| Postharvest fungal diseases cause substantial economic losses world-widely. In view of the fungicidetoxicity on the environment and human health, and the development of fungicide resistance bypathogens, great efforts have been made to exploit alternatives to the synthetic fungicides. Thebiological control by using antagonistic yeasts has been considered to be one of the most promisingnon-fungicidal methods. Strains of Cryptococcus laurentii are well known as postharvest biocontrolyeasts, which have been shown to have high antagonistic activity in various fruit. Unfortunately, atpresent, the effectiveness and stability of yeast antagonists have not been reached an adequate controllevel when compared with the synthetic fungicides. Therefore, the objective of this research was toexplore the potential utilization of such ways as delaying harvested fruit senescence, inducing harvestedfruit resistance, using natural bioactive substance or stimulating yeast physiology for improving theefficacy ofC. laurentii in inhibiting the postharvest fungal diseases in harvested fruit.The main research findings were summarized as follows:1. Salicylic acid (SA) had little direct antifungal activity against Penicillium expansum and Botrytiscinerea in vivo. However, SA at 10μ/ml or 100μg/ml could enhance the efficacy of C. laurentii inreducing the blue and gray mold rots in apple or pear fruit, which might be supposed to be related to thefruit natural resistance induced by SA. Moreover, the application of SA at 10μg/ml or together with C.laurentii resulted in inhibition of the ethylene production but in promotion of the activities ofsuperoxide dismutase, catalase, peroxidase, phenylalanine amonialyase, polyphenol oxidase andlipoxygenase of apple fruit. On the other hand, treatment of pear fruit with SA at 100μg/ml was alsofound to elicit the fruit peroxidase activity. Anyway, SA at 10μg/ml or 100μg/ml had no influence onthe growth of C. laurentii in apple or pear fruit wounds.2. Cytokinin (N6-benzyladenine, 6-BA) at 500 to 2000μg/ml was effective in inhibiting the P.expansum infections in apple or pear fruit wounds, but its efficacy was declining rapidly with theincubation time. Integrated application of C. laurentii and 6-BA at 20μg/ml or 1000μg/ml broughtabout a more effective inhibition of the blue and gray mold rots in apple or pear fruit wounds than thatof the 6-BA or C. laurentii alone. Moreover, 6-BA at 20μg/ml or together with C. laurentii stimulatedthe superoxide dismutase activity but it inhibited the increase of the peroxidase activity of apple fruit.On the other hand, treatments of pears with 6-BA at 1000μg/ml or with C. laurentii led to an increasein the catalase activity but in an inhibition of the activities of both peroxidase and lipoxygenase as well as the ethylene production. Similarily, 6-BA from 20 to 2000μg/ml did not affect the population growthof C. laurentii in apple or pear fruit wounds.3. Auxins including indole-3-acetic acid (IAA), indole-3-buyric acid (IBA) and2, 4-dichlorophenoxyacetic acid (2, 4-D) were shown to have little direct antifungal activity against P.expansum and B. cinerea in vivo. However, IAA at 20μg/ml or 100μg/ml augmented the efficacy of C.laurentii in reducing the blue and gray mold rots in apple or pear fruit. Moreover, it was found that IAAcould induce the apple and pear fruit resistance to molds infection, which was dependent on the IAAconcentration, the time interval between the IAA treatment and fungal pathogens inoculation, thepathogen inoculum concentration and the incubation time. In the apple wounds, IAA at the optimalconcentration (20μg/ml) and the optimal time interval (24 h) significantly inhibited the sporegermination of P. expansum and the initial mold infections, however, its efficacy was decreasing withthe increase of both of the pathogen inoculum concentration and the incubation time. The results fromthe present paper also revealed that IAA at 20μg/ml alone or together with C. laurentii could stimulatethe superoxide dismutase, catalase, peroxidase activities, whereas it inhibited the lipoxygenase activityof apple fruit. Just the same, IAA at 100μg/ml or together with C. laurentii also motivated the catalase,peroxidase and polyphenol oxidase activities of pear fruit. In addition, IAA at 20μg/ml or lower wasnot found to have any influence on the population growth of C. laurentii in fruit wounds but IAA wasshown to have toxic effect on C. laurenn'i when its concentration was increased to 200μg/ml or abovein vitro and in vivo.4. A combination of C. laurentii and gibberellic acid (GA3) at 200μg/ml or 2000μg/ml produced amore effective control of blue and gray mold rots in apple or pear wounds than C. laurentii alone,although GA3 was not found to have toxic effect on P. expansum and B. cinerea as well as C. laurentiiin vivo. Moreover, it was shown that GA3 at 2000μg/ml induced the pear fruit resistance to P.expansum infections, which was associated with the increased peroxidase and polyphenol oxidaseactivities and with the inhibition of the lipoxygenase activity and lipid peroxidation. On the other hand,in apple fruit, GA3 at 200μg/ml alone or with C. laurentii enhanced the activities of both superoxidedismutase and catalase but inhibited the increase in peroxidase activity.5. Application of CaCl2 made C. laurentii more effective in delaying the blue and gray mold rots inpear fruit. Although CaCl2 had little antifungal activity against P. expansum or B. cinerea as well as C.laurentii in vivo, it induced the fruit resistance to P. expansum in pear wounds and activated theperoxidase activity of pear fruit.6. Furthermore, the results from the present research showed that a combination of 0.2％chitosan and 1％CaCl2 and 20μg/ml SA was a most effective approach to improvement of the effectiveness andstability of C. laurentii against P. expansum and Rhizopus stolonifer in apple or pear fruit. Our resultsalso showed that jasmonic acid at 10μg/ml, IAA at 10μg/ml or GA3 at100μg/ml could enhance theefficacy of C. laurentii against P. expansum infection in peach fruit. Application of CaCl2 at 2％, SA at20μg/ml, GA3 at 100μg/ml also enhanced the efficacy of C. laurentii against the fungus Pestalotiadiospyri infection in persimmon fruit.7. Chitosan had the strength to inhibit the blue mold rot in apple or pear wounds, and its efficacywas shown to increase with the increase in its concentration and with the decrease in its viscosity, andalways declined with the incubation time. Combination of chitosan and C. laurentii exhibited asynergistic effect on the blue mold rot, which was the most effective at the optimal concentration ofchitosan at 0.1％or 0.5％in apple or pear fruit. Furthermore, an addition of CaCl2 (1～2％) to thesuspension of C. laurentii with chitosan (0.5％) could further reduce the blue mold rot of pear fruit andthe blue and soft mold rots of peach or nectarine fruit. In addition, application of chitosan at 0.2％induced tomato fruit resistance to the fungus Alternaria alternate, and a combination of 0.2％chitosanand C. laurentii resulted in a synergistic inhibitive effect on the natural decay of tomato fruit.8. In grape fruit, application of C. laurentii, heat treatment, chitosan or ethanol alone was shown tobe effective in reducing the decay of grape fruit. And heat treatment, chitosan, or calcium chloride couldimprove the efficacy of C. laurentii against B. cinerea infection. Moreover important, it was found thata most effective means of controlling the decay of grape was by using a combination of 15～30％ethanol,0.2％chitosan and 1～2％calcium chloride, which efficacy was shown to be equal to that of SO2.9. The physiology manipulation of biocontrol yeasts may also be a useful method of enhancingthe efficacy of postharvest biocontrol yeasts. The results from this research showed that application ofchitin or chitosan could induce C. laurentii antagonistic activity to P expansum infection in apple, pearor peach fruit after cultivation of the yeast in a liquid media amended with chitin at 1.0％or chitosan at0.002％, which might be related to enhancement of the population growth of C. laurentii in fruit wounds,stimulation of the chitinase activity and augment action of the capacity of inducing fruit naturalresistance after cultivation by chitin or chitosan. The proteomic analysis and comparison of C. laurentiiin response to chitin was analyzed based on two-dimensional liquid chromatography (ProteomeLabTMPF-2D).In conclusion, the results from the present paper showed that the biocontrol efficacy of C.laurentii could be improved by use of the methods of elicit of fruit resistance, retard of fruit senescenceor by chitosan, and combination of these approaches above and other non-fungicidal methods, such asheat treatment, sodium bicarbonate and ethanol could effectively control postharvest fungal diseases of various fruit. Moreover, the efficacy of C laurentii could be augmented by cultivation of chitin-orchitosan-amended liquid media, which might provided a novel strategy for enhancement of thebiological activity of C. laurentii against postharvest fungai pathogens. |
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