Integration of rapid hot water treatments and biocontrol agents to control postharvest pathogens of tomato.

Abstract

Anthracnose and sour rot caused by Colletotrichum and Galactomyces (syn. Geotrichum) species, respectively, are major fungal postharvest pathogens causing significant losses of tomato fruit. The growing public concern over human health and environmental risks posed by pesticides, the accumulation of chemical residues in fruit, and the production of secondary effects on fruit, as well as the development of resistant strains has reduced the available options of synthetic fungicides to control these pathogens. Finding alternatives or integrated approaches to provide disease control comparable to the use of synthetic fungicides is therefore needed, especially for the control of postharvest diseases, while maintaining a high quality of fruit during storage and marketing. The overall objective of this study was to develop an integrated treatment that combined rapid hot water treatments with biological control agents to control two postharvest pathogens of tomato, Colletotrichum and Galactomyces spp., and to track their impact on the postharvest quality of tomato fruit. The mechanisms of rHWTs and antagonist yeasts involved in decay control were also investigated, in passing. Isolation and Identity of the Pathogens Isolation and identification of fungal pathogens associated with tomato fruits were carried out to determine the most common fungi associated with tomato spoilage in South Africa. A total of 55 isolates were recovered from symptomatic tomato fruits with typical symptoms of anthracnose and sour rot. The cultural and morphological characteristics of all isolates were observed and compared with standard descriptions to establish their identity. Pathogenicity tests were performed. The effects of wound and non-wound inoculation methods on the infection process and disease development were studied by scanning electron microscopy (SEM). The identities of one of the most pathogenic isolates of each pathogen were then determined using the consensus sequences and the nucleotide Basic Local Alignment Search Tool (BLASTn) on The National Center for Biotechnology Information (NCBI) website. Out of the 55 isolates, 33 were Colletotrichum spp., and the other 22 isolates were Galactomyces speciesp. Colletotrichum isolates were further classified into Colletotrichum gloeosporioides and Colletotrichum acutatum, based on cultural and morphological analyses. All the Galactomyces isolates were similar and were identified as strains of Galactomyces candidum. Among the isolated strains, C24 and C37A from the Colletotrichum isolates, and G18, G23 and G29 from the Galactomyces isolates, were extremely pathogenic. SEM results showed that all wound and non-wound Colletotrichum inoculated fruits developed anthracnose, whereas non-wound Galactomyces inoculated fruits failed to develop sour rot, indicating that Galactomyces requires a wound for infection to occur. Molecular analyses confirmed the identities of the pathogens as Colletotrichum gloeosporioides (Penz.) and Galactomyces candidum Butler & Petersen (anamorph: Geotrichum candidum Link). The detection of these predominant fungal pathogens in this study indicated that both fungal pathogens are widely distributed on tomato fruit in KwaZulu-Natal. There is therefore a need to roll out effective and sustainable control strategies. Isolation, screening and identification of yeast strains A total of 148 yeast isolates were recovered from the surface of tomato fruits and were screened for antifungal activity in vitro using a dual culture assay. Only 25 isolates had strong antifungal activity against C. gloeosporioides and G. candidum. These isolates were then screened for phytotoxicity on healthy tomato fruits. Subsequently ten yeast isolates, which were non-phytotoxic to tomato fruits and which inhibited both pathogens, were selected for in vivo testing of their antifungal activity and their effects on tomato quality. The effects of delays between pathogen inoculation after yeast treatment, as well as the mechanism of decay control, were studied using SEM. Out of these 25 isolates, 4 were excluded for showing phytotoxic effect on the fruits. Isolates Y108, Y121 and Y124 showed strong antagonistic effects against both pathogens with no detrimental effect on the fruit. However, the application of the best 10 antagonist yeasts had no effect on the general quality parameters of the tomato fruits. The identity of the best three antagonist yeast isolates was then determined using molecular analysis of their sequences of the internal transcribed spacer (ITS) regions, which identified the best three isolates as strains of Meyerozyma guilliermondii (Wick) Kurtzman. The biocontrol efficacy of the yeast isolates was affected by the timing of their application. The yeast cells needed time to multiply, and thereby provide preventative protection. The sooner the application of the yeast treatments, the better was the biocontrol efficacy of the antagonist yeasts. Competition for nutrients, attachment to fungal hyphae and production of an extracellular matrix were among the probable modes of action of the antagonist yeasts in this study. The best isolates of M. guilliermondii, especially isolate Y108, were

effective as biocontrol agents against C. gloeosporioides and G. candidum and could provide a sustainable alternative to the use of chemical pesticides. Hot water treatments with temperatures of 20, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71 and 80℃ were applied to tomato fruit for periods of 10, 20 and 30s on non-inoculated and inoculated fruit, in order to determine the optimal temperature x time combinations on pathogen control and postharvest quality traits of fruits. The effect of shorter times at the best working temperatures were also tested. The mechanism of heat treatments on decay control was then studied using the SEM. The temperature regimes at which no heat damage occurred on the skin of tomato fruits were 20℃, and from 44℃ to 59℃, at all exposure times, and at 62℃ for 20s. With increased temperature x time combinations above these levels, all treatments caused heat damage, which appeared as peeling, scalding, cracking and ageing either at the same time of treatment, or after 10 days of storage at 25℃. The best combinations of the rHWTs significantly reduced disease incidence, while maintaining fruit quality. These were: 56℃ x 20s, 59℃ x 10s and 62℃ x 10s. Moreover, the combinations of 56℃ x 15s, and 62℃ x 8s were even more effective. Heat treatments caused the melting of the wax platelets of the fruit, sealing cracks in the wax cover of fruit, which remained highly visible on control fruits. Induction of host defence, and inhibition of sporulation and mycelial growth were among the possible modes of action of HWTs in this study. The results have demonstrated the high potential of rHWTs to control C. gloeosporioides and G. candidum, while maintaining postharvest quality during storage, thus prolonging the shelf-life of tomato fruit. Therefore, rHWTs should be considered as a viable technology for the control of postharvest diseases of tomato fruits on a commercial level. rHWT, equivalent to pasteurization, is a rapid process, and avoids introducing a delay in the processing time of large volumes of fruit going through a commercial packhouse. The application of rHWTs and antagonist yeasts each provided significant control of both C. gloeosporioides and G. candidum. The combination of these two treatments enhanced the efficacy of both individual treatments. The integration of rHWTs at 62 x 8s with the yeast M. guilliermondii isolate Y108 resulted in the best disease control against both C. gloeosporioides and G. candidum, and delivered enhanced tomato fruit quality postharvest. This enhanced effect of rHWTs in combination with antagonistic yeasts could be the result of various interactions between the heat treatments, antagonist yeasts and the fruit.

The results presented in this thesis highlight the potential to use biological and physical disease control management strategies, as stand-alone treatments or in combination, as alternative control measures against postharvest tomato anthracnose and sour rot. Although both rHWTs and antagonist yeasts reduced both C. gloeosporioides and G. candidum incidence, the combined treatment provided the best disease control with the best fruit quality. Heat treatments partially disinfect fruit, allowing for the successful colonization of the fruit surfaces and wound sites with antagonist yeasts, which then provide a residual disease control effect for the fruits. Integration of these treatments enhanced persistence and stability of each single treatments, which would be valuable in the tomato industry as part of an effective disease management strategy, which would be economically viable, readily implemented and environmentally sound. Further research is required to implement the technology at an industrial scale.