新月弯孢菌营养体亲和群鉴定方法探讨

 采用菌株直接配对、显微镜观察菌丝融合和硝酸盐缺陷型(nit)突变体互补测试法,对44个来自不同地区的玉米弯孢菌叶斑病叶中的新月弯孢菌(Curvularia lunata)菌株进行了营养体亲和性分析。在野生型菌株的两两配对中,C.lunata菌株接触产生4种反应类型,但无明显的抗衡反应产生。在菌丝融合显微观察中,光学显微镜下只见大多数菌丝反应为一菌丝向另一菌丝无限靠近,未见融合,但在电子显微镜下,发现两菌丝细胞已不再是独立的。这些结果表明,因两菌落间的抗衡反应和菌丝融合特征都不明显,故不宜采用直接配对和显微观察来划分C.lunata的营养体亲和群。在nit突变体互补测试中,菌株在KClO₃浓度1.5%~3.0%的KPS中诱导表明,2.0%~3.0% KClO₃适宜于大多数菌株。抗氯酸盐突变体在Czapek培养基中能鉴定出nit突变体,但在MM上却不能。全部3640抗氯酸盐突变体在Czapek中共鉴定出2 40个稳定的nit突变体,其中nit1占59.2%,nit3占39.2%,NitM占0.8%,nit D (3种氮源都不能利用)占0.8%。44个菌株中,24个(55%)获得了nit突变体,20个(45%)还没有,仅2个菌株(124和155)各获得了1个NitM突变体。各菌株突变体类型间只有NitM与nit1或nit3是异核亲和的,其余均不能产生亲和反应。菌株124和155都是营养体自身亲和菌株。用2个NitM作测试菌株,初步划分出2个VCGs,5个归入VCG1,另有5个归入VCG2,其它菌株因未获得NitM或nit突变体还不能鉴定。以上结果表明,硝酸盐缺陷型突变体互补测试法可用于鉴定C.lunata菌株的营养体亲和群。

Study on vegetative compatibility of Curvularia lunata isolated from corn in China

Forty four isolates of Corn Curvularia leaf spot fungus (Curvularia lunata) were isolated from infected corn leaves collected from different regions in China. Vegetative compatibility of these isolates was assessed using different methods. By pairing isolates on potato dextrose agar (PDA) and observing contact reaction between expanding colonies, four reaction types were observed, but no clear barrage formations were found. Hyphal anastomosis patterns were examined by microscopy and electron microscopy. Of all pairing combinations, very few complete hyphal anastomosis was observed, but the anastomosis-like types that one hyphae was infinitely close to another were commonly found under microscopy. However, under electron microscopy, a combination interaction could be observed where the two hyphal contacted. The results showed that no clear barrage formation and anastomosis signs occurred by isolate pairing. Nitrate non utilizing(nit) mutants of C. lunata were used to study vegetativecompatibility of C. lunata. The isolates were cultured on potato sucrose agar(PSA) containing KClO₃(1.5%-3.0%) to generate chlorate resistant mutants(sectors). The result indicated that 2.0%-3.0% chlorate in KPS was effective for most isolates. Nit mutants could be selected from chlorate reistant sectors on Czapek but not on minimal medium(MM). A total of 240 stable nit mutants were selec ted from 3640 sectors on Czapek, in which 59.2% were classed as nit 1,39.2% as nit 3,0.8% as NitM and 0.8% as nit D (unable to utilize nitrate, nitrite and hypoxanthine). Among the 44 tested isolates, 24(55%)generated nit mutants, and only two isolates(isolate 124 and 155) generated NitM mutants. The genetically different mutants from each isolate were complementally paired in all possible combinations. The results indicated that NitM was complementary with both nit1 and nit3 derived from the same parental isolate, and the remaining mutant combinations were unable to complement. Both isolate 124 and 155 were self-compatible. Employing the two NitM mutants as tester, 5 isolates were classed to VCG1 while other 5 to VCG2.The remains did not belong to any VCG because of the absence of their nit or NitM mutants. These results suggest that complementation tests between nitrate non-utilizing mutants may be more suitable to assign vegetative compatibility groups of C. lunata.