CN116606780A - 一株假黄单胞菌属细菌njau-t129及其应用 - Google Patents
一株假黄单胞菌属细菌njau-t129及其应用 Download PDFInfo
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Abstract
本发明公开了一株假黄单胞菌属细菌NJAU‑T129,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2023年3月24日,保藏编号为CGMCC No.26906。本发明也公开了由其制备的菌剂及在促进作物生长、消减作物种植土壤中抗生素抗性基因含量中的应用。本发明假黄单胞菌属细菌NJAU‑T129对番茄种植土壤抗生素抗性基因有显著消减效果,也对番茄的生长有良好的促进效果。
Description
技术领域
本发明涉及生物防控技术领域,具体涉及一株兼具消减土壤抗生素抗性基因和促生功能的假黄单胞菌属细菌NJAU-T129及其应用。
背景技术
土壤抗生素抗性基因的爆发与水平转移会引起土壤中许多致病微生物携带抗生素抗性基因,该现象在作物的整个生长期均可发生。由于病原菌能在土壤中大量存活,一旦获得抗生素抗性,存在从食物链移动到餐桌,对人体健康造成危害的潜在威胁。另外,施用畜禽粪污及大量施用其制造而成的有机肥也会引入外源抗生素抗性基因,不合理的耕作施肥容易导致病原菌爆发造成土壤抗性基因激增。我国已发现抗生素抗性基因含量过高的土壤,因此,探索消减土壤抗生素抗性基因新型技术,成为当前研究的热点。目前市面上大都是普通腐熟有机肥、化肥和促生或防控土传病害生物有机肥产品,对于能消减土壤抗生素抗性基因生物有机肥产品很少提及。基于此,本发明从番茄植株根际土壤分离筛选出一株能够有效消减土壤中抗生素抗性基因含量的功能细菌,将其添加至具有良好改土增产效果的腐熟有机肥作为基肥的土壤中去,对其消减土壤中抗生素抗性基因及增产效果进行了测定,以期创制消减土壤抗生素抗性基因的新型生物有机肥产品。
发明内容
本发明的目的是提供一株兼具消减土壤抗生素抗性基因和促生功能的假黄单胞菌属细菌NJAU-T129及其应用,以解决现有技术的不足。
本发明的目的可通过以下技术方案实现:
本发明第一方面提供了一株假黄单胞菌属细菌NJAU-T129,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2023年3月24日,保藏编号为CGMCCNo.26906。
本发明第二方面提供了上述假黄单胞菌属细菌NJAU-T129制备的菌剂。
进一步地,所述菌剂通过以下方法制备得到:将保藏编号为CGMCC No.26906的假黄单胞菌属细菌NJAU-T129进行液体发酵,发酵结束后离心获得菌体,用无菌生理盐水重悬菌体,使菌悬液中菌含量≥106个/ml。
更进一步地,所述菌剂通过以下方法制备得到:将保藏编号为CGMCC No.26906的假黄单胞菌属细菌NJAU-T129接种至R2A液体培养基进行液体发酵,发酵条件:pH为7.0,温度为30℃,转速为170-190r/min,发酵时间为48-72h;发酵结束后6000-8000r/min离心8-10min获得菌体,用无菌生理盐水重悬菌体,使菌悬液中菌含量≥106个/ml。
本发明第三方面提供了上述菌剂在促进作物生长、消减作物种植土壤中抗生素抗性基因含量中的应用。
进一步地,所述作物包括番茄。
进一步地,所述抗生素抗性基因包括四环素耐药基因tetW。
进一步地,应用时,以腐熟有机肥作为基肥,作物定植后一段时间施入所述菌剂。
更进一步地,所述腐熟有机肥包括腐熟鸡粪有机肥。
更进一步地,盆栽作物时,上述菌剂施入量为20-50mL·kg-1土壤,优选30-35mL·kg-1土壤。
本发明的有益效果:
本发明针对有效消减番茄作物种植土壤已有的高抗生素抗性基因含量和遏制由于施肥不当带入或病原菌爆发引起的土壤抗生素抗性基因持续增加现象,筛选出一株兼具消减土壤抗生素抗性基因和促生功能的假黄单胞菌属细菌NJAU-T129,为降低土壤抗生素抗性基因含量和增加作物生物量提供技术支持及应用。
本发明盆栽试验结果表明,使用NJAU-T129功能菌研制的生物有机肥能够良好促进番茄植株的生长,显著降低土壤中四环素耐药基因tetW含量。四环素耐药基因是土壤最常见的抗生素抗性基因之一,四环素耐药基因的消减对土壤抗生素抗性基因的消减意义巨大。可见,本发明假黄单胞菌属细菌NJAU-T129对番茄种植土壤抗生素抗性基因有显著消减效果,也对番茄的生长有良好的促进效果。
附图说明
图1为基于菌株NJAU-T129 16SrRNA基因序列构建的系统发育树。
图2为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Pseudomonas sp.C1R15)平板对峙结果。
图3为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Pseudomonas sp.C4R11)平板对峙结果。
图4为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Bacillus sp.C35)平板对峙结果。
图5为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Pseudomonas sp.C4R16)平板对峙结果。
图6为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Bacillus sp.C16)平板对峙结果。
图7为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Bacillus sp.C30)平板对峙结果。
图8为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Bacillus sp.C40)平板对峙结果。
图9为菌株NJAU-T129与土壤常见携带抗生素抗性基因细菌(Bacillus sp.S62)平板对峙结果。
图10为接种菌株NJAU-T129菌悬液对番茄种植土壤中四环素耐药基因含量的影响。
生物材料保藏信息
菌株NJAU-T129,分类命名为Pseudoxanthomonas sp.,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏日期为2023年3月24日,保藏编号为CGMCC No.26906。
具体实施方式
下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明,但并不用来限定本发明的实施范围。
实施例1 功能菌株的分离与筛选
1.供试材料:
供试菌种:土壤常见高含量抗生素抗性基因菌芽孢杆菌属细菌Bacillus sp.、假单胞属细菌Pseudomonas sp.、大肠杆菌Escherichia coli由江苏省固体有机废弃物资源化高技术研究重点实验室分离和保存。
供试培养基:R2A培养基(g/L):酵母浸出粉0.5g,蛋白胨0.5g,葡萄糖0.5g,酪蛋白水解物0.5g,可溶性淀粉0.5g,磷酸氢二钾0.3g,无水硫酸镁0.024g,丙酮酸钠0.3g,琼脂粉18g,最终pH7.2±0.2。液体培养基去除琼脂粉。
2.菌株的分离:
将所采集番茄根际土壤样品用无菌水将根际土洗下经2000r/min、30s离心处理后,取1g离心得到的根际土,以10ml无菌水重悬,按100倍、1000倍、10000倍稀释涂布于固体R2A平板上,30℃培养箱中培养2-4天之后,分别挑取形态差异菌落纯化待用。
3.功能菌株初筛:
以固体R2A平板对称轴画线,分别将所分离待测菌株与土壤常见高含量抗生素抗性基因菌株一一接种于对称轴线两边,给每种菌株接种5个点,共计10个点,呈V字形,接种量为1.5uL/点,于30℃恒温培养箱中培养3-5天。挑选其中抑制效果较强的菌落,于试管斜面划线保存和超低温冰箱甘油管冷冻保存待用。
4.功能菌株复筛:
复筛采用初筛同样的方法,但以相同条件下不接种功能菌株的固体R2A平板作为对照,并设置3个重复,置于30℃培养箱中培养5天后,观察各功能菌株对携带高抗生素抗性基因菌株的抑制能力。选取其中抑制效果最好的功能菌株暂命名为NJAU-T129,如图2-图9所示,其能显著抑制Bacillus sp.(C40、S62、C16、C30、C35)和Pseudomonas sp.(C4R16、C1R15、C4R11)。
实施例2 菌株NJAU-T129的鉴定
菌株NJAU-T129在R2A固体培养基上于30℃培养箱中恒温培养72小时后,其菌落形态为圆形饼状,颜色乳白偏淡黄,菌落外缘较厚,中心较透明。菌落边缘光滑整齐,表面湿润,容易从培养基上挑取。16S序列构建的发育树比对分析结果表明,菌株NJAU-T129与Pseudoxanthomonas wuyuanensis同源性最高,与Xanthomonassp.同源性最远,结合16S序列构建的发育树比对分析结果和生理生化性状,将菌株NJAU-T129鉴定为假黄单胞菌属细菌(Pseudoxanthomonassp.)(图1)。通过发育树结果在ncbi中查询到该菌属对作物无害,对人和动物无致病性。菌株NJAU-T129已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.26906。
实施例3 利用盆栽实验研究菌株NJAU-T129结合有机肥对番茄植株生物量及土壤典型抗生素抗性基因含量的影响
盆栽实验地点位于江苏省南京市南京农业大学玻璃温室,试验时间为2022年3月至5月,供试番茄品种为常规矮生红番茄。将番茄种子在室温下消毒清洗,并于30℃培养箱中遮光催芽至露白后分别埋入一定含水量的基质中,分别长至两叶一心后移栽入盆栽。供试菌剂为NJAU-T129菌悬液(取活化后的NJAU-T129加入R2A液体培养基中于摇床中发酵,发酵条件为pH7.0,温度30℃,转速为170-190r/min,发酵时间为48-72h,发酵结束后6000-8000r/min离心8-10min获得的菌体用无菌生理盐水重悬,使菌悬液中菌含量≥106个/ml)。供试土壤为长期(5年以上)浇灌养鸡场养殖粪污的土壤。
盆栽试验共设置6个处理,分别为1)OF1,施用鸡粪有机肥(腐熟);2)F,施用鲜鸡粪(鲜鸡粪按照含水量换算,保证其干物质量与鸡粪有机肥一致);3)OF1+NJAU-T129,施用鸡粪有机肥(腐熟)和NJAU-T129菌悬液;4)F+NJAU-T129,施用鲜鸡粪(鲜鸡粪按照含水量换算,保证其干物质量与鸡粪有机肥一致)和NJAU-T129菌悬液;5)CF,施用化肥(单质化肥,按照1个盆添加的有机肥的N、P、K含量换算出所需的单质化肥的量添加);6)CF+NJAU-T129,施用化肥(单质化肥,按照1个盆添加的有机肥的N、P、K含量换算出所需的单质化肥的量添加)和NJAU-T129菌悬液。每个盆装土300g,肥料按照土壤质量的1wt%作基肥拌土加入,以单质化肥将各个施肥处理养分(N、P、K)补充平衡,移栽7天后,有NJAU-T129菌悬液的处理组接种NJAU-T129菌悬液,NJAU-T129菌悬液接种量为每盆10mL,直接施入至番茄根际,接菌结束再生长21天后,每个处理分别取4盆长势一致的番茄进行生物量测定。
各处理组生物量如表1所示,从株高、茎粗上看,接种NJAU-T129菌悬液处理的番茄植株略优于不接菌对照处理中的番茄植株,虽没有产生显著性差异,但普遍长势更好,且多数加菌的处理长势更加均一,而不接种NJAU-T129菌悬液的植株长势参差不齐;从地上部干重上看,加入了NJAU-T129菌悬液的化肥处理显著优于不接菌的对照处理,其他加入了NJAU-T129菌悬液的处理和对照相比,也明显有更高的干物质量;从地上部鲜重看来,化肥处理间加入NJAU-T129菌悬液的处理鲜重也是显著高于纯化肥,鸡粪有机肥处理间加NJAU-T129菌悬液的处理也大大增加了生物量,其他处理间加入了菌悬液的处理也有更好的生物量。结合地上部鲜重、地上部干重、株高、茎粗可以明显看出,加入NJAU-T129菌悬液的所有处理都长的更加均一整齐,普遍更好。综上所述,相对于不接菌对照处理,接种NJAU-T129菌悬液对盆栽番茄植株体现出良好的促生效果。
表1 番茄促生试验生物量情况
处理 | 株高(cm) | 茎粗(cm) | 地上部鲜重(g) | 地上部干重(g) |
CF+NJAU-T129 | 15.46±0.94ab | 0.56±0.05b | 18.56±2.15bc | 5.14±1.63ab |
CF | 14.60±2.91ab | 0.55±0.04b | 12.74±4.39d | 2.64±0.99c |
OF1+NJAU-T129 | 16.02±1.30a | 0.69±0.06a | 26.42±3.89a | 6.51±1.66a |
OF1 | 14.00±1.79ab | 0.66±0.12a | 20.96±7.28abc | 5.45±2.33ab |
F+NJAU-T129 | 15.16±1.13ab | 0.55±0.07b | 17.37±1.35bcd | 5.07±0.88ab |
F | 13.58±2.05b | 0.57±0.09b | 15.69±4.37cd | 4.32±1.60bc |
注:CF代表化肥处理,OF1代表鸡粪有机肥处理,F代表鲜鸡粪处理,NJAU-T129代表接菌处理即接种NJAU-T129菌悬液处理。表中数据表示平均值±标准偏差。
取盆栽试验1)OF1,施用鸡粪有机肥(腐熟);2)F,鲜鸡粪;3)OF1+NJAU-T129,施用鸡粪有机肥(腐熟)和NJAU-T129菌悬液3个处理的土体(接菌结束再生长21天后的土体)进行抗生素抗性基因含量的测定。
结果如图10所示,腐熟的鸡粪有机肥处理比未腐熟的鲜鸡粪处理土壤所携带的四环素耐药基因tetW显著更少,降低了约四倍。加入NJAU-T129菌悬液的鸡粪有机肥在消除番茄种植土壤四环素耐药基因tetW的效果极其明显,相比于施用鲜鸡粪的处理,四环素耐药基因tetW下降了约2个数量级,比单独的鸡粪有机肥的效果更加显著。四环素耐药基因属于土壤中最常见的抗生素抗性基因之一,经常由于添加在畜禽饲料中随畜禽粪便进入土壤,是土壤中占比较大的抗生素抗性基因。加入NJAU-T129菌悬液的鸡粪有机肥能显著消减土壤中四环素耐药基因含量,这对消减土壤抗生素抗性基因含量有极大贡献。
Claims (10)
1.一株假黄单胞菌属细菌NJAU-T129,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2023年3月24日,保藏编号为CGMCC No.26906。
2.权利要求1所述的假黄单胞菌属细菌NJAU-T129制备的菌剂。
3.根据权利要求2所述的菌剂,其特征在于,所述菌剂通过以下方法制备得到:将保藏编号为CGMCC No.26906的假黄单胞菌属细菌NJAU-T129进行液体发酵,发酵结束后离心获得菌体,用无菌生理盐水重悬菌体,使菌悬液中菌含量≥106个/ml。
4.根据权利要求3所述的菌剂,其特征在于,所述菌剂通过以下方法制备得到:将保藏编号为CGMCC No.26906的假黄单胞菌属细菌NJAU-T129接种至R2A液体培养基进行液体发酵,发酵条件:pH为7.0,温度为30℃,转速为170-190r/min,发酵时间为48-72h;发酵结束后6000-8000r/min离心8-10min获得菌体,用无菌生理盐水重悬菌体,使菌悬液中菌含量≥106个/ml。
5.权利要求2或3或4所述的菌剂在促进作物生长、消减作物种植土壤中抗生素抗性基因含量中的应用。
6.根据权利要求5所述的应用,其特征在于,所述作物包括番茄。
7.根据权利要求5所述的应用,其特征在于,所述抗生素抗性基因包括四环素耐药基因tetW。
8.根据权利要求5所述的应用,其特征在于,应用时,以腐熟有机肥作为基肥,作物定植后一段时间施入所述菌剂。
9.根据权利要求8所述的应用,其特征在于,所述腐熟有机肥包括腐熟鸡粪有机肥。
10.根据权利要求8所述的应用,其特征在于,盆栽作物时,所述菌剂施入量为20-50mL·kg-1土壤。
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