CN117070421A - 一种复合菌群及其在促进猕猴桃生长中的应用 - Google Patents
一种复合菌群及其在促进猕猴桃生长中的应用 Download PDFInfo
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- CN117070421A CN117070421A CN202311262502.7A CN202311262502A CN117070421A CN 117070421 A CN117070421 A CN 117070421A CN 202311262502 A CN202311262502 A CN 202311262502A CN 117070421 A CN117070421 A CN 117070421A
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
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- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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Abstract
本发明公开了一种复合菌群及其在促进猕猴桃生长中的应用。本发明的复合菌群含有根瘤菌F1、中华根瘤菌2854‑3和慢生根瘤菌DYQJB2，将该复合菌群与枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌混配成菌剂，可以应用于改良碱性土壤、降低猕猴桃植株对土壤中氮肥的需求量和促进猕猴桃植株生长。因此，本发明的复合菌群在猕猴桃栽培和生产中具有重要的应用价值。
Description
技术领域
本发明属于农业微生物技术领域，具体涉及一种复合菌群及其在促进猕猴桃生长中的应用。
背景技术
外施微生物菌群可以与土壤中原有的有益微生物共同形成优势菌群，促进土壤生态系统中碳、氮、氧等元素的良性循环，不但能修复土壤生态环境系统，还能在减少无机肥料使用的同时增加作物产量，从而促进绿色农业发展。另外，在农业生产中微生物菌肥生产成本适中，利于农民接受。
猕猴桃是近代以来人工驯化最成功的4种野生果树之一，其对土壤酸碱度要求不是很严格，但是以在酸性及微酸性土壤（pH5.5～6.5）最为适宜。因此，研发适合猕猴桃生产的微生物菌群及施用技术，不但可以实现减肥增效，保护生态环境，还能促进猕猴桃产业的可持续发展。
研发加速经济作物生长的菌肥，不但能改良土壤，促进植被恢复，还是水源地生态绿色产业、保护水质、护好水源的必然选择。
发明内容
本发明的目的是提供一种能适用于改良碱性土壤、减施氮肥且促进猕猴桃快速生长的复合菌群，并提供该复合菌群在促进猕猴桃生长中的应用。本发明是以丹江口水源地库区周边猕猴桃种植地的土壤为基础开发的复合菌群。
本发明的第一个目的是提供根瘤菌（Rhizobiumsp.）F1，其保藏编号为GDMCCNO.63694。
本发明还提供中华根瘤菌（Sinorhizobiumsp.）2854-3，其保藏编号为GDMCCNO.63695。
本发明还提供慢生根瘤菌（Bradyrhizobiumsp.）DYQJB2，其保藏编号为GDMCCNO.63696。
本发明的第二个目的是提供一种复合菌群，其含有所述的根瘤菌F1、中华根瘤菌2854-3和慢生根瘤菌DYQJB2。
本发明的第三个目的是提供一种菌剂，其含有枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌，以及所述的根瘤菌F1、中华根瘤菌2854-3和慢生根瘤菌DYQJB2。
优选，所述的枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌为北海亦强生物科技有限公司生产。
优选，所述的菌剂中各菌的菌体数量比是枯草芽孢杆菌：地衣芽孢杆菌：胶冻样芽孢杆菌：根瘤菌F1：中华根瘤菌2854-3：慢生根瘤菌DYQJB2为270：270：90：7：7：7。
本发明的第四个目的是提供一种生物菌肥，其含有所述的菌剂和肥料载体。
本发明的第五个目的是提供所述的菌剂在如下（1）-（3）中至少一种中的应用：
（1）改良碱性土壤；
（2）降低植物对土壤中氮肥的需求量；
（3）促进植物生长。
优选，所述的植物为猕猴桃。所述的应用，包括将所述的菌剂接种至植物根部或植物根际土壤的步骤。
本发明的根瘤菌F1、慢生根瘤菌DYQJB2在含碳水化合物的培养基上产酸，可促进碱性土壤中猕猴桃嫁接苗的生长发育。本发明的含有根瘤菌F1、中华根瘤菌2854-3和慢生根瘤菌DYQJB2的复合菌群及菌剂在猕猴桃栽培和生产中具有重要的应用价值。
保藏说明：
本发明的Rhizobiumsp. F1（根瘤菌F1）于2023年08月21日保藏于广东省微生物菌种保藏中心（GDMCC），保藏编号：GDMCC NO. 63694，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所。
本发明的Sinorhizobiumsp.2854-3（中华根瘤菌2854-3）于2023年08月21日保藏于广东省微生物菌种保藏中心（GDMCC），保藏编号：GDMCC NO. 63695，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所。
本发明的Bradyrhizobiumsp. DYQJB2（慢生根瘤菌DYQJB2）于2023年07月28日保藏于广东省微生物菌种保藏中心（GDMCC），保藏编号：GDMCC NO. 63696，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所。
附图说明
图1是菌株F1的形态学及基本生物学特征；其中，图1中的A：菌株F1对不同碳源的利用及不同pH条件下的生长情况；图1中的B：菌株F1在不同抗生素培养基上的生长情况；图1中的C：菌株F1产酸产碱能力、解磷解钾能力检测；图1中的D：菌株F1在不同NaCl浓度条件下的生长；图1中的E：菌株F1对温度的耐受性分析。
图2是菌株F1的16S rDNA基因序列系统发育树。
图3是菌株2854-3的形态学及基本生物学特征；其中，图3中的A：菌株2854-3在不同碳源条件下的生长状态；图3中的B：菌株2854-3在不同pH条件下的生长状态；图3中的C：菌株2854-3在不同NaCl浓度条件下的生长；图3中的D：菌株2854-3对有机磷无机磷及磷酸钾的利用分析；图3中的E：菌株2854-3产酸产碱能力检测。
图4是菌株2854-3的16S rDNA基因序列系统发育树。
图5是菌株DYQJB2的形态学及基本生物学特征；其中，图5中的A：菌株DYQJB2对不同碳源的利用情况；图5中的B：菌株DYQJB2在不同pH条件下的生长状态；图5中的C：菌株DYQJB2在不同抗生素培养基上的生长情况；图5中的D：菌株DYQJB2产酸产碱能力检测；图5中的E：菌株DYQJB2在不同NaCl浓度条件下的生长；图5中的F：菌株DYQJB2对温度的耐受性分析；图5中的G：菌株DYQJB2对有机磷无机磷及磷酸钾的利用分析。
图6是菌株DYQJB2的16S rDNA基因序列系统发育树。
图7是不同菌群处理5个月后猕猴桃的长势。
图8是不同菌群处理5个月后植株株高及光合指标；其中，图8中的A：不同处理后植株状况；图8中的B：植株高度统计结果；图8中的C：植株叶片的光合速率结果；图8中的D；植株叶片叶绿素含量结果。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
以下实施例中所用的枯草芽孢杆菌（产品名称：枯草芽孢杆菌，活菌数量1000亿/g，剂型：水溶性粉剂，购买网址：https://item.jd.com/10034826879550.html）、胶冻样芽孢杆菌（产品名称：胶冻样芽孢杆菌（全水溶型），活菌数量100亿/g，购买网址：https://item.jd.com/10042467225005.html）、地衣芽孢杆菌（产品名称：地衣芽孢杆菌，活菌数量1000亿/g，剂型：水溶型粉剂，购买网址：https://item.jd.com/10034827357945.html）均由北海亦强生物科技有限公司生产。
实施例1
1. 菌株F1、DYQJB2和2854-3的分离获得
取来源于南方海滨的豆科植物海刀豆、大叶千斤拔、田菁等豆科植物的根瘤，用75%酒精消毒30 s，再用2%NaClO水溶液消毒5 min，无菌水漂洗5-6遍，加适量YMA液体培养基（按照表1的YMB培养基配方，将其中的琼脂粉成分去掉后即为YMA液体培养基），用无菌镊子捣碎根瘤，在YMB平板划线纯化，28℃恒温培养2-3 d。
编号为F1的菌株从海南省文昌市丹场村海岛路边荒地的海刀豆根瘤中分离获得，编号为DYQJB2的菌株从大叶千斤拔的根瘤中分离获得，编号为2854-3的菌株从南沙珊瑚岛礁的田菁根瘤中分离获得。
2. 菌株F1、DYQJB2和2854-3的基本生物学特性分析
2.1 NaCl耐受特性分析
（1）提前一天配制YMB培养基（分别为正常的YMB培养基或添加质量分数1%、2%、3%、4%、5% NaCl的YMB培养基）。根据表1配方称取各组分并混合，加水定容至1 L，用5% NaOH溶液或5% HCl溶液调节pH至7.0，121℃灭菌30 min；然后分装成YMB培养基平板。
表1 YMB培养基配方（1 L）
（2）将不同来源的菌液分别稀释102后备用（用2 mL灭菌的离心管加1 mL YMB后加入相应的分离菌10 µL，混匀，酒精灯旁操作）。
（3）吸取稀释后的菌液5 µL点在平板上，在平板写上菌株号和日期，放置28℃培养箱培养，3天后，对照正常YMB培养基上的长势观察在不同盐浓度下各菌的长势，记录并拍照。
2.2 pH实验
配制YMB培养基（pH3、pH5、pH7、pH9、pH11、pH14的培养基），吸取稀释后的菌液5 µL点在不同pH的平板上，3天后观察长势，拍照。
2.3 解磷能力
配制蒙金娜有机磷(卵磷脂)、蒙金娜无机磷(磷酸三钙)、解钾活性测定培养基平板，每个平板划分6个区域，依次吸取5 μL菌液点在每个区域的中心（取样确保精确，不要让菌液流动），在28℃培养箱培养3 d，观察菌株是否生长以及是否形成解磷、解钾圈。
培养基配方如下：①蒙金娜有机磷(卵磷脂) 细菌培养基(1 L)：葡萄糖10.0 g，(NH4)2SO40.5 g，NaCl 0.3 g，MgSO4·7H2O 0.3 g，FeSO40.03 g，MnSO4·H2O 0.03 g，KCl0.3 g，CaCO31.0 g，卵磷脂0.3 g，琼脂20 g，pH 7.0。其中卵磷脂用体积分数75 %乙醇水溶液加热溶解，单独灭菌，与灭菌冷却至60℃的培养基混合后倒平板。
②蒙金娜无机磷(磷酸三钙)细菌培养基(1 L)：葡萄糖10 g，(NH4)2SO40.5 g，NaCl0.3 g，MgSO4·7H2O 0.3 g，FeSO4·7H2O 0.03 g，MnSO4·H2O 0.03 g，CaCO35.0 g，KCl 0.3g， Ca3(PO4)25.0 g，琼脂20 g，pH7.0。
③解钾活性测定培养基（1 L）：蔗糖2.0 g，(NH4)2SO40.5 g，Na2HPO41.5 g，MgSO4·7H2O 0.5 g，钾长石粉5.0 g，pH7.2。
2.4 菌株在培养基上的产酸或产碱能力检测
配制酸碱（溴百酚蓝）培养基。根据表2配方称取各组分并混合，加水定容至1 L，用5% NaOH溶液或5% HCl溶液调节pH至7.0，121℃灭菌30 min。然后分装平板。
表2 酸碱培养基配方（1 L）
灭菌后接种前述的各分离菌5 µL，28℃培养3-5天，观察培养基颜色。培养基变蓝色，代表该菌产碱；培养基变黄色，代表该菌产酸；培养基不变色，为绿色，代表不产酸碱。
结果表明，菌株F1在YMB平板28℃恒温培养3 d后，菌落边缘平滑，具有凸起，菌落颜色微黄。菌株F1可以在以甘露醇、肌醇、甘油、丙二酸、草酸钠、淀粉、柠檬酸钠、葡萄糖、果糖、木糖、阿拉伯糖、乳糖、蔗糖、麦芽糖为碳源的平板生长，适宜生长的pH范围为pH 7-pH9，具有红霉素、氯霉素、磷霉素、庆大霉素、卡那霉素、金霉素、链霉素、四环素、氨苄青霉素、壮观霉素抗性，产酸（代谢产物使溴百酚蓝培养基变成黄色，表明其代谢物为酸性），不具有解有机磷、无机磷的能力，不具有解钾能力，最高可耐2%NaCl，在28℃-37℃下均可生长（图1）。
菌株2854-3在YMB平板28℃恒温培养3 d后，菌落透明，微白，边缘光滑，具有凸起。菌株2854-3可以在以甘露醇、肌醇、甘油、丙二酸、草酸钠、淀粉、葡萄糖、木糖、乳糖、蔗糖、麦芽糖为碳源的平板生长，适宜生长的pH范围为pH5-pH11，中性（代谢产物不能使溴百酚蓝培养基变色，表明其代谢物为中性），具有解有机磷、无机磷的能力，不具有解钾能力，最高可耐3% NaCl（图3）。
菌株DYQJB2在YMB平板28℃恒温培养7 d后才能出现边缘平滑的菌落，菌落透明，边缘光滑，具有凸起。菌株DYQJB2可以在以甘露醇、肌醇、甘油、柠檬酸钠、丙二酸、草酸钠、淀粉、葡萄糖、果糖、木糖、阿拉伯糖、乳糖、蔗糖、麦芽糖为碳源的平板生长，适宜生长的pH范围为pH5-pH11，具有红霉素、磷霉素、庆大霉素、卡那霉素、链霉素、四环素、氨苄青霉素、壮观霉素抗性，产酸（代谢产物使溴百酚蓝培养基变成黄色，表明其代谢物为酸性），不具有解有机磷、无机磷的能力，不具有解钾能力，最高可耐5%NaCl，在28℃和37℃下均可生长（图5）。
3. 菌株F1、DYQJB2和2854-3的分子生物学鉴定
通过16S rDNA对菌株F1、DYQJB2和2854-3菌株进行分子学鉴定，利用引物（F:AGAGTTTGATCCTGGCTCAG; R:TACGGCTACCTTGTTACGACTT）分别对3个菌株基因组DNA进行PCR扩增，分别扩增出约为1.4 kb大小的片段，其中扩增的菌株F1的16S rDNA的核苷酸序列如SEQ ID NO.1所示，1385 bp；扩增的菌株DYQJB2的16S rDNA的核苷酸序列如SEQ ID NO.2所示，1388 bp；扩增的菌株2854-3的16S rDNA的核苷酸序列如SEQ ID NO.3所示，1347 bp；将扩增序列经DNAMAN软件进行序列比对，SNAPGENE软件进行序列拼接，在NCBI (https://blast.ncbi.nlm.nih.gov/) 基因库进行同源性比对。采用MEGA5.0软件用邻接法(Neighbor-Joining)构建系统发育树，进行系统发育分析。
发现F1菌株属于根瘤菌属（Rhizobium），F1菌株与Rhizobiumsp.亲缘关系最近（图2）。对照菌株F1的形态特征、生理生化特性以及通过16S rDNA基因序列构建的系统发育树分析，鉴定菌株F1属于Rhizobium属，因此将F1菌株命名为Rhizobiumsp. F1（根瘤菌F1）。
2854-3菌株属于中华根瘤菌属（Sinorhizobium），与Sinorhizobium alkalisoli亲缘关系最近（图4）。对照菌株2854-3的形态特征、生理生化特性以及通过16S rDNA基因序列构建的系统发育树分析，鉴定菌株2854-3属于Sinorhizobium属，因此将2854-3菌株命名为Sinorhizobiumsp.2854-3（中华根瘤菌2854-3）。
DYQJB2菌株属于慢生根瘤菌属（Bradyrhizobium），与Bradyrhizobiumsp.亲缘关系最近（图6）。对照菌株DYQJB2的形态特征、生理生化特性以及通过16S rDNA基因序列构建的系统发育树分析，鉴定菌株DYQJB2属于Bradyrhizobium属，因此将DYQJB2菌株命名为Bradyrhizobiumsp. DYQJB2（慢生根瘤菌DYQJB2）。
实施例2：菌群促进猕猴桃嫁接苗幼苗期的生长
1. 盆栽猕猴桃种植
取丹江口土样（pH 7.98）敲碎、去杂，定量（称重）装盆，花盆大小为26 cm × 24cm × 29 cm，按照正常猕猴桃种植所需肥料量中氮肥减半的原则，进行肥料配制，即磷酸二氢钾10.8 g/盆，尿素减半为4.8 g /盆进行拌土。共装150盆用来种植150株长势一致的猕猴桃嫁接苗（接穗高均为30 cm），嫁接苗接穗为东红，砧木为米良一号。
2. 菌群组合设计及接种处理方法
盆栽猕猴桃嫁接苗在温室种植两周后进行菌剂接种处理，各处理的菌剂组合如表3，共设计了3个菌剂组合，分别为T1、T2和T3。每组菌剂组合处理7盆苗，设3组生物重复，共计每种菌剂组合处理21株苗。
表3 进行盆栽猕猴桃处理的3种菌剂组合
T1处理为：每盆在植株根际周围轻轻浇灌500 mL自来水（不加任何菌，自来水于使用前已放置一晚，消除氯气），确保没有多余的液体流出花盆。
制备芽孢杆菌的菌剂组合溶液：将枯草芽孢杆菌、地衣芽孢杆菌、胶冻样芽孢杆菌的接种量参照说明书，即枯草芽孢杆菌和地衣芽孢杆菌按照150 g/亩，胶冻样芽孢杆菌按照500 g/亩的用量混合。具体为：枯草芽孢杆菌（活菌数量1000亿/g）称取1.5 g，地衣芽孢杆菌（活菌数量1000亿/g）称取1.5 g，胶冻样芽孢杆菌（活菌数量100亿/g），称取5 g，将上述称好的三种菌混合在一起，溶于12 L水中，制备得到芽孢杆菌的菌剂组合溶液。
T2处理为：取制备的芽孢杆菌的菌剂组合溶液30.86 mL（即其中的活菌数量为9×108个菌），加自来水（于使用前已放置一晚，消除氯气）补足配制成总体积为500 mL的T2处理液，每盆在植株根际周围轻轻浇灌500 mL T2处理液，确保没有多余的液体流出花盆。
将菌株F1、2854-3、DYQJB2分别接种于YMA液体培养基（按照表1的YMB培养基配方，将其中的琼脂粉成分去掉后即为YMA液体培养基）中28℃培养3-7天至OD600值在0.6左右，于3000g离心10分钟，弃上清后，用无菌氯化镁（浓度为10 mM/L）重悬沉淀，定容至10 mL，然后用水调整菌液浓度并测定OD600值。按照OD600=1时，菌液浓度为2×109个/mL计算。
T3处理为：取OD600=0.005的菌株F1、2854-3、DYQJB2的菌液各1 mL（即其中每种菌株的活菌数量均为1×107个菌），然后取前述制备的芽孢杆菌的菌剂组合溶液30.86 mL（即其中的活菌数量为9×108个菌），混合，并加自来水（于使用前已放置一晚，消除氯气）补足配制成总体积为500 mL的T3处理液，每盆在植株根际周围轻轻浇灌500 mL T3处理液，确保没有多余的液体流出花盆。
在进行上述的T1、T2、T3接种处理的2个月后，各处理植株对应相同处理方式再重复接种一次，5个月后测定相关指标。
3. 植株生理指标测定
种植5个月后对植株的株高，光合速率及叶绿素含量进行测定。
猕猴桃的光合参数由便携式光合作用仪（LI-6400XT，USA）于天气晴朗时的上午9：00~11：00测定，测定时叶片选择完全展开的由上到下第三片功能叶，每3株的平均值作为一个重复，每个处理3个重复。测定时，光照强度设定为1000 μmol /(m2·s)，空气流速400mmol/s，设定叶温28℃，平均温度29.5±2℃，外界CO2浓度394±14 µmol/mol。
猕猴桃叶绿素含量采用型号为SPAD-502plus便携式叶绿素仪于晴天分别测定植株上中下三部分，每个部分2个叶片，共6个叶片的SPAD值，每个处理随机选取9株测量，每个处理上、中、下部共18个数值，然后取平均值作图。
结果表明，T3菌群处理后植株长势一致，株高显著高于其他处理的植株（图7），且T3处理后植株叶片的光合速率及叶绿素含量均显著高于其他处理的株系（图8）；而T2相对于T1，在株高、植株叶片的光合速率及叶绿素含量方面均无显著差异。上述结果表明，T2处理中仅仅利用现有商业菌株（枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌）没有明显改善效果，而结合本实施例的新的菌株F1、2854-3、DYQJB2及现有商业菌株（枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌）的T3处理对猕猴桃嫁接苗苗期的生长具有显著的促进作用。
Claims (10)
1. 根瘤菌（Rhizobium sp.）F1，其特征在于，保藏编号为GDMCC NO.63694。
2. 中华根瘤菌（Sinorhizobium sp.）2854-3，其特征在于，保藏编号为GDMCCNO.63695。
3. 慢生根瘤菌（Bradyrhizobium sp.）DYQJB2，其特征在于，保藏编号为GDMCCNO.63696。
4.一种复合菌群，其特征在于，含有权利要求1所述的根瘤菌F1、权利要求2所述的中华根瘤菌2854-3和权利要求3所述的慢生根瘤菌DYQJB2。
5.一种菌剂，其特征在于，含有枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌，以及权利要求1所述的根瘤菌F1、权利要求2所述的中华根瘤菌2854-3和权利要求3所述的慢生根瘤菌DYQJB2。
6.根据权利要求5所述的菌剂，其特征在于，所述的枯草芽孢杆菌、地衣芽孢杆菌和胶冻样芽孢杆菌为北海亦强生物科技有限公司生产。
7.根据权利要求5所述的菌剂，其特征在于，所述的菌剂中各菌的菌体数量比是枯草芽孢杆菌：地衣芽孢杆菌：胶冻样芽孢杆菌：根瘤菌F1：中华根瘤菌2854-3：慢生根瘤菌DYQJB2为270：270：90：7：7：7。
8.一种生物菌肥，其特征在于，含有权利要求5-7任一项所述的菌剂和肥料载体。
9.权利要求5-7任一项所述的菌剂在如下（1）-（3）中至少一种中的应用：
（1）改良碱性土壤；
（2）降低植物对土壤中氮肥的需求量；
（3）促进植物生长。
10.根据权利要求9所述的应用，其特征在于，所述的植物为猕猴桃。
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