The Rlm1 transcription factor in Candida oleophila contributes to abiotic stress resistance and biocontrol efficacy against postharvest gray mold of kiwifruit

https://doi.org/10.1016/j.postharvbio.2020.111222Get rights and content

Highlights

  • Candida oleophila effectively controlled postharvest gray mold of kiwifruit.

  • Two Rlm1 mutants (ΔRlm1-1 and ΔRlm1-2) of C. oleophila were generated.

  • The two mutants were more sensitive to a variety of stresses, compared to WT.

  • The two mutants exhibited lower biocontrol efficacy against gray mold.

  • The study shed light on the function of Rlm1 in a postharvest biocontrol yeast.

Abstract

Biological control utilizing antagonistic yeasts has been actively pursued as an alternative to synthetic fungicides for the management of postharvest diseases. Abiotic stress resistance is an important attribute for antagonistic yeasts, directly associated with their biocontrol efficacy. The MADS-box transcription factor, Rlm1, has been reported to regulate the response of model yeasts to cell wall stress. Rlm1 in the antagonistic yeast, Candida oleophila, was found to play a role in resistance to salt, heat, and oxidative stress. Two Rlm1 mutants (ΔRlm1-1 and ΔRlm1-2) were generated. Compared to the wild-type (WT), C. oleophila I-182, ΔRlm1-1, and ΔRlm1-2 were more sensitive to a variety of stresses, including heat, salt, and oxidative stress. The mutants also exhibited lower biocontrol efficacy against gray mold caused by Botrytis cinerea, and slower growth in kiwifruit wounds with respect to the WT. This study provided the information to understand the relationship between the Rlm1 transcription factor, stress resistance, and biocontrol efficacy of antagonistic yeasts used for the biocontrol of postharvest diseases.

Introduction

Kiwifruit, “the king of fruits”, is popular with consumers due to its pleasurable taste and high concentration of vitamins, minerals and other nutrients (Drevinskas et al., 2017; Wu et al., 2017). Kiwifruit, however, is susceptible to various fungal pathogens, including Botrytis cinerea (Di Francesco et al., 2018; Liu et al., 2018), Penicillium expansum (Luo et al., 2019; Zhu et al., 2016), Diaporthe phaseolorum (Kai et al., 2020), and Alternaria alternata (Li et al., 2017), and Nigrospora sphaerica (Li et al., 2018), with B. cinerea being the most prevalent postharvest pathogen throughout the world (Bardas et al., 2010; Pei et al., 2019). Yield loss due to gray mold caused by B. cinerea can be up to 20 %, or even 30 % under extreme circumstances in certain areas (Michailides and Elmer, 2000).

The use of antagonistic yeasts as biocontrol agents to manage postharvest diseases of fruits has been actively explored (Droby et al., 2016; Liu et al., 2013; Perez et al., 2016; Rivas-Garcia et al., 2019; Tian et al., 2004; Vilaplana et al., 2020; Wisniewski et al., 2016). Among them, Candida oleophila has been extensively studied due to its broad spectrum of activity against a variety of pathogens in various fruits, including apple (Lahlali and Jijakli, 2009), pear (Nie et al., 2019), citrus (Liu et al., 2019), and banana (Bastiaanse et al., 2010). However, the biocontrol efficacy of yeast antagonists can be affected by various environmental conditions, including high temperature, salt stress, and oxidative stress (Cheng et al., 2016; Ippolito and Nigro, 2000; Liu et al., 2012; Wang et al., 2018). Previous studies have explored methods to enhance the stress tolerance of antagonistic yeasts, including the use of sugar protectants (Abadias et al., 2001; Zheng et al., 2019), glycine betaine (Sui et al., 2012; Zhang et al., 2017a), glutathione (Zhang et al., 2017b), and L-ascorbic acid (Liu et al., 2009), as well as the adaptation of the yeast to sublethal stress, including heat stress (Cheng et al., 2016; Liu et al., 2011), oxidative stress (Liu et al., 2012), and salt stress (Wang et al., 2018). Additionally, antioxidant genes in C. oleophila I-182, such as a peroxisomal catalase (NCBI accession: JN615130), cytochrome c peroxidase (NCBI accession: JN615131), and a thioredoxin reductase (NCBI accession: JN615133), have been reported to be upregulated during the exposure of the yeast to a mild H2O2 stress (5 mM H2O2, 30 min) (Liu et al., 2012). Although the gene CoEXG1 that encodes for a secreted 1,3-β-glucanase has been cloned and characterized in C. oleophila I-182 (Bar-Shimon et al., 2004; Segal et al., 2002; Yehuda et al., 2003), knowledge about the function of specific genes in response to stress via genetic mutation is lacking.

The MADS-box transcription factor, Rlm1, has been reported to confer resistance to the negative effects of the constitutive activity of the Mpk1 mitogen-activated protein kinase (MAPK) pathway in the model yeast, Saccharomyces cerevisiae. Deletion of Rlm1 reduces the negative impact of cell wall disruptors and reduced flocculation (Dodou and Treisman, 1997; Jung et al., 2002). More recently, Slt2 MAPK association with chromatin was required for the transcriptional activation of Rlm1-dependent genes in response to cell wall stress (Sanz et al., 2018). Rlm1 has been reported to participate in cell wall biogenesis, virulence, and carbon adaptation in Candida albicans (Delgado-Silva et al., 2014; Oliveira-Pacheco et al., 2018). The functional role of Rlm1 for yeast antagonists, however, has not been investigated.

The objective of the present study was to determine the role of Rlm1 transcription factor in stress tolerance and biocontrol efficacy of C. oleophila. Thus, the study compared (i) the survival of wild-type (WT) C. oleophila and two mutants of Rlm1Rlm1-1 and ΔRlm1-2) when the yeast cells were subjected to salt, heat, and oxidative stress, and (ii) the ability of the different mutants to protect kiwifruit from postharvest development of gray mold, caused by B. cinerea. It was hypothesized that the dysfunction of Rlm1 transcription factor of C. oleophila may lead to impairment of stress tolerance and biocontrol efficacy.

Section snippets

Fruit

Kiwifruits (Actinidia chinensis cv. Chuhong) were harvested at commercial maturity from a research planting located on the grounds of Chongqing University of Arts and Sciences, Yongchuan, China (29.351N, 105.895E). Fruits without wounds or rot were selected based on uniformity of size, disinfected with 2% (v/v) sodium hypochlorite for 2 min, rinsed with sterile tap water, and air-dried.

Fungal plant pathogen

The fungal plant pathogen, B. cinerea strain HFXC-16, was isolated from infected kiwifruits (Chen et al., 2015

Verification of ΔRlm1 mutants

Sequences of the two identified Rlm1 genes, Rlm1-1 and Rlm1-2, were deposited in GenBank of the NCBI under the accession Nos. MH603069 and MH603070, respectively. Deletion of the targeted genes was conducted based on the protocol of Sun et al. (2019) to examine the potential function of the two genes. The primers rlm1-sqF and rlm1-sqR were used to identify ΔRlm1-1 mutants, based on the complete cds of C. oleophila Rlm1-1. The calculated size of the PCR product in WT I-182 is 2860 bp, while the

Conclusions

The present study demonstrated that the Rlm1 transcription factor in C. oleophila I-182 played an important role in the adaptation of this antagonistic yeast to abiotic stress (heat, salt, and oxidative stress) and, as a consequence, in biocontrol efficacy against gray mold of kiwifruit. The results provided insight into understanding the potential functional role of the Rlm1 transcription factor in biocontrol yeasts. The mechanism how Rlm1 mediated stress tolerance and biocontrol efficacy

Author contribution statement

JL and HW conceived and designed the experiments. YS, ZS, YZ, WL, MJ and YL performed the experiments. WL, YW and XG analyzed the data. YS, JL and HW drafted the manuscript. All authors read and approved the final manuscript.

Declaration of Competing Interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service

Acknowledgments

This work was supported by National Natural Science Foundation of China (31870675), Natural Science Foundation of Chongqing Science and Technology Bureau (cstc2018jcyjAX0129), Natural Science Foundation of Jiangsu Province (grant BK20181322), Specific Research Project of Guangxi for Research Bases and Talents (AD18126005), Open Project Program of Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, and Scientific Research Program for Graduate Students of Chongqing University

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