Survey of fungi associated with cassava root rot from different producing regions in Brazil

Although root rot is one of the major diseases affecting Brazilian cassava (Manihot esculenta Crantz.), little is known about the diversity of root rot pathogens. In this study, diseased plants exhibiting root rot symptoms were collected from cassava-producing regions in five Brazilian states: Bahia, Sergipe, Paraíba, Maranhão, Tocantins, and Paraná. Seventy isolates were obtained and assigned to species complexes based on rDNA's ITS (internal transcribed spacer of the ribosomal DNA) region (ITS1, ITS2 and 5.8S). A total of seven species complexes was found belonging to the genus Fusarium (56/74), followed by Lasiodiplodia (8/74), Neoscytalidium (6/74), and Diaporthe/Phomopsis complex (2/74), Phytophthora, and Corallomycetella (1/74 each). These species were distributed differently according to sample locations and states, but overall, the F. solani species complex (FSSC) was the most prevalent. A number of phylogenetic lineages had not been previously reported as being associated with cassava-root rot disease, such as: F. graminearum (FGSC), F. incarnatum-equiseti (FIESC) and F. chlamydosporum (FCSC) complexes, and a phylogenetic lineage most closely related to P. phaseolorum. Results suggest the need to improve knowledge of the species associated with cassava, including multilocus phylogeny for a more specific characterization, and differences in the resistance background associated with these species, as a strategy to incorporate resistance to multiple pathogens in cassava breeding programs

Caracterização molecular de espécies de Colletotrichum associadas a frutos no Brasil

Colletotrichum species are considered one of the most economically important plant pathogens. They cause many losses in tropical, subtropical and temperate regions affecting a wide range of plant species. In tropical and subtropical regions C. gloeosporioides and C. acutatum are associated with significant losses on pre and post-harvest anthracnoses. There are still many features to understand about Colletotrichum biology and its systematics. The accurate identification of species involved with each anthracnose is of high relevance to establish management strategies to control the disease. Although the great advances on Colletotrichum systematics, species complex such as C. gloeosporioides and C. acutatum are used in a broad sense in Brazil. These complexes were recently investigated and showed to be highly genetic and geographic variable. In this study multigene analysis were carried out based on ITS, GAPDH, CHS-1, TUB2 and CAL or HIS3 partial sequences for strains of C. gloesporioides and C. acutatum complexes collected from fruit crops in Brazil. Strains from different countries and exepitypes and others sequences available on GenBank from the species accepted on both complexes were added on dataset. Six strains from C. gloeosporiodes complex and five for C. acutatum were selected based on multigene phylogeny to investigate the pathogenicity through inoculations on detached fruit. The multigene phylogenies showed the occurrence of species in Brazil related to those complexes with a high genetic variability among them. The phylogeny of Brazilian strains belonging to the C. gloeosporioides complex showed that C. siamense represents the most genetically and host-specific variable clade. In contrast, C. asianum clade grouped only strains isolated from mango. The strains from this clade used on pathogenic test were not able to infect avocado and one of the strains caused symptoms only on mango. All strains from Brazil grouped in one subclade within the C. fructicola clade and seem to represent a genetically distinct group. C. theobromicola is first reported causing anthracnose on acerola fruit. Three new species (C. polyphialidicum, C. paranaense and C. pruni) belonging to the C. acutatum complex were recognized and their morphologic descriptions were provided. The pathogenic test for the strains in the C. acutatum complex showed their cross infection ability, but in some cases the larger lesions were produced on the original host. Most brazilian strains from C. acutatum complex grouped in one subclade within the C. nymphaeae clade and seem to be genetically distinct.Fungos do gênero Colletotrichum são considerados um dos mais importantes economicamente na Fitopatologia. Espécies desse gênero são encontradas amplamente disseminadas e estão associadas a diversas espécies de plantas hospedeiras. Em regiões tropicais e subtropicais, espécies dos complexos C. gloeosporioides e C. acutatum são a principal causa das antracnoses em pré e póscolheita de frutos e consequentemente causam significantivas perdas. Ainda há muitos aspectos a serem compreendidos sobre o gênero Colletotrichum, como a biologia e a sistemática. A acurada identificação das espécies associadas a antracnoses é de suma importância para o estabelecimento de estratégias de controle. No entanto, apesar dos grandes avanços na sistemática desse gênero, complexos de espécies como aquelas citadas acima são tratados de modo genérico no Brasil. Estes complexos de espécies foram recentemente estudados e considerados geneticamente e geograficamente variáveis. Neste sentido, o presente trabalho teve como objetivo caracterizar isolados de Colletotrichum spp. associados a diferentes frutos e regiões do Brasil por meio de análise filogenética. Para análise multilocus, foram utilizadas sequências parciais dos genes ITS, GAPDH, CHS-1, TUB2 and CAL ou HIS3. Sequências de espécies-tipos disponíveis no GenBank e de isolados de diferentes países foram adicionadas ao conjunto de dados. Com base nos resultados obtidos por meio de filogenia multilocus, seis isolados do complexo C. gloeosporiodes e cinco do complexo C. acutatum foram escolhidos para testes de patogenicidade cruzada. A espécie C. siamense, pertencente ao complexo C. gloeosporioides, foi a mais variável geneticamente e quanto ao hospedeiro de origem. Diferentemente, apenas isolados obtidos de manga se agruparam no clado C. asianum. Isolados agrupados neste clado não infectaram abacate e um dos isolados (CPC 20969) causou sintomas apenas em manga. No clado C. fructicola, isolados coletados no Brasil se agruparam em um subclado e parecem representar um grupo geneticamente distinto. A espécie C. theobromicola é relatada pela primeira vez em acerola. Foram identificadas três novas espécies, C. polyphialidicum, C. paranaense e C. pruni, pertencentes ao complexo C. acutatum. Isolados brasileiros agrupados no clado C. nymphaeae parecem representar um grupo geneticamente distinto, todos se agruparam em um subclado. Isolados do complexo C. acutatum utilizados no teste de patogenicidade provocaram sintomas nos hospedeiros testados, porém, em algumas inoculações, as lesões foram maiores no hospedeiro de origem

Sensitivity of Hemileia vastatrix to the fungicides azoxystrobin and tebuconazole

O monitoramento da sensibilidade de patógenos a fungicida é etapa necessária para compreender os processos associados ao surgimento da resistência, a distribuição de isolados resistentes, a evolução de populações insensíveis e seus impactos no controle das doenças. O manejo da ferrugem do cafeeiro (Hemileia vastatrix), doença mais destrutiva desta cultura, é baseado no controle químico, porém não havia informações quanto à sensibilidade do patógeno a fungicidas. Coletaram-se isolados de H. vastatrix nas principais regiões produtoras de Minas Gerais e Espírito Santo, e determinou-se a sensibilidade dos isolados aos fungicidas azoxistrobina e tebuconazol, por meio da estimativa da dose efetiva para inibir 50% da germinação ou crescimento do patógeno (DE50). Os valores de DE50 variaram entre 0,004 e 0,12 μg/ml para azoxistrobina e 0,01 a 5,11 μg/ml, para tebuconazol. Não houve associação dos valores de DE50 com a procedência dos isolados ou com o histórico de aplicação de fungicidas, tanto para azoxistrobina (χ2 = 0,63; P=0,42 e χ2 = 0,15; P=0,70) quanto para tebuconazol (χ2 = 3,23; P=0,20 e χ2 = 0,24; P=0,89). Não houve correlação entre distância geográfica e DE50 dos fungicidas. Portanto, não houve evidência de agrupamento espacial quanto à sensibilidade dos isolados aos dois fungicidas. Ambos, azoxistrobina e tebuconazol, reduziram a germinação e elongação do tubo germinativo. Não foi possível amplificar e sequenciar fragmento do gene CYP51 relacionadas à redução da sensibilidade de tebuconazol. Para azoxistrobina, obteve-se sequência parcial do gene CYTB, porém, em conformidade com o teste fenotípico, não se constataram as mutações que conferem insensibilidade ao fungicida. Não há evidência de resistência de H. vastatrix aos fungicidas azoxistrobina e tebuconazol nas populações de Minas Gerais e Espírito Santo.Monitoring the sensitivity of pathogens to fungicides is a crucial step to understand the processes associated with the emergence of resistance, distribution of resistant isolates, the evolution of insensitive populations, and the impacts on disease control. The management of coffee leaf rust (Hemileia vastatrix), the most destructive coffee disease, is based on chemical control, however there was no information about sensitivity of H. vastatrix to fungicides. Isolates of H. vastatrix were collected in the main producing regions of Minas Gerais and Espírito Santo states, and the sensitivity to azoxistrobin and tebuconazole was determined by estimating either the effective dose to inhibit 50% germination or fungal growth (ED50). The ED50 values for azoxistrobin varied between 0.004 and 0.12 μg/ml and for tebuconazole between 0.01 to 5.11 μg/ml. There was no association of ED50 values with isolate origin or fungicide regimes for both azoxistrobin (χ2 = 0.63; P=0.42 e χ2 = 0.15; P=0.70) and tebuconazole (χ2 = 3.23; P=0.20 e χ2 = 0.24; P=0.89). There was no association between geographic distance and ED50 values, therefore there was no spatial clustering of sensitivity of isolates to both fungicides. Both fungicides reduced germination and germ tube elongation. The primers used did not allow amplification and sequencing of the genomic regions associated with tebuconazole action site in the pathogen. For azoxistrobin, partial sequence was obtained for the CYTB gene, but, consistent with the results from the phenotypic test, no mutations known to confer fungicide insensitivity were detected. There is no evidence of resistance in H. vastatrix to both azoxistrobin and tebuconazole.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Survey of fungi associated with cassava root rot from different producing regions in Brazil

ABSTRACT: Although root rot is one of the major diseases affecting Brazilian cassava (Manihot esculenta Crantz.), little is known about the diversity of root rot pathogens. In this study, diseased plants exhibiting root rot symptoms were collected from cassava-producing regions in five Brazilian states: Bahia, Sergipe, Paraíba, Maranhão, Tocantins, and Paraná. Seventy isolates were obtained and assigned to species complexes based on rDNA's ITS (internal transcribed spacer of the ribosomal DNA) region (ITS1, ITS2 and 5.8S). A total of seven species complexes was found belonging to the genus Fusarium (56/74), followed by Lasiodiplodia (8/74), Neoscytalidium (6/74), and Diaporthe/Phomopsis complex (2/74), Phytophthora, and Corallomycetella (1/74 each). These species were distributed differently according to sample locations and states, but overall, the F. solani species complex (FSSC) was the most prevalent. A number of phylogenetic lineages had not been previously reported as being associated with cassava-root rot disease, such as: F. graminearum (FGSC), F. incarnatum-equiseti (FIESC) and F. chlamydosporum (FCSC) complexes, and a phylogenetic lineage most closely related to P. phaseolorum. Results suggest the need to improve knowledge of the species associated with cassava, including multilocus phylogeny for a more specific characterization, and differences in the resistance background associated with these species, as a strategy to incorporate resistance to multiple pathogens in cassava breeding programs