Integration of continuous biofumigation with Muscodor albus with pre-cooling fumigation with ozone or sulfur dioxide to control postharvest gray mold of table grapes

Abstract

An integrated approach was evaluated that combined biological and chemical fumigation of table grapes to control postharvest gray mold caused by Botrytis cinerea. After fumigation of the grapes with ozone or sulfur dioxide during pre-cooling, the fruit were then exposed to continuous biofumigation by the volatile-producing fungus Muscodor albus during storage. Biofumigation was provided by in-package generators containing a live grain culture of the fungus. This grain formulation of M. albus survived the initial ozone or sulfur dioxide fumigation, but sulfur dioxide reduced its production of isobutyric acid, an indicator of the production of antifungal volatiles. Gray mold incidence was reduced among inoculated ‘Autumn Seedless’ grapes from 91.7 to 19.3% by 1 h fumigation with 5000 μL L⁻¹ ozone, and further reduced to 10.0% when ozone fumigation and M. albus biofumigation were combined. The natural incidence of gray mold among organically grown ‘Thompson Seedless’ grapes after 1 month of storage at 0.5 °C was 31.0%. Ozone fumigation and M. albus biofumigation reduced the incidence of gray mold to 9.7 and 4.4, respectively, while the combined treatment reduced gray mold incidence to 3.4%. The use of commercial sulfur dioxide pads reduced the incidence to 1.1%. The combination of ozone and M. albus controlled decay significantly, but was less effective than the standard sulfur dioxide treatments. Although less effective than sulfur dioxide treatment, ozone and M. albus controlled decay significantly, and could be alternatives to sulfur dioxide, particularly for growers complying with organic production requirements.

Introduction

Gray mold, caused by Botrytis cinerea Pers., the most important postharvest disease of table grapes, is controlled by sulfur dioxide fumigation and storage at −0.5 °C. It is controlled by sulfur dioxide fumigation either at field temperature in fumigation chambers or during initial forced-air cooling of the grapes, followed by 2- to 6-h-long weekly fumigation during cold storage (Harvey and Uota, 1978, Luvisi et al., 1992). In export packages, sulfur dioxide generator sheets are used, which continuously emit a low concentration of gas within the packages during storage when hydrated by water vapor (Droby and Lichter, 2004). Generator pads typically protect the grapes from decay for a period of 2 months, and sometimes longer (Zutahy et al., 2008). While the sulfite residue tolerance is rarely exceeded in commercial practice (Austin et al., 1997), excessive residues can accumulate in wounded or detached berries (Smilanick et al., 1990b). Sulfur dioxide can cause unacceptable bleaching injuries to berries (Crisosto and Mitchell, 2002) and compromise their flavor (Chervin et al., 2005), and can cause food allergies to humans (Tayler et al., 2000). In the USA, it is prohibited from use on certified organic grapes.

Because of issues associated with sulfite residues, sulfur dioxide emissions, and its negative impact on grape quality, safe, effective, and economical alternative strategies to control gray mold are needed (Lichter et al., 2006). Alternatives requiring additional processing are unlikely to be implemented by California table grape growers, who normally pack their fruit into their final commercial packages in the vineyard (Crisosto and Mitchell, 2002).

A novel alternative for controlling fungal diseases is biological fumigation, or biofumigation, with the volatile-producing fungus Muscodor albus Worapong, Strobel, and Hess (Strobel et al., 2001, Strobel, 2006, Mercier et al., 2007). Isolate 620 of this fungus, which was the first Muscodor isolate discovered (Worapong et al., 2001), is currently being developed by AgraQuest Inc., Davis, CA as a biofumigant for agricultural uses (Mercier et al., 2007). The volatiles from M. albus isolate 620 are fungicidal to most postharvest pathogens and were used successfully to control storage decay in a number of commodities such as apples (Mercier and Jiménez, 2004), grapes (Mlikota Gabler et al., 2006, Mlikota Gabler et al., 2007), peaches (Mercier and Jiménez, 2004, Schnabel and Mercier, 2006) and lemons (Mercier and Smilanick, 2005).

Continuous biofumigation with M. albus effectively controlled gray mold of grapes in many types of packages and storage conditions (Mlikota Gabler et al., 2006, Mlikota Gabler et al., 2007). A developed formulation of M. albus consisting of desiccated rye grain colonized by the fungus has to be activated for postharvest use by rehydration (Mercier et al., 2007). This formulation was used in a pad or sachet delivery system for the fumigation of individual shipping boxes containing peaches (Schnabel and Mercier, 2006), grapes (Mercier et al., 2005), as well as cherries and raspberries (J. Mercier, unpublished data). The treatment could be applied passively by simply placing activated M. albus sachets within packages of grapes as is now done with sulfur dioxide generator pads. M. albus produces a “musky” odor that declines rapidly within packages after the sachets are removed. The level of biofumigation activity is directly affected by the storage temperature, and therefore, larger doses may be required at lower storage temperatures. As M. albus releases volatiles slowly at low temperatures, this technology does not provide a fast postharvest sanitation as achieved with other gases such as sulfur dioxide (SO₂) or ozone (O₃), but its continuous long-term release of active volatiles can protect the grapes during storage and transport.

Ozone is another alternative fumigant that has been tested to control postharvest decay of grape. Ozone is a natural substance in the atmosphere and one of the most potent sanitizers against a wide spectrum of microorganisms (Khadre et al., 2001). It is classified as GRAS (generally recognized as safe) for food contact applications in the USA (US Food and Drug Administration, 2001). The product of ozone degradation is oxygen; therefore it leaves no residues on treated commodities. A single fumigation with 200 μL L⁻¹ ozone for 4 h (Mlikota Gabler et al., 2002) or overnight fumigation with 500 μL L⁻¹ ozone (Shimizu et al., 1982) reduced gray mold decay in stored table grapes. A single fumigation of grapes with high concentrations (up to 10,000 μL L⁻¹ × h) during the pre-cooling of grapes significantly reduced gray mold in storage (Mlikota Gabler et al., 2007). Continuous fumigation during storage with a low dose of ozone (0.1–0.3 μL L⁻¹) for 7 weeks at 5 °C, prevented aerial mycelial growth (nesting) from B. cinerea among ‘Thompson Seedless’ grapes, but did not decrease the number of gray mold infections (Palou et al., 2002), even when used in combination with modified atmosphere packaging (Artes-Hernandez et al., 2004, Artes-Hernandez et al., 2007). As ozone does not have residual activity, it would be desirable to combine it with another treatment for more prolonged decay control.

Our objectives were to evaluate the novel integrated treatment to control postharvest gray mold of table grapes which consisted of an initial fumigation with high concentrations of ozone (5000 μL L⁻¹ for 1 h) during the pre-cooling phase followed by the continuous in-package biofumigation with M. albus during cold storage of grapes, as a way to replace sulfur dioxide fumigation. Another approach consisted of an initial fumigation of grapes with sulfur dioxide during pre-cooling followed by a continuous in-package biofumigation with M. albus during cold storage, as a way to replace weekly sulfur dioxide fumigation during cold storage or sulfur dioxide generator pads. Integrated treatments were evaluated in larger semi-commercial tests and compared to conventional sulfur dioxide treatments. The compatibility of M. albus with ozone and sulfur dioxide fumigations was evaluated by measuring volatile production and fungicidal activity of M. albus after exposure to those fumigants.