Elsevier

Food Control

Volume 120, February 2021, 107499
Food Control

Antifungal activity of volatile organic compounds produced by Pseudomonas fluorescens ZX and potential biocontrol of blue mold decay on postharvest citrus

https://doi.org/10.1016/j.foodcont.2020.107499Get rights and content

Highlights

  • Production of VOCs is an important biocontrol mechanisms of P. fluorescens ZX.

  • Culture conditions have effects on types and antifungal activity of VOCs from BCAs.

  • SMOAs are promising for controlling blue mold on citrus fruits at appropriate dose.

  • DMDS and DMTS show the highest inhibitory effects among all VOCs.

Abstract

In this research, postharvest citrus fruits with blue mold caused by Penicillium italicum were treated with volatile organic compounds (VOCs) produced by Pseudomonas fluorescens ZX incubated on NA and in NB. Treatments with pure individual components of the VOCs were also conducted. The VOCs from P. fluorescens ZX inhibited mycelial growth and conidial germination of P. italicum by 42.14% and 77.86%, respectively. In vivo experiments revealed that blue mold disease incidence and lesion size upon fruits were significantly suppresed by VOCs from P. fluorescens ZX incubated on NA, in NB and on healthy fruits. In addition, the VOCs produced by bacterial fluid provided higher biocontrol efficacy than did that from NA plates. The exposure to VOCs from bacterial fluid of P. fluorescens ZX resulted in morphological abnormalities of the conidia and hyphae of P. italicum. In vitro testing of the individual pure compounds comprising the VOCs suggested that organic acids and sulfur compounds are the active components of VOCs, with dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) exhibiting the highest antifungal activity: concentrations of only 100 and 10 μL/L, respectively, afforded complete inhibition of blue mold on citrus fruits in vivo. Organic acids showed promise for controlling blue mold, but only in sufficiently low concentrations so as not to promote physiological disease or damage in the fruits.

Introduction

Citrus is one of the most popular fruits throughout the world due to their delicious taste, fragrant flavor, nutritive value and relatively low price (Li et al., 2010). However, citrus fruits are highly perishable and easy to suffer a wide variety of postharvest fungal diseases caused by Penicillium digitatum, Penicillium italicum, Colletotrichum gloeosporioides, Geotrichum citri-aurantii, Alternaria citri, etc (Araújo et al., 2019; Ferraz, da Cunha, da Silva, & Kupper, 2016; Li et al., 2010; Mercier & Smilanick, 2005; Vilanova et al., 2014). Particularly, green mold, caused by P. digitatum, and blue mold, caused by P. italicum, are the most devastating postharvest diseases affecting citrus fruits, significantly reducing fruit value, and shelf-life and inflicting severe economic losses (Talibi, Boubaker, Boudyach, & Ait Ben Aoumar, 2014). Blue mold is particularly pernicious due to its worldwide prevalence, especially in oranges, and its exceptionally rapid spread among packed cartons of oranges stored at low temperatures (Chalutz & Wilson, 1990; Demirci, 2011).

For a long time, traditional physical methods, including transient heat treatment, refrigerated atmosphere storage, modified atmosphere, ionizing radiation, etc., have been applied for the control of postharvest decay, while none of these provided complete control (Hong, Lee, & Kim, 2007; Padova, Kahan, & Barkai-Golan, 1969; Talibi, Boubaker, Boudyach, & Ait Ben Aoumar, 2014). On the other hand, chemical fungicides, such as imazalil, prochloraz, and carbendazim have been offered some degree of control over postharvest diseases, however their efficacy has been diminished in recent years due to the emergence of pathogen resistance (Sánchez-Torres & Tuset, 2011). Worse yet, in order to achieve a sufficient effect for disease control, much more synthetic fungicides are usually required, which can be harmful to non-targeted microorganisms, food safety and human health (Conner, McAndrew, Kiehn, Chapman, & Froese, 2004; Martins et al., 2019). Therefore, it is of great importance to find alternative methods for controlling postharvest decay in a nontoxic and eco-friendly way.

Recently, the use of biological control agents (BCAs) to inhibit pathogens has attracted more attention. BCAs can display diverse modes of action, making pathogen resistance unlikely (Wallace, Hirkala, & Nelson, 2018a). Thus, BCAs are considered an effective, safe, and environmentally friendly approach against phytopathogens (Dukare et al., 2018; Lugtenberg, Rozen, & Kamilov, 2017; Talibi, Boubaker, Boudyach, & Ait Ben Aoumar, 2014; Wallace et al., 2018a). It is generally believed that BCAs exhibit various biocontrol mechanisms, including parasitism (Dukare et al., 2018; Sahu, Singh, Shankar, & Pradha, 2018; Wallace, Hirkala, & Nelson, 2017; 2018a; 2018b), competition for limiting nutrients and niches (Lugtenberg, Rozen, & Kamilov, 2017; Wallace et al., 2017; Wang et al., 2019; 2020) and induced resistance (Dukare et al., 2018; Patel et al., 2015; Zhao et al., 2018). In addition, antimicrobial substances are also found such as phenazine-1-carboxylicacid, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, siderophore, chitinase and β-1,3-glucanase from BCAs (Arseneault, Goyer, & Filion, 2016; Dukare et al., 2018; Prabhukarthikeyan, Keerthana, & Raguchander, 2018; Suganthi, Senthilkumar, Arvinth, & Chandrashekara, 2017; Wallace et al., 2017). Compared with diffusible antimicrobial substances, investigations related with volatile organic compounds (VOCs) produced by BCAs on disease suppression have gained popularity, as they are biodegradable (Qin, Xiao, Cheng, Zhou, & Si, 2017). Moreover, bio-fumigation with VOCs produced by microorganisms circumvents direct contact between fruit and antagonist, and therefore does not leave toxic residues on the fruit surface (Mercier & Smilanick, 2005; Toffano, Fialho, & Pascholati, 2017). Some of the reported examples include VOCs produced by Streptomyces globisporus JK-1 against P. italicum on postharvest Citrus microcarpa (Li et al., 2010); by Bacillus subtilis CF-3 against C. gloeosporioides on litchi fruits (Zhao et al., 2019); and by Aureobasidium pullulans L1 and L8 for biocontrol of brown rot caused by Monilinia spp. on stone fruits (Di Francesco, Di Foggia, & Baraldi, 2020).

Recent decades have witnessed global research insights into the antibacterial and antifungal activities of Pseudomonas fluorescens, a strongly colonizing, mesophilic, heterotrophic, gram-negative rod-shaped bacterium with polar flagella (Sahu, Singh, Shankar, & Pradha, 2018). VOCs-producing P. fluorescens have also shown promise in previous work. Wallace et al. (2017) reported that VOCs produced by P. fluorescens 1–112, 2–28 and 4–6 could completely inhibit spore germination of Penicillium expansum. Proteomics analysis conducted by Raza et al. (2016) indicated that VOCs emitted by P. fluorescens WR-1 inhibited protein metabolism of Ralstonia solanacearum to significantly reduce its virulence. Additionally, the VOCs produced by P. fluorescens UM showed strong antagonism against Botrytis cinerea during confrontation assays. They also significantly increased Medicago truncatula biomass and chlorophyll content (Hernandez-Leon et al., 2015).

P. fluorescens ZX has been proposed as a BCA to control postharvest decay in grape caused by B. cinerea (Jiang et al., 2019), or in citrus caused by P. italicum and P. digitatum (Wang et al., 2019; 2020). Investigations carried out by Wang et al. (2019; 2020) provided experimental evidence that VOCs produced by P. fluorescens ZX played significant roles in inhibition of fungal growth. These VOCs were isolated and identified by using headspace solid phase microextraction (SPME) and gas chromatography with mass spectrometric detection (GC-MS), and respectively, 20 and 16 types of VOCs were identified from nutrient broth agar (NA) culture and nutrient broth (NB) culture where P. fluorescens ZX grew (Wang et al., 2020). However, it remains unclear which precise VOC of P. fluorescens ZX are responsible for fungicidal effects against P. italicum.

Therefore, based on the results of our earlier work, the present study aimed to evaluate the bio-fumigation action of P. fluorescens ZX VOCs on citrus fruits, and to investigate the roles of individual VOC components in controlling the development of blue mold on postharvest citrus fruits. The insights provided by this work could provide an effective basis to develop biopreservation agents to efficiently control P. italicum and, thus, to broaden biological protection pathways of postharvest fruits.

Section snippets

Fruit

Freshly hand-harvested, mature and healthy Newhall navel orange fruits (Citrus sinensis Osbeck) were obtained from a local orchard. Citrus fruits used in this study did not received any pre-harvest fungicide treatment. Fruits with similar shape, color and size were selected and randomly divided into groups (27 fruits per group) for the experiments. Before each trial, fruits were washed with tap water, and surface-sterilized with 75% ethanol, followed by air drying at room temperature. After

In vitro antifungal activity of VOCs produced by P. fluorescens ZX

The experimentally determined in vitro antifungal activities of VOCs produced by P. fluorescens ZX are presented in Fig. 1. The fungus P. italicum showed significant (P < 0.05) inhibition of mycelial and CFU growth in presence of VOCs incubation. Moreover, the initial concentration of bacterial inoculation exerted a significant (P < 0.05) effect on inhibition rate, with VOC-induced inhibition strengthened under bacterial suspensions applied to give higher initial inoculation. When the bacterial

Discussion

Extensive study has been conducted on various modes of action related to inhibition of plant pathogenic fungi by BCAs. However, only recently have researchers recognized the important role of antifungal VOCs (Toffano et al., 2017). Previously, VOCs synthetized by P. fluorescens have been studied against some plant soil-borne diseases (Hernandez-Leon et al., 2015; Raza et al., 2016) and apple postharvest pathogens (Wallace et al., 2017; 2018a; 2018b), which yielded satisfactory results. In

Conclusions

This study demonstrated that P. fluorescens ZX-produced VOCs—specifically their pure constituent components of acetic acid, butyric acid, isobutyric acid, 2-MBA, 3-BMA, and especially DMDS and DMTS—were effective for controlling P. italicum, the pathogen that causes blue mold on postharvest citrus fruits. The active compounds identified in this work are promising as widely deployable agents for the biocontrol of blue mold. In addition, we demonstrated that the antifungal activity of VOCs

CRediT authorship contribution statement

Zhirong Wang: Conceptualization, Investigation, Data curation, Formal analysis, Resources, Methodology, Software, Writing - original draft, Writing - review & editing, Supervision. Tao Zhong: Investigation, Data curation, Formal analysis, Software, Writing - original draft. Kewei Chen: Data curation, Writing - original draft, Writing - review & editing. Muying Du: Resources, Writing - original draft, Funding acquisition. Guangjing Chen: Investigation, Data curation. Xuhui Chen: Investigation,

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We gratefully acknowledged the Chinese-Hungarian Intergovernmental Scientific and Technological Industrial R + D Program (National Key R&D Program of China, project number in China is 2016YFE0130600; in Hungary is TET_16_CN-1-2016-0004) and A Project Funded by Ministry of Science and Technology of China (No. G20190022022) for supplying the testing materials and related services.

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