Abstract
Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens’ AGL-1 concentration (OD600), 0.8; concentration of Colletotrichum conidium, 1×106 conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25°C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300–400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant’s pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).
Highlights
Stylosanthes guianensis, a diverse tropical and subtropical forage legume, is native to South America, Central America, and Africa
Based on the constructed anthracnose genetic transformation system of S. guianensis, this study aimed to clone genes related to pathopoiesia of pathogenic bacteria
Results of the differences in concentrations of A. tumefaciens and Colletotrichum spore liquid were analyzed by ANOVA
Summary
Stylosanthes guianensis, a diverse tropical and subtropical forage legume, is native to South America, Central America, and Africa. It is used for grazing cattle and raising livestock. To China from Australia, Africa, and South America began in the late 1960s and has continued to the present. Anthracnose of Stylosanthes, mainly caused by Colletotrichum gloeosporioides (Penz.) Penz. Fungal colonization on the leaf tissue follows and is associated with host cell necrosis, leading to a blight-like symptom and the formation of spore masses as acervuli [6,7]. Research in Australia, Colombia, Brazil, and China has identified two biotypes of C. gloeosporioides infecting Stylosanthes sp. The diversity among strains pathogenic on Stylosanthes and their relationship with other strains were analyzed doi:10.1371/journal.pone.0111172.g001
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
