Botrytis cinerea Pers:Fr. is an important pathogen on many vegetable crops grown under greenhouse conditions as well as under field conditions. Under high humidity conditions or when free moisture is present on the plant surface, the pathogen infects fruits, flowers, leaves, and stems causing tissue decay. This is followed by prolific sporulation of the pathogen, producing a gray mold appearance. Wounded tissues are especially susceptible to this pathogen. Much of the research activity to achieve biological control of B. cinerea on vegetable crops has centered around the use of T. harzianum, followed by Ulocladium spp. and a number of yeasts, as described later.
4.1.1 Trichoderma As a Biocontrol Agent of Botrytis Cinerea
Isolate T-39 of T. harzianum (marketed as Trichodex™) provided control of gray mold as well as a number of other fungal diseases of cucumber under commercial greenhouse conditions (Elad 2000a). T. harzianum T-39 was applied as part of a gray mold management program in alternation with chemical fungicides. The biocontrol agent was effective when applied in formulations containing two concentrations of the active ingredient (0.2 and 0.4 g/l), at around 1010cfu/g of T. harzianum (Elad et al. 1993). A number of other research studies have confirmed the efficacy of T. harzianum strains in reducing development of B. cinerea on crops such as cucumber and tomato under laboratory conditions and on greenhouse-grown plants (Dik and Elad 1999; Dik et al. 1999; O'Neill et al. 1996; Utkhede et al. 2000).
Mechanisms involved in the biological suppression of infection and inoculum potential of B. cinerea by Tricho-derma are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several studies. Reported combinations include antibiosis with enzyme degradation of B. cinerea cell walls and parasitism (Belanger et al. 1995); competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis (Elad 1996). Since, germinating B. cinerea conidia are dependent on the presence of nutrients to initiate pathogenesis, competition for nutrients is important in biocontrol. Pathogen conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by Trichoderma and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degrade B. cinerea cell walls. The latter has been demonstrated much more convincingly in vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation of Botrytis on necrotic plant tissues is a means to minimize secondary spread of pathogen inoculum. Zimand et al. (1996) also demonstrated that the presence of T. harzianum at the site where B. cinerea infects can have an adverse effect upon activity of pathogen enzymes involved in pectin degradation and host cell wall destruction, e.g., pectinase, cutinase, and pectate lyase. Since such enzymes are intimately involved in the infection process by B. cinerea, the effect of the biocontrol agent in reducing their activity in vitro and on the surface of plant leaves could also limit disease development by the pathogen (Kapat et al. 1998).
The inhibition of pathogen enzymes was proposed to be due to the secretion of serine proteases by T. harzianum (Elad and Kapat 1999), which could also inhibit pathogen spore germination. The presence of protease inhibitors was found to reduce the biocontrol activity. The potential role of induced plant resistance by T. harzianum for control of B. cinerea was demonstrated by De Meyer et al. (1998), wherein application of the biocontrol agent to roots or leaves of a number of different plant species was observed to provide protection against the pathogen on leaves that were spatially separated from the site of application of T. harzianum. This was attributed to induction of systemic resistance that delayed or suppressed spreading lesion formation (De Meyer et al. 1998).
4.1.2 Saprophytic Fungi and Yeasts As Biocontrol Agents of Botrytis Cinerea
The leaf surface of plants (phylloplane) is frequently colonized by a range of saprophytic fungi and yeasts, which rely on plant nutrient exudates and a range of other carbon/ nitrogen sources for their survival, e.g., damaged or senescing tissues, pollen grains, insect honeydew. If present in the same niche as plant pathogenic fungi, these saprophytes may compete with pathogens for nutrients, infection sites, or reduce growth and sporulation of the pathogen on host tissues through competition or antagonism (Fokkema 1993). Recovery of selected fungi and yeasts and reapplication to the leaf or stem surface has identified a number of potential biological control agents that can reduce diseases caused by B. cinerea. On onion leaf tissues, the saprophytic fungi Alternaria alternata, Chaetomium globosum, Ulocladium atrum, and U. chartarum suppressed sporulation of the pathogen significantly when applied after pathogen inoculation (Kohl et al. 1995; 1999). A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been described to detect and quantify U. atrum in colonized plant tissues (Karpovich-Tate and Dewey 2001) and could be useful in monitoring of this biocontrol agent. Application of the saprophytic fungus Cladosporium cladosporioides to wounds on tomato stems was reported to reduce infection by B. cinerea in laboratory and greenhouse experiments (Eden et al. 1996).
The yeast-like fungi Aureobasidium pullulans and Cryptococcus albidis significantly reduced sporulation of B. cinerea on pruning wounds and stems of cucumber and tomato under laboratory and greenhouse conditions (Dik and Elad 1999; Dik et al. 1999). Another yeast, Rhodosporidium diobovatum, when applied to tomato stems, reduced lesion size due to B. cinerea and the treated plants yielded higher fruit when compared to the untreated controls (Utkhede et al. 2000). Both C. albidus and Rhodotorula glutinis reduced sporulation of B. cinerea on bean and tomato leaves and reduced disease levels (Elad et al. 1994).
4.1.3 Mechanisms of Action of Yeasts Against Botrytis Cinerea
Yeasts can compete effectively against B. cinerea for nutrients, such as glucose and fructose (Filonow 1998; Filonow et al. 1996), thereby reducing pathogen colonization of plant tissues and sporulation (Elad et al. 1994). Yeasts such as Aureobasidium have also been reported to produce mycotoxins in culture (Schrattenholz and Flesch 1993). Yeast cells may attach to pathogen hyphae, as demonstrated for B. cinerea, and cause them to degrade (Cook et al. 1997) through secretion of cell wall degrading enzymes
(Wisniewski et al. 1991). The production of cell wall degrading enzymes, such as b-1,3-glucanases has also been documented in yeasts such as Pichia anomola that are effective biocontrol agents against B. cinerea as a postharvest treatment (Jijakli and Lepoivre 1998). In vivo studies with Candida saitoana in apple demonstrated that B. cinerea hyphae had degenerated (El-Ghaouth et al. 1998), implicating the possible role of toxins and/or enzymes. In addition, plant cells in the vicinity of the yeasts appeared to have enhanced structural defense responses, suggesting an induction of defense in the host plant may have occurred. Stimulation of host cell defenses by the yeast C. oleophila was recently described (Droby et al. 2002).
Yeasts with biocontrol potential against gray mold have been characterized using molecular techniques to provide a method to distinguish between closely-related strains and to identify and monitor survival of strains after application (Schena et al. 2000; 2002). These techniques include arbitrarily primed polymerase chain reaction (AP-PCR), random amplified polymorphic DNA (RAPD-PCR) analysis, and sequence-characterized amplification region (SCAR) analysis. In addition, transformation of the yeast Metschnikowia with green fluorescent protein (GFP) was achieved and colonies could be visualized under epifluorescence (Nigro et al. 1999). The transformed strains behaved similarly to the wild-type strains in biocontrol activity against B. cinerea and in growth rates. Another yeast, A. pullulans, was also transformed with GFP and colonies were readily visible on apple leaf surfaces when subjected to fluorescence and could be quantified (Wymelenberg et al. 1997).
Genetic transformation of the yeast Saccharomyces to express a cecropin A-based peptide with antifungal activity was recently described (Jones and Prusky 2002). The transformants inhibited the growth of Colletotrichum and reduced fungal decay of tomato fruits when applied prior to pathogen inoculation. The expression of the antifungal peptide in the biological control agent suggests a new approach for disease control.
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