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Diversity, characterization and application of banana endophytic bacteria from Cu-contaminated paddy fields in Changhua, Taiwan
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植物內生細菌已廣泛應用在植物保護領域，如病害防治、促進生長、適應逆境等。本研究比較台灣彰化銅污染與非污染水稻田內生長之香蕉內生細菌多樣性與菌相差異，進一步分析耐銅特性與促進植物生長的特性，最後評估香蕉內生細菌對水稻生長的效果與銅逆下對水稻生長的影響。自台灣彰化大肚溪流域和洋仔厝溪流域之間採集香蕉組織樣本，於3處銅污染地與3處非污染地內各分離到193株菌株，分析結果得知，銅污染地之內生細菌多樣性較低 (Shannon index = 2.781; Simpson index = 6.826)，然土壤銅濃度與多樣性指數無明顯相關，屬豐富度亦無明顯差異。本研究中屬優勢菌屬之Bacillus 屬內生菌易自銅污染農地中分離得到，分離率為 35.2 %；此外6處農地之內生細菌以香蕉地下部所分離之比率較高，進一步比較銅污染地與非污染地之地上部與地下部菌株分離，顯示銅污染地之地上部菌株分離率 (36.8 %) 高於非污染地 (21.2 %)。由耐銅性測試得知，銅污染地內之耐銅菌株比率顯著高於非污染地，然地上部與地下部之耐銅菌株比率無明顯差異，顯示銅污染地之內生細菌的耐銅能力和其於植物體內的分佈並沒有相關。於93株耐銅菌株中，37株菌株具有促進油菜與水稻生長的潛力，並挑選對種子活力指數最高之5株菌株，Lysobacter sp. R5-43、Herbaspirillum sp. P5-6、Rhizobium sp. R6-6-1、Micromonospora sp. R6-22 及 Paenibacillus sp. PS6-4，測試對水稻生長的影響，結果指出，處理內生細菌之水稻種子，能促進幼苗之生長。分析可促進水稻生長之因子指出，除 R6-22 菌株外，其餘4者可以生合成生長素、螯鐵蛋白或具溶解磷酸鹽之能力。於不同銅濃度下處理內生細菌，結果顯示於1 ppm銅濃度下，R5-43與 P5-6菌株具促進水稻幼苗高度與重量之趨勢；而於2.5 ppm銅濃度下，則以R5-43、R6-6-1、R6-22 及 PS6-4 菌株對幼苗株高有顯著促進效果；另於含 120 ppm 銅之 0.8 % WA中，水稻根部皆無法正常發育，唯處理R6-6-1與PS6-4菌株之株高有顯著增加。此外於 50 ppm 銅濃度下添加P5-6、R6-6-1及PS6-4菌株後，水稻種子發芽率可顯著提升11.9 ~ 20.4 %。未來仍需持續探討供試菌株在水稻植體內的纏據能力與位置，測試其他的促進植物生長與溶解難溶重金屬銅之特性，並分析供試菌株在促進水稻生長的同時，是否會增加水稻對重金屬銅的生物可利用性，以期能將植生萃取法應用於田間，增加土壤整治策略的多元性。
Endophytic bacteria have been known for plant protection, including disease control, growth promotion, stress adaptation, etc. This study aimed to A) compare diversity and bacterial flora of banana endophytic bacteria from copper-contaminated and uncontaminated paddy fields in Changhua, Taiwan, B) analyze copper-tolerant and plant growth promoting traits, and C) evaluate the effect of endophytic bacteria on rice growth with or without copper. After collecting banana tissue samples from the Dadu and Yangzaicuo basin, 193 and 193 strains were respectively isolated from samples of copper-contaminated and uncontaminated sites. Though diversity of banana endophytic bacteria was lower in contaminated sites (Shannon index = 2.781; Simpson index = 6.826), there was no correlation between soil copper concentrations and diversity indexes, and no difference in genus richness. Of these strains, Bacillus sp. was the predominant group in contaminated sites and had the highest isolation frequency (35.2 %). In addition, strains in 6 sites were mainly collected from the root systems of banana, and strains from root or shoot systems were further analyzed. The percentage of strains from shoot systems in contaminated sites (36.8 %)was and significantly higher than the percentage of those in uncontaminated sites (21.2 %). According to the copper tolerance test, the percentage of copper-tolerant strains in contaminated sites was higher than the percentage of those in uncontaminated sites; however, there was no significant difference in the percentages of copper-tolerant strains between root and shoot systems, which revealed that there was no correlation between copper-tolerant traits and distribution of endophytic bacteria in banana. Among 93 copper-tolerant strains, 37 strains had the potential to promote growth of both rape and rice. Five strains with high relative vigor indexes, including Lysobacter sp. R5-43, Herbaspirillum sp. P5-6, Rhizobium sp. R6-6-1, Micromonospora sp. R6-22, and Paenibacillus sp. PS6-4, were examined for there effectiveness on rice growth. The results indicated that rice seeds treated with endophytic bacteria could promote seedlings growth and it might be attributed to production of auxins, siderophores, or compounds solubilizing phosphate. Rice seeds treated with endophytic bacteria was examined by different concentrations of copper. Under 1 ppm of copper, rice seedlings treated with strains R5-43 and P5-6 became higher and heavier. Under 2.5 ppm of copper, leaves of rice seedlings treated with strains R5-43, R6-6-1, R6-22 and PS6-4 were significantly longer. 0.8 % WA with 120 ppm of copper was used to simulate the copper contaminated field condition, and the results indicated that strains R6-6-1 and PS6-4 could promote the rice seedlings growth to overcome the stunting symptom under copper stress. Besides, strains P5-6, R6-6-1 and PS6-4 could accelerate rice seeds germination under 50 ppm of copper. We expect to put phytoextraction into practice, and enrich soil remediation strategies; therefore, we suggest that some concepts below be cleared: A) the colonization ability and location of strains in rice, B) another plant growth-promoting and heavy metals solubilization traits, and C) effect of endophytic bacteria on bioavailability of heavy metals.
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