定量分离土壤绿僵菌的多果定高效选择性培养基

摘要

This paper is to determine an efficiently selective medium and its efficiency for quantitative evaluation of the fungusMetarhizium anisopliae as a biocontrol agent applied in soil. The selectivity and detection rate of dodine was investigated at different concentrations. The results showed that 50-200 mg/L of dodine in medium could effectively inhibit non-target fungi in soil and highlight M. anisopliae, while preventing bacterial growth by chloromycetin. But lower dodine concentration, 25 mg/L, could not completely preclude non-target fungi with residues accounted for about 50% of the total colonies. When increasing dodine concentration, M. anisopliae would slow growth, yellow conidiation and decrease colonies. At 50 mg/L of dodine, detection rates get to 69.20% and 71.43% after inoculation ofM. anisopliae conidia for 4 d and 6 d. The values were significantly higher than 45.45% and 61.24% detected on 200 mg/L concentration, though significantly lower than 95.56% and 94.72% detected on control medium without dodine in the same inoculation and incubation. Finally, a favorable medium including basic nutrition supplemented with 50 mg/L dodine as selective agent was determined, which can effectively inhibit non-target fungi and extract stablyM. anisopliaein 70% efficiency. Inhibition and selection efficiency of selective agent should not be ignored in quantitative detection of microorganism in soil. Properly quantitative assessment of M. anisopliae in field is a necessary foundation for analysis of population dynamics, ecological effects and sustained biocontrol.%为了定量评估生防真菌绿僵菌在土壤中的存活能力和种群数量，需要确定高效的选择性培养基及其分离效率。本文研究了不同浓度多果定对绿僵菌的选择作用和分离检出率，结果表明，在以氯霉素抑制土壤细菌的基础上，多果定浓度在50～200 mg/L时能够有效抑制土壤中的非目标真菌而选择出可清晰鉴别和计数的绿僵菌；当浓度为25 mg/L时，对杂菌的抑制不完全，残留杂菌数占总数50%左右。随着多果定浓度增高，绿僵菌生长减缓、产孢颜色变黄、分离检出率降低。在多果定50 mg/L浓度下，接种培养绿僵菌4 d和6 d的菌落检出率分别69.20%和71.43%，显著低于对照培养基的检出率95.56%和94.72%；在200 mg/L的高浓度下，4 d和6 d检出率分别只有45.45%和61.24%，又显著低于前者低浓度下的检出率。试验确定了在蔗糖基础培养基中添加50 mg/L多果定为选择剂，能够有效抑制土壤中非目标真菌99%以上，对绿僵菌的分离效率稳定在70%左右。选择剂的抑制作用和选择效率是土壤微生物定量检测中不可忽略的因子。正确地评估田间绿僵菌数量是研究其种群动态、生态效应和持续生防作用的必要基础。