The major objective of this study was to isolate yeasts and Bacillus spp.
antagonistic to P. digitatum and to assess their potential ability for biological control of green mould of citrus. This approach has been reported by others (Wilson and Chalutz 1989; Chalutz and Wilson, 1990; Smilanick et al., 1993;
Bouzerda et al., 2003). However, it is the first reported study in which yeasts and Bacillus have been isolated in South Africa and their potential antagonistic ability assessed against P. digitatum. Isolation of yeast and Bacillus isolates from a particular geographical location may be more effective and specific against the pathogen isolates present in that region (Vero et al., 2002;
Bouzerda et al., 2003).
A pilot study on the microorganisms present on the surfaces of mature citrus was conducted. Dense bacterial and yeast populations were found, with little growth of filamentous fungi. This indicates that microorganisms present on fruit in orchards may be used as a rich source of yeast and Bacillus isolates (Figure 3.1). The presence of microorganisms on mature fruit reflect the microorganisms‟ abilities to tolerate the hostile conditions present on the surface of fruit, namely: low nutrient availability, UV radiation, rapid climatic Isolate B13 (× 105) Isolate Grape (× 105)
Cells ml-1
a
ab ab ab ab
bc
ab ab ab ab
d
0 10 20 30 40 50 60 70 80
0 1 10 25 100 1000 1 10 25 100 1000
Lesion diameter (mm)
Fokkema, 1998); and reflect their ability to colonize and survive on the target host tissue.
All 60 yeasts and 92 Bacillus isolates recovered from the surface of the fruit reduced disease development by P. digitatum on navel oranges in the initial screening. However, only 20 isolates (10 yeast and 10 Bacillus isolates) reduced the fruit surface area infected by P. digitatum on navel oranges by
≤50% (Table 3.1). Isolates of yeast and Bacillus which failed to reduce infection levels by P. digitatum to ≤50 on the fruit surface were considered inadequate to warrant further screening (Table 3.1).
In subsequent screenings, yeast isolates provided superior control of P.
digitatum compared with the best Bacillus isolates (Table 3.3). In general, yeast isolates effectively reduced disease development by P. digitatum on navel and Valencia oranges, and lemons. In a preventative trial with 20 yeast and Bacillus isolates, yeast isolates B13 and Grape, originally isolated from the skin of a Valencia orange and a grapefruit, respectively, exhibited the highest antagonistic activity against P. digitatum infection on navel oranges and lemons (Table 3.3). On Valencia oranges, Isolate Grape was more effective than Isolate B13. However, in the dosage trial, which compared the performance of these two isolates at a range of cell concentration, Isolate B13 consistently, provided better control of green mould on lemons than Isolate Grape (Figure 3.3). Isolate B13 was therefore selected for further studies.
In these trials, yeast antagonists were effective when applied preventatively but not when applied curatively. This is in agreement with the results obtained by Qing and Shiping (2000), who showed that the yeast Pichia membranefaciens Hansen was only effective when applied to nectarine fruit 24-48 hours before inoculation with Rhizopus. Similarly, de Capdeville et al. (2002) showed that yeasts reduced the progress of Penicillium expansum (Link) Thom. on apple fruit more effectively when applied to the fruit 24 hours or 96 hours before inoculation with the pathogen than when applied 24 hours after inoculation.
Chalutz and Wilson (1990) also found that the efficacy of a yeast isolate of Debaryomyces hansenii (Zopf) Lodder and Kreger-van Rij against green and blue moulds of grapefruit was reduced if application was delayed by three hours
The lack of curative disease control by yeast antagonists could be due to the pathogen penetrating into the fruit tissues and therefore being out of reach of the antagonist, as suggested by Mercier and Smilanick (2005). An alternative hypothesis is that biocontrol yeasts act by competitively colonizing wounds faster than Penicillium. Once established in a fruit wound, the yeasts utilize all the nutrients released from the wound. In this scenario, no nutrients are left to stimulate Penicillium spores to germinate. However, if the Penicillium spores have access to wound nutrients for three hours before the yeasts are applied, then the stimulation of Penicillium spores to germinate will have already occurred before the yeasts are applied. Hence, yeasts isolates would not be effective in a curative role.
The study of the biocontrol activities of different concentrations of the two yeast isolates B13 and Grape against P. digitatum demonstrated that both yeasts reduced the lesion diameters of P. digitatum on lemon, irrespective of the yeast concentrations. Isolate B13 demonstrated greater biocontrol efficacy and completely controlled P. digitatum development at 1 × 108 cells mℓ-1. Similar findings were reported by Tian et al. (2002) with Candida guilliermondii (Castellani) Langeron and Guerra and P. membranefaciens at a concentration of 108 cells mℓ-1, which completely controlled infection by Rhizopus stolonifer on peaches and nectarines. Chanchaichaovivat et al. (2007) were able to control anthracnose on chilli (6.5% disease incidence) caused by Colletotrichum capsici, with a yeast P. guilliermondii Strain R13, applied at 108 cells mℓ-1. Zheng et al. (2005) were also able to control green mould of oranges with yeast Rhodotorula glutinis (Harrison), applied at a concentration of 1 × 109 cells mℓ-1. Reports by Janisiewicz (1988) and Hong et al. (1998) demonstrated that a direct relationship exists between the population density of the biocontrol agents and the effectiveness of the postharvest biological control treatment.
Isolate B13 is an isolate of Candida fermentati, which was previously thought to be the anamorph of Pichia guilliermondii, cosmopolitan yeast associated with numerous habitats (Kurtzman and Fell, 2000). However, low DNA base sequence relatedness, different electrophoretic karotypes and nucleotide divergence of the D1/D2 region have all lead to the reinstatement of Candida fermentati as a separate species (Vaughan-Martini et al., 2005). To the best of
because its activities as an effective biocontrol agent have been credited to P.
guilliermondii.
Candida fermentati has been isolated from the gut of beetles (Suh and Blackwell, 2004). It has not been reported to pose a risk to human health.
Therefore, rigorous toxicological tests would be needed before its use as a biocontrol agent could be commercialized.