拮抗北细辛菌核病木霉菌的分离、鉴定及生防效果

Isolation, identification and biocontrol effect of Trichoderma antagonistic to Sclerotinia disease of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag.

摘要:

[背景] 菌核病是北细辛根部主要病害之一，木霉菌作为目前应用最广泛的生物防治真菌，利用木霉菌防治北细辛菌核病是目前研究的热点。[目的] 通过稀释分离法对健康北细辛植株根际土壤进行菌株分离，以期筛选出有效拮抗北细辛菌核病的生防木霉菌。[方法] 以北细辛菌核病菌为靶标菌，采用平板对峙培养、挥发性与非挥发性物质抑菌的方法对分离得到的木霉菌进行筛选，采用生长速率法对筛选出的木霉菌的发酵液进行抑菌效果测定，并采用硫代巴比妥酸法测定筛选出的木霉对北细辛菌核病菌的丙二醛（Malondialdehyde，MDA）含量、紫外吸收法测定过氧化氢酶（Catalase，CAT）活性、氮蓝四唑法测定超氧化物歧化酶（Superoxide Dismutase，SOD）活性、愈创木酚法测定过氧化物酶（Peroxidase，POD）活性的影响。[结果] 从土壤中分离出木霉菌共14株，通过形态学和ITS-RPB2双基因联合构建系统发育树，鉴定其为哈茨木霉（Trichoderma harzianum）、钩状木霉（Trichoderma hamatum）、拟康氏木霉（Trichoderma koningiopsis）、深绿木霉（Trichoderma atroviride）、短密木霉（Trichoderma brevicompactum）和装絮木霉（Trichoderma tomentosum）。对峙培养试验表明，钩状木霉A26、拟康氏木霉B30、钩状木霉C6、哈茨木霉A17对北细辛菌核病菌抑制率均在90%以上，挥发性物质抑制测定结果显示钩状木霉C6抑制率最高，为53.73%±0.07%，木霉菌的非挥发性物质抑菌作用较强，哈茨木霉A17、钩状木霉A26、钩状木霉C6的非挥发性物质对细辛菌核病菌的抑制率均在75%以上，而拟康氏木霉B30抑制率可达100%。因此，筛选出的哈茨木霉A17、钩状木霉A26、拟康氏木霉B30、钩状木霉C6为拮抗效果较强的生防木霉菌，这4株木霉菌的发酵液对北细辛菌核病菌的抑制率分别为56.33%±0.12%、77.22%±0.06%、82.28%±0.03%、46.20%±0.04%。经这4株木霉菌的非挥发性物质处理7 d后，菌核病菌MDA含量显著增加，钩状木霉A26是对照组的7.7倍，最为显著；菌核病菌抗氧化酶活性均降低，与对照组相比，CAT、SOD、POD活性分别下降了19.67%-75.84%、4.71%-68.71%和3.57%-67.86%。[结论] 从北细辛健康植株根际土壤中分离的木霉菌株哈茨木霉A17、钩状木霉A26、拟康氏木霉B30、钩状木霉C6对北细辛菌核病菌均有较好的抑制效果，可用于北细辛菌核病的生物防治。

Abstract:

[Background] Sclerotinia disease is one of the main root diseases of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. Trichoderma is extensively used for biocontrol owing to the fungistatic activity. Recently, the control of Sclerotinia asari by Trichoderma has attracted the interest of scholars. [Objective] Trichoderma strains were isolated from the rhizosphere soil of healthy A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. with the dilution-plate method and those antagonistic to S. asari were screened out. [Methods] The inhibition of the isolated strains (plate confrontation method), the volatile and non-volatile strain metabolites, and strain fermentation broth (growth rate method) on S. asari was respectively determined. The malondialdehyde (MDA) content (thiobarbituric acid method), catalase (CAT) activity (ultraviolet absorption method), superoxide dismutase (SOD) activity (nitroblue tetrazolium assay), and peroxidase (POD) activity (guaiacol method) of S. asari treated with the selected Trichoderma were determined. [Results] A total of 14 Trichoderma strains were isolated and identified based on morphological observation and ITS-RPB2 sequence alignment as T. harzianum, T. hamatum, T. koningiopsis, T. atroviride, T. brevicompactum, and T. tomentosum, respectively. The inhibition rates of T. hamatum A26, T. koningiopsis B30, T. hamatum C6, and T. harzianum A17 on S. asari were all above 90%. The volatile metabolites of T. hamatum C6 demonstrated the highest inhibition rate (53.73%±0.07%), and the non-volatile metabolites showed stronger inhibitory effect, particularly those of A17 (inhibition rate: >75%), A26 (inhibition rate: >75%), C6 (inhibition rate: >75%), and B30 (inhibition rate: 100%). Thus, strains A17, A26, B30, and C6 had the strongest control effect and the inhibition rates of the fermentation broth of these 4 Trichoderma strains against S. asari were 56.33%±0.12%, 77.22%±0.06%, 82.28%±0.03%, and 46.20%±0.04%, respectively. After being treated with the non-volatile metabolites of the 4 strains for 7 days, S. asari saw significantly increased MDA content, particularly the S. asari strains treated with the non-volatile metabolites of A26 (MDA content was 7.7 times that of the control). Besides, the antioxidant enzyme activity of S. asari decreased. To be specific, the activity of CAT, SOD, and POD declined by 19.67%-75.84%, 4.71%-68.71%, and 3.57%-67.86%, respectively, as compared with that of the control. [Conclusion] Trichoderma A17, A26, B30, and C6 isolated from the rhizosphere soil of healthy plants of A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. can be used for the biocontrol of S. asari.