幾株分離自麻天牛的白僵菌的生長(zhǎng)、產(chǎn)孢特性及產(chǎn)酶能力分析

摘要

昆蟲病原真菌白僵菌Beauveria spp.是害蟲生防產(chǎn)品研發(fā)的主要真菌之一，其生長(zhǎng)及產(chǎn)酶特性是高毒力菌株篩選的重要依據(jù)。本研究對(duì)15株分離自麻天牛僵蟲的白僵菌進(jìn)行了鑒定，并對(duì)其生長(zhǎng)、產(chǎn)孢以及胞外水解酶活性進(jìn)行了測(cè)定分析。rDNAITS序列進(jìn)化關(guān)系分析結(jié)果顯示，15株白僵菌中11株為球孢白僵菌B.bassiana，4株為硫酸白僵菌B.sulfurescens。不同溫度及培養(yǎng)基條件下菌株生長(zhǎng)與產(chǎn)孢分析結(jié)果表明，25℃，PPDA培養(yǎng)基適合大部分白僵菌的生長(zhǎng)，溫度下降至20℃時(shí)各菌株產(chǎn)孢量更高;CN004、CN011、CN012及CN055菌株為產(chǎn)孢優(yōu)勢(shì)候選菌株，其中CN055菌株產(chǎn)孢量可達(dá)到（1.99±0.57）×106 個(gè)/mm2。胞外水解酶活性測(cè)定結(jié)果表明，CN006、CN012、CN040及CN055的幾丁質(zhì)酶活性較強(qiáng)，且?guī)锥≠|(zhì)酶活性呈現(xiàn)先上升后下降趨勢(shì)，培養(yǎng)至第5天酶活性最高。CN009、CN019、CN040及CN055菌株的蛋白酶活性較強(qiáng)，蛋白酶活性呈現(xiàn)先急劇上升、再平緩上升的趨勢(shì)，CN055菌株第9 天的酶活性為（472.5±0.39） U/mL，顯著高于其他菌株（Plt;0.05）。CN040和CN055菌株在產(chǎn)孢、產(chǎn)幾丁質(zhì)酶和蛋白酶能力上兼具優(yōu)勢(shì)，可作為后續(xù)研發(fā)的重點(diǎn)菌株。

關(guān)鍵詞

白僵菌; 產(chǎn)孢; 幾丁質(zhì)酶; 蛋白酶; 篩選

中圖分類號(hào)：

S 476.12

文獻(xiàn)標(biāo)識(shí)碼： A

DOI： 10.16688/j.zwbh.2024523

Characteristics of growth， sporulation and enzyme production capacity of Beauveria strains isolated from Thyestilla gebleri

CAI Ni1#， QIAO Zhijun1，2#， LIU Fang1， FANG Wei1， LIU Shaofang3， NONG Xiangqun4， WANG Kaimei1*

（1. Hubei Biopesticide Engineering Research Centre， Wuhan 430064， China; 2. School of Life Science and Technology，

Wuhan Polytechnic University， Wuhan 430030， China; 3. Jiangxi Provincial Key Laboratory of Natural Microbial

Medicine Research， Nanchang 330013， China; 4. State Key Laboratory for Biology of Plant Diseases and Insect

Pests， Institute of Plant Protection， Chinese Academy of Agricultural Sciences， Beijing 100193， China）

Abstract

The entomopathogenic fungus Beauveria spp. is one of the most important fungal genera for the development of biocontrol agents. Growth and enzyme production traits are key indicators for selecting highvirulence strains. In this study， 15 Beauveria strains were isolated from naturally infected Thyestilla gebleri and identified via rDNAITS sequence analysis. Among them， 11 strains were identified as B. bassiana， and four as B.sulfurescens. The effects of temperature and media on fungal growth and sporulation were evaluated. Most strains exhibited optimal growth at 25℃ on PPDA medium， while sporulation peaked at 20℃. Strains CN004， CN011， CN012 and CN055 exhibited superior sporulation， with CN055 producing up to （1.99±0.57）×106 conidia/mm2. Extracellular enzyme activity assays showed that CN006， CN012， CN040 and CN055 had strong chitinase activity， which peaked on the 5th day after inoculation before declining. CN009， CN019， CN040 and CN055 showed high protease activity， with a sharp initial increase followed by a slower rise. On the 9th day， CN055 displayed the highest protease activity （472.5±0.39 U/mL）， significantly higher than other strains （Plt;0.05）. In summary， CN040 and CN055 demonstrated superior traits in terms of sporulation and hydrolytic enzyme production， making them promising candidates for future development of fungal biopesticides.

Key words

Beauveria spp.; sporulation; chitinase; protease; strain selection

白僵菌 Beauveria spp.是重要的昆蟲病原真菌，其生態(tài)友好，致病力強(qiáng)，對(duì)人畜、作物無(wú)毒，在農(nóng)業(yè)、林業(yè)和醫(yī)學(xué)等領(lǐng)域有重要應(yīng)用價(jià)值，尤其在生物防治中表現(xiàn)突出。白僵菌屬真菌已報(bào)道的有20 多個(gè)種［1］，其中以球孢白僵菌Beauveria bassiana與布氏白僵菌B.brongniartii昆蟲致病性研究最多。例如，張瀚文等［2］用 1×108 個(gè)/mL 球孢白僵菌孢子懸浮液分別侵染長(zhǎng)林小蠹Hylurgus ligniperda幼蟲和成蟲，接種后7 d，長(zhǎng)林小蠹幼蟲和成蟲的校正死亡率均達(dá)到 100%;林華峰等［3］用布氏白僵菌處理斜紋夜蛾Spodoptera litura 2齡幼蟲，其致死中時(shí)（LT50）為2.95 d，累計(jì)校正死亡率達(dá)到100%。……