Monitoring and analysis of rice pathogen Ustilaginoidea virens isolates with resistance to sterol demethylation inhibitors in China

Xiayan Pan,Mingli Yong,Huijuan Cao,Xiaole Yin,Junjie Yu,Yan Du,Yongfeng Liu,Tianqiao Song,Mina Yu,Zhongqiang Qi,Rongsheng Zhang

doi:10.1186/s42483-020-00062-x

Abstract

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is an important fungal disease of rice. In China, sterol demethylation inhibitors (DMIs) are common fungicides used to control RFS. In a previous study, we detected two propiconazole-resistant U. virens isolates in 2015 in Huai’an city, Jiangsu Province, China. In the current study, we detected six propiconazole-resistant isolates out of 180 U. virens isolates collected from rice fields in Jiangsu Province in 2017, and found they were from three different places (Xuzhou, Huai’an and Jintan). All these six propiconazole-resistant isolates were cross-resistant to three other sterol demethylation inhibitor (DMI) fungicides, i.e. difenoconazole, tebuconazole, and epoxiconazole. Among them, two isolates (2017–61 and 2017–170) had high fitness. Through sequencing and RT-qPCR analysis, we found that the expression levels of CYP51 and its encoded protein were significantly increased in the propiconazole-resistant isolates with a “CC” insertion mutation upstream of the CYP51 coding region compared to the propiconazole-sensitive isolates. In addition, propiconazole stimulated CYP51 expression in all isolates. Propiconazole also stimulated the accumulation of CYP51 protein in propiconazole-sensitive isolates and propiconazole-resistant isolates without mutation, but not in propiconazole-resistant isolates with the “CC” mutation. According to JASPAR database analysis, the predicated functional binding sites for propiconazole-resistant isolates with a “CC” insertion mutation and propiconazole-sensitive isolates were different. Given the high fitness of the propiconazole-resistant isolates, the development of resistance to DMIs in U. virens should be monitored. Furthermore, we speculated that the over-expression of CYP51 may contribute to DMI resistance in U. virens with the “CC” insertion mutation.

Highlights

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is a major fungal disease in most ricegrowing areas all over the world
Frequency and distribution of propiconazole-resistant isolates of U. virens at the sampling regions A total of 180 field isolates of U. virens were used to monitor the resistance of this pathogen to propiconazole, and six of them (2017–2, 2017–6, 2017–61, 2017–170, 2017– 176, and 2017–179) could grow on Potato sucrose agar (PSA) plates containing 1 μg/mL of propiconazole
The propiconazole sensitivity (EC90) of these resistant isolates significantly increased compared to that of the sensitive isolate 2017–11. All these resistant isolates were lowly resistant to propiconazole with resistance factor (RF) values less than 10.0 (Table 2)

Summary

Introduction

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is a major fungal disease in most ricegrowing areas all over the world. DMIs are broad-spectrum fungicides that inhibit ergosterol biosynthesis by targeting sterol 14α-demethylase CYP51, a key enzyme in sterol biosynthesis. The common mechanisms of fungal resistance to DMI include mutations in the target CYP51 gene; overexpression of the CYP51 gene, and enhanced efflux pumps that are encoded by ATPbinding cassette (ABC) transporter (Ma et al 2006; Kretschmer et al 2009; Cools et al 2010, 2012; Wang et al 2015; Tsao et al 2016). In U. virens, up-regulation of CYP51 and increased ergosterol biosynthesis were reported to be associated with propiconazole resistance (Zhou et al 2019). Due to the lack of resistant field isolates, the resistance mechanism of U. virens to DMI fungicides requires further investigation

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