Elsevier

Biological Control

Volume 149, October 2020, 104325
Biological Control

Proteomic analysis reveals the mechanisms involved in the enhanced biocontrol efficacy of Rhodotorula mucilaginosa induced by chitosan

https://doi.org/10.1016/j.biocontrol.2020.104325Get rights and content

Highlights

  • R. mucilaginosa induced by chitosan for 24 h showed the best biocontrol efficacy.

  • Proteins involved in growth and reproduction, energy metabolism were up-regulated.

  • Proteins related to stress response were induced by chitosan.

  • The expression of CTS2 (chitinase) was also increased by chitosan.

  • The ROS accumulation was decreased in R. mucilaginosa incubated with chitosan.

Abstract

The biocontrol efficacy of the yeast, Rhodotorula mucilaginosa against postharvest diseases of strawberries can be significantly enhanced by culturing the yeast in a medium containing chitosan. The molecular mechanisms involved in the enhanced biocontrol efficacy of R. mucilaginosa, however, have not been comprehensively explored. Results of the present study indicate that the biocontrol efficacy of the yeast against gray mold (Botrytis cinerea) of strawberry was greatest when the yeast was cultured in a medium containing 0.5% (w:v) chitosan for 24 h before use. Proteomic and bioinformatic analyses revealed that proteins involved in growth and reproduction, energy metabolism, antioxidant response, response to abiotic stress, and degradation of pathogen cell walls were all up-regulated by chitosan resulting in the enhanced antagonistic activity of R. mucilaginosa against B. cinerea. The decrease in ROS accumulation in R. mucilaginosa cultured in a medium containing chitosan was in agreement with the increased abundance of proteins related to antioxidant defense. Key differentially expressed proteins (DEPs) were identified and 12 randomly-selected DEPs were validated by parallel reaction monitoring. The present results provide a theoretical reference for further study of the molecular mechanisms involved in the improvement of biocontrol efficacy of antagonistic yeast by elicitor molecules and also provide a novel approach for improving biological control of postharvest diseases.

Introduction

Pathogen-induced decay is a primary reason for postharvest losses of fruits. Postharvest infections of strawberry by Rhizopus stolonifer and Botrytis cinerea result in Rhizopus decay and gray mold, respectively (Zhang et al., 2014). Biological control of postharvest decay with antagonistic yeasts is an alternative strategy to the use of chemical fungicides which pose a risk to human health and the environment. A previous study reported that antagonistic yeast are a natural inhibitor of fungi (Liu et al., 2013). Since antagonistic yeast are susceptible to unstable environmental factors, their use alone may not effectively control postharvest diseases of fruits compared to synthetic fungicides. Biological control can be markedly improved, however, if an elicitor is added to the growth medium of the antagonistic yeast (Wang et al., 2011).

Several previous studies have explored the mechanisms involved in the enhancement of biocontrol efficacy of antagonistic yeast by the use of elicitors. Yeast cells harvested from nutrient yeast dextrose broth (NYDB) amended with β-glucan can multiply in fruit wounds more rapidly than antagonistic yeast harvested from NYDB alone, which enables them to better compete with pathogens in wounded tissues (Wang et al., 2018). Cell wall hydrolases, such as chitinase, secreted by yeast are one of the major antipathogenic factors against mold fungi. Yu et al. (2008) reported that cell-free filtrate of Cryptococcus laurentii cultured in a chitin amended medium, exhibits a high level of chitinase activity, which has direct antifungal activity against Penicillium expansum in pear fruit wounds. Lu et al. (2014) found that the antioxidant enzyme activity, including catalase (CAT) and superoxide dismutase (SOD), in the treatment group (yeast grown in NYDB + elicitor) was significantly higher than it was in the control group (yeast grown in NYDB alone). Thus, they reported that the elicitor enhanced the antioxidant capacity of the yeast. Furthermore, antioxidant substances, such as trehalose and glutathione, are also advantageous to the enhancement of antioxidant capacity in yeast (Fernando et al., 2012, Liu et al., 2015). The abundance of some proteins associated with stress response and regulation, such as serine/threonine protein kinase, are also increased by elicitors (Zhang et al., 2017).

Natural decay of strawberries by R. stolonifer and B. cinerea has been reported to be significantly reduced by the application of the yeast R. mucilaginosa grown in NYDB amended with chitosan (Zhang et al., 2014). The population of yeast that had been cultured in NYDB amended with chitosan in wounds of strawberries was more than three-fold higher than the control group (yeast incubated in NYDB without chitosan). Chitinase activity was also significantly higher in the treated yeast than in the control group of yeast. Two-dimensional electrophoresis and identification by MALDI-TOF/TOF revealed that some basic cell metabolism-related proteins exhibited increased abundance in the yeast and may have played a role in the increase in biocontrol efficacy (Zhang et al., 2014). Other differentially expressed proteins (DEPs) that are critical to the potential molecular mechanisms responsible for the chitosan enhancement of biocontrol by yeast, however, have not been systematically characterized. Proteomics has been widely used to identify mechanisms responsible for biological control of postharvest diseases. Chan et al. (2007) utilized a proteomic approach to study the mechanisms involved in enhanced resistance of peach fruits induced by Pichia membranefaciens and salicylic acid. Liu et al. (2018) identified differentially accumulated proteins in kiwifruit response to B. cinerea in their investigation of the interactions that occur between kiwifruit and B. cinerea.

In the present study, a qualitative and quantitative analysis of proteins by Tandem mass tag (TMT) was performed on a test group of R. mucilaginosa (yeast grown in NYDB + chitosan) and a control group (yeast grown in NYDB). Differentially expressed proteins associated with the enhanced efficacy of the yeast against postharvest pathogens were identified using a bioinformatic analysis and then verified based on parallel reaction monitoring (PRM) technology of a random selection of differentially expressed proteins.

Section snippets

Fruits

‘Fengxiang’ strawberries (Fragaria ananassa Duch.) were purchased from “Jiangxin” island in Zhenjiang, Jiangsu province. Berries were selected on the basis of uniformity of color (80–90% red), size (15–18 g), firmness (1.8–2.2 kg/cm2), total soluble solids (9–10%), titratable acidity (0.7–1.0%). Strawberries used in the study were free from injury and infection.

Chitosan

Chitosan with 90% deacetylation was purchased from Sangon Company (Shanghai, China).

Strains and culture conditions

The antagonistic yeast, R. mucilaginosa, used in

Effect of time of incubation in chitosan-amended NYDB on the biocontrol efficacy of R. mucilaginosa against gray mold of strawberry

R. mucilaginosa harvested from NYDB media amended with chitosan controlled gray mold of strawberries more effectively than yeast that were cultured in NYDB alone (Fig. 1). This was especially true for R. mucilaginosa cultured for 24 h in NYDB supplemented with chitosan, where disease incidence was only 35%. This level of efficacy was greater than the level of biocontrol exhibited by yeast cultured in the chitosan-containing medium for 18 and 36 h. These results indicate that the antagonistic

Discussion

In this study, the ability of chitosan to enhance the biocontrol efficacy of R. mucilaginosa against gray mold of strawberry, along with underlying mechanism associated with the enhanced efficacy, were investigated. Results showed that the biocontrol efficacy of R. mucilaginosa was significantly improved by culturing the yeast in NYDB containing 0.5% (w:v) chitosan, especially for 24 h. A proteomic analysis by TMT and a bioinformatic analysis of DEPs were conducted to better understand the

Conclusion

R. mucilaginosa cultured in NYDB amended with chitosan (0.5% w/v) for 24 h exhibited the best biocontrol efficacy against gray mold of strawberry. The mechanisms involved in the enhanced biocontrol efficacy brought about by exposure of the yeast cells to chitosan are diagrammed in Fig. 7 and are based on a bioinformatic analysis of proteomic data obtained by TMT. Proteins involved in a MAPK signaling pathway and the synthesis of cell walls, including BNI1 and CHS3, exhibited increased abundance

CRediT authorship contribution statement

Ning Gu: Investigation, Methodology, Data curation, Formal analysis, Software, Writing - original draft. Xiaoyun Zhang: Investigation, Methodology, Funding acquisition, Writing - original draft. Xiangyu Gu, Lina Zhao and Solairaj Dhanasekaran: Writing - review & editing. Xin Qian: Methodology, Software. Hongyin Zhang: Project administration, Supervision, Funding acquisition, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China (31772369, 31772037) and the Agricultural Independent Innovation Fund in Jiangsu Province (CX (18)2028). We thank Dr. Michael Wisniewski (Adjunct Professor, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA) for his critical reading and editing of the manuscript.

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