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Selection of High Efficient Fungi for Litter Degradation in Firebreak Belt

  • Received Date: 2022-07-18
    Accepted Date: 2022-08-09
  • Objective To screen and optimize the strain and apply it to the degradation of dead leaves of landscape tree species in forest firebreak belt for recycling of dead leaves and forest fire prevention. Method The highly degrading strains were screened out by the degradation methods of leaf strips of tree species in color bio-fireproof forest belt and the degradation weight loss methods. The highly degrading strains and their combinations were selected by the experimental analysis of the degradation weight loss effect of the highly degrading strains and their combinations. In addition, their degradation rules on the dead branches and leaves were analyzed. Result Through the degradation experiments of 11 strains on deciduous leaves and dead branches and leaves of 12 colorful landscape trees in the firebreak belt, Postia Placenta (Pp),Trichoderma Koningii (Tk1) and Polystictus versicolor (Pv) were found to have strong degradation ability and weight loss effect. The weight loss of Sassafras tsumu, Photinia × fraseri and Schima superba were 54.3% ± 2.3% and 62.1% ± 3.3% respectively. Conclusion Pp + Pv, Pv + Tk1 and Pp + Tk1 have significant degradation effect on the tree species in the forest firebreak belts, which can be widely used as in the forest firebreak belts in the south of Jiangsu Province.
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    [6] 陈书峰, 赵 亮, 刘德华. 绿色木霉在稻壳和鼓皮混合基质上固态发酵生产纤维素酶的研究[J]. 食品与发酵工业, 2004, 30(1):9-12. doi: 10.3321/j.issn:0253-990X.2004.01.003

    [7] 李大婧, 刘春泉, 王振宇. 纤维素酶及其在天然产物开发中的应用[J]. 江苏农业科学, 2005, 33(6):140-142. doi: 10.15889/j.issn.1002-1302.2005.06.049

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    [13] 徐 明, 郑华英, 李沛峰. 彩色树种抗火性测定[J]. 江苏林业科技, 2022, 49(5):1-5.

    [14] 李阳阳, 陈帅民, 范作伟, 等. 水稻秸秆降解复合菌系的筛选构建及其田间应用效果[J]. 植物营养与肥料学报, 2021, 27(12):2083-2093. doi: 10.11674/zwyf.2021154

    [15] 林俊芳, 刘志明, 陈晓阳, 等. 真菌漆酶的酶活测定方法评价[J]. 生物加工工程, 2009, 7(4):1-8.

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Selection of High Efficient Fungi for Litter Degradation in Firebreak Belt

  • Jiangsu Academy of Forestry, Nanjing, P. R. China

Abstract:  Objective To screen and optimize the strain and apply it to the degradation of dead leaves of landscape tree species in forest firebreak belt for recycling of dead leaves and forest fire prevention. Method The highly degrading strains were screened out by the degradation methods of leaf strips of tree species in color bio-fireproof forest belt and the degradation weight loss methods. The highly degrading strains and their combinations were selected by the experimental analysis of the degradation weight loss effect of the highly degrading strains and their combinations. In addition, their degradation rules on the dead branches and leaves were analyzed. Result Through the degradation experiments of 11 strains on deciduous leaves and dead branches and leaves of 12 colorful landscape trees in the firebreak belt, Postia Placenta (Pp),Trichoderma Koningii (Tk1) and Polystictus versicolor (Pv) were found to have strong degradation ability and weight loss effect. The weight loss of Sassafras tsumu, Photinia × fraseri and Schima superba were 54.3% ± 2.3% and 62.1% ± 3.3% respectively. Conclusion Pp + Pv, Pv + Tk1 and Pp + Tk1 have significant degradation effect on the tree species in the forest firebreak belts, which can be widely used as in the forest firebreak belts in the south of Jiangsu Province.

  • 近年来,随着林业生态建设的不断发展,森林资源和蓄积量的不断增加,森林防火面临的形势愈发严峻。江苏苏南丘陵山区名胜古迹众多,人文活动频繁,森林火灾潜在隐患巨大。亟待构建彩色树种防火林带,可在有效阻减森林火灾的同时,形成彩色景观的防火林带,对于实现我省丘陵山区防火树种“珍贵化、彩色化、效益化”,保护森林生态环境,筑牢生态防火屏障,促进美化景观、发展旅游和丘陵山区乡村振兴的意义重大。

    防火林带林下可燃物是影响林火发生与蔓延的重要因子。目前,美国、加拿大等国均研制出了加快可燃物的降解及循环技术[1-3]。地球纤维素每年通过光合作用更新量为4.0 × 1010亿吨,但其中只有极少部分为人们所利用[4]。目前,降解纤维素的方法较多,有生物、化学等方法,但微生物降解是最为关注的。降解纤维素的微生物有真菌、细菌和放线菌等微生物;国内外主要的研究集中在木霉属、青霉属、曲霉属、根霉属、漆斑霉属等丝状真菌上[5-8]。目前主要是用真菌来发酵生产纤维素酶,国内外已经对这几种菌进行比较透彻的研究,尤其是对木霉属菌的研究,普遍认为它是纤维素酶的高产菌株。另外,绿色木霉(Trichoderma viride)和黑木霉(Aspergillus niger)被认为是产纤维素酶最稳定和无毒安全的菌种。康氏木霉(T. koningii)、拟康氏木霉(T. pseudokoningii)也是目前较好的纤维素酶生产菌。因此,从自然界中分离生长快,产酶活力高的纤维素降解菌尤为重要[9-11]。为了充分利用这些可再生资源,本研究立足于防火林带景观树种,引进并筛选出一批木纤维素降解真菌,通过单因素和联用试验和优化,以期通过优化菌株及其联用组合加快防火林带景观树种的枯枝落的降解速度,达到进一步有效利用资源和减少森林火灾的目的。

    • 试验地点位于南京市南郊牛首山南的江苏省林业科学研究院实验林场(118°25′63″ E,32°01′88″ N)。该区属北亚热带季风气候,光热条件兼有南北之长。年均气温15.4 ℃、年日照2 116 h,全年无霜期229 d,年降水量1 000~1 050 mm,秋冬降水量少,较为干燥,是林火高发期。南京市南郊常见乡土木本植物品种有355余种[12]

    • 木荷(Schima superba Gardn. et Champ.)、红楠( Machilus thunbergii Sieb. et Zucc.)、檫树(Sassafras tsumu Hemsl.)、北美枫香(Liquidambar styraciflua L.)、红果冬青(Ilex purpurea Hassk)、油樟(Cinnamomum longipaniculatum (Gamble) N.Chao ex H.W.Li) 、红花深山含笑(Michelia maudiae Dunn)、杨梅(Myrica rubra (Lour.) S. et Zucc.)、桂花(Osmanthus fragrans (Thunb.) Lour.)、油茶(Camellia oleifera Abel.)、茶树(Camellia sinensis (L.) O. Ktze)、红叶石楠(Photinia × fraseri Dress)共12种景观树种[13]

    • 采样时间为2021年4月8日;采样地点为南京南郊丘陵山区院实验林场;样品采集地的立地条件要求基本一致。在林带下层收集和修剪枯枝、落叶,装入保鲜袋并带回实验室;枯枝落叶为实验取样选择材料,落叶剪取落叶条,枯枝落叶剪成2 ~ 3 cm后烘干、粉碎处理后冷藏待用。

    • 绵皮卧孔菌(Postia placenta 5608)(Pp)、云芝 (Polystictus versicolor 83971)(Pv) 、黑曲霉 (Aspergillus niger var. niger 86958)(An)、雷斯青霉 (Penicillium raistrickii 87589)(Pr)、绳状青霉(Penicillium funiculosum 82491)(Pf)、哈茨木霉 (Trichoderma harzianum 51675)(Th)、木霉(Trichoderma atroviride 88573)(Ta)、木霉(Trichoderma citrinoviride 81905)(Tc)、木霉(Trichoderma erinaceum 51932)(Te)、康宁木霉(Trichoderma koningii 84489)(Tk1)、康宁木霉(Trichoderma koningii 87659)(Tk2)等菌种由中国微生物菌种保藏管理委员会林业微生物中心(cfcc)提供。

    • (1)培养基:①PDA培养基:去皮马铃薯200.0 g·L−1、葡糖糖20.0 g·L−1、KH2PO4 3.0 g·L−1、MgSO4·7H2O 1.5 g·L−1、琼脂15.0 g·L−1;②PDA液体培养基:PDA培养基不添加琼脂;③赫奇逊氏无机盐培养基:KH2PO4 1.0 g·L−1、 NaCl 0.1 g·L−1、 MgSO4·7H2O 0.3 g·L−1、NaNO3 2.5 g·L−1、FeCl3 0.01 g·L−1、CaCl2·2H2O 0.1 g·L−1,pH值7.2左右。④液体发酵培养基:蛋白胨3 g·L−1、NH4NO3 2 g·L−1、酵母粉0.5 g·L−1、KH2PO4 4 g·L−1、MgSO4·7H2O 0.3 g·L−1、CaCl2·2H2O 0.3 g·L−1、吐温-80 0.2 g·L−1、CMC-Na 10 g·L−1, pH值6。以上所有培养基均需1×105 Pa灭菌30 min后冷却待用。(2)培养条件:将保存于PDA斜面的各供试菌株分别移植于90 mm的PDA平板,在25 ℃无光照恒温箱内生长4 d。用8 mm的灭菌打孔器切取边缘生长旺盛的带菌培养基块,接种至铺有灭菌隔离膜的PDA平板上,于上述同一条件下生长4 d。

    • 将11种菌株接种到液体PDA培养基中制备菌液,30 ℃、160 r·min−1摇床培养5 d,在装有1 cmх5 cm的红叶石楠等12种供试树种的落叶条的赫奇逊氏无机盐培养基(30 mL)中接入1 mL菌液,30 ℃、160 r·min−1摇床培养10 d;对照为无菌赫奇逊氏无机盐培养基培养的落叶条[14]。根据落叶的断裂程度判断降解效果(分五级指数):0级为落叶边缘膨胀;1级为落叶整齐膨胀并弯曲变形;2级为落叶膨胀加大并少量断裂;3级为落叶1/2块状膨胀并断裂;4级为整块状膨胀并断裂。防火林带景观树种落叶降解效果/%=(对照ID−处理ID)/对照IDх100计算,ID为试验防火林带景观树种落叶降解指数。

    • ①不同菌株对枯枝落叶失重筛选试验。接种11种菌株到液体发酵培养基中制备菌液,30 ℃、160 r·min−1摇床培养5 d;将12种供试枯枝落叶在105 ℃烘至质量恒定,以烘干的1 g供试枯枝落叶为唯一碳源配制液体发酵培养基,接入1 mL菌液,30 ℃、160 r·min−1摇床培养10 d,将发酵液过滤并将残留纤维素烘干称量质量,用减重法计算出失重率;②单种和联用混用组合菌株对枯枝落叶失重效果试验。用从11种菌株中选出的3株强降解菌株,分别设1、2、3、1 + 2、2 + 3和1 + 3共6个单种和联用混用组合菌株,对照为无菌赫奇逊氏无机盐培养基培养的枯枝落叶,并将6个组合菌株接种到液体发酵培养基中制备菌液,30 ℃、160 r·min−1摇床培养5 d;将选出的檫树、红叶石楠、木荷3抗火树种[13]的供试枯枝落叶在105 ℃烘至质量恒定,以烘干的1 g供试枯枝落叶为唯一碳源配制液体发酵培养基,接入1 mL菌液,30 ℃、160 r·min−1摇床培养10 d,将发酵液过滤并将残留纤维素烘干称量质量,用减重法计算出失重率[15-16]。③菌株联用混用与单用对枯枝落叶降解失重增效果/%=(混用失重率-单用失重率)/单用失重率)计算。

    • 所有试验数据均为重复3次的平均值,采用Duncan法作处理间差异多重比较。

    2.   结果与分析
    • 通过对12种防火林带景观树种落叶的降解实验比较,研究11株菌株对落叶的降解能力。经为期10 d的落叶降解实验发现:绵皮卧孔菌 (Pp)对12种防火林带景观树种落叶的降解能力最高,其降解率为44.62% ± 2.93%,康宁木霉(Tk1)和云芝 (Pv) 对12种防火林带景观树种落叶的降解能力次之,其降解率分别为43.88% ± 2.50%和43.44% ± 1.71%,试验说明3株菌株对12种防火林带景观树种落叶均有很强的降解效果,且降解能力明显高于其他8株菌株(表1)。

      菌株Strain树种 Species
      木荷
      S.
      superba
      红楠
      M.
      thunbergii
      檫树
      S.
      tsumu
      红果冬青
      I.
      purpurea
      油樟
      C.
      longipaniculatum
      北美枫香
      L.
      styraciflua
      红花深山含笑
      M.
      maudiae
      桂花
      O.
      fragrans
      杨梅
      M.
      rubra
      油茶
      C.
      oleifera
      红叶石楠
      P. ×
      fraseri
      茶叶树
      C.
      sinensis
      Pp45.2a44.60a49.2a46.0a39.9ab46.20a42.90a43.8a47.2a42.1a48.1a40.2a
      Pv43.8a43.3a44.6a44.8a39.5ab42.7a42.7a44.3a41.2a45.0a45.3a44.1a
      An33.8b37.3ab37.0ab34.7b32.7b35.2ab31.7ab35.4b31.9b33.2b30.5b31.5b
      Pr29.6c31.5b31.5b29.0c29.4bc32.8b30.6b27.7c27.8c28.9c30.9b26.6c
      Pf38.3ab29.4b38.3ab35.6ab35.5ab34.7ab34.7ab36.7ab34.7ab34.3ab40.1a33.9ab
      Th34.0b31.3b31.0b34.9b33.1b29.9b33.6b35.2b36.0b35.1b36.8ab34.0b
      Ta32.3b33.7b33.7b28.9bc31.9b35.3b31.3b35.1b25.7bc29.7bc38.3ab29.9bc
      Tc28.8c29.1bc24.3c33.0bc30.8bc26.7c27.0c29.3c34.3bc32.8bc21.9c31.0bc
      Te24.7c23.9c29.7bc28.0c25.7c27.3c25.7c26.3c25.7c26.8c28.7c23.3c
      Tk142.7a45.7a46.9a45.2a43.1a47.3a38.9ab45.3a40.8a44.0a44.8a41.9a
      Tk238.5b34.8b35.3b36.1b30.3b36.7b32.3b34.6b34.9b33.3b36.9b28.8bc
      注:表中数据经邓肯氏新复极差检验法(DMRT)检验在P<0.01水平差异显著。
        Note: Data is tested by Duncan multiple range test, P<0.01.

      Table 1.  Degradation effect of different strains on deciduous leaves of tree species in colorful landscape fire-resistant belt %

    • 经为期10 d的枯枝落叶的降解失重实验,结果表明,11株菌株对12种防火林带景观树种枯枝落叶均有一些的降解失重效果;但康宁木霉(Tk1) 对12种防火林带景观树种枯枝落叶的降解失重效果最高,其降解失重率为54.33% ± 2.79%;绵皮卧孔菌(Pp) 和云芝(Pv)2株菌株次之,其对12种防火林带景观树种枯枝落叶的降解失重率分别为53.22% ± 4.02%和53.91% ± 2.28%,说明3菌株对12种防火林带景观树种枯枝落叶均有明显的降解失重能力,其它8菌株的降解失重效果较差(表2)。

      菌株Strain树种Species
      木荷
      S.
      superba
      红楠
      M.
      thunbergii
      檫树
      S.
      tsumu
      红果冬青
      I.
      purpurea
      油樟
      C.
      longipaniculatum
      北美枫香
      L.
      styraciflua
      红花深山含笑
      M.
      maudiae
      桂花
      O.
      fragrans
      杨梅
      M.
      rubra
      油茶
      C.
      oleifera
      红叶石楠
      P. ×
      fraseri
      茶叶树
      C.
      sinensis
      Pp55.1a52.1a58.7a56.0a47.3ab59.3a50.7a50.0a54.2a49.5ab56.8a48.9ab
      Pv56.0a55.7a56.3a53.9a49.8ab57.1a53.6a52.8a52.8a54.3a54.4a50.2a
      An44.1b38.0bc45.0ab43.9b39.8b46.9ab36.9bc38.1bc41.0b39.5bc44.7b41.3b
      Pr40.7b37.2bc42.6b41.1b44.3b40.3b35.8bc36.6bc39.8bc37.3bc41.3b39.8bc
      Pf45.1ab45.4b44.8ab43.6b38.9bc45.3ab44.1b39.8bc44.3b37.0bc46.7ab39.2bc
      Th41.9b36.0bc40.7b40.8b36.1bc42.3b35.9bc35.3bc38.8bc33.8bc42.0b35.3bc
      Ta42.2b41.2b43.9b41.0b34.8bc41.7b43.1b38.8bc42.4b36.7bc40.9b35.1bc
      Tc35.7c30.0c35.7c36.8c35.4c36.3c28.8c30.2c35.9c35.1c36.3c36.0c
      Te38.8bc31.8bc38.3bc37.7bc31.8bc39.8bc29.9c28.3c31.6bc29.8c39.1bc27.4c
      Tk153.7a54.8a57.2a54.8a50.9a58.7a51.7a53.5a55.3a52.1a55.9a49.7ab
      Tk241.9b37.8bc43.1b40.4b35.0bc40.7b36.9bc33.7bc38.4bc34.2bc42.8b33.8bc
      注:表中数据经邓肯氏新复极差检验法(DMRT)检验在P<0.01水平差异显著。
        Note: Data is tested by Duncan multiple range test, P<0.01.

      Table 2.  The rate of weight loss of deciduous leaves of tree species degraded by different strains in color landscape fire-resistant belt %

    • 通过3强降解菌株单用及联用对防火林带3种景观树种[16]的枯枝落叶降解失重实验,研究它们对枯枝落叶的失重效果。10 d的降解失重实验表明,3种防火林带景观树种的枯枝落叶降解失重效果明显。①檫树的枯枝落叶经Pp、Pv和Tk1的单一菌株液处理,降解失重率分别为57.8%、53.8%和58.1%,经Pp + Pv、Pv + Tk1和Pp + Tk1联用混合菌株处理,降解失重率分别为65.2%、63.9%和67.3%,试验说明菌株混合联用能明显提高对檫树枯枝落叶降解失重效果。②红叶石楠的枯枝落叶经Pp、Pv和Tk1的单一菌株液处理,降解失重率分别为54.9%、51.8%和54.7%,经Pp + Pv、Pv + Tk1和Pp + Tk1联用混合菌株处理,降解失重率分别为62.4%、60.7%和63.563.5%,试验也说明菌株混合联用能明显提高对红叶石楠枯枝落叶降解失重效果。③木荷的枯枝落叶经Pp、Pv和Tk1的单一菌株液处理,降解失重率分别为53.1%、52.0%和52.7%,经Pp + Pv、Pv + Tk1和Pp + Tk1联用混合菌株处理,降解失重率分别为59.0%、56.3%和60.8%,试验也说明菌株混合联用能明显提高对木荷枯枝落叶降解失重效果。强降解菌株及其联用对三防火林带景观树种枯枝落叶降解失重效果试验也同样说明,落叶树种檫树的降解失重率略高于常绿树种红叶石楠和木荷(图1)。

      Figure 1.  weight loss of three highly degraded strains and their combination on the branches and leaves of three fire-resistant tree species

    3.   讨论
    • 林木腐朽真菌广泛存在于各种枯死的树干、倒木、枯枝落叶及人工木制品上,通过分泌产生各种生物酶,降解木材中的纤维素、半纤维素和木质素,在森林生态系统中起着关键作用,是生态系统分解者中的重要成员。但同时它们也能引起林木病害,造成林业生产上的损失[46];而通过选择和优化菌株加快防火林带景观树种枯枝落叶的降解,减少林地枯枝落叶载量,有利于预防森林火灾的发生。不同菌株降解实验表明绵皮卧孔菌 (Pp)、康宁木霉 (Tk1)和云芝 (Pv)对防火林带景观树种落叶均有很强的降解效果,相对其它8菌株的降解效果略差一些,因此,研究发现强降解真菌和对其进一步优化组合非常必要。

      森林土壤、林下枯落物和腐烂木材中具有丰富的微生物多样性,其中很多类型的微生物可能降解植物主要部分并将营养成分释放到环境中,从而参与养分循环。当Laccase和MnP分别单独存在时,都不能有效的降解木质素,而2种同时存在则木质素得到了有效的降解,这表明2种酶在催化木质素生物降解反应中具有协同作用[17-18]。本研究发现的3强降解菌株通过联用也能明显提高对檫树、红叶石楠和木荷枯枝落叶降解失重效果,说明协同作用的存在,且明显高于Pp、Pv和Tk1的单一菌株处理的降解效果;强降解菌株单用及联用对落叶树种檫树的降解效果略高于常绿树种红叶石楠和木荷,这可能与常绿树种革质层厚和叶面蜡质层厚有关。

      平板对峙试验表明,自主筛选复配的复合生防菌剂 (Trichoderma viride. XP1, Bacillus amyloliquefaciens. XP2)对Foc具有显著的抑菌效果。通过盆栽试验探讨复配复合生防菌剂防控香蕉枯萎病发生的效果,为其生物防治机制提供理论支撑[19]。本研究发现的Pp + Pv、Pv + Tk1和Pp + Tk1 3组混合联用菌株能明显提高对枯枝落叶的降解效果,也为研制复合生防菌剂打下了良好基础。

    4.   结论
    • 通过11菌株对12种防火林带景观树种枯枝落叶的降解实验,筛选出绵皮卧孔菌 (Pp)、康宁木霉(Tk1)和云芝 (Pv)3株强降解菌株;3菌株单用及其联用对檫树、红叶石楠和木荷枯枝落叶降解失重率分别为54.3% ± 2.3%和62.1% ± 3.3%。筛选出的3菌株单用(Pp、Pv和Tk1)及其联用组合,Pp + Pv、Pv + Tk1和Pp + Tk1 3组混合联用菌株对枯枝落叶的降解效果明显提高,广大应用能为复合生防菌剂研发和大规模有效降解林间枯落物,减轻森林火灾,其前景广阔。

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