TW200946477A

TW200946477A - Novel species of Azospirillum rugosum and use thereof as biofertilizer

Info

Publication number: TW200946477A
Application number: TW97117919A
Authority: TW
Inventors: Chiu-Chung Young; Wei-An Lai; P D Rekha
Original assignee: Chiu-Chung Young
Priority date: 2008-05-15
Filing date: 2008-05-15
Publication date: 2009-11-16

Abstract

The invention relates to an isolated, purified and cultured microorganism strain named Azospirillum rugosum IMMIB AFH-6. The microorganism strain has the following eight functions: biological nitrogen fixation, mineral phosphorous dissolution, enzyme activities of phosphatase, nitrate reductase, gelatin protease, urease, production of plant growth hormone indole-3-acetic acid, and production of extracellular polysaccharides and so on. The microorganism is demonstrated to be safe and is not known pathogen by analysis of 16S rRNA gene sequence, composition of fatty acid methyl ester of whole cell hydrolysates, and phenotype features. The effects of the microorganism strain for facilitating plant growth, soil improvement, and as a microorganism fertilizer are demonstrated by analysis of its enzyme activities, carbon source utilization features, seed germination bioanalysis, and crop pot tests.

Description

200946477 九、發明說明：【發明所屬之技術領域】本發明係有關於一種經分離純化培養之微生物株，特別是一種具有（1)生物固氮（nitrogen fixation )、（2)溶解礦物鱗(minerai solubilization)、（3)磷酸水解酶(phosphatase)、（4)硝酸還原酶(nitrate reductase)、（ 5 )動物膠蛋白酶(gdatinase)、（ ό )尿素酶(urease)的活性、（7 ) 可產生植物生長激素丨嗓-3-醋酸（indole-3- acetic acid ; IAA)、（ 8 )可產生胞外多醣(extracellular polysaccharide)等至少8種功能之新穎皺紋固氮螺旋〇菌 η/go⑽w) IMMIB AFH-6。【先前技術】按，習知農田大量使用化學肥料固然可以快速提供作物初期所需養分’但是會導致土壤酸化、肥力不均與地下水源污染。已知土壤微生物中若具有固氮作用、無效性營養溶解及分解土壤中有機質的功能’可供為微生物肥料，而微生物肥料可取代部份化學肥料，以解決長期施用化學肥料所衍生出土壌劣化的問題。植物生長促進根圈細菌〇 (plant growth promoting rhizobacteria ; PGPR)依照其有益的作用機制有四：（1)生物肥沃作用（biofertilization)，（2)生物防治（bi〇_contr〇l)、（3) 生物復育作用（bioremediation)，（4)植物激素作用（phytostimulati〇n)。已知植物根分泌物(root exudates)的組成通常包括有醣類(carb〇hydrate)、胺基酸 (amino acids)、維生素(vitamins)、有機酸(organic acids)和其他各樣的化合物，這些根分泌物有利於微生物的利用，並被認為是根圈中微生物數量及活性增加的主要理由。因此細菌對各種碳源的利用能力也被認為是細菌成功拓殖化根圈的因素之一。· 固氮螺旋菌屬)為用於促進青草與穀類具大潛力的的接種劑， 200946477 固氮螺旋菌屬可藉由分泌植物激素、固氮作用、增進植物對礦物的攝取、硝酶還原作用等機制而促進植物生長（James，2000)，然而此些作用的前提為其菌體對植物根的拓殖化作用。細胞多形性使其可於改變的環境條件下適應良好。固氮螺旋菌在低氧時為類螺旋狀的營養細胞’偏好利用果糖當碳源；在限制氧及高碳氮比下，活性生長之菌體則產生聚-β-羥丁酸(p〇1y_P_hydrQxybutyrate ; PHB) ’細胞則分化為球狀的似胞囊細胞（CySt-Uke ceii)，胞外多糖類(extraceilular polysaccharide)造成細胞凝絮(cell aggregation) ’偏好PHB做為細胞利用之碳源 (Tarrand 等人，1978 ; Sadasivan 和 Neyra，1985 ; Sadasivan 和 Neyra，1987)。 ©由上可推知’固氮螺旋菌在農業上的運用具有其眾多優點：（1)目前尚未發現有任何病原菌屬於該屬的種類；（2)無似根瘤菌（rhiz〇bia)需與結根瘤（nodule)的豆科植物宿主交互作用才有固氮功能，固氮螺旋菌對植物宿主的選擇性不限於豆科作物；（3)可以產生似胞囊細胞(cyst_iike cell) ’此結構有利於根拓殖化作用(root colonization)及固氮作用(nitrogen fixation) (Katupitiya 等人，1995 ; PeregGerk 等人，2000)。緣是，本創作人有感於開發新穎菌種的重要性，乃潛心研究並配合學理之應用’終於提出一種合理且產生似胞囊細胞且可有多種活性的 ❹固氣螺旋菌。固氮螺旋菌屬（Genus Jzo印的生物分類地位為：門 (phylum)名為原細菌門（proteobacteria)，綱（class)名為阿爾法原細菌 (alphaproteobacteria) ’ 目（order)名為紅螺旋細菌（Rhodospirillales)，科（family)名為紅螺旋菌（Rhodospirillaceae)，目此屬已發表的包括 10個種（species ):亞馬遜河固氮螺旋菌（y4zas/wW//Mm amazonense ) (MagalhSes，1984)、巴西固氮螺旋菌(Tarrand 等人 ’ 1979)、加拿大固氣螺旋菌（』zos/?z>77/wm camw/mse )(Mehnaz 等人，2007 )、多貝賴納固氮螺旋菌(^ζίλφίπΥ/Μ/η cfoeZiereiwmjfe ) (Eckert 等人，2001)、高鹽 200946477 固氮螺旋菌(Azospirilhim halopraeferens) ( Reinhold 等人，1987 )、伊拉克固氮螺旋菌z'ra灸mse) ( Khammas等人，1997 )、遲緩固氮螺旋菌 /arg/woMe) (Sly 和 Stackebandt，1999)、生脂固氮螺旋菌 (Azospirillum Hpoferum )(Skerman 等人，19SQ)、糖蜜草靣 I螺旋菌(Azospirillum we/i«is)(Peng 等人，2006)、稻草固氮螺旋菌（也〇职>77/«»|<^>^^) (Xie 和 Yokota，2005)。固氮螺旋菌屬可由溫帶、亞熱帶及熱帶環境中青草、穀類和食用作物的根及土壤分離而得。至今，固氮螺旋菌「屬」（genus)之菌株在台灣尚未有專利之前案，而在 ©國外則有專利共12筆，如表1所示，包括有巴西固氮螺旋菌（乂、生脂固氮螺旋菌〇4. //po/erww)、糖蜜草固氮螺旋菌（』. we/im··?)等不同種類的菌株，也有人工基因改造的菌株（engineering strains)，其功能包括有：果膠質分解活性(pectinolytic activity)、抗農藥（resistance to pesticide)、固氮（nitrogen fixation)、產生植物激素 (phytostimulation)、溶破物填（phosphate solubiliz- ation)、对酸等活性。其應用的範圍包含：生物肥料（biofertilizer)或細菌肥料（bacterial fertilizer)、生物防治（biological control )、飼料添加物（added to feedstuff ❹ of livestock)、處理家.畜糞便（treating excreta of livestock)、細菌廢肥 (bacterial manure)等，而施用的作物對象包括有：糖蜜草（molasses weed)、無芒雀麥(brome grass)、稻米（rice)、玉米（corn)、小麥(wheat) 等禾本科作物（gramineous crops)。本發明菌種至今在國内外台灣尚未有申請通過的專利資料，而在國際專利中共12筆有同屬（genus)但不同種(species)的菌株：但是本發明的菌株為生物學上新穎的細菌菌種。【發明内容】本發明之主要目的係提供一種新穎的皺紋固氮螺旋菌 rwgosww) IMMIB AFH-6，其係由台灣高雄之污染土壤中 200946477 經培養基之培養、分離及純化後選取之菌株，經菌種鑑定結果顯示與加拿大固氮螺旋菌細菌最相似，因此命名為皺紋固氮螺旋菌（七〇>57^>"/«»^«发〇>5«所），其係於96年12月11日寄存於臺灣新竹的食品工業發展研究所，寄存編號為BCRC 910373。本發明之次一目的，係在於提供一種新穎的皺紋固氮螺旋菌 (JzomzWZ/m/w rMgOSM/w) IMMIB AFH-6之製劑，其主要係具有生物固氮 (nitrogen fixation)、溶解礦物鱗(mineral phosphate solubilization)、靖酸水解酶（phosphatase)、确酸還原酶(nitrate reductase)、動物膠蛋白酶〇 (gelatinase)的活性、可產生植物生長激素-⑼哚-3-醋酸（indole-3-acetic acid ; IAA )、胞外多醣(extracellular polysaccharide)等8種功能之菌種，可供開發具有促進植物生長、改善土壤物理性質、有機質廢棄物處理之微生物製劑。【實施方式】本發明係有關於一種具有（1)生物固氮（nitr〇genflxation)、（2) 溶解礦物磷（mineral phosphate solubilization)、（ 3 )有磷酸水解酶 ❹（phosphatase)、（ 4 )硝酸還原酶(nitrate㈣⑽咖）、（5 )動物膠蛋白酶 (gelatinase )、（6)尿素酶(urease)的活性、可產生植物生長激素_ 吲°木_3_醋酸（indole-3- acetic acid ; IAA)、（8)可產生胞外多醣(extracellular polysaccharide)等至少8種功能之新穎皺紋固氮螺旋菌謂 ⑽仍謂）ΙΜΜΙΒ AFH_6，其係於中華民國卯年^月^曰寄存於臺灣新竹的食品卫業發展研究所’寄存編號為BCRC9l〇373。本發明菌株IMMIBAFH-6的基本特性如下： 1·培養及形態學的特徵本發明g株革蘭氏染色為陰性@ G(_)，無孢子，兼性嫌氣菌，在 200946477 生長對數期時之細胞呈桿狀至微曲桿狀，具有鞭毛，故屬移動性。於腦心浸出物瓊脂（Brain heart infusion agar ; ΒΗΙ )、胰蛋白酶分解大豆瓊脂（Tryptic soy agar ; TSA)或營養璦脂（Nutrient agar ; ΝΑ)培養基生長時菌落形狀為完整邊緣、表面平滑，隨時間變為不規則的縐折外形、亮橘色。 2·培養性狀的特徵本發明菌株之生長溫度在於20-37°C間，化學異營性，生長pH值為 6-10 ’ 在含 2%NaCl 的 NB (HIMEDIA)可生長。 © 3·菌株之生理特性如下：本發明菌株具有下列酵素活性：觸酶（catalase)、鹼性磷酸酯酶 (alkaline phosphatase)、酸性鱗酸醋酶(acid phosphatase)、萘紛-AS-BI-填水解酶（naphthol-AS- ΒΙ-phosphhydrolase)、丁酸 S旨酶（butyrate esterase ’有四個碳）、辛酸醋酶（carprylate esterase，有八個碳）、白胺酸芳香基酿胺酶（leucine arylamidase)、棟胺酸芳香基醯胺酶（valine arylamidase)、脱胺酸芳香基酿胺酶(cystine arylamidase)、冷-葡萄糖發酶（召-glucosidase)、冷-半乳糖普酶（β-galactosidase)、動物膠冰解酶 φ (gelatinase)、尿素酶(urease)。 4·碳源的利用性本發明菌株可利用之碳源，如下列碳源：N-乙醯半乳糖胺 (N-acetyl-D-galactosamine) 、N-乙醯葡萄糖胺（N-acetyl-D-glucosamine)、岩蕪 SI (fucose)、葡萄糖（glucose)、阿拉伯糖 (D-arabinose)、麥芽糖（maltose)、甘露糖（D-mannose)、蔑糖（sucrose)、海藻糖（trehalose)、檸檬酸(citric acid)、葡萄糖酸(gluconic acid)、半乳糖搭酸(D-galacturonic acid)、β_经丁酸（β-hydroxylbutyric acid)、葡萄糖搭酸(D-glucuronic acid)、葡萄糖二酸(D-saccharic acid)、對起笨 200946477 醋酸（p-hydroxy-phenyl-acetic acid)、奎寧酸（quinic acid)、丙胺基甘胺酸（L-alanylglycine)、天門冬醯胺(L-asparagine)、甘胺醯基谷胺酸 (glycyl-L-glutamic acid)、甘胺醯基天門冬胺酸（giyCyi_L-aspartic acid)、甘油（glycerol)和 m-肌醇（m-inositol)。本發明之皺紋固氮螺旋菌rwgosww) IMMIB AFH-6包括有下列特徵：一、本發明菌株為自污染土壤環境分離之天然野生菌種，未經任何人工基因改造的處理。二、本發明菌種為一新穎菌株，具備生物固氮、礦物磷溶解、蛋白質分解等數種功能活性之菌種。三、 Ο本發明菌株所屬之菌屬（genus)依照美國國家衛生研究院（National Institutes of Health ； NIH)的Guidelines生物危害性分級中，不屬於已知之人體病原菌。基於上述特徵，本菌可開發為微生物製劑。本發明之菌株可以做以下的應用：（1)微生物肥料，可施用於根圈， (2) 土壤改良劑，施用於土壤，（3)堆肥或有機質品質改良，施用於未腐熟或已腐熟的堆肥或有機質。本發明之菌株具有多形性(polymorphism)的性質，在低氧、高碳氮〇比(high C/N ratio)時，菌體為營養細胞(vegetative cells)，可合成聚-β-經丁酸(poly-p-hydroxybutyrate ; ΡΗΒ)、具有極性鞭毛(polar flagellum)，有趨化性運動能力(chemotactic mobility)。在低破氮比（C/N ratio)、高氧的生長環境中將分化(differentiate)為囊胞狀的細胞（cyst-like cell)，可以產生胞外多 St (extracellular polysaccharide ; EPS)、發生細胞凝聚作用（cell aggregation);此對於本發明菌株之有關促進土壤團粒的形成（formation of soil aggregation)、根拓殖化作用（root colonization)、固氮作用 (nitrogen fixation)所需要的微氧(microaerobic)條件、菌體抵抗環境逆境(stress resistance)的能力有莫大幫助。 200946477 根據本發明，可使用皺紋固氮螺旋菌IMMIB AFH_6之組成物處理。本發明之具體實施例為將皺紋固氮螺旋菌IMMIB AFH 6先行培養於液體培養基中，利用所製備之菌劑將其接種至作物根圈土壤中，直接做為作物之微生物肥料之功效。另外，本發明之較佳實施例為將所製備之菌劑與有機質混合後施用於植物根圈土壤中，此方法可提高生物肥料及有機質對於植物生長的促進效果。茲以下列實例予以詳細說明本發明，唯並不意味本發明僅侷限於此等實例所揭示之内容。 Ο 【固氮螺旋菌株之分離篩選】自台灣尚雄市之污染土壤以營養填脂平板培養基(nutrient agar ; NA)(HIMEDIA)分離及純化培養之菌株。【以多項分類方法分析本發明之IMMIB AFH-6菌株】 1.基因型的分類分析以16S rRNA基因定序鑑定本發明之菌株，其係利用M〇 BIO公司 O ( USA )所提供的微生物染色體DNA分離套組（UltraCleanTM Microbial Genomic DNA Isolation Kit)進行 DNA 萃取，以大腸桿菌(Escherichia coli)之次單元(small_subunit，SSU) Rma基因之高度保留序列設計細菌專一性引子(bacterial universal primer)，正向引子 IF (5，-GAG TTT GAT CAT GGC TCA G-3，)及反向引子 9R (5’-AAG G AG GTG ATC CAA CCG CA-3’）（如表 2 所示)(Brosius et al.， 1978 ; Edwards et al.，1989)進行聚合酶鏈反應(PCR)，PCR反應液中分別添加細菌染色體DNA約20-50 ng做為模版、兩股引子各20 pmo卜1.5 U Taq DNA聚合酶、200 // M dNTPs、緩衝溶液使總體積為25 /z L，反應條件為95 °C 5分鐘， 95 °C 1分鐘、50 °C 1分30秒、72 °C 2分鐘、共35循環，72°C 7分鐘。以 200946477 聚合酶鏈反應進行16S_A之增殖。再利用QIAquiekGdEx福-套組回收 DNA後’進打欲定序基因片段之熱循環反應，選用之定序引子為高度保留之⑽ rDNA專-性引子(表2) ’將經細菌專一性引子放大之片段以定序用引子進行染200946477 IX. Description of the Invention: [Technical Field] The present invention relates to a microorganism strain which has been isolated and purified, in particular, having (1) nitrogen fixation, and (2) dissolved mineral scale (minerai solubilization) ), (3) phosphatase, (4) nitrate reductase, (5) animal gelatinase (gdatinase), (ό) urease (urease) activity, (7) can produce plants Growth hormone indole-3-acetic acid (IAA), (8) can produce at least 8 kinds of novel extracellular polysaccharides (extracellular polysaccharide), etc. 皱/go(10)w) IMMIB AFH- 6. [Prior Art] According to the conventional use of chemical fertilizers, it is possible to quickly provide the nutrients needed at the beginning of the crop, but it will lead to soil acidification, uneven fertility and groundwater source pollution. It is known that if soil microbes have the function of nitrogen fixation, inefficient nutrient dissolution and decomposition of organic matter in soil, it can be used as microbial fertilizer, and microbial fertilizer can replace part of chemical fertilizer to solve the problem of soil degradation caused by long-term application of chemical fertilizer. problem. Plant growth promoting rhizobacteria (PGPR) has four beneficial mechanisms according to its beneficial mechanism: (1) biofertilization, (2) biological control (bi〇_contr〇l), (3) ) bioremediation, (4) phytohormone action (phytostimulati〇n). It is known that the composition of plant root exudates usually includes carb〇hydrate, amino acids, vitamins, organic acids and various other compounds. Root exudates are beneficial to the use of microorganisms and are considered to be the main reason for the increase in the number and activity of microorganisms in the root ring. Therefore, the ability of bacteria to utilize various carbon sources is also considered to be one of the factors for the successful colonization of bacteria. · Azospirillum is an inoculant for promoting the potential of grasses and cereals. 200946477 Azospirillum can be used to secrete plant hormones, fix nitrogen, enhance plant uptake of minerals, and reduce nitrate activity. Promote plant growth (James, 2000), however, the premise of these effects is the colonization of plant roots by their cells. The pleomorphism of the cells allows them to adapt well under changing environmental conditions. Nitrogen-fixing bacteria are spiral-like vegetative cells in hypoxia. Preference is given to the use of fructose as a carbon source; in the limiting oxygen and high carbon-nitrogen ratios, the active growth of the cells produces poly-β-hydroxybutyrate (p〇1y_P_hydrQxybutyrate). ; PHB) 'Cells differentiate into globular cystic cells (CySt-Uke ceii), extracellular oligosaccharides cause cell aggregation 'Preference PHB as a carbon source for cell utilization (Tarrand Et al., 1978; Sadasivan and Neyra, 1985; Sadasivan and Neyra, 1987). © It can be inferred that 'the use of nitrogen-fixing bacteria in agriculture has many advantages: (1) no pathogens have been found to belong to this genus; (2) no rhizobium (rhiz〇bia) needs to be associated with nodule (nodule) legume host interaction has nitrogen fixation function, the selectivity of nitrogen-fixing bacteria to plant hosts is not limited to legumes; (3) can produce cystic cells (cyst_iike cell) 'this structure is conducive to root colonization Root colonization and nitrogen fixation (Katupitiya et al., 1995; PeregGerk et al., 2000). The reason is that this creator has the impression that the importance of developing novel strains is to study and cooperate with the application of science. Finally, a rational and cytoplasmic cell-like bacterium can be found that can produce multiple activities. The taxonomic status of Genus Jzo (Genus Jzo) is: phylum is called proteobacteria, and class is called alphaproteobacteria. The order is called red spiral bacteria. Rhodospirillales), family name Rhodospirillaceae, which has been published to include 10 species: Aatricium aeruginosa (y4zas/wW//Mm amazonense) (MagalhSes, 1984), Azospirillum brasiliensis (Tarrand et al. '1979), S. aureus ("zos/?z" 77/wm camw/mse) (Mehnaz et al., 2007), Doberina Helicobacter aeruginosa (^ζίλφίπΥ/ Μ/η cfoeZiereiwmjfe ) (Eckert et al., 2001), high salt 200946477 Azospirilhim halopraeferens (Reinhold et al., 1987), Azospirillum z'ra moxibustion mse) (Khammas et al., 1997), slow Azospirillum/arg/woMe) (Sly and Stackebandt, 1999), Azospirillum Hpoferum (Skerman et al., 19SQ), Azospirillum we/i«is (Peng et al) People, 2006), Nitrogenase grass pylori (also square level > 77 / «» | < ^ > ^^) (Xie and Yokota, 2005). Azospirillum can be isolated from the roots and soil of grasses, cereals and food crops in temperate, subtropical and tropical environments. So far, the strain of the genus strain of genus has not been patented in Taiwan, and there are 12 patents in foreign countries, as shown in Table 1, including the Brazilian nitrogen-fixing bacteria (乂, fat) Different strains such as nitrogen-fixing sputum 〇 4. //po/erww), Helminthosporium aeruginosa (". we/im··?), and artificial strains (engineering strains), whose functions include: Pectinolytic activity, resistance to pesticide, nitrogen fixation, phytostimulation, phosphate solubiliation, acid and other activities. The scope of application includes: biofertilizer or bacterial fertilizer, biological control, added to feedstuff ❹ of livestock, treating excreta of livestock, Bacterial manure, etc., and the crop subjects applied include: molasses weed, brome grass, rice, corn, wheat, etc. Gracineous crops. The strain of the present invention has not yet been patented by Taiwan at home and abroad, and 12 strains of the same species (genus) but different species in the international patent: but the strain of the present invention is biologically novel. Bacterial species. SUMMARY OF THE INVENTION The main object of the present invention is to provide a novel S. aureus strain Rwgosww) IMMIB AFH-6, which is a strain selected from the cultured, isolated and purified medium of 200946477 contaminated soil of Kaohsiung, Taiwan. The identification results showed that it was most similar to the S. aureus bacterium, so it was named as S. cerevisiae (seven 〇 > 57^>"/«»^«发发>5«), which was in 1996. Stored at the Food Industry Development Research Institute in Hsinchu, Taiwan on December 11 with the registration number BCRC 910373. A second object of the present invention is to provide a novel preparation of N. japonicus (JzomzWZ/m/w rMgOSM/w) IMMIB AFH-6, which mainly has nitrogen fixation, dissolved mineral scales (mineral) Phosphate solubilization), phosphatase, nitrate reductase, gelatinase activity, plant growth hormone-(9) indole-3-acetic acid Eight kinds of functional strains such as IAA) and extracellular polysaccharide can be used to develop microbial preparations that promote plant growth, improve soil physical properties, and treat organic waste. [Embodiment] The present invention relates to (1) biological nitrogen fixation (nitr〇genflxation), (2) mineral phosphate solubilization, (3) phosphoric acid hydrolyzing enzyme phosphatase, (4) nitric acid Reductase (nitrate (4) (10) coffee), (5) animal gelatinase (gelatinase), (6) urease (urease) activity, can produce plant growth hormone _ 吲 ° wood _3_ acetic acid (indole-3- acetic acid; IAA (8) A novel wrinkle-fixing nitrogen-fixing spore bacterium that can produce at least 8 functions such as extracellular polysaccharide (10) is still called ΙΜΜΙΒ AFH_6, which is stored in the Hsinchu, Taiwan, in the Republic of China. The Institute for Health Development's registration number is BCRC9l〇373. The basic characteristics of the strain IMMBAFH-6 of the present invention are as follows: 1. Characteristics of culture and morphology The Gram stain of the g strain of the present invention is negative @G(_), spore-free, facultative anaerobic bacteria, when the logarithmic phase is grown in 200946477 The cells are rod-shaped to slightly curved, with flagella, so they are mobile. When the brain heart infusion agar (ΒΗΙ), tryptic soy agar (TSA) or nutrient rouge (Nutrient agar; ΝΑ) medium grows, the colony shape is a complete edge, the surface is smooth, with The time becomes an irregular shape and a bright orange color. 2. Characteristics of culture traits The growth temperature of the strain of the present invention is between 20 and 37 ° C, chemically heterogeneous, and the growth pH is 6-10 ′. It can be grown in NB (HIMEDIA) containing 2% NaCl. The physiological characteristics of the strains are as follows: The strain of the present invention has the following enzyme activities: catalase, alkaline phosphatase, acid phosphatase, naphthalene-AS-BI- Hydrolyzing enzyme (naphthol-AS- ΒΙ-phosphhydrolase), butyrate esterase (butyrate esterase 'four carbons), caprylate esterase (eight carbons), leucine aryl amylase ( Leucine arylamidase), valine arylamidase, cysteine arylamidase, cold-glucosease, cold-galactosidase (β- Galactosidase), animal gelatinase φ (gelatinase), urease (urease). 4. Utilization of carbon source The carbon source of the strain of the present invention can be utilized, such as the following carbon sources: N-acetyl-D-galactosamine, N-acetylglucosamine (N-acetyl-D) -glucosamine), fucose, glucose, D-arabinose, maltose, D-mannose, sucrose, trehalose, lemon Citric acid, gluconic acid, D-galacturonic acid, β-hydroxylbutyric acid, D-glucuronic acid, glucose diacid ( D-saccharic acid), p-hydroxy-phenyl-acetic acid, quinic acid, L-alanylglycine, L-asparagine , glycyl-L-glutamic acid, gyiCyi_L-aspartic acid, glycerol and m-inositol. The present invention has the following characteristics: 1. The strain of the present invention is a natural wild strain isolated from a contaminated soil environment, and is not treated by any artificial genetic modification. 2. The strain of the present invention is a novel strain, and has several kinds of functional activities such as biological nitrogen fixation, mineral phosphorus dissolution, and protein decomposition. 3. The genus of the strain of the present invention belongs to the known human pathogens according to the Nationals Institute of Health (NIH) Guideline biohazard classification. Based on the above characteristics, the bacteria can be developed as a microbial preparation. The strain of the present invention can be used for the following applications: (1) microbial fertilizer, can be applied to the root ring, (2) soil conditioner, applied to the soil, (3) compost or organic quality improvement, applied to unfertilized or decomposed Compost or organic matter. The strain of the present invention has polymorphism properties, and in the case of low oxygen, high carbon/azepine ratio (high C/N ratio), the cells are vegetative cells, and poly-β-jing can be synthesized. Acid (poly-p-hydroxybutyrate; ΡΗΒ), with polar flagellum, chemotactic mobility. In the low-nitrogen ratio (C/N ratio), hyperoxic growth environment, differentiated into cyst-like cells, can produce extracellular polysaccharide (EPS), occur Cell aggregation; this is microaerobic required for the strains of the present invention to promote formation of soil aggregation, root colonization, and nitrogen fixation. Conditions, the ability of the bacteria to resist environmental stress, can be of great help. According to the present invention, the composition of S. cerevisiae IMMIB AFH_6 can be treated. A specific embodiment of the present invention is to first culture the A. oxysporum IMMIB AFH 6 in a liquid medium, and inoculate it into the root zone soil of the crop by using the prepared microbial agent, thereby directly serving as a microbial fertilizer for the crop. Further, in a preferred embodiment of the present invention, the prepared microbial agent is mixed with an organic substance and applied to the root zone soil of the plant, and the method can improve the promoting effect of the biological fertilizer and the organic matter on the growth of the plant. The invention is illustrated by the following examples, which are not intended to be construed as limiting the invention. Ο [Separation and Screening of Nitrogen-Solid Spiral Strains] The cultured strains were isolated and purified from the contaminated soil of Shangxiong City, Taiwan, using nutrient agar (NA) (HIMEDIA). [Analysis of the IMMIB AFH-6 strain of the present invention by a plurality of classification methods] 1. Classification analysis of genotypes The strain of the present invention was identified by 16S rRNA gene sequencing using a microorganism chromosome provided by M〇BIO O (USA) The DNA extraction kit (UltraCleanTM Microbial Genomic DNA Isolation Kit) was used for DNA extraction, and the bacterial universal primer was designed with the high retention sequence of the Rma gene of Escherichia coli (Small_subunit, SSU). Primer IF (5,-GAG TTT GAT CAT GGC TCA G-3,) and reverse primer 9R (5'-AAG G AG GTG ATC CAA CCG CA-3') (as shown in Table 2) (Brosius et al. , 1978 ; Edwards et al., 1989) Polymerase chain reaction (PCR), adding about 20-50 ng of bacterial chromosomal DNA to the PCR reaction solution as a template, two primers each 20 pmo 1.5 U Taq DNA polymerase , 200 // M dNTPs, buffer solution to make the total volume 25 / z L, the reaction conditions are 95 °C for 5 minutes, 95 °C for 1 minute, 50 °C for 1 minute and 30 seconds, 72 °C for 2 minutes, a total of 35 cycles , 72 ° C for 7 minutes. Proliferation of 16S_A was carried out by polymerase chain reaction in 200946477. After using QIAquiekGdEx Fu-set to recover DNA, the thermocycling reaction of the gene fragment is selected, and the sequence primer is selected as the highly retained (10) rDNA-specific primer (Table 2) 'The bacteria-specific primer will be amplified. The fragment is dyed in sequence with primers

劑終止循環疋序(dye terminator cycle sequencing)反應。最後以 ABI® 310 PRISM 定序儀進·光之_及相觸，制本個細自IMMIB…脱之16S rDNA 1，448鹼基對(表3)。將GeneBank取得的固氮螺旋屬的菌種（species of g_ ▲哪W//謂)加入 ARBdatabase (Ludwig et al. ’ 2004)，並利用的工具進行排比 © (align)。利用鄰近連接(neighbour-joining) (Saitou和Nei，1987)方法推論演化樹 (evolutionary trees) ’利用Jukes和cant〇r (1969)的校正計算演化距離矩陣 (Evolutionaiy distance matrix)。最後演化樹的拓樸學以基於1〇〇〇次隨機分析 (re-sampling)的鄰近連接法(neighbour-joining method)的自舉分析(bootstrap analyses)評估（Felsenstein, 1985)。經演化分析指出本發明菌株屬於固氮螺旋菌，其序列相似度與加拿大固氮螺旋菌及多伯固氮螺旋菌cfoMemherae)最高(97.0°/。），同屬的其他菌種的相似度明顯較低。圖2為此本發明的菌株IMMIB AFH-6以最大似然法〇 (maximum-likeliho〇d method)構築的樹狀圖，分支以顯示數字代表1〇⑻次隨機分析後仍歸於同一群叢之百分比例，顯示本發明菌株IMMIB AFH-6與加拿大固氮螺旋菌最有相關性。本發明菌株之16S rDNA序列已送存至美國國家生技資訊中心基因資料庫(NCBI Genbank)得到登錄號碼(accession number)為AM419042。將本發明菌株IMMIB AFH-6與A LMG 23617T、A DSM 1690τ 及乂 cfo功em’werae DSM 131317的染色體分別做DNA-DNA的雜合作用，顯示本發明菌株IMMLB AFH-6與此二菌株的再結合數值(reassociation values)分別為 17.0%、25.0%及19.0%，均低於60%，顯示本發明菌株為一新穎菌種。 12 200946477 2.細胞脂肪酸組成分析測試接種皺紋固氮螺旋菌IMMIB AFH-6與固氮螺旋菌屬之其他菌種的細胞壁脂肪酸之型態(profile)，將菌株於含Bffl液體培養基的錐形瓶，於37°C 振盪培養一週後測定其化學分類性質，將菌體以1〇/0 (v/v)甲醛殺死 (formaldehyde) ’離心收集、蒸餾水清洗、冷凍乾燥菌體。依照如Minnikin等人 (1980)所述，使用酸性甲醇分解萃取脂肪，如Yassin (1988)的方法純化脂肪酸，並利用Shimadzu GC-MS (Typ QP2010)分離、鑑定及定量。細胞脂肪酸分析顯示存在有 n-C14: 0 (1.48%)、n-C15 : 0 (1.96%)、n-C16: 1 cw7c (18.02%)、n-C16 : 〇 0 (12.40%)、2 個異構物的 9，10-methyl- -ene-hexadecanoate (0.55%和 1.28%)、 n- C17 : 0 (0.43%)、n-C18 : 2ω6，9c (2.0%)、n-C18 : l«)7c (39.61%)、n-C18 : 〇( 1.77% )及n-C19: lco9c (0.54%)為主要的非羥基的脂肪酸甲酯(non-hydroxylated fatty acid methyl esters)。主要的羥基的脂肪酸(hydroxylated fatty acids )為 n-C14 : 0 3ΟΗ(5·6%)和n-C16 30H(3.2%)。顯示本發明菌株的脂肪酸組成具有固氮螺旋菌典型的脂肪酸。將本發明菌株IMMIB AFH-6的類異戊二烯（isoprenoid)以質譜分析(mass spectral analysis)顯示：在具有 1〇個isoprene單位的普醌(ubiqUinone ; q_1〇)。 ❹ 【菌體生長形態及化學性質分析】依Cowan ( 1974)的修改方法將進行革蘭氏（Gram)染色，本發明菌株革蘭氏染色為陰性菌G(-)。將本發明菌的菌體於移動性液體培養基（mobility)内在30 °C下生長3天，以顯微鏡檢測其移動性 (mobility)。本發明細菌無孢子，兼性嫌氣菌，在生長對數期時之細胞呈桿狀至微曲桿狀，具有鞭毛’故屬移動性。於腦心浸出物瓊脂(Brain heart infusion agar ; Bffl )、胰蛋白酶分解大豆瓊脂（Tryptic soy agar ； TSA)或營養瓊脂(Nutrient agar ; ΝΑ)培養基生長時菌落形狀為完整邊 13 200946477 緣、表面平滑，隨時間變為不規則的縐折外形、亮橘色。本發明菌株之生長溫度在於20-37°C間，化學異營性，生長PH值為6_10，在含 2%NaCl 的 NB (HIMEDIA)可生長。【以BIOLOG分析皺紋固氮螺旋菌imMIB AFH-6之碳源利用能力】將皺紋固氮螺旋菌IMMIB AFH-6菌株畫線接種至無氮培養基…比Agar)培 ' 養孤，於30°c培養丨6-24小時，將菌落懸浮於GN/GP-IF接種劑’調整菌液濃 ❹度至波長590 nm下之透光度65%，取150μ1調整懸浮液注至GN2 BI〇L〇G 96 孔槽(wells)之微量平盤，置於3〇°C培養72小時，以BIOL〇G判讀機測定其波長595 nm的吸光值，經由判讀機的判讀，會將各培養井内的不同深淺的呈色反應的OD值（人1 = 590，以=750)減去八-1培養井（空白試驗，不含碳源）的數值，可分為強反應、弱反應及無反應三種。由BIOLOG分析結果如表5所示，得知本發明分離之皺紋固氮螺旋菌IMMIBAFH_6菌種可利用的碳源共計20種’其中（1)醣類(carbohydrate)有9種可被利用，包括：N-乙醯半乳糖胺(N-acetylgalactosamine)、N-乙醯葡萄糖胺(N-acetylglucosamine)、岩藻醣(focose)、葡萄糖(glucose)、阿拉伯糖（D-arabinose )、麥芽糖(maltose)、甘露〇糖（D-mannose)、蔗糖(sucrose)、海藻糖（trehalose) ; (2)羰酸(carboxylic acid) 有9種可被利用；其中包括檸檬酸(citric acid)、葡萄糖酸(gluconic acid)、半乳糖醛酸(D-galacturonic acid)、β·羥丁酸（β-hydroxylbutyric acid)、葡萄糖醛酸 (D-glucuronic acid) ' 葡萄糖二酸（D-saccharic acid)、對經苯醋酸 (p-hydroxy-phenyl-acetic acid)、奎寧酸(quinic acid) ; (3 )胺基酸有 3 種可被利用：丙胺基甘胺酸(L-alanylglycine)、天門冬醯胺(L-asparagine)、甘胺醯基谷胺酸 (Glycyl-L-glutamic acid)、甘胺醯基天門冬胺酸(Glycyl-L-aspartic acid) ; (4)醇類有 2 種：甘油(glycerol)、m-肌醇(m-inositol)。由BIOLOG分析結果可知皺紋固氮螺旋菌IMMIB AFH-6可利用如上所列 200946477 知9種醣類，3種胺基酸，9種有機酸，3種醇類。習知，植物根圈的分泌物含有多量聽類與叛酸，可預期將本發明菌株施用於植物根圈，其將可^根部拓殖作用的優勢，對於植物可發揮細菌本身具有的生理功效。 ° 【以ΑΡΙ20Ε、ΑΡΙ20ΝΕ、API-ΖΥΜ套組測定皺紋固氮螺旋菌ΙΜμιβ AFH-6之生理性質】 - 1.氧化酶及觸酶活性測試： ❹ 依據bioMerieux廠商提供的說明書測試其氧化酶活性，將菌落直接以氧化酶試劑（OX reagent)滴一滴，呈紫色為正反應（p〇sitive reaction)，表示本發明細菌菌種可分泌氧化酶（oxidase)，以3〇/〇過氧化氫(hydrogen peroxide)滴於菌落上可產生氣泡，表示本發明菌種可分泌觸酶（catalase)。 2.API-20E 分析：以API-20E(bioMerieux， Inc.公司）測定本發明菌株之酵素活性。以接種環挑一個於無氮璦脂（Nfb agar)培養基生長的菌落 (colony)’懸浮於5ml生理食鹽水’攪拌均勻。將菌液滴入測試條(strip) ®的管子内，蓋好蜂巢形培養盤，置於37°C培養室培養18-24小時，依 bioMerieux，Inc.公司之產品進行下列反應：（1)VP測試：加VPI， VP2試劑各一滴於VP孔槽中，需十分鐘後觀察變色反應。（2)TDA測試：加一滴TDA試劑於TDA孔槽中反應，立即觀察顏色變化。(3)IND 測試：加一滴IND試劑於IND孔槽中反應，立刻觀察顏色變化。依照bioMerieux，Inc·，Hazelwood，MO公司提供的說明書所述之方法進行判讀，結果如第3圖所示，得知本發明菌株IMMIB AFH-6具有/5-半乳糖苦酶（β-galactosidase)、動物膠分解酶(gelatinase)、尿素酶 (urease)的活性，可將反應產生3-經丁酮（acetoin)。 15 200946477 對甘露糖醇(manitol)、蔗糖(sucrose)、蜜二糖(melibi〇se)有發酵/氧化的反應，可利用檸檬酸（citrate)。無下列酵素活性：精氨酸雙水解酶 (arginine dihydrolase)、離氨酸脫羧酶（lySine decarb〇Xylase)、鳥氨酸脫羧酶（ornithine decarboxylase)、色氨酸脫醯氨酶（trypt〇phan de_ carbamylase)，不產生硫化氫及INDOLE，對於對葡萄糖（gluc〇se)、肌醇（inositol)、山梨醇（sorbitol)、鼠李糖（rhamnose)、苦杏仁普 (amygdalin)和阿拉伯糖(arabiose)等無氧化/發酵的能力。 3. API 20NE ：以API 20 NE測域本發明菌株之生化活性，如套組之操作說明 (bioM0rieux，France )。其步驟如下所述：將細菌劃線於適合生長的固體培養基上’於30 C培養。將純菌洛加入2 ml生理食鹽水，混合均勻，將菌體懸浮液吸光值Αβοο調整為0.5。將無菌水補滿於培養盤的凹槽，以保持培養盤的溼氣。將API 20 ΝΕ反應條(strip)置於培養盤中。將菌體懸浮液滴入Ν〇3至pnpg試驗孔的tube中。迅旧、ADii、URE試驗孔的cupule覆蓋無菌礦物油。取200μ1 之菌體懸浮液加入AUX medium中’培養基的組成分為：2 g/L硫酸銨、10.5 ml/L. VITAMIN solution、10 ml/L 微量元素(trace element)、6.24 g/L 磷酸鈉 ❹（monos〇dium phosphate)、1.5 g/L 氣化鉀(potassium chloride)、1.5 g/L 竣脂(agar)， pH 7.0-7.2，均勻混勻。將所調配的菌體懸浮液加入GLU至pAC 12個試驗孔的 tube及cupule中。將培養盤蓋子蓋上後，於30乞培養48小時。加入MT1和 NIT2試劑各一滴至N03試驗孔中’反應5分鐘後判讀◊加入一滴JAMES試劑於TRP試驗孔中，可立即判讀。由ApI2〇NE測試結果如第4圖所示，得知 IMMIB AFH-6具有下列活性：還原确’酸為亞确酸(Re(jucti〇n 〇f nitrate to nitrite)' 精胺酸雙水解酶(y\rginine dihydrolase) ' β-胃萄糖芽酶(β-glucosidase) ' β-半乳糖苷酶(β-galactosidase)、動物膠分解酶（gelatinase)，具有同化 (assimilation)葡萄糖(glucose)、阿拉伯糖(心此㈤祀）、甘露糖(mann〇se)、甘露醇 16 200946477 (mannitol)、N-乙醯葡萄糖胺(N-acetyl- glucosamine)、麥芽糖(Maltose)、葡萄糖酸 (Potassium-gluconate)、蘋果酸(Malate)、已二酸(Adipic acid)、檸檬酸(citrate)的能力。無色胺酸酶(tryptophanase)的活性、無發酵葡萄糖(giucose)的能力、無同化苯醋酸(Phenylacetic acid)的能力。 4. API ZYM : 以API-ΖΥΜ套組（bioMerieux，Inc_公司）測試本發明菌株皺紋固氮螺旋菌IMMIB AFH-6之專一酵素活性。首先以接種環取培養|上的菌落後，溶於5 ml無菌水中，經調整菌液濃度使透光度接近50%，〇取調整好之菌液65 //1加入每個反應孔槽内後，將樣品置於30。〇中反應24小時，再加入API-ΖΥΜ套組A液與B液各一滴，靜置5分鐘後觀察顏色變化。由API-ΖΥΜ分析得知IMMIB AFH-6具有下列活性：（1 )構酸水解酶（phosphatase):驗性碟酸酯酶（alkaline phosphatase)、酸性鱗酸醋酶（acid phosphatase)、萘紛-AS-ΒΙ-碟水解酶 (naphthol-AS- ΒΙ-phosphhydrolase) ; ( 2 )醋酶（esterase) : 丁酸酿酶 (butyrate esterase)、辛酸醋酶（carprylate esterase)、（3)蛋白酶：白胺酸芳香基醯胺酶（leucine arylamidase)、擁胺酸芳香基酿胺酶（valine Q arylamidase)、胱胺酸芳香基醯胺酶（cystine arylamidase)、（ 4)糖基水解酶(glycyl hydrolase) : /3-葡萄糖苦酶（/3-glueosidase)活性；肉莖寇酸醋酶(myristate esterase)、胰蛋白酶（trypsin)、胰糜蛋白酶（α -chymotrypsin)、α-半乳糖苦酶（a -galactosidase)、召-半乳糖甘酶（冷 -galactosidase)、召-葡糖苷酸酶（p_glucuronidase)、α _ 甘露糖苷酶 (α-mannosidase)、Ν-乙醯-β-葡糖醯胺酶（N-acetyl-β- glucosamidase)、 α-岩藻糖脊酶（α-fucosidase)則無反應活性。 17 200946477 【測試皺紋固氮螺旋菌IMMIB AFH-6的功能】為了瞭解本發明菌株皺紋固氮螺旋菌IMMIB AFH-6的可能用途，吾人選擇固氮、礦物磷溶解、吲哚-3-醋酸產生等活性對於促進植物生長、改善土壤理化性質等有關的功能予以測試。 1.生物固氮活性：將本發明菌種皺紋固氮螺旋菌IMMIB AFH-6先塗抹於固體Nfb 培養基：填酸二氫鉀（dipotassium hydrogen phosphate [K2HP〇4]) 0.5 g/1，硫酸鎮含七分子水(magnesium sulfate [MgS04.7H20])，氣化〇納（sodium chloride [NaCl]) 0.1 g/1，氣化药含二分子水（calcium chloride [CaCl2 . 2H20]) 0.02 g/1，鉬酸二鈉含二分子水（disodium molybdate [Na2Mo〇4 · 2H20]) 0.002 g/1» 蘋果酸(L-malic acid) 5.0 g/1，氫氧化鉀（potassium hydroxide [KOH]) 4.5 gA，硫酸鎮含一分子水 (Manganese sulfate (MnS〇4 . H20) 0.01 g/1 ’ 1.64% 乙二銨四醋酸 (ethylenediamine tetraacetic acid ； EDTA-Fe ) 4ml/l， 0.5%溴瑞香草藍（Bromothymol blue) 3 ml，生物素(biotin) 0.1 mg/1，遭脂（agar) 17.5 g/1 。將本發明菌株以穿刺法接種至裝有無氮Nfb半固體培養基〇 (agar I.75 g/Ι，其餘與固體Nfb成分同）之試管，接菌後3〇°c靜置培養3天。依洪（1995)所述之乙炔還原法測定其菌種固氮酵素活性之乙烯產生量’以無菌之處理之樣品為空白組，以氣相層析-火焰離子摘測儀(GC-FID)測試乙烯量，最後計算固氮活性，其表示方式為每小時產生乙烯ΗΓ6莫耳數(QH2 nmole / h)。經測定本發明菌種可達到739 9+ 24.8 C2H2 nmole / h之固氮酵素活性。 2.礦物磷的溶解：依照Nautiyal (1999)所述的方法，測定本發明菌株皺紋固氮螺旋菌 IMMIB AFH-6的溶磷能力：以無菌竹籤將菌體接種於Ca_P培養基，其配方 18 200946477 為：10 g/l 葡萄糖(glucose)、5 g 鱗酸三弼（tiicalciumphosphate [ Ca3(P〇4)2])、 5 g/1 含六分子水的氣化鎮(magnesium chloride [ MgCh · 6H2O ] )、0.2 g/1 氯化卸 (potassium chloride [ KC1 ] )、0.1 g/1 含七分子水的硫酸鎮（magnesium sulfate[MgS〇4.7H2O]) ’ 0·1 g/1 硫酸敍（ammonium sulfate [ NH4)2S〇4] )，0.002 g/1 含六分子水的硫酸猛(manganese sulfate [ M11SO4 ♦ 6H2O ] )、0.0〇2 g/1 含七分子水的硫酸亞鐵（ferrous sulfate [ FeS04 · 7H20 ]、15 g/1 瓊脂(agar))，於 30°C 下培養4天。本發明菌株在Ca-P瓊脂培養m上生長的菌落周圍可產生透明環 (clear zone)，此表示具有溶解磷酸三鈣的能力。 Ο 3.0弓丨嗓醋酸(Indole-3-acetic acid ; IAA)的產生：接種本發明菌株皺紋固氮螺旋菌1]^^[18八？11-6的菌體至於含8|111 無菌NB液體培養基的通氣試管中，於30°C 180 rpm振盈至隔夜，取 8 ml菌液以5，000 g離心10分鐘，倒去上清液（supernatant)，將菌體沉澱(cell pellet)懸浮於4 ml無菌生理食鹽水(0.85% NaCl)，取10yl 的菌體懸浮液（cells suspension)(相當於log CFU 12_0的起始生菌數）於分別裝有5 1111含0、50、100、200、500//§/1111色胺酸([-11>>^〇卩11汪11) 的NB及Nfb液體培養基的通氣玻璃試管（2直徑xl5cm長度）中，於〇怪溫箱中振盪培養30°c 180 rpm連續48小時，依照Patten和Glick (2002 )所述的方法並予部分修改測定菌液中的IAA含量。取出菌液 1.4 ml，於4°C，5，500 X g離心10 min，取上清液lml到新的試管並加入4mL Salkowski’s試劑（需另作Blank) ’劇烈振盪(vortex) ’於室溫下並避光靜置20 分鐘，測波長535 nm的吸光值(A535)，必須以未接菌的無菌培養基當作空白組 (blank)。以五種已知濃度的0-100 /zg的IAA當做標準液(standard)進行線性迴歸(linear regression)，所得到的決定係數(coefficient of determination ; 為0.9916，此數值大於0.99，此表示本試驗資料組(data set)中的變化性比例 (proportion of variability)被一個統計模式所解釋，標準曲線可被接受。以此標準 19 200946477 取線來計算菌液中ΙΑΑ的含量。結果如第3圖所示，發現：本發明菌株皺紋固氣螺旋菌IMMIB AFH-6菌體於添加色胺酸的無氮培養基(Nfb)可以產生 IAA，且隨著其添加量的提高而增加，其中以添加5〇〇//g/ml色胺酸的無氮培養基中培養的產生最多35.5±7.5eg，此時生菌數為LogCFU 10.0。但於液體營養培養基(包括添加與不添加色胺酸)培養的均無法偵測到IAA的產生’此表示添加色胺酸的無氮培養基（Nfb+Trp)是有利於本發明菌株產生IAA 的生長條件。 4.細菌產生胞外多醣能力的測試： © 參考如payne和Finkelstein (1977)並予以修改的方法，將菌體稀釋塗抹於營養瓊脂（nutrient agar;簡稱NA)添加0.01%剛果紅 (Congo red，diphenyldiazo-bis- a -naphthyl- amino- sulfonate ;簡稱 CR)培養基，於30°C靜置培養3天，觀察菌落與剛果紅結合的情形，有、、’〇〇剛果紅表不菌體具有凝集（flocculation ; cell aggregation)的能力’而凝集能力與細菌產生胞外多聽（extraceiiuiar p〇iySacharide)有關’對於細菌對根部的附著作用（ro〇t attachment)很重要，是根拓殖作用的重要過程（Gerk et al.，2000)。本發明菌株IMMIB AFH-6於 © ΝΑ+0.01% CR培養基上可長出粗糙的菌落並有結合剛果紅的能力（如第5圖所示）’因此得知具有產生胞外多聽，具有細胞凝聚的能力預期其施用於植物根圈後根拓殖化作用’並改良土壤物理性質。【測試敏紋固氮螺旋菌IMMIB AFH-6促進植物生長之活性】將本發明菌株皺紋固氮螺旋菌IMMIB AFH-6進行種子發芽生物分析(seed germination bioassay)、溫室盆栽試驗以瞭解菌株接種於根圈對於作物生長的影響： 20 200946477 1. 種子發芽生物分析：依照Leather和Einheling ( 1988)所述之方法進行種子發芽生物分析。將2 ml的各不同稀釋的處理液添加至裝入9 〇 cm直徑的塑膠培養皿(petrf也也)的 Advantev no.l濾紙（70 mm直徑)上。本試驗以結球萵苣(head leftuce ;學名 Ζαβ咖_V var.如細〇及小蘿蔔(radish;學名知^應_·)的種子分別進行生物分析。種子之表面殺菌處理方法為將種子浸潰於〇 5% Na〇cl水溶液3〇分鐘，再以無菌蒸餾水清洗3次，再以無菌水浸潰3小時。依時鐘方向、種子尖 0% 端向内方式’將12粒浸泡的種子置於已加溶液的每張濾紙上。每一處理各四重複。以無菌蒸镏水為控制組（control)。每一個處理各四重覆。將培養皿組放置於控制生長|目中，於24 C黑暗培養72小時。培養後測量胚根(ro〇t)與胚芽(sh〇〇t) 的長度(mm)。以控制組為100% ’計算相對根延長率(reiative r〇〇t ei〇ngati〇n ; RR)、相對地上部伸長率(relative shoot elongation ; RS)。結果如表6所示：與控制組比較，接種本發明菌株皺紋固氮螺旋菌IMMIB AFH-6的根部長度有 45/〇(萵苣)及52% (小蘿蔔)的提高’而地上部(sh〇〇t)的長度較控制組的有24%(結 Ο 1 球萵苣）及27%(萵苣)的增長。 2. 盆栽試驗的效果：The agent terminates the cycle terminator cycle sequencing reaction. Finally, using the ABI® 310 PRISM sequencer, the light and the touch, made this fine from IMMIB... 16S rDNA 1,448 base pairs (Table 3). The strains of the genus Nitrogen from GeneBank (species of g_ ▲W//) were added to the ARBdatabase (Ludwig et al. '2004) and the tools used were compared to © (align). The evolutionary trees are inferred using the neighboring-joining (Saitou and Nei, 1987) method using the correctional distance matrix of Jukes and Cant〇r (1969). The topology of the last evolutionary tree was evaluated by bootstrap analyses based on a re-sampling neighbor-joining method (Felsenstein, 1985). The evolutionary analysis indicated that the strain of the present invention belongs to the nitrogen-fixing strain, and its sequence similarity is the highest (97.0°/.) of the nitrogen-fixing bacteria and the cfoMemherae of the genus, and the similarity of other strains of the same genus is significantly lower. Figure 2 is a tree diagram of the strain IMMIB AFH-6 of the present invention constructed by the maximum-likeliho〇d method, and branches are shown to represent the number of 1〇(8) random analyses and still belong to the same cluster. In percentage, it is shown that the strain IMMIB AFH-6 of the present invention has the most correlation with S. aureus. The 16S rDNA sequence of the strain of the present invention has been deposited in the National Biotechnology Information Center (NCBI Genbank) to obtain an accession number of AM419042. The chromosomes of the present strain IMMMB AFH-6 and A LMG 23617T, A DSM 1690τ and 乂cfo work em'werae DSM 131317 were separately subjected to DNA-DNA hybridization, and the strain of the present invention, IMMLB AFH-6, and the two strains were shown. The reassociation values were 17.0%, 25.0%, and 19.0%, respectively, both lower than 60%, indicating that the strain of the present invention is a novel strain. 12 200946477 2. Analysis of cellular fatty acid composition The cell wall fatty acid profile of inoculated P. aeruginosa IMMIB AFH-6 and other strains of the genus Azospirillum was applied to a conical flask containing Bffl liquid medium. After 37 °C shaking culture for one week, the chemical classification properties were determined, and the cells were killed by 1 〇/0 (v/v) formaldehyde. The cells were collected by centrifugation, washed with distilled water, and lyophilized. The fat was extracted by acidic methanolysis as described by Minnikin et al. (1980), and the fatty acid was purified by the method of Yassin (1988) and separated, identified and quantified by Shimadzu GC-MS (Typ QP2010). Cellular fatty acid analysis showed the presence of n-C14: 0 (1.48%), n-C15: 0 (1.96%), n-C16: 1 cw7c (18.02%), n-C16: 〇0 (12.40%), 2 9,10-methyl--ene-hexadecanoate (0.55% and 1.28%), n-C17: 0 (0.43%), n-C18: 2ω6, 9c (2.0%), n-C18: l« 7c (39.61%), n-C18: 〇 (1.77%) and n-C19: lco9c (0.54%) are the main non-hydroxylated fatty acid methyl esters. The main hydroxylated fatty acids are n-C14: 0 3 (5.6%) and n-C16 30H (3.2%). The fatty acid composition of the strain of the present invention is shown to have a fatty acid typical of nitrogen-fixing spirochetes. The isoprenoid of the present strain IMMIB AFH-6 was shown by mass spectral analysis: in ubiqUinone; q_1〇 having one isoprene unit. ❹ [Analysis of cell growth morphology and chemical properties] Gram staining was performed according to the modified method of Cowan (1974), and the strain of the present invention was Gram stained as a negative bacteria G(-). The cells of the present invention were grown in a mobile liquid culture at 30 ° C for 3 days, and their mobility was examined by a microscope. The bacteria of the present invention have no spores and faint anaerobic bacteria, and the cells in the logarithmic phase of growth are rod-shaped to micro-curved rods, and have flagella' On the brain heart infusion agar (Bffl), tryptic soy agar (TSA) or nutrient agar (Nutrient agar; ΝΑ) medium, the colony shape is intact. 13 200946477 Edge, smooth surface It changes into an irregular shape and bright orange over time. The growth temperature of the strain of the present invention is between 20 and 37 ° C, chemically heterogeneous, and has a growth pH of 6 to 10, and can be grown in NB (HIMEDIA) containing 2% NaCl. [Analysis of the carbon source utilization ability of A. striata immiB AFH-6 by BIOLOG] Inoculate the strain of Ammonia spirulina IMMIB AFH-6 into a nitrogen-free medium...Agar), grow at 30 °C 6-24 hours, suspend the colony in the GN/GP-IF inoculant' adjust the concentration of the bacterial solution to a transmittance of 65% at a wavelength of 590 nm, and take a 150 μl adjusted suspension into the GN2 BI〇L〇G 96 well. The microplates of the wells were cultured at 3 °C for 72 hours, and the absorbance at 595 nm was measured by a BIOL〇G reader. The interpretation of the readers revealed the different depths in each well. The OD value of the color reaction (human 1 = 590, = 750) minus the value of the 8-1 culture well (blank test, no carbon source) can be divided into three types: strong reaction, weak reaction and no reaction. The results of the BIOLOG analysis are shown in Table 5. It is found that there are 20 kinds of carbon sources available for the isolation of the A. cerevisiae IMMBAFH_6 strain of the present invention. Among them, (1) 9 kinds of carbohydrates can be utilized, including: N-acetylgalactosamine, N-acetylglucosamine, focose, glucose, D-arabinose, maltose, D-mannose, sucrose, trehalose; (2) 9 kinds of carboxylic acid can be used; including citric acid, gluconic acid ), D-galacturonic acid, β-hydroxylbutyric acid, D-glucuronic acid 'D-saccharic acid, phenylacetic acid ( P-hydroxy-phenyl-acetic acid, quinic acid; (3) 3 kinds of amino acids can be used: L-alanylglycine, L-asparagine ), Glycyl-L-glutamic acid, glycine-aspartic acid (Glycyl-L-aspar) Tic acid); (4) Alcohol There are two kinds of alcohol: glycerol, m-inositol. From the results of BIOLOG analysis, it can be seen that N. sphaeroides IMMIB AFH-6 can utilize 9 kinds of saccharides, 3 kinds of amino acids, 9 kinds of organic acids and 3 kinds of alcohols as listed above in 200946477. It is known that the secretion of the plant root ring contains a large amount of auditory and tickic acid, and it is expected that the strain of the present invention can be applied to the plant root ring, which will have the advantage of colonization, and the physiological function of the bacteria itself can be exerted on the plant. ° [Determination of the physiological properties of S. cerevisiae ΙΜμιβ AFH-6 by ΑΡΙ20Ε, ΑΡΙ20ΝΕ, API-ΖΥΜ set] - 1. Oxidase and catalase activity tests: 测试 Test oxidase activity according to the instructions provided by bioMerieux manufacturer, The colony is directly dropped with a oxidase reagent (OX reagent) to form a purple purple positive reaction (p〇sitive reaction), indicating that the bacterial strain of the present invention can secrete an oxidase, which is 3 〇/〇 hydrogen peroxide (hydrogen peroxide). Dropping on the colony can generate bubbles, indicating that the species of the present invention can secrete catalase. 2. API-20E analysis: The enzyme activity of the strain of the present invention was measured by API-20E (bioMerieux, Inc.). A colony (colony) grown in a nitrogen-free rouge (Nfb agar) medium was suspended in 5 ml of physiological saline with the inoculating loop and stirred uniformly. Drop the bacteria into the strip of the test strip ®, cover the honeycomb culture tray, and incubate in a 37 ° C culture chamber for 18-24 hours. Perform the following reactions according to the products of bioMerieux, Inc.: (1) VP test: Add VPI, one drop of VP2 reagent in the VP well, and observe the color change reaction after ten minutes. (2) TDA test: A drop of TDA reagent was added to the TDA well to immediately observe the color change. (3) IND test: Add a drop of IND reagent to the IND cell and immediately observe the color change. The interpretation was carried out according to the method described in the specification provided by bioMerieux, Inc., Hazelwood, MO, and as shown in Fig. 3, it was found that the strain IMMMB AFH-6 of the present invention has a 5-galactosidase (β-galactosidase). The activity of animal gelatinase and urease can produce 3-acetone (acetoin). 15 200946477 Fermentation/oxidation reaction for manitol, sucrose, melibi〇se, citrate can be used. No enzyme activity: arginine dihydrolase, lySine decarb〇Xylase, ornithine decarboxylase, tryptophan deaminase (trypt〇phan de_ Carbamylase), does not produce hydrogen sulfide and INDOLE, for glucose (gluc〇se), inositol, sorbitol, rhamnose, amygdalin and arabose No ability to oxidize/ferment. 3. API 20NE: The biochemical activity of the strain of the invention is measured by API 20 NE, such as the operating instructions of the kit (bioM0rieux, France). The procedure is as follows: the bacteria are streaked onto a solid medium suitable for growth' culture at 30 C. The pure fungus was added to 2 ml of physiological saline, mixed evenly, and the absorbance of the bacterial suspension Αβοο was adjusted to 0.5. Sterile water is filled in the grooves of the culture tray to maintain moisture in the culture tray. The API 20 ΝΕ reaction strip was placed in a culture dish. The bacterial suspension was dropped into the tube of Ν〇3 to the pnpg test well. The cupule of the old, ADii, and URE test wells is covered with sterile mineral oil. 200 μl of the bacterial suspension was added to the AUX medium. The composition of the medium was divided into 2 g/L ammonium sulfate, 10.5 ml/L. VITAMIN solution, 10 ml/L trace element, and 6.24 g/L sodium phosphate. Monosium dium phosphate, 1.5 g/L potassium chloride, 1.5 g/L agar, pH 7.0-7.2, uniformly mixed. The prepared bacterial suspension was added to the tube and cupule of GLU to 12 test wells of pAC. After the lid of the culture plate was capped, it was cultured at 30 Torr for 48 hours. One drop of each of MT1 and NIT2 reagents was added to the N03 test wells. After 5 minutes of reaction, the test was performed and a drop of JAMES reagent was added to the TRP test well for immediate interpretation. As a result of the test of ApI2〇NE, as shown in Fig. 4, it was found that IMMIB AFH-6 has the following activities: Re (jucti〇n 〇f nitrate to nitrite) arginine dihydrolase (y\rginine dihydrolase) 'β-glucosidase ' β-galactosidase, gelatinase, assimilation glucose, Arabinose (heart (5) 祀), mannose (mann〇se), mannitol 16 200946477 (mannitol), N-acetyl-glucosamine, maltose, gluconic acid (Potassium-gluconate ), Malate, Adipic acid, citrate, tryptophanase activity, giucose-free, phenylacetic acid (Phenylacetic acid) 4. API ZYM: The specific enzyme activity of the strain S. cerevisiae IMMIB AFH-6 of the present invention was tested with an API-ΖΥΜ kit (bioMerieux, Inc.). Dissolved in 5 ml of sterile water and adjusted to the concentration of the liquid to make the light Nearly 50%, after taking the adjusted bacterial solution 65 //1 into each reaction well, put the sample in 30. The reaction in the sputum for 24 hours, then add one drop of API-ΖΥΜ set of liquid A and liquid B The color change was observed after standing for 5 minutes. It was found by API-ΖΥΜ analysis that IMMIB AFH-6 has the following activities: (1) phosphatase: alkaline phosphatase, acid lactic acid Acid phosphatase, naphthol-AS-ΒΙ-phosphhydrolase; (2) esterase: butyrate esterase, octyl acetate (carprylate) Esterase), (3) protease: leucine arylamidase, valine Q arylamidase, cystine arylamidase, (4) Glycyl hydrolase: /3-glucosesidase activity; myristate esterase, trypsin, trypsin (α-chymotrypsin), Alpha-galactosidase (a-galactosidase), gamma-galactosidase (cold-galact) Osidase), p-glucuronidase, α-mannosidase, N-acetyl-β-glucosamidase, α-fucoid The sugar-chidase (α-fucosidase) is not reactive. 17 200946477 [Testing the function of S. cerevisiae IMMIB AFH-6] In order to understand the possible use of the strain S. aureus IMMIB AFH-6, we have selected activities such as nitrogen fixation, mineral phosphorus dissolution, and indole-3-acetic acid production. Test related functions such as promoting plant growth and improving soil physical and chemical properties. 1. Biological nitrogen fixation activity: The strain S. aureus IMMIB AFH-6 of the present invention is first applied to a solid Nfb medium: dipotassium hydrogen phosphate [K2HP〇4] 0.5 g/1, and the sulfuric acid town contains seven Molecular water (magnesium sulfate [MgS04.7H20]), gas chloride (sodium chloride [NaCl]) 0.1 g / 1, gasification drug containing two molecules of water (calcium chloride [CaCl2. 2H20]) 0.02 g / 1, molybdenum Disodium molybdate [Na2Mo〇4 · 2H20] 0.002 g/1» L-malic acid 5.0 g/1, potassium hydroxide [KOH] 4.5 gA, sulfuric acid The town contains one molecule of water (Manganese sulfate (MnS〇4. H20) 0.01 g/1 ' 1.64% ethylenediamine tetraacetic acid (EDTA-Fe) 4ml / l, 0.5% bromothymol blue (Bromothymol blue) 3 ml, biotin 0.1 mg/1, lipid (agar) 17.5 g/1. The strain of the present invention was inoculated by puncture into a nitrogen-free Nfb semi-solid medium (agar I.75 g/Ι, the rest) The test tube was the same as the solid Nfb component, and it was statically cultured for 3 days at 3 °C after inoculation. The acetylene reduction method described in Yihong (1995) The amount of ethylene produced by the nitrogen-fixing enzyme activity of the strain was determined as a blank group, and the amount of ethylene was measured by gas chromatography-flame ion spectrometer (GC-FID), and finally the nitrogen fixation activity was calculated. The ethylene ΗΓ6 moles (QH2 nmole / h) is produced every hour. The strain of the present invention can be determined to have a nitrogen-fixing enzyme activity of 739 9+ 24.8 C2H2 nmole / h. 2. Dissolution of mineral phosphorus: According to Nautiyal (1999) The method described is used to determine the phosphorus solubilizing ability of the strain S. cerevisiae IMMIB AFH-6: the cells are inoculated into Ca_P medium with a sterile bamboo stick, and the formula 18 200946477 is: 10 g/l glucose (glucose), 5 g Triicalciumphosphate [Ca3(P〇4)2], 5 g/1 gas chloride town containing six molecules of water (magnesium chloride [McCh · 6H2O ] ), 0.2 g/1 chlorination (potassium chloride [ KC1 ]), 0.1 g/1 sulfuric acid town containing seven molecules of water (magnesium sulfate [MgS〇4.7H2O]) '0·1 g/1 sulphate (ammonium sulfate [NH4)2S〇4] ), 0.002 g/1 Sulfuric acid containing six molecules of water (manganese sulfate [ M11SO4 ♦ 6H2O ] ), 0.0〇2 g/1 with seven points Aqueous ferrous sulfate (ferrous sulfate [FeS04 · 7H20], 15 g / 1 agar (agar)), cultured for 4 days at 30 ° C. The strain of the present invention can produce a clear zone around the colonies grown on the Ca-P agar culture m, which means that it has the ability to dissolve tricalcium phosphate. Ο 3.0 Production of Indole-3-acetic acid (IAA): Inoculation of the strain S. aureus of the present invention 1]^^[18? The cells of 11-6 were incubated in a ventilated tube containing 8|111 sterile NB liquid medium at 30 ° C for 180 rpm until overnight. 8 ml of the bacterial solution was centrifuged at 5,000 g for 10 minutes, and the supernatant was decanted. (supernatant), suspend the cell pellet in 4 ml of sterile physiological saline (0.85% NaCl), and take 10yl of cell suspension (equivalent to the number of starting bacteria of log CFU 12_0) Ventilation glass tubes containing 5 1111 NB and Nfb liquid media containing 0, 50, 100, 200, 500//§/1111 tryptophan ([-11>>^〇卩11wang11) In the diameter of xl5 cm, the culture was shaken at 30 ° C for 180 hours in a temperature tank for 48 hours, and the IAA content in the bacterial liquid was determined according to the method described by Patten and Glick (2002). Remove 1.4 ml of the bacterial solution, centrifuge at 5,500 x g for 10 min at 4 ° C, take 1 ml of the supernatant into a new tube and add 4 mL of Salkowski's reagent (need to be Blank) 'violent oscillation (vortex) ' at room temperature After standing for 20 minutes in the dark and measuring the absorbance at 535 nm (A535), the unsterilized sterile medium must be used as a blank. Linear regression was performed using five known concentrations of 0-100 /zg IAA as the standard. The coefficient of determination (0.991, which is greater than 0.99, indicates the test). The proportion of variability in the data set is explained by a statistical model, and the standard curve can be accepted. The standard 19 200946477 is used to calculate the content of strontium in the bacterial liquid. The result is shown in Figure 3. As shown, it was found that the strain of the present invention S. cerevisiae IMMIB AFH-6 can produce IAA in a nitrogen-free medium (Nfb) supplemented with tryptophan, and increases with the addition amount thereof, wherein 5 is added. The culture in the nitrogen-free medium of 〇〇//g/ml tryptophan produced up to 35.5±7.5eg, and the number of bacteria at this time was LogCFU 10.0. However, it was cultured in liquid nutrient medium (including adding and not adding tryptophan). None of the production of IAA was detected. This indicates that the addition of tryptophan-free nitrogen-free medium (Nfb+Trp) is a growth condition that facilitates the production of IAA by the strain of the present invention. 4. Test for the ability of bacteria to produce extracellular polysaccharides: © Reference Pay Ne and Finkelstein (1977) and modified methods, the bacteria were diluted and applied to nutrient agar (NA) to add 0.01% Congo red (Digodiazo-bis-a-naphthyl-amino- sulfonate; referred to as CR The culture medium was statically cultured at 30 ° C for 3 days, and the colony was observed to bind to Congo red. There was , ' 'Congo red non-bacterial body has the ability to flocculate cell aggregation' and the agglutination ability and bacteria Extracellular auditory (extraceiiuiar p〇iySacharide) is important for the attachment of bacteria to the roots (ro〇t attachment) and is an important process for root colonization (Gerk et al., 2000). The strain of the invention IMMIB AFH -6 on © ΝΑ+0.01% CR medium can grow coarse colonies and have the ability to bind Congo red (as shown in Figure 5). Therefore, it is known that it has the ability to produce extracellular multi-audio and has cell aggregation. Application to post-root colonization of plant roots' and improve soil physical properties. [Testing activity of Ammonia-fixing Spirulina IMMIB AFH-6 to promote plant growth] Wrinkle-fixing nitrogen-fixing spiral of the present invention IMMIB AFH-6 for seed germination bioassay, greenhouse pot experiment to understand the effect of strain inoculation on root growth on crop growth: 20 200946477 1. Seed germination bioanalysis: according to Leather and Einheling (1988) Methods Seed germination biological analysis was performed. 2 ml of each of the differently diluted treatment solutions was added to an Advantev no.l filter paper (70 mm diameter) packed in a 9 〇 cm diameter plastic Petri dish (petrf also). This experiment uses the seeds of the head lettuce (the head name Ζαβ咖_V var. such as fine 〇 and radish (radish; scientific name know _ _)) for biological analysis. The surface sterilizing treatment method is to soak the seeds. Leave 5% Na〇cl in water for 3 minutes, then rinse with sterile distilled water for 3 times, then immerse in sterile water for 3 hours. Place 12 seeds soaked in the clock direction, seed tip 0% end inward way Each filter has been added to each filter paper. Each treatment is repeated four times. The sterile steamed water is used as the control group. Each treatment is treated with four replicates. The culture dish group is placed in the control growth | C was cultured for 72 hours in the dark. The length (mm) of the radicle (ro〇t) and the germ (sh〇〇t) was measured after the culture. The relative root elongation rate was calculated by the control group of 100% 'reiative r〇〇t ei〇 Ngati〇n; RR), relative shoot elongation (RS). The results are shown in Table 6. Compared with the control group, the root length of the strain S. aureus IMMIB AFH-6 inoculated with the strain of the present invention was 45/ 〇 (lettuce) and 52% (small radish) increase 'the length of the upper part (sh〇〇t) The control group had an increase of 24% (successful 1 ball lettuce) and 27% (lettuce) 2. The effect of the pot experiment:

本盆栽試驗之作物採用萵苣(lettuce ; ZaiMca iai/va L.)作物進行，試驗時間為2006年4月-5月。土壤為採集自崙背土壤，屬於黏壤土(day1〇am)，經風乾、過篩(4 mm mesh)後，將每kg 土壤裝入一塑膠盆(1〇 cm深度xl2cm頂部直徑x9 cm底部直徑）。使用的處理為：⑴對照組（blank)，未施用任何處理、 [2]半量化肥（1/2 CF )、[3]全量化肥(1CF)、[4]接種本發明菌株IMMIB AFH-6、[5]1/2CF+ IMMIB AFH-6、[6] 1CF+ IMMIB AFH-6、[7] PMF、 21 200946477 [8] PMF+1/2CF、[9]PMF+1CF、[10]PMF+ + IMMIB AFH-6、[11] 1/2CF+PMF+ IMMIB AFH-6、[12]1CF+PMF+ IMMIB AFH-6。依照作物施肥手冊推薦量做為全量化肥(1CF)，1.043 g/kg尿素(urea)，0.222 g/kg過磷酸妈(calcium perphosphate)，0.08 g/kg 氣化卸(potassium chloride)，以此當做全量化肥。其深度為30cm。盆栽試驗採逢機取樣設計（RCBD，random complete block design)。每處理4重覆。將種子先浸泡於蒸餾水後，播種 5顆種子至每一盆栽内土壤〇.5 cm以下。每日澆水。播種7天後將幼苗拔至剩〇下2棵。每週間測量株高。4〇天後拍照，採集地上部及根圈土壤樣品。灌溉水採用自動澆水滴管》種植後40後收穫採取地上部稱量鮮重，並經80 C烘乾後稱其乾重，以Duncan’s multiple range test統計是否有顯著差異（P=0.005)，結果如表7所示。在生質量方面，經40天的生長情形如第4圖所示。1CF、1CF+ IMMIB AFH_6處理之間無明顯差異(ρ<0·01)。本實施例的結果以接種皺紋固氮螺旋菌 @ IMMIB AFH-6並添加有機質肥及半量化肥的效果較佳。上述實施例僅為說明本發明之原理及其功效，而非限制本發明。因此’習於此技術之人士對上述實施例所做之修改及變化仍不違背本發明之精神。本發明之權利範圍應如後述之申請專利範圍所列。【圖式簡單說明】第1圖為本發明之皺紋固氮螺旋菌IMMIB AFH-6菌株的生長菌落圖。第2圖係以距離矩陣分析皺紋固氮螺旋菌imMIB AFH-6菌株與 22 200946477 其相似之菌株之16SrRNA基因序列，以CLUSTALX軟體進行多序列比對並繪製親源演化樹狀圖。第3圖為本發明之皺紋固氮螺旋菌IMMIB AFH-6菌株之ΑΠ20Η 分析套組的測試結果。第4圖為本發明之皺紋固氮螺旋菌IMMIB AFH-6菌株之 API20NE分析套組的測試結果。第5圖為本發明之皺紋固氮螺旋菌IMMIB AFH-6菌株於添加 0.01%剛果紅的營養瓊脂固體平板培養基上靜置培養3天後的菌落形 ©態及結合剛果紅的情形。第6圖為本發明之皺紋固氮螺旋菌IMMIB AFH-6菌株於〇 5〇〇 yg/mlNfb液體培養基中振盪培養42天後產生的吲哚各醋酸的量第7圖係溫室盆栽土試驗萵苣之根圈經含本發明之皺紋固氮螺旋菌IMMIB AFH_6接種後，經過4〇天生長的情形。、 ❹ 23 200946477 表1 :國内外已申請通過之有關固I螺旋菌「屬」之專利資料。___ 公告編號專利名稱 f $ 公告日國豕 W020070907 JP2007084476 Ο YU14004 CN1810953 CN1552846 〇 R0117333 FR2806420 KR20010096494The crops were tested on lettuce (Letuce; ZaiMca iai/va L.) crops from April to May 2006. The soil is collected from the back soil, belonging to the clay loam (day1〇am). After air drying and sieving (4 mm mesh), each kg of soil is filled into a plastic pot (1〇cm depth xl2cm top diameter x9 cm bottom diameter) ). The treatments used were: (1) control group (blank), no treatment, [2] semi-quantitative fertilizer (1/2 CF), [3] full-quantity fertilizer (1CF), [4] inoculation of the present strain IMMIB AFH-6, [5] 1/2CF+ IMMIB AFH-6, [6] 1CF+ IMMIB AFH-6, [7] PMF, 21 200946477 [8] PMF+1/2CF, [9]PMF+1CF, [10]PMF+ + IMMIB AFH -6, [11] 1/2CF+PMF+ IMMIB AFH-6, [12] 1CF+PMF+ IMMIB AFH-6. According to the recommended amount of crop fertilization manual, it is used as full-quantity fertilizer (1CF), 1.043 g/kg urea (urea), 0.222 g/kg calcium perphosphate, 0.08 g/kg of potassium chloride. Fully quantified fertilizer. Its depth is 30cm. The potted test sampling design (RCBD, random complete block design). 4 repetitions per treatment. After soaking the seeds in distilled water, seed 5 seeds to a depth of less than 5 cm in each pot. Watering daily. After 7 days of sowing, the seedlings were pulled to the remaining 2 trees. Plant height was measured weekly. 4 days later, take pictures and collect soil samples from the shoots and roots. Irrigation water is automatically sprinkled with water-dropping tube. After planting 40, the fresh weight is weighed in the ground, and the dry weight is weighed after drying at 80 C. Is there a significant difference in the statistics of Duncan's multiple range test (P=0.005)? As shown in Table 7. In terms of quality, the growth after 40 days is shown in Figure 4. There was no significant difference between 1CF, 1CF+ IMMIB AFH_6 treatment (ρ < 0·01). The results of this example are better at inoculating W. aeruginosa @ IMMIB AFH-6 with the addition of organic fertilizer and semi-quantitative fertilizer. The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Therefore, modifications and variations of the embodiments described above will be made without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing the growth colony of the S. cerevisiae IMMIB AFH-6 strain of the present invention. Fig. 2 is a 16SrRNA gene sequence of a strain similar to that of 22 200946477, and a multi-sequence alignment with CLUSTALX software and a parental evolution tree diagram. Fig. 3 is a test result of the Η20Η analysis kit of the S. aureus strain IMMIB AFH-6 of the present invention. Fig. 4 is a test result of the API20NE analysis kit of the S. aureus strain IMMIB AFH-6 of the present invention. Fig. 5 is a view showing the colony form of the A. faecalis strain IHMIB AFH-6 of the present invention after standing on a nutrient agar solid plate medium supplemented with 0.01% Congo red for 3 days and in combination with Congo red. Fig. 6 is a graph showing the amount of acetic acid produced after shaking culture of the A. sphaeroides IMMIB AFH-6 strain in the liquid medium of 〇〇5〇〇yg/ml Nfb for 42 days in the present invention. Fig. 7 is a greenhouse potted soil test lettuce The root ring was inoculated for 4 days after inoculation with the S. aureus strain IMMIB AFH_6 of the present invention. , ❹ 23 200946477 Table 1: Patent information on the "genus" of the genus I. ___ Announcement No. Patent Name f $ Announcement Date International 豕 W020070907 JP2007084476 Ο YU14004 CN1810953 CN1552846 〇 R0117333 FR2806420 KR20010096494

Novel bacterium and method for control of plant disease using the same. (利用新穎細菌及其方法於控制植物疾病。） Control method and symbiotic bacterium of Aphelenchoides besseyi. (葉芽線蟲的控制方法及共生細菌。） Mikro-organisms for the treatment of soil and process for obtaining them. (處理土壤的微生物及其獲得它們的程序。） Molasses weed nitrogen fixing spirillum and its application. (糖蜜草固氮螺旋菌及其應用。） Engineering strain of Brasil diazotrophic spirillum DraT* containing multiple copied nifA gene. (含有多複本w/A基因的變異菌株巴西固氮螺旋菌DraT*。） Mutant strain of Azospirillum lipoferum for producing mixed nitrogen-fixing and phytostimulating bacterial fertilizer. ^可產生混合固It及植物激素的生脂固氣螺旋菌的變異株。） Microbial mixture for fixing nitrogen and increasing solubility of phosphorus compounds to improve fertility of soil, comprises the co-fermentation of Azotobacter chroococcum, Azospirillum brasiliense and Bacillus megaterium. (因固氮及增進溶磷而促進土肥之包含共同發酵之褐球涿扇岸磨、巴西固氮螺旋菌及巨大芽孢桿菌的微生物混合物。） Microbial preparartion added to feedstuff of livestock or used for treating excreta of livestock. (加入家畜的飼料或處理家畜糞便的微生本本匈牙利中國中國羅馬尼亞斯洛伐克韓國 2007/9/7 2007/4/5 2006/8/17 2006/8/2 2004/12/8 2002/1/30 2001/9/21 2001/11/7 24 200946477 物製劑。）Novel bacterium and method for control of plant disease using the same. (Control method and symbiotic bacterium of Aphelenchoides besseyi.) Control method and symbiotic bacteria of M. elegans.) Mikro-organisms for The treatment of soil and process for obtaining them. Molasses weed nitrogen fixing spirillum and its application. Engineering strain of Brasil diazotrophic spirillum DraT Mutant strain of Azospirillum lipoferum for producing mixed nitrogen-fixing and phytostimulating bacterial fertilizer. ^Can produce mixed solid It and plants A mutant strain of the hormone-producing Helicobacter pylori. Microbial mixture for fixing nitrogen and increasing solubility of phosphorus compounds to improve fertility of soil, including the co-fermentation of Azotobacter chroococcum, Azospirillum brasiliense and Bacillus megaterium. (Promoting the soil and containing the co-fermented brown ball due to nitrogen fixation and phosphorus solubilization) Microbial preparartion added to feedstuff of livestock or used for treating excreta of livestock. (Additional feed to livestock or used to treat livestock manure. Hungarian China China Romania Slovakia South Korea 2007/9/7 2007/4/5 2006/8/17 2006/8/2 2004/12/8 2002/1/30 2001/9/21 2001/11/7 24 200946477 Preparations.)

KR960002865B HU69943 SU1796603 〇 ZA8307965, AU2263283 A fertilizer and the preparing process thereof(肥料及其製備程序。） Azospirillum NCIMB 40487 and NCIMB 40488 active in bio- stimulation of plant growth. (具刺激植物生長活性的固氮螺旋菌 NCIMB 40487 和 NCIMB 40488。） Strain of bacterium azospirillum lipoferum for production of fertilizer for perennial rye-grass and smooth brome-grass. (生產供多年生裸麥草及雀麥草之肥料的菌株-生脂固氮螺旋菌。） Novel strain of Azospirillum, methods of growing the strains, compositions containing them and use thereof as biofertilizer. (固氮螺旋菌新穎菌株、菌株的養殖方法、含菌株的組成並用做生物肥料。）韓國義大利英國南非、澳洲 1996/2/27 1995/09/28 1993/03/30 1984/7/25 1984/5/22 表2.以聚合酶鏈反應(PCR)增量16S rRNA所採用之引子 (primers)_ 引子代號核酸序列（5’至3’）核酸位置# 1F GAGTTTGATCATGGCTCAG 9-27 3F CCTACGGGAGGCAGCAG 341-357 4F GTGCCAGCAGCCGCGGTAA 515-533 4R TTACCGCGGCTGCTGGCAC 533-515 5F AAACTCAAATGAATTGACGGGG 907-928 6F GCAACGCGAAGAACCTTACC 966-985 7R TGACGGGCGGTGTGTACAA 1408-1390 9R AAGGAGGTGATCCAACCGCA 1541-1522 :核酸位置係指大腸桿菌之16S rRNA核酸序列之位置 25 200946477KR960002865B HU69943 SU1796603 〇ZA8307965, AU2263283 A fertilizer and the preparation process thereof. Azospirillum NCIMB 40487 and NCIMB 40488 active in bio- stimulation of plant growth. (Nylon-fixing bacteria NCIMB 40487 and stimulating plant growth activity) NCIMB 40488.) Strain of bacterium azospirillum lipoferum for production of fertilizer for perennial rye-grass and smooth brome-grass. (Producing strains for perennial ryegrass and bromegrass fertilizers - S. aureus) Novel strain of Azospirillum, Methods of growing the strains, compositions containing them and use thereof as biofertilizer. (N. atrophic strains, strain culture methods, and composition of strains used as biofertilizers.) Korea Italy, South Africa, Australia 1996/2/27 1995 /09/28 1993/03/30 1984/7/25 1984/5/22 Table 2. Primers used to increase 16S rRNA by polymerase chain reaction (PCR) _ primer nucleic acid sequence (5' to 3') Nucleic acid position # 1F GAGTTTGATCATGGCTCAG 9-27 3F CCTACGG GAGGCAGCAG 341-357 4F GTGCCAGCAGCCGCGGTAA 515-533 4R TTACCGCGGCTGCTGGCAC 533-515 5F AAACTCAAATGAATTGACGGGG 907-928 6F GCAACGCGAAGAACCTTACC 966-985 7R TGACGGGCGGTGTGTACAA 1408-1390 9R AAGGAGGTGATCCAACCGCA 1541-1522: Nucleic acid position refers to the position of the 16S rRNA nucleic acid sequence of Escherichia coli 25 200946477

表3.皺紋固氮螺旋菌IMMIB AFH-6菌株的16S rDNA之核酸序列 (已登錄於美國NCBI database號碼AM419042)_ 1 ggcatgcctaacacatgcaagtcgaacggaggcttcggcctcagtggcgcacgggtgagt 61 aacacgtgggaacctgcctttcggttcgggataacgtctggaaacggacgctaacaccgg 121 atgcgcccttcgggggaaagtttacgccgagagaggggcccgcgtccgattaggtagttg 181 gcggggtaacggcccaccaagccgacgatcggtagctggtctgagaggatgatcagccac 241 actgggactgagacacggcccagactcctacgggaggcagcagtggggaatattggacaa 301 tgggggcaaccctgatccagcaatgccgcgtgagcgatgaaggccttcgggttgtaaagc 361 tctttcgcacgcgacgatgatgacggtagcgtgagaagaagccccggctaacttcgtgcc 421 agcagccgcggtaatacgaagggggctagcgttgttcggaattactgggcgtaaagggcg 481 cgtaggcggcctgttcagtcagaagtgaaagccccgggctcaacctgggaatggcttttg 541 atactggcaggcttgagttccggagaggatggtggaattcccagtgtagaggtgaaattc 601 gtagatattgggaagaacaccggtggcgaaggcggccatctggacggacactgacgctga 661 ggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacga 721 tgaatgctagacgctggggtgcatgcacttcggtgtcgccgctaacgcattaagcattcc 781 gcctggggagtacggccgcaaggttaaaactcaaaggaattgacgggggcccgcacaagc 841 ggtggagcatgtggt t taat tcgaagcaacgcgcagaacct taccaaccct tgacatgtc 901 cact t tcggcaccagagatggtgct 11cggt tcggccgggtggaacacaggtgctgcatg 961 gctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccccca 1021 ccgccagttgccatcattcagttgggcactctggcggaaccgccggtgacaagccggagg 1081 aaggcggggatgacgtcaagtcctcatggcccttatgggttgggctacacacgtgctaca 1141 atggcggtgacagtgggacgcgaagccgcgaggtggagccaatccccaaaagccgtctca 1201 gttcggattgcactctgcaactcgggtgcatgaagttggaatcgctagtaatcgcggatc 1261 agcacgccgcggtgaatacgt tcccgggcct tgtacacaccgcccgtcacaccatgggag 1321 t tggct t tacccgaagacggtgcgctaacccgcaaggggggcagccggccacggtcaggt 1381 cagcgactggggtgaagtcgtaacacggtagccgtaggggaacctgcggctggatcacct 1441 cctttcta 26 200946477 Ο ❹ 酵素活性項目酵素種類反應性 1 1·碟酸水解酶驗性碟酸水解酶(alkaline phosphatase) + (phosphatase) 酸性碟酸水解酶（acid phosphatase) + 萘紛-AS-ΒΙ-麟酸水解酶(phosphohydrolase) + 2.酯酶丁睃酿酶（四碳）（butyrate esterase) + (esterase) 辛酸 S旨酶（八碳)(caprylate esterase) + 肉豆謹酸醋酶(十四碳)(myristate esterase) - 3.胺基肽酶白胺酸酿胺酶(leucine aminopeptidase) + (aminopeptidase) 擁胺酸酿胺酶(valine aminopeptidase) - 胱胺酸酿胺酶(cystine aminopeptidase) - 4.蛋白酶胰蛋白水解酶(trypsin) - (protease) 騰糜蛋白酶(chymotrypsin) - 動物膠水解酶（gelatinase) + 5.脫胺酶精胺酸雙水解酶(arginine dihydrolase) (deaminase) (tryptophan dehydrogenase) - 尿素水解酶(urease) + 6. 脫羧酶離胺酸脫叛酶(lysine decarboxylase) - (decarboxylase) 鳥胺酸脫叛酶(ornithine decarboxylase) - 色胺酸水解酶(tryptophanase) - 7.糖基水解酶 α_ 半乳糖苦酶(α-galactosidase) (glycosyl β-半乳糖苦酶(β-galactosidase) hydrolase) β_葡萄糖酸糖普酶(β-glucuronidase) - α_ 葡萄糖普酶(a-glucosidase) - β_ 葡萄糖普酶(β-glucosidase) Ν-乙酿β·葡萄糖酿胺喊N_acetyi_p_giucosaminidase) α-甘露糖普酶(a-mannosidase) - 1. ! 达 7T C 虚. α_岩藻糖普酶(a-fbcosidase) - 27 200946477 ΟTable 3. The nucleic acid sequence of 16S rDNA of wrinkles nitrogenase pylori IMMIB AFH-6 strain (NCBI database are logged in U.S. numbers AM419042) _ 1 ggcatgcctaacacatgcaagtcgaacggaggcttcggcctcagtggcgcacgggtgagt 61 aacacgtgggaacctgcctttcggttcgggataacgtctggaaacggacgctaacaccgg 121 atgcgcccttcgggggaaagtttacgccgagagaggggcccgcgtccgattaggtagttg 181 gcggggtaacggcccaccaagccgacgatcggtagctggtctgagaggatgatcagccac 241 actgggactgagacacggcccagactcctacgggaggcagcagtggggaatattggacaa 301 tgggggcaaccctgatccagcaatgccgcgtgagcgatgaaggccttcgggttgtaaagc 361 tctttcgcacgcgacgatgatgacggtagcgtgagaagaagccccggctaacttcgtgcc 421 agcagccgcggtaatacgaagggggctagcgttgttcggaattactgggcgtaaagggcg 481 cgtaggcggcctgttcagtcagaagtgaaagccccgggctcaacctgggaatggcttttg 541 atactggcaggcttgagttccggagaggatggtggaattcccagtgtagaggtgaaattc 601 Gtagatattgggaagaacaccggtggcgaaggcggccatctggacggacactgacgctga 661 ggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacga 721 tgaatgctagacgctggggtgcatgcacttcggtgtcgccgctaacgcattaagcattcc 781 gcctggggagtacggccgcaaggttaaaactcaaa ggaattgacgggggcccgcacaagc 841 ggtggagcatgtggt t taat tcgaagcaacgcgcagaacct taccaaccct tgacatgtc 901 cact t tcggcaccagagatggtgct 11cggt tcggccgggtggaacacaggtgctgcatg 961 gctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccccca 1021 ccgccagttgccatcattcagttgggcactctggcggaaccgccggtgacaagccggagg 1081 aaggcggggatgacgtcaagtcctcatggcccttatgggttgggctacacacgtgctaca 1141 atggcggtgacagtgggacgcgaagccgcgaggtggagccaatccccaaaagccgtctca 1201 gttcggattgcactctgcaactcgggtgcatgaagttggaatcgctagtaatcgcggatc 1261 agcacgccgcggtgaatacgt tcccgggcct tgtacacaccgcccgtcacaccatgggag 1321 t tggct t tacccgaagacggtgcgctaacccgcaaggggggcagccggccacggtcaggt 1381 cagcgactggggtgaagtcgtaacacggtagccgtaggggaacctgcggctggatcacct 1441 cctttcta 26 200946477 enzymatic kind of the reactive Ο ❹ enzyme activity item 11 Acid phosphatase + acid phosphatase + phosphatase Enzyme (four carbon) (butyrate) Esterase) + (esterase) caprylate esterase + myristate esterase - 3. aminopeptidase leucine aminopeptidase + (aminopeptidase) valine aminopeptidase - cystine aminopeptidase - 4. protease trypsin - (protease) chymotrypsin - animal glue Gelatinase + 5. Deaminase arginine dihydrolase (deaminase) (tryptophan dehydrogenase) - urea hydrolase (urease) + 6. decarboxylase lysine decarboxylase - (decarboxylase) ornithine decarboxylase - tryptophanase - 7. glycosyl hydrolase α_galactosidase (glycosyl β-galactosidase) -galactosidase) hydrolase) β-glucuronidase - α_ a-glucosidase - β_glucosidase Ν-ethyl β-glucose-enamined N_acetyi_p_giucosami Nidase) α-mannosidase (a-mannosidase) - 1. ! up to 7T C imaginary. α_fucose-enzyme (a-fbcosidase) - 27 200946477 Ο

表5.皺紋固氮螺旋菌imMIB AFH-6對破源之茯謝活性碳源分類碳源種類氧化同化套組1 1.醣類 N· 葡綠(N_acetyi_D-glucosamine) + + B，N (carbohydrate) 即sj-acetyl-D-galactosamine) + + B 阿拉伯糖(L-arabinose) + + Β,Ν 葡萄糖·(glucose) + + Β,Ν 甘露糖(mannose) + + Ε,Ν,Β 甘露醇(mannitol) + + Β,Ν 麥芽糖(maltose) + + Β,Ε,Ν 嚴糖(sucrose) + + Β,Ν 岩藻糖(trehalose) + Β 2.羧酸己二酸(adipic acid) ? + Ν (carboxylic 羊壤酸(capric acid) - Ν acid) 檸檬酸(citric acid) + + Β,Ν 對經基苯醋酸(p-hydroxyphenylacetic acid) + 9 Β /3 -經基丁酸(/3 -hydroxybutyric acid) + 9 Β 葡萄糖二酸(D-saccharic acid) + ? Β 葡萄糖酸(gluconic acid) + + Β,Ν D-葡萄糖搭酸(glucuronic acid) + ? Β 奎寧酸(quinic acid) + ? Β 己二酸(adipic acid) + Ν 蘋果酸(malic acid) + Ν 3.胺基酸甘^feSTL^^^Glycyl-L-glutamic acid) + ? Β (amino acid) 甘胺醯基心天門冬胺酸(Glycyl-L-aspartic 9 acid) 丙胺基甘胺酸(L-alanyl-lycine) + 9 Β 4.麵甘油(glycerol) + 9 Β (alcohol) 肌醇(m-inositol) + ? Ε/Β 使用的測試套組代號，Β : Biolog, Ε: API 20Ε，Ν: API 20ΝΕ。 +為正反應，-為無反應，？為尚未測試或未知。 28 200946477 表6.種子生物分析接種皺紋固氮螺旋菌IMMIB八1?1_1_6對於植物生長的影響田相對生長(Relative elongation)% 植物種類（plant) 根(root) 地上部（Shoot) 控制組(control) 100 100 小蘿蔔（Radish) 152 124 結球萵苣（Head lettuce) 145 127Table 5. MI 固固 im im im im im im im im im im im im im im im im im im im im im 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. That is, sj-acetyl-D-galactosamine) + + B arabinose (L-arabinose) + + Β, 葡萄糖 glucose · (glucose) + Β, 甘 mannose + Ε Ε, Ν, Β mannitol + + Β, 麦 maltose + + Β, Ε, 严 sucrose + + Β, 岩 trehalose + Β 2. adipic acid + + Ν ( Carboxylic acid (capric acid) - Ν acid) citric acid + + Β, Ν p-hydroxyphenylacetic acid + 9 Β /3 - butylbutyric acid (/3 -hydroxybutyric acid + 9 D D-saccharic acid + Β gluconic acid + + Β, Ν D-glucose acid + Β quinic acid + ? Β Adipic acid + Ν malic acid + Ν 3. amino acid ^feSTL^^^Glycyl-L-glutamic acid) + ? Β (amino acid) glycine 心-aspartic acid aspartate (Gl ycyl-L-aspartic 9 acid) L-alanyl-lycine + 9 Β 4. Glycerol + 9 Β (alcohol) Inositol (m-inositol) + ? Ε/Β used Test kit code, Β: Biolog, Ε: API 20Ε, Ν: API 20ΝΕ. + is a positive reaction, - is no reaction,? Not yet tested or unknown. 28 200946477 Table 6. Seed bioassay inoculation of P. aeruginosa IMMIB VIII 1 1_1_6 on plant growth. Relative growth % plant type root (root) above ground control group (control) 100 100 Radish 152 124 Head lettuce 145 127

G 表7.盆栽試驗添加皺紋固氮螺旋菌immib AFH-6對於萵苣之生長的促進效果地上部重量（shoot weight) _鮮重(Fresh)__ 乾重(Dry) (g/p〇t) % (g/pot) % BK 12.6±3.3 bc 67.3 0.9±0.2 bc 64.3 PMF 9.7±l.l c 51.9 0.7±0.2 bc 50.0 In 15.0±5.3 abc 80.2 1.3±0.3 ab 92.9 In-PMF 8.5±1.〇c 45.5 0.7±0.1 c 50.0 1/2CF 16.3±6.3 abc 87.2 1.2±0.6 abc 63.2 PMF+1/2CF 14.0±3.3 bc 74.9 1.2±0.3 abc 85.7 In-1/2CF 18.1±6.6 abc 96.8 1.3土0.4 abc 92.9 PMF+In-1/2CF 25.6±2.0 a 136.9 1.9±0.1a 135.7 1CF 18.7±2.7 abc 100 1.4士0.4 abc 100 PMF+1CF 10.0±3.1c 53.5 0.8±0.2 b 57.1 In-ICF 22.4±2.7 ab 119.8 1.6±0.3 ab 114.3 PMF+In-ICF 14.4士7.9 abc 77.0 l.l±0.5abc 78.6G Table 7. Pot experiment to increase the growth of lettuce nitrogen-fixing Imib AFH-6 for lettuce growth. Shoot weight _ Fresh weight __ Dry weight (Dry) (g/p〇t) % ( g/pot) % BK 12.6±3.3 bc 67.3 0.9±0.2 bc 64.3 PMF 9.7±ll c 51.9 0.7±0.2 bc 50.0 In 15.0±5.3 abc 80.2 1.3±0.3 ab 92.9 In-PMF 8.5±1.〇c 45.5 0.7± 0.1 c 50.0 1/2CF 16.3±6.3 abc 87.2 1.2±0.6 abc 63.2 PMF+1/2CF 14.0±3.3 bc 74.9 1.2±0.3 abc 85.7 In-1/2CF 18.1±6.6 abc 96.8 1.3 soil 0.4 abc 92.9 PMF+In- 1/2CF 25.6±2.0 a 136.9 1.9±0.1a 135.7 1CF 18.7±2.7 abc 100 1.4士0.4 abc 100 PMF+1CF 10.0±3.1c 53.5 0.8±0.2 b 57.1 In-ICF 22.4±2.7 ab 119.8 1.6±0.3 ab 114.3 PMF+In-ICF 14.4 7.9 abc 77.0 ll±0.5abc 78.6

* BK :對照組。 1CF :「作物施肥手冊」推薦量之NPK。* BK: control group. 1CF: NPK recommended by the Crop Fertilization Manual.

In :含本案菌種皺紋固氮螺旋菌IMMIB AFH-6。 29In : Contains the strain S. aureus IMMIB AFH-6. 29

Claims

200946477 X. Patent application scope: 1 · A microbial strain isolated and purified, named as Immune Azospirillum IMMIB AFH-6, which was deposited at the Food Industry Development Research Institute in Hsinchu, Taiwan on December 11, 1996. The deposit number is BCRC 910373. 2 A composition comprising the microbeads of claim 1 of the patent application. 3. The composition of claim 2, the composition having at least biological nitrogen fixation, dissolved mineral phosphorus, phosphohydrolase, nitrate reductase, or animal protease activity. 4. The composition of claim 2, which can produce phytohormone < 丨丨丨 ind 醋酸醋酸醋酸醋酸醋酸 ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind ind The composition of claim 2 can be developed as a microbial preparation. The composition of claim 2 can be applied to the root ring. For a microbial fertilizer. 7. The composition of claim 2, which is applied to the soil as a soil amendment. ❹8. The composition of claim 2 of the patent application can be applied to unfertilized or decomposed compost or organic matter as a compost or quinone organic quality quality improver. 9. The composition of claim 2, which composition can be used to promote plant growth, soil improvement or the use of microbial fertilizers. A method for promoting plant growth, comprising at least the following steps: propagating a microorganism strain as in claim 1 of the patent application; and inoculating the microorganism strain to about a seedling root ring or a seed. u. The method of claim 1G of the patent application wherein the vaccination is carried out by inoculating the cells containing the microorganism. The method of claim 10, wherein the plant comprises at least one of a lettuce and a radish seed organism.

31 200946477 VII. Designated representative map: (1) The representative representative of the case is: Figure 1. (2) A brief description of the symbol of the representative figure: none

8. If there is a chemical formula in this case, please reveal the chemical formula that best shows the characteristics of the invention: ❹ 4