The characterization of fungicide resistance, population structure, and aggressiveness of fungal species associated with ascochyta blight of pulse crops in Montana

Abstract

Ascochyta blight (AB) of pulse crops causes yield loss in Montana, where 1.24 million acres were planted to pulses in 2018. Pyraclostrobin and azoxystrobin, quinone outside inhibitor (QoI) fungicides, have been the choice of farmers for the management of AB in pulses. QoI-fungicide-resistant Didymella rabiei isolates were found in one chickpea seed lot each received from Daniels, McCone and Valley Counties, MT, from seed produced in 2015 and 2016. Multiple alignment analysis of amino acid sequences showed a mutation that replaced the codon for amino acid 143 from GGT to GCT, introducing an amino acid change from glycine to alanine (G143A), which is reported to be associated with QoI resistance. Under greenhouse conditions, disease severity was significantly higher on pyraclostrobin-treated chickpea plants inoculated with QoI-resistant isolates of D. rabiei (QoI-R) than sensitive isolates (QoI-S) (p-value = 0.001). D. rabiei-specific PCR primer pair and probes were developed to discriminate QoI-R and QoI-S isolates. In North America, AB of dry pea is caused by a complex of fungal pathogens (Didymella pisi, Peyronellaea pinodes, and Peyronellaea pinodella). D. pisi is the predominant causal pathogen of AB of dry pea in Montana resulting in yield losses. Thirty-three microsatellite markers (SSR) were developed and used to analyze the genetic diversity and population structure of 205 D. pisi isolates from four geographical regions of Montana. Unweighted Neighbor-joining, principal coordinate, and population structure analyses grouped these 205 isolates into two major sub-groups. The clusters did not match the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 15% among populations. There was moderate genetic variation in the total populations (PhiPT = 0.153). Recently, a shift in pathogen composition has been observed in Montana from D. pisi to P. pinodes and P. pinodella. Also, a Phoma sp. was found associated with AB contaminated dry pea seeds and included in this study. Mycelial growth and sporulation were evaluated at different temperatures. Also, the pathogenicity of Phoma sp. and the difference in aggressiveness among the fungal pathogens was evaluated. At all temperatures, Phoma sp. had the highest growth rate (p-value = < 0.001) and produced more spores than the other species (p-value = < 0.001). P. pinodes caused greater disease severity than the other species when inoculated on pea plants (cv. Carousel, p-value < or = 0.001). The Phoma sp. was not pathogenic. Peameal agar was used to visually discriminate between fungal species. Diagnosis of AB of dry pea is challenging because of the complex of pathogens involved. Also, they have slow growth rate and different morphotypes. Currently, there are no PCR-based assays developed for D. pisi or any of the fungal pathogens associated with the AB complex of dry pea. D. pisi specific SYBR green SSR-qPCR and conventional SSR-PCR assays were developed for rapid detection and quantification of D. pisi both in-planta and in-vitro.