水稻内生联合固氮细菌的筛选,鉴定及其分布特性

利用乙炔还原法和固定１５Ｎ２ 活性测定法对分离自水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）“越富”种子、根、茎和叶的内生细菌进行了筛选,获得２９ 株具有体外固氮能力的水稻内生联合固氮细菌。鉴定结果表明它们分属于根癌土壤杆菌（ Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ （Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ） Ｃｏｎｎ） ,放射土壤杆菌（ Ａ． ｒａｄｉｏｂａｃｔｅｒ （Ｂｅｉｊｅｒｉｎｃｋ ｅｔ ｖａｎ Ｄｅｌｄｅｎ） Ｃｏｎｎ） ;阴沟肠杆菌（ Ｅｎｔｅｒｏｂａｃｔｅｒ ｃｌｏａｃａｅ （Ｊｏｒｄａｎ） Ｈｏｒｍａｅｃｈｅ ｅｔ Ｅｄｗａｒｄｓ） ,成团肠杆菌（ Ｅ． ａｇｇｌｏｍｅｒａｎｓ （Ｂｅｉｊｅｒｉｎｃｋ） Ｅｗｉｎｇ ｅｔ Ｆｉｆｅ） ,坂崎肠杆菌（ Ｅ． ｓａｋａｚａｋｉｉ Ｆａｍｅｒ ｅｔ ａｌ．） ;皮氏产碱菌（ Ａｌｃａｌｉｇｅｎｅｓ ｐｉｅｃｈａｕｄｉｉ Ｋｉｒｅｄｊｉａｎ ｅｔ ａｌ．） ,反硝化产碱菌（ Ａ． ｄｅｎｉｔｒｉｆｉｃａｎｓ （Ｌｅｉｆｓｏｎ ｅｔ Ｈｕｇｈ） Ｒｕｇｅｒ ｅｔ Ｔａｎ） ;类产碱假单胞菌（ Ｐｓｅｕｄｏｍｏｎａｓ ｐｓｅｕｄｏａｌｃａｌｉｇｅｎｅｓ Ｓｔａｎｉｅｒ） ,产碱假单胞菌（ Ｐ． ａｌｃａｌ     

Biochemical Characteristics of Rice Endophytic DiazotrophicBacteria and Their Infection to Tobacco and Maiz

Some biochemical properties of rice endophytic diazotrophic bacteria N1 ( Bacillus pumilus), N2 ( Enterobacter cloacae) and N3 ( Enterococcus gallinarum) were characterized in this study. The plasmids harboring the reporter gene lacZ fused to the promoters of nifH and nifHDK, and the reporter gene gfp as well, were first transformed into rice endophytic diazotrophic bacteria respectively through triparental mating. After the conjugated bacteria were inoculated into tobacco Yunyan 85 and maize Jitian 6, the staining of β-galactosidase was carried out on the tobacco and maize roots and the observation was made under the confocal laser scanning microscope, transmission electron microscope and scanning electron microscope. The results showed that the rice endophytic diazotrophic bacteria were not only present in epidermal cells, cortex cells and intercellular spaces of tobacco and maize roots, but also found in vascular tissue cells of maize stem also, indicating that the bacteria had migrated from the roots into the stem. Data showed that the the fresh weight of maize plants inoculated with rice endophytic diazotrophic bacteria N3 was 18.52% more than that of the control.     

玉米内生细菌

<正>拮抗内生细菌对寄主植物具有促生、防病、杀虫及固氮等多方面的生物学作用,是植物病害防治的天然资源菌,并且能够作为外源基因载体,为杀虫、防病、固氮等外源植物基因导入植物体内提供了一种很好的     

Endophytic Establishment of Diazotrophic Bacteria in Auxin-Induced Tumors of Cereal Crops

Gramineous crops such as wheat (triticum oestivum), maize (zea mays), and rice (oryza sativa) develop tumorous structures (para-nodules) along primary and secondary roots when treated with low concentrations of various auxins. Rice forms additional tumors along its hypocotyle. Histologically, auxin-induced tumors appear as cancerous grown out root meristems and thus are comparable in origin and structure to stem nodules of the legume sesbania rostrata. Auxin-affected root meristems do not recover and develop further to large nodule-like organs. Introduced diazotrophs (Azospirillum spp., Azorhizobium caulinodans, Rhizobium spp.) potentially inhabit tissues of both stem and root tumors with the central meristem as a major colonization niche. Evidence is given that infecting bacteria follow a ‘crack entry’ invasion at sites where developing tumors have emerged through the root cortex and epidermis. Bacteria are shown to establish with high cell numbers inside intercellular spaces of cortical and meristematic tissues. Plant-cell infection of tumor cells takes place with bacteria found inside the cell-cytoplasm surrounded by membrane-like structures. Once inhabiting induced tumor tissues introduced diazotrophs colonize endophytically with high cell numbers. Mutant, ammonium-excreting and thus ecologically disadvantaged A. brasilense is shown to survive inside para-nodulating maize and rice plants with a dense population. Micro-aerobic nitrogenase activities of tumor inhabiting diazotrophic bacteria (A. brasilense, Azotobacter vinelandii, A. caulonidans) are in general highly increased when compared with untreated control plants. Additionally, bacterial nitrogenase activity is less sensitive to an increased oxygen tension in the root environment. The host plants benefit from the enhanced nitrogen fixation in their para-nodulating roots. Highest rates of incorporation of fixed nitrogen into host plant material is reported for para-nodule inhabiting ammonium excreting A. brasilense strain C3. The host plant potentially stimulates the nitrogenase activity of endophytically colonizing diazotrophs by providing energy in the form of a suitable carbon source. In conclusion, it is demonstrated that gramineous plants are potentially capable of developing an endophytical diazotrophic symbiosis through para-nodule formation.     

防治烟草赤星病有益内生细菌的筛选及抑菌作用

从健康烟草的叶、茎中分离到302株非病原内生细菌,通过平板对峙培养,筛选出对烟草赤星病菌［Alternaria alternata（Fr.）Keissl］不同致病力的4个代表菌株均有拮抗作用的11个菌株。室内测定其对赤星病菌抑菌带的宽度达5.5～13.2mm;拮抗、防病试验测定,来自叶片内的内生菌株Itb162对赤星病菌有较强和稳定的拮抗作用,对赤星病有52.0%的防病效果。无菌滤液实验表明,拮抗内生细菌Itb162无菌滤液在一定浓度范围内均能有效地抑制菌丝生长,减少孢子萌发,且浓度越高,抑制能力越强。     

白肋烟内生细菌的分离鉴定及降低N-亚硝胺含量研究

以表面消毒法分离白肋烟TN90和TRM品种 30个样品的内生细菌 ,共分离到 33株内生细菌。对这些菌株进行了初步分类鉴定 ,它们属于假单胞菌属、黄单胞菌属、节杆菌属、葡萄球菌属、棒杆菌属、黄杆菌属、气球菌属和利斯特氏菌属。筛选出TEB1 1、TEB1 7、TEB2 3、TEB2 6、TEB30、TEB34等 6株还原硝酸盐和亚硝酸盐能力较强的菌株 ,以粉碎烟叶接种、叶柄浸泡接种和叶面喷雾接种 3种方式处理调制后 ,化验分析结果表明 ,接种内生细菌能降低2 7 5 6 %～ 99 88%白肋烟TSNA含量 ,降低百分率以粉碎烟叶接种最高 ,其次是叶柄浸泡接种 ,叶面喷雾接种最低     

烟草内生细菌防治烟草黑胫病及促生作用研究

筛选烟草内生细菌防治烟草黑胫病, 获得了对烟草黑胫病有很好防效的内生细菌118、57和93等菌株。在温室控病实验中它们的防效分别可达69.23%、61.53%和65.38%。118菌株对烟草疫霉(Phytophthora parasitica var. nicotianae)菌丝生长有明显的拮抗作用。118菌株具有较广的抗菌谱, 且对烟草有促生效果, 烟草的鲜重增产率为13.10%。     

烟草内生细菌种群动态研究

经过对 7个田间种植的烟草品种不同栽培时期、不同组织内生细菌种群动态研究表明 ,不同品种内生细菌种群有一定程度差异。同一品种中有的内生细菌为常住菌群 ,有的为暂居菌群 ,带菌量根中最高 ,茎次之 ,叶中最低。在整个生育期中 ,7个品种内生细菌数量表现出从种子到出苗期大幅增加 ,从出苗期到十字期又大幅度下降 ,随后从缓苗期到伸根期再一次急剧增加并维持在一个较高水平。通过对烟草这一重要经济作物内生细菌种群动态变化研究 ,可为烟草病虫害生物防治和促生增产研究提供理论基础。     

烟草内生细菌防治烟草黑胫病及促生作用研究

筛选烟草内生细菌防治烟草黑胫病,获得了对烟草黑胫病有很好防效的内生细菌118、57和93等菌株.在温室控病实验中它们的防效分别可达69.23%、61.53%和65.38%.118菌株对烟草疫霉(Phytophthora parasitica var.nicotianae)菌丝生长有明显的拮抗作用.118菌株具有较广的抗菌谱,且对烟草有促生效果,烟草的鲜重增产率为13.10%.     

玉米苗期内生细菌的种群初探及有益内生细菌的筛选

从3个供试玉米品种(川单13、川单418、川单416)苗期分离、纯化内生细菌68株,经形态及理化特征鉴定,分属5个不同的属,微球菌属和芽孢杆菌属为优势属.芽孢杆菌属细菌鉴定出5个种.供试品种中内生细菌种群数量各不相同,种群数量由多到少依次是川单13、川单418和川单416.通过抑菌谱和拮抗菌发酵液对种子萌芽率的影响,筛选出两株抑菌谱广,对玉米种子萌发无抑制作用的内生芽孢细菌BH和B98.盆栽实验显示,两株细菌均对玉米植株具有促生作用,对玉米纹枯病具有防治效果.