Cnidoscolus blistering yellow mosaic virus: a new begomovirus isolated from Cnidoscolus urens in Brazil

Members of the genus Begomovirus have circular single-strand DNA genome encapsidated into quasi-icosahedral particles, which are transmitted by the Bemisia tabaci complex, with non-cultivated hosts acting as reservoirs for these viruses. In this study a new begomovirus infecting Cnidoscolus urens (Euphorbiaceae) from Brazil has been characterized. The complete DNA-A sequence was used for species demarcation, phylogenetic and recombination analyzes with other previously reported begomoviruses. The DNA-A (2585 nt) has a genome organization that is typical of the New World bipartite begomovirus, most closely related to tomato yellow spot virus, with 80.6% nucleotide identity. Based on the identity criterion for DNA-A established by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses of ≥ 91%, this isolate should be considered a member of a new species, for which the name “Cnidoscolus blistering yellow mosaic virus” (CnBYMV) is proposed. Phylogenetic analysis revealed that CnBYMV grouped with begomoviruses described in cultivated and non-cultivated plants from Brazil. Results of recombination analysis indicated that the novel begomovirus was a recombinant, with cnidoscolus mosaic leaf deformation virus and sida mottle virus as probable parents

Three species of Colletotrichum are associated with anthracnose of pomegranate in Brazil

Pomegranate is a fruit rich in vitamins and secondary metabolites used in traditional medicine and industry. However, production losses have been associated the anthracnose, disease caused by Colletotrichum species. This is an important disease of the pomegranate, as it affects the yield and the quality of the fruits. The present study aimed to investigate which species of Colletotrichum are associated with anthracnose disease in pomegranate in the Northeast region of Brazil, using multi-locus phylogenetic analysis and morpho-cultural characteristics. The total DNA extracted was amplified with GAPDH, TUB2, CAL, ACT genes and the ITS-rDNA region. The sequences obtained were used for the construction of phylogenetic trees of Bayesian inference. The mycelial growth rate, size and shape of the conidia and appressories were evaluated for the morpho-cultural characterization of the species. Six isolates were analysis and three species belonging to the C. gloeosporioides complex were identified in this study. This is the first report of C. theobromicola (2) in pomegranate fruit in Brazil and C. siamense (2) and C. fructicola (2) in the world.Pomegranate is a fruit rich in vitamins and secondary metabolites used in traditional medicine and industry. However, production losses have been associated the anthracnose, disease caused by Colletotrichum species. This is an important disease of the pomegranate, as it affects the yield and the quality of the fruits. The present study aimed to investigate which species of Colletotrichum are associated with anthracnose disease in pomegranate in the Northeast region of Brazil, using multi-locus phylogenetic analysis and morpho-cultural characteristics. The total DNA extracted was amplified with GAPDH, TUB2, CAL, ACT genes and the ITS-rDNA region. The sequences obtained were used for the construction of phylogenetic trees of Bayesian inference. The mycelial growth rate, size and shape of the conidia and appressories were evaluated for the morpho-cultural characterization of the species. Six isolates were analysis and three species belonging to the C. gloeosporioides complex were identified in this study. This is the first report of C. theobromicola (2) in pomegranate fruit in Brazil and C. siamense (2) and C. fructicola (2) in the world

Efficiency of essential oils to control Colletotrichum theobromicola in vitro

Essential oils promote the inhibitory control of several fungi, including those within the genus Colletotrichum, the causal agent of Anthracnose, a disease which may occur at any stage of development in various crops, reducing up to 70% of crop production in some cases. Thus, the use of alternative products constitutes an important strategy for the integrated management, promoting less persistent molecules in the environment and lower toxicity rates, providing health benefits to producers and consumers of agricultural products. In this context, the present study evaluated the in vitro fungitoxic effect of essential oils from Java citronella (Cymbopogon winterianus), clove (Syzygium aromaticum), eucalyptus (Eucalyptus globulus) and rose pepper (Schinus terebinthifolius) on the mycelial growth of Colletotrichum theobromicola. The experiment was performed under completely randomized design, in a 4x5 factorial scheme (4 essential oils x 5 concentrations), with five replications, and the experimental unit consisting of a Petri dish. The treatments were generated by combining the concentrations (0, 5, 10, 15, 25 and 50 μL mL-1) of essential oils (citronella, clove, eucalyptus and rose pepper). The plates were inoculated with the pathogen C. theobromicola and incubated for seven days at 25 ± 2 °C. To verify the difference between treatments, the percentage of mycelial growth inhibition (PGI) was estimated. The mycelial growth of C. theobromicola was significantly reduced with increasing concentrations of essential oils. At a concentration of 50 μL mL-1, the essential oil from S. terebinthifolius showed the best result inhibiting 54.57% of mycelial growth, followed by the oil from S. aromaticum (49.26%), C. winterianus (23.70%) and E. globulus (17.90%). All the studied oils showed antifungal activity

Resistance to tospovirus, cloning and molecular characterization of Sw-5 alleles from different Lycopersicon species

O presente trabalho objetivou: 1) avaliar a resistência de acessos de Lycopersicon spp. a tospovírus; 2) estudar a herança da resistência em algumas dessas fontes e 3) clonar e caracterizar molecularmente alelos do loco Sw-5 provenientes de Lycopersicon spp. Plantas de 12 acessos de tomateiro, pertencentes às espécies L. peruvianum, L. chilense e L. hirsutum foram inoculadas com cinco isolados de tospovírus, pertencentes às espécies TSWV, TCSV, CSNV e GRSV. A reação das plantas foi avaliada periodicamente por 30 dias. Ao final desse período, as plantas assintomáticas foram confirmadas como resistentes, mediante DAS-ELISA. A resistência na maioria dos materiais foi de amplo espectro (efetiva contra todos isolados), porém em alguns casos a resistência foi do tipo isolado-es pecífica. Os materiais mais promissores foram os acessos PI 126928, LA 444/1, LA 371 e PI 126944 de L. peruvianum e LA 130 e LA 2753 de L. chilense. Esses materiais constituem fontes de resistência alternativas para o manejo das tospoviroses do tomateiro. O estudo de herança da resistência em L. hirsutum PI 134417 indicou que a resistência nessa fonte é condicionada por dois genes independentes, um dominante e um recessivo. Num teste de alelismo, o gene dominante segregou independentemente do gene Sw-5, indicando que são genes distintos. Em L. peruvianum PI 126928, a resistência segregou como uma característica condicionada por dois ou mais genes dominantes não ligados. Na terceira etapa do trabalho, comparou-se o nível de conservação de alelos do loco Sw-5 em Lycopersicon spp. Os alelos foram obtidos mediante PCR de longo alcance, utilizando-se o sistema ELONGASE TM (Gibco-BRL) para amplificação. Nas reações de amplificação foram utilizadas três combinações de oligonucleotídeos desenhados com base na seqüência do gene Sw-5. Como molde foi utilizado DNA extraído de plantas cuja reação a tospovírus já era conhecida. Foram obtidos 16 alelos do loco Sw-5, provenientes de quatro espécies do gênero Lycopersicon. Após uma pré-caracterização mediante clivagem com enzimas de restrição, 10 clones foram selecionados para seqüenciamento. A análise de seqüência dos alelos revelou identidade de nucleotídeos superior a 91% para a ORF completa. Quando a comparação foi realizada para os diferentes domínios que compõem o gene Sw-5, observou-se que as maiores divergências residem nas extremidades 5’ e 3’ do gene. A comparação entre as proteínas codificadas pelo alelo Sw-5 1 (confere resistência) e seu alelo sw-5 2 (confere suscetibilidade) revelou 53 substituições ao nível de aminoácidos. Destas substituições, 14 são não-sinônimas e se concentram na extremidade amino e nas LRR, indicando o provável envolvimento dessas regiões na especificidade da resistência. A análise funcional dos alelos do loco Sw-5 e a construção de quimeras entre os alelos Sw-5 e sw-5 2 estão em andamento e auxiliarão a estabelecer as bases moleculares da resistência do tomateiro a tospovírus.Lycopersicon peruvianum, L. chilense and L. hirsutum germplasm was inoculated with five isolates from the tospovirus species Tomato spotted wilt virus , Tomato chlorotic spot virus , Chrysanthemum stem necrosis virus, and Groundnut ringspot virus. Several introductions showed resistance to all isolates. However, in some cases, resistance isolate-specific was also observed. PI 126928, LA 444/1, LA 371 and PI 126944 of L. peruvianum and LA 130 and LA 2753 of L. chilense were the most resistant materials. Inheritance studies showed that two independent genes, one dominant and one recessive, control the PI 134417 resistance. Allelism tests revealed that this dominant gene is not an allele of the Sw-5 locus. The resistance derived from PI 126928 segregated as a trait controlled by two or more non-linked dominant genes. Sixteen alleles of the Sw-5 locus were PCR-amplified from four Lycopersicon species, cloned and sequenced. The alleles possess more than 91% of identity at nucleotide level; the largest divergence was observed in the 5 ́and 3 ́ extremities. Sequence comparisons between the resistance allele Sw-5 1 and the susceptible allele Sw-5 2, revealed 53 amino acid substitutions; fourteen substitutions are non-synonymous and located at the amino and carboxi terminus of the protein. The functional analysis of the other alleles and the construction of chimeras between Sw-5 1 and Sw-5 2 will allow to identify the protein region involved in the resistance specificity.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

PROGRESSO E ARRANJO ESPACIAL DO MOSAICO DOURADO EM FAVA

The Golden mosaic, caused by Bean golden mosaic virus (BGMV), causes severe losses to faba beans (Phaseolus lunatus L.) growers in the Northeastern region of Brazil. This study aimed at analyzing the temporal and spatial aspects of the disease at two experimental plots (A and B with 1360 m2 each) cultivated with faba beans located in Rio Largo, Alagoas. The evaluation of the disease incidence was done by visual in-spection of all plants at 30, 60, 90, 120 and 150 days after planting (DAP). The epidemics were compared with respect to initial incidence (yo), maximum incidence (ymax), rate of disease progression (RDP), and area under the disease progress curve (AUDPC). The spatial pattern of the disease was analyzed by isopath mapping, ordi-nary runs, fitting the beta-binomial distribution and spatial autocorrelation. The analysis of the progress curves showed that the epidemic was significantly (P≤0.05) faster in plot B, probably associated with increased expo-sure to whitefly populations from areas external to assay. The within rows aggregation increased along with the period after planting and the increase in the disease incidence in both areas. In plot A, there was a slight pre-dominance of aggregation within the rows while, in plot B, the across rows aggregation of sick plants was prevalent

Identificação e caracterização de espécies de Colletotrichum associadas à antracnose de anonáceas no estado de Alagoas

No Brasil, Colletotrichum gloeosporioides é a única espécie associada à antracnose de anonáceas. Contudo, apenas características morfológicas têm sido utilizadas na identificação. Assim, o objetivo do trabalho foi identificar e caracterizar espécies de Colletotrichum que causam a antracnose nas culturas da pinha e da graviola no Estado de Alagoas. Cinquenta e um isolados, obtidos de folhas de pinheira e gravioleira com sintomas típicos da doença, foram coletados em Maceió, Palmeira dos Índios e União dos Palmares. Os isolados foram inoculados em folhas destacadas de ambas as culturas e caracterizados morfologicamente através da morfometria de conídios e apressórios, e molecularmente por meio do sequenciamento da região ITS. Verificou-se que todos os isolados foram patogênicos a ambas as espécies. Na caracterização morfológica, os isolados foram agrupados em três grupos: M1, M2 e M3. O grupo M1 foi formado por 32 isolados com características relacionadas a C. gloeosporioides. No grupo M2, constituído de 15 isolados, predominaram características de C. boninense, enquanto, no grupo M3, composto por quatro isolados, as características foram típicas de C. fragariae. A análise filogenética, da mesma forma, também resultou em três grupos (F1, F2 e F3), os quais, de modo geral, estiveram em concordância com os dados da morfologia. O grupo filogenético F1 concentrou os isolados do grupo morfológico M1 e sequências de referências de C. gloeosporioides e C. fragariae. O grupo F2, que agrupou as sequências de C. boninense, concentrou os isolados do grupo morfológico M2. Finalmente, o grupo F3 incluiu sequências de C. magna e outros quatro isolados desse estudo. Assim, foi possível comprovar que quatro espécies de Colletotrichum são responsáveis pela antracnose em pinheira e gravioleira em Alagoas: C. gloeosporioides, C. boninense, C. fragariae e C. magna

Contrasting genetic structure between two begomoviruses infecting the same leguminous hosts

Begomoviruses are whitefly-transmitted, ssDNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as ‘mixing vessels’ where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared with cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, Phaseolus lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were sampled from two regions of Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. We observed a higher degree of genetic variation in isolates from MaYSV populations than BGMV populations, which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography