CN114450391A - 新型温度优化的芽孢杆菌纲 - Google Patents
新型温度优化的芽孢杆菌纲 Download PDFInfo
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Abstract
本发明涉及具有提高的生长速率和植物生长促进特性的副地衣芽孢杆菌新型菌株,以及与它们所衍生的亲代菌株相比具有提高的生长速率的副地衣芽孢杆菌新型菌株。
Description
发明内容
本发明涉及包含在不同温度条件下具有提高的生长速率和较高的生物量产量的芽孢杆菌纲(Bacilli)和副地衣芽孢杆菌(Bacillus paralicheniformis)的组合物、其用途、其制备方法,以及在不同温度条件下具有提高的生长速率和较高的生物量产量的芽孢杆菌纲和副地衣芽孢杆菌用于防治、对抗植物有害生物和/或赋予对植物有害生物的特异抗性的用途。具体地,本发明涉及由BioF和HrcA基因中的一者或多者编码的蛋白质功能发生改变的芽孢杆菌纲和副地衣芽孢杆菌的菌株。
本发明的发明人已经生成了在不同温度范围内显示出提高的生长速率和较高的生物量产量的菌株。
此外,在模型植物生物体拟南芥中,本发明选出的新型菌株显示与原始菌株相比,在植物生长实验中诱导轻微的提高。
对与表型变化相关的基因型变异进行了描述。已在植物实验***中对衍生菌株进行了测试,并且已证明其与原始菌株相比促进了植物生长的增加。此外,在本文中公开并讨论了解释基因修饰影响的两种不同机制。
技术领域
在当今注重环境保护的现代生态社会背景下,人们认为生物防治对于常规防治方法来说是一种有吸引力的替代或补充。生物防治是指使用一种生物(捕食者、寄生虫或病原体),其攻击另一种对作物造成经济损害的生物。这在农业生态***以及依赖于有害生物综合治理(IPM)的常规农业中是一种非常常见的策略。
尽管生物防治在减少或杜绝杀虫剂使用以及提高农民收入方面带来了积极效果,但是对世界范围内的一系列实验的分析显示,生物防治带来的效果仍集中在仅少数作物和气候条件促进芽孢杆菌、特别是像副地衣芽孢杆菌这种植物促生根际细菌的生长速率的特定地理区域内。在病虫害防治领域仍存在许多需要发展的地方。
已非常重视利用定植于植物根部的细菌(称为根际细菌)进行生物防治的研究。用于促进生长和/或对植物病原菌起生物防治作用的有益根际细菌被称为植物促生根际细菌或PGPR。
PGPR生物防治成功的关键因素之一是(例如凭借生长速率和生物量)在生境成功定殖。因此,生物膜的成功形成可增强PGPR的保护作用。
概括的说,经过了适应性实验室进化实验,冷适应衍生物副地衣芽孢杆菌DSM33110菌株生成。选出了14种改良的衍生物,并对它们的基因组进行测序以鉴定所获得的基因型变化。对衍生菌株进行生理性表征,并在植物生长实验中测试其性能。基于本文的结果,我们提出了解释观察到的与亲代菌株的表型差异的机制。此外,这些进化实验还贡献了在植物生长促进方面显示出改良的特性的新型芽孢杆菌纲菌株。
现有技术
用环境可接受的产品或生态技术取代化学品的社会压力促使寻求促进植物健康的替代方法。在这种情况下,生物防治被认为是综合方法中的替代方法之一,在综合方法中寻求确保农业的可持续发展。
使用合成杀虫剂对人类和环境带来的风险强调,在优化可持续农业***方面需要例如生物防治的工具。
基于在不同温度条件下提高的生长速率和生物量形成可以增强芽孢杆菌纲的生物保护作用的想法,本发明的发明人选出了在不同温度条件下显示出生长速率和生物量形成增加的芽孢杆菌纲衍生菌株。
据我们所知,对于芽孢杆菌纲,尤其是副地衣芽孢杆菌菌株,还从未描述过与在不同温度条件下提高的生长速率和生物量形成相关的特定遗传特征以及相关作用方式。
附图说明
图1—冷适应菌株的ALE生成:实验方法。A.ALE实验方法用于选择冷适应DSM33110衍生物,进行12周。每天对12个独立的过夜培养物进行5%-10%稀释,并转移至新鲜培养基中(在两种不同的生长培养基中进化出6个独立的培养物)。每天进行OD(600nm)测定,并计算每周的传代数。每周将各管的样品以甘油储备液的形式冷冻保存。在整个ALE过程中,温度从25℃逐渐降低至18℃。B.经过12周的适应性实验室进化后,将各培养物的连续稀释液平板接种,并选出2×960个独立的克隆,在LB肉汤中生长,并在96孔微量滴定板中以甘油储备液的形式冷冻保存。首先在21℃时用生长曲线测量仪960比较单个克隆的生长曲线。选出14个生长速度较快的克隆,并在不同的生长培养基和温度条件下分析它们的生长模式。
图2—选出的冷适应衍生物对比亲代菌株DSM33110的生长曲线。A.在MSgg生长培养基中进化的冷适应衍生物在21℃在MSgg培养基中生长,并与亲代菌株DSM33110进行比较。B.在MS-Rex生长培养基中进化的冷适应衍生物在21℃在MS-Rex培养基中生长,并与亲代菌株DSM33110进行比较。测定不同菌株达到的最大生物量产量。
图3—各衍生菌株和亲代菌株DSM33110的最大生长速率计算值(μmax)。A.在21℃在MSgg培养基中达到的μmax值。B.在21℃时MS-Rex中达到的μmax值。
图4—冷适应衍生物在30℃在不同生长培养基中的生长曲线。选择的冷适应衍生物与亲代菌株DSM33110的生长曲线。(A-B)冷适应衍生物在30℃在LB培养基中生长,并与亲代菌株DSM33110进行比较。(C-D)冷适应衍生物在30℃在BHI培养基中生长,并与亲代菌株DSM33110进行比较。X轴对应于实验的时间尺度,为小时。测定不同菌株达到的最大产量。A-C图对应于在MSgg生长培养基中进化的衍生物。B-D图对应于在MS-Rex生长培养基中进化的衍生物。
图5—植物生长促进实验。DSM33110和14个冷适应改良衍生菌株CA1-CA7和CAREX1-CAREX7(分别对应于CHCC32528-CHCC32529-CHCC36494-CHCC36497和CHCC36751-36757)的植物生长和适应性促进定量结果。结果对应于在体外琼脂***中进行的实验。左图对应于每株植物计算的总叶面积测量平均值(每个菌株接种48株植物)。中间图对应于每株植物的平均鲜重。右图对应于通过鲜重归一化的叶绿素定量结果。误差条对应于样品之间的标准偏差。
图6—A.BioF同源物的一级氨基酸序列比对(大肠杆菌K12、枯草芽孢杆菌168和副地衣芽孢杆菌DSM33110,如所示)。星号标记的位置对应于被鉴定为导致氨基酸变化的SNP的残基。方形标记的位置对应于催化残基。B.BioFDSM33110蛋白质的3D结构模型。红色圆圈标记催化残基的位置。绿色方框对应于鉴定的氨基酸变化的位置。
图7—示出枯草芽孢杆菌中描述的HrcA调节子的示意图。HrcA作为七顺反子dnaK操纵子(hrcA-grpE-dnaK-dnaJ-3xorfs)和双顺反子groE(groES-groEL)这两个操纵子的转录抑制子。对DSM33110中存在的同源基因组区域的分析证实了这些操纵子组织的保守性。
图8—比较在盆栽土中生长的拟南芥中的冷适应衍生物对植物生长的促进作用。亲代菌株DSM33110和2个冷适应改良衍生菌株(分别对应于CHCC36494-CHCC36496的CA4和CA6)的植物生长和适应性促进定量结果。A-B.亲代菌株(DSM33110)和冷适应衍生物CA4和CA6分别与枯草芽孢杆菌菌株DSM33015和DSM32938的两种组合的植物生长和适应性促进定量结果。将在盆栽土中预生长7天的拟南芥幼苗通过蘸根法接种在重悬于10mM MgSO4缓冲液(OD600 0.01)中的细菌培养物上。在收集数据前,让幼苗再生长14天。误差条对应于平行测定之间的标准偏差,并且假设两个样品的方差相等,通过进行t检验(p<0.05)来确定统计显著性。A.植物生长实验在22℃在植物生长室中进行。B.植物生长实验在15℃在植物生长室中进行。
具体实施方式
根际细菌
土壤是复杂生物群落的家园,在该生物群落中,微生物、原核生物和真核生物在数量和多样性上都占了大多数。一些原核生物以植物的根际和/或根面作为生态位,在这里繁殖、生存并保护自身免受其他的土壤微生物群落的拮抗作用。这些生物体被统称为根际细菌。
在与植物结合的过程中,根际细菌可能产生有害效果,无效果或产生有益效果。那些实施有益效果(生长促进和病害生物防治)的根际细菌被称为PGPR(“植物促生根际细菌”)。据估计,仅有1%至2%的根际细菌对与其所结合的植物具有一定的有益效果。
作为生物防治剂的PGPR
PGPR已用于植物病害的生物防治,进而提高作物产量。如何以及为何开展这种生物防治仍然是一个需要补充研究的课题。
在一些情况下,有可能通过PGPR对病原体产生的直接拮抗作用来进行生物防治,其中涉及已知的抗生机制:抗微生物物质的产生、直接寄生、对营养物质和生态位的竞争。研究已表明,某些PGPR似乎作为ISR(诱导***抗性)的诱导子起作用,也就是说,植物变得被***性保护以抵抗多于一种的病原体,这与旨在更具体地实施防治的经典生物防治不同。
影响PGPR感染和定殖于植物表面的能力的重要参数是PGPR在其所暴露的条件下生长的能力。因此,本公开的发明人力求提高在不同的温度、最优选降低的温度条件下的生长速率和生物量形成,从而增强本文公开的芽孢杆菌纲的植物生长促进能力。
根际环境
将根际细菌引入根系环境的最简便的方法之一是在播种前施用于种子上。种子发芽过程以种子分泌物的形式释放大量的碳水化合物和氨基酸。因此,这些与种子一起引入土壤中的生物体利用分泌物作为营养源,并随着生根而定殖于根。在不同温度下利用种子的根分泌物的能力较强的根际细菌分离物可在定殖于根时具有选择性优势。
芽孢杆菌属的PGPR已与线虫防治产生联系。Sikora,R.A.(Interrelationshipbetween plant health promoting rhizobacteria,plant parasitic nematodes andsoil microorganisms,Medicine Faculty Landbouww Rijksuniv Gent,Landbouww,第53卷,第2b期,第867-878页,1988年)观察到用芽孢杆菌属菌株处理各种作物的种子时,花生根结线虫(Meloidogyne arenaria)、南方根结线虫(M.incognita)和肾形线虫(Rotylenchulus reniformis)的感染减少约60%-65%。
根际细菌的商业应用优势
与化学杀虫剂或者甚至其他生物防治剂相比,根际细菌具有以下几个优点:易于大量生产、易于储存以及适用于制剂技术。
可通过处理基质、将幼苗根系浸入细菌悬浮液中、用细菌悬浮液浇灌植物,通过将种子浸渍/包被在根际细菌的悬浮液中或通过用种子造粒施用PGPR来施用根际细菌。
芽孢杆菌属细菌
芽孢杆菌属物种为革兰氏阳性菌,其特征在于具有厚的细胞壁并且不具有外膜,这与革兰氏阴性菌不同。革兰氏阳性菌的细胞壁的大部分由肽聚糖组成。
革兰氏阳性菌物种根据其形态和生化特征分成了不同的群。芽孢杆菌属属于产孢菌群。细菌孢子是适应性最强的细胞类型之一;它们能抵抗许多环境变化,耐受干热和某些化学消毒剂,并且可以在旱地上存活数年。
当芽孢杆菌纲,例如副地衣芽孢杆菌在种子或土壤附近施用时,其有益效果可能不仅仅由于其对病原体的拮抗作用。PGPR对发芽、发育和作物产量具有积极影响,这也是由于产生了通过磷的溶解促进生长和植物营养增加的物质。
在本发明的上下文中,生物肥料是活体微生物的混合物,这些活体微生物在施用于种子、植物或土壤时促进营养物质供给(诸如NH4 +、SO4 2-、K+或PO4 3-)的增加(Vessey,2003年)。
在本发明的上下文中,植物生物刺激素是施用于植物并旨在提高营养效率、非生物胁迫耐受性和/或作物品质性状的任何物质或微生物,无论其营养物质含量。推而广之,植物生物刺激素也指含有此类物质和/或微生物的混合物的商业产品(du Jardin,2015年)。
在本发明的上下文中,植物生长促进剂或植物生长促进微生物是能够定殖于根和/或内部植物组织并通过充当生物肥料、生物刺激素或经由植物病害的生物防治来促进植物生长和健康的微生物。该试剂或微生物是一种可以大量(105-107CFU/g新鲜根)定殖于植物根部,并以积极的方式影响植物生长的土壤和根际栖息微生物(Spaepen等人,2009年;Antoun和Prevost,2005年)。
因此,本发明的第一方面涉及本文所述的新型菌株或其突变体。
本发明的组合物除了活性组分之外,还可包含其农业化学上可接受的赋形剂和/或媒介物。本发明的组合物还包含农业化学上可接受的载体、媒介物和/或佐剂。
植物的主要作物有甘蔗、咖啡、大豆、棉花、玉米、马铃薯、番茄、烟草、香蕉、水稻、小麦、鳄梨、菠萝、西葫芦、可可豆、椰子、燕麦、洋葱、莴苣、甜菜头、胡萝卜、木薯、豆类、向日葵、胡椒、芜菁、苹果、草莓、秋葵、萝卜和洋葱。
对于水果种植:柑橘、葡萄、番石榴、番木瓜、无花果、桃、李和枇杷是特别相关的,并且对于园艺学,茄子和十字花科植物。
对于花卉栽培:玫瑰、菊花、洋桔梗、大丁草、孤挺花、秋海棠和鸡冠花。
本发明的组合物可以包被在植物种子上,并且可以包含约1.0×102CFU/种子至约1.0×109CFU/种子的量的芽孢杆菌纲(例如副地衣芽孢杆菌)的孢子。
植物种子可包括但不限于单子叶植物、双子叶植物、谷类、玉米、甜玉米、爆米花玉米、种子玉米、青贮玉米、田地玉米、水稻、小麦、大麦、高粱、芸苔属蔬菜、花椰菜、卷心菜、菜花、球芽甘蓝、羽衣甘蓝、无头甘蓝、芥菜、球茎甘蓝、鳞茎类蔬菜、洋葱、大蒜、小葱、果蔬、胡椒、番茄、茄子、地樱桃、绿番茄、秋葵、葡萄、香草/香料、葫芦科蔬菜、黄瓜、哈密瓜、甜瓜、香瓜、西葫芦、西瓜、南瓜、茄子、叶用蔬菜、莴苣、芹菜、菠菜、欧芹、菊苣、豆科植物/蔬菜(多汁的干豆子和豌豆)、豆类、青豆、豆荚、荚豆、大豆、干豆、鹰嘴豆、利马豆、豌豆、三角豆、裂荚豌豆、扁豆、油籽作物、芥花、蓖麻、棉花、亚麻、花生、油菜、红花、芝麻、向日葵、大豆、根/块茎和球茎类蔬菜、胡萝卜、马铃薯、甘薯、甜菜头、生姜、辣根、萝卜、人参、芜菁、甘蔗、甜菜、草或草皮草的种子。
进一步,植物种子可包括干豆、玉米、小麦、大豆、油菜、水稻、黄瓜、胡椒、番茄、西葫芦、棉花、草和草皮草的种子。
在一个可选的实施方案中,本发明的芽孢杆菌纲或组合物可加入到:植物周围的土壤或生长培养基;在将植物种子播种到土壤或生长培养基中之前的该土壤或生长培养基;或在土壤或生长培养基中种植植物、植物扦插物、植物嫁接物或植物愈伤组织之前的该土壤或生长培养基。
在一个或多个实施方案中,植物可包括大豆、豆类、豆荚、小麦、棉花、玉米、胡椒、番茄、马铃薯、木薯、葡萄、草莓、香蕉、花生、西葫芦、南瓜、茄子和黄瓜。
在本发明的组合物和方法中,病原体感染可以由多种植物病原体引起,这些植物病原体例如包括但不限于植物真菌病原体、植物细菌病原体、锈菌、葡萄孢属、灰葡萄孢、葱鳞葡萄孢、欧文氏菌属、胡萝卜软腐欧文氏菌、解淀粉欧文氏菌、狄克氏菌属、达旦提狄克氏菌、茄狄克氏菌、农杆菌属、根癌农杆菌、黄单胞菌属、白纹黄单胞菌、野油菜黄单胞菌胡萝卜致病变种、桃李黄单胞菌、鹅掌藤黄单胞菌、稻黄单胞菌稻生致病变种、木杆菌属、苛养木杆菌,韧皮杆菌属、韧皮杆菌,镰孢属、黄色镰孢、禾谷镰孢、尖孢镰孢、尖孢镰孢古巴专化型、尖孢镰孢番茄专化型、北美大豆猝死综合症病菌、核盘菌属、核盘菌、小核盘菌、银斑核盘菌、尾孢属/尾孢菌属、钩丝壳属、葡萄钩丝壳(白粉菌)、叉丝单囊壳属(白粉菌)、白叉丝单囊壳、秘密叉丝单囊壳、拟茎点霉属、霜霉病拟茎点霉菌、链格孢菌属、极链格孢菌、葱链格孢菌、交链孢、茄链格孢、细极链格孢、假单胞菌属、丁香假单胞菌番茄致病变种、疫霉属、致病疫霉、寄生疫霉、大豆疫霉菌、辣椒疫霉菌、肉桂疫霉菌、草莓疫霉菌、疫霉属、栎树疫霉菌、棕榈疫霉菌、烟草疫霉菌、层锈菌属、豆薯层锈菌、山马蝗层锈菌、曲霉菌属、黄曲霉、黑曲霉、单孢锈菌属、疣顶单孢锈菌、枝孢属、多主枝孢、根霉属、少根根霉、青霉属、丝核菌属、立枯丝核菌、玉蜀黍丝核菌、稻枯斑丝核菌、褐色丝核菌(Rhizoctonia caritae)、禾谷丝核菌、紫纹羽核菌、草莓丝核菌、多枝丝核菌、Rhizoctonia rubi、豆类丝核菌、壳球孢菌、稻痕病菌、球腔菌属、禾生球腔菌、斐济球腔菌(香蕉黑条叶斑病)、苹果斑点球腔菌、柑橘球腔菌、稻瘟菌属、稻瘟病菌、链核盘菌属、桃褐腐病菌、Monilinia vacciniicorymbosi(蓝莓干枯病菌)、核果链核盘菌、炭疽菌属、胶孢炭疽菌、尖孢炭疽菌、Colletotrichum Candidum、间座壳属、柑橘间座壳、棒孢属、多主棒孢菌、裸孢子囊菌属、美洲苹果锈病菌、裂盾菌属、粟酒裂殖杆菌、粘壳孢菌属、仁果粘壳孢菌、葡萄座腔菌属、杨树溃疡病菌、明孢盘菌属、Wilsonomyces的种、穿孔病菌(Wilsonomyces carpophilus)、单丝壳属、斑点单囊壳、蔷薇单囊壳、白粉菌属、壳多胞菌属、颖枯壳多孢、腐霉属、极腐霉、瓜果腐霉、不规则腐霉、胡萝卜腐霉、鲁特里亚腐霉、森林腐霉、黑星菌属、苹果黑星菌、轮枝孢属、黑粉菌属、大麦散黑粉菌、玉米黑粉菌、甘蔗黑粉菌、麦角菌属、麦角菌、腥黑粉菌属、小麦腥黑粉菌、小麦光腥黑粉菌、刚毛腥黑粉菌、小麦矮化腥黑粉菌、茎点霉属、大豆生茎点霉、短小茎点霉、甘蓝茎点霉、禾旋孢腔菌、小麦全蚀病菌、毛盘孢属、喙孢属、黑麦喙孢、平脐蠕孢属、长螺孢属、大麦长蠕孢、玉米长蠕孢、马铃薯长蠕孢和小麦褐斑长蠕孢或它们的组合。
在一些实施方案中,病原体感染可由以下项的一种或组合引起:大豆锈菌(豆薯层锈菌、山马蝗层锈菌)并且植物包括大豆;灰葡萄孢(灰霉病)并且植物包括葡萄;灰葡萄孢(灰霉病)并且植物包括草莓;灰葡萄孢(灰霉病)并且植物包括番茄;链格孢属(例如茄链格孢)并且植物包括番茄;链格孢属(例如茄链格孢)并且植物包括马铃薯;菜豆锈病(疣顶单胞锈菌)并且植物包括菜豆;扩散叉丝壳(大豆白粉病)并且植物包括大豆;斐济球腔菌(香蕉黑条叶斑病)或尖孢镰孢古巴专化型(巴拿马病)并且植物包括香蕉;黄单胞菌属或稻黄单胞菌稻生致病变种并且植物包括水稻;白纹黄单胞杆菌并且植物包括木薯;野油菜黄单胞菌并且植物包括番茄;灰葡萄孢(胡椒灰霉病)并且植物包括胡椒;白粉菌并且植物包括葫芦;核盘菌(白霉病)并且植物包括豆荚;核盘菌(白霉病)并且植物包括马铃薯;银斑核盘菌(币斑病)并且植物包括草皮草;南方白霉并且植物包括花生;叶斑病(尾孢属/尾孢菌属)并且植物包括花生;禾谷镰孢(小麦赤霉病)并且植物包括小麦;禾生球腔菌(小麦叶枯病)并且植物包括小麦;颖枯壳多孢(稃枯病和小麦颖枯病)并且植物包括小麦;解淀粉欧文氏菌并且植物包括苹果、梨和其他仁果;苹果黑星菌并且植物包括苹果、梨和其他仁果;或立枯丝核菌并且植物包括小麦、水稻、草皮草、大豆、玉米、豆科植物和蔬菜作物。包含如本文所述的杆菌菌株的组合物可以是液体、油分散剂、粉剂、干燥的可湿性粉剂、可涂抹粒剂或干燥的可湿性粒剂的形式。更具体地,该组合物可以是例如乳油(EC)、悬浮剂(SC)、悬乳剂(SE)、胶囊悬浮剂(CS)、水分散粒剂(WG)、乳粒剂(EG)、油包水乳剂(EO)、水包油乳剂(EW)、微乳剂(ME)、油分散剂(OD)、油悬浮剂(OF)、油溶性液剂(OL)、可溶性液剂(SL)、超低容量悬浮剂(SU)、超低容量液剂(UL)、可分散液剂(DC)、可湿性粉剂(WP)或任何技术上可行的制剂,与农业上可接受的助剂组合。
因此,本发明涉及包含副地衣芽孢杆菌DSM 33238、DSM 33239、DSM 33240、DSM33241、DSM 33242、DSM 33243、DSM 33244或其突变体的组合物,并且涉及包含该组合物或通过制备该组合物的方法制备的试剂盒,以及说明和合适的容纳器。因此,本发明还涉及一种制备组合物的方法,该组合物包含副地衣芽孢杆菌DSM 33238、DSM 33239、DSM 33240、DSM 33241、DSM 33242、DSM 33243、DSM 33244或其突变体以及农业化学上可接受的载体、媒介物和/或佐剂,并且还涉及该组合物用于防治、对抗植物线虫和/或赋予对植物线虫的特异抗性的用途。
此外,本发明涉及有效量的副地衣芽孢杆菌DSM 33238、DSM 33239、DSM 33240、DSM 33241、DSM 33242、DSM 33243、DSM 33244或其突变体在制备在植物培养中具有植物生长促进作用的农业化学品组合物中的用途,以及促进植物健康的方法。
在一个优选的方面,本发明涉及bioF基因具有突变的芽孢杆菌属,其中该突变与bioF基因具有突变的芽孢杆菌属的亲代菌株相比,改变了由bioF编码的蛋白质的酶动力学。
在另一个优选的方面,本发明涉及hrcA基因具有突变的芽孢杆菌属,其中该突变与其亲代菌株相比,使由hrcA编码的蛋白质功能失调,例如丧失功能。
此外,本发明涉及以下方面:
方面1.一种芽孢杆菌属,其在bioF和/或hrcA基因中具有突变。
方面2.一种芽孢杆菌属,当与以DSM33110保藏的副地衣芽孢杆菌中的hrcA和bioF的相应直系同源基因相比时,所述芽孢杆菌属在hrcA和/或bioF基因中具有突变。
方面3.一种芽孢杆菌属,其当与SEQ ID NO:1相比时在hrcA基因中具有突变,和/或当与SEQ ID NO:3相比时在bioF基因中具有突变。
方面4.一种芽孢杆菌属,其选自以保藏号DSM 33238、DSM 33239、DSM 33240、DSM33241、DSM 33242、DSM 33243、DSM 33244保藏在德国微生物保藏中心(Deutsche Sammlungvon Mikroorganismen und Zellkulturen)的菌株。
方面5.根据前述方面中任一项所述的芽孢杆菌属,其中所述芽孢杆菌属的所述bioF基因的最近直系同源物与SEQ ID NO:1具有小于100%,例如小于99%、小于98%的序列同一性。
方面6.根据前述方面中任一项所述的芽孢杆菌属,其中所述芽孢杆菌属的所述bioF基因的最近直系同源物与SEQ ID NO:1具有至少95%,例如至少96%、至少97%、至少98%、至少99%的序列同一性。
方面7.根据前述方面中任一项所述的芽孢杆菌属,其中所述副地衣芽孢杆菌的所述hrcA基因的最近直系同源物与SEQ ID NO:3具有小于100%,例如小于99%、小于98%的序列同一性。
方面8.根据前述方面中任一项所述的芽孢杆菌属,其中所述芽孢杆菌属的所述hrcA基因的最近直系同源物与SEQ ID NO:3具有至少95%,例如至少96%、至少97%的序列同一性。
方面9.根据前述方面中任一项所述的芽孢杆菌属,其中所述突变为缺失、取代或***。
方面10.根据前述方面中任一项所述的芽孢杆菌属,其中所述突变导致移码、引入终止密码子或影响编码蛋白质的动力学。
方面11.根据前述方面中任一项所述的芽孢杆菌属,其中由所述hrcA和/或bioF基因中的一个或多个或它们的最近直系同源物中的一个或多个编码的蛋白质在结构上受所述突变影响。
方面12.根据前述方面中任一项所述的芽孢杆菌属,其中所述菌株的基因组与以保藏号DSM33110保藏在德国微生物保藏中心的菌株的基因组具有至少99%,例如至少99.5%,例如至少99.8%,例如至少99.9%的同一性。
方面13.根据前述方面中任一项所述的芽孢杆菌属,其中所述芽孢杆菌属选自:地衣芽孢杆菌、解淀粉芽孢杆菌、副地衣芽孢杆菌、蜡状芽孢杆菌、贝莱斯芽孢杆菌、巨大芽孢杆菌。
方面14.根据前述方面中任一项所述的芽孢杆菌属,其中所述菌株衍生于以保藏号DSM33110保藏在德国微生物保藏中心的菌株,或与以保藏号DSM33110保藏在德国微生物保藏中心的菌株共享表型或基因型特征的菌株。
方面15.根据前述方面中任一项所述的芽孢杆菌属,其中所述芽孢杆菌属具有以保藏号DSM 33238、DSM 33239、DSM 33240、DSM 33241、DSM 33242、DSM 33243、DSM 33244保藏在德国微生物保藏中心的副地衣芽孢杆菌菌株中的一个或多个的表型或基因型特征。
方面16.根据前述方面中任一项所述的芽孢杆菌属,当在MSgg和/或MS-rex培养基中于21℃生长时,所述芽孢杆菌属与以保藏号DSM33110保藏在德国微生物保藏中心的副地衣芽孢杆菌相比,显示提高的生长速率。
方面17.根据方面16所述的芽孢杆菌属,其中所述生长速率是如本文实施例2中所述被测定的。
方面18.根据方面16或17中任一项所述的芽孢杆菌属,其中所述生长速率比亲代菌株的生长速率高至少20%,例如至少40%,例如至少60%,例如至少80%,例如至少100%。
方面19.一种组合物,所述组合物包含根据前述方面中任一项所述的芽孢杆菌属。
方面20.一种组合物,所述组合物包含根据前述方面中任一项所述的芽孢杆菌属及其农业化学上可接受的赋形剂和/或载体。
方面21.根据方面19或20中任一项所述的组合物,所述组合物进一步包含以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在的微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂、除草剂、植物提取物、植物生长调节剂或肥料中的一种或组合,载体、表面活性剂、分散剂或酵母提取物。
方面22.根据方面19至21中任一项所述的组合物或根据方面1至18中任一项所述的芽孢杆菌属作为生物刺激素和/或生物杀线虫剂的用途。
方面23.根据方面19至21中任一项所述的组合物或根据方面1至18中任一项所述的芽孢杆菌属用于防治、对抗植物线虫和/或赋予对植物线虫的特异抗性的用途。
方面24.根据方面22或23中任一项所述的用途,其中所述植物线虫选自由以下组成的组:根结线虫属、短体线虫属、异皮线虫属、孢囊线虫属、茎线虫属、垫刃线虫属、剑线虫属、穿孔线虫属、肾形线虫属、螺旋线虫属和刺线虫属。
方面25.根据方面22至24中任一项所述的用途,其中所述植物线虫选自由以下组成的组:南方根结线虫、爪哇根结线虫、短小根结线虫、巴拉那根结线虫、大豆孢囊线虫和玉米短体线虫。
方面26.根据方面22至25中任一项所述的用途,其中将根据方面19至21中任一项所述的组合物或根据方面1至18中任一项所述的芽孢杆菌属施用于植物、种子上或植物的生境中。
方面27.根据方面26所述的用途,其中所述植物选自由以下组成的组:玉米、水稻、甘蔗、大豆、马铃薯、胡萝卜、咖啡和香蕉。
方面28.一种赋予提高的对植物线虫的抗性的方法,所述方法包括将有效量的根据方面1至18中任一项所述的芽孢杆菌属或根据方面19至21中任一项所述的组合物施用于植物和/或它们的生境。
方面29.一种试剂盒,所述试剂盒包括如方面19至21中任一项所定义的组合物、使用说明和合适的容器。
方面30.一种植物种子,所述植物种子包被有根据方面19至21中任一项所述的组合物,所述组合物以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在。
方面31.根据方面30所述的植物种子,其中所述组合物包含约1.0×102CFU/种子至约1.0×109CFU/种子的量的根据方面1至18中任一项所述的芽孢杆菌属的孢子。
方面32.根据方面30或31所述的植物种子,其中所述组合物进一步包含以适于有利于植物生长和/或在易感植物中赋予免遭病原体感染的保护的量存在的微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂或植物生长调节剂中的一种或组合。
方面33.根据方面1至18中任一项所述的芽孢杆菌属,其中BioF由SEQ ID NO:1的DNA序列或SEQ ID NO:2的氨基酸序列或其同源物编码,和/或HrcA由SEQ ID NO:3的DNA序列或其氨基酸序列同源物编码。
项目1.一种副地衣芽孢杆菌,当与以保藏号为DSM33110保藏在德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌中hrcA和/或bioF的相应直系同源基因相比,所述副地衣芽孢杆菌在hrcA和/或bioF基因中具有突变。
项目2.一种副地衣芽孢杆菌,当与例如以DSM33110保藏在德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌中由SEQ ID NO:1编码的hrcA和/或由SEQ ID NO:3编码的bioF的相应直系同源基因相比,所述副地衣芽孢杆菌在hrcA和/或bioF基因中具有突变。
项目3.根据项目1或2中任一项所述的副地衣芽孢杆菌,其中所述副地衣芽孢杆菌为衍生于保藏为DSM33110的副地衣芽孢杆菌的突变体。
项目4.一种副地衣芽孢杆菌,所述副地衣芽孢杆菌以DSM 33238、DSM 33239、DSM33240、DSM 33241、DSM 33242、DSM 33243或DSM 33244保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)。
项目5.根据项目1至4中任一项所述的副地衣芽孢杆菌,其中所述bioF基因中的所述突变引起基因型变化,所述基因型变化导致Pro314至Ser、Ala244至Val、Asp300至Gly和/或Arg37至Trp的氨基酸变化。
项目6.根据项目1至5中任一项所述的副地衣芽孢杆菌,其中所述hrcA基因中的所述突变引起基因型变化,所述基因型变化导致终止密码子,从而使HrcA抑制子失活。
项目7.根据前述项目中任一项所述的副地衣芽孢杆菌,当在MSgg中于21℃生长时,所述副地衣芽孢杆菌与以保藏号DSM33110保藏在德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌相比,显示提高的最大生长速率。
项目8.根据项目7所述的副地衣芽孢杆菌,其中所述最大生长速率是如本文实施例2中所述被测定的。
项目9.根据前述项目中任一项所述的副地衣芽孢杆菌,其中根据前述项目中任一项所述的副地衣芽孢杆菌菌株的生长速率比以保藏号DSM33110保藏在德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌的生长速率高至少20%,例如至少40%,例如至少60%,例如至少80%,例如至少100%。
项目10.根据前述权利要求中任一项所述的副地衣芽孢杆菌,所述副地衣芽孢杆菌在限菌植物***(gnotobiotic plant system)中施用于拟南芥Col-0植物并于22℃和/或15℃生长时,能够增加所述拟南芥的总叶面积、鲜重或叶绿素含量。
项目11.一种组合物,所述组合物包含根据前述项目中任一项所述的副地衣芽孢杆菌及其农业化学上可接受的赋形剂和/或载体。
项目12.根据项目11所述的组合物,所述组合物进一步包含以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在的微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂、除草剂、植物提取物、植物生长调节剂或肥料中的一种或组合,载体、表面活性剂、分散剂或酵母提取物。
项目13.根据项目11至12中任一项所述的组合物或根据项目1至9中任一项所述的副地衣芽孢杆菌作为生物刺激素、生长促进剂和/或生物杀线虫剂的用途。
项目14.根据项目13所述的用途,其中所述植物线虫选自由以下组成的组:根结线虫属、短体线虫属、异皮线虫属、孢囊线虫属、茎线虫属、垫刃线虫属、剑线虫属、穿孔线虫属、肾形线虫属、螺旋线虫属和刺线虫属,例如南方根结线虫、爪哇根结线虫、短小根结线虫、巴拉那根结线虫、大豆孢囊线虫和玉米短体线虫。
项目15.根据项目13至14中任一项所述的用途,其中将根据项目11至12中任一项所述的组合物或根据项目1至10中任一项所述的副地衣芽孢杆菌施用于植物、种子上或植物的生境中,例如土壤中。
项目16.根据项目13至15中任一项所述的用途,其中所述植物选自由以下组成的组:玉米、水稻、甘蔗、大豆、马铃薯、胡萝卜、咖啡和香蕉。
项目17.一种植物种子,所述植物种子包被有根据项目11至12中任一项所述的组合物,所述组合物以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在。
项目18.根据项目17所述的植物种子,其中所述组合物包含约1.0×102CFU/种子至约1.0×109CFU/种子的量的根据项目1至10中任一项所述的芽孢杆菌属的孢子。
项目19.根据项目18或18所述的植物种子,其中所述组合物进一步包含微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂或植物生长调节剂中的一种或组合,所述一种或组合以适于有利于植物生长和/或在易感植物中赋予免遭病原体感染的保护的量存在。
下文中给出的说明性实施例用于更好地描述本发明。然而,所述的制剂仅仅涉及本发明的一些实施方案的一些方式,不应视为对本发明范围的限制。
如本公开中所使用的,菌株描述符号根据下表可互换地使用:
实施例
生长培养基组合物:
MSgg培养基(pH7)(过滤除菌):
1×MOPS溶液:
100mM吗啉丙磺酸(MOPS)(pH7)
5mM KH2PO4/K2HPO4
1×微量元素溶液:
50μM MnCl2
1μM ZnCl2
100μM FeCl3
2mM MgCl2
700μM CaCl2
2μM硫胺素
氨基酸补充剂
50μg/mL苏氨酸、色氨酸和/或苯丙氨酸
碳源
0.5%谷氨酸盐
0.5%甘油
MS-REX(pH7)过滤除菌
Murashige和Skoog(MS)培养基是在实验室中用于植物或植物细胞培养物培养的植物生长培养基。MS中补加了从水培玉米植物培养物收集的玉米根分泌物(1×)。根分泌物已收集在水中,并进行冷冻干燥以获得根分泌物的浓缩物(25×)。
MS组合物
主要盐(常量营养素)/1L
硝酸铵(NH4NO3)1,650mg/L
氯化钙(CaCl2·2H2O)440mg/L
硫酸镁(MgSO4·7H2O)370mg/L
磷酸二氢钾(KH2PO4)170mg/L
硝酸钾(KNO3)1,900mg/L
次要盐(微量营养素)/1L
硼酸(H3BO3)6.2mg/L
氯化钴(CoCl2·6H2O)0.025mg/L
硫酸亚铁(FeSO4·7H2O)27.8mg/L
硫酸锰(II)(MnSO4·4H2O)22.3mg/L
碘化钾(KI)0.83mg/L
钼酸钠(Na2MoO4·2H2O)0.25mg/L
硫酸锌(ZnSO4·7H2O)8.6mg/L
乙二胺四乙酸铁钠(NaFe-EDTA),构成每升水中含5.57g FeSO4·7H2O和7.45gNa2-EDTA的5mL/L储备溶液。
硫酸铜(CuSO4·5H2O)0.025mg/L
维生素和有机化合物/1L
肌醇100mg/L
烟酸0.5mg/L
盐酸吡哆醇0.5mg/L
盐酸硫胺素1.0mg/L
甘氨酸2mg/L
实施例1:适应性实验室进化(ALE)
我们进行了适应性实验室进化(ALE)活动,以培育在低温条件下生长速率提高的副地衣芽孢杆菌DSM33110衍生物。选择了两种不同的生长培养基平行进行该进化过程。12个独立的培养物(每种生长培养基6个)生长过夜,并每天稀释并转移至新鲜培养基(参见图1)。首先,从副地衣芽孢杆菌DSM33110细胞培养物(OD600 1)的储备液接种MSgg或MS-Rex生长培养基。每天监测DSM33110传代培养物的光密度,之后稀释并转移至新鲜的生长培养基中。每周将每管的样品制备成两份甘油储备液。一份用于新一轮的生长传代的接种,另一份则冷冻保存。在ALE活动的12周内,温度从25℃逐渐降低至18℃。计算达到的总传代数,经过12周的适应性进化,培养物生长了120-140代。将进化实验的最终样品连续稀释并平板接种在LB琼脂平板上以获得分离的菌落。用菌落挑选器机器人挑取2×960个克隆,并在96孔板中生长以生成单独的甘油储备液。
实施例2:生长速率分析
为了比较适应衍生物和亲代菌株之间的生长曲线和生长速率,将2×958个克隆(加上亲代菌株DSM33110)接种到含有MSgg或MS-Rex培养基的96深孔板中。在21℃时在生长曲线测量仪中进行生长实验。获得的数据允许鉴定生长较快的衍生物以及选择30个最佳的单独克隆。然后对生长曲线进行一式三份测定以确保再现性。将斜率函数应用于指数期OD600记录值,计算生长速率。Umax值对应于OD600单位每小时(h-1)的变化。图2示出了从各生长培养基中培育的七个最佳衍生物的平均生长数据。图3示出了衍生物和亲代菌株DSM33110的最大生长速率计算值(μmax)。
实施例3:改良衍生物的基因组测序和SNP分析
最终选出的克隆(2×7)保藏在CHCC保藏中心中并进行基因组测序。通过将选出的冷适应菌株的基因组序列与DSM33110基因组序列进行比较来进行单核苷酸多态性(SNP)分析。
表1.SNP分析和ORF靶点鉴定。表中包含将冷适应衍生ID/名称与相应的CHCC编号联系起来的信息。总结了SNP的特性和出现频率。
在14个已测序的冷适应DSM33110衍生物中,有8个显示出靶向bioF orf的突变。bioF编码8-氨基-7-氧代壬酸合酶(EC 2.3.1.47),其为生物素辅因子生物合成途径中的酶。参见下文对鉴定出的SNP的生理学解释。
基因组测序结果显示,编码热诱导转录抑制子HrcA的orf hrcA内存在若干个SNP(在14个衍生物中发现5个)。在大多数情况下,鉴定出的突变对应于终止密码子,并且我们仅在其中一个突变株中鉴定出Pro314至Thr的氨基酸变化。最有可能的情况是,所有这些突变都使抑制子失活。
实施例4:生长优势特异性分析
为了评估生长改良表型是否局限于特定的生长培养基组成或低温,我们比较了冷适应衍生物与亲代菌株DSM33110在30℃时在复合培养基(LB和BHI)中的生长曲线。
在冷适应衍生物中观察到更高的生物量产量和更快的生长速率,这表明突变不仅有助于菌株在低温下生长。在适应性进化过程中,我们选择了快速生长者和达到更高产量的衍生物。
实施例5:植物生长促进实验
在以使用填充有琼脂固化的植物生长培养基的24孔板为基础的限菌***中,使用拟南芥Col-0植物在22℃和15℃时比较衍生菌株之间的植物生长促进效率。基于三次独立实验的结果,衍生菌株CA4、CA6和CAREX1(分别为CHCC36494、CHCC36496和CHCC36751)在土壤和琼脂***中均被选为最佳冷适应DSM33110衍生物。结果示于图5中。
为了进一步评估冷适应选出的衍生物的植物适应性促进作用,应用团队进行了一系列实验。在土壤***中使用拟南芥植物比较衍生菌株之间的植物生长促进效率。在植物生长室中进行实验。将含有冷适应改良的衍生物CA4和CA6的两种不同菌株组合与亲代菌株促进拟南芥植物生长的能力进行比较。如图19所示,副地衣芽孢杆菌CHCC36494(CA4)和CHCC36496(CA6)与2种不同的枯草芽孢杆菌菌株共同接种。接种前,使拟南芥幼苗发芽并在盆栽土中预生长7天。细菌培养物在接种前一天生长过夜。在接种当天的早晨,细菌培养物进行稀释并在LB肉汤中生长至OD600 1。然后将细菌细胞洗涤并以OD600 0.01重悬于10mMMgSO4缓冲液中。以1e6 CFU接种植物(每个菌株接种8株植物),将未接种对照植物与亲代菌株和冷适应衍生物的结果进行比较。在两个不同温度(22℃和15℃)以及16h光照期/8h暗期的光周期的条件下,植物在生长室中生长2周以评估这些菌株在较低的次优Ta条件下的性能。接种后2周进行芽鲜重定量。获得的结果显示,与未接种植物相比,所有处理的植物生长都有增加,其中在含有冷适应改良衍生物CA6的两种组合中观察到更相关的增加(在22℃和15℃温度下分别增加19%-40%和36%-36%)。含有衍生物CA6(CHCC36496)的菌株组合在22℃和15℃下均具有最佳结果。然而,与未接种的植物相比,仅有一种菌株组合在15℃下显示统计学显著的结果。因此,选择菌株组合CA6(CHCC36496)加枯草芽孢杆菌DSM33015在作物和田间试验中进行另外的测试。
实施例6:与鉴定出的SNP相关的MoA的生理学解释
BioF-生物素(也称为维生素H)是所有形式的生命都需要的共价结合的酶辅因子。在8个突变序列中,有5个的基因型变化使蛋白质具有Pro314至Ser的氨基酸变化。在其余3个突变序列中,氨基酸变化是不同的(Ala244至Val、Asp300至Gly和Arg37至Trp)。BioFDSM33110氨基酸序列的同源性检索、ClustalW比对和蛋白质3D结构建模表明,选出的SNP导致的氨基酸变化很可能不是催化位点的一部分(1)(图6)。这些SNP如何影响BioF酶的活性和细胞内的生物素水平目前还在研究中。
生物素是在脂肪酸合成、氨基酸代谢和糖原异生过程中进行羧化和脱羧反应的多种关键代谢酶所需的必需辅因子(2)。据报道,在蜡状芽胞杆菌中,有限量的生物素限制了生长并改变了细胞膜脂肪酸组成(3)。在芽孢杆菌属中,脂肪酸和磷脂合成中的第一中间体是丙二酰辅酶A。丙二酰辅酶A是通过accBC基因上编码的乙酰辅酶A羧化酶由乙酰辅酶A合成的。芽孢杆菌的accBC操纵子编码乙酰辅酶A羧化酶的两个亚基,即生物素羧基载体和生物素羧化酶。此外,还报道了枯草芽孢杆菌中脂肪酸accBC基因的转录水平与细胞生长速率之间的直接相关性(4)。
HrcA-HrcA先前在枯草芽孢杆菌中被描述为I类热激基因的转录抑制子,即HrcA调节子(5)。HrcA调节子仅由七顺反子dnaK操纵子和双顺反子groE这两个操纵子组成(图7)。这两个操纵子均编码分子伴侣并对热应激作出反应。HrcA与位于两个操纵子中并称为CIRCE(伴侣表达控制反向重复序列)元件的反向重复序列结合。DnaK、DnaJ、GrpE和GroES-GroEL是对低温和高温处理响应而过表达的伴侣(Schumann,W.,2003年)。枯草芽孢杆菌hrcA基因敲除导致两个I类热激操纵子在不存在热激的情况下具有高组成型表达。此外,枯草芽孢杆菌中hrcA的缺失有助于菌株在温度变化(热或冷激)后更快地恢复生长。相应地,HrcA过表达具有相反的作用,并且细胞对温度变化的反应能力较差(6)。
对冷适应衍生物的基因组分析鉴定出了影响hrcA orf的突变。在大多数情况下,这些SNP对应于终止密码子,并且我们仅在其中一个突变菌株中鉴定出Pro314至Thr的氨基酸变化。最有可能的情况是,所有这些突变都使抑制子失活,从而使调节子中的两个操纵子过度表达。
结论
综上所述,经过了适应性实验室进化活动,从副衣地芽孢杆菌(DSM33110)中培育出了在不同温度下具有提高的生长速率和更高的生物量产量的菌株。选择了14种不同的衍生物,并对它们的基因组进行测序以鉴定所获得的基因型变化。对衍生菌株进行生理性表征,并在植物生长实验中测试其性能。
基于这些结果,提出了解释观察到的与亲代菌株的表型差异的具体机制。
此外,这些进化实验还提供/贡献了在植物生长促进方面显示改良特性的新型芽孢杆菌属菌株。
在一个优选的方面,本文所用的术语“bioF基因”意指BioF区8-氨基-7-氧代壬酸合酶(EC_2.3.1.47)。
在另一个优选的方面,本文所用的术语“hrcA基因”意指热诱导转录抑制子HrcA。
保藏和专家方案
申请人要求,在授予专利之日前,下文所述的保藏微生物的样品仅可由专家获得。
申请人于2019年5月8日将副地衣芽孢杆菌亲代菌株以DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心。
申请人于2019年8月14日将衍生于亲代菌株的菌株以如下保藏号保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心:
副地衣芽孢杆菌CHCC32530=DSM 33238
副地衣芽孢杆菌CHCC36494=DSM 33239
副地衣芽孢杆菌CHCC36496=DSM 33240
副地衣芽孢杆菌CHCC36751=DSM 33241
副地衣芽孢杆菌CHCC36753=DSM 33242
副地衣芽孢杆菌CHCC36754=DSM 33243
副地衣芽孢杆菌CHCC36755=DSM 33244
根据国际承认用于专利程序的微生物保存布达佩斯条约进行保藏。
序列
所附的序列表构成本说明书的一部分。
如序列表中所指明的,序列
SEQ ID NO:1定义hrcA核苷酸序列
SEQ ID NO:2定义HrcA蛋白质序列
SEQ ID NO:3定义bioF核苷酸序列
SEQ ID NO:4定义BioF蛋白质序列。
序列表
<110>科·汉森有限公司
<120> P6888新型冷适应芽孢杆菌纲
<130> P6888
<160> 7
<170> BiSSAP 1.3.6
<210> 1
<211> 1032
<212> DNA
<213>副地衣芽孢杆菌(Bacillus paralicheniformis)
<220>
<223>副地衣芽孢杆菌CHCC3809中的热诱导转录抑制子HrcA核苷酸序列
<400> 1
atgttaacaa atcgtcagct gttaatcttg caggttatcg tcaacgattt cattcgttca 60
gctcagccgg taggatcaag aacgctttcc aaaaaggaag atatcacatt cagctcagca 120
acgatcagaa acgaaatggc tgacttggaa gagctcggtt ttattgaaaa aacccactca 180
tcttcaggcc ggattccttc tgaaaaaggc tatcgctatt atgtcgatca tctgctttca 240
cccggaaagc tgtcaaaaac ggacttgaac attattcatt cggttttcaa agaaaaaatc 300
tttgaactcg aaaaagcggt gcagaagtcg gctcaagtgc tgtctgatct gacaaattat 360
acatcgattg tcctcggtcc gagactgagc gaaaatcatc tcaaacagat ccagattgtg 420
ccgattcagc ctaagaaggc cgttgccatt ctagtaacga ataccggcca tgtcgagaat 480
aaaacgatca actttccggc ggaggtcaat ctttccgatc tcgaaaagct ggtgaatata 540
ttaaatgaac gccttagagg cgtgccgatc tcagagctga aagacaggat tttcaaagag 600
gtcgtcatct tcttaaagtc gcatatccaa aattacgata cgattttaca cgggctcggc 660
gcaacgctgg attcatctgt tcaaaccgac cggctgtttt tcggcggcaa gattaatatg 720
ctgaatcagc ccgaatttca cgatattgac agagtgaaat cgctattgtc gctcattgag 780
aaagaacagg agcttctccg gctctttcag tcgactgagt ccggaattac cattaaaatc 840
ggcaaggaaa acgactatga agaaatggaa aactgcagcc tgattaccgc gacatacacg 900
gtcggttcaa aacagatcgg ctccatcgcg gtcatcgggc cgacgcgcat ggactactcc 960
cgcgtcgtcg gtttgcttca gcacgtatca tctgacttgt caaaagcgtt gacaagtttg 1020
tatgatgggt aa 1032
<210> 2
<211> 343
<212> PRT
<213>副地衣芽孢杆菌
<220>
<223>副地衣芽孢杆菌CHCC3809中的热诱导转录抑制子HrcA蛋白质序列
<400> 2
Met Leu Thr Asn Arg Gln Leu Leu Ile Leu Gln Val Ile Val Asn Asp
1 5 10 15
Phe Ile Arg Ser Ala Gln Pro Val Gly Ser Arg Thr Leu Ser Lys Lys
20 25 30
Glu Asp Ile Thr Phe Ser Ser Ala Thr Ile Arg Asn Glu Met Ala Asp
35 40 45
Leu Glu Glu Leu Gly Phe Ile Glu Lys Thr His Ser Ser Ser Gly Arg
50 55 60
Ile Pro Ser Glu Lys Gly Tyr Arg Tyr Tyr Val Asp His Leu Leu Ser
65 70 75 80
Pro Gly Lys Leu Ser Lys Thr Asp Leu Asn Ile Ile His Ser Val Phe
85 90 95
Lys Glu Lys Ile Phe Glu Leu Glu Lys Ala Val Gln Lys Ser Ala Gln
100 105 110
Val Leu Ser Asp Leu Thr Asn Tyr Thr Ser Ile Val Leu Gly Pro Arg
115 120 125
Leu Ser Glu Asn His Leu Lys Gln Ile Gln Ile Val Pro Ile Gln Pro
130 135 140
Lys Lys Ala Val Ala Ile Leu Val Thr Asn Thr Gly His Val Glu Asn
145 150 155 160
Lys Thr Ile Asn Phe Pro Ala Glu Val Asn Leu Ser Asp Leu Glu Lys
165 170 175
Leu Val Asn Ile Leu Asn Glu Arg Leu Arg Gly Val Pro Ile Ser Glu
180 185 190
Leu Lys Asp Arg Ile Phe Lys Glu Val Val Ile Phe Leu Lys Ser His
195 200 205
Ile Gln Asn Tyr Asp Thr Ile Leu His Gly Leu Gly Ala Thr Leu Asp
210 215 220
Ser Ser Val Gln Thr Asp Arg Leu Phe Phe Gly Gly Lys Ile Asn Met
225 230 235 240
Leu Asn Gln Pro Glu Phe His Asp Ile Asp Arg Val Lys Ser Leu Leu
245 250 255
Ser Leu Ile Glu Lys Glu Gln Glu Leu Leu Arg Leu Phe Gln Ser Thr
260 265 270
Glu Ser Gly Ile Thr Ile Lys Ile Gly Lys Glu Asn Asp Tyr Glu Glu
275 280 285
Met Glu Asn Cys Ser Leu Ile Thr Ala Thr Tyr Thr Val Gly Ser Lys
290 295 300
Gln Ile Gly Ser Ile Ala Val Ile Gly Pro Thr Arg Met Asp Tyr Ser
305 310 315 320
Arg Val Val Gly Leu Leu Gln His Val Ser Ser Asp Leu Ser Lys Ala
325 330 335
Leu Thr Ser Leu Tyr Asp Gly
340
<210> 3
<211> 1152
<212> DNA
<213>副地衣芽孢杆菌
<220>
<223>副地衣芽孢杆菌CHCC3809中的BioF区8-氨基-7-氧代壬酸合酶(EC_2.3.1.47)核苷酸序列
<400> 3
gtgcccattg acgaatggct cagcagtcgg ctcgccagga cgaaggcagc cgggctttac 60
cgctcgctta aaccgcctca agctgtcgcg gaagcgaaac gtacgaatcg ggcgtcaaac 120
gattatttga gcctggcgaa cgacaaacgg ctgattcacg ccgcagaaac ggcgcttcgc 180
cgctttggag ccgggagcac agggtcaagg ctgacgtccg gcaataccga atggcatgaa 240
aaacttgagc ggaaaatcgc cggttttaag cagacagaag ccgctctctt gttttcaagc 300
ggatatttgg ccaatatcgg cgtgctttcg tctttgcctg aaaaaggaga cgtcatatta 360
agcgatcaat tgaaccatgc gagcatcatc gacggctgcc gcctttcaaa agctgatacg 420
gttgtttacc gccacacgga tatgaatgat cttgaagaaa aactccgcac cgcacagagc 480
cgtgctcgct gttttattgt gacagacggc gtgttcagca tggacggcac aattgcaccg 540
cttgacgaaa tcatgctcct tgccaagcgg taccgggcct ttgtcatcgt tgatgatgcc 600
cacgcaacag gggttttggg agatgccggc aggggaacgg gcgagtattt cggcgtctcc 660
ccagatgttg tgatcggcac gttaagtaaa gctgtaggcg cggaaggcgg cttcgtcgcg 720
ggctctaaag cattgatcga ttttttgctg aatcatgcga gaacgtttat ctttcaaacg 780
gctgtcccgc ccgcaagctg tgcggcggca tgcagagctt tagacatcat taaagacagc 840
cgggataaac gccggctttt gcaatcatcg gtgaccacga tcaaacgggg tctcgaggat 900
atcgggttta cggtcaaggg agaagatacg ccgatcatac ccgttatgat cggagaccct 960
caaaaagccg ttcgatttgc aaacggcctc aaggagaaag ggattgaggc cccggccatc 1020
cgcccgccga ctgttgcgga aggagagagc cgaatcaggc tgaccgtcac cgcagatcgc 1080
aagttaagag atattgaagc tctattagag gggtttaaat tagtgggaag agagttgaac 1140
ttggtgaaat ga 1152
<210> 4
<211> 383
<212> PRT
<213>副地衣芽孢杆菌
<220>
<223>副地衣芽孢杆菌CHCC3809中的BioF区8-氨基-7-氧代壬酸合酶(EC_2.3.1.47)蛋白质序列
<400> 4
Met Pro Ile Asp Glu Trp Leu Ser Ser Arg Leu Ala Arg Thr Lys Ala
1 5 10 15
Ala Gly Leu Tyr Arg Ser Leu Lys Pro Pro Gln Ala Val Ala Glu Ala
20 25 30
Lys Arg Thr Asn Arg Ala Ser Asn Asp Tyr Leu Ser Leu Ala Asn Asp
35 40 45
Lys Arg Leu Ile His Ala Ala Glu Thr Ala Leu Arg Arg Phe Gly Ala
50 55 60
Gly Ser Thr Gly Ser Arg Leu Thr Ser Gly Asn Thr Glu Trp His Glu
65 70 75 80
Lys Leu Glu Arg Lys Ile Ala Gly Phe Lys Gln Thr Glu Ala Ala Leu
85 90 95
Leu Phe Ser Ser Gly Tyr Leu Ala Asn Ile Gly Val Leu Ser Ser Leu
100 105 110
Pro Glu Lys Gly Asp Val Ile Leu Ser Asp Gln Leu Asn His Ala Ser
115 120 125
Ile Ile Asp Gly Cys Arg Leu Ser Lys Ala Asp Thr Val Val Tyr Arg
130 135 140
His Thr Asp Met Asn Asp Leu Glu Glu Lys Leu Arg Thr Ala Gln Ser
145 150 155 160
Arg Ala Arg Cys Phe Ile Val Thr Asp Gly Val Phe Ser Met Asp Gly
165 170 175
Thr Ile Ala Pro Leu Asp Glu Ile Met Leu Leu Ala Lys Arg Tyr Arg
180 185 190
Ala Phe Val Ile Val Asp Asp Ala His Ala Thr Gly Val Leu Gly Asp
195 200 205
Ala Gly Arg Gly Thr Gly Glu Tyr Phe Gly Val Ser Pro Asp Val Val
210 215 220
Ile Gly Thr Leu Ser Lys Ala Val Gly Ala Glu Gly Gly Phe Val Ala
225 230 235 240
Gly Ser Lys Ala Leu Ile Asp Phe Leu Leu Asn His Ala Arg Thr Phe
245 250 255
Ile Phe Gln Thr Ala Val Pro Pro Ala Ser Cys Ala Ala Ala Cys Arg
260 265 270
Ala Leu Asp Ile Ile Lys Asp Ser Arg Asp Lys Arg Arg Leu Leu Gln
275 280 285
Ser Ser Val Thr Thr Ile Lys Arg Gly Leu Glu Asp Ile Gly Phe Thr
290 295 300
Val Lys Gly Glu Asp Thr Pro Ile Ile Pro Val Met Ile Gly Asp Pro
305 310 315 320
Gln Lys Ala Val Arg Phe Ala Asn Gly Leu Lys Glu Lys Gly Ile Glu
325 330 335
Ala Pro Ala Ile Arg Pro Pro Thr Val Ala Glu Gly Glu Ser Arg Ile
340 345 350
Arg Leu Thr Val Thr Ala Asp Arg Lys Leu Arg Asp Ile Glu Ala Leu
355 360 365
Leu Glu Gly Phe Lys Leu Val Gly Arg Glu Leu Asn Leu Val Lys
370 375 380
<210> 5
<211> 383
<212> PRT
<213>大肠杆菌(Escherichia coli)K12
<400> 5
Met Ser Trp Gln Glu Lys Ile Asn Ala Ala Leu Asp Ala Arg Arg Ala
1 5 10 15
Ala Asp Ala Leu Arg Arg Arg Tyr Pro Val Ala Gln Gly Ala Gly Arg
20 25 30
Trp Leu Val Ala Asp Asp Arg Gln Tyr Leu Asn Phe Ser Ser Asn Asp
35 40 45
Tyr Leu Gly Leu Ser His His Pro Gln Ile Ile Arg Ala Trp Gln Gln
50 55 60
Gly Ala Glu Gln Phe Gly Ile Gly Ser Gly Gly Ser Gly His Val Ser
65 70 75 80
Gly Tyr Ser Val Val His Gln Ala Leu Glu Glu Glu Leu Ala Glu Trp
85 90 95
Leu Gly Tyr Ser Arg Ala Leu Leu Phe Ile Ser Gly Phe Ala Ala Asn
100 105 110
Gln Ala Trp Ile Ala Ala Met Met Ala Lys Glu Asp Arg Ile Ala Ala
115 120 125
Asp Arg Leu Ser His Ala Ser Leu Leu Glu Ala Ala Ser Leu Ser Pro
130 135 140
Ser Gln Leu Arg Arg Phe Ala His Asn Asp Val Thr His Leu Ala Arg
145 150 155 160
Leu Leu Ala Ser Pro Cys Pro Gly Gln Gln Met Val Val Thr Glu Gly
165 170 175
Val Phe Ser Met Asp Gly Asp Ser Ala Pro Leu Ala Glu Ile Gln Gln
180 185 190
Val Thr Gln Gln His Asn Gly Trp Leu Met Val Asp Asp Ala His Gly
195 200 205
Thr Gly Val Ile Gly Glu Gln Gly Arg Gly Ser Cys Trp Leu Gln Lys
210 215 220
Val Lys Pro Glu Leu Leu Val Val Thr Phe Gly Lys Gly Phe Gly Val
225 230 235 240
Ser Gly Ala Ala Val Leu Cys Ser Ser Thr Val Ala Asp Tyr Leu Leu
245 250 255
Gln Phe Ala Arg His Leu Ile Tyr Ser Thr Met Pro Pro Ala Gln Ala
260 265 270
Gln Ala Leu Arg Ala Ser Leu Ala Val Ile Arg Ser Asp Glu Gly Asp
275 280 285
Ala Arg Arg Glu Lys Leu Ala Ala Leu Ile Thr Arg Phe Arg Ala Gly
290 295 300
Val Gln Asp Leu Pro Phe Thr Leu Ala Asp Ser Cys Ser Ala Ile Gln
305 310 315 320
Pro Leu Ile Val Gly Asp Asn Ser Arg Ala Leu Gln Leu Ala Glu Lys
325 330 335
Leu Arg Gln Gln Gly Cys Trp Val Thr Ala Ile Arg Pro Pro Thr Val
340 345 350
Pro Ala Gly Thr Ala Arg Leu Arg Leu Thr Leu Thr Ala Ala His Glu
355 360 365
Met Gln Asp Ile Asp Arg Leu Leu Glu Val Leu His Gly Asn Gly
370 375 380
<210> 6
<211> 388
<212> PRT
<213>枯草芽孢杆菌(Bacillus subtilis)
<400> 6
Met Lys Ile Asp Ser Trp Leu Asn Glu Arg Leu Asp Arg Met Lys Glu
1 5 10 15
Ala Gly Val His Arg Asn Leu Arg Ser Met Asp Gly Ala Pro Val Pro
20 25 30
Glu Arg Asn Ile Asp Gly Glu Asn Gln Thr Val Trp Ser Ser Asn Asn
35 40 45
Tyr Leu Gly Leu Ala Ser Asp Arg Arg Leu Ile Asp Ala Ala Gln Thr
50 55 60
Ala Leu Gln Gln Phe Gly Thr Gly Ser Ser Gly Ser Arg Leu Thr Thr
65 70 75 80
Gly Asn Ser Trp Val His Glu Lys Leu Glu Lys Lys Ile Ala Ser Phe
85 90 95
Lys Leu Thr Glu Ala Ala Leu Leu Phe Ser Ser Gly Tyr Leu Ala Asn
100 105 110
Val Gly Val Leu Ser Ser Leu Pro Glu Lys Glu Asp Val Ile Leu Ser
115 120 125
Asp Gln Leu Asn His Ala Ser Met Ile Asp Gly Cys Arg Leu Ser Lys
130 135 140
Ala Asp Thr Val Val Tyr Arg His Ile Asp Met Asn Asp Leu Glu Asn
145 150 155 160
Lys Leu Asn Glu Thr Gln Arg Tyr Lys Arg Arg Phe Ile Val Thr Asp
165 170 175
Gly Val Phe Ser Met Asp Gly Thr Ile Ala Pro Leu Asp Gln Ile Ile
180 185 190
Ser Leu Ala Lys Arg Tyr His Ala Phe Val Val Val Asp Asp Ala His
195 200 205
Ala Thr Gly Val Leu Gly Asp Ser Gly Gln Gly Thr Ser Glu Tyr Phe
210 215 220
Gly Val Cys Pro Asp Ile Val Ile Gly Thr Leu Ser Lys Ala Val Gly
225 230 235 240
Ala Glu Gly Gly Phe Ala Ala Gly Ser Ala Val Phe Ile Asp Phe Leu
245 250 255
Leu Asn His Arg Thr Phe Ile Phe Gln Thr Ala Ile Pro Pro Ala Ser
260 265 270
Cys Ala Ala Ala His Glu Ala Phe Asn Ile Ile Glu Ala Ser Arg Glu
275 280 285
Lys Arg Gln Leu Leu Phe Ser Tyr Ile Ser Met Ile Arg Thr Ser Leu
290 295 300
Lys Asn Met Gly Tyr Val Val Lys Gly Asp His Thr Pro Ile Ile Pro
305 310 315 320
Val Val Ile Gly Asp Ala His Lys Thr Val Leu Phe Ala Glu Lys Leu
325 330 335
Gln Gly Lys Gly Ile Tyr Ala Pro Ala Ile Arg Pro Pro Thr Val Ala
340 345 350
Pro Gly Glu Ser Arg Ile Arg Ile Thr Ile Thr Ser Asp His Ser Met
355 360 365
Gly Asp Ile Asp His Leu Leu Gln Thr Phe His Ser Ile Gly Lys Glu
370 375 380
Leu His Ile Ile
385
<210> 7
<211> 383
<212> PRT
<213>副地衣芽孢杆菌
<220>
<223> DSM33110
<400> 7
Met Pro Ile Asp Glu Trp Leu Ser Ser Arg Leu Ala Arg Thr Lys Ala
1 5 10 15
Ala Gly Leu Tyr Arg Ser Leu Lys Pro Pro Gln Ala Val Ala Glu Ala
20 25 30
Lys Arg Thr Asn Arg Ala Ser Asn Asp Tyr Leu Ser Leu Ala Asn Asp
35 40 45
Lys Arg Leu Ile His Ala Ala Glu Thr Ala Leu Arg Arg Phe Gly Ala
50 55 60
Gly Ser Thr Gly Ser Arg Leu Thr Ser Gly Asn Thr Glu Trp His Glu
65 70 75 80
Lys Leu Glu Arg Lys Ile Ala Gly Phe Lys Gln Thr Glu Ala Ala Leu
85 90 95
Leu Phe Ser Ser Gly Tyr Leu Ala Asn Ile Gly Val Leu Ser Ser Leu
100 105 110
Pro Glu Lys Gly Asp Val Ile Leu Ser Asp Gln Leu Asn His Ala Ser
115 120 125
Ile Ile Asp Gly Cys Arg Leu Ser Lys Ala Asp Thr Val Val Tyr Arg
130 135 140
His Thr Asp Met Asn Asp Leu Glu Glu Lys Leu Arg Thr Ala Gln Ser
145 150 155 160
Arg Ala Arg Cys Phe Ile Val Thr Asp Gly Val Phe Ser Met Asp Gly
165 170 175
Thr Ile Ala Pro Leu Asp Glu Ile Met Leu Leu Ala Lys Arg Tyr Arg
180 185 190
Ala Phe Val Ile Val Asp Asp Ala His Ala Thr Gly Val Leu Gly Asp
195 200 205
Ala Gly Arg Gly Thr Gly Glu Tyr Phe Gly Val Ser Pro Asp Val Val
210 215 220
Ile Gly Thr Leu Ser Lys Ala Val Gly Ala Glu Gly Gly Phe Val Ala
225 230 235 240
Gly Ser Lys Ala Leu Ile Asp Phe Leu Leu Asn His Ala Arg Thr Phe
245 250 255
Ile Phe Gln Thr Ala Val Pro Pro Ala Ser Cys Ala Ala Ala Cys Arg
260 265 270
Ala Leu Asp Ile Ile Lys Asp Ser Arg Asp Lys Arg Arg Leu Leu Gln
275 280 285
Ser Ser Val Thr Thr Ile Lys Arg Gly Leu Glu Asp Ile Gly Phe Thr
290 295 300
Val Lys Gly Glu Asp Thr Pro Ile Ile Pro Val Met Ile Gly Asp Pro
305 310 315 320
Gln Lys Ala Val Arg Phe Ala Asn Gly Leu Lys Glu Lys Gly Ile Glu
325 330 335
Ala Pro Ala Ile Arg Pro Pro Thr Val Ala Glu Gly Glu Ser Arg Ile
340 345 350
Arg Leu Thr Val Thr Ala Asp Arg Lys Leu Arg Asp Ile Glu Ala Leu
355 360 365
Leu Glu Gly Phe Lys Leu Val Gly Arg Glu Leu Asn Leu Val Lys
370 375 380
PCT/RO/134表
Claims (19)
1.一种副地衣芽孢杆菌(Bacillus paralicheniformis),所述副地衣芽孢杆菌当与以保藏号DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌中hrcA和/或bioF的相应直系同源基因相比时,在hrcA和/或bioF基因中具有突变。
2.一种副地衣芽孢杆菌,所述副地衣芽孢杆菌当与由SEQ ID NO:1编码的hrcA和/或由SEQ ID NO:3编码的bioF的相应直系同源基因相比时,在hrcA和/或bioF基因中具有突变,所述由SEQ ID NO:1编码的hrcA和/或由SEQ ID NO:3编码的bioF的相应直系同源基因在例如以DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌中。
3.根据权利要求1或2中任一项所述的副地衣芽孢杆菌,其中所述副地衣芽孢杆菌为衍生于以DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌的突变体。
4.一种副地衣芽孢杆菌,所述副地衣芽孢杆菌以DSM 33238、DSM33239、DSM 33240、DSM33241、DSM 33242、DSM 33243或DSM33244保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)。
5.根据权利要求1至4中任一项所述的副地衣芽孢杆菌,其中所述bioF基因中的所述突变引起基因型变化,所述基因型变化导致Pro314至Ser、Ala244至Val、Asp300至Gly和/或Arg37至Trp的氨基酸变化。
6.根据权利要求1至5中任一项所述的副地衣芽孢杆菌,其中所述hrcA基因中的所述突变引起基因型变化,所述基因型变化导致终止密码子,从而使HrcA抑制子失活。
7.根据前述权利要求中任一项所述的副地衣芽孢杆菌,所述副地衣芽孢杆菌当在MSgg培养基中于21℃生长时,当与以保藏号DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌相比,显示提高的最大生长速率。
8.根据权利要求7所述的副地衣芽孢杆菌,其中所述最大生长速率是如本文实施例2中所述被测定的。
9.根据前述权利要求中任一项所述的副地衣芽孢杆菌,其中根据前述权利要求中任一项所述的副地衣芽孢杆菌菌株的生长速率比以保藏号DSM33110保藏在布伦瑞克因霍芬街7B,D-38124的莱布尼茨研究所DSMZ-德国微生物保藏中心(DSMZ)的副地衣芽孢杆菌的生长速率高至少20%,例如至少40%,例如至少60%,例如至少80%,例如至少100%。
10.根据前述权利要求中任一项所述的副地衣芽孢杆菌,所述副地衣芽孢杆菌在限菌植物***中施用于拟南芥Col-0植物并于22℃和/或15℃生长时,能够增加所述拟南芥的总叶面积、鲜重或叶绿素含量。
11.一种组合物,所述组合物包含根据前述权利要求中任一项所述的副地衣芽孢杆菌及其农业化学上可接受的赋形剂和/或载体。
12.根据权利要求11所述的组合物,所述组合物进一步包含以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在的微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂、除草剂、植物提取物、植物生长调节剂或肥料中的一种或组合,载体、表面活性剂、分散剂或酵母提取物。
13.根据权利要求11至12中任一项所述的组合物或根据权利要求1至10中任一项所述的副地衣芽孢杆菌作为生物刺激素、生长促进剂和/或生物杀线虫剂的用途。
14.根据权利要求13所述的用途,其中所述植物线虫选自由以下组成的组:根结线虫属、短体线虫属、异皮线虫属、孢囊线虫属、茎线虫属、垫刃线虫属、剑线虫属、穿孔线虫属、肾形线虫属、螺旋线虫属和刺线虫属,例如南方根结线虫、爪哇根结线虫、短小根结线虫、巴拉那根结线虫、大豆孢囊线虫和玉米短体线虫。
15.根据权利要求13至14中任一项所述的用途,其中将根据权利要求11至12中任一项所述的组合物或根据权利要求1至10中任一项所述的副地衣芽孢杆菌施用于植物、种子上或植物的生境中,例如土壤中。
16.根据权利要求13至15中任一项所述的用途,其中所述植物选自由玉米、水稻、甘蔗、大豆、马铃薯、胡萝卜、咖啡和香蕉组成的组。
17.一种植物种子,所述植物种子包被有根据权利要求11至12中任一项所述的组合物,所述组合物以适于有利于植物生长和/或在易感病植物中赋予免遭病原体感染的保护的量存在。
18.根据权利要求17所述的植物种子,其中所述组合物包含约1.0×102CFU/种子至约1.0×109CFU/种子的量的根据权利要求1至10中任一项所述的芽孢杆菌属的孢子。
19.根据权利要求17或18所述的植物种子,其中所述组合物进一步包含微生物的、生物的或化学的杀虫剂、杀真菌剂、杀线虫剂、杀细菌剂或植物生长调节剂中的一种或组合,所述一种或组合以适于有利于植物生长和/或在易感植物中赋予免遭病原体感染的保护的量存在。
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CN118048282A (zh) * | 2024-04-16 | 2024-05-17 | 云南农业大学 | 一株解淀粉芽孢杆菌及其应用 |
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WO2024047025A1 (en) * | 2022-08-30 | 2024-03-07 | Chr. Hansen A/S | Derivative bacillus strains for high abiotic stress |
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CN118048282A (zh) * | 2024-04-16 | 2024-05-17 | 云南农业大学 | 一株解淀粉芽孢杆菌及其应用 |
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