23.2.1 Use of Naturally Occurring Antagonists for Colonization of Infection Sites
So far, the biological control of postharvest diseases with naturally occurring microorganisms has relied essentially on the inundative approach, that is, the mass introduction of a microbe to establish an antagonistic population on wounds and other possible infections sites on fruits or tubers. Little work has been done on other approaches such as the enhancement of the existing surface microflora. While many antagonistic microbes were initially selected from the fruit microflora , their natural populations are likely to be too low to have a significant impact on plant pathogens . Mass introduction of yeasts or bacteria permits achieving an instant antagonistic population that would never be attained under normal conditions. This approach has several advantages since a drench or spray treatment of several commodities can easily be applied as the harvested commodities are brought in from the field before packing or storage. If the process is performed in a timely fashion, most wounds sustained during harvesting and handling should become protected before significant pathogen development can occur. Treatment after harvest can also be more economical than treating a whole orchard or field and allows use of more concentrated cell suspensions.
The purpose of antagonists is to colonize rapidly possible infection sites and protect them from infections. Usually, populations of effective antagonists increase rapidly initially and stabilize thereafter. Such a colonization pattern can be seen in fruit wounds treated with yeasts such as Candida oleophila [3,4], Cryptococcus albidus , C. laurentii , and Pichia membranefaciens  and bacteria such as Pseudomonas syringae . However, wounds can have a narrow window for optimal colonization as they dry out . This requires that application takes place as soon as possible after harvest and handling, as antagonists usually have little curative activity . Also, wounds on oil glands of citrus fruit were found to be more difficult to colonize by C. oleophila, resulting in poor decay control .
Research has shown that there may be limitations to the postharvest use of antagonists , although results comparable to those obtained with synthetic fungicides can sometimes be achieved . Often, a lack of curative activity is the main problem, as efficacy becomes much reduced or nil when antagonists arrive on wounds after pathogens [2,5,10]. Also, antagonists may have different efficacy depending on the fruit species  or the type of decay . These limitations likely result from the mode of action of a given antagonist or differing ability to colonize and establish on various commodities. For these reasons, antagonistic yeasts or bacteria cannot be considered as "silver bullets,'' and as biological systems are more sensitive to environmental conditions than chemical agents. Depending on the disease system, improvements in formulation or combination with other treatments may help increase decay control and meet the requirements of the horticultural industry. Most probably, further improvements in efficacy are likely to come from a better understanding of the mode of action and ecology of these antagonists.
While most of the research on naturally occurring antagonists has focused on postharvest treatment, there have been positive reports recently on the use of preharvest applications of antagonists to control postharvest diseases [13-15]. Such application can be done periodically during the growth of the fruit, up to the day of harvest. Field application can help achieve an early colonization of possible infection sites and reduce incipient infections from the field. Also, it can make biological control possible in crops that are too fragile or incompatible with postharvest drenching or spraying, such as grapes and soft fruits. However, field application of antagonists will expose them to possibly adverse environmental conditions such as desiccation and solar radiation, which they will have to withstand in order to be effective. In this situation, the selection process must be different than for postharvest application and take into account the ability of antagonists to survive on the intact fruit surface. Benbow and Sugar showed that certain yeasts are naturally adapted to field conditions and could maintain their populations on pear fruit for three weeks . Using another strategy, Teixido et al. used culture media with low water activity to help Candida sake adapt to water stress . Such physiologically modified yeasts were better adapted to colonize apples in the orchard. Our own field research on preharvest appli-
cations with Bacillus subtilis (Serenade AS) has shown promise on stone fruit for control of monilinia. On apricots artificially inoculated after harvest with Monilinia, there was only 8% infection when sprayed preharvest with Serenade (applied as 4Qt in 100 gal of water per acre) compared to 52% infection in untreated fruit. A rate of 4oz per 100 gal of Elite 45WP (tebuconazole) had 0% infection. In a second test, there was 14% infection with Elite, 28% with Serenade, and 71% in the untreated fruit.
Inoculated trials are the most severe test, and in an actual postharvest situation the results are likely to be better. A trial was conducted with the University of California at Davis on Bing cherries inoculated postharvest with brown rot (monilinia) and gray mold (Botrytis cinerea) after preharvest treatment (air blast sprayer, 100 gal water per acre) with Serenade (6 lb) and an adjuvant (Sylgard) and chemical pesticides. The incidence of brown rot decay with the Serenade treatment was 5.6%, compared to 4.5% for triflumizole (Procure 4SC) (rate of 12 fl oz), 0.4% for iprodione (Rovral) 4F (1 Qt), and 32% in the untreated fruit. Against gray mold, there was a 1% incidence of decay (percent of fruit with decay) with Serenade treatment compared to 0% incidence for both Procure and Rovral, and 2.9% incidence for untreated fruit.
There has been no systematic study of the mode of action of any given postharvest biocontrol agent, and most possible inhibition mechanisms remain unproven at this time. Antagonists could act in passive ways, simply using space or nutrients needed by pathogens, or directly interact with the pathogen to cause inhibition through parasitism or the synthesis of inhibitory molecules, such as antibiotics or hydrolytic enzymes. Finally, the triggering of defense responses in the host, resulting in enhanced resistance, could also be part of the biocontrol mechanism. It is quite possible that in many cases, a number of active and passive mechanisms are involved and act together which make it even more difficult to decipher the basis of the biocontrol phenomenon.
Many antagonistic yeasts that are good wound colonizers are not associated with any obvious inhibitory mechanism. By simply colonizing and forming a cell layer on the surface of wounds, these antagonists may act by competitive or pre-emptive exclusion, blocking access to the infection site. This mechanism is difficult to prove but its occurrence is plausible, especially when a colonization period is required for the antagonist to be effective. Along with competitive exclusion, competition for nutrients is often claimed in the absence of other more obvious or active mechanisms and is supported by the fact that yeasts or yeast-like organisms were able to remove amino acids or sugars in nutrient wells or in wounds [16,17]. Also, the addition of nutrients was shown to cancel antagonistic activity . However, in many cases, the question remains as to whether nutrients in wounds are really limiting for pathogens. Furthermore, yeasts that effectively remove nutrients in wounds are not necessarily good antagonists . As in leaf surface bacteria, where nutrient-regulated reporter genes were used to study nutrient consumption on leaves [19,20], such molecular tools in antagonists or pathogens could be useful for elucidating the question of nutrient competition in wounds.
The involvement of active mechanisms such as antibiotic production by Pseudomonas syringae , production of cell wall degrading enzymes by Aureobasidium pullulans  or Pichia anomala , or attachment to pathogens by various bacteria and yeasts [23,24] have been associated with biocontrol activity on fruits. Again, the definite role of these mechanisms in biocontrol is difficult to demonstrate, and the use of molecular tools might be the best approach to elucidate the role of those antifungal factors. Such a molecular approach was used by Grevesse et al. to elucidate the role of p-1,3-glucanase produced by the antagonistic yeast P. anomala . The biocontrol activity of the yeast remained unaffected by the shut down of p-1,3-glucanase production from the disruption of a gene involved in the production of the enzyme, thus dismissing its role in antagonism.
So far, the induction of disease resistance in stored fruits and vegetables by antagonists has been little studied. In most cases, it is not known whether biocontrol agents can induce such defense responses. Defense enzymes such as p-1,3-glucanase, chitinase, and peroxidase were induced in apple wounds by A. pullulans . In oranges, Arras reported the accumulation of the phyto-alexins scoparone and scopoletin in response to Candida famata . While these defenses could contribute at least in part to the biocontrol activity, the importance of induced resistance in postharvest biocontrol remains unknown and its possible role is yet to be demonstrated. It is possible that many more antagonists can trigger defense responses and enhance host resistance. It is likely that biocontrol action relying on induced defenses would be rather host-specific, as harvested fruits and vegetables vary in their ability to respond to elicitors and produce defense responses. More advances in the development of biofungicides are likely to come when we better understand mechanisms of biological control on stored commodities.
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