連作對結球甘藍(Brassica oleracea var. capitata)根部可培養型內生細菌之影響及其功能性研究

The Effect of Continuous Cropping on Culture-dependent Bacterial Endophytes of Cabbage (Brassica oleracea var. capitata) Roots and Their Functional Characterization

丁俊榮(Jyun-Rong Ding)

指導教授 ： 楊禮亘

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2013年)

https://doi.org/10.6827/NFU.2013.00130

摘要

連作障礙是農業栽種上影響作物產量最常遇到的瓶頸。在台灣一些蔬菜瓜果類作物更是首當其衝，如結球甘藍、小白菜等十字花科、辣椒、番茄等茄科及洋香瓜等葫蘆科植物。其中，十字花科的結球甘藍在台灣年產量約32~34萬公噸佔台灣蔬菜類總產量的11%~12%，市場需求相當高但也受連作障礙所害最重。
本研究於雲林縣西螺鎮的農地以連作三期之方式種植結球甘藍(每作期約50~58天)，並於每作收成後採集其根部樣品進行可培養型內生細菌之分離，以了解連作對植物根部內生細菌菌相之影響。本實驗從結球甘藍根部總共篩得310株細菌菌株，菌數量約為2.3x104 (CFU/g)。經16S rRNA基因序列定序得40種不同之菌種，分別為2株Actinobacteria、2株Bacteroidetes、10株Firmicutes、3株α-Proteobacteria、1株β-Proteobacteria及22株γ-Proteobacteria菌綱之菌種。比較結球甘藍各作期之根部菌種分布發現其內生菌種分布會因連作使得屬於革蘭氏陽性菌之比例從原本45%降至25%，而革蘭氏陰性菌種所佔比例從原本的55%增加至75%。文獻指出許多植物內生菌具有合成促進植物生長物質或抵抗病原菌之代謝活性以利於未來農業生技之應用，例如溶磷作用、植物荷爾蒙IAA之合成能力、鐵載體合成能力、分泌分解酵素(蛋白酶、纖維素酶、澱粉酶)等。因此本實驗針對結球甘藍40種不同之內生菌種進行以上功能分析並同時測試其對5種常見作物病原真菌(齊整小核菌、十字花科炭疽刺盤孢菌、立枯絲核菌、甘藍鏈隔孢菌及疫病菌屬)之培養皿對峙試驗。試驗結果顯示，具有蛋白分解功能菌株有21株；具有澱粉分解功能菌株有9株；具有纖維素分解功能菌株有5株；具有溶磷功能菌株有7株；具有鐵載體功能菌株有26株；具有生產IAA功能菌株有30株。在病原真菌對峙試驗方面，總共有7株能抑制病原真菌的生長，其中又以LS0171、LS0156、LS0363菌株能同時抑制三種病原真菌為最佳。值得一提的是LS0363菌株同時具有合成鐵載體、IAA能力及抑制三種病原真菌之功效。我們將進一步篩選菌種危害等級1且具促進植物生長功能的菌株進行水稻種子處理試驗，結果發現編號LS0161和LS0126菌株能增加根部長度及編號LS0183菌株能增加植株鮮重。編號LS0388、LS0410及LS0258菌株能增加水稻葉片中葉綠素a含量。此研究可望在未來發展出新穎之生物製劑，有利於農業作物生長及對抗病害之發生，減少或替代化學性肥料的依賴以降低環境污染。

關鍵字

結球甘藍 ； 內生菌 ； 病原真菌 ； 16S rRNA ； 連作 ； 促進植物生長物質

並列摘要

Continuous cropping obstacle (CCO) is the most common bottleneck that affects crop yield in agricultural plantation. Several important vegetable and melon crops in Taiwan are highly susceptible to CCO, such as cabbage (Brassica oleracea var. capitata), Brassica rapa chinensis, pepper (Capsicum annuum), tomato (Lycopersicon esculentum Mill.), muskmelon (Cucumis melo L.) etc. The annual production of the highly demanding Cruciferae vegetable Brassica oleracea var. capitata is around 320 to 340 thousand metric tons and accounts for 11-12% of the total foliage crops in Taiwan, yet its production in particular is highly hampered by CCO. To characterize CCO effects on the endophytic microflora in cabbage root tissues, three consecutive harvests from continuous cropping (each harvest period of about 50 to 58 days) on a farmland in Yunlin County, Siluo district was performed. Root samples were harvested and culturable endophytic bacteria were isolated and identified. The bacterial colony-forming unit (CFU) in cabbage root tissues were 2.3x104 (CFU/g), and among them, 310 bacterial strains were isolated for 16S ribosomal RNA gene amplification and sequence analysis. A total of 40 different bacterial species were identified, 2 strains of Actinobacteria, 2 strains of Bacteroidetes ,10 strains of the Firmicutes, 3 strains of the α-Proteobacteria, 1 strains of the β-Proteobacteria and 22 strains of the γ-Proteobacteria. A comparison of cabbage root endophytic bacteria revealed that continuous cropping increased gram-negative bacteria numbers (55% to 75%) while gram-positive bacteria decreased relatively (45% to 25%). Previous studies indicated that plant endophytes confer plant growth promoting substances (PGPS) and disease resistance activities, such as the production of IAA (indole-3-acetic acid), siderophores, and the ability to produce degrading enzymes (protease, cellulase, amylase) which could be applied as agricultural biotechnological products. We have tested the 40 different endophytic bacterial strains for the above-mentioned functions as well as their antagonistic effect on five common crop fungal pathogens (Sclerotium rolfsii Sacc., Colletotrichum higginsianum Sacc., Rhizoctonia solani Kuhn, Alternaria brassicae and Phytophthora spp.). The results showed 21 strains with proteolytic ability, 9 strains with starch degrading ability, 5 strains with cellulose degrading ability, 7 strains with phosphate solubilization ability, 26 strains with siderophore producing and 30 strains with IAA producing capabilities. Seven strains showed antagonistic activities against phytopathogenic fungi, in which LS0171, LS0156 and LS0363 strains exhibited antagonistic effects on three different phytopathogenic fungi. It is worth mentioning that LS0363 strain has the ability to synthesis siderophores, IAA and also antagonize three different phytopathogenic fungi. We have selected individual endophytic isolates that were classified as Biosafety Level 1 and possessed multiple PGP promoting activities for their functional studies by inoculating on rice seeds. Inoculation of endophytes in rice showed that LS0161 and LS0126 strains could increase the length of rice roots and LS0183 strain could increase the rice fresh weight. In addition, LS0388, LS0410 and LS0258 strains could increase chlorophyll a content of rice leaf. This study is expected to develop novel environmental friendly biological agents for their PGP and disease resistance activities in attempt to reduce or substitute the use of chemical fertilizers and to reduce environmental pollution.

並列關鍵字

Cabbage (Brassica oleracea var. capitata) ； Endophytes ； Phytopathogenic fungi ； 16S ribosomal RNA ； Continuous cropping ； PGPS

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