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摘要:
目的 杉木赤枯病是杉木的重要病害之一,严重威胁杉木苗木生产及幼树成林。为了更好保护杉木,防治杉木赤枯病,从杉木叶片分离到内生细菌SM-1,探讨其对杉木赤枯病菌(Pestalotiopsis apiculatus)的拮抗作用。 方法 经形态观察、生理生化特性观察、16S rRNA及Biolog系统鉴定其种类;含毒平板及牛津杯法检测SM-1无菌发酵液对赤枯病菌的抑制率,并观察SM-1对赤枯病菌菌丝形态的影响;离体枝条的叶片接种试验检测SM-1无菌发酵液的防病效果。 结果 SM-1为萎缩芽孢杆菌(Bacillus atrophaeus),其无菌发酵液对赤枯病菌的抑制率可达58.80%;显微观察显示SM-1能够使赤枯病菌菌丝生长受到抑制,细胞膨大成球形;离体枝条的叶片接种试验揭示SM-1无菌发酵液对杉木赤枯病的抑制效率达95%,能够极显著减轻叶片发病程度(P<0.01)。 结论 杉木内生芽孢杆菌SM-1对杉木赤枯病具有良好的生物防治潜力。 Abstract:Objective Endophytic bacteria that could effectively control the copper blight, one of the most significant diseases that seriously infects the seedlings and saplings, of Chinese fir (Cunninghamia lanceolate) were investigated. Methods Candidate endophytic bacteria were cultured and isolated from leaves of Chinese fir followed by morphological, physiological, and biochemical analyses as well as 16S rRNA sequencing for identification. Pathogen inhibition against Pestalotiopsis apiculatus by the sterile fermentation filtrate of potential specimens was tested on toxic plate and Oxford cup and observed under a microscope. Effectiveness of the disease control by the selected bacteria was further verified in vitro on fir leaves. Results The endophytic bacterium coded SM-1 showed a significant antagonistic effect against P. apiculatus. It was isolated and identified as Bacillus atrophaeus. On the toxic plate and Oxford cup tests, the SM-1 fermentation filtrate yielded a P. apiculatus inhibition rate of 58.80%. Morphologically, the bacterium inhibited the mycelial growth and caused cellular swelling that changed the cells spherical in appearance of the pathogen. The efficacy of the SM-1 filtrate on copper blight on the fir leaves was 95% with significantly reduced disease severity (P<0.01). Conclusion The SM-1, identified as B. atrophaeus, appeared to be a potential biocontrol agent to deter copper blight on Chinese fir. -
表 1 生理生化鉴定结果
Table 1. Physiological and biochemical characteristics of SM-1
项目 Items 1 2 3 4 5 6 7 氧化酶活性 Oxidase activity − + − + − + + 糖原 Glycogen − Nd − − + + − 乳糖 Lactose − + − − + − − 蜜二糖 Melibiose − + − − − − + 甲基葡萄糖苷 Methyl α-d-glucoside − + − − + + + 杜-杜拉诺糖 d-Turanose − + − − − − + 注:① “+”表示阳性,“−”表示阴性; ②1. SM-1, 2. 解淀粉芽孢杆菌, 3.萎缩芽孢杆菌, 4.摩加夫芽孢杆菌, 5.暹罗芽胞杆菌, 6.贝莱斯芽孢杆菌, 7. B. subtilis subsp. Stercoris。
Note: ① ‘+’ means positive; ‘−’ means negative; ②1. SM-1, 2. B. amyloliquefaciens, 3. B. atrophaeus, 4. B. mojavensis, 5. B. siamensis, 6. B. velezensis, 7. B. subtilis subsp. Stercoris.表 2 与菌株SM-1最为相似的细菌种类
Table 2. Bacterial species with closest similarity to SM-1
种类 Species 菌株编号 Strain 16S 序列注册号 Accession number 相似性 Similarity/% 贝莱斯芽孢杆菌 Bacillus velezensis CR-502 AY603658 99.29 B. subtilis subsp. Stercoris D7XPN1 JHCA01000027 99.21 暹罗芽孢杆菌 B. siamensis KCTC 13613 AJVF01000043 98.97 枯草芽孢杆菌枯草亚种 B. subtilis subsp. Subtilis NCIB 3610 ABQL01000001 98.69 解淀粉芽孢杆菌 B. amyloliquefaciens DSM 7 FN597644 98.69 B. nakamurai NRRL B-41091 LSAZ01000028 98.69 B. subtilis subsp. inaquosorum KCTC 13429 AMXN01000021 98.49 耐盐短杆菌 B. halotolerans ATCC 25096 LPVF01000003 98.49 特基拉芽孢杆菌 B. tequilensis KCTC 13622 AYTO01000043 98.49 萎缩芽孢杆菌 B. atrophaeus JCM 9070 AB021181 98.49 莫海威芽孢杆菌 B. mojavensis RO-H-1 JH600280 98.42 死亡谷芽孢杆菌 B. vallismortis DV1-F-3 JH600273 98.42 枯草芽孢杆菌斯氏亚种 B. subtilis subsp. Spizizenii NRRL B-23049 CP002905 98.35 B. glycinifermentans GO-13 LECW01000063 97.45 副地衣芽孢杆菌 B. paralicheniformis KJ-16 KY694465 97.38 B. licheniformis 地衣芽孢杆菌 ATCC 14580 AE017333 97.18 B. haynesii NRRL B-41327 MRBL01000076 97.11 表 3 Biolog系统对细菌菌株SM-1的鉴定
Table 3. Identification of bacterial strain SM-1 by Biolog system
排序 Rank 相似性 Similarity 位距 Distance 种类 Species 1 0.631 5.311 萎缩芽孢杆菌 Bacillus atrophaeus 2 0.163 5.752 解淀粉芽孢杆菌 B. amyloliquefaciens 3 0.062 6.336 摩加夫芽孢杆菌 B. mojavensis 4 0.046 6.511 简单芽胞杆菌 B. simplex -
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