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標題: Azospirillum rugosum新種與耐鹽根圈細菌的植物生長促進特性之篩選及對植物接種的效益
Screening for plant-growth promoting traits and the inoculation effect on plants of Azospirillum rugosum sp. nov. and salt-tolerant rhizosphere bacteria
作者: 賴威安
Lai, Wei-An
關鍵字: 植物生長促進根圈細菌;plant-growth promoting rhizobacteria (PGPR);生物肥料;固氮作用;溶磷作用;ACC 脫胺酶相對養分吸收率;biofertilizer;nitrogen fixation;mineral phosphate solubilization;ACC deaminase;relative nutrient absorption rate (RNAR).
出版社: 土壤環境科學系所
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土壤中有益的游離生活細菌常與植物根結合,稱為「植物生長促進根圈細菌」(PGPR)。PGPR 與植物作用的機制包含生物肥料、生物防治、植物刺激及生物復育等。為了篩選可促進作物生長之有益微生物,本研究自台灣土壤及含鹽地多種植物的根圈土壤中分離本土微生物、鑑定並針對多種植物生長促進(PGP)的特徵進行一系列之研究。本研究自台灣台中縣市、嘉義縣、南投縣、霧峰鄉、高雄縣等地區的土體樣品中分離54株細菌,其中IMMIB AFH-6 菌株為高雄縣油煉廠油汙汙染的土壤的分離菌,經16S rDNA 序列分析比對結果與 Azospirillum canadense 標準菌株最相似 (96.8%),與Azospirillum 其他標準菌株的相似度均低於97%;染色體DNA-DNA 雜合顯示IMMIB AFH-6 與A. brasilense、 A. canadense 和 Azospirillum doeberinerae 的標準菌株關連性為25%、17%及19%。菌體脂肪酸組成中主要的非羥基脂肪酸為n-C18:1ω7c(39.61%)、羥基脂肪酸為n-C14:0 3-OH(5.6%),基於化學分類、表現型、親緣型的多相分類的證據,IMMIB AFH-6 菌株為新種命名為Azospirillum rugosum。目前已有許多關於Azospirillum刺激植物生長發育的研究資料,但對於其與植物結合過程的機制仍不甚了解,因此本研究選擇屬於Azospirillum 的IMMIB AFH-6進行此菌株之植物生長促進特徵之研究,並探討微生物接種配合化學肥料及有機質肥料,對作物生長之影響。IMMIB AFH-6 菌株具有游離固氮、磷酸三鈣溶解、吲哚3-乙酸(IAA)生成、硝酸還原酶、磷酸水解酶等促進作物生長之特徵,產生胞外多醣體,有利於根拓殖化作用,種子發芽生物分析接種IMMIB-AFH6,可促幼苗胚根生長44%(萵苣)及52%(小蘿蔔),促進胚莖長度27%(萵苣)及24%(小蘿蔔)。萵苣盆栽試驗結果顯示,接種IMMIB AFH-6 菌株並施用半量化肥(NPK)及猪糞有機肥(PMF),對於地上部的生質量有明顯促進之效益,除產量可明顯提高58.3%外,植體中之N、P、K、Zn 的相對養分吸收率亦顯著提高,分別達200%、138%、162%及237%。
含鹽地12 種植物根圈土壤中分離出63 個菌株,並測試其固氮、溶解磷酸鈣、產生IAA、1-胺基環丙烷-1-羧酸(ACC)去胺酶、胞外多醣(EPS)的能力。以微鹽分範圍0.13%氯化鈉濃度,大油菜的胚根與胚莖具有最大的生長長度,0.88%氯化鈉濃度下則胚根與胚莖均受到抑制(28%),本研究選擇20株具植物生長促進特徵的耐鹽性分離菌株及6株耐鹽性的Azospirillum標準菌株,測試其在0.13%及0.88%鹽度下接種對於大油菜種子發芽生長2週的影響。結果顯示在0.13%鹽濃度下,接種不具生產植物激素IAA能力的菌株包括Bacillus sp. CC-CCM15-5、Brachybacterium sp. CC-LVL15-1 及Shinella sp. CC-CCM15-2菌株,對植物生長有抑制的作用,但在0.88%鹽濃度則接種反而有顯著促進生長(分別提高胚莖長度278%、132%、287%,提高胚根長度64%、150%、101%)的效果。另接種具產生IAA 能力的3菌株Arthrobacter sp. CC-HBB15-2、Brevibacterium CC-SN15-10、Staphylococcus sp. CC-SN15-6 在0.88%鹽濃度下卻有抑制大油菜的生長(胚莖長度被抑制26%、24%及35%;胚根長度被抑制12、17%及10%)的效果,其他植物生長促進相關特徵如固氮、溶磷酸鈣、ACC 去胺酶活性、產生乙烯能力與菌株在0.88%鹽度下促進幼苗生長無必然相關。此結果暗示:特別是在鹽逆境下包含乙烯抑制主根生長,及誘導側根與不定根生長,其合成受到相對高量的IAA所誘導的已知機制應加以考慮。

Beneficial free-living bacteria in soil are usually associated with root of plant, mentioned as plant-growth promoting rhizobacteria (PGPR).。Mechanisms in the action of PGPR on plant were classified into biofertilization, biological control, phytostimulation, bioremediation, etc. In order to screen beneficial bacteria which show the promotion to plant growth, the indigenous strains from bulk soil and salinated rhizosphere samples were isolated, identified and characterized with multiple plant growth promoting (PGP) traits. The 54 isolates were from bulk soil of Taichung County, Chiayi county, Nantou county, Wufong County and Kaushiung County in Taiwan; among them the strain IMMIB AFH-6 was isolated from refinery oil contaminated soil, and highest similarity (96.8%) was shown to type strain of Azospirillum canadense based on 16S rDNA sequence. The similarity level are lower than 97% to other type strain of Azospirillum; chromosomal DNA-DNA pairing study showed that IMMIB AFH-6 displayed 25%, 17% and 19% relatedness to the type strain of A. brasilense, A. canadense and A. doeberinerae. Major non-hydroxylated fatty acids are n-C18:1ω7c(39.61%) and the major hydroxylated fatty acid are n-C14:0 3-OH (5.6%). Based on chemotaxonomic, phenotypic and phylogenic evidences, isolate IMMIB AFH-6 are proposed as novel species and named A. rugosum. A lot of experimental data about stimulation of plant growth and development by Azospirillum, but the mechanisms during association is not realized. The PGP traits of Azospirillum rugosum IMMIB AFH-6 and the effects of inoculation with IMMIB AFH-6 combined with fertilizer and organic fertilizer on crop growth was studied. IMMIB AFH-6 has free-living dinitrogen fixation, tricalcium phosphate solubilization, indole-3-acetic acid (IAA) production, nitrate reductase, phosphatase activities and can produce exo-polysaccharide to colonize root and promote plant growth. Inoculation with IMMIB-AFH6 can improve radicle length by 44%(lettuce) and 52%(radish); hypocotyl length by 27% (lettuce) and 24% (radish) under seed germination assay. Hence, we select IMMIB AFH-6 belong to genus Azospirillum to study about PGPR. An integrated strategy was used to promote plant growth by a combination of bacterial inoculation, mineral fertilizer and organic fertilizer application. The results demonstrated that shoot biomass (yield) was increased by 58% and the relative nutrient absorption rate (200%、138%、162%、237%) of N, P, K, Zn in shoot also apparently increased after application of Azpspirillum rugosum IMMIB AFH-6 in combination with half-dose of mineral fertilizer and pig manure organic fertilizer (PMF).
Sixty three bacterial strains were isolated from salinated rhizospheres of twelve plants. The bacterial ability of nitrogen-fixation, tricalcium phosphate solubilization, IAA production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and extracellular polysaccharide (EPS) production were measured individually. Canola seed germination bioassay introduced in 0-0.88% saline water and best growth in 0.13% sodium chloride are shown and growth was inhibited by 28% in 0.88% sodium chloride. Twenty strains with plant-growth promoting traits among isolates from salinated rhizosphere was chosed as bioinoculants to tested canola growth in 0.13% and 0.88% sodium chloride during 2 weeks. And the results demonstrated that growth of seeds inoculated with IAA-producing bacteria involved Bacillus sp. CC-CCM15-5, Brachybacterium sp. CC-LVL15-1 and Shinella sp. CC-CCM15-2 were inhibited in 0.13% sodium chloride by 75%, 69% and 72% in hypocotyl and 60%, 36% and 51% in radicle respectively, but was improved, by 278%, 132% and 287% in hypocotyl and by 64%, 150% and 101% in radicle respectively, in 0.88% sodium chloride. While the growth of seeds inoculated with IAA producing strains Arthrobacter sp. CC-HBB15-2, Brevibacterium CC-SN15-10 and Staphylococcus sp. CC-SN15-6 were inhibited by 26%, 24% and 35% in hypocotyl and 12, 17% and 10% in radicle under 0.88% sodium chloride. The other PGP traits including nitrogen fixation, tricalcium phosphate solubilization, ACC deaminase, and ethylene production seems not shown indispensable for bacteria to improve seedling growth in 0.88% sodium chloride in this research. This result impliedthat known mechanism including inhibition of primary root growth, and induction of lateral and adventious root growth by ethylene and synthesis of which induced by relatively high concentration IAA should be considered especially in salt stress.
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