| Summary: | 碩士 === 國立中興大學 === 植物病理學系所 === 103 === Mango is one of the most important fruit crops and is the highest value of exported fresh fruits in Taiwan. Colletotrichum gloeosporioides causes mango anthracnose and is one of the major pathogens in mango. It can infect mango leaves, flowers, branches and fruit, and causes considerable fruit disqualified during postharvest period. Currently, the disease management strategies majorly rely on fruit bagging and chemical fungicides application. However, fungicide application can result in fungicide resistance and excessive chemical residues. The purpose of this study is to screen and apply microbe- and plant-derived materials to control mango anthracnose. Since mango fruit is seasonally available, a stable inoculation platform for using mango leaf as inoculation material was established in the beginning in my thesis study. Mango (cv. Irwin) leaf with age within 8-days is highly susceptible to the infection of C. gloeosporioides TYC-2 and the infection is stable and repeatable. In addition, anthracnose lesions appeared earlier on the abaxial than the adaxial surface of a leaf, which is not related to the ability of germination and appressorial formation of the pathogen on both surfaces. Among 17 tested plants, ethanol extracts from 4 different plants showed nearly 50% of inhibition on mycelial growth of TYC-2. Plant A ethanol-extracts showed complete inhibition on TYC-2 spore germination at 0.08 mg/ml in vitro. The anthracnose lesion was not appeared when mango leaf and fruit were co-inoculated with TYC-2 and plant A ethanol-extracts (0.08 mg/ml). Total 45 yeast isolates and 83 bacterial isolates were isolated from the soil, leaves or flowers of mango and showed inhibitory ability to the mycelial growth of TYC-2. Among all yeast isolates, isolate 3H-4 showed strong competition against the growth of C. gloeosporioides in PDA medium. Among all bacterial isolates, strain A and B has significant inhibitory effect to 15 Colletotrichum isolates which cause the anthracnose of mango, Chinese cabbage and chili pepper. Strain A and B were identified as Bacillus amyloliquefaciens based on the comparison of 16S rDNA sequence and Biolog analysis. The 100-fold dilution of 4-day-old culture liquid of strain A or B cultured in culture medium (strain A-SSM or strain B-SSM) could completely inhibit anthracnose lesion production on detached leaves and fruit. Moreover, it showed 80% lesion area reduction when treated with 600-fold dilution of strain B-SSM on detached leaf. The bacteria population of strain B -SSM could increase slightly from 1.48 × 109cfu/ml to 2.6 × 109cfu/ml after stored for 20 weeks under room temperature. In addition, the antagonistic activity of strain B -SSM was resistant to heat treatment, 100℃ for 20 minutes. It indicates that strain B -SSM had great stability during short-time storage assay. To improve the antagonistic activity of strain B, various plant oils were added into SSM to culture this bacterial strain. The results showed that 0.5% (v/v) plant oil A amended SSM could increase the antagonistic activity of strain B by increasing the lesion area reduction from 43% to 100% when 200-fold diluted bacterial culture was applied. The active ingredient of strain B -SSM remained in the culture filtrate but not the culture pellets after the bioactivity assay on the detached leaf. Culture filtrate of strain B -SSM could inhibit spore germination and cause abnormal swelling of germ tubes of TYC-2 in vitro and in planta under the examination of light microscopy as well as scanning electron microscopy. There were many vacuole-like structures formed in hyphal 32 h after treated with strain B -SSM, and no necrosis lesion was observed on fruit 72 h after treatment. Thin layer chromatographic (TLC) analysis revealed that two regions (Rf 0.045 and 0.38) with antifungal activity were identified in strain B -SSM and strain B -SSM with 0.5% plant oil A. The bioactive components were recovered from the TLC plate and analyzed by high-performance liquid chromatography (HPLC). The data revealed that the bioactive components contain iturin A. Based on the results presented in this study, the ethanol-extracts of plant A and bacterial strain B has great potential for further development of biological control agents in field applications to control mango anthracnose.
|
|---|
