CN116515673A - 假单胞菌及其菊花秸秆发酵产物以及菊花秸秆发酵产物促进植物生长的应用 - Google Patents
假单胞菌及其菊花秸秆发酵产物以及菊花秸秆发酵产物促进植物生长的应用 Download PDFInfo
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- CN116515673A CN116515673A CN202310104240.5A CN202310104240A CN116515673A CN 116515673 A CN116515673 A CN 116515673A CN 202310104240 A CN202310104240 A CN 202310104240A CN 116515673 A CN116515673 A CN 116515673A
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- pseudomonas
- fermentation
- pse13
- chrysanthemum
- straw
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株温哥华假单胞菌PSE13,分类命名为温哥华假单胞菌(Pseudomonas vancouverensis),于2022年12月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCC No.26170。本发明公开了温哥华假单胞菌PSE13与贵州木霉NJAU4742在制备菊花秸秆发酵产物的应用。以菊花秸秆为底物、贵州木霉NJAU4742具有较好的秸秆降解能力,为温哥华假单胞菌PSE13生长提供功能物质,发酵结束后温哥华假单胞菌PSE13的数量级在109‑1010之间;同时,发酵后代谢物质可以极显著地发挥促生作用,促进植物的生长,使植株生物量显著增加。
Description
技术领域
本发明属于微生物领域,涉及假单胞菌与木霉的菊花秸秆发酵产物在植物促生方面的应用,具体涉及假单胞菌的菊花秸秆发酵产物在植物促生方面的应用。
背景技术
微生物发酵技术是功能性微生物利用底物合成某种或某类化合物的重要手段之一,在医学、食品以及农业等诸多领域有着广泛的应用。以往,绝大部分化合物依赖于化工合成和植物提取,在很大程度上能够满足需求,但不可避免的也会对环境和生态造成严重破坏。随着社会的进步和人们对高生活质量的迫切需求,微生物发酵技术成为研究热点,开始逐渐取代上述两种手段。
秸秆的构成使其成为一种很好的固体发酵介质,而其中的可溶性碳水化合物、植物蛋白以及微量元素又可为周围的微生物生长繁殖提供营养。以秸秆为基质构建发酵系统进行制肥具有广泛的应用潜力。目前,秸秆发酵方式有:以单菌直接发酵,如加入强降解菌绿色木霉直接发酵;多种降解菌共同发酵,如拟康宁木霉和黄孢原毛平革共同发酵为主,发酵周期多超过40天,周期较长、耗时耗力。目前,用于微生物发酵的秸秆多以小麦、玉米等主要粮食作物秸秆为主,缺乏对药用、观赏用作物秸秆资源化利用。秸秆发酵产物利用多以饲料化、肥料化等为主,缺乏发酵产物多用途、高附加值利用。秸秆发酵菌种多以强降解菌、产酵母菌为主,缺乏促生菌、抑病菌等可调控土壤微生物环境、调控植物生长发育的菌种参与。
利用有益微生物改善土壤微生物环境、调控植物生长发育,已成为当今农业可持续发展的重要科学策略之一。生活在植物根际并且有利于植物生长的细菌称为植物根际促生菌(plant growth promoting rhizobacteria,PGPR),假单胞菌作为其中一种重要的植物根际促生菌,许多研究证明假单胞菌等根际促生菌对包括但不限于蓝莓、甘蓝型油菜、紫金草、大豆、甘蔗、小麦、樱桃等多种作物具有一定促进生长作用。但利用假单胞菌进行秸秆固体发酵及其利用秸秆后发酵产物的作用效果尚没有研究。
发明内容
发明人采用假单胞菌和木霉接力发酵的方式,探究温哥华假单胞菌PSE13不同秸秆的发酵产物对植物的促生效果,简单分析促进作物生长的功能性物质。发明人发现:菊花秸秆经温哥华假单胞菌PSE13与贵州木霉NJAU4742接力发酵后,其浸提液可以极显著地促进番茄的生长;进一步结合室内实验分析发现,菊花秸秆经温哥华假单胞菌PSE13与贵州木霉NJAU4742发酵后,吲哚乙酸和铁载体两类与植物促生相关的次级代谢产物显著富集。
本发明的目的是通过以下技术方案实现的:
一株温哥华假单胞菌PSE13,分类命名为温哥华假单胞菌(Pseudomonasvancouverensis),于2022年12月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCC No.26170。
所述的温哥华假单胞菌PSE13在秸秆发酵中的应用。
优选的,所述的温哥华假单胞菌PSE13在秸秆固体发酵中的应用。
所述的温哥华假单胞菌PSE13与贵州木霉NJAU4742在制备菊花秸秆发酵产物的应用。
优选的,所述的温哥华假单胞菌PSE13与贵州木霉NJAU4742通过固体发酵在制备菊花秸秆发酵产物中的应用。
本发明的另一个目的是提供一种菊花秸秆发酵产物,所述的菊花秸秆发酵产物是以温哥华假单胞菌PSE13与贵州木霉NJAU4742为功能微生物,以菊花秸秆为底物,经固体发酵和浸提制得的。
所述的菊花秸秆发酵产物含有IAA、铁载体。
所述的菊花秸秆的品种不限。
本发明的另一个目的是提供一种所述的菊花秸秆发酵产物的制备方法,包括:将菊花秸秆烘干、磨粉过筛、灭菌,接入贵州木霉NJAU4742孢子液,调节菊花秸秆粉含水率至70%~80%,进行固体发酵5~9天,再接入温哥华假单胞菌PSE13菌液进行固体发酵5~9天,固体发酵后,用水浸提,得到发酵产物。
温哥华假单胞菌PSE13菌悬液的OD600=0.1~1,每1~5g菊花秸秆接入0.1~2mL温哥华假单胞菌PSE13菌悬液;贵州木霉NJAU4742孢子液的浓度为105CFU/mL~108CFU/mL,每1~5g菊花秸秆接入3~15mL贵州木霉NJAU4742孢子液。
具体的,温哥华假单胞菌PSE13菌悬液的OD600=0.5,每1.2g菊花秸秆接入0.1mL温哥华假单胞菌PSE13菌悬液;贵州木霉NJAU4742孢子液的浓度为106CFU/mL,每1.2g菊花秸秆接入3.6mL贵州木霉NJAU4742孢子液。
所述的温哥华假单胞菌PSE13菌悬液是由以下方法制得的:将温哥华假单胞菌PSE13菌株在LB平板上划线,置于恒温培养箱中,20~30℃培养直至出现单菌落;挑取LB平板上的单菌落于液体LB培养基中,置于摇床中,20~30℃、150~170r·min-1过夜培养,获得种子液,按照接种量为0.1~1%(v/v)将种子液转接于100mL液体LB培养基中,20~30℃、150~170r·min-1振荡培养8~10h,用无菌水稀释至目标浓度,得到温哥华假单胞菌PSE13菌悬液。
贵州木霉NJAU4742,分类名称为贵州木霉(Trichoderma guizhouense),于2016年4月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCCNo.12166。
所述的贵州木霉NJAU4742孢子液是由以下方法制得的:将贵州木霉NJAU4742菌株点接于PDA平板上,置于恒温培养箱中,30℃培养7天,用无菌水洗下孢子液,用两层无菌纱布将孢子液过滤,经无菌水稀释至目标浓度,获得贵州木霉NJAU4742孢子液。
所述的固体发酵(即接入贵州木霉NJAU4742后的固体发酵,以及接入温哥华假单胞菌PSE13后的固体发酵)的环境温度为20~30℃。
所述的浸提的条件为:在温度20~30℃、转速150~170r·min-1下浸提0.5~1h。
本发明的另一个目的是提供所述的温哥华假单胞菌PSE13与木霉NJAU4742的菊花秸秆发酵产物在植物促生的应用或制备促进植物生长的肥料或制剂的应用。
本发明的有益效果:
本发明以菊花秸秆为底物、温哥华假单胞菌PSE13作为目标菌株,同时贵州木霉NJAU4742具有较好的秸秆降解能力,为温哥华假单胞菌PSE13生长提供功能物质以更好进行发酵过程。
菊花秸秆经木霉NJAU4742与温哥华假单胞菌PSE13接力发酵获得发酵产物,发酵结束后温哥华假单胞菌PSE13的数量级在109-1010之间;同时,发酵后代谢物质可以极显著地发挥促生作用,促进植物的生长,使植株生物量显著增加。
附图说明
图1为不同秸秆及发酵方式对假单胞菌生物量的影响。
图2为各发酵方式浸提液对种子萌发的影响。
图3为各发酵方式产IAA和铁载体情况。
图4为各发酵方式浸提液对株高、根长、茎粗影响结果。
图5为各发酵方式浸提液对地上部干鲜重、地下部干鲜重影响结果。
图6为各发酵方式浸提液对SPAD值影响结果。
生物材料保藏信息
PSE13,分类名称为温哥华假单胞菌(Pseudomonas vancouverensis),于2022年12月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院,中国科学院微生物研究所,菌种保藏号为CGMCC No.26170。
NJAU4742,分类名称为贵州木霉(Trichoderma guizhouense),于2016年4月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院,中国科学院微生物研究所,菌种保藏号为CGMCC No.12166。
具体实施方式
下面结合具体实施方式对本发明的技术方案作进一步说明。
菊花秸秆:烘干、磨粉过20目筛、1.2g秸秆封装三角瓶,115℃灭菌30min,待用。
MS缓冲溶液:50mmol/L Tris-HCl(pH 7.5),100mmol/L NaCl,10mmol/L MgSO4,0.01%明胶。
1/10倍稀释的胰蛋白胨大豆琼脂培养基(1/10TSA):每升含1.5g胰蛋白胨,0.5g大豆蛋白胨,0.5g氯化钠,15g琼脂,pH 7.0。
胰蛋白胨大豆液体培养基(TSB):每升含有15g胰蛋白胨,5g大豆蛋白胨,5g氯化钠,pH 7.0。
30%甘油:纯净甘油30mL、氯化钠0.5g,碱性磷酸钠(磷酸氢二钠)1.0g,蒸馏水定容至100mL,115℃灭菌30min。
温哥华假单胞菌PSE13的筛选与鉴定:取1g番茄根际土壤(南京市江宁区麒麟镇后村蔬菜大棚),与9mL MS缓冲溶液混合于50mL三角瓶,将三角瓶置于旋转摇床中,在转速为170r·min-1、温度为30℃下处理30分钟,使用无菌水将土壤悬浊液稀释至10-5-10-6悬浊液,往100μL稀释土壤悬浊液加入含有1/10倍稀释的胰蛋白胨大豆琼脂培养基的培养皿中涂布均匀,在恒温(30℃)培养箱中黑暗培养48h;随后使用无菌牙签从每个样品中随机挑取单菌落,划线于TSA平板纯化;挑取纯的单菌落在96孔板中于100μL胰蛋白胨大豆液体培养基中,将孔板放入摇床(30℃,170r·min-1)中孵育过夜,加入100μL30%甘油并充分混合,然后保存于-80℃冰箱,最后从根际土样中分离、纯化得到菌株。
将纯化得到的菌株根际细菌分别划线于TSA平板上挑取单克隆。菌株总DNA的提取按TIANGamp细菌DNA Kit(天根生物科技有限公司,北京)操作步骤进行。总DNA在0.8%琼脂糖凝胶上电泳检测。细菌16S rRNA基因扩增的通用引物:27F(5’-AGAGTTTGATCCTGGCTCAG-3’);1492R(5’-GGTTACCTTGTTACGACT T-3’)(Eden et al.,1991)。PCR反应体系(25μL):DNA模板1μL,反应混合物12.5μL,前后端引物各1μL,ddH2O 9.5μL。PCR反应条件:95℃预变性5min后,进入热循环:94℃变性30s,58℃退火30s,72℃延伸1min 30s,共30个循环。最后72℃延伸10min。PCR产物在1%的琼脂糖凝胶上电泳后检测。PCR扩增产物由上海生物工程技术有限公司纯化后测序。根据16S rRNA的基因序列的测序结果,在http://www.ncbi.nlm.nih.gov在线查询分析。
基于16S rRNA的系统发育分析结果显示,根际分离细菌主要隶属于为变形菌门(Proteobacteria)、假单胞菌属(Pseudomonas)。分离纯化得到的细菌记为PSE13,于2022年12月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心,分类名称为温哥华假单胞菌PSE13(Pseudomonas vancouverensis),菌种保藏号为CGMCC No.26170。
温哥华假单胞菌PSE13菌悬液的制备:将-80℃冰箱保存的温哥华假单胞菌PSE13菌株在LB平板上划线,并置于恒温培养箱(28℃)培养直至出现单菌落;挑取LB平板上的单菌落于液体LB培养基中,置于摇床(28℃、170r·min-1)中过夜培养,获得种子液,按照接种量为1%(v/v)将种子液转接于含有100mL液体LB培养基的250mL三角瓶中扩大培养,28℃、170r·min-1振荡培养10h,用无菌水稀释至OD600=0.5,得到温哥华假单胞菌PSE13菌悬液,待用。
贵州木霉NJAU4742孢子液的制备:将实验室保存的贵州木霉NJAU4742点接于PDA平板上,置于恒温培养箱(贵州木霉NJAU4742培养温度为30℃)中培养7天,用无菌水洗下孢子液,并用两层无菌纱布将孢子液过滤至2mL离心管中,吸取一定量的孢子液于血球计数板中计数,经无菌水稀释,获得孢子浓度为106CFU/mL的孢子液,离心管收集待用。
按不同发酵方式、发酵类型、菌株接种形式组合秸秆进行发酵。
不同发酵配方如表1所示。
表1.不同发酵方式
实施例1
菊花秸秆及发酵方式对有益菌生物量的影响
秸秆:菊花秸秆。
发酵方式:温哥华假单胞菌PSE13单独发酵7天(A1)、贵州木霉NJAU4742和温哥华假单胞菌PSE1同时接入,共同发酵14天(A2)、温哥华假单胞菌PSE13单独发酵14天(A3)、贵州木霉NJAU4742发酵7天后加入温哥华假单胞菌PSE13接力发酵7天(A4)、不同发酵配方如表1所示。
取菊花植株,去根、叶,烘干,磨粉,过20目筛。
单菌发酵(A1):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中接入100μL温哥华假单胞菌PSE13菌悬液OD600=0.5,并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养7天;取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
共同发酵(A2):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中同时接入3.6mL 106CFU/mL的木霉孢子液、100μL温哥华假单胞菌PSE13菌悬液OD600=0.5,并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养14天;取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
单菌发酵(A3):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中接入100μL温哥华假单胞菌PSE13菌悬液OD600=0.5,并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养14天;取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
接力发酵(A4):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中接入3.6mL 106CFU/mL的木霉孢子液并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养7天;从培养箱中取出三角瓶,并在超净台中接入100μL温哥华假单胞菌PSE13菌悬液OD600=0.5,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养7天,接力发酵(A4)发酵结束后,取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
单菌发酵(A5):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中接入3.6mL 106CFU/mL的木霉孢子液并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养7天;取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
单菌发酵(A6):称取1.20g菊花秸秆粉于150mL三角瓶中,用封口膜进行封口,在温度115℃下灭菌30min;在超净工作台中接入3.6mL 106CFU/mL的木霉孢子液并用无菌水调节含水率至75%,置于培养箱中,在空气相对湿度80%、温度30℃下静置发酵培养14天;取出三角瓶,加入24mL无菌水,并在温度28℃、转速170r·min-1下浸提1h。
发酵方式A1-A4浸提结束后,进行以下处理:静置半个小时,取上清液。在固体LB平板上稀释涂布,平板在温度30℃、空气相对湿度80%下培养约20h计数。
以温哥华假单胞菌PSE13作为目标菌株,分析发酵方式A1、A2、A3、A4对假单胞菌生物量的影响,结果如图1、表2所示。由结果可知,与其他三种发酵方式相比,采用假单胞菌和木霉接力发酵的方式,木霉NJAU4742发酵7天后加入温哥华假单胞菌PSE13继续发酵7天的接力发酵方式(A4),发酵后温哥华假单胞菌PSE13生物量增长显著,PSE13的有效活菌数可以达到约3×109个/g。说明贵州木霉NJAU4742具有较好的菊花秸秆降解能力,能为温哥华假单胞菌PSE13生长提供功能物质更好进行发酵过程,且采用接力发酵后温哥华假单胞菌PSE13生物量增长显著。
表2.不同发酵方式对温哥华假单胞菌PSE13生物量的影响
注:不同小写字母表示处理间差异显著(P<0.05)。
实施例2
不同发酵方式浸提液对产IAA、铁载体情况的影响
结合实施例1,将发酵方式(A4)定为最佳发酵组合,菊花秸秆最佳组合接力发酵后显著富集代谢产物IAA、铁载体。选取接力发酵方式(A4)发酵浸提液进行IAA、铁载体产量测定,选取单菌发酵方式(A1)、单菌发酵方式(A5)进行对比分析;同时,考虑到接力发酵方式(A4)的发酵过程中贵州木霉NJAU4742在单独发酵7天后仍然继续进行7天发酵过程,发酵总过程为14天,故选取单菌发酵方式(A6)贵州木霉NJAU4742单独发酵14天过程进行对比分析。
实施例1中发酵方式A1、A4-A6浸提结束后,进行以下处理:静置半个小时,取上清液,用0.22μm滤膜过滤,得到无菌浸提液。
铁载体产量测定采用实验室常规方法,即CAS检测液测定法。本方法基于检测液中物质能与铁载体结合产生显色反应(Himpsl et al.,2019)。按无菌浸提液和CAS检测液的体积比1:1将无菌浸提液与CAS检测液混合,静置反应1h,用酶标仪测定630nm波长下的吸光度值,得出值A。用无菌MKB培养基作为对照,按无菌MKB培养基和CAS检测液的体积比1:1将无菌MKB培养基与CAS检测液混合,静置反应1h,用酶标仪测定630nm波长下的吸光度值,得到值Ar。铁载体相对含量(SU)计算参考(Schwyn et al.,1987),公式为:
式中,SU表示铁载体相对含量,A表示细菌检测值,Ar表示对照值。
采用双抗体夹心法测定无菌浸提液中吲哚乙酸(IAA)水平。用纯化的吲哚乙酸(IAA)捕获抗体包被微孔板,制成固相抗体,往包被的微孔中依次加入吲哚乙酸(IAA),再与HRP标记的检测抗体结合,形成抗体抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深钱和样品中的吲哚乙酸(IAA)呈正相关。采用酶标仪,在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中吲哚乙酸(IAA)含量。
由图2可知,接力发酵方式(A4)获得的发酵液中,IAA、铁载体含量分别为36.3nmol/L、9.9SU(%);该接力发酵方式(A4)IAA含量与单菌发酵(A1)无显著差异,但低于单菌发酵(A6);该接力发酵方式(A4)铁载体含量与单菌发酵(A1)无显著差异,但显著高于发酵方式(A5、A6)。
综合目标功能菌株(温哥华假单胞菌PSE13)的生物量、发酵液供能物质含量的数据,接力发酵方式(A4)较好。
实施例3
不同发酵方式浸提液对种子萌发的影响
实施例1中发酵方式A1、A4-A6浸提结束后,进行以下处理:静置半个小时,取上清液,用0.22μm滤膜过滤,得到无菌浸提液。
种子:红矮生番茄种子。
种子清水浸泡3h后使用5%次氯酸钠消毒1min,超纯水冲洗6遍后,将种子放入装有1层滤纸的平板中(记为实验第1天),加入5mL不同发酵方式的无菌浸提液,以5mL无菌水代替浸提液作为空白对照(CK);每日观察并记录每个培养皿中发芽种子数,实验开始5天后所有处理均再无新种子萌发,因此计算第5天的种子发芽率(发芽种子数占供试种子数的百分比,%)。
结果如图3所示,与空白对照(CK)相比,各发酵浸提液处理(A1、A4、A5、A6)均略有增加,说明浸提液对种子萌发无明显抑制作用。
实施例4
菊花秸秆发酵后浸提液盆栽实验分析
实施例1中发酵方式A1、A4-A6浸提结束后,进行以下处理:静置半个小时,取上清液,用0.22μm滤膜过滤,得到无菌浸提液。
采用盆栽促生实验对菊花秸秆发酵后浸提液进行实验。选取大小一致的红矮生番茄,进行移栽,每个盆栽容量300g基质,单个盆栽移栽一颗番茄苗,当番茄幼苗达到三片真叶期时(移栽后第3天)加入不同处理发酵液(采用灌根处理,每20mL/300g基质),所有植物均生长在自然温度变化范围为25℃至35℃的温室中,并定期浇水。每隔3天,随机调整托盘位置,于第28天进行取样。
设置4个处理:接力发酵方式(A4)无菌浸提液,单菌发酵(A5)无菌浸提液、单菌发酵(A6)无菌浸提液,使用等量无菌水替换浸提液作为空白对照(CK)。每处理随机选取30株番茄植株,每株分离为地上和地下部分,进行株高的测量;最后测定根、干鲜物质量与地上部干鲜物质量、SPAD值(相对叶绿素含量)用日本柯尼卡美能达公司产便携式SPAD-502型叶绿素仪测定,结果如图4、图5、图6所示。
图4展示了经不同发酵方式浸提液灌根处理后植株的株高、根长、茎粗情况。图5展示了经不同发酵方式浸提液灌根处理后植株的地上部干重、地下部干重、地上部鲜重、地下部鲜重情况。图6展示了经不同发酵方式发酵液灌根处理后植株的SPAD值情况。
由图4可知,接力发酵方式(A4)的浸提液使植物株高显著增高、根部长度显著增长、茎粗显著增加。由图5可知,接力发酵方式(A4)的浸提液使植物地上部干鲜重、地下部干鲜重均提升显著。由图6可知,接力发酵方式(A4)的浸提液使植物SPAD值显著提升。
基于经不同发酵方式发酵后温哥华假单胞菌的生物量、产IAA、铁载体情况、发酵液灌根处理后植株的株高、根长、茎粗、地上部干鲜重、地下部干鲜重、SPAD值情况,发现菊花秸秆经温哥华假单胞菌PSE13与贵州木霉NJAU4742接力发酵方式(A4),温哥华假单胞菌的生物量显著提升;发酵浸提液产IAA、铁载体能力较强;发酵液灌根处理后植株的株高、根长、茎粗在高度、长度、粗细方面均有显著提升;植物地上部干鲜重、地下部干鲜重,重量提升显著;植物SPAD值显著提升。故选择发酵方式(A4)作为最佳发酵配方,其浸提液可以极显著地促进番茄的生长。
Claims (10)
1.一株温哥华假单胞菌PSE13,分类命名为温哥华假单胞菌(Pseudomonasvancouverensis),于2022年12月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCC No.26170。
2.权利要求1所述的温哥华假单胞菌PSE13在秸秆发酵中的应用。
3.权利要求1所述的温哥华假单胞菌PSE13与贵州木霉NJAU4742在制备菊花秸秆发酵产物的应用。
4.一种菊花秸秆发酵产物,其特征在于:所述的菊花秸秆发酵产物是以权利要求1所述的温哥华假单胞菌PSE13与贵州木霉NJAU4742为功能微生物,以菊花秸秆为底物,经固体发酵和浸提制得的。
5.根据权利要求4所述的菊花秸秆发酵产物,其特征在于:菊花秸秆发酵产物含有IAA、铁载体。
6.根据权利要求4所述的菊花秸秆发酵产物,其特征在于:贵州木霉NJAU4742,保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCC No.12166。
7.一种权利要求5所述的菊花秸秆发酵产物的制备方法,其特征在于:包括:将菊花秸秆烘干、磨粉过筛、灭菌,接入贵州木霉NJAU4742孢子液,调节菊花秸秆粉含水率至70%~80%,进行固体发酵5~9天,再接入温哥华假单胞菌PSE13菌悬液进行固体发酵5~9天,用水浸提,得到发酵产物。
8.根据权利要求7所述的菊花秸秆发酵产物的制备方法,其特征在于:温哥华假单胞菌PSE13菌悬液的OD600=0.1~1,每1~5g菊花秸秆接入0.1~2mL温哥华假单胞菌PSE13菌悬液;贵州木霉NJAU4742孢子液的浓度为105CFU/mL~108CFU/mL,每1~5g菊花秸秆接入3~15mL贵州木霉NJAU4742孢子液。
9.根据权利要求7所述的菊花秸秆发酵产物的制备方法,其特征在于:所述的固体发酵的环境温度为20~30℃;所述的浸提的条件为:在温度20~30℃、转速150~170r·min-1下浸提0.5~1h。
10.权利要求4所述的菊花秸秆发酵产物在植物促生的应用或制备促进植物生长的肥料或制剂的应用。
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