European apple canker: morphophysiological variability and pathogenicity in isolates of Neonectria ditissima in southern Brazil / Cancro europeu da macieira: variabilidade morfofisiológicas e patogenicidade de isolados de Neonectria ditissima no sul do Brasil

ABSTRACT: European apple canker (EC) is caused by Neonectria ditissima, a pathogen officially registered as a quarantine pest in 2012. Thirty-five isolates of N. ditissima of different geographical regions of southern Brazil from apple branches showing symptoms of EC were identified by the specific pair primers Ch1 and Ch2 and analyzed concerning the virulence on Gala apple cultivar and morphophysiological characteristics. The disease symptoms were characterized and the isolates compared based on average mycelium growth (AMG), mycelium growth index (MGI), colony color, conidia type, dimensions and growth on potato dextrose agar (PDA), malt agar (AM), and synthetic SNAY (SN) culture media. Nineteen isolates showed the greatest AMG on PDA, forming three growth groups of 35.56 (GI), 52.71 (GII), and 62.67mm (GIII). Seven isolates showed MGI greater than 4.0mm diameter on PDA compared with that on AM and SN. The highest conidia production was on SN, and the predominant colony color in all media was white to beige with central pigmentation of brown and borders colored in shades of beige. There were significant differences among the average dimensions of micro- and macroconidia on PDA, AM, and SN. The pathogenicity was confirmed for all isolates despite of different morphophysiological characteristics. There was no correlation among isolates morphophysiological variability, virulence, and geographical origin.

RESUMO: O cancro europeu (CE) da macieira é causado pelo fungo Neonectria ditissima e foi oficialmente registrado como praga quarentenária em 2012. Foram avaliados 35 isolados de N. ditissima provenientes de ramos de macieira com sintomas típicos de CE de diferentes regiões do sul do Brasil. Os isolados foram identificados pelos primers específicos Ch1 e Ch2 e analisados quanto as características morfofisiológicas e virulência na cultivar de macieira Gala. Os sintomas foram caracterizados e os isolados comparados com base no crescimento micelial médio (CMM), índice de crescimento micelial (ICM), coloração das colônias e tipos, dimensões e produção de conídios em meios de cultura batata-dextrose-ágar (BDA), malte-ágar (MA) e meio sintético SNAY (SS). Dezenove isolados tiveram os maiores CMM sobre BDA, formando três grupos de crescimento de 35.56 (GI), 52.71 (GII) e 62.67mm (GIII). Sete isolados tiveram ICM maior de 4mm de diâmetro sobre BDA quando comparados com os meios MA e SS. A maior produção de conídios ocorreu sobre o meio SS e a coloração predominante foi de branco-à-bege com pigmentação central marrom e bordas de colônia em tons de bege. Foram observadas diferenças significativas nas dimensões médias dos micro- e macroconídios sobre os meios de culturas BDA, MA e SS. Todos os isolados foram patogênicos na cultivar de macieira gala, independentemente das diferenças morfofisiológicas. Não foram observadas correlações entre a variabilidade morfofisiológicas, virulência e origem geográfica dos isolados.

Metodologia para determinação da zona de abscisão foliar em pomáceas / Methodology to determine abscission zone in apple trees

Estudos anatômicos e de ontogênese da abscisão foliar em pomáceas são escassos. A queda foliar promovida após formada a zona de abscisão é um processo natural nas pomáceas, e prolonga-se durante o outono, até que ocorram as primeiras geadas no Sul do Brasil. Ainda não se conhece até quando as folhas de macieiras permanecem funcionais. O objetivo deste trabalho foi desenvolver uma metodologia que permita estudar o início e o fim das zonas de abscisão foliar de macieiras. O trabalho foi realizado nas cultivares 'Galaxy' e 'Fuji' em pomares comerciais de macieira, de Vacaria, Rio Grande do Sul. O método proposto consistiu em coletar,ramos do ano, com 10cm a 20cm de comprimento, e utilizar um segmento contendo uma parte do caule, uma gema axilar e a base do pecíolo, descartando a parte das quatro folhas do ápice. Os segmentos foram fixados, seccionados e corados para a observação da zona de abscisão em microscopia óptica. Os resultados mostraram que a metodologia utilizada permitiu descrever a zona de abscisão em macieiras e que ela é formada por células alongadas com parede celular delgada, apresentando lignificação junto à parede celular. O processo manteve a gema intacta e ocorreu no sentido adaxial para o abaxial. A abscisão foliar natural, em plantas de Galaxy e Fuji em sintomas de doença e em condições ambientais favoráveis, pode ser acompanhada 7 e 14 dias antes e depois da colheita, e após, com intervalos de 14 e 21 dias até maio. A partir de maio, as coletas devem ser feitas semanalmente. Essas informações serão úteis na tomada de decisão para realizar práticas culturais e/ou de controle químico, para a manutenção das folhas sadias, resultando no maior acúmulo de reservas de carboidratos e/ou redução do número de aplicações de fungicidas.

Studies about anatomical and ontogeny of leaf abscission in pome fruit trees are scarce. The leaf fall occurs after the abscission zone is promoted, being a natural process in pome fruit, and it lasted from the fall until the first frost in southern Brazil. It is not known yet how long the leaves of apple trees remained functional. The aim of this study was to develop a methodology to study the beginning and the end zones of leaf abscission of Galaxy and Fuji apple trees. The study was conducted in commercial apple orchards in Vacaria, Rio Grande do Sul. The proposed method consisted of collecting branches to 10cm to 20cm in length, and use a segment containing a portion of the stem, an axillary bud and the base of the petiole, discarding the part of the four-leaf apex. The segments were fixed, sectioned and stained for observation of the abscission zone in optical microscopy. The methodology allowed the understanding of the abscission zone on apple trees. The abscission zone was formed by elongated cells with thin walls, presenting cell wall lignification. The process kept the bud intact and occurred from the adaxial to abaxial surface. The natural leaf abscission in Galaxy and Fuji plants without symptoms of disease and favorable environmental conditions , can be accompanied by 7 and 14 days before and after harvest, and after intervals of 14 and 21 days until May. From May, collections should be weekly. This information will be useful in making a decision to carry out cultural practices and / or chemical control, for the maintenance of healthy leaves, resulting in greater accumulation of carbohydrate reserves and / or reducing the number of fungicide applications.

Meio semiseletivo para recuperação e quantificação de Cryptosporiopsis perennans em maçãs / Semi-selective medium for recovery and quantification of Cryptosporiopsis perennans from apple fruits

A podridão "olho de boi", causada pelo fungo Cryptosporiopsis perennans, é uma das doenças de pós-colheita da macieira mais importantes no Brasil. Os objetivos deste trabalho foram desenvolver e avaliar um meio semiseletivo para a recuperação e mensuração da população epífita de C. perennans em maçãs "Fuji". A suplementação de BDA com tetraciclina, nistatina e iprodiona, nas respectivas concentrações de 12,5; 1 e 1µg p.a mL-1, proporcionou o desenvolvimento de colônias de C. perennans estatisticamente igual (P>0,05) ao observado no meio BDA. Em condições de infecção natural e em amostras pré-inoculadas com C. perennans, o meio semiseletivo permitiu detectar e quantificar a população do fungo na superfície dos frutos, além de ter inibido o crescimento de fungos leveduriformes, dematiáceos e leveduras. Em lavagens de frutos previamente inoculados, o número de conídios recuperados com o meio semiseletivo apresentou correlação (r=0,88; P<0,05) com a concentração do inóculo aspergido sobre os frutos.

Bull's eye rot, caused by Cryptosporiopsis perennans, is one of the most important apple's posharvest diseases in Brazil. The aim of this research was to develop and evaluate a semi-selective media to recovery and quantification of the epiphytic population of C. perennans on the apple fruit surface. A PDA medium amended with tetracycline, nistatine and iprodione in concentrations of 12.5; 1 and 1µg i.a.mL-1, respectively, allowed the development of C. perennans colonies statistically similar (P>0.05) to those in PDA media. Nistatine concentrations higher than 1µg.mL-1 inhibited the growth of the pathogen. Under natural infection conditions and pre-inoculation with C. perennans, the media allowed the detection and quantification of the fungal population on the fruit surface without affecting C. perennans development. Number of conidia observed in pre-inoculated fruits was correlated (r=0.88; P<0.05) to inoculum concentration sprayed on the fruit surface.

On the origin and spread of the Scab disease of apple: out of central Asia.

BACKGROUND: Venturia inaequalis is an ascomycete fungus responsible for apple scab, a disease that has invaded almost all apple growing regions worldwide, with the corresponding adverse effects on apple production. Monitoring and predicting the effectiveness of intervention strategies require knowledge of the origin, introduction pathways, and population biology of pathogen populations. Analysis of the variation of genetic markers using the inferential framework of population genetics offers the potential to retrieve this information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a population genetic analysis of microsatellite variation in 1,273 strains of V. inaequalis representing 28 orchard samples from seven regions in five continents. Analysis of molecular variance revealed that most of the variation (88%) was distributed within localities, which is consistent with extensive historical migrations of the fungus among and within regions. Despite this shallow population structure, clustering analyses partitioned the data set into separate groups corresponding roughly to geography, indicating that each region hosts a distinct population of the fungus. Comparison of the levels of variability among populations, along with coalescent analyses of migration models and estimates of genetic distances, was consistent with a scenario in which the fungus emerged in Central Asia, where apple was domesticated, before its introduction into Europe and, more recently, into other continents with the expansion of apple growing. Across the novel range, levels of variability pointed to multiple introductions and all populations displayed signatures of significant post-introduction increases in population size. Most populations exhibited high genotypic diversity and random association of alleles across loci, indicating recombination both in native and introduced areas. CONCLUSIONS/SIGNIFICANCE: Venturia inaequalis is a model of invasive phytopathogenic fungus that has now reached the ultimate stage of the invasion process with a broad geographic distribution and well-established populations displaying high genetic variability, regular sexual reproduction, and demographic expansion.

Endophytic yeasts and filamentous fungi associated with southern Brazilian apple (Malus domestica) orchards subjected to conventional, integrated or organic cultivation.

Endophytic fungi were isolated from leaves, flowers and fruit of healthy apple trees (Malus domestica , Borkh.) growing in southern Brazilian orchards under three different cultivation systems (conventional, integrated and organic), during two vegetative cycles. The greatest total number of endophytic isolates was obtained from the orchards under organic cultivation when compared to integrated and conventional cultivation systems. Filamentous fungi from the genera Colletotrichum , Xylaria and Botryosphaeria were the most frequent ones and the most representative yeast genera were Sporobolomyces , Rhodotorula , Debaryomyces and Cryptococcus . It is suggested that some isolates may be used as indicators of the different management systems.