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標題: Isolation and characterization of bacteria associated with tomato (Solanum lycopersicum L.) leaves
作者: Sheng-Chen Peng
關鍵字: 番茄;葉圈;拮抗試驗;植物生長促進特性;碳源代謝潛勢;tomato;phyllosphere;antagonistic test;plant growth promoting characteristics;carbon catabolic potential
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本研究利用肉湯抽出物培養基從番茄葉圈中篩出44個分離株,利用甲醇培養基篩選出68個分離株,全部共112個分離株,經16S rDNA序列鑑定後,分別屬於3種菌門、4種菌綱,共有Acinetobacter, Bacillus, Brachybacterium, Exiguobacterium, Lysinibacillus, Methylobacterium, Microbacterium, Paenibacillus, Pantoea, Paracoccus, Pseudomonas, Rhizobium, Sphingomonas及Xanthomonas等14個菌屬。肉湯抽出物固體培養基篩選到數量最多的菌屬依序是Xanthomonas的 27%、Pseudomonas 的20%及Bacillus 的20%;甲基培養基篩選到數量最多的菌屬依序是Xanthomonas的 44%、Pseudomonas的 22%及Pantoea 的18%。從以上菌株中挑選出23個不同菌屬菌種的分離株,與番茄病原菌Xanthomonas campestri pv. vesicatoriar進行拮抗試驗,共有5個分離株對病原菌表現出拮抗圈。並從5個分離株中挑出2個拮抗病原菌的分離株Pantoea sp. MYEO 12及 Bacillus sp. NYPO 7進行番茄抗病的生物試驗,將番茄植株種植在生長箱中無菌的環境下,接種病原菌及NYPO 7的處理,能有效將番茄的罹病指數從4.3降到2.4,而接種病原菌及MYEO 12的處理,番茄的罹病指數仍維持在3.8,無法有效防治病原菌,但是無論是接種MYEO 12及NYPO 7都能有效提高番茄幼苗的生質量,幫助其生長。接著對葉圈分離株進行植物生長促進能力的分析試驗,在游離固氮活性及溶磷活性試驗中,分別各有5個分離株表現較佳的活性,其固氮活性介於0.136-0.456 ηmol/(tube x hr);溶磷能力介於225-1430 μg/mL;在IAA合成能力的試驗中,23個分離株都表現出IAA的合成能力,且IAA濃度高於50 μg/mL的菌株有6株,可以推測葉圈分離株主要藉由合成IAA來促進植物生長。從以上試驗中,挑出6個具有植物生長促進潛力的分離株Lysinibacillus sp. NYPO 3、Bacillus sp. NYPO 7、Methylobacterium sp. MYET 17、Microbacterium sp. MYEO 1、Pantoea sp. MYEO 12及Rhizobium sp. MYEO 9進行番茄溫室的盆栽試驗。雖然接種NYPO 3的盆栽處理在乾重上能高於對照組,接種MYEO 1及接種MYEO 12的盆栽處理在株高上也高於對照組,但都沒有顯著的提高。另外對於番茄盆栽微生物群落的碳代謝潛勢與群落結構分析,結果顯示接種不同的菌株,對番茄盆栽介質的菌相有明顯的改變,但菌相的改變對於番茄植株而言沒有顯著的影響。

In response to the increasing population around the world, food production largely relies on the application of chemical fertilizers and chemical pesticides. However, the high amount of agrochemical input has led to many environmental problems such as soil degradation and ecological imbalance. Development of microbial agents used to increase nutrient availability or control plant pathogen provides alternatives to sustain plant growth and health. Plant rhizosphere and phyllosphere have been demonstrated to harbor highly diverse microorganisms with various functions. However, our knowledge of the microbiology of phyllospheric bacteria has historically lagged behind our knowledge of the microbiology of rhizospheric bacteria. This motivates us to isolate and characterize bacteria associated with leaves of tomato, the most significant horticultural crop worldwide. In the present study a total of 44 and 68 isolates were obtained from phyllosphere of tomato using nutrient agar and methanol medium agar, respectively. 16S rDNA sequences assigned them to 14 genera namely Acinetobacter, Bacillus, Brachybacterium, Exiguobacterium, Lysinibacillus, Methylobacterium, Microbacterium, Paenibacillus, Pantoea, Paracoccus, Pseudomonas, Rhizobium, Sphingomonas and Xanthomonas. Among them, genera Xanthomonas and Pseudomonas were the dominant bacterial groups obtained from phyllosphere no matter which medium was used for isolation. Twenty-three isolates were selected and used for further studies. The antagonistic test demonstrated that 5 isolates were able to inhibit the growth of tomato pathogen Xanthomonas campestri pv. vesicatoriar. Inoculation of Bacillus sp. NYPO 7 was successfully used to control plant disease, as can be seen from the decrease of disease index from 4.3 to 2.4. Higher biomass of tomato seedlings were also recorded in Bacillus sp. NYPO 7 or Pantoea sp. MYEO 12 inoculating treatment after 6 weeks of cultivation. Considered for plant growth promoting traits, the highest free-living nitrogen fixing activities ranging from 0.136-0.456 ηmol/ (tube x hr) were obtained. Five isolates showed promising tricalcium phosphate solubilizing activities, and the soluble P ranged from 225 to 1430 μg mL-1 after 4 days of cultivation. All 23 isolates possessed IAA producing abilities. Six isolates were used separately as inoculants in pot experiment to evaluate their performance on tomato growth. The results demonstrated that dry weight of stems and leaves was slightly higher in half dose of chemical fertilization along with Lysinibacillus sp. NYPO 3 inoculating treatment than that in half dose of chemical fertilization treatment only. Distinct community level physiological profiles were obtained after bacterial inoculation, demonstrating that soils under different treatments harbored microorganisms with different carbon catabolic potential.
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