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林业科学 ›› 2020, Vol. 56 ›› Issue (8): 89-97.doi: 10.11707/j.1001-7488.20200811

• 论文与研究报告 • 上一篇    下一篇

纤维素高效降解真菌的筛选及其降解森林地表可燃物的效果

孙思琦,瓮岳太,邸雪颖,刘志华,杨光*   

  1. 东北林业大学林学院 森林生态系统可持续经营教育部重点实验室 哈尔滨 150040
  • 收稿日期:2019-10-27 出版日期:2020-08-25 发布日期:2020-09-15
  • 通讯作者: 杨光
  • 基金资助:
    "十三五"国家重点研发计划子课题(2017YFD0600106-2);国家自然科学基金项目(31870644);中央高校基本科研业务费专项资金资助(2572019CP10)

Screening of Efficient Cellulose-Degrading Fungi and Their Effects on Degradation of Forest Surface Fuel

Siqi Sun,Yuetai Weng,Xueying Di,Zhihua Liu,Guang Yang*   

  1. Key Laboratory of Sustainable Forest Ecosystem Management of Ministry of Education College of Forestry, Northeast Forestry University Harbin 150040
  • Received:2019-10-27 Online:2020-08-25 Published:2020-09-15
  • Contact: Guang Yang

摘要:

目的: 筛选纤维素的降解真菌,探究其对森林地表可燃物的分解效果,以期利用纤维素高效降解真菌促进降解森林地表可燃物,从而降低森林火灾风险。方法: 采集东北林业大学帽儿山实验林场的兴安落叶松、胡桃楸、水曲柳和云杉人工林内的可燃物,采用孟加拉红培养基进行真菌的分离培养,采用刚果红染色法,根据测定菌株的纤维素分解指数筛选高活性纤维素酶菌株,对其进行形态学及分子鉴定;以阔叶、针叶与针阔混交可燃物碎块为分解基质,接入高活性纤维素酶菌株菌液,恒温培养80天,定期取样测定综纤维素含量,分析纤维素分解过程并筛选出对森林地表可燃物中纤维素降解效果最好的菌株;结合扫描电镜超微结构观察结果进行验证。选取筛选出的纤维素高效降解真菌菌株,制取菌饼并接入麦芽浸粉培养基中,恒温振荡培养5天,得到的菌悬液作为单一菌剂,2菌种菌悬液等体积混合为混合菌剂,分低、中、高剂量喷洒到不同样地中装有胡桃楸、兴安落叶松以及胡桃楸-兴安落叶松混交可燃物的凋落物袋内,每月采集凋落物袋测定综纤维素质量分数,分析不同菌剂对可燃物中综纤维素降解效果。结果: 根据孟加拉红氯霉素培养基筛选出的15株真菌在CMC-Na培养基上产生的水解圈大小,筛选出8株高活性纤维素酶菌株,菌株B2(枝细枝孢)的纤维素分解指数最大;根据地表可燃物样品中的纤维素降解结果,菌株A4(紧密帚枝霉)对3种可燃物基质中综纤维素的降解能力最强,其次是菌株A2(肉色隔孢伏革菌);根据扫描电镜观察结果,菌株A4的菌丝可以黏附在叶片表面并侵入叶片组织,通过分泌纤维素酶降解叶片中的综纤维素;不同剂量使可燃物基质综纤维素降解率由大到小呈现大剂量、中剂量、小剂量的规律,且施加菌剂的3种基质综纤维素降解率均高于对照;施加菌剂的3种可燃物基质综纤维素降解率由大到小均表现为:菌剂C(混合菌剂)>菌剂B(菌株A4)>菌剂A(菌株A2)。结论: 菌株A4为室内降解试验筛选出的高效纤维素降解真菌,在野外对地表可燃物中综纤维素也表现出较强降解能力,且与菌株A2构建的混合菌剂对可燃物中综纤维素降解效果更佳,可在后续研究中摸索其最佳产酶条件,以实现林区地表可燃物更大程度上的降解。

关键词: 纤维素, 真菌, 筛选, 降解能力, 森林可燃物

Abstract:

Objective: In this study, efficient cellulose-degrading fungi to degrade surface fuel of Maoer Mountain plantation were screened for investigating the effect of cellulose-degrading fungi on forest surface fuel decomposition, so as to reduce the fuel accumulations in the forest and reduce the potential risk for forest fire. Method: In this study, the fuels in Larix gmelinii, Juglans mandshurica, Fraxinus mandshurica and Picea asperata plantations were collected from Maoer Mountain Experimental Forest Station of Northeast Forestry University. The fungi were isolated and cultured using the rose bengal chloramphenicol agar medium, and then Congo red staining was used to screen strains with high cellulase activity according to the cellulose decomposition index. Morphological and molecular identification were performed. The suspensions of different strains with high cellulase activity were inoculated into flasks with three kinds of decomposition substrates (coniferous, broad-leaved and broadleaf-conifer mixed) and then cultured in a constant temperature incubator for 80 days. Samples were taken regularly, and the content of holo-cellulose was determined for analyzing the cellulose decomposition rule. The strain with the most efficient degradation effect on natural cellulose in forest fuel was selected, which was verified by scanning electron microscope. The screened efficient cellulose-degrading fungi were used to prepare the fungal disks, which were put into the malt extract medium, and the shaking culture was performed for 5 days. The fungal suspension was used as a single microbial agent, and the mixed microbial agent was obtained by mixing two fungal suspensions with equal volume. The microbial agents were sprayed into litter bags with different fuels of J. mandshurica, L. gmelinii and their mix in low, medium and high dosages. Litter bags were collected each month to determine holo-cellulose content in the fuels, and analyze the degradation effect of different microbial agents on the holo-cellulose in fuels. Result: Among fifteen fungi selected by RBC medium, eight had high cellulase activity, according to the distinct hydrolytic circles they produced on CMC-Na medium. Fungus B2 (Cladosporium ramotenellum) had the highest cellulolytic index. Fungus A4 (Sarocladium strictum) had the strongest degradation capable of holo-cellulose in the three fuel substrates based on the cellulose decomposition result, followed by fungus A2 (Peniophord incarnata). Further, the scanning electron micrographs indicated that the mycelia of isolate A4 could adhere to the leaf surface and invade the leaf tissue to degrade holo-cellulose in leaves by secreting cellulolytic enzymes. The degradation rate of holo-cellulose in the three substrates with different dosages was higher than that of the control, and the order was high concentration, medium concentration, low concentration and control. The degradation rate of holo-cellulose in the three fuel substrates with microbial agents was as follows:microbial agent C(mixed microbial agent)> microbial agent B(fungus A4)> microbial agent A(fungus A2). Conclusion: Fungus A4 is a highly effective fungus selected in this study. It also has a strong degradation ability in the field test, and the mixed microbial agent constructed with strain A2 has a better degradation effect on the holo-cellulose in the fuels. The optimal conditions of enzyme production can be explored in the follow-up study, so as to realize the degradation of forest surface fuels to a greater extent.

Key words: cellulose, fungi, screening, degradation ability, forest fuel

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