Abate Dawit (PhD)Kefialew Yonas2018-07-172023-11-082018-07-172023-11-082014-06http://etd.aau.edu.et/handle/123456789/8865Anthracnose 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.enBiorational ManagementThesis