Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits

Hu et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(4):275-285 275 Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits* Hao HU, Yang XU, Huang-ping LU, Rui XIAO, Xiao-dong ZHENG†‡, Ting YU (Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute for Food Science, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China) † E-mail: Received June 10, 2014; Revision accepted Dec. 10, 2014; Crosschecked Mar. 27, 2015 Abstract: A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with 8 increasing concentrations of incubated cells. The best concentration was 1×10 cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits. Key words: Biocontrol, Postharvest, Rhodotorula mucilaginosa, Tibetan yeast isolates, Pear doi:10.1631/jzus.B1400162 Document code: A CLC number: S476+.9 1 Introduction The pear (Pyrus spp.) is one of the most important and popular fruits in China (Liu et al., 2013; Yue et al., 2014). During postharvest storage, blue mold is a major disease of pear (Sanderson and Spotts, 1995; Jurick et al., 2010). Currently, the application of synthetic fungicides is the main method adopted to control the disease (Lennox et al., 2004; Sugar and Basile, 2011). However, with the application of postharvest fungicides, the resistance of the pathogen ‡ Corresponding author Project supported by the Doctoral Program Foundation of the Ministry of Education of China (No. 20100101110087) and the National Natural Science Foundation of China (No. 30972051) ORCID: Xiao-dong ZHENG, http://orcid.org/0000-0002-0307-7754; Hao HU, http://orcid.org/0000-0003-3020-1930 © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 * gradually increases, and fungicidal residues that raise food safety issues for consumers are also of great concern. Therefore, there is an urgent need to explore other methods to replace synthetic fungicides for the control of postharvest diseases of pear (Wilson and Wisniewski, 1989; Wisniewski and Wilson, 1992). The use of microbial antagonists, yeasts in particular, to control postharvest decay is regarded as a promising alternative (Wilson et al., 1991; Spadaro and Gullino, 2004; Sharma et al., 2009). Among numerous antagonists, antagonistic yeasts show great promise (Zhang et al., 2007; Xu et al., 2008; Wang et al., 2009; 2011; Lutz et al., 2011; Janisiewicz et al., 2014; Lu et al., 2014). Because most yeasts do not produce antibiotics, they are considered to be safer than other kinds of microbial antagonists (Wisniewski et al., 1991). Furthermore, the colonization of fruit wounds by yeasts is rapid 276 Hu et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(4):275-285 (Castoria et al., 2005). One of the main mechanisms by which antagonistic yeasts may control postharvest decay is by competing for space and nutrients (Droby et al., 2009; Jamalizadeh, 2011). Finally, many biocontrol yeasts show the ability to be compatible with fungicides and other chemicals (An et al., 2012; Yu et al., 2012; D'Aquino et al., 2013). Antagonistic yeasts have been isolated from soil, fruit surfaces, and fruit wounds (Janisiewicz and Korsten, 2002). Due to the unique geographical environment of Tibet Autonomous Region of China, there has been little research on the use of Tibetan yeasts for the control of postharvest diseases of fruit. Zhao et al. (2012) reported that a Streptomyces sp. was selected to control gummy stem blight. In this study, the yeast Rhodotorula mucilaginosa was selected from Tibetan fermented products, following the screening of twenty yeast isolates for their effectiveness in biocontrol. The aim of this study was to investigate and evaluate the efficacy of R. mucilaginosa from Tibet in inhibiting postharvest blue mold decay in pear caused by Penicillium expansum. Although some studies have been conducted on R. mucilaginosa as a biocontrol agent (Li et al., 2011; Lutz et al., 2011; Robiglio et al., 2011), this is the first report of R. mucilaginosa from Tibet being used as an agent for controlling the blue mold of pear. 2 Materials and methods 2.1 Fruits Nashi pear fruits (Pyrus pyrifolia Nak., cultivar “Shuijing”), of uniform ripeness and size and without any apparent damage or infection, were harvested at commercial maturity in Hangzhou, Zhejiang Province, China. After being disinfected with a solution of 0.1% (v/v) sodium hypochlorite for 1 min, the fruits were rinsed with tap water and air-dried at room temperature (20 °C) prior to experiments. 2.2 Yeasts The Tibetan yeasts for screening were provided by the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education (Inner Mongolia Autonomous Region, China). They were isolated from yak yoghourt, identified by large-subunit (26S) ribosomal DNA (rDNA) D1/D2 domain sequence analysis, stored in ampoule tubes in lyophilized form at −80 °C, and activated on nutrient yeast dextrose agar (NYDA: 8 g nutrient broth, 5 g yeast extract, 10 g glucose, and 20 g agar in 1 L of distilled water) for two generations at 28 °C in the dark. Before the experiments, yeasts were inoculated by immersing a loop in 50 ml NYD broth (NYDA without agar) and incubated at 28 °C on a rotary shaker at 200 r/min for 24 h. To remove the medium, the cells were centrifuged at 3500g for 10 min and rinsed twice in sterile distilled water. Then, the pellets were re-suspended in sterile distilled water and adjusted to the required concentrations by counting cells with a hemocytometer. 2.3 Pathogen P. expansum was originally isolated from symptomatic pear fruits. The pathogen was cultu (...truncated)