CN109112072B - 一种木霉菌在促进植物生长中的应用 - Google Patents
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Abstract
本发明公开了一种木霉菌在促进植物生长中的应用。本发明的木霉菌为棘孢木霉(Trichoderma asperellum)菌,菌株号:HTTA‑Z0002,保藏编号CGMCC No.15677。本发明的HTTA‑Z0002菌株能降解磷钾和纤维素、产植物激素和铁载体等多方面促进植物生长,并能诱导植物抗逆、抗盐,是一株对植物生长具有多功能促进作用的菌株,可减少化肥使用。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种木霉菌在促进植物生长中的应用。
背景技术
农业生产离不开肥料,化肥的滥用增加了生产成本,导致土壤板结,有机质贫乏,耕地肥力下降大面积缺氮磷钾,不但降低农产品的品质,更严重的是污染土壤和环境,土壤的次生盐渍化也越来越重,因此,发展微生物肥料是目前国家解决农业用肥和保护土壤及环境的战略发展规划。
木霉菌因其丰富的酶类和植物促生长作用及环境安全,成为最受关注的肥料益生菌,生境不同则直接影响这类活菌对植物生长的功能和作用,相对陆地生境木霉,海洋生境木霉开发研究较少,属于新生境微生物,由于海洋生境较陆地恶劣,在此特殊生境生活的木霉对营养要求较低,人工培养成本较低,对植物的促进作用多样化,显示出良好的开发应用前景。
我国耕地现有超过0.2亿hm2面积的盐碱地,并有逐年增加的趋势,因人多地少的国情,提高盐渍土壤的生产力一直受到从国家到地方政府的大力提倡,微生物肥料是微生物在植物附近代谢起作用,大多为活菌制剂,来自陆地的微生物因为盐渍土壤盐分含量较高,影响其生长而不能正常发挥作用,海洋尤其极地独特的地理位置和酷寒、高盐等极端环境,造就了微生物的多种适应性生存,能更好的在盐渍地发挥作用,同时产生的对植物的功能也多于陆地微生物。
微生物肥料主要是微生物生命活动产生酶和有益物质发挥作用,磷钾是植物生长必需的大量元素,土壤存在有大量无机磷和硅酸盐矿物形式的钾,由于其溶解性差,难于被植物吸收,一些微生物产生酶和有益物质可将难溶的磷、钾元素转变为可溶态供植物利用,能产植酸酶的微生物也可以将植物不能利用的植酸转化成植物可以利用的有效磷元素;吲哚乙酸(IAA)是一种植物内源生长素,参与细胞生长和形成层***等多种生理生化过程的调节与控制,在植物生长中不可缺乏,植物周围的微生物产生IAA也将对植物有很好的补充作用;自然界中铁元素大多数以不溶解状态存在,植物难以利用,铁载体是一类具有很强特异螯合Fe3+的小分子化合物,许多植物根际益生微生物可通过合成这类物质来摄取环境中的铁,与病原菌争夺铁并将多余的铁提供给植物利用。铁载体还可螯合锰、铅、汞、铬、镉等金属离子,对土壤环境修复、食品安全等领域具有重要意义。
纤维素是地球上分布最广,但其它生物包括植物无法直接利用的资源,我国仅农作物废弃物每年就可高达0.7×109吨,这些原料大部分被焚烧,不但利用率低,而且给环境保护带来巨大压力。利用有益微生物产生的纤维素酶来降解纤维素效率高、无污染,具有双重的经济和生态价值。纤维素酶是由内切葡聚糖酶、纤维二糖水解酶等组成的复合酶系,Eg1基因是微生物合成纤维素酶的关键基因,其敏感和准确性成为人们评价菌株是否产纤维素酶的有效指标。
肥料菌株为了更好的为人类所利用和产业化生产,菌株要满足肥料及生产发酵性状均达标的条件,微生物菌株通过各种筛选模型获得后,通常都进行诱变育种,如操作的成功,其经济价值可在野生菌株的基础上提高几百甚至上千倍,微生物液体发酵是体现低成本和容易工业化生产的基础,但木霉在液态培养产孢量少,固态培养又制约了大规模生产,而木霉菌株的活菌制剂施用的是分生孢子,即分生孢子数量与肥效成正比。本发明针对木霉肥料菌株最重要的抑菌活性和液体培养产孢量特性,诱变育种获得的菌株HTTA-Z0002,不但能促进植物生长和抗逆,抑制土壤病原菌,还具有良好的人工发酵培养生产的特性。
发明内容
本发明的目的是提供一种木霉菌在促进植物生长中的应用。
本发明的棘孢木霉(Trichoderma asperellum)菌保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏日期2018年5月17日,菌株号: HTTA-Z0002,保藏编号CGMCC No.15677;中国北京朝阳区北辰西路3号。
为了实现上述目的,本发明提供如下技术方案:
一种木霉菌在促进植物生长中的应用,所述木霉菌为棘孢木霉 (Trichodermaasperellum)菌,菌株号:HTTA-Z0002,保藏编号CGMCC No.15677。
进一步地,所述应用包括降解磷、钾、植酸钙或纤维素。
进一步地,所述应用包括促进植物种子的萌发、幼苗的生长。
一种化肥,包含上述木霉菌。
本发明在两种常规测试磷钾的培养基上,HTTA-Z0002木霉菌株水解圈很明显,表明菌株具有良好的降解磷钾的作用;在含有植酸钙的培养基上水解圈也很明显,即能水解植酸钙,表明具有产植酸酶能力,可将植物不能利用的植酸转化成植物可以利用的有效磷元素;菌株在纤维素作为唯一碳源的培养基上能生长,同时PCR也扩增出菌株合成纤维素酶的关键基因-内切葡聚糖酶基因 Eg1,双重证明具有降解纤维素的能力;木霉菌的发酵液经Salkowski比色液显色检测,结果显示红色,采用HPLC法也测定到吲哚乙酸,均说明菌株能产生 IAA,通过内标法得出木霉菌HTTA-Z0002菌株吲哚乙酸的产量为2.09mg/L;菌株可使CAS检测培养基变***,表明具有产铁载体能力,铁载体的相对含量随着培养时间而增加,第7天时到达最大值,为83.9%。
HTTA-Z0002菌株对植物种子的萌发和幼苗的生长都具有促进作用,菌液处理的番茄植株苗比清水对照处理的苗健壮,叶片大,叶色浓绿,茎秆粗,根毛长,幼苗的平均株高增长70.%;根长增长96.6%;茎粗增长85.7%,番茄叶片叶绿素a、叶绿素b和总叶绿素分别比对照分别提高了50.0%-125.0%,平均鲜重、干重增加86.0%-128.6%
在盐渍土壤栽培,所述的HTTA-Z0002菌株对番茄种子萌发促进作用较大,胚根、胚轴都接近正常土壤培养,发芽指数明显高于对照,提高56.7%。植株与抗逆有关的酶类SOD、POD、CAT、PPO、PAL及脯氨酸、可溶性糖等含量显著提高,提高幅度为39.7%-87.5%,与CK相比差异显著(P<0.05)。经HTTA-Z0002 菌处理后,番茄根、叶内Na+含量都有不同程度的降低,与对照相比,Na+在根和叶内的积聚降低了43.1%-65.2%,根和叶片中K+含量升高,根、叶中K+含量提高了37.0%-46.8%。
本发明的应用效果在于:HTTA-Z0002菌株能降解磷钾和纤维素、产植物激素和铁载体等多方面促进植物生长,并能诱导植物抗逆、抗盐,是一株对植物生长具有多功能促进作用的菌株,可减少化肥使用。人工培养发酵生产容易操作,成本低,对环境安全。
附图说明
图1亲缘关系分枝树。
图2HPLC检测IAA吸收峰
具体实施方式
以下具体实施例是对本发明提供的方法与技术方案的进一步说明,但不应理解成对本发明的限制。
实施例1:木霉菌株的分类鉴定
棘孢木霉HTTA-Z0002菌株,在固体MPDA培养基(马铃薯浸汁200-250ml, 蛋白胨1-2g,葡萄糖25-30g,NaCl 5-8g,MgCl 26H2O 0.1-0.3g,KCl 0.1g,琼脂18g水 1000ml),20℃-23℃,培养3-5天,连续观察记录菌落形态、颜色等变化,挑取菌丝于光学显微镜下观察产孢结构。菌株在MPDA人工培养基生长旺盛,产孢层与分生孢子梗密集如毡状,青绿色,间或白色,分生孢子梗有隔膜,产生侧向分枝,分枝垂直对生,基部较宽,末端较细,呈瓶状,分生孢子单孢,球形至卵形,无色,在尖端形成较明显的分生孢子团。
菌株经上述方法培养,利用真菌ITS基因的通用引物,常规方法PCR扩增产物测序,并通过NCBI的BLAST程序进行对比,分析序列同源性及做亲缘关系分枝树比较近缘种类(见图1)。结合生物学菌落形态、颜色及产孢结构的显微构造,菌株的分子生物学信息,参考国内外相关木霉分类鉴定专著,鉴定菌株为棘孢木霉Trichoderma asperellum。
实施例2:木霉菌株的诱变育种
经多次肥料活性测试筛选出的木霉菌株,采用常规紫外-微波复合诱变,选择生长旺盛的单克隆菌落,先在木霉常用的MPDA培养基、酵母蔗糖(ESA)培养基、理查德(Richard)培养基,测试生长和产孢情况,选择生长和产孢普遍较好的MPDA培养基,在固态MPDA培养基培养5天,淘汰生长异常的菌株,然后接入液态MPDA,分别在20℃、23、25℃培养7天,显微计数并比较其产孢数量,选择产孢数量超过50%的菌株,选出的菌株连续液体培养5代,采用上述同样的产孢数量测试方法,观察其生长和产孢的稳定性,最终选出的 HTTA-Z0002木霉菌株,最大产孢量为4.25×108cfu/cm2,较野生菌提高了150.7%。
实施例3:对植物作用的实验室检测
采用已报道的特殊测试培养基培养HTTA-Z0002菌株,观察并计算的方法测试:根据菌落与水解圈的大小、用十字相乘法分别测量菌落直径(d/mm)与水解圈直径(D/mm),计算D/d值,检测其溶磷解钾、降解纤维素及产植酸酶的能力;采用常规的HPLC法测定产吲哚乙酸能力,并计算吲哚乙酸的产量;通过已报道的半PDA和CAS培养基,检测木霉菌的产铁载体能力,以其吸光值作为参比值(Ar),计算嗜铁素的相对含量(%)=[(Ar-As)/Ar]×100%。同时利用引物 XWS-F/XWS-R PCR扩增,对其是否具有降解内切葡聚糖酶Eg1基因进行验证,对获得的Eg1基因进行序列比对分析。
在两种测试磷钾的培养基上,HTTA-Z0002木霉菌株菌落周围水解圈很明显,说明具有较强的溶磷解钾能力。
表1.木霉降解磷钾及产植酸酶和纤维素酶
作用 | 水解圈直径(D)(mm) | 菌落直径(d)(mm) | D/d |
降解磷 | 40.4±0.3 | 24.8±0.4 | 1.63 |
降解钾 | 45.1±0.4 | 28.2±0.3 | 1.59 |
产植酸酶 | 40.0±0.2 | 17.2±0.3 | 2.37 |
产纤维素酶 | 71.2±0.5 | 46.1±0.4 | 1.76 |
在含有植酸钙的培养基上,木霉菌菌落周围有明显的水解圈产生,即能水解植酸钙,表明具有产植酸酶能力;HTTA-Z0002菌株可使CAS检测培养基变***,表明均具有产铁载体能力,铁载体的相对含量随着培养时间而增加,第7天时到达最大值,为83.9%。
木霉菌的发酵液经Salkowski比色液显色检测,结果显示红色,说明菌株能产生IAA;HPLC检测发酵液样品,样品在第18min位置出现吸收峰,通过微积分计算得峰面积,结合吲哚乙酸标准曲线,通过内标法得出木霉菌吲哚乙酸的产量为2.09mg/L。
HTTA-Z0002菌株在纤维素作为唯一碳源的培养基上,能生长,说明能利用解纤维素作为碳源,即具有降解纤维的能力,利用引物XWS-F/XWS-R PCR扩增,特异性条带约为750bp,特异性条带在NCBI经对比分析,与木霉属Eg1基因同源性达到97%,也证明菌株有合成纤维素酶的关键基因-内切葡聚糖酶基因Eg1,即具有产纤维素酶的能力。
实施例4:对普通土壤栽培植物的促生长作用
采用常规方法测试HTTA-Z0002菌株对植物的促生长作用,分别用稀释500 倍的HTTA-Z0002菌株发酵液和清水浸番茄种(对照),栽种在装有灭菌土的盆钵中,每个处理12盆,3个重复,2片真叶时,用同浓度发酵液灌根1次,培养25天,连根拔出,常规方法统计根长、株高、鲜重、干重、叶绿素含量与对照相比计算增长率。
表2HTTA-Z0002菌株对番茄苗的促生长作用
番茄苗 | CK | 处理 | 增长率% |
根长/cm | 2.9 | 5.7 | 96.6 |
株高/cm | 9.9 | 16.9 | 70.7 |
茎粗/cm | 0.7 | 1.3 | 85.7 |
鲜重/g | 16.3 | 31.9 | 86.0 |
干重/g | 2.1 | 4.8 | 128.6 |
叶绿素a/mg/g·FW | 0.4 | 0.6 | 50.0 |
叶绿素b/mg/g·FW | 0.3 | 0.6 | 100.0 |
总叶绿素/mg/g·FW | 0.4 | 0.9 | 125.0 |
由表2可以看出,经HTTA-Z0002菌株对番茄种子的萌发和幼苗的生长都具有明显促生作用,菌液处理的番茄植株苗比清水处理的健壮,叶片大,叶色浓绿,茎秆粗,根毛长,幼苗的平均株高增长70.%;根长增长96.6%;茎粗增长85.7%,番茄叶片叶绿素a、叶绿素b和总叶绿素分别比对照分别提高了 50.0%-125.0%,81.2%,平均鲜重、干重增加86.0%-128.6%。
实施例5:对盐渍土壤栽培植物的促生长作用及诱导植物抗盐
将番茄苗栽种在自然海滨盐渍土壤(NaCl浓度为0.4%),设栽种在普通耕作土与无木霉菌盐渍土番茄苗2个对照,处理2、4、8、10天后,常规方法测试并与对照比较胚根、胚轴长,计算发芽指数;幼苗生长20天,收获完整的植株,常规方法测定幼苗茎粗,株高、根长、展开叶片数等计算壮苗指数,同时测试植株与抗逆有关的超氧化物歧化酶(SOD),过氧化物酶(POD)、过氧化氢酶 (CAT)、多酚氧化物酶(PPO)、苯丙酸解氨酶(PAL)、脯氨酸、可溶性糖等含量,与抗盐有关的植物根、叶内Na+、K+含量。
结果证明,番茄的种子在盐渍土壤中萌发受到抑制,胚根、胚轴都较短,加入HTTA-Z0002菌株对番茄种子萌发促进作用较大,胚根、胚轴都接近正常,发芽指数明显高于对照,提高56.7%。植株的SOD、POD、CAT、PPO、PAL、脯氨酸、可溶性糖含量显著提高,提高幅度为100%-180%
表3木霉处理对盐胁迫下番茄种子胚根、胚轴及发芽指数的影响
*小写英文字母表示多重比较α=0.05的差异显著性,无相同字母的同列数据间差异显著。
如下表4所示,与对照相比,木霉菌处理株高、叶长、展开叶数、茎粗分别提高87.5%、39.7%、45.1%和69.0%,与CK相比差异显著(P<0.05)。
表4木霉菌处理对番茄幼苗形态的影响
表5经木霉菌处理对番茄根叶内Na+、K+含量的影响
从表5可以看出,与对照相比,经HTTA-Z0002菌处理后,番茄根、叶内 Na+含量都有不同程度的降低,与对照相比,Na+在根和叶内的积聚降低了 43.1%-65.2%,根和叶片中K+含量升高,根、叶中K+含量提高了37.0%-46.8%。番茄生长20天后,对照的干重含水量为920.7%,经菌株处理干重含水量分别为1200.1%,处理能够促进番茄植株对水分的吸收,保持体内较高的水分含量,降低Na+在植物体内的积聚,提高植物体内K+含量,从而不同程度地减轻或缓解盐伤害,番茄苗生长情况也显示,对照番茄苗叶片失水干枯面积较大,显现出较重的盐害症状,经菌株处理的番茄苗,第叶片盐害症状明显减轻,比对照盐害指数降低了61.7%。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求保护范围内。
Claims (2)
1.一种木霉菌在促进植物生长中的应用,其特征在于,所述木霉菌为棘孢木霉(Trichoderma asperellum )菌,菌株号:HTTA-Z0002,保藏编号CGMCC No.15677;所述应用包括降解磷、钾、植酸钙或纤维素和促进植物种子的萌发、幼苗的生长。
2.一种化肥,包含如权利要求1所述的木霉菌。
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