Identificação molecular e patogenicidade de espécies de Berkeleyomyces associadas a hortaliças no Brasil com ênfase na cultura da alface

Abstract

Lettuce (Lactuca sativa L.; Asteraceae) is commonly consumed and widely cultivated around the world, being the most important crop within the group of leafy vegetables. Black root rot (caused by Berkeleyomyces species) has affected the production of this crop in several countries in recent years. The genus Berkeleyomyces is currently represented by two species B. basicola and B. rouxiae (a cryptic sister species of B. basicola). However, no information is available about the Berkeleyomyces species present in association with the lettuce in neotropical areas. The present study is the first attempt to characterize Berkeleyomyces species associated with lettuce and other leafy vegetables via molecular approches, including specific PCR primers able to discriminate the two currently described Berkeleyomyces species. The host range of the Berkeleyomyces isolates from Brazil was also assessed. The molecular identification of the isolates was initially performed based upon the sequence information of the genomic regions LSU (large subunit rDNA) e MCM7 (DNA replication licensing factor MCM7). The sequences of 53 Berkeleyomyces isolates obtained from five different hosts were compared with 26 sequences of reference isolates from members of the Ceratocystidaceae family as well as with isolates from B. basicola and B. rouxiae available in the GenBank database. In the LSU phylogenetic tree, all isolates clustered to Berkeleyomyces species. The MCM7 region was informative and gave support to distinguish the two Berkeleyomyces species. Species-specific fragments of approximately 110 bp were generated via PCR when employing primers specific for B. basicola (basi60s_F and basi60s_R) and for B. rouxiae (roux60s_F and roux60s_R). These two primer pairs displayed similar results to the identification obtained via phylogeny. In lettuce, 21 isolates were classified as B. basicola and 24 isolates as B. rouxiae. This is the first formal record of B. basicola infecting lettuce in the world and the first identification of B. rouxiae in Brazil. Two isolates of each pathogen species (B. basicola and B. rouxiae) and 26 cultivars of ten vegetable crops were used in host range assays. The seedlings were produced in polystyrene trays and kept under greenhouse conditions. Plantlets were irrigated twice a day. At 22 days, they were transplanted to trays of 60 cells, filled with a third of the substrate colonized with each isolate at a concentration of 7.5 x 105 conidia/g of substrate. The experimental design was completely randomized in a 4 x 26 factorial arrangement, with six replicates plus controls (= mock-inoculated plants). The evaluations were carried out 22 days after inoculation using as criterium a disease severity scale (ranging from 1 = absence of symptoms to 5 = more than 90% of the roots severely affected). Lettuce, chicory, radicchio, and endive were identified as novel natural hosts of B. basicola and B. rouxiae for the first time in Brazil and in the world. Furthermore, new potential hosts of these pathogens were also identified (Swiss chard cultivars ‘Verde Escura’ and ‘Artemis’, lettuce cultivars ‘Aurélia’, ‘Elisa’ and ‘Leila’, chicory cultivar ‘Spadona’, ‘Folha Amarela’, ‘Cabeça-Pão-de-Açúcar’ and ‘Cabeça Vermelha’, endive cv. ‘Catalogna Folha Larga’ and coriander cv. ‘Verdão’).