为深入了解细菌HT-6对致病疫霉(Phytophthora infestans)菌丝生长的抑制机理,采用琼脂柱对接法观察细菌HT-6对致病疫霉空间位点的竞争,铬天青(CAS)培养基观察嗜铁素的产生,硫酸-蒽铜比色法和水杨酸浓硫酸比色法分别比较葡萄糖和硝态氮的利用率,同时以致病疫霉及其细胞壁制剂(CWP)为诱导物,采用平板透明圈法观察HT-6产生的纤维素酶和β-1,3-葡聚糖酶,DNS法测定酶活并评价含有2种酶的无菌体发酵液对致病疫霉的抑制活性.结果显示,单独接种在PDA琼脂柱上的致病疫霉能沿琼脂柱生长并迅速扩展至黑麦固体培养基(Rye)表面生长,与致病疫霉菌饼对接的HT-6也能沿着琼脂柱生长并扩展至Rye培养基表面迅速蔓延,而与HT-6对接的致病疫霉菌丝体仅在PDA琼脂柱上生长,没有扩展至Rye表面,被HT-6的生长所阻止;HT-6能产生嗜铁素,致病疫霉不产生嗜铁素;HT-6对葡萄糖和硝态氮的利用率(75.66%和69.05%)均显著高于致病疫霉(55.97%和56.87%);HT-6菌株单独培养时不产生纤维素酶和β-1,3-葡聚糖酶,但致病疫霉菌体和CWP均能诱导HT-6产生这2种酶,其中菌体诱导后的2种酶活,分别为6.638、2.913 U,均显著高于CWP的诱导处理(1.326、1.289 U,P<0.05);CWP诱导后含有2种酶的HT-6无菌体发酵液对致病疫霉菌丝生长具有明显抑制作用(抑菌率为33.25%),但显著低于单独培养的HT-6菌液原液的抑菌活性(抑菌率为81.33%).这表明HT-6能够通过空间和营养竞争以及致病疫霉诱导产生的细胞壁降解酶抑制致病疫霉,其中以空间和营养竞争为主.
In order to deeply understand the antagonistic mechanism of bacterium HT-6 against Phytophthora infestans, the agar column method was used to observe the competition in spatial position between HT-6 strain and P. infestans. Chrome Azurol Sulphonate(CAS)medium was used to survey and determine the production of siderophore. Sulfuric acid anthrone colorimetric method and salicylic acid-concentrated sulfuric acid colorimetry method were used to compare the utilization rates of glucose and nitrate nitrogen, respectively. At the same time, P. infestans and its cell wall preparation(CWP)were used as inducers to induce the cellulase and β-1,3-glucanase produced by HT-6, which were observed based on the plate transparent circle method and measured with DNS method. The inhibition of HT-6 free cell fermentation liquid induced by CWP on P. infestans mycelia growth was evaluated with dual culture method. The results showed that P. infestans mycelia inoculated on PDA column alone could grow along column and spread quickly onto Rye medium, HT-6 docked with P. infestans on the agar column could rapidly grow and spread on the surface of the Rye medium, as well, while P. infestans docked with HT-6 on the agar column grew only in agar column and was inhibited by HT-6; HT-6 could produce siderophore, while P. infestans could not. The utilization rates of glucose(75.66%)and nitrate nitrogen(69.05%)by HT-6 were significantly higher than those of P. infestans(55.97% and 56.87%), respectively. And HT-6 strain could not produce cellulase and β-1,3-glucanase as it was cultured alone, but both P. infestans strain and its CWP could induce HT-6 to produce these two enzymes. Activities of cellulase and β-1,3-glucanase induced by P. infestans strain were 6.638,2.913 U, while those by CWP were 1.326,1.289 U, respectively. There was a significant difference between two induction treatments(P<0.05); HT-6 free cell fermentation liquid containing two enzymes after CWP induction had a significant inhibitory effect on P. infestans mycelia growth, inhibition rate was 33.25%, but it was significantly lower than that of the HT-6 bacterial solution, inhibition rate reached up to 81.33%. It indicated that the bacterium HT-6 can inhibit the P. infestans through both the competition of space and nutrition,and producing the cell wall degrading enzymes induced by P.infestans, in which space and nutrition competition are dominant.
[1] OMPANT S, SAMAD A, FAIST H, et al. A review on the plant microbiome: Ecology, functions, and emerging trends in microbial application[J]. Journal of Advanced Research, 2019,19(5): 29-37. DOI: org/10.1016/j.jare.2019. 03.004.
[2] VANDANA U K, RAJKUMARI J, SINGHA L P, et al.The endophytic microbiome as a hotspot of synergistic interactions, with prospects of plant growth promotion[J]. Biology,2021,10(2): 101-129. DOI: org/10.3390/biology 10020101.
[3] FERREIRA M J, SILVA H, CUNHA A. Siderophore-producing rhizobacteria as a promising tool for empowering plants to cope with iron limitation in saline soils: a review[J]. Pedosphere, 2019,29(4): 409-420. DOI: org/10.1016/S1002-0160(19)60810-6.
[4] 刘云鹏.根际促生解淀粉芽孢杆菌根际定殖和诱导植物系统抗性的机理研究[D].北京:中国农业科学院, 2019.
[5] 贾丽艳,高娟娟,荆旭,等.一株产细菌素细菌CGMCC 6624分离鉴定及其生物学特性[J].中国食品学报, 2019, 19(12): 243-249. DOI:10.16429/j.1009-7848.2019.12.030.
[6] 丁昭仪.沼泽红假单胞菌GJ-22的叶际定殖与植物促生、抗病毒诱导作用[D].长沙:湖南农业大学, 2017.
[7] 王智荣,梅小飞,杜木英,等.荧光假单胞菌ZX对采后锦橙绿霉病的防治及其抑菌机制[J].微生物学报, 2019, 59(5): 950-964. DOI:10.13343/j.cnki.wsxb.20180490.
[8] 虢雅洁,李小龙,彭旭云,等.淡紫紫孢菌PLF-1对感染尖刀镰孢菌百合种球的促生作用及其相关防御酶活性变化[J].生物资源, 2020, 42(1): 143-150. DOI:10.14188/j.ajsh.2020.01.020.
[9] 林睿.土壤链霉菌拮抗致病疫霉及其对植物促生特性的研究[D].呼和浩特:内蒙古农业大学, 2019.
[10] 马强.粘细菌菌株E10和E12抗马铃薯晚疫病菌活性成分的作用机理及其初步分离[D].呼和浩特: 内蒙古农业大学, 2018.
[11] 王游游.致病疫霉拮抗菌WL2及其脂肽的防病促生作用研究[D].保定:河北大学, 2018.
[12] 张聪颖,蒋继志,梁娇,等.细菌HT-6的鉴定及其对马铃薯致病疫霉抑制稳定性的探究[J].作物杂志,2019(6):162-167. DOI:10.16035/j.issn.1001-7283.2019.06.026.
[13] 黄英菊,蒋继志,冯丽娜,等.几种拮抗菌对致病疫霉抑制作用的比较研究[J].河北农业大学学报, 2014, 37(4): 80-85. DOI:10.13320/j.cnki.jauh.2014.0093.
[14] 张红霞,蒋继志,张荷花,等.致病疫霉诱导细菌HT-6产生纤维素酶发挥抑菌作用[J].河北农业大学学报, 2019, 42(4): 82-87. DOI:10.13320/j.cnki.jauh.2019.0082.
[15] OHTSU Y, OSAKI H. Antagonism of Bacillus subtilis against Rosellinia necatrix and its strong antagonism on the medium of soy, wheat bran and agar(SWBA)[J]. Japanese Journal of Phytopathology,2000,66(2): 178-179.
[16] 余贤美.海南岛橡胶根际嗜铁细菌B.subtilis CAS15筛选及嗜铁素基因dhbC克隆、表达与功能分析[D].泰安:山东农业大学, 2009.
[17] 邵红涛,许艳丽.具有生防能力的木霉菌(Trichoderma spp.)与两株大豆根腐病病原菌(Fusarium oxysporum、Rhizoctonia solani)对碳、磷、铁的竞争研究[J].黑龙江大学自然科学学报, 2007, 24(1): 126-129. DOI:10.3969/j.issn.1001-7011.2007.01.027.
[18] 侯宁.HT-6抑制致病疫霉生化机制的初步探索[D].保定:河北大学, 2019.
[19] 赵倩,石晓勇,陈月红,等.水杨酸浓硫酸比色法测定浒苔中硝酸盐含量[J].海洋学报(中文版), 2017, 39(2): 112-119. DOI:10.3969/j.issn.0253-4193.2017.02.011.
[20] 李永才,毕阳,胡洁,等.真菌菌丝细胞壁提取物对马铃薯块茎组织抗干腐病的诱导效应[J].西北植物学报, 2009, 29(4): 749-753. DOI:10.3321/j.issn: 1000-4025.2009.04.016.
[21] 郭荣君,李世东,张晶,等.基于营养竞争原理的大豆根腐病生防芽孢杆菌的筛选及其特性研究[J]. 植物病理学报, 2010, 40(3): 307-314. DOI:10.13926/j.cnki.apps.2010.03.009.
[22] CELAR F. Competition for ammonium and nitrate forms of nitrogen between some phytopathogenic and antagonistic soil fungi[J]. Biol Control, 2003, 28(1): 19-24. DOI:10.1016/S1049-9644(03)00049-5.
[23] 陈刚,杨谦,刘志华,等.病原菌细胞壁诱导下球毛壳菌葡聚糖酶活测定及抑菌试验[J].西南林学院学报, 2010, 30(3): 50-53,58. DOI:10.3969/j.issn.2095-1914.2010.03.012.
[24] 殷福娇.木霉几丁质酶和β-葡聚糖酶基因的诱导及其cDNA片段的克隆[D].昆明:西南林学院,2008. (
