Biorational Management of Postharvest Anthracnose on Tropical Fruits and Gummy Stem Blight on Cucurbits Biorational Management of Postharvest Anthracnose on Tropical Fruits and Gummy Stem Blight on Cucurbits
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Date
2014-06
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Addis Ababa Universty
Abstract
Anthracnose caused by Colletotrichum gloeosporioides and Colletotrichum acutatum and
Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) are
among the most important diseases of fruit and cucurbits. Currently, the control of fruit and
vegetable diseases relies mainly on the use of synthetic fungicides. Elucidating non-chemical
control methods to reduce postharvest decay is becoming more important. This study
investigated biological based approaches to controling these diseases. The first objectives of this
study were to analyze morphological, physiological and molecular methods in the differentiation
of Colletotrichum isolates obtained from banana, mango and papaya fruits and to evaluate the
biocontrol potential of antagonistic bacteria, yeasts and fungal isolates to manage anthracnose
disease of banana, mango and papaya during storage. In the first phase, a phenotypic analysis of
Colletotrichum isolated from banana, mango and papaya were carried out to identify the species
responsible for anthracnose disease on these hosts. A total of 45 isolates from three hosts were
used. The overall similarity among different isolates of Colletotrichum was determined using
cultural characteristics. According to the results fungal isolates could be divided in to 12 distinct
groups based on morphological similarity. Subsequent identification based on ITS sequence lead
to the identification of Colletotrichum isolates as C. acutatum and C. gloeosporioides. Isolates
from mango were C. gloeosporioides while isolates from banana and papaya was C. acutatum.
Further, the study confirmed the cross infection potential of Colletotrichum isolates and absence
of host specificity. Effect of different temperature, pH level, culture media, light intensity,
carbon and nitrogen sources were tested against the growth of C. acutatum and C.
gloeosporioides. Results indicated that the growth of these isolates varied with the different
environmental and nutritional conditions tested.
The second phase of this study was conducted to isolate, screen and identify indigenous
microorganisms found on fruit surfaces in order to find a suitable biocontrol agent against
postharvest fruit anthracnose caused by C. gloeosporioides and C. acutatum. Bacteria, yeast and
fungal isolates recovered from leaf and fruit surfaces of banana, mango and papaya were tested
In vitro and In vivo against C. gloeosporioides and C. acutatum. The microbial antagonists
inhibited mycelial growth in the dual culture assay and conidial germination of C. acutatum and C. gloeosporioides isolates in vitro. Studies were carried out to analyze the ability of the
antagonists to produce extracellular enzymes on an amended solid media. Fourteen (14) isolates
used produced cellulose and chitinase on amended media but only four isolates showed
glucanase pectinase and protease activity on solid media. Additional experiments were
conducted to extract and determine the nature of antifungal substances produced by antagonists
that were inhibitory towards Colletotrichum isolates. Microbial antagonist isolates differ in their
preference of culture media. The results of In vivo experiments under artificial infection
conditions showed that suspensions containing unwashed cells of antagonists provided the
highest levels of inhibition of anthracnose, while the washed cell suspension and autoclaved
culture filtrates provided less protection against the disease after 30 d. Nineteen (19) different
antagonists were evaluated on their own and in combination with fungicide and hot water for
their ability to reduce postharvest fruit anthracnose diseases in vivo under natural infection
conditions. Integrating fungicides or hot water with antagonists controlled anthracnose more
effectively than fungicide control. Nine antagonists were more effective than other isolates in the
control of postharvest anthracnose when fruit were treated under natural infection conditions.
After phenotypic and molecular analysis, the bacterial isolates were identified as six
Pseudomonas, three Bacillus and a Paenibacillus species. The Pseudomonas and Bacillus strains
can neither be sufficiently re-solved by MALDI-TOF MS nor by 16S rRNA gene sequence
analysis. The most effective yeast (M-23-L-1) and filamentous fungal (M-30-F-2) isolates were
identified as Candida rogousa and Trichoderma longibrachiatum, respectively. This experiment
identified six Pseudomonas and three Bacilli antagonist isolates as novel strains to be used as
biological control agents against anthracnose of tropical fruits.
The second objectives of this study were to characterize populations of Didymella bryoniae from
commercial watermelon and other cucurbit hosts from different parts of the USA on the basis of
their biological and molecular diversity and to evaluate the effect of tiadinil and two thymolbased
formulations against D. bryoniae and Gummy Stem Blight (GSB) development. In the first
phase, morphological characteristics and rDNA Internal Transcribed Spacer (ITS) sequences
were analyzed to identify the causal organism of this disease. Thirty five isolates of Didymella
spp. and Phoma spp. associated with GSB on watermelon, canary melon, muskmelon and squash
from Florida and Georgia were characterized based on morphology on culture media, pathogenicity assays and genetic characterization using ITS sequence analysis. All the isolates
were pathogenic on watermelon cv. Melody, but to a varying degree. RAPD and ITS sequence
analysis indicated genetic variation between the isolates. The ITS region analysis showed the
presence of two isolates, DB-05 and DB-33, which showed a higher similarity to D. bryoniae
isolates from China. This is the first description of an isolate with this unique sequence in Florida
and Georgia. The present study brings insights into the current genetic profile of D. bryoniae
isolates in Florida and Georgia, and its similarity with international isolates.
During the second phase, direct antifungal activity of tiadinil and the effect of two-thymol
formulations on D. bryoniae were evaluated in vitro. All test materials used in the study affected
fungal growth, with tiadinil at 10 ppm and thymol-based formulations at 0.1 ppm significantly
(P 0.05) reducing mycelial growth, conidia germination and germ tube elongation. Foliar
application of tiadinil (before and after inoculation) on artificially infected watermelon seedlings
at 10 ppm significantly reduced the disease severity compared to the untreated controls
(P 0.05). The disease severity on seedlings treated with tiadinil at 3000 ppm was statistically
comparable to chlorothalonil control (P 0.05). Plants with foliar applications of tiadinil at
1000 ppm (before pathogen inoculation) had significantly lower disease severity than plants with
drench application (P 0.05). Foliar application of tiadinil was affected by chemical
concentration and frequency of application. Thus, based on this study, it is prudent to say that
tiadinil and thymol-based formulations are potential materials for use in watermelon production
for effective GSB disease suppression.
This study represents a novel report dealing with the biocontrol of anthracnose in banana, mango
and papaya fruit by the application of possibly new antagonist bacterial strains and a yeast
isolate. It strongly recommends the use of a combination of biological control agents with
commercial treatments as a safe and effective disease management option against the postharvest
anthracnose of tropical fruits. The study also highlighted the possible utilization of tiadinil and
thymol-based formulations against GSB as a management strategy.
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Keywords
Biorational Management