MfOfd1 is crucial for stress responses and virulence in the peach brown rot fungus Monilinia fructicola.

Ming-Ming Zhang,Xiao Xu,Wei-Xiao Yin,Chao‐Xi Luo,Zuo-Qian Wang,Song Huang

doi:10.1111/mpp.12933

Abstract

Monilinia fructicola is the most widely distributed species among the Monilinia genus in the world, and causes blossom blight, twig canker, and fruit rot on Rosaceae fruits. To date, studies on genomics and pathogenicity are limited in M. fructicola. In this study, we identified a redox‐related gene, MfOfd1, which was significantly up‐regulated at 1 hr after inoculation of M. fructicola on peach fruits. We used the clustered regulatory inter‐spaced short palindromic repeats (CRISPR)/Cas9 system combined with homologous recombination to determine the function of the MfOfd1 gene. The results showed that the sporulation of knockdown transformants was reduced by 53% to 83%. The knockdown transformants showed increased sensitivity to H2O2 and decreased virulence on peach fruits compared to the wild‐type isolate Bmpc7. It was found that H2O2 could stimulate the expression of MfOfd1 in the wild‐type isolate. The transformants were also more sensitive to exogenous osmotic stress, such as glycerol, d‐sorbitol, and NaCl, and to dicarboximide fungicides (iprodione and dimethachlon). These results indicate that the MfOfd1 gene plays an important role in M. fructicola in sporulation, oxidative response, osmotic stress tolerance, and virulence.

Highlights

Monilinia fructicola is an important Ascomycota pathogen in peach production that causes blossom blight, twig canker, and brown rot of fruits, with severe yield losses during both field production and post-harvest processing (Luo, 2017)
Some genes were found to be related to the redox state and played an important role in pathogenesis, for example an endopolygalacturonase-encoding gene was demonstrated to be crucial for pathogenicity in M. fructicola and Botrytis cinerea (Have et al, 1998; Chou et al, 2015)
Mean ± SD; values within the same column followed by the same letters are not significantly different based on one-way analysis of variance with the least-significant difference (LSD) test in SPSS 21.0 software at p =

Summary

| INTRODUCTION

Monilinia fructicola is an important Ascomycota pathogen in peach production that causes blossom blight, twig canker, and brown rot of fruits, with severe yield losses during both field production and post-harvest processing (Luo, 2017). As time progressed, the virulence difference between the wild-type isolate and the transformants gradually decreased These results indicate that the MfOfd gene is important for the virulence of M. fructicola, especially in the early stage of infection. To elucidate the roles of MfOfd to oxidative stress in M. fructicola, mycelial growth on PDA amended with H2O2 was measured for the wild-type isolate and knockdown transformants. | 825 fluffy and the mycelia were off-white, while the colonies of transformants were dense and dark brown, and produced large amounts of conidia, which showed concentric sporodochia (Figure 7a) These results indicate that the deletion of MfOfd did not affect the integrity of the M. fructicola cell wall, but may have an impact on the membrane protein. TA B L E 2 Sensitivity of Bmpc and ΔMfOfd strains to glycerol, d-sorbitol, NaCl, H2O2, sodium dodecyl sulphate (SDS), Congo Red, and MgSO4 Mycelial growth inhibition (%)

M d-sorbitol

| DISCUSSION

Findings

| EXPERIMENTAL PROCEDURES