Botrytis cinerea, the causal agent of grey mould or botrytis bunch rot in grapes is responsible for significant economic damage in vineyards worldwide. During the last 50 years, management of B. cinerea has relied heavily upon the use of synthetic chemicals (Rosslenbroich & Stuebler, 2000). This approach, however, is not regarded as sustainable, because of the relative ease with which fungicide-resistant strains of B. cinerea emerge within vineyard populations (Latorre et al., 2002; Sergeeva et al., 2002; Leroux, 2004) and increasing public discussion about pesticides and human and environmental health (Janisiewicz & Korsten, 2002; Spadoro & Gullino, 2005). As a consequence, more stringent regulations governing botryticide residue have severely restricted chemical control options in conventionally managed vineyards, particularly during the preharvest period. Ironically, it is during this period that rapidly ripening berries become increasingly susceptible to B. cinerea infection. In organically managed vineyards there are very few options for pathogen suppression, and disease control is dependent upon cultivars with inherent resistance (Topfer & Eibach, 2002), canopy management (Zoecklein et al., 1992) and the application of ‘soft control options’ such as compost teas (Ryan et al., 2005).
There are naturally occurring alternatives to synthetic botryticides, including plant defence stimulants to increase host resistance and microorganisms to suppress disease epidemics. However, the complexities of interactions between plants and microbes in the field present an enormous challenge for the practical implementation of biological control methods. This review discusses the biological suppression of B. cinerea in grapes and considers potential constraints for the practical implementation of biological control methods in vineyards.
Successful implementation of biosuppressive methods for control of B. cinerea is dependent upon an intimate knowledge of the ecology and epidemiology of the disease in vineyards (Elmer & Michailides, 2004; Holz et al., 2004). Botrytis cinerea survives saprophytically through the winter on a diverse range of host species (Bisiach et al., 1984; Sutton, 1991). Within the vineyard, several sources of overwintering inoculum have been identified, including sclerotia (Nair et al., 1995), grape vine prunings (Thomas et al., 1983) and other necrotic grape tissues in the vine (Emmett & Nair, 1991; Fowler et al., 1999; Elmer & Michailides, 2004) and on the ground (Seyb, 2004). Release of fresh conidia from these sources in the spring provides an abundance of inoculum for infection of floral tissues and tender young shoots and leaves in the grapevine (McClellan & Hewitt, 1973; Nair & Hill, 1992; Nair et al., 1995). Apothecia of the sexual state (Botryotinia fuckeliana) have been reported, but their occurrence is sporadic (Beever & Weeds, 2004). Senescing floral tissues are highly susceptible to B. cinerea (Keller et al., 2003) and profuse sporulation is frequently observed on these tissues when conditions favour pathogen development in the spring. During active growth, B. cinerea produces a range of hydrolytic enzymes and metabolites to facilitate penetration and colonization of host tissues (Kars & van Kan, 2004). Physical and chemical host defences limit the spread of early-season infections and the pathogen enters a quiescent or latent phase in the host tissue. In berries, B. cinerea remains in a latent state until the postveraison (change of berry colour and commencement of berry ripening) period, and then resumes pathogenic development as host defences naturally begin to decline (Pezet & Pont, 1992; Nunan et al., 1998; Holz et al., 2003; Pezet et al., 2003). Latent infection of berry pedicels, and to a lesser extent grape bunch rachii, also accounts for fruit infection at vintage (Michailides et al., 2000; Holz et al., 2003). Late-season berry infections can also arise, particularly in wounded or cracked berries, from direct infection by airborne conidia or from mycelia growing out from saprophytic bases within aborted flowers, aborted fruitlets and calyptras trapped within developing bunches (Nair & Parker, 1985; Nair & Hill, 1992; Latorre & Rioja, 2002; Seyb, 2004). Prolonged wet periods during the late season period encourages pathogen development and leads to proliferation of B. cinerea on necrotic grape leaf debris and in the ripening grape bunch, resulting in substantial crop losses at vintage. More detailed discussion on the relative importance of early-, mid- and late-season berry infections has recently been published (Elmer & Michailides, 2004).
Microbial suppression of Botrytis cinerea
Many nonpathogenic microorganisms suppress the growth of plant pathogens through competition for nutrients, the production of inhibitory metabolites and/or parasitism, thereby naturally limiting plant disease in the environment. Numerous studies have described the isolation of antagonistic microorganisms with a view to exploiting their potential for biological disease suppression. However, despite many reports of successful biocontrol of B. cinerea in laboratory conditions, only a small proportion of these have demonstrated field efficacy and an even smaller subset have been developed into commercial products (Table 1). The following section discusses the use of disease-suppressive microorganisms with particular emphasis on their potential to control B. cinerea in grapes.
Table 1. Examples of microbial antagonists of Botrytis cinerea on grape tissues
|Filamentous fungi and oomycetes|
| Gliocladium spp.|| ||Grape berry assays||Machowicz-Stefaniak (1998)|
| Epicoccum nigrum|| ||Necrotic grape leaf discs||Stewart et al. (1998)|
|Grape rachii||Fowler et al. (1999)|
| Pythium radiosum|| ||In vitro grape vine plantlets||Paul (1999a)|
| P. periplocum|| ||In vitro grape vine plantlets||Paul (1999b)|
| Trichoderma atroviride (LC52)||Sentinel (Agrimm Technologies, NZ)||Grapevine field studies||http://www.agrimm.co.nz|
| T. harzianum (T39)||Trichodex (Makhteshim-Agan, Israel)||Multi-country grapevine field studies||O’Neill et al. (1996); Elad (2001); Elad & Stewart (2004)|
| T. harzianum (P1)|| ||Grapevine field studies||Latorre et al. (1997)|
| T. viride (Td50)||Trichopulvin 25WP (Research Institute of Plant Protection, Romania)||Grapevine field studies||Sesan et al. (1999)|
| Ulocladium atrum (U385)|| ||Multi-country grapevine field studies||Lennartz et al. (1998); Schoene & Kohl (1999); Roudet & Dubos (2001); Schoene et al. (2000); Metz et al. (2002)|
| Ulocladium oudemansii (HRU3)||Botry-Zen (Botry-Zen Ltd, NZ)||Grapevine field studies||Reglinski & Kingston (2001); Elmer et al. (2003); Reglinski et al. (2005); Elmer et al. (2005)|
| Ulocladium spp.|| ||Grape rachii||Fowler et al. (1999)|
|Yeasts and yeast-like fungi|
| Acremoniumcephalosporium (B11)|| ||Grape berry assays||Zahavi et al. (2000)|
| Aureobasidium pullulans|| ||Grape berry assays and field studies||Lima et al. (1996, 1997, 1999); Schena et al. (1999); Castoria et al. (2001)|
| Candida guilliermondii|| ||Grape berry assays||McLaughlin et al. (1992)|
|Grapevine field studies||Zahavi et al. (2000)|
| C. saitoana||Bio-Coat and Biocure||Grapevine field studies||Schena et al. (2004)|
| Cryptococcus laurentii LS-28|| ||Grape berry assays||Lima et al. (1998, 1999)|
| Metschnikowia spp. LS15|| ||Grape berry assays and grapevine field studies||Schena et al. (2000)|
| Metschnikowia fructicola||Shemer (AgroGreen, Minrav Group, Israel)||Grapevine field studies||Kurtzmann & Droby (2001); Keren-zur et al. (2002)|
| Metschnikowia pulcherrima 320|| ||Grape berry assays||Nigro et al. (1999)|
| Pichia membranifaciens (FY 101)|| ||In vitro grape vine plantlets||Masih et al. (2000, 2001)|
| Rhodotorula glutinis (LS-11)|| ||Grape berry assays||Lima et al. (1998, 1999)|
| Saccharomyces chevalieri||Saccharopulvin 25 PU (RIPP) Bucharest, Romania||Grapevine field studies||Sesan et al. (1999)|
| Trichosporon pullulans (RB9)|| ||Grape berry assays Grapevine field studies||Schmidt et al. (1996); Holz & Volkmann (2002)|
| Bacillus spp.|| ||Grape berry assays||Ferreira (1990)|
|In vitro grape vine plantlets||Paul et al. (1998)|
| Bacillus subtilis (QST-713)||Serenade (Agra Quest, USA)||Grapevine field studies||Esterio et al. (2000); Schilder et al. (2002)|
| Bacillus circulans|| ||In vitro grape vine plantlets||Paul et al. (1997)|
| Brevibacillus brevis|| ||Grape berry assays||Ellis (1996); Seddon et al. (2000)|
|Grapevine field studies||Schmitt et al. (2002)|
| Pseudomonas fluorescens|| ||Grape berry assays||Krol (1998)|
| Serratia liquefaciens|| ||Necrotic grape leaf disc assays||Whiteman & Stewart (1998)|
No other single fungal genus has received as much attention as the Trichoderma spp. for biocontrol of plant pathogens. Biocontrol research with Trichoderma spp. against B. cinerea in grapes commenced nearly three decades ago (Dubos et al., 1978, 1982) and the best results were achieved when disease pressure in the vineyard was low to moderate (Bisiach et al., 1985b; Gullino & Garibaldi, 1988; Garibaldi et al., 1989). An isolate of T. harzianum (T39), originally isolated from cucumber, was the first Trichoderma sp. to be specifically formulated into a commercial product for control of B. cinerea (Elad, 2001). Manufactured as Trichodex by Makhteshim-Agan (Israel), the efficacy of this product was comprehensively evaluated in 139 field experiments in commercial vineyards over 19 countries on 34 varieties between 1988 and 1994. The control efficacy was 36%, compared with 52% with standard botryticides, when applications were made at the four growth stages; end of flowering, bunch closure, veraison, 2–3 weeks postveraison, at a rate of 4 kg ha−1. Efficacy declined when the interval between the last preharvest application and vintage was extended out to 5 weeks, indicating that late-season protection of ripening fruit was important for B. cinerea control (O’Neill et al., 1996).
An isolate of T. harzianum was specifically selected for its ability to colonise senescent floral debris (stamens, calyptra and aborted fruitlets) and the green structural tissues of the grape bunch, since these were potential sites for latent B. cinerea infections (Holz et al., 1997, 2003). Colonization capability of T. harzianum was superior to that of Gliocladium roseum, Ulocladium atrum and Trichosporon pullulans, in bunches of table (cv. Dauphine) and wine (cv. Chardonnay) grapes in South African vineyards (Holz & Volkmann, 2002). Antagonist establishment fluctuated between vineyards and between seasons, but only T. harzianum effectively colonized monitored positions within bunches during the season. However, efficacy of botrytis bunch rot control was not possible due to the sporadic nature of the pathogen in the experimental vineyards over two growing seasons.
The product T-22 (Bioworks Inc., USA) contains T. harzianum (1295-22), and is primarily used to control soilborne pathogens and as a plant growth stimulant (Harman, 2000; Dissevelt & Ravensberg, 2002). In grapes, suppression of B. cinerea with T-22 was equivalent to a standard botryticide programme (Harman et al., 1996; Wilson, 1997). Trichoderma harzianum isolates (code S10B and P1) have been evaluated on table grape cv. Thompson Seedless in Chile. Over a range of disease pressure conditions (1992–96), isolate P1 provided effective control of botrytis bunch rot and efficacy was equivalent to a botryticide programme based upon the dicarboximide fungicide, vinclozolin. The effectiveness of P1 as a formulated BCA was significantly better compared with the unformulated treatment (Latorre et al., 1997).
Trichopulvin 25 PU (T. viride isolate code Td50) was evaluated against three other biological control agents for botrytis bunch rot control in field-grown grapes in Romania (Sesan et al., 1999). Four applications of Trichopulvin 25 PU per season resulted in high levels of botrytis bunch rot suppression (range 70–96%) and compared well with a Trichodex programme (range 51–96%) and a dicarboximide fungicide-based programme. Sentinel, a new biofungicide containing T. atroviride (LC52) is manufactured by Agrimm Technologies Ltd and is registered in New Zealand for control of B. cinerea in grapes.
Mechanisms of B. cinerea suppression by different Trichoderma spp. are diverse and include antibiosis (Cutler et al., 1996; Cooney et al., 1997; Rey et al., 2001), competition (Elad et al., 1999), mycoparasitism (Dubos et al., 1982; Papavizas, 1985) and induction of plant defence mechanisms (Elad, 2000; Hanson & Howell, 2004). Some isolates exhibit multiple modes of action: for example, the T39 isolate used in Trichodex is an effective nutrient competitor, but also interferes with B. cinerea pectolytic enzymes and induces host resistance (Elad & Stewart, 2004).
Species of Ulocladium spp. are closely related to Alternaria and Stemphylium spp. (Simmons, 1967), and while some species are reportedly pathogenic (e.g. U. cucurbitae), others (e.g. U. atrum) are predominantly saprophytic. An isolate of U. atrum (U385) was first identified as an antagonist of B. cinerea over a decade ago (Kohl et al., 1993) and, since then, has been shown to suppress this pathogen in several field and glasshouse crop systems (Kohl et al., 1995a, 1995b, 2001). In field tests in grapes, applications of this isolate reduced botrytis bunch rot by up to 67% in different wine-growing regions in Germany (Lennartz et al., 1998; Schoene & Kohl, 1999; Schoene et al., 2000) and similar levels of botrytis bunch rot control were reported from studies with U385 in French vineyards (Roudet & Dubos, 2001). Subsequent field studies in German vineyards confirmed that, under moderate pathogen pressure, U. atrum (385) has the potential to control botrytis bunch rot of grapes. However, it was proposed that when vineyard conditions were highly conducive to infection, the efficacy of this BCA would decline to the point where it could not completely replace synthetic botryticides (Metz et al., 2002).
Ulocladium atrum (385) was evaluated in South African vineyards between 1996 and 2000 in comparative tests with G. roseum, T. harzianum and Trichosporon pullulans. Unfortunately, the occurrence of B. cinerea during that period was sporadic and conclusions on the relative performance of the BCAs was not possible (Holz & Volkmann, 2002). Several Ulocladium spp. isolates have been investigated as potential B. cinerea antagonists on necrotic grape leaf discs in New Zealand (NZ) laboratories (Stewart et al., 1998). One isolate (U13) suppressed B. cinerea conidiophore production by up to 90% on necrotic leaf discs that had been preinoculated with B. cinerea, then exposed to field conditions in a NZ vineyard (Stewart et al., 1998). In separate studies, U13 was field evaluated for suppression of B. cinerea overwintering inoculum potential on grape rachii in the canopy. Some reduction in B. cinerea inoculum potential was reported 2 months after harvest at one site, but the reduction in pathogen inoculum was not maintained through to early spring (Fowler et al., 1999).
Laboratory screening of saprophytic isolates of Alternaria spp., Epicoccum nigrum, Cladosporium spp., Trichoderma spp. and Ulocladium spp. from a diverse range of necrotic host tissues identified several isolates capable of surviving interrupted wet periods and effectively suppressing B. cinerea conidial production on necrotic leaf discs that had been preinoculated with a conidial suspension of B. cinerea (Elmer et al., 1995). Selected isolates from these studies were comprehensively evaluated in 11 repeated field assays in two regions of NZ during 1996–97. Three Ulocladium spp. isolates consistently and repeatedly reduced B. cinerea spore production by 90–100% (Michailides & Elmer, 2000). One of these isolates was subsequently identified as U. oudemansii by the Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands in 1998. Multisite field evaluation of this isolate in grapes demonstrated that early season establishment of U. oudemansii in floral bunch trash (calyptra and aborted fruitlets) significantly reduced the incidence and severity of B. cinerea on these tissues prior to bunch closure, resulting in a significant reduction of botrytis bunch rot at vintage (Reglinski & Kingston, 2001; Elmer et al., 2003). A commercial formulation, based upon this isolate (Botry-Zen; http://www.botryzen.co.nz) was recently developed for early-season control of B. cinerea in grapes (International Patent Application number PCT/NZ01/00111). Field efficacy of Botry-Zen was demonstrated over a wide range of botrytis disease pressure conditions and ranged from 81 to 91% in multisite vineyard trials (cv. Chardonnay) over three seasons (2002–05) in the Hawke's Bay region of NZ (Elmer et al., 2005).
Competition for senescent and necrotic host tissues is considered the primary mechanism for Botrytis spp. suppression by U385 (Kohl et al., 1997; Kessel et al., 2002, 2005). In grapes, studies have shown colonization of senescent grape floral tissues and establishment in juvenile berries via floral abscission wounds by U385 as the primary mode of action (Roudet & Dubos, 2001). It was also proposed that production of extracellular hydrolytic enzymes could also assist biocontrol of B. cinerea in necrotic strawberry tissues by U385 (Berto et al., 2001). Aggressive saprophytic colonization of grape floral tissues prior to bunch closure was proposed as the primary mode of action for the U. oudemansii isolate used in Botry-Zen (Elmer et al., 2003).
Other fungal antagonists
Several commonly occurring fungi (e.g. Alternaria spp., Cladosporium spp., E. nigrum) and basidiomycetous yeasts (e.g. Aureobasidium pullulans.) have been isolated from grape tissues (Dugan et al., 2002) and found to be antagonistic to B. cinerea. The Cladosporium spp. are one of the commonest saprophytic fungi colonizing senescent and necrotic tissues on above-ground plant parts, and Wood (1951) and Newhook (1957) were the first to demonstrate the biocontrol potential of Cladosporium spp. against B. cinerea. Subsequent studies with this antagonist have been reported in tomatoes (Eden et al., 1996), kiwifruit (Harvey et al., 1991; Elmer et al., 1995), onions (Kohl et al., 1995b) and grapes (Bisiach et al., 1985a). Alternaria spp. are also reported to be effective antagonists of Botrytis spp. (Peng & Sutton, 1991; Elmer et al., 1995; Michailides & Elmer, 2000; Elad & Stewart, 2004). There are no known reports of Alternaria spp. being tested in vineyards and only one report for Cladosporium spp. (Bisiach et al., 1985a). Interestingly, both fungal species were the most prevalent saprophytes on necrotic floral debris in both sprayed and unsprayed vineyards (PAG Elmer & PN Wood, The Horticulture and Food Research Institute of New Zealand, unpublished data).
There have been many reports of successful suppression of B. cinerea with isolates of E. nigrum (syn. E. purpurascens) (Hill et al., 1999; Elmer et al., 2001; Szandala & Backhouse, 2001). In grapes, E. nigrum effectively suppressed B. cinerea on leaf discs that had been preinoculated with the pathogen in laboratory and vineyard assays (Stewart et al., 1998). Application of E. nigrum to grape rachii significantly reduced B. cinerea inoculum production on these tissues over a range of incubation temperatures (10–20°C), indicating that this BCA had the potential to effectively reduce overwintering B. cinerea in the vineyard (Fowler et al., 1999). Further experiments demonstrated that E. nigrum reduced B. cinerea inoculum on overwintering rachii 2 months after harvest, but the reduction in inoculum potential was not maintained through to early spring. Field applications of an isolate of E. nigrum (HRE2) suppressed botrytis bunch rot by 60% (cv. Chardonnay) when evaluated in a NZ vineyard in separate studies (PAG Elmer, unpublished data). Although some isolates of E. nigrum produce antimicrobial metabolite(s) capable of completely suppressing germination of B. cinerea conidia, this isolate did not produce assay-detectable antimicrobials, and it was concluded that the primary mode of action of this isolate was aggressive saprophytic colonization of necrotic vine tissues (Elmer et al., 2001).
Gliocladium roseum (reclassified as Clonostachys rosea) has effectively suppressed B. cinerea in both field and glasshouse crops (Sutton et al., 1997; Kohl et al., 1998; Morandi et al., 2001). Published comparative field evaluations of this BCA in viticulture are sparse, but in one report Gliocladium spp. were reported to be less effective than Trichoderma spp. when tested on grapes (Machowicz Stefaniak, 1998). In contrast, an unnamed Gliocladium spp. was as effective as the dicarboximide fungicide vinclozolin against B. cinerea in vineyard tests (Cherif et al., 1998). Mode of action studies indicate antibiosis, and mycoparasitism of conidia and germ tubes are important biocontrol mechanisms (Kohl et al., 1997; Li et al., 2002).
Two aggressive oomycetous mycoparasites, identified as Pythium radiosum, protected detached grapevine leaves in vitro (Paul, 1999b), and in a separate study P. periplocum completely protected 2-month-old grapevine plants (c.v. Chardonnay and Pinot noir) from B. cinerea. When coinoculated with B. cinerea in vitro, the mycelium was easily penetrated, resulting in widespread destruction of the pathogen hyphae, confirming P. periplocum as a BCA (Paul, 1999a).
Yeast and yeast-like fungi
Yeasts and yeast-like fungi are a major component of the epiphytic microbial community on the surfaces of fruits and vegetables. A diverse range of yeasts (e.g. Rhodotorula glutinis, Candida spp., Pichia membranifaciens, Kloeckerea apiculata, Saccharomyces spp.) and yeast–like species (e.g. A. pullulans and T. pullulans) have shown efficacy against B. cinerea. Rhodotorula glutinis (LS-11) and Cryptococcus laurentii (LS-28) were reported to be effective B. cinerea antagonists in vineyards. LS-28 was regarded as more promising based upon its biosuppression capabilities over a broad range of experimental conditions (Lima et al., 1998). These antagonists also demonstrated low sensitivities to copper oxychloride and dicarboximide fungicides, but were classed as being highly sensitive to the demethylation inhibitor (DMI) fungicides, penconazole (Topas) and tebuconazole (Folicur).
The formulated yeast product Saccharopulvin 25 PU (Saccharomyces chevalieri) was applied at the end of flowering, petal fall, berry formation, bunch closure and 3 weeks before harvest for three seasons (1995–97) at 6 × 106 CFU mL−1 (Sesan et al., 1999). Average botrytis bunch rot incidence was 40% in the untreated controls and Saccharopulvin treatment efficacy was 91% when averaged over three seasons. Zahavi et al. (2000) evaluated Candida guilliermondii A42 and Acremonium cephalosporium B11 against B. cinerea in field-grown table and wine grapes. Two to five applications of A42, at 7- to 10-day intervals, to both crops from veraison (1996–98), reduced the incidence of rots from B. cinerea and Aspergillus niger at harvest (wine grapes) and after postharvest storage (table grapes) in two of the three growing seasons: B11 reduced both rot-inducing pathogens in the wine grapes but not in the table grapes. Interestingly, in the 1996 growing season, none of the BCA treatments or the chemical controls effectively reduced incidence of rot in Sauvignon blanc grapes compared with the untreated controls. On this occasion B. cinerea may have been established in the bunches as a consequence of conditions favourable to the pathogen over the flowering period (Elmer & Michailides, 2004). Consequently, the postveraison BCA and chemical treatments could only be expected to protect rapidly ripening berries from conidial infections and not from aggressive B. cinerea infections from a saprophytic base residing in floral debris and aborted fruitlets within the bunch.
The ascosporic yeast, Metschnikowia pulcherrima (anamorph: Candida pulcherrima) isolate 320, was identified as an effective antagonist against botrytis storage rot in table grapes (Nigro et al., 1999). An isolate of the yeast M. fructicola was also evaluated by Kurtzman & Droby (2001), and Keren-zur et al. (2002) reported good disease control against both B. cinerea and A. niger. A water-dispersible granule with a shelf life of 1 year has been successfully formulated and marketed as Shemer and registered in Israel by AgroGreen Minrav Group (http://www.agrogreen.co.il). Pichia membranifaciens (FY 101), an isolate from grapes, was an effective antagonist of B. cinerea on grapevine plantlets grown in vitro and in coinoculation studies on grape berries (Masih et al., 2000, 2001). The so-called ‘pink yeasts’, Sporobolomyces spp., are commonly found on fruit surfaces, including grapes (De la Torre et al., 1999). Although selected isolates of S. roseus have been reported as very effective antagonists of B. cinerea (Janisiewicz et al., 1994), it is thought that this BCA has not been tested in grapes.
The primary mode of action of yeast-based BCAs is generally considered to be competition for B. cinerea infection sites and germination stimulating nutrients (Filonow, 1998). Other modes of action include elicitation of host defences (Adikaram et al., 2002), production of antifungal hydrolytic enzymes (Masih & Paul, 2002) and attachment to B. cinerea hyphae, thereby debilitating subsequent growth and development (Cook et al., 1997). There are two commercialized yeast products, Aspire (based upon Candida oleophila) and Yield Plus (based upon Cryptococcus albidus), registered for use against postharvest pathogens (including B. cinerea) of pome and citrus fruits.
The ubiquitous yeast-like fungus A. pullulans is well documented as an antagonist of fungal and bacterial pathogens (Janisiewicz & Korsten, 2002). It is readily isolated from the phylloplane of many diverse plant species, and in grapes it has been isolated as an endophyte in dormant canes (Bugaret & Lafon, 1989), grape buds and grape berries, often concentrated at the grape berry/receptacle base (Dugan et al., 2002). Several BCA screening programmes have included A. pullulans as a potential antagonist of B. cinerea in grapes (Bisiach et al., 1985a; Lima et al., 1996; Schena et al., 1999). Two isolates of A. pullulans (L47 and LS-30) are reported to be highly effective against B. cinerea on table grapes (Lima et al., 1996, 1997; Castoria et al., 2001). Interestingly, there are very few reports of field-based evaluation of isolates of A. pullulans in wine grapes.
Rapid wound colonization was identified as the primary mode of action of A. pullulans against B. cinerea (Lima et al., 1999), since antibiosis and direct interference with hyphae of B. cinerea were not involved (Castoria et al., 2001). Some isolates of A. pullulans induce a host resistance response (see later) and/or produce antimicrobial metabolites of low-level acute toxicity, such as the aureobasidins (Takesako et al., 1991; De Lucca & Walsh, 1999). A commercial BCA product (Blossom-Protect fb; http://www.bio-protect.de) based upon A. pullulans is registered for use against the causal agent of bacterial fireblight bacterium (Erwinia amylovora), but its efficacy against botrytis bunch rot in grapes is not known.
The yeast-like fungus T. pullulans (code RB9) was evaluated in a commercial table grape vineyard and performed at least as well as other BCAs and the fungicide vinclozolin. Enhancement of host defence was proposed as a possible mechanism of action (Schmidt et al., 1996). Evaluation of the comparative efficacy of T. pullullans in South African vineyards was inconclusive due to low B. cinerea inoculum levels over the duration of the study (Holz & Volkmann, 2002).
Many studies have investigated the potential of bacteria as B. cinerea antagonists in a wide range of fruit crops, including tomatoes (Daggas et al., 2002; McHugh et al., 2002), strawberries (Helbig, 2001; Guetsky et al., 2002), apples (Janisiewicz & Jeffers, 1997) and pears (Seddon et al., 2000; Nunes et al., 2001). Bacterial BCAs with reported activity against B. cinerea on grape tissues include Bacillus spp. (Ferreira, 1990; Krol, 1998; Paul et al., 1998), Bacillus circulans (Paul et al., 1997), Brevibacillus brevis (formerly Bacillus brevis; Seddon et al., 2000), Bacillus subtilis (Esterio et al., 2000), Pseudomonas fluorescens (Krol, 1998), and Serratia liquefaciens (Whiteman & Stewart, 1998).
Four applications of a new formulation of Serenade (B. subtilis strain QST-713) were compared with a traditional spray programme used to treat table grapes (cv. Thompson Seedless) for B. cinerea management in Chile. Higher rates of the product (15 kg ha−1) resulted in postharvest disease control equivalent to a traditional botryticide programme (Esterio et al., 2000). Up to 90% disease control was reported when green table grapes were artificially inoculated with B. cinerea then treated with a suspension of B. brevis (Ellis, 1996; Seddon et al., 2000). When B. brevis was used in combination with Milsana (a.i. Reynoutria sachalinensis extract) and Myco-sin (consisting of diatomaceous earth, sulphuric basalt, silicic acid, Equisetum extract) in an integrated disease control programme, botrytis bunch rot was significantly reduced in organically grown grapes (Schmitt et al., 2002). Using a different approach and focusing on reducing B. cinerea inoculum potential on necrotic tissues, Whiteman & Stewart (1998) demonstrated that S. liquefaciens effectively protected leaf discs from B. cinerea. In laboratory assays with 2-month-old grapevine plantlets (cvs Chardonnay and Pinot noir), Paul et al. (1997) reported that a culture of B. circulans or its filtrate completely suppressed symptoms of B. cinerea infection in vitro.
It has been proposed that bacteria are generally less tolerant of fluctuations in available water compared with filamentous fungi and yeasts (Fokkema & Shippers, 1986; Rousseau & Doneche, 2001) and may not survive on the phylloplane. However, the spore-forming bacilli are able to withstand harsh environmental conditions (Seddon et al., 2000; Elad & Stewart, 2004). Commercialized bacterial-based BCAs for pre- and postharvest botrytis control include P. syringae (Biosave 100 and Biosave 1000), and B. subtilis (Serenade and Rhapsody). However, Serenade is the only bacterial-based BCA with a label claim for use against B. cinerea in grapes. Further advances in formulation technology specifically focused on Bacillus spp. are likely to improve the suppressiveness of bacterial-based BCAs and extend their performance over a wide range of field and postharvest conditions (Schisler et al., 2004).
The primary mode of action for bacterial BCAs varies and includes modification of the wettability of plant surfaces by Bacillus brevis (Edwards & Seddon, 1992); competition for B. cinerea-stimulating nutrients, e.g. Pseudomonas spp. (Elad & Stewart, 2004); production of cell wall-degrading enzymes such as endochitinase, e.g. S. plymuthica (Kamensky et al., 2003); secretion of inhibitory compounds, e.g. P. cepacia (Janisiewicz & Roitman, 1988), P. antimicrobia (Walker et al., 2001), B. subtilis (Leifert et al., 1995) and B. brevis (Seddon et al., 2000); reduction of B. cinerea inoculum production, e.g. S. liquefaciens (Whiteman & Stewart, 1998); and induction of systemic resistance, e.g. P. aeruginosa and Bacillus spp. Some isolates (e.g. S. plymuthica) are reported to reduce B. cinerea germination and development via several mechanisms of antifungal activity (Kamensky et al., 2003).
Mycoviruses and isolates of B. cinerea
Interest in mycoviruses as potential biocontrol agents has increased recently, especially where it can be clearly demonstrated that their presence in the pathogen cell reduces pathogenic growth and sporulation (Tsai et al., 2004). Mycoviruses of B. cinerea have been reported (Howitt et al., 2001; Castro et al., 2003), but their practical application for suppression of B. cinerea epidemics has yet to be demonstrated. Considerable genetic diversity exists among wild populations of B. cinerea (Giraud et al., 1999; Beever & Weeds, 2004; Martinez et al., 2005), including isolates with nil to very low levels of host pathogenicity. The potential for nonpathogenic isolates to be used as biocontrol agents against pathogenic isolates of B. cinerea in grapes has been proposed (van der Cruyssen & Kamoen, 1995) and field evaluation of nonpathogenic isolates in grapes is currently being evaluated (R. Beever, LandCare, NZ, personal communication).
Induction of resistance to Botrytis cinerea
Grapevines rely upon preformed and inducible resistance mechanisms to defend against infection by B. cinerea (Gabler et al., 2003; Keller et al., 2003). The cuticle and cell wall physically resist hyphal penetration whilst tannins and phenolics in the cell wall inhibit fungal enzymes implicated in pathogenesis (Sarig et al., 1998; Goetz et al., 1999). Early inducible responses include the deposition of new cell wall material, the release of reactive oxygen species (ROS) and hypersensitive cell death (HR) at the infection site (Hammerschmidt, 1999). However, the role of these defence responses in resistance to B. cinerea is controversial since recent evidence indicates that B. cinerea can exploit ROS production during plant colonization (Lyon et al., 2004) and that the HR may actually facilitate infection by necrotrophic fungi (Govrin & Levine, 2000).
The best characterized inducible resistance mechanisms to B. cinerea in grapevines are the accumulation of antimicrobial phytoalexins and the synthesis of pathogenesis-related (PR) proteins (Jeandet et al., 1995; Sbaghi et al., 1995). The major phytoalexins produced in grapevines are resveratrol and its derivatives piceid, pterostilbene and ɛ-viniferin (Jeandet et al., 2002). Resveratrol accumulation occurs in the abaxial surface of leaves and in berry skins at concentrations ranging from 40 to 400 µg per g fresh weight (Jeandet et al., 1991; Adrian et al., 2000). Studies on grape plantlets demonstrated a positive correlation between UV-induced resveratrol synthesis in leaves and field resistance to B. cinerea among 11 Vitis spp. (Sbaghi et al., 1995). Fungitoxicity studies in vitro showed that resveratrol concentrations of 90 µg mL−1 inhibited conidial germination by 50% whilst concentrations ranging from 60 to 140 µg mL−1 reduced mycelial growth and caused cytological modifications typically associated with stress (Adrian et al., 1997). In berries, resveratrol levels steadily decline during fruit maturation and this is concomitant with increased susceptibility to grey mould (Jeandet et al., 1991; Sarig et al., 1997). Resveratrol levels are generally lower in flowers than in leaves and berries and this has been proposed as a contributory factor to their higher susceptibility to B. cinerea (Jeandet et al., 2002). Furthermore, the relative concentrations of phenolic compounds vary in different parts of the flower with lowest levels in the receptacle area, typically where disease is highest (Keller et al., 2003).
Pathogenesis-related proteins, including chitinases, glucanases and thaumatin-like proteins, accumulate in berries and leaves in response to pathogen attack and are thought to contribute to grapevine resistance by degrading structural components in fungal cell walls (Giannakis et al., 1998). The pattern of PR-protein expression varies depending on the tissue type, developmental stage and also on the type of infecting pathogen (Busam et al., 1997b; Derckel et al., 1999; Robert et al., 2002). In grape berries PR-protein levels are also produced constitutively as a normal part of the ripening process (Robinson et al., 1997; Tattersall et al., 1997; Davies & Robinson, 2000) and this has been proposed to represent a prophylactic measure against environmental stress and pathogen attack (Davies & Robinson, 2000). Chitinase and thaumatin-like protein represent the predominant PR-proteins in grape berries and together can account for half of the soluble protein in ripe grapes (Waters et al., 1998). Bezier et al. (2002) showed that polygalacturonase inhibitor proteins (PGIPs) were expressed in vine leaves and berries in response to infection by B. cinerea: PGIPs are thought to contribute to disease resistance by inhibiting the degradation of the plant cell wall by fungal polygalacturonases.
Various biotic and abiotic agents have been shown to activate defence mechanisms associated with resistance to B. cinerea in grape cell suspensions, leaves and berries, so indicating their potential to elevate disease resistance (Table 2).
Table 2. Examples of inducing agents with demonstrated cellular and biological activity on grape tissues
|Aluminium chloride||Phytoalexin accumulation in grape leaves||Adrian et al. (1996); Borie et al. (2004)|
|Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (Syngenta – formerly Novartis)||Chitinase class III transcript accumulation in cell suspensions Increased levels of resveratrol and anthocyanin in berries and induced resistance to B. cinerea||Busam et al. (1997b) Iriti et al. (2004)|
|Chitosan||Induction of phenylalanine ammonia-lyase activity and elevation of resistance to B. cinerea in berries||Romanazzi et al. (2002)|
|Field efficacy against B. cinerea||Amborabe et al. (2004)|
|Chitogel (Ecobulle, France)||Reduced B. cinerea infection on plantlet leaves||Ait Barka et al. (2004)|
|5-Chlorosalicylic acid||Field efficacy against B. cinerea||Reglinski et al. (2005)|
|2,6-Dichloroisonicotinic acid||Accumulation of stilbene synthase transcripts||Busam et al. (1997a)|
|Accumulation of class III chitinase transcripts||Busam et al. (1997b)|
|Laminarin (β-1,3-glucan from Laminaria digitata)||Induction of oxidative bust, induction of defence-related genes in cell suspensions, and elevation of resistance to B. cinerea in plantlets and detached leaves||Aziz et al. (2003)|
|Methyl jasmonate||Phytoalexin accumulation in leaves and berries||Larronde et al. (2003)|
|Induction of the oxidative burst and accumulation of several defence-related proteins||Repka et al. (2004)|
|Milsana (Reynoutria sachalinensis extract, KHH BioScience Inc., USA)||Field efficacy against B. cinerea||Schilder et al. (2002); Schmitt et al. (2002)|
|Oligogalacturonide (Cermav, France)||H2O2 production, induction of defence-related genes in cell suspensions, and elevation of resistance to B. cinerea in detached leaves||Aziz et al. (2004)|
|Phosphate salts||Induction of peroxidase activity in berries||Reuveni & Reuveni (1995)|
|Salicylic acid||Induction of PR-proteins||Renault et al. (1996)|
|Induction of chitinases in berries||Derckel et al. (1996, 1998)|
|Accumulation of chitinase class I and III transcripts in cell suspensions||Busam et al. (1997b)|
|Synermix (a.i. AlCl3, Goemar, France)||Field efficacy against B. cinerea||Jeandet et al. (2000)|
Salicylic acid (SA) plays a regulatory role in the coordination of plant disease resistance (Hammerschmidt & Smith-Becker, 1999). Exogenous application of SA and functionally related analogues, including 2,6-dichloroisonicotinic acid (INA) and benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl esther (BTH), have been shown to induce resistance to viral, fungal, oomycetous and bacterial pathogens in both dicotyledonous and monocotyledonous plants (Renault et al., 1996; Reglinski et al., 1997, 1998). Treatment with SA induced chitinase expression in cv. Pinot Noir cells (Busam et al., 1997b) and stimulated accumulation of PR-proteins in grape leaves (Renault et al., 1996), stems, roots and berries (Derckel et al., 1996, 1998). Ten field applications of 0·2% SA (∼10 mm) also reduced severity of downy mildew (Plasmopara viticola) by 30% on leaves and by up to 40% on berries, and caused a slight increase in SA residues on grapes and in wines (Kast, 2000). Salicylic acid levels measured in wines prepared from grapes (cvs Reisling, Muller-Thurgau and Pinot Noir) receiving between eight and 13 applications of SA (0·18–0·48 kg ha−1) (Pour-Nikfardjam et al., 1999) had no effect on the SA content of 23 commercially available German wines from six different grape-growing regions. White wines on average had lower SA concentrations (∼50 µg L−1) than red wines (∼160 µg L−1).
Because SA is a plant hormone, it affects grapevine physiology. In vineyard studies on Shiraz grapes, berry ripening was retarded by 2–4 weeks when SA (7·2 mm) was injected into berries 2–3 weeks before veraison (Kraeva et al., 1998). This affect was thought to be due to the inhibition of abscisic acid (ABA), a plant hormone that is thought to play an important role in triggering veraison. The SA treatment also caused limited cell necrosis on the berry skin that may be indicative of phytotoxicity. Indeed, field application of SA may be impractical because of the narrow margin separating rates at which it is efficacious and the rate at which it is phytotoxic. Foliar application of SA, at concentrations greater than 2 mm, caused leaf chlorosis and burning of leaf margins on kiwifruit (Reglinski et al., 1997) and grapes (unpublished data). These SA concentrations are lower than those used in the studies reported above and phytotoxicity may have been exacerbated by addition of an adjuvant (0·1% Pulse).
5-Chlorosalicylic acid (5CSA) is a chlorinated synthetic derivative of salicylic acid and has been shown to be a more potent inducer of general plant defence mechanisms than salicylic acid itself (Conrath et al., 1995). Reglinski et al. (2005) evaluated the use of 5CSA for controlling botrytis bunch rot in cv. Chardonnay grapes in New Zealand. Regular applications (every 14–28 days) of 1 mm 5CSA in 0·1% Pulse significantly reduced the incidence and severity of botrytis bunch rot. In more recent studies on cvs Chardonnay and Merlot grapevines, disease severity was reduced by 60–90% compared with control vines that had no botryticide treatments (unpublished data). Efficacy was generally as good as or better than the recommended fungicide programme. However, some problems accompanied frequent and repeated application of 5CSA, including reduced berry weight, leaf chlorosis and detectable chemical residues on grapes and in wines. Furthermore, when cv. Cabernet Sauvignon vines were treated with 0·8 mm 5CSA, at 3-weekly intervals from fruit-set to harvest, there was a 15% reduction in total phenolics extraction during vinification and a decrease in wine quality index (Duxbury et al., 2004).
The synthetic ‘SA mimic’ BTH produced by Syngenta (Switzerland) is marketed as Bion in Europe and was originally registered for controlling powdery mildew on cereals. In the USA the product is called Actigard or Blockade and is registered for use on tobacco, lettuce, tomato, and spinach. Treatment with BTH induced expression of PR-proteins in grapevine cell cultures (Busam et al., 1997a, 1997b). Foliar applications of Bion, 4 and 7 days before powdery mildew (Erysiphe necator) inoculation, reduced disease incidence and severity on 1-year-old grapevines cvs Carmenere, Chardonnay and Merlot under glasshouse conditions (Campbell & Latorre, 2004). The level of powdery mildew control was equal to that obtained with a single application of kresoxim methyl (Stroby; BASF, Germany). In field studies on cv. Merlot three applications of 0·3 mm BTH, applied over a 1-week period during veraison, induced a 40% increase in trans-resveratrol and a 100% increase in anthocyanins in berry skins (Iriti et al., 2004). At harvest, BTH-treated clusters were more resistant to infection following B. cinerea inoculation than untreated controls. There was a reduction in both disease incidence and severity on BTH-treated berries compared with untreated clusters. None of the clusters from BTH-treated grapevines exceeded 25% disease severity (% of infected berries per cluster) whereas over 90% of untreated control clusters showed a disease severity of > 50%.
The nonprotein amino acid, β-aminobutyric acid (BABA), has been reported to stimulate plant defences and to induce resistance against a broad range of fungal, oomycetous and bacterial plant pathogens (Cohen, 2002). Pretreatment of grape leaf discs (cvs Reisling and Chardonnay) with BABA (12·5–100 µg mL−1) reduced subsequent downy mildew sporulation by 85–94% (Cohen et al., 1999). Higher BABA concentrations (> 500 µg mL−1) were required to provide similar levels of control on intact potted plants, possibly due to the reduced uptake of active ingredient. A 25% wettable powder formulation containing BABA (2000 µg mL−1 a.i.) provided systemic resistance to downy mildew when applied via the roots or lower leaves of potted plantlets (Cohen et al., 1999). Formulations containing BABA have been field-evaluated for the control of downy mildew in grapes (Reuveni et al., 2001). Two foliar sprays of BABA (25% WP, 100 or 200 g per 100 L applied at 2500 L ha−1) or a mixture of BABA and different fungicides at reduced rates effectively controlled (> 90%) downy mildew in leaves of field-grown Chardonnay and Cabernet Sauvignon grapevines. BABA was as efficacious as metalaxyl-Cu (Ridomil-Copper) or dimethomorph + mancozeb (Acrobat Plus). A BABA–Cu complex manufactured by Makhteshim-Agan was highly effective and reduced downy mildew by 83–98% over two consecutive seasons, indicating an additive interaction between BABA and fungicides.
The use of BABA against B. cinerea in grapes has not been reported, as far as is known. However, BABA-treated Arabidopsis were found to be less sensitive to two different strains of B. cinerea (Zimmerli et al., 2001), indicating the compound's potential to elevate resistance to necrotrophs.
Jasmonic acid and methyl jasmonate are lipid-based compounds of wide distribution in the plant kingdom. It is well established that jasmonates are important regulators of many plant developmental processes, including seed germination, flower and fruit development, growth, and senescence (Staswick & Lehman, 1999). More recently, jasmonates have been shown to play a central signalling role in plant defence responses against insect attack and some necrotrophic pathogens. Furthermore, along with ethylene, jasmonic acid is thought to mediate the activation of induced systemic resistance (ISR), an induced resistance response typically associated with root colonization by certain nonpathogenic rhizobacteria (Pieterse et al., 2001).
There are many studies describing the use of jasmonates to elicit the expression of defence-related genes that are normally activated by wounding or in response to insect and/or pathogen attack (Inbar et al., 1998; Staswick & Lehman, 1999; Thaler, 1999). The addition of methyl jasmonate to grapevine cell cultures stimulated the accumulation of PR-proteins (Repka, 2001) and stilbene phytoalexin biosynthesis (Krisa et al., 1999). In glasshouse studies, treatment of grapevine leaves (Limberger) with droplets (50 µm) of methyl jasmonate (MeJA) resulted in localized lesion formation and the expression of PR-proteins both locally and systemically (Repka et al., 2001b). More recently, it was reported that exposure of grape leaves and berries to MeJA vapour for 7 days (400 nmol L−1) caused elicitation of stilbene biosynthesis (Larronde et al., 2003). Interestingly, the main stilbene produced in the leaves was trans-piceid, whilst trans-resveratrol accumulated in the berries but infection studies of treated tissues were not conducted. However, given the reported association of phenolics in resistance to B. cinerea, it is not unreasonable to anticipate some efficacy against botrytis bunch rot (Jeandet et al., 1991, 2000, 2002).
NPK fertilizers have been reported to stimulate growth and induce systemic resistance in cucumber and maize, and to be effective in wine grapes for the control of powdery mildew (Reuveni & Reuveni, 1998). Phosphonates are proposed to activate plant defences (Guest & Grant, 1991) and have been proposed as an alternative to copper for controlling downy mildew in grapes. Foliar applications of potassium phosphate salts reduced the severity of powdery mildew on Chardonnay leaves and grape clusters (Reuveni & Reuveni, 1995). Particularly effective powdery mildew control was achieved when phosphonate was applied in combination with Bordeaux mixture and a sulphur fungicide (Soyez, 2002). In extensive field trials at 13 sites, potassium phosphonate provided downy mildew control but resulted in significant phosphonate residues in the wine (Speiser et al., 2000). Commercially available products that contain phosphonates include Phytogard (Intrachem Bio, France), which contains potassium phosphonate (K2HPO3), and Aliette (Aventis, USA), which contains fostetyl-Al (aluminum tris O-ethyl phosphonate). Copper is widely used in viticulture for the control of downy mildew, and copper hydroxide has been reported to elicit the level of peroxidase, phenol, resveratrol and anthocyanins in grapevines (Coulomb et al., 1998). Studies on Arabidopsis mutants with impaired defence signalling pathways demonstrate that these fungicides and copper hydroxide may mediate an induced resistance response (Molina et al., 1998).
Calcium is considered to play an important role in maintaining the stability and integrity of the cell wall and plasma membrane in grape skins by chelating pectic components and so reducing susceptibility to enzymic digestion (Chardonnet et al., 1997). It has been proposed that the decline in calcium levels during berry ripening may contribute to the observed increase in susceptibility to infection. Externally applied calcium has been shown to increase calcium content in berries and to increase resistance to botrytis rots both in bunches on the vines and in bunches stored in the cold room (Miceli et al., 1999). The cell walls of treated berries had increased levels of cellulose and higher amounts of both oxalate-soluble and alkali-soluble pectins. The increased resistance to B. cinerea was attributed to the involvement of calcium ions in the stabilization of the cell wall structure. Potassium silicate has also been implicated in strengthening of the cell walls in grape leaves (Bowen et al., 1992).
Plant and microbial extracts
Inducible host defences are activated upon perception of elicitor-active oligosaccharides that are released at the plant/pathogen interface during attempted infection. Elicitor-active oligosaccharides such as glucans can also be extracted from fungal cell walls by hot water extraction or by partial acid hydrolysis (Sharp et al., 1984) and these extracts have been used in plant defence studies. Glucan fragments are among the most potent and best characterized (Cote & Hahn, 1994). Plants vary in their response to particular fungal glucans (Yamaguchi et al., 2000), and this has been proposed as evidence for the involvement of glucans in pathogen recognition. β-Glucan elicitors from Phytophthora spp. are effective at low concentrations (10−9m) and rely on specific structural characteristics for bioactivity (Sharp et al., 1984). Specific binding sites for β-glucan elicitors have been identified in several plant species (Cote et al., 2000). Although synthetic glucan elicitors seem an attractive option for crop protection, their production cost, on an industrial scale, has probably hindered commercialization to date. However, new methods for preparation of 3,6 branched glucans have recently been developed that are considered suitable for larger scale production (Ning et al., 2002). Crude extracts prepared from B. cinerea cell walls stimulated the accumulation of PR proteins (Repka et al., 2001a) and induced the accumulation of resveratrol and other fungistatic derivatives (Liswidowati et al., 1991) in grapevine cell cultures. Use of these extracts in the vineyard is not known.
In France, Laboratoires Goëmar have developed a method for extracting laminarin, a linear β−1,3 glucan, from marine brown alga (Laminaria digitata). Laminarin at 1 g L−1 induced the activation of 11 selected defence-related genes in grape cells and induced resistance to B. cinerea on detached cv. Chardonnay leaves (Aziz et al., 2003) with lesions on detached leaves being reduced in size by approximately 50%. A laminarin extract has been registered by Goëmar in France for use on winter wheat under the trademark Iodus 40 and the company has commercially developed a similar product containing algal β-1,3 glucan, VacciPlant (Agrimar Corp., USA) for registration in the USA. It is reported that foliar application of VacciPlant shows efficacy against a range of diseases affecting annual and perennial crops including downy mildew and B. cinerea on grapes (see http://www.goemar.com/).
Oligogalacturonides (OGAs) from plant cells walls were recently shown to induce multiple defence mechanisms in grape cell suspensions, including the induction of nine defence-related genes and stimulation of chitinase and glucanase activities (Aziz et al., 2004). OGA also induced resistance to B. cinerea in detached leaves in a dose-dependent manner. Metabolic inhibitor studies indicated that OGA-induced resistance to B. cinerea is mediated by signalling pathways involving the oxidative burst and protein phosphorylation. Elicityl (http://www.elicityl.fr/) are developing plant-derived oligosaccharides for use in viticulture. Elicityl is associated with the Centre de Recherche sur les Macromolecules Vegetales in the Centre National de la Recherche Scientifique (CNRS), Grenoble, France.
Chitosan, a mostly deacetylated β-1,4-linked d-glucosamine polymer, is a structural component of fungal cell walls. Chitosan has been reported to enhance disease resistance against many fungal diseases, when applied as either a pre- or a postharvest treatment (Wilson et al., 1994; El Ghaouth, 1997; Reglinski et al., 2004). In addition, chitosan can be directly antimicrobial and has been shown to interfere with the germination and growth of several phytopathogenic fungi, including Botrytis spp. (Ben Shalom et al., 2003). Structure-activity studies using purified chitosan oligomers have shown that the degree of polymerization and also the degree of acetylation have an influence on elicitor activity. Because chitosan fragments have a linear structure and show less structural variation specific to their origin other than chain length, they may play a role in recognition of a broad spectrum of potential pathogens and predators (Yamaguchi et al., 2000) and thereby may enable chitosan-based products to elicit broad-spectrum resistance.
Chitosan has been field-tested in a small number of studies on grapes. Aqueous solutions of 0·01–0·1% chitosan (molecular mass of 2–4 kDa), applied shortly after flowering and then again 14 days later, were reported to induce resistance to powdery mildew in grapes under field conditions (Gorbatenko et al., 1996). Application of 0·1–1% chitosan, 21 days and/or 5 days before harvest, reduced botrytis bunch rot severity on cv. Italia table grapes to ∼20% (Romanazzi et al., 2002). This compares with ∼75% infection on untreated grapes and ∼10% on grapes treated with the fungicide procymidone. A postharvest chitosan dip reduced incidence and severity of botrytis bunch rot in a concentration-dependent manner, with 1% chitosan the most effective treatment. Amborabe et al. (2004) reported that chitosan (2% ChitN15) application caused a sequential induction of PAL and chitinase activity in Chardonnay vines and also provided both protective and curative control of B. cinerea. In vineyard studies by the current authors, application of 0·01% chitosan every 10 days from flowering through to harvest reduced botrytis bunch rot by approximately 80% on Chardonnay grapes (unpublished data). This compared with 40% disease control using an industry standard fungicide programme.
There is increasing commercial interest in chitosan-based products. A formulated chitosan solution, Chitogel (Ecobulle, France), has been reported to stimulate the growth of Chardonnay plantlets and to enhance their resistance to challenge inoculation with B. cinerea (Ait Barka et al., 2004). In 1999, Safescience Inc. (USA) introduced a chitosan-based product Elexa that was registered with the EPA as a ‘reduced risk’ pesticide with no withholding period. In vineyard trials in the USA, eight applications of Elexa over the season reduced the incidence of downy mildew by 50% and powdery mildew by 75% compared with untreated controls (Schilder et al., 2002). A more recent formulation containing 4% chitosan, Elexa 4 Plant Defense Booster (Plant Defense Boosters Inc., USA) has been submitted for organic registration in the USA (http://www.plantdefenseboosters.com).
Extracts from leaves of the giant knotweed (Reynoutria sachalinensis) have been shown to be a potent defence elicitor and to control cucumber powdery mildew (Fofana et al., 2002). Commercialized formulations of R. sachalinensis (Milsana; BASF) were produced in the early 1990s. In 2000 KHH BioScience Inc. (USA) registered Milsana Bioprotectant Concentrate in the USA for use on glasshouse-grown ornamental (nonfood) plants to control powdery mildew. This product contains 5% of the ethanolic extract of R. sachalinensis. In vineyard trials in Germany between 1999 and 2001, Milsana applied every 7–10 days reduced the incidence of powdery mildew and botrytis bunch rot on grape berries to the same degree or better than sulphur and the copper-containing agent FW 450 (Dow Agro, USA; Schmitt et al., 2002). In the USA, eight applications of Milsana over the season reduced leaf powdery mildew severity by 75% compared with untreated controls, whilst four applications, applied between prebunch closure and harvest, reduced incidence of B. cinerea in grape clusters by 50% (Schilder et al., 2002).
Synermix (Laboratoires Goëmar) contains seaweed extract and AlCl3 and has been shown to elicit resveratrol accumulation in detached grape leaves of cvs Pinot noir and Rupestris du Lot (Jeandet et al., 1996, 2000). Resveratrol accumulation was greater in cv. Rupestris du Lot, indicating that the elicited response is cultivar-dependent. In vineyard trials carried out over an 8-year period Synermix enhanced the efficacy of iprodione against B. cinerea (Jeandet et al., 1996, 2000).
Compost-amended soils have been shown to reduce the severity of root rots, vascular wilts and nematode diseases. Composts are believed to control disease through direct antifungal activity and also indirectly through the induction of host plant defences, but little is known about the specific interactions between chemical and microbial components in compost that affect the activation of plant resistance. Aqueous extracts of composts, from both animal and plant sources, have been used to control B. cinerea, downy mildew and powdery mildew (Elad & Shtienberg, 1994). Efficacy of some compost extracts was reduced by pasteurization (Elad & Shtienberg, 1994) and sterile composts do not induce plant resistance, so microbial populations appear to play a crucial role (Hoitink et al., 1997). Compost extracts from cattle manure, horse manure and grape marc successfully suppressed B. cinerea in detached grape leaves and berries (Ketterer et al., 1992; Elad & Shtienberg, 1994). Disease severity was reduced by up to 95% on detached leaves (Ketterer et al., 1992) and by up to 70% on grape berries incubated at high humidity (Elad & Shtienberg, 1994). The extracts were less effective when used in the field to control botrytis bunch rot. However, efficacy was improved when extract was mixed with casein or pine oil and disease suppression was equal to that obtained with a conventional fungicide (Ketterer et al., 1992). More recently, compost extracts (compost tea) have been applied as foliar sprays or soil drenches, as a natural fertilizer and to aid disease control in Californian vineyards (Ryan et al., 2005). However, much of the evidence to date is anecdotal and more rigorous evaluations are necessary to enable an assessment of the value of these extracts for implementation of induced resistance in viticulture.
Disease-suppressive microorganisms, including Trichoderma spp., Aureobasidium pullulans, Pichia guilliermondii and Candida spp., described earlier, have also been reported to enhance plant disease resistance through the activation of host defences (Wilson et al., 1994; Elad, 2000; Ippolito & Nigro, 2000; Adikaram et al., 2002; Yedidia et al., 2003). Trichoderma spp. produce many extracellular metabolites and it has been suggested that some of these may elicit the plant defence response (Elad, 2000; Hanson & Howell, 2004). Cellulase from T. viride was reported to stimulate resveratrol production and the hypersensitive response in grape cell cultures (Calderon et al., 1993) and to induce the accumulation of peroxidase isozymes in grape leaves and stems (Barcelo et al., 1996). Another hydrolytic enzyme, xylanase, is produced extracellularly by T. viride and A. pullulans and it is possible that the degradation of xylan in plant cell walls releases elicitor-active fragments that trigger the host defence response. Xylanase has been shown to activate defence-related responses in tobacco cells (Yano et al., 1998) and to induce PR-protein synthesis in tobacco leaves (Lotan & Fluhr, 1990). More recently it was proposed that enzyme activity is not necessary for xylanase elicitor activity and that bioactivity resides in the protein itself (Enkerli et al., 1999).
Soil application of T. harzianum T39 was shown to induce resistance in the stems and leaves of pepper plants to infection by B. cinerea (De Meyer et al., 1998). Recently, it was reported that root application of T. asperellum (T203) induced foliar resistance to Pseudomonas syringae pv. lachrymans in cucumber (Yedidia et al., 2003) and that the resistance response was mediated via the jasmonic acid/ethylene pathway (Shoresh et al., 2005). The induction of systemic resistance in plants by soil microorganisms is more commonly associated with certain root-colonizing bacteria, mainly nonpathogenic fluorescent Pseudomonas spp. (Hoffland et al., 1996; Wei et al., 1996). Siderophores secreted by some rhizobacteria, as well as outer membrane lipopolysaccharides, appear to play important roles in the activation of disease resistance (De Meyer & Hofte, 1997; Bakker et al., 2003). There are few reports describing the use of rhizobacteria for disease control in grapes. Chardonnay explants co-cultured with Pseudomonas sp. (strain PsJN) grew faster and were more resistant to infection by B. cinerea than nonbacterized controls (Ait Barka et al., 2000, 2002). The authors suggest that the integration of such bacteria into grapevine propagation systems can reduce the need for fungicides and at the same time enhance plant vigour. Localized leaf inoculation of 2-month-old Chardonnay plantlets with the soil bacterium Bacillus sp. induced resveratrol accumulation and enhanced resistance to subsequent inoculation with Botrytis sp. (Paul et al., 1998).
Efficacy of treatments
Variable performance of biological-based control strategies in the field is recognized to constitute a significant constraint for their practical implementation (Fravel, 1999; Stewart, 2001; Shtienberg & Elad, 2002). The complexities of host/pathogen/environment interactions in the vineyard system present an enormous challenge to achieving a consistent level of disease control in viticulture. Efficacy is dependent upon timely delivery of the BCA to ensure early establishment relative to the pathogen (Elad & Stewart, 2004). Thereafter, it is the ability of the BCA to survive environmental extremes common in many vineyards and resume rapid growth when conditions favour B. cinerea which will determine the level of biosuppression. Poorly timed BCA applications compromise efficacy, especially where gaps in epidemiological knowledge and B. cinerea-infection pathways are not well understood. Other vineyard practices that may have a negative impact upon BCA efficacy include incompatibility with vineyard pesticides and substandard canopy management (Elmer & Michailides, 2004). Induced resistance relies upon a physiological and biochemical response by the plant and so elicitor efficacy may be affected by different climatic and agronomic factors that influence general plant health. Indeed, there is evidence that both constitutive and inducible resistance mechanisms are affected by light, temperature, soil type and soil nutrient level or availability, particularly nitrogen (Falkhof et al., 1988; Stout et al., 1998; Genoud et al., 2002; Wiese et al., 2003; Dietrich et al., 2004). However, relatively little is known about the way external factors influence the plasticity of grapevine defence mechanisms to B. cinerea.
The efficacy of biological control methods that rely on the use of a single BCA can be variable, depending upon B. cinerea inoculum levels and local environmental conditions (O’Neill et al., 1996; Shtienberg & Elad, 2002). One potential way to reduce this variability and improve efficacy is to combine compatible biocontrol treatments that occupy different environmental niches and/or have different modes of action. Several studies have demonstrated that combinations of rhizobacterial biocontrol agents can provide greater, more reliable and broader spectrum disease control than that obtained using individual strains (Jetiyanon & Kloepper, 2002; De Boer et al., 2003; Jetiyanon et al., 2003). Biosuppression of grey mould on strawberries by P. guillermondii and B. mycoides was more effective and more consistent across a range of environmental conditions when these ecologically different BCAs were combined than when applied separately (Guetsky et al., 2001, 2002). Efficacy of the mixture reflected the sum of their activities and it was proposed that P. guillermondii competed with B. cinerea for nutrients and B. mycoides secreted inhibitory compounds (Guetsky et al., 2002).
Improvements in biocontrol efficacy have also been achieved by applying BCAs in combination with different chemicals. Suppression of root diseases (Chen et al., 1996; Vogt & Buchenauer, 1997; Benhamou et al., 1998) and postharvest fruit decay (El Ghaouth et al., 2000a, 2000b; Qin et al., 2003; Qin & Tian, 2005) was enhanced when BCAs were applied in combination with chemicals such as calcium chloride, chitosan, 2-deoxy-d-glucose, salicylic acid and silicon. Calcium and magnesium salts enhanced the biocontrol efficacy of antagonistic yeasts against postharvest rots caused by B. cinerea on apples (Wisniewski et al., 1995) and table grapes (Schmidt et al., 1996). In vineyard studies carried out during 1999–2001, plant health enhancers (Milsana and Myco-Sin) and a bacterial antagonist (B. brevis), were tested in combination for their ability to control powdery mildew, downy mildew and grey mould (Schmitt et al., 2002). Application of the biocontrol treatments at 10-day intervals throughout the season reduced incidence of B. cinerea on grape berries to 29·8%, compared with 89·7% on control plots. Disease control was comparable to that obtained using wettable sulphur and the copper-containing agent FW 450. Two biocontrol products under commercial development (BioCoat, a combination of C. saitoana with chitosan) and Biocure (C. saitoana combined with a lytic enzyme) were tested for efficacy in semicommercial field test in table grapes (cv. Italia) in Italy. Preharvest applications reduced botrytis bunch rot in cool storage by 33–46%, a level of control comparable to a conventional fungicide treatment (Schena et al., 2004).
Antagonistic fungi (U. oudemansii and E. nigrum) have been tested in combination with a chemical inducer (5CSA). This strategy is based on the premise that fungal antagonists will displace B. cinerea on necrotic tissues in the grapevine, so reducing inoculum potential, whilst 5CSA enhances resistance in leaves and developing berries to attempted infection. Applications every 10 days of 1 mm 5CSA and U. oudemansii alone and in combination significantly reduced postharvest development of botrytis bunch rot in Chardonnay grapes (Reglinski et al., 2005). After 14 days incubation in high humidity, botrytis bunch rot severity was significantly lower on bunches treated with 5CSA/U. oudemansii than on those treated with either component alone. The current authors observed similar results in vineyard studies using 5CSA/E. nigrum combinations.
The isolate of U. oudemansii used in these trials has now been registered for early season control of B. cinerea in grapes in New Zealand (Botry-Zen). Botry-Zen is primarily an early-season protectant but may not provide protection of ripening fruit postveraison. Furthermore, application of Botry-Zen at ∼350 L ha−1 did not distribute sufficient quantity of the antagonist to the grape bunch inflorescences, indicating that the current trend in viticulture towards low-volume application may be unsuitable for microbial-based BCAs (P. N. Wood & P. A. G. Elmer, The Horticulture and Food Research Institute of New Zealand Ltd, unpublished data).
Biocontrol treatments, whether operating by antibiosis, competition, and/or elevation of host resistance, can be highly effective against B. cinerea in grapes under controlled conditions and on occasions in vineyards. Levels of disease suppression can equal those achieved with synthetic botryticides, particularly under low-to-moderate disease pressure conditions. However, more commonly, reliance on a single mode of action biocontrol agent has suffered from variable efficacy under field conditions. This may, in part, explain why only a small proportion of the BCAs that show efficacy under laboratory conditions have been commercially developed for crop protection. Other constraints on the uptake and commercialization of biologically based strategies, including production costs and regulatory issues, have been reviewed elsewhere (Fravel, 1999; Stewart, 2001; Gerhardson, 2002; Spadoro & Gullino, 2005).
The development of more efficacious biological control product formulations will not guarantee uptake by vineyard managers, particularly where there is the perception that biocontrol is inferior to chemical control. In those cases, adoption of biological control methods will largely be driven by the withdrawal of traditional fungicides, and governed by product (biocontrol) availability and choice, and also a demonstrated financial and/or environmental benefit. However, given the differences between the modes of action of conventional and biological control methods, traditional application strategies and vineyard practices may, on occasion, be incompatible with BCAs and plant resistance stimulants. Fungicide programmes for botrytis bunch rot control specifically target critical periods during the growing season: flowering, bunch closure, veraison and preharvest. While timing is equally critical for biologically based strategies, further research is recommended to determine when it is appropriate to substitute biological control treatments for fungicides, and when changes in protocol are required. Recent studies in South Africa indicate that a single late-flowering application is inadequate for effective botrytis bunch rot suppression, since B. cinerea infection has already occurred (Holz et al., 2003). Furthermore, it may be inappropriate to apply inducing agents preharvest because there is evidence that inducible resistance responses decline in ripening berries (Creasy & Coffee, 1988; Jeandet et al., 1995; Bavaresco et al., 1997; Bais et al., 2000). Product delivery is especially important for antagonists where competitive exclusion of B. cinerea is the primary mode of action. Research is required to determine more accurately the interaction between water rates and sprayer technology on antagonist establishment on target tissues. The potential sensitivity of B. cinerea antagonists to other commonly used fungicides in the vineyard should also be considered.
Biosuppression of B. cinerea appears to be most effective under low-to-moderate inoculum pressure (O’Neill et al., 1996; Lennartz et al., 1998; Schoene & Kohl, 1999; Schoene et al., 2000), indicating that good canopy hygiene (e.g. bunch trash removal, leaf plucking, shoot thinning) to create canopy conditions less conducive to survival of, and infection by, B. cinerea may assist biocontrol. Pruning practices are known to reduce the severity of disease by altering the canopy microclimate. However, leaf removal dramatically reduced the formation of phenolic compounds in grape floral tissues (Keller et al., 2000) and could potentially decrease overall resistance during the critical bloom period when B. cinerea can infect flowers and remain latent in the developing berry until veraison. The impact of defence-inducing agents on grape and wine quality requires further research since it was recently reported that 5CSA-treated Cabernet Sauvignon vines produced lower quality wine than fungicide-treated controls (Duxbury et al., 2004). Reasons for the reduction in wine quality are unknown but resource allocation costs cannot be discounted. A more pertinent issue for wine quality relates to a possible elevation in PR-proteins in elicited grapes. PR-proteins in ripe grape berries are reported to be responsible for clouding or haze in wines and it is not known if late-season inducer application would significantly augment the problems associated with protein instability in wines (Waters et al., 1996, 1998; Tattersall et al., 1997).
The development of combinatory approaches that involve two or more biocontrol components, each occupying a different environmental niche and/or expressing a different mode of action, will improve consistency and efficacy (Schmitt et al., 2001, 2002; Guetsky et al., 2002; Reglinski et al., 2005). The integration of this approach with new epidemiological knowledge, prediction models and viticultural practices will significantly advance the development of strategies for biosuppression of B. cinerea.