Integration of rapid hot water treatments and biocontrol agents to control postharvest pathogens of tomato.
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Date
2019
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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.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.