Maria A. Ferreira

Species or Genotypes? Reassessment of Four Recently Described Species of the Ceratocystis Wilt Pathogen, Ceratocystis fimbriata, on Mangifera indica.

Ceratocystis wilt is among the most important diseases on mango (Mangifera indica) in Brazil, Oman, and Pakistan. The causal agent was originally identified in Brazil as Ceratocystis fimbriata, which is considered by some as a complex of many cryptic species, and four new species on mango trees were distinguished from C. fimbriata based on variation in internal transcribed spacer sequences. In the present study, phylogenetic analyses using DNA sequences of mating type genes, TEF-1α, and β-tubulin failed to identify lineages corresponding to the four new species names. Further, mating experiments found that the mango isolates representing the new species were interfertile with each other and a tester strain from sweet potato (Ipomoea batatas), on which the name C. fimbriata is based, and there was little morphological variation among the mango isolates. Microsatellite markers found substantial differentiation among mango isolates at the regional and population levels, but certain microsatellite genotypes were commonly found in multiple populations, suggesting that these genotypes had been disseminated in infected nursery stock. The most common microsatellite genotypes corresponded to the four recently named species (C. manginecans, C. acaciivora, C. mangicola, and C. mangivora), which are considered synonyms of C. fimbriata. This study points to the potential problems of naming new species based on introduced genotypes of a pathogen, the value of an understanding of natural variation within and among populations, and the importance of phenotype in delimiting species.

Movement of Genotypes of Ceratocystis fimbriata Within and Among Eucalyptus Plantations in Brazil

Ceratocystis wilt on eucalyptus, caused by Ceratocystis fimbriata, was first recognized in 1997 in the state of Bahia, Brazil, but is now known in five other states and in four other countries. C. fimbriata is a native, soilborne pathogen in some parts of Brazil but we hypothesized that genotypes of the pathogen have been moved among plantations in rooted cuttings collected from diseased trees and within plantations on cutting tools. We used six microsatellite markers to identify 78 genotypes of C. fimbriata among 177 isolates from individual trees in 20 eucalyptus plantations. The highest gene and genotypic diversity values were found in plantations on formerly wild Cerrado forest in Minas Gerais, suggesting that the fungus was in the soil prior to planting eucalyptus. In contrast, one or only a few genotypes were found in plantations on previous pastureland (with no woody hosts) in Bahia and São Paulo, and most of these genotypes were found in a Bahian nursery or in one of two Bahian plantations that were sources for rooted cuttings. Sources of cuttings tended to be dominated by one or a few genotypes that may have been spread within the plantation on cutting tools.

Genetic Analyses Trace the Yunnan, China Population of Ceratocystis fimbriata on Pomegranate and Taro to Populations on Eucalyptus in Brazil

Genotypes of the Latin American wilt pathogen Ceratocystis fimbriata have been moved around the world in vegetatively propagated material of various crop plants, including Ipomoea batatas (sweet potato), Colocasia esculenta (taro), and Eucalyptus spp. When compared to a worldwide collection of isolates of C. fimbriata, isolates from taro, Punica granatum (pomegranate), and Eriobotrya japonica (loquat) from Yunnan Province, China were found to have sequences of internal transcribed spacer (ITS) rDNA and mating type genes that were identical to isolates from Eucalyptus in Brazil. Analyses of 35 isolates with 14 microsatellite markers revealed that the Yunnan population was nearly uniform, consisting of only 19 alleles and seven closely related genotypes, suggesting that the population is not natural and is the result of an introduction. As in comparisons of sequences of ITS rDNA and mating type genes, the microsatellite alleles of the Yunnan isolates were most similar to those of Eucalyptus isolates from Minas Gerais and Bahia, Brazil, where C. fimbriata is native, soilborne, and commonly infects cuttings of Eucalyptus spp. used for rooting in nurseries. Thus, the Yunnan population, which is causing severe losses on pomegranate, may have been indirectly derived from introductions of C. fimbriata in contaminated Eucalyptus cuttings from Brazil.

Genetic variability suggests that three populations of Ceratocystis fimbriata are responsible for the Ceratocystis wilt epidemic on kiwifruit in Brazil

Ceratocystis fimbriata is a native, soilborne pathogen in South America that causes a lethal wilt disease on a broad range of economically important plants. Ceratocystis wilt on kiwifruit (Actinidia spp.) was first recognized in 2010 in the state of Rio Grande do Sul, Brazil. The genetic variation among kiwifruit isolates was analized to determine if a single introduced strain of the pathogen was responsible for the epidemic or if there was substantial genetic variation in the population, suggesting that the fungus was soilborne and indigenous to the region. We used 14 microsatellite (simple sequence repeat, SSR) markers to identify 18 genotypes of C. fimbriata among 76 isolates from eight kiwifruit farms. The 18 genotypes clustered into three groups based on UPGMA analysis of the microsatellite alleles. The largest group comprised 60 isolates of 11 closely-related microsatellite genotypes obtained from seven of the eight farms. These genotypes appeared to have originated from a single farm that had supplied cuttings for grafting to the other farms. The population of the pathogen from the farm that supplied the cuttings had the highest level of genotypic diversity and relatively high gene diversity, suggesting that this source population represented an indigenous, soilborne population. Phylogenetic analyses of the DNA sequences of the mating type locus (including portions of MAT1-1-2 and MAT1-2-1) placed the isolates into three groups, corresponding to the three microsatellite groups. Most of the isolates, including all the tested isolates from the farm that supplied the cuttings, had mating type gene sequences that were distinct from other Brazilian populations of C. fimbriata. A second group comprised isolates from one farm that had mating type gene sequences typical of Mata Atlântica (Rio de Janeiro and São Paulo) populations of C. fimbriata on Colocasia esculenta and Mangifera indica. Three farms purchased kiwifruit plants or rootstocks from commercial nurseries in Brazil as well as scions from the source farm, and some of the isolates from these farms were genetically similar to Eucalyptus isolates of C. fimbriata from Bahia and Minas Gerais, Brazil. The kiwifruit epidemic in Rio Grande do Sul is the southern-most report of C. fimbriata in Brazil, and the primary pathogen population on kiwifruit appears to be indigenous and originated from a single farm that distributed the pathogen in grafting material. In addition, commercial nursery stock was also implicated as sources of C. fimbriata genotypes. The disease is a major limiting factor for kiwifruit production in southern Brazil, and the results suggest that clean planting stock will be important to successful production.

Genetic variation, morphology and pathogenicity of Ceratocystis fimbriata on Hevea brasiliensis in Brazil

Ceratocystis fimbriata causes diseases on a wide variety of plants in Brazil, including rubber tree (Hevea brasiliensis), on which it causes gray mold or moldy rot on tapping panels affecting latex yield. However, C. fimbriata isolated from rubber tree have not been critically studied. Phylogenetic analyses of sequences of ITS rDNA and a mating type gene placed rubber tree isolates from Acre and Bahia among Brazilian isolates of C. fimbriata from other hosts. In the analyses of 14 microsatellite loci, the rubber tree isolates from Bahia were identical to each other and had alleles similar to those of Brazilian isolates from mango and eucalyptus. The microsatellite alleles of the Acre rubber tree isolates were identical to each other but distinct from other Brazilian isolates. The rubber tree isolates were morphologically indistinguishable from each other and very similar to the isolates of C. fimbriata on Ipomoea batatas, on which the species was originally described. Based on inoculation experiments results, the Bahia and Acre rubber tree isolates do not appear to be host specialized, which is typical for Brazilian isolates of C. fimbriata sensu stricto.

Murcha-bacteriana: disseminação do patógeno e efeitos da doença sobre a clonagem do eucalipto

Este trabalho objetivou estudar a disseminacao de Ralstonia solanacearum por meio de mudas e avaliar os efeitos da murcha-bacteriana sobre a produtividade na clonagem do eucalipto. Amostras de minicepas, miniestacas e mudas de sete clones foram avaliadas quanto a presenca de infeccoes bacterianas. Em experimento especifico, avaliou-se o substrato de enraizamento como fonte de inoculo do patogeno. Em intervalos mensais, determinaram-se o enraizamento medio e o indice de produtividade (IP), visando avaliar os efeitos da doenca sobre a produtividade de minicepas. Constatou-se a presenca do patogeno em minicepas de cinco clones, enquanto todas as mudas de todos os setes clones avaliados apresentaram a doenca. Essa diferenca comprovou que a disseminacao do patogeno e a transmissao da doenca ocorreram durante o processo de clonagem do eucalipto. O substrato de enraizamento foi eficiente para a sobrevivencia do patogeno e como fonte de inoculo para a doenca. As mudas sem sintomas e com infeccoes sistemicas provaram o potencial de transmissao do patogeno para o campo no sistema de propagacao clonal do eucalipto. Os estresses fisiologicos causados pelas infeccoes bacterianas reduziram quatro vezes o IP dos minijardins clonais. As medidas adotadas foram eficientes para a erradicacao da doenca no viveiro e para evitar a disseminacao do patogeno para o campo.

Avaliação da resistência do eucalipto à murcha-bacteriana causada por Ralstonia solanacearum

The use of resistant eucalyptus clones is the most important strategy to disease control on the field. Thus, this work aimed to evaluated methods of Ralstonia solanacearum inoculation in order to select eucalyptus resistant to bacterial wilt. The inoculation methods were selected due to ease of operation and tested in order to evaluate four clones (hybrids E. urophylla x E. grandis): i) application of 5 mL pathogen inoculum (108 cfu/mL) on the root collar of seedlings; ii) application of 5 mL of pathogen inoculum (108 cfu/mL) on the root collar of seedlings after wound of the radicular system; iii) cut of 1/3 basal of the radicular system and immersion of the roots into pathogen inoculum, at the same concentration, for 1 min; and iv) conduction of seedlings transplanted to infested sand maintained in container with application at 15 days intervals (rate of 0.25 L/m3) and bacterial suspension (108 cfu/mL) and submitted to collection of sprouting, similarly as it is done in commercial clonal mini-garden. The seedlings transplant to infected sand presented a higher incidence of the disease (bacterial exudation), ranging from 81 to 100%. The second trial aimed to evaluate resistance of different eucalyptus species, using the method of transplanting seedlings to the infected sand. The evaluation of the infection by exudation bacterial method was more appropriate to compare the genotypes. The bacterial wilt intensity was affected by plant and environment conditions. Every species showed disease symptoms, but E. tereticornis and E. grandis showed, respectively, the lowest (33.3%) and the highest (91.7%) percentage of infected genotypes (susceptible). The results of this work are important tools (inoculation method) and resistance sources (eucalyptus species) to bacterial wilt disease control in eucalyptus.

Ocorrência de Ceratocystis fimbriata em Kiwi (Actinidia deliciosa) no sul do Brasil

In a routine inspection on a plantation of kiwi (Actinidia deliciosa) plants were found with wilt symptoms, internal browning and death of the stem tissues. Isolations from infected tissues and soil rhizosphere, allowed to obtain a culture of fungus with morphological characteristics similar to Ceratocystis fimbrita whose identification was confirmed based on the sequencing of the ITS region of rDNA. The pathogenicity test was conducted in Monty and Farroupilha varieties. It was found that the causal agent of the disease in kiwi belong to the C. fimbriata complex and to the group of Latin America and the isolates inoculated were pathogenic to two varieties tested.

Procedimento estatístico para validação de escalas diagramáticas na quantificação de doenças

RESUMO – Na validacao de escalas diagramaticas de doencas e comum utilizar a regressao linear simples para testar a identidade entre dados estimados e valores reais de severidade de doenca. Nestes casos, apos ajustar a regressao linear, as hipoteses H01: β 0 = 0 e H02: β 1 = 1 ao testadas, mas nao de forma simultânea, ou seja, se β 0 = 0 ao mesmo tempo em que β 1 = 1, ou, se β 1 = 1 ao mesmo tempo em que β 0 = 0. O objetivo deste trabalho foi propor um novo procedimento estatistico para validacao de escalas diagramaticas, denominado L&O. Este procedimento resulta da combinacao do teste F, modificado por Graybill (1976), teste t para o erro medio e analises do coeficiente de correlacao linear. Este procedimento foi aplicado para testar a identidade entre valores estimados e valores reais de severidade da doenca causada por Quambalaria eucalypti em eucalipto. Os resultados mostraram que o procedimento estatistico foi eficiente, nao subjetivo e permitiu tambem testar a identidade entre quaisquer dois vetores ou grupos de dados quantitativos.

Canker disease caused by Chrysoporthe doradensis and C. cubensis on Eucalyptus sp. and Tibouchina spp. in Brazil

Chrysoporthe species are known to cause canker on hosts belonging to Myrtaceae and Melastomataceae families. Cankers occur on tree trunks and branches and may reduce growth and lead to plant death. In this study, the incidence and pathogenicity of Chrysoporthe cubensis and other species causing canker on Eucalyptus sp., Tibouchina heteromalla and T. granulosa were examined. The isolates were collected in Maranhão (MA) and Minas Gerais (MG) in Brazil. Sequence analysis of beta-tubulin and actin genomic regions confirmed the presence of C. cubensis and C. doradensis on clones of the hybrid Eucalyptus grandis x E. urophylla, T. granulosa, and T. heteromalla in Brazil. Morphological characterization enabled the identification of the isolates from both genera primarily based on differences in conidial size and shape. The isolates were pathogenic to ten Eucalyptus clones and Tibouchina plants. Our results contribute new knowledge of Chrysoporthe species causing diseases of woody plants in Brazil of importance to Eucalyptus breeding programs when screening for resistance. Furthermore, this is the first report of C. doradensis infecting Eucalyptus and T. granulosa, as well as the first record of C. cubensis infecting T. heteromalla in Brazil.