CN110050831B - 用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液 - Google Patents
用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液 Download PDFInfo
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Abstract
本发明公开了一种季也蒙毕赤酵母J‑SS‑CX,为Meyerozyma guilliermondii,保藏编号为CGMCC NO.17240。本发明还同时公开了一种用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液,其制备方法为:将NYDA固体培养基活化好的季也蒙毕赤酵母J‑SS‑CX接种到NYDB培养基中培养一代后,再接种到含12%NaCl的NYDB培养基中培养,所得的菌体用无菌蒸馏水洗涤、稀释。本发明能实现在不使用化学杀菌剂的前提下能有效控制果实采后病害,该酵母制剂操作方便,效果好,成本低且安全、对环境友好。
Description
技术领域
本发明涉及果实采后病害防治技术领域,具体是一种通过直接作用来抑制果实采后病害的生物保鲜技术。
背景技术
受采后病害、物流技术和多种非生物因素的影响,水果采后的损失较为严重。其中,病原真菌病原真菌侵染是造成水果果实采后腐烂的主要原因,常见的病原真菌主要有青霉属(Penicillum spp.)、葡萄孢属(Botrytis spp.)和链格孢属(Alternaria spp.)等。据资料显示,我国每年有近8000万吨水果受采后病害影响无法销售,经济损失达千亿元,严重限制了我国水果产业的发展。
目前,化学杀菌剂仍然是我国控制水果采后病害的主要措施。以柑橘为例,中国发明专利《柑橘长效保鲜剂》,公开号CN101766219A,公开了一种由赤霉素、瑞毒霉、2,4-D钠盐、托布津、柠檬酸、多氯苯甲酸、水混合组成的柑橘长效保鲜剂。中国发明专利《一种含有抑霉唑的杀菌组合物》,公开号CN102150677A,公开了一种由5~79%抑霉唑、10~79%甲基硫菌灵或多菌灵复配而成的抑霉唑杀菌组合物。中国发明专利《柑橘保鲜剂》,公开号CN101766217A,公开了由纤维素、甲基托布津、多菌灵、对羟基苯甲酸酯、维生素C、青鲜素、水混合组成的一种柑橘保鲜剂。中国发明专利《一种多菌灵和高良姜素复合杀菌剂在柑橘防腐保鲜中的应用》,公开号CN108935653A,公开了一种多菌灵和高良姜素组成的复合杀菌剂。
化学杀菌剂的残留容易对人体健康及环境安全造成严重危害,同时会导致病原微生物产生耐药性,影响病害防治效果。因此,寻找新型的、安全有效的病害控制方法来代替杀菌剂日益成为人们关注的焦点。
季也蒙毕赤酵母酵母(Meyerozyma guilliermondii)是国内外较广泛研究的采后生物防治酵母菌株。中国授权发明专利《一种生防酵母菌活性冻干粉及其制备方法》,公开号CN109055248A,公开了一种由季也蒙毕赤酵母、海藻糖、谷胱甘肽复合使用的生防酵母菌活性冻干粉及其制备方法。中国授权发明专利《一种提高草莓采后贮藏性的生物防治方法》,公开号CN103392792A,公开了一种基于葡萄有孢汉逊酵母菌悬液及季也蒙毕赤酵母菌悬液混合施用的草莓生物保鲜剂。中国授权发明专利《一株控制梨果采后病害的季也蒙毕赤酵母》,公开号CN107460134A,公开了一株由土壤分离得到的能够控制梨果采后病害的季也蒙毕赤酵母及其制备与使用方法。中国授权发明专利《防治苹果果实轮纹病的生防菌季也蒙毕赤酵母Y#1及其生防制剂》,公开号CN107937285A,公开了一株苹果果实轮纹病防治用途的生防制剂。由此可见,生防酵母可以被考虑广泛应用于果实采后病害预防工作中。
发明内容
本发明要解决的技术问题是提供一种用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液及所用的季也蒙毕赤酵母J-SS-CX。本发明能实现在不使用化学杀菌剂的前提下能有效控制果实采后病害,该酵母制剂操作方便,效果好,成本低且安全、对环境友好。
为了解决上述技术问题,本发明提供一种季也蒙毕赤酵母J-SS-CX,为Meyerozymaguilliermondii,保藏编号为CGMCC NO.17240。
本发明还同时一种用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液,该季也蒙毕赤酵母菌悬浮液的制备方法为:
将季也蒙毕赤酵母J-SS-CX(NYDA固体培养基活化好的季也蒙毕赤酵母J-SS-CX)接种到NYDB培养基中,于(200±20)rpm、(28±1)℃条件下培养(24±1)h后(即,培养一代后),再接种到含12%NaCl的NYDB培养基中,于(200±20)rpm、(28±1)℃条件下传代培养(24±1)h,离心,离心所得的菌体用无菌蒸馏水进行洗涤,然后用无菌蒸馏水稀释,得季也蒙毕赤酵母菌悬浮液;所述季也蒙毕赤酵母菌悬浮液中酵母细胞含量为(0.8~1.2)×108spores/mL;
含12%NaCl的NYDB培养基为1000mL NYDB培养基中含有120g NaCl。
含12%NaCl的NYDB培养基,具体为:牛肉浸膏8g,酵母粉5g,葡萄糖10g,氯化钠120g,用水定容至1000mL,高压蒸汽灭菌(121℃灭菌20min)。
作为本发明的用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液的改进:所述季也蒙毕赤酵母菌悬浮液中酵母细胞含量为1×108spores/mL。
本发明首先获得了季也蒙毕赤酵母J-SS-CX,将其于NYDB液体培养基活化一代后于12%NaCl逆境传代培养;所得的培养液离心、收集菌体,无菌水清洗得到菌体,菌体用无菌水稀释制备成0.8~1.2×108spores/mL(优选1.0×108spores/ml)的菌悬液。
本发明实际使用时,可选择以下任意一种方式对果实进行处理:
处理方式一:选择无机械损伤、未感染、大小和成熟度等外观品质基本一致的果实,用自来水冲洗并在室温(25℃)下放置干燥。将果实在本发明的季也蒙毕赤酵母菌悬浮液中浸泡10分钟后,取出放置干燥,装箱贮藏。
处理方式二:采用采前喷壶喷洒的方法处理,将本发明的季也蒙毕赤酵母菌悬浮液喷在果实表面(湿润即可),待自然风干(不再滴水为止)后取下果实,装箱贮藏。
本发明具有如下技术优势:
(1)本发明中的季也蒙毕赤酵母资源丰富、培养方便、成本低廉、性价比高,具有生物可降解性;
(2)本发明可以显著降低樱桃番茄果实的采后灰霉病发病率;降低水果的腐烂率,从而达到水果贮藏保鲜的目的。
(3)本发明对环境及人体健康无任何毒害作用,具有经济实用、安全高效、环境友好等特点;
(4)经过高盐诱导培养后提高了季也蒙毕赤酵母对樱桃番茄果实采后灰霉病的直接抑制作用。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为季也蒙毕赤酵母J-SS-CX在不同NaCl浓度逆境培养所得的OD值对比图;
图2为季也蒙毕赤酵母J-SS-CX(CGMCC NO.17240)与公开号CN107460134A专利中使用的季也蒙毕赤酵母(CCTCC NO:M2017270)经普通培养及盐诱导培养后菌悬液对樱桃番茄果实灰霉病的抑制作用对比图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、季也蒙毕赤酵母酵母的筛选:
选用海南省三沙群岛海沙;
培养试剂为虎红(孟加拉红)培养基;
培养筛选条件为:称取10g海沙,加入100mL无菌水,200r/min震荡30min后吸取上清液,随后将上清液梯度用无菌水稀释10、102、103、104倍,分别从每个梯度吸取100μL均匀涂布于虎红培养基平板,培养箱中28℃培养3天,根据菌落形态进行挑选并划线纯化,镜检及PCR扩增16s rDNA测序确定为酵母后,接入NYDA培养基斜面保存。
最后筛选出菌株J-SS-CX,16s rDNA鉴定该菌株为Meyerozyma guilliermondii,其菌落特征如下:圆形,乳白色,边缘整齐,表面光滑湿润。
将该活化好的菌株J-SS-CX接种在50mL NYDB培养基中200rpm、28℃条件下培养24h后,再吸取1mL菌液加入不同NaCl浓度的50mL NYDB培养基中,200rpm、28℃条件下培养24h,收集1mL培养液,在4000rpm、4℃条件下离心15min,对离心收集所得的菌体用无菌蒸馏水洗涤2次,加入1mL无菌水,于600nm条件下测定OD值判断其生长情况。本酵母在不同NaCl浓度逆境培养条件下的生长OD值结果如图1所示。结果表明,该酵母经12%NaCl NYDB培养基逆境培养后仍可生长。
注:12%NaCl NYDB培养基为1000mL NYDB培养基中含有120g的NaCl;其余依次类推。
将菌株J-SS-CX进行保藏,保藏信息如下:
保藏名称:Meyerozyma guilliermondii,保藏单位: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: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,保藏编号:CGMCC NO.17240,保藏日期:2019年01月25日。
实施例2、季也蒙毕赤酵母菌悬浮液的制备方法:
季也蒙毕赤酵母J-SS-CX(CGMCC NO.17240)在低温(4℃)下保存于NYDA培养基中,活化时取出在28℃下于NYDA培养基中培养48小时,重复传代培养2次后,用接种环将活化好的季也蒙毕赤酵母接到NYDB培养基中,在200rpm、28℃条件下培养24h,再吸取培养基1/50体积的菌液到含12%NaCl的NYDB培养基(即,菌液:含12%NaCl的NYDB培养基=1:50的体积比)中,在200rpm、28℃条件下传代培养24h,收集培养液,在4000rpm、4℃条件下离心15min,对离心收集所得的菌体用无菌蒸馏水洗涤2次,以除去培养介质。用无菌蒸馏水稀释重新悬浮酵母细胞,光学显微镜下使用血球记数板确定其浓度后用无菌蒸馏水调整到所需要的浓度(0.8~1.2)×108spores/mL,优选浓度为1.0×108spores/mL,从而得到相应浓度的季也蒙毕赤酵母菌悬液。
NYDA培养基为:牛肉浸膏8g,酵母粉5g,葡萄糖10g,琼脂20g,用水定容至1000mL,高压蒸汽灭菌(121℃灭菌20min)。
含12%NaCl的NYDB培养基为:牛肉浸膏8g,酵母粉5g,葡萄糖10g,氯化钠120g,用水定容至1000mL,高压蒸汽灭菌(121℃灭菌20min)。
实验1、季也蒙毕赤酵母对樱桃番茄果实灰霉病的直接抑制作用
1、实验材料:
果实为樱桃番茄果实。
病原菌:灰葡萄孢(Botrytis cinerea),25℃活化7天备用。
2、处理:
选取外观整齐、色泽均匀、无病虫害、无机械损伤的樱桃番茄果实,使用自来水清洗后,用浓度为0.1%的次氯酸钠溶液浸泡消毒3min,取出,随后用自来水冲洗去除果实表面残余次氯酸钠,晾干备用。
将准备好的樱桃番茄果实放入季也蒙毕赤酵母菌悬浮液(1.0×108spores/mL)中,浸泡10分钟取出风干(不再滴水为止),放在塑料筐中,用保鲜膜密封后即可。
用无菌打孔器在樱桃番茄果实的表面制造一个伤口(5mm直径×3mm深)。分别设置以下5个实验组:
组别1-1、伤口内加入30μL浓度1×108spores/mL的Mey(CGMCC NO.17240);
组别1-2、伤口内加入30μL浓度1×108spores/mL的12Mey(12%NaCl培养的CGMCCNO.17240);
组别2-1、伤口内加入30μL浓度1×108spores/mL的Mey10(CCTCC NO:M2017270);
组别2-2、伤口内加入30μL浓度1×108spores/mL的12Mey10(12%NaCl培养的CCTCC NO:M2017270);
对照、伤口内加入等量(30μL)的无菌水;
将上述实验组在常温(25℃)放置2小时后,再在每个伤口处加入20μL浓度为1×104spores/mL病原菌孢子悬浮液(Botrytis cinerea),在常温(25℃),90%湿度条件下贮藏,并用PE塑料膜密封作保湿处理。
每个组别的样本量为20个樱桃番茄果实,试验重复3次,第6天观察结果(取平均值)。
注:
1)、浓度1×108spores/mL的12Mey(12%NaCl培养的CGMCC NO.17240),即为实施例2制备所得的季也蒙毕赤酵母菌悬液;
2)、浓度1×108spores/mL的Mey(CGMCC NO.17240)的制备方法为:将实施例2中的“含12%NaCl的NYDB培养基”改成“NYDB培养基”,其余等同于实施例2。
NYDB培养基为:牛肉浸膏8g,酵母粉5g,葡萄糖10g,用水定容至1000mL,高压蒸汽灭菌(121℃灭菌20min)。
3)、浓度1×108spores/mL的12Mey10(12%NaCl培养的CCTCC NO:M2017270)的制备方法为:将活化后的CCTCC NO:M2017270替代活化后的季也蒙毕赤酵母J-SS-CX(CGMCCNO.17240),其余等同于实施例2。
4)、浓度1×108spores/mL的Mey10(CCTCC NO:M2017270)的制备方法为:将活化后的CCTCC NO:M2017270替代活化后的季也蒙毕赤酵母J-SS-CX(CGMCC NO.17240),且将“含12%NaCl的NYDB培养基”改成“NYDB培养基”,其余等同于实施例2。
3、结果:
如图2所示:
在接入病原菌后第6天,无菌水对照组樱桃番茄果实灰霉病发病率为98.3%;
经过12%NaCl逆境培养后该酵母对樱桃番茄果实处理后的发病率明显降低,为21.67%(组别1-2);
普通培养方式的三沙分离季也蒙毕赤酵母处理的樱桃番茄果实灰霉病发病率为63.3%(组别1-1);
季也蒙毕赤酵母(CCTCC NO:M2017270)经过12%NaCl逆境培养以及未经逆境培养酵母对樱桃番茄果实处理后的发病率比较接近,分别为43.33%和50.00%。
发病率的计算公式如下:发病率(%)=(发病的果实数/果实总数)×100%。
对比实验、将季也蒙毕赤酵母(CCTCC NO:M2016719)按照实验1所述方式分别设定含12%NaCl的NYDB培养基的培养,以及常规NYDB培养基的培养,其余等同于该实验;所得结果为:所得的2种菌悬液(浓度1×108spores/mL,分别对应12%NaCl的NYDB培养基培养所得、常规NYDB培养基培养所得)对樱桃番茄果实处理后的发病率比较接近,无显著差异;且效果远远不如本发明的12Mey。
季也蒙毕赤酵母(CGMCC2.3943)、生防菌季也蒙毕赤酵母Y#1(CCTCC NO:M2015814)按照上述方式进行实验,所得结果也均为:所得的2种菌悬液(浓度1×108spores/mL,分别对应12%NaCl的NYDB培养基培养所得、常规NYDB培养基培养所得)对樱桃番茄果实处理后的发病率比较接近,无显著差别;且效果远远不如本发明的12Mey。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (2)
1.用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液,其特征是该季也蒙毕赤酵母菌悬浮液的制备方法为:
将季也蒙毕赤酵母J-SS-CX接种到NYDB培养基中,于(200±20)rpm、(28±1)℃条件下培养(24±1)h后,再接种到含12%NaCl的NYDB培养基中,于(200±20)rpm、(28±1)℃条件下传代培养(24±1)h,离心,离心所得的菌体用无菌蒸馏水进行洗涤,然后用无菌蒸馏水稀释,得季也蒙毕赤酵母菌悬浮液;所述季也蒙毕赤酵母菌悬浮液中酵母细胞含量为(0.8~1.2)×108 spores/mL;所述季也蒙毕赤酵母J-SS-CX为Meyerozyma guilliermondii,保藏编号为CGMCC NO.17240。
2.根据权利要求1所述的用于控制樱桃番茄果实采后病害的季也蒙毕赤酵母菌悬浮液,其特征是:
所述季也蒙毕赤酵母菌悬浮液中酵母细胞含量为1×108 spores/mL。
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