Identification for the Causal Agent of Lettuce Black Leaf Spot and Its Microbial Control Experiments

• Main Authors: Yu-Hsiu Kao, 高郁琇
• Other Authors: 黃振文
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/54823175064537905430

碩士 === 國立中興大學 === 植物病理學系所 === 101 === During spring season in 2011, a disease previously unreported in Taiwan was observed on commercial plantings of lettuce (Lactuca sativa L.) at Erlun in Yunlin County. A lot of black-brown spots showed on leaves of diseaed head lettuce plants. When spots are so numerous, they coalesce to form areas extending whole leaf and resulting in poor standing of head lettuce with leaf yellow, drying and death. Disease incidence and the rate of poor standing of head lettuce seedlings occurred in cultivation trays at Erlun were 98% and 16%, respectively. A species of Alternaria was consistently isolated from diseased portions of the infected leaves on 2% (w/v) water agar. Spore suspensions (105 conidia/ml) of the pathogen were used to inoculate fourteen crops to confirm its host range. The results showed that leaf spots were similar to the original symptoms developed only on inoculated plants of ten lettuce cultivars. However, inoculated tomato and cabbage plants did show hypersensitive-like reaction. Other inoculated plants remained symptomless. Based on ribosomal DNA internal transcribed spacer (ITS) sequencing analysis, the ITS sequence of the pathogen was similar to A. solani. The ITS sequence identity between the pathogen and A. solani (BCRC No. 32123) obtained from Food Industry Research and Development Institute was 99%. On V8 juice agar, colonies of the pathogen are dark-brown with concentric rings. Most of conidiophores with single conidiogenous site and a few with 2-4 conidiogenous sites. Conidia borne singly from each conidiogenous site on the conidiophores, long-ellipsoid or long-ovoid, 6-10 transverse septa and 0-3 longitudinal septa, 1-2 beaks, 82-90 × 13 μm. Beaks are narrow-tapered and become up to 163-176 μm long. The optimum temperature for mycelial growth, conidial germination and infection on lettuce of Alternaria sp. isolates AL1-2 and AL3-11 was 24℃. The results indicated that the fungus may be a new species of Alternaria on Lactuca sativa in the world and we named it Alternaria lactucae Kao & Huang sp. nov.. Fifteen antagonistic bacteria were evaluated for their effects on mycelial growth and conidial germination of A. lactucae isolates AL1-2 and AL3-11. Among tested bacteria, Bacillus amyloliquefaciens isolate BA01 was an effective biocontrol agent which was able to inhibit 55% of mycelial growth and 80% of conidial germination and did also cause germ tubes swelling of A. lactucae. Application of detached head lettuce leaf for evaluating the efficacy of BA01 on controlling lettuce black leaf spot showed that BA01 was able to reduce 89% of development of black spot on detached head lettuce leaf. The fermented broth of BA01 cultured in Soybean meal-Molasses (SYM) medium was analyzed by bioassay method of detached head lettuce leaf for disease control. 100-fold dilution of fermented broth of BA01 cultured in SYM media could inhibit 36% and 41% of development of black spot caused by A. lactucae isolates AL1-2 and AL3-11, respectively. Adding different plant oils into SYM media to evaluate their effects on the control efficacy of BA01, the results indicated that fermented broth of BA01 cultured in SYM media amended with 2.5% (w/v) peanut oil did show to be the most effective in controlling lettuce black leaf spot. Inhibition rate of 200-fold dilution of the fermented broth was more than 46%. In addition, we also found that SYM media amended with 0.125% (w/v) emulsified agent could enhance the ability of BA01 for controlling lettuce black leaf spot. Inhibition rate of 200-fold dilution of the fermented broth reached more than 56%. In greenhouse test, spraying two fermented broths of BA01 at 250-fold dilution to head lettuce plants one day before inoculating with the pathogen (105 conidia/ml) did show to be the most effective in controlling the disease. Analysis of antifungal compounds produced from cultural filtrate of BA01 by high pressure liquid chromatography (HPLC) showed that BA01 could not only produce surfactin but also produce iturin A. This study demonstrates that the two fermented broths of B. amyloliquefaciens BA01 have the potential to be developed into microbial plant protectants for controlling lettuce black leaf spot.