[目的]研究二郎山林场华山松针叶凋落初期针叶上的真菌多样性,分析凋落物分解初期的真菌结构组成及其功能性,揭示真菌多样性与凋落物分解的关系以及根球壳孢菌在针叶分解中的产酶特性.[方法]采用研磨法对分别从海拔2 750,2 650,2 510 和2 460 m 采集的华山松新近凋落针叶进行真菌分离,结合形态学和真菌ITS序列的分子生物学对分离物鉴定分类.选取3 株在形态学上存在明显差异的根球壳孢菌以及1 株分离自华山松的二郎山散斑壳为分解菌株,以华山松针叶为唯一底物,采用试管培养法进行发酵,探讨根球壳孢菌的腐生性能.[结果]分离鉴定结果显示,从华山松凋落针叶中获得了 23 个属于不同分类单元的真菌,其中半知菌数量最多,共有15 个分类单元,子囊菌次之,为5 个.所有分离菌株隶属15 属,毛霉属、木霉属、根球壳孢菌属、青霉属和蓝状菌属在4 个海拔凋落物中均存在,为华山松凋落物前期的丰富物种.木霉属和青霉属等丝状真菌在数量上的优势以及种类上的多样性,说明其在凋落物分解过程中发挥着重要作用,而担子菌在数量以及种类上的缺乏说明其在分解前期参与少,推测其后期将从环境进入参与木质素的分解.通过测定4 株真菌分解华山松凋落针叶时的纤维素酶活,4 株真菌分泌的内切纤维素酶和 β-葡聚糖酶活性在第13 天达到峰值,外切纤维素酶活性在第17 天达到峰值,但4 株真菌菌株之间的各纤维素酶活力差异不大,引起的针叶质量损失率也并无显著差异.根球壳孢菌和二郎山散斑壳对针叶的纤维素成分具有分解作用,内切纤维素酶、外切纤维素酶、β -葡萄糖苷酶之间存在协同效应.二郎山散斑壳分解的华山松针叶的平均质量损失率为15. 74%,该菌多存在于落地华山松针叶上,而活体针叶中鲜有发现,且未能造成华山松的落针病害; 根球壳孢菌在25 天内引起华山松针叶质量损失率为12. 00% ~13. 40%.[结论]木霉属和青霉属等丝状真菌在凋落物初期发挥主要作用,且在不同海拔均为优势物种.二郎山散斑壳与根球壳孢菌均存在弱腐生性,但其产纤维素分解酶能力是否与其致病力相关仍有待进一步研究.%[Objective]Pinus armandii distributes widely in the Erlang Mountain and is one of the dominant species in the community. The Erlang Mountain Forest Farm is located in Sichuan Province and it contains rich microbial resources. This article was intended to study the fungal diversity and analyze the relationship between fungal structure and their functions at the beginning of the P. armandii litter decomposition period.[Method]The grinding method was used in this paper to isolate fungi on the P. armandii litter from the altitude of 2 750,2 650,2 510,and 2 460 m. The isolates were further indentified according to the morphology combined with PCR amplification and sequencing using the universal primers based on the ITS sequence of fungi. In addition,the Rhizosphaera was for the first time isolated from P. armandii,and the relevant research on the fungi is still insufficient. There remains controversy on the fungal role as a pathogen or weak parasitic fungus in foreign reports. The fungus was reported to cause disease on P. sylvestris var. mongolica in China in 2013. In this study,12 strains of Rhizosphaera were isolated from the four elevations. To study the saprophytic performance,three strains of Rhizosphaera sp. with different morphological features,namely R. sp. 1,R. sp. 2 and R. sp. 3,and one strain of Lophodermium erlangshanense were selected for decomposition test in vitro,in which the P. armandii litter is the sole substrate. [Result]The isolation results showed that 23 fungi taxa were obtained. The imperfecti and ascomycetes were the major components that occupy 15 taxa and 5 taxa,respectively. All of the fungi belonged to 15 genera. Mucor,Trichoderma,Rhizosphaera,Penicillium and Talaromyces were isolated from the plots over the four different elevations, and were the abundant species. The filamentous fungi, such as Trichoderma, and Penicillium,which had the quantitative superiority and diversity,undoubtedly would play an important role in the process of litter decomposition. However,basidiomycete fungi,without abundant amount and species,might not participate in the decomposition at the early stage. The basidiomycete fungi originated from the environment may participate in the further lignin decomposition at the late stage. Through the determination of cellulose enzyme produced by the fungi,the activities of endoglucanase and β-glucosidase reach the maximum at the 13th day. The activities of Cellobiohydrolase reach the maximum at the 17th day. In general,there was no significant difference in the enzyme activities as well as the mass loss among the four strains. Those results indicated that Rhizosphaera sp. and L. erlangshanense could decompose the cellulose components,and there was a synergistic effect from the different cellulase enzymes ( endoglucanase,cellobiohydrolase,β-glucosidase). The mass loss decomposed by L. erlangshanense was 15. 74%,while the loss caused by Rhizosphaera sp. ranged from 12. 00% to 13. 40%. According to the previous researches,L. erlangshanense was usually found in the litter and rarely in alive needles,suggesting that it may not cause the needle disease. [Conclusion]The results indicated that filamentous fungi, such as Trichoderma, and Penicillium, played an important role at the beginning of the litter decomposition period. They were both the dominant species in the four different altitudes. Rhizosphaera sp. and L. erlangshanense were weak saprotrophic fungi in the P. armandii litter. However,it needs to be further verified that whether the capacity of cellulose decomposing enzyme is related to their pathogenicity.
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