Unveiling the association of Colletotrichum species with soybean anthracnose using histological, populational and genomic approaches

Abstract

The large soybean cultivation in Brazil, mostly under monoculture and non-tillage system, affects the intensity of diseases. Amongst them, anthracnose is the most prominent, as soybeans are susceptible to infection at all stages of development. Frequent reports on the increase incidence in the northern and mid-western regions indicate that chemical control program for fungal diseases in soybean has not been effective against anthracnose. Up to 2007, only C. truncatum had been reported as the causal agent of the disease in Brazil; an additional association of C. cliviae was reported from 2012 on in Mato Grosso State. To better understand the association of pathogenic Colletotrichum species to soybean anthracnose, this proposal aims: a) developing a LAMP-based test to detect C. cliviae in asymptomatic soybean seeds and plant samples; b) elucidating the direction of transmission of C. cliviae and C. truncatum from soybean seeds and plants; c) testing in vitro sensibility of C. cliviae (and C. truncatum?) to fungicides; d) accessing the genetic structure of C. truncatum populations, based on SSR markers and multilocus sequencing; d) inferring the predominant reproductive mode in these populations; e) fine-scale investigating demographic processes, using SNP markers in a population genomic approach; f) examining the influence on the development of anthracnose symptoms by pathogenic C. cliviae and C. truncatum strains through inoculations on soybean plants; g) studying the histology of colonization of soybean plants by C. cliviae and C. truncatum strains; h) identifying differentially expressed genes by pathogenic C. cliviae and C. truncatum strains, based on RNAseq, in distinct soybean growth stages. Knowing the direction of C. cliviae and C. truncatum transmission could indicate if there is need for seed treatment with the evaluated fungicides, and a rapid LAMP test could diagnose C. cliviae in soybean seeds and plants. Based on efficient mechanisms of pathogen dissemination over long distances and the levels of recombination previously reported, we tested the hypotheses: (i) that C. truncatum populations are non-differentiated across soybean fields; (ii) that this pathogen is recombinant in structure (iii) that the current geographic distribution of C. truncatum reflects the historical expansion of soybean crops in Brazil, from South towards Central-West and North regions. The genomic approach might provide clues of what makes C. truncatum such a successful pathogen. Investigating the histology of soybean plants infected by C. cliviae and C. truncatum will enable us to better understand the strategy of cellular colonization selected by these distinct Colletotrichum species. Through the identification of differentially expressed genes between pathogenic C. cliviae and C. truncatum strains by comparison of transcriptome profiles expressed in each fungal species might indicate what biochemical pathways are recruited in each step of plant growth stage. A clear understanding of the factors responsible for infection and colonization of C. cliviae and C. truncatum is essential to develop long-term control measures to manage anthracnose. (AU)