heterozygosis; 33 F2 plants showed the Sw-5 gene according to their DNA analysis. Three F2 plants (number 65, 68 and 78) showed the Ty-3 and Sw-5 genes in homozygosis besides the Ty-1 gene (resistance to TYLCV) from their female parent. Their productivity ranged from 6.0 to 6.8 kilograms per plant. These are the first Cuban results in which resistance genes for two important diseases in the tomato crop are obtained in a same genotype by the early and simultaneous molecular screening for resistance and the productivity evaluation under field conditions, Both tools, when simultaneously used, are efficient in reducing selection time and space. [ABSTRACT FROM AUTHOR]

The current classification scheme of the causal agent of the bacterial wilt [Ralstonia solanacearum) (Smith) Yabuuchi] is based on the analysis of the endoglucanase gene sequence of the bacterium. The objective of this paper was to optimize the PCR amplification of this gene. PCR parameters were determined using the OLIGO software version 7.53 for in silico analyses. The effect of cell concentration on amplification efficiency was assessed by using serial dilutions of R. solanacearum suspensions. For the in vitro assays, a two step-PCR protocol was evaluated with the GoTaq® DNA Polimerasa kit. The cell concentration was a critical factor on amplification efficiency with an optimal value of 108 CFU.ml-1. Time was reduced for the initial denaturalization and final extension phases and hybridization and extension were combined in a single step resulting in a 57 minute shorter-PCR program than the original protocol. The phylogenetic analysis of this gene sequence will allow performing diversity studies of Cuban isolates of R. solanacearum, establishing comparative relationships with isolates of other geographical locations and updating the disease management strategies. [ABSTRACT FROM AUTHOR]

The common bean (Phaseolus vulgaris L.) is one of the most important grain legumes for human consumption. In Cuba, there are different factors that limit its production, among which the bean golden yellow mosaic disease, caused mainly by the begomovirus Bean golden yellow mosaic virus (BGYMV). Genetic improvement for resistance to this virus is one of the most effective strategies for disease control. The objective of this work is to evaluate the behavior of bean genotypes against the bean golden yellow mosaic disease and the identification of the resistance gene bgm-1 using the codominant SCAR marker S2 in genotypes from the INCA working collection. The severity of the viral symptoms was evaluated in each genotype in flowering stage according to the scale proposed by CIAT (1991). DNA extraction was performed on 61 genotypes. From the PCR analysis with the S2 primers it was possible to identify 19 genotypes with presence of the bgm-1 gene in recessive state, which confers the resistance to the BGYMV. 32 genotypes with 1 or 2 symptoms degree caused by begomovirus were detected, without the presence of the bgm-1 recessive gene. The results of this work constitute a great contribution to the common bean improvement program in Cuba, due to the fact that analyzed genotypes are materials from the genetic improvement programs of the region and from cultivars that belong to national production and constitute new resistance sources. [ABSTRACT FROM AUTHOR]

In order to identify the mite species infesting Morus alba L. (mulberry), systematic samplings were carried out on mulberry plants belonging to the "Los Mangos" farm of the Science, Technology and Innovation Entity "Sierra Maestra", Playa municipality, La Habana, Cuba, from January to June 2018. In the samplings, 30 plants were selected at random, from which three leaves were extracted and examined under a Stemi DV4 stereo microscope. The adult mites were rinsed and mounted on slides. For their identification, they were observed under an Axioskop A1 microscope, and the corresponding dichotomous keys were used. As a result of the inventory, it was reported the presence of the phytophagous mite Tetranychus sp. (Acari: Tetranychidae) and the predators Neoseiulus longispinosus, Euseius hibisci and Amblyseius curiosus (Acari: Phytoseiidae). [ABSTRACT FROM AUTHOR]

With the aim of detecting and identifying Xanthomonas axonopodis pv. phaseoli, the causal agent of the disease known as common bacterial blight (CBB) on common bean, 30 leaf and pod samples were taken from naturally infected plants in different localities of Mayabeque province. The samples were examined and the causal agent isolated and identified by using the following techniques: isolation on semiselective media (YDCA y XCP1), pathogenicity test in a susceptible cultivar (BAT 304), and the PCR technique with specific primers (p7X4c yp7X4e). Of the 30 isolates obtained, 29 caused symptoms on the cultivar used in the pathogenicity test. Different symptomatologies of the disease were shown by the artificially inoculated plants. The amplification product of the PCR was a DNA fragment with 800 bp, the size reported for the isolates of this pathogen. These results showed the presence of X. axonopodis pv. phaseoli affecting the bean growing areas of the province, a frequent problem in these areas. [ABSTRACT FROM AUTHOR]

The genetic diversity among 30 Cuban strains of Ralstonia solanacearum isolated from potato and tomato and belonging to biovars 1 and 2, was assessed by the repetitive sequence based polymerase chain reaction (rep-PCR) method with ERIC primer sets. The method defined 11 genotypes from all the strains. The primary groupings obtained by cluster analysis corresponded with the strain biovar, while the subdivisions found within each group were related to the host from which the strains were obtained. Potato isolates showed a 37.2% similarity and the presence of specific variants within each biovar for each geographic region. Isolates from tomato showed a similarity of 71.3 % with the cross presence of the same genotype in different geographic regions and the incidence of different genetic variants in the same region. The Principal Component Analysis explained 81.32 % of isolate variability, which corroborated the groupings according to the host and biovar and allowed to establish relationships based on the geographic location. The differential distribution of the genetic diversity among Cuban R. solanacearum strains from potato and tomato crops may be useful information to establish specific measures to control the bacterial wilt disease in Cuba. [ABSTRACT FROM AUTHOR]

The bacterial isolates SUR - 6, DiP/2012 and Oct/Y were isolated from silkworms (Bombyx mori L.) infected naturally, which presented evident symptoms of bacterial flacheria. The goal of this study was to identify the bacterial pathogens associated with symptoms observed in silkworm breeding areas of Cuba, to establish control and sanitation measures. The morphological, biochemical and physiologic study allowed to identify these isolates as Enterobacter cloacae (SUR - 6), Serratia marcescens (DiP /2012), and Acinetobacter baumanii (Oct/Y), which are informed widely in the literature like pathogens of insects. [ABSTRACT FROM AUTHOR]

The mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is affected by numerous diseases like Grassery, caused by the nuclear polyhedrosis virus (BmNPV). The management and control of these diseases mainly consist of prevention and early diagnosis. The objective of this work was aimed at the diagnosis of the virus BmNPV associated with the symptoms of Grassery observed in the production areas of the Mulberry silkworm in Cuba. Molecular diagnostic methods for BmNPV based on radioactive and non-radioactive hybridization of nucleic acids (HNA), from larvae's polyhedra extractions in the 3rd and 4th instars, were developed. In both hybridization methods, BmNPV presence was detected. Positive results of 5.5% of samples analyzed in the 3rd larval stage were obtained using radioactive hybridization, which agreed with non- radioactive hybridization results. Nevertheless, in the 4th stage, 28.8% of positive samples were detected using radioactive hybridization and 22.2% of total analyzed samples for non-radioactive hybridization. Although radioactive hybridization showed higher sensibility, both methods allowed to confirm that it was possible to detect BmNPV from the 3rd larval stage, which allows to establish the enhancement of preventive treatment and management measures of mulberry litters with the objective of minimizing production loses. [ABSTRACT FROM AUTHOR]

The 'Tomato yellow leaf curl disease', caused by Tomato Yellow Leaf Curl Virus (TYLCV-IL [CU]) has caused extensive damage to tomato crops in Cuba. There are several resistance genes that have been introgresed to tomato from wild Solanum species. The objective of this study was to assess the segregation of two such genes, Ty-1 and Ty-2, as the basis for selection in improving resistance to TYLCV-IL [CU]. Two F2 populations, susceptible cultivar 'Amalia' x resistant cultivar 'H-24' (Ty-2), TY-2F2 and 'Amalia' x resistant cultivar 'LA3473' (Ty-1), TY-1F2, were obtained. Plants F2 and plants of susceptible controls, cvs. 'Amalia' and 'STY-1'. The plants, 25 days after sowing, were inoculated with viruliferous (Cuban isolate TYLCV-IL [CU]) whiteflies (Bemisia tabaci, biotype B) for 48 hours before transplantation to the field. The disease severity index (DSI, 0-4 scale) and viral DNA (dot blot hybridization) were evaluated 30 days after the inoculation. The plant frequency distribution of disease severity and viral DNA concentration changed according to TY-2F2 and TY-1F2 plants. Therefore, we should select as resistant plants to TYLCV those with 1 score in TY-1F2 and 0 scored in TY-2F2, when the improvement program is performed by selecting phenotypic resistance TYLCV-IL [CU] conferred by Ty-1 and Ty-2 resistance genes, respectively. [ABSTRACT FROM AUTHOR]

Tomato Yellow Leaf Curl disease is one of the most important problems affecting tomato cultivation in most part of the Cuban growing areas and in other countries. The casual agent is the TomatoYellow Leaf Curl Virus (TYLCV). At the moment several genes related with the resistance have been described. The genes Ty-1, Ty-2 and Ty-3 are those that more they have been used in the plant breeding programs of the species. In Cuba, Vyta is only variety that had Ty-1 gene and other accessions carrying the Ty-2 gene. The amplification of the gene Ty-3 was carried out for PCR and were used the specific primers FLUW-25R and FLUW-25F. In this work the presence of the band related with the resistant pattern of the gene Ty-3 was identified in one that six characterized accessions. [ABSTRACT FROM AUTHOR]

La marchitez bacteriana, causada por Ralstonia solanacearum (Smith) Yabuuchi, es una devastadora enfermedad que afecta a varios cultivos de importancia económica a nivel mundial. La capacidad de supervivencia del patógeno y la ocurrencia de infecciones latentes hace que el diagnóstico tenga importante función en la prevención de la enfermedad. El objetivo de este trabajo fue desarrollar técnicas serológicas y de detección de ácidos nucleicos para el diagnóstico sensible del patógeno, realizar un análisis comparativo de los parámetros de desempeño y estudiar la diversidad genética de 30 aislados de R. solanacearum, obtenidos de papa y tomate (pertenecientes a los biovares 1 y 2) mediante la amplificación de las secuencias con sensorrepetitivas intergénicas de enterobacterias (ERIC-PCR). El análisis de la probabilidad postest, en función de la prevalencia, permitió decidir con mayor precisión las técnicas apropiadas para el diagnóstico y demostró la factibilidad del uso combinado del ELISA y la PCR para la detección de R. solanacearum, teniendo en cuenta el orden de aplicación en función de la prevalencia de la enfermedad. Por otra parte, los estudios de variabilidad permitieron distinguir 11 genotipos entre los aislados cubanos. Las agrupaciones primarias obtenidas mediante el análisis de conglomerados se correspondieron con el biovar de los aislamientos, mientras que las subdivisiones encontradas dentro de cada grupo se correspondieron con el hospedero del cual se obtuvieron. Los aislados obtenidos de papa mostraron un coeficiente de similitud del 37,2% y la presencia de variantes específicas para cada región geográfica dentro de cada biovar. Los aislados obtenidos del cultivo del tomate registraron una similitud del 71,3% con la presencia cruzada del mismo genotipo en diferentes regiones geográficas y la incidencia de diferentes variantes genéticas en una misma región. Desde el punto de vista geográfico, el Análisis de Componentes Principales mostró relaciones entre los aislamientos para el 25% de la variabilidad observada. La distribución diferencial de la diversidad genética entre los aislados cubanos de R.solanacearum, obtenidos de los cultivos de papa y tomate, puede ser una herramienta útil para el establecimiento de medidas específicas para el control de la Marchitez Bacteriana en Cuba. [ABSTRACT FROM AUTHOR]

El frijol común ( Phaseolus vulgaris L.) es la leguminosa comestible de mayor importancia en el mundo, proporciona una fuente significativa de proteínas, vitaminas y minerales a la dieta humana. La baja productividad de este cultivo se debe a numerosos factores, entre ellos están los problemas fitosanitarios, como son las enfermedades causadas por bacterias. En Cuba encontramos el denominado Tizón común o Bacteriosis común, provocado por Xanthomonas axonopodis pv. phaseoli que ocasiona grandes pérdidas en el rendimiento y en la calidad de la semilla. Con el objetivo de elaborar una estrategia que permita la detección temprana de dicho patógeno en semillas y en la fase temprana de la enfermedad en campo se emplearon hojas y frutos procedentes de plantas con síntomas típicos de la enfermedad. Para ello, se procedió al aislamiento en medios de cultivo semiselectivos (YDC y XCP1), la prueba de patogenicidad en cultivos susceptibles, así como la Reacción en Cadena de la Polimerasa (PCR) con los cebadores específicos p7X4c yp7X4e. Los aislados manifestaron diferentes sintomatologías de la enfermedad en las plantas inoculadas artificialmente y con el empleo de la PCR se obtuvo, como producto amplificado, un fragmento de ADN cuya talla corresponde con la informada en la literatura para los aislados pertenecientes a este patógeno. Esta metodología permitió detectar la presencia de este agente en las regiones productoras de frijol en la provincia Mayabeque. Se mostró que la estrategia trazada puede emplearse como método de diagnóstico para la detección temprana de dicho patógeno en semillas o en las fases tempranas de la enfermedad; de esta forma se contribuye a controlar, disminuir o evitar las pérdidas causadas por este agente en el cultivo del frijol. [ABSTRACT FROM AUTHOR]

Las epifitias causadas por geminivirus son un factor limitante para la productividad de los cultivos de frijol y de soya en Cuba. La detección y la caracterización de los agentes causales es una necesidad para el manejo de estas enfermedades. Desde la década de los setenta del pasado siglo se ha podido evidenciar en Cuba una emergencia de enfermedades virales en plantaciones de frijol común. Durante las campañas de producción 2012-2015 se ha comenzado a observar una diversidad de síntomas asociados a la presencia de poblaciones de mosca blanca, debido a la alta tasa de mutaciones y al intercambio genético que presentan los geminivirus como mecanismos evolutivos. En este trabajo se colectaron muestras con síntomas diferenciales en varias localidades productoras, se realizaron extracciones de ADN, las que se amplificaron mediante el sistema de círculo rodante. Los genomas circulares amplificados fueron digeridos con diferentes combinaciones de enzimas de restricción. Los resultados revelaron diversos patrones polimórficos, los cuales pueden estar asociados a la presencia de especies de begomovirus no informadas previamente en el país, tanto en infecciones simples como en concomitantes. En el trabajo se discuten los resultados relacionados con la diversidad de especies presentes y la presencia de infecciones mixtas. [ABSTRACT FROM AUTHOR]

Blackleg, caused by Pectobacterium spp. and Dickeya spp., is an important disease of potatoes. During the period from November 20 and March 2021, stems of potato plants showing necrosis and rot symptoms, and chlorotic leaves, were collected from commercial production areas of the Mayabeque province of Cuba (Fig. 1A). After disinfestation of affected stems, small fragments of the stem were cut and macerated in a sterile 0.85% NaCl solution. Serial dilutions of bacterial suspension were prepared and streaked onto nutrient agar in Petri plates. Two colonies per sample showing the characteristic of "fried egg" were selected for further investigation, and an isolated was selected and named D7. The isolated bacterium was rod shaped, gram-negative, motile, oxidase and indole production negative, with anaerobic growth, and able to use lactose as carbon source in Mac Conkey Agar medium. One colony of the isolate D7 was selected and multiplied. Total DNA of the bacteria cells was extracted and used to amplify the genes pelADE (Nassar et al., 1996) and gapA (Cigna et al., 2017), to differentiate Dickeya from Pectobacterium. The sequence obtained showed 99.75% and 99.88% nucleotide identity with Dickeya solani for pelADE (Genbank accession number ON644347) and gapA (Genbank accession number ON644346), respectively. To confirm the pathogenicity of the isolate D7, four 15-day-old potato plants, including two plants of each 'Otolia' and 'Naima' potatoes were inoculated with a bacterial suspension of the isolate D7 (108 CFU/ml) in sterile water by stabbing. Control plants were stabbed with sterile water. Inoculated plants were maintained at 28°C, relative humidity of about 90%, and at 12 h light/12 h dark, as described by (Chen et al. 2014). After 3 to 5 days, typical blackleg disease symptoms (water-soaked lesions and necrosis) developed at the inoculated areas of plants (Fig. 1B). No symptom was observed in the control plants. Bacterium was re-isolated from symptomatic plants and the isolates had the same cultural, physiological, and biochemical characteristics to the isolate D7. To our knowledge, this is the first report of D. solani causing blackleg in potato fields in Cuba. Further studies to determine the spread of this pathogen in potato producing areas in Cuba is underway.