拮抗菌HT-6对致病疫霉的竞争及诱导性抑制

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.

Competition and induction inhibition of bacterium HT-6 on mycelia growth of Phytophthora infestans

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.