CN116694531A - 一种芽孢杆菌hlh_19及其应用 - Google Patents
一种芽孢杆菌hlh_19及其应用 Download PDFInfo
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- CN116694531A CN116694531A CN202310783322.7A CN202310783322A CN116694531A CN 116694531 A CN116694531 A CN 116694531A CN 202310783322 A CN202310783322 A CN 202310783322A CN 116694531 A CN116694531 A CN 116694531A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
本发明公开了一种芽孢杆菌HLH_19及其应用,该芽孢杆菌HLH_19已于2023年3月8日保藏于中国典型培养物保藏中心(CCTCC),地址:湖北省武汉市武昌区八一路299号武汉大学,保藏号为CCTCCNO:M2023266。本发明以烤烟烟花为分离材料,进行高效PGPR内生细菌的分离筛选和鉴定,筛选出的芽孢杆菌菌株HLH_19不仅溶磷能力较强,产IAA较强,并经过实验验证,该菌株还可以明显抑制植物病原细菌的生长,还能促进植物生长,提高烟苗的叶片数、茎粗、株高和鲜重。
Description
技术领域
本发明涉及一种芽孢杆菌,具体涉及一种芽孢杆菌HLH_19及其应用。
背景技术
烤烟(Flue cured Tobacco)是茄科一年生草本植物,是我国重要的经济作物,除了可作为卷烟的原材料以外,其所含的大量的天然化学物质,如茄尼醇、蛋白质、烟碱、尼古丁,都是优质的化工和医药原料。目前,在种植生产中,产量低、病害多已经成为了制约农业发展的主要因素。传统的化肥及农药虽然能在短期内提高农作物产量,但长期使用会对生态环境造成一定的破坏。因此开发高效环保并且适宜烤烟栽培的菌剂肥料能够有效提高烟叶的产量和品质。
长期以来,促进植物生长以及防治植物病害的主要方式是化肥和农药,虽然这种方法在促进生长和防治疾病的上效果较为显著,但是与此同时也引起了土壤板结、植物病原抗药性、农药残留等问题,造成了一定的环境污染和生态破坏。因此,改进现有种植方式,加强农作物自身微生物资源挖掘与应用,对于提高农作物产量和品质、保护生态环境具有重要的意义。
植物内生菌是指存在于植物组织中的细菌、放线菌和真菌等,在与植物长期的共存过程中,构建了和谐共处生存关系,并进化出了多种人类可以利用的有益功能,包括促进植物生长、抵抗植物病虫害、提高植物适应环境的能力。使用植物内生菌制备的微生物制剂可以降低农药、化肥施用给人类健康和生态环境带来的隐患,保护植物根际微生态平衡。因此,植物内生细菌在当前的微生物学领域研究有重要研究价值。
发明内容
本发明的目的是提供一种芽孢杆菌HLH_19及其应用,解决了化肥及农药对环境污染的问题,能够促进植物生长,抑制植物病原菌,而且能够有效溶溶磷源,可以用于生物有机肥的开发,对于种植业化肥减施增效具有重要价值。
为了达到上述目的,本发明提供了芽孢杆菌(Bacillus)HLH_19,该芽孢杆菌HLH_19已于2023年3月8日保藏于中国典型培养物保藏中心(CCTCC),地址:湖北省武汉市武昌区八一路299号武汉大学,保藏号为CCTCC NO:M2023266。
本发明的另一目的是提供所述的芽孢杆菌HLH_19在防治青枯假单胞菌、金黄色葡萄球菌引起的植物病害中的应用。
优选地,所述植物为烤烟。
优选地,所述芽孢杆菌HLH_19作为防治作物病害的药物。
本发明的另一目的是提供所述的芽孢杆菌HLH_19在生物有机肥料方面的应用。
优选地,所述芽孢杆菌HLH_19具有溶磷和/或产IAA的作用。
本发明的另一目的是提供所述的芽孢杆菌HLH_19在作为植物生长调节剂中的应用。
优选地,在作为烟草生长调节剂中的应用。
优选地,在调节烟草株高、茎直径、叶片数和鲜重中任意一项或两项以上中的应用。
本发明的另一目的是提供所述的芽孢杆菌HLH_19在降解有机磷和/或无机磷中的应用。
本发明的芽孢杆菌HLH_19及其应用,解决了化肥及农药对环境污染的问题,具有以下优点:
本发明以烤烟的花为分离材料,进行高效PGPR内生细菌的分离筛选和鉴定,筛选出的芽孢杆菌菌株HLH_19可以用于新型生物有机肥的研发,对于种植业化肥减施增效具有重要价值。
本发明的芽孢杆菌HLH_19不仅溶磷能力较强,产IAA较强,并经过实验验证,该菌株还可以明显抑制植物病原细菌的生长,还能促进植物生长,提高烟苗的叶片数、茎粗、株高和鲜重。
附图说明
图1为本发明菌株HLH_19的代谢产物正离子流图。
图2为本发明菌株HLH_19的代谢产物负离子流图。
图3为本发明菌株HLH_19前10种代谢物热图。
图4本发明菌株HLH_19在盆栽试验中对烤烟苗期素质的影响;其中CK为不接种本菌株的烟苗,HLH_19为接种本菌株的烟苗。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实验例1芽孢杆菌(Bacillus)HLH_19的分离与鉴定
1、菌株来源
本实验例样品取于凉山州彝族自治区会理市龙河村的烤烟烟花,随机采集烤烟烟花,装入灭菌密封塑料袋,贴上标签,带回实验室保存。
2、内生细菌的分离
以凉山州烤烟烟花为材料,通过LB培养基进行烤烟内生细菌的选择性培养和纯化培养。将烤烟烟花先用0.1%Tween20清洗30s,无菌水冲洗三次;75%乙醇浸泡2min,无菌水冲洗三次;2%次氯酸钠浸泡2min,无菌水冲洗三次;10%NaHCO3漂洗5min,无菌水冲洗三次。用研钵研磨压碎,用镊子去植物组织块涂抹在LB培养基表面,于35℃培养箱中培养,待长出明显细菌菌落后,根据菌落大小、形态和色泽等表观特征挑取细菌进行纯化,最终分离得到97株菌株。
进一步将这些菌株点接于相应的无机磷以及有机磷平板上,培养7d,观察透明圈情况,挑选出具有溶磷能力的细菌28株。
3、芽孢杆菌HLH_19的鉴定
对HLH_19菌株用细菌基因组提取试剂盒进行细菌DNA提取,稀释10倍作为PCR反应的模板进行细菌16S扩增,扩增引物为27F(5'-AGAGTTTG ATCCTGGCTCAG-3')和1492R(5'-GGTTACCTTGTTACGACTT-3'),然后将扩增产物回收并进行测序,测得16S rRNA基因的序列,16S rRNA基因的NCBI基因登录号为OP048044。
基于以上特征,将菌株HLH_19鉴定为芽孢杆菌(Bacillus sp.)。该菌株已于2023年3月8日保藏于中国典型培养物保藏中心(CCTCC),地址:湖北省武汉市武昌区八一路299号武汉大学校内,保藏号为CCTCC NO:M2023266。该菌株菌落为米黄色,圆形或类圆形,直径在1.0mm以上,表面隆起光滑,有树状纹。
实验例2有机磷和无机磷溶解能力初筛
将分离得到菌株接种于有机磷和无机磷固体培养基中,28℃培养3天,测定溶解圈的大小以初步定性测定菌株是否具备有机磷和无机磷的溶解能力。结果见表1和表2。
上述有机磷固体培养基的成分包含:10g葡萄糖、0.5g硫酸铵、0.5g酵母浸粉、0.3g氯化钠、0.3g氯化钾、0.3g硫酸镁、0.03g硫酸亚铁、0.03g硫酸锰、0.2g卵磷脂、1g碳酸钙、20g琼脂和1L水。
上述无机磷固体培养基的成分包含:10g葡萄糖、0.5g硫酸铵、0.5g酵母浸粉、0.3g氯化钠、0.3g氯化钾、0.3g硫酸镁、0.03g硫酸亚铁、0.03g硫酸锰、5g磷酸三钙、20g琼脂和1L水。
表1烤烟内生细菌有机磷降解能力初筛
注:nd:not detected,为该活性未检测到;表中有机磷溶解圈的数值单位均为cm。
表2烤烟内生细菌无机磷溶解能力初筛
注:nd:not detected,为该活性未检测到;表中无机磷溶解圈的数值单位均为cm。
根据溶磷圈的直径大小,从表1和表2挑选出具有明显溶磷效果的BJ_1、BJ_2、BJ_5、BJ_11、BJ_13、BJ_24、BJ_30、ZCJ_3、ZCJ_4、ZCJ_5、ZCJ_10、ZCJ_11、ZCJ_15、ZCJ_17、HLH_4、HLH_5、HLH_11、HLH_14、HLH_15、HLH_19、HLH_22、HLH_23、MYH_5、MYH_7、MYH_11、MYH_14、MYH_22、MYH_23共28个菌进行后续溶磷能力的定量测定。
实验例3无机磷溶解能力的定量测定
将实验例2分离得到28株菌株接种于无机磷液体培养基中,于30℃,160r/min振荡培养5d,取菌株培养液,8000rpm离心20min,上清液采用钼锑抗比色法测定培养液中有效磷含量。
表3烤烟内生细菌无机磷溶解能力
对具有溶磷能力的28株菌株进行无机磷溶解能力定量测定(表3),烤烟顶花中部分细菌具有较好的溶磷能力,其中溶磷能力高于200mg·L-1的菌株有10株,占分离总菌株数的35.7%,IAA产量高于250mg·L-1的菌株有7株,占25.0%。溶磷能力最强的菌株是BJ_2,为334.81mg·L-1。
实验例4产吲哚乙酸(IAA)测定
将实验例1分离得到28株菌株接种到金氏培养基中培养到对数期,在30℃、200r/min振荡培养24h,取50μL于洗净的白瓷板中,加50μL显色剂,37℃下避光30min,如果出现粉红色,表明产生IAA,以没有接菌的金氏培养基为对照。
将金氏培养基中培养的菌株,12000r/min离心15min,取上清液1mL加入2mLSalkowski显色剂,28℃下避光30min后,530nm下测定吸光度值。以空白培养基作对照,并以IAA的标准样品对应的光密度做标准曲线,计算IAA的产量(mg/L),不同菌株的IAA产生量见表4。
表4烤烟内生细菌产IAA能力
对具有溶磷能力的28株菌株进行产IAA测定(表4),烤烟烟花中部分细菌具有较好的IAA产生能力,其中IAA产量高于30mg·L-1的菌株有5株,占分离总菌株数的17.9%,IAA产量高于40mg·L-1的菌株有3株,占10.7%。IAA产量最高的菌株是BJ_24,产量为76.37mg·L-1。
实验例5抗菌活性测定
采用平板对峙法测定了分离菌株对番茄早疫病菌(Afternaria solani(Ell.etMart).Joneset Grout)、黄瓜晚疫病菌(Phytophthora infestans)、金黄色葡萄球菌(Staphylococcus aureusRosenbach)和大肠杆菌(Staphylococcus aureusRosenbach)、青枯假单胞菌(Pseudomonas solanacearum)等5种指示菌的拮抗效果。测定抑菌圈直径(HD)与菌落直径(CD),通过计算HD/CD的比值来筛选出有抑菌效果的菌株,具体如表5。
表5烤烟内生细菌抑菌能力
注:表5中,1.番茄早疫病菌(Afternariasolani(Ell.etMart).JonesetGrout),2.黄瓜晚疫病菌(Phytophthora infestans),3.金黄色葡萄球菌(StaphylococcusaureusRosenbach),4.大肠杆菌(StaphylococcusaureusRosenbach),5.青枯假单胞菌(Pseudomonassolanacearum)。表中++:HD/CD值介于2-3;+:HD/CD值介于1–2;-:HD/CD值小于1。
由表5可知,14株对金黄色葡萄球菌有拮抗效果,12株对大肠杆菌有拮抗效果,12株对青枯假单胞菌有拮抗效果。能同时抑制两种病原菌以上的占分离总菌株数的46.43%,其中HLH_19菌株能够同时抑制金黄色葡萄球菌和青枯假单胞菌2种病原菌,且对青枯假单胞菌有较好的效果。
最终,筛选出兼具溶磷、产IAA、抑菌能力均较好的HLH_19。
实验例6 HLH_19的代谢产物研究
次生代谢物分离纯化:将分离得到菌株接种到LB液体培养基中,在30℃、200r/min的条件下恒温摇床中培养,培养48h后,于常温5000r/min离心10min,保留上清发酵液。将上述发酵液用加入10倍体积的预冷90%甲醇(即100mg样本加入1mL 90%甲醇),匀浆,干燥。乙腈水溶液处理后取上清液进样分析。用Agilent 1290lnfinity LC系统进行色谱分析,全程4℃自动进样。采用正、负离子双模式进行质谱鉴定。原始数据经Proteo Wizard转换成mzML格式,然后经XCMS和SIMCA-P 14.1软件处理得到数据,数据经Pareto-scaling预处理后,分别进行单维和多维统计分析。在KEGG数据库中匹配和聚类分析并用JavaTree View软件绘图。Fisher精确检验(Fisher's Exact Test)用于比较各通路在代谢物集合中的分布,评价通路代谢物富集的显著性
代谢产物结果:以VIP>1和P<0.05为条件,对鉴定的代谢物进行筛选,得到37种主要差异代谢物(图1和图2),排名前10种代谢物见图3,分别为精氨酸(Arginine)、唑吡坦(Zolpidem)、2-氨基-1-苯乙醇(2-amino-1-phe nylethanol)、次黄嘌呤(Hypoxanthine)、DL-精氨酸(DL-Arginine)、DL-苯丙氨酸(DL-phenylalanine)、二岩藻糖基乳糖-N-六糖(Dflnha)、3,5-二甲氧基-α-甲基-4-丙氧基苯乙胺(Benzeneethanamine,3,5-dimethoxy-.alpha.-methyl-4-propo xy)、甘油磷酰胆碱(Glycerophosphocholine)、D-鸟氨酸(D-ornithine)。
实验例7促生性能的盆栽验证
基于上述实验例的测定结果,将具有溶磷促生功能的细菌HLH_19活化后接种于LB液体体培养基中,在35℃、180r/min的恒温摇床中培养,培养48h后,作为种子液以1:1比例接种LB液体培养基,然后于35℃、180r/min的恒温摇床中震荡培养24h,使成为1×108cfu/mL的菌悬液,取出备用。
将长势一致的烟苗种植于花盆中,每个花盆中的土样为2.5kg,土壤为采集自凉山州烟区的紫色土。采用浇灌的方式将菌株HLH_19菌剂接种于烟苗根部。每一个星期重复加一次菌悬液(1×108cfu/mL),每次40mL,总共浇灌2次,以无菌水作对照(CK)。30d测定植株生长情况(如株高、叶片数、径直径、鲜重),结果见表6和附图4。
表6菌株HLH_19对烟苗种子生长的影响
从表6和附图4,可以看出,根据IAA产量选择了HLH_19做了盆栽实验(地点四川农业大学教学农场),用菌处理的植株较CK有明显效果,具体各方面数据见表6。施加HLH_19菌株的烟苗植株的株高、叶片数、茎直径、鲜重均显著增加,增幅分别达到5.84%、13.75%、21.46%、10.17%。
综上所述,本发明综合评价烤烟烟茎内生细菌的产IAA能力、抗植物病原细菌特性、促进植物生长和溶磷能力,选择出了HLH_19菌株,HLH_19菌株可以促进植物的生长,抑制植物病原细菌,提高烟苗的叶片数、茎粗、株高,溶解土壤无机磷。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (10)
1.芽孢杆菌(Bacillus)HLH_19,其特征在于,该芽孢杆菌HLH_19已于2023年3月8日保藏于中国典型培养物保藏中心(CCTCC),地址:湖北省武汉市武昌区八一路299号武汉大学,保藏号为CCTCC NO:M2023266。
2.如权利要求1所述的芽孢杆菌HLH_19在防治青枯假单胞菌、金黄色葡萄球菌引起的植物病害中的应用。
3.根据权利要求2所述的应用,其特征在于,所述植物为烤烟。
4.根据权利要求2所述的应用,其特征在于,所述芽孢杆菌HLH_19作为防治作物病害的药物。
5.如权利要求1所述的芽孢杆菌HLH_19在生物有机肥料方面的应用。
6.根据权利要求5所述的应用,其特征在于,所述芽孢杆菌HLH_19具有溶磷和/或产IAA的作用。
7.如权利要求1所述的芽孢杆菌HLH_19在作为植物生长调节剂中的应用。
8.根据权利要求7所述的应用,其特征在于,在作为烟草生长调节剂中的应用。
9.根据权利要求8所述的应用,其特征在于,在调节烟草株高、茎直径、叶片数和鲜重中任意一项或两项以上中的应用。
10.如权利要求1所述的芽孢杆菌HLH_19在溶解有机磷和/或无机磷中的应用。
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