辣椒白绢病拮抗放线菌SD-64的鉴定及作用机制研究

doi:10.16420/j.issn.0513-353x.2023-0058

摘要/Abstract

摘要：

通过平板对峙培养法从辣椒根际土壤中筛选到1株对辣椒白绢病菌（Sclerotium rolfsii）具有抑制活性的放线菌株SD-64。利用形态学观察和分子生物学方法对其进行了鉴定，研究其对辣椒白绢病的防效以及对白绢病菌的作用机制，并对菌株SD-64发酵液粗提物进行了提取。结果表明，菌株SD-64对白绢病菌的平板抑制率达到99.27%。基于形态和16S rDNA鉴定SD-64为灰橙链霉菌（Streptomyces griseoaurantiacus）。SD-64能产生黑色素和纤维素酶，其发酵产物可以影响辣椒白绢病菌菌核的产生与萌发，影响菌丝形态，破坏细胞膜的完整性，增加菌丝内活性氧的积累。辣椒离体叶片和活体盆栽防效试验显示SD-64发酵液能明显降低辣椒白绢病的发生。SD-64发酵液粗提物对辣椒白绢病菌的EC₅₀为78.281 μg · mL^-1。综上所述，菌株SD-64对辣椒白绢病菌具有的良好防治效果，具有开发应用潜力。

关键词: 辣椒, 白绢病, 生物防治, 放线菌, 16S rDNA, 作用机制

Abstract:

In this study，a strain of Streptomyces SD-64 was screened from the rhizosphere soil of peppers by plate confrontation method，which has strong antagonistic effects on Sclerotium rolfsii. It was identified by morphological observation and molecular biology method，and its control effect on pepper southern blight and its mechanism of action were evaluated. The crude extract from the fermentation broth of strain SD-64 was extracted. The plate bioassay showed that strain SD-64 the inhibition rate on S. rolfsii 99.27%. Based on morphology and 16S rDNA identification，SD-64 was identified as Streptomyces griseoaurantiacus. SD-64 could produce melanin and cellulase. Its fermentation products could affect the formation and germination of sclerotium，affect hyphal morphology，destroy the integrity of cell membrane，and increase the accumulation of reactive oxygen species（ROS）in hyphae. The test of control effect of in vitro and pot experiment showed that the fermentation solution of SD-64 could significantly reduce the incidence of pepper southern blight. The EC₅₀ of the crude extract from SD-64 fermentation broth against S.rolfsii was 78.281 μg · mL^-1. In conclusion，the strain SD-64 has good control effect on pepper southern blight and has the potential for development and application.

Key words: pepper, Sclerotium rolfsii, biological control, actinomyces, 16S rDNA, mode of action

刘仕达, 邱泽澜, 李晓刚, 钟杰, 朱俊子. 辣椒白绢病拮抗放线菌SD-64的鉴定及作用机制研究[J]. 园艺学报, 2024, 51(1): 175-189.

LIU Shida, QIU Zelan, LI Xiaogang, ZHONG Jie, ZHU Junzi. Identification and Mechanism Characterization of Antagonistic Actinomyces SD-64 Against Pepper Southern Blight[J]. Acta Horticulturae Sinica, 2024, 51(1): 175-189.

图/表 15

图1 菌株SD-64与辣椒白绢病菌的平板对峙试验

Fig. 1 Plate confrontation experiment between strain SD-64 and Sclerotium rolfsii

图2 不同浓度SD-64菌株发酵液对白绢病菌菌丝生长的抑制作用

Fig. 2 Inhibiting effect of strain SD-64 fermentation broth on mycelial growth of Sclerotium rolfsii

表1 不同浓度SD-64菌株发酵液对辣椒白绢病菌的抑菌率

Table 1 Inhibition rate of strain SD-64 on Sclerotium rolfsii

浓度/% Concentration	菌落直径/cm Colony diameter	抑菌率/% Inhibition rate
0（对照Control）	5.42 ± 0.07	—
1	3.92 ± 0.34 b	32.54 ± 0.07 d
2	4.52 ± 0.50 a	19.55 ± 0.11 e
5	2.68 ± 0.13 c	59.24 ± 0.03 c
10	1.88 ± 0.12 d	76.55 ± 0.03 b
20	1.17 ± 0.05 e	92.06 ± 0.01 a

图3 不同浓度SD-64发酵液对辣椒白绢病菌菌核产生（A）与萌发（B）的影响

Fig. 3 Sclerotium production（A）and germination（B）of strain SD-64 against Sclerotium rolfsii

图4 菌株SD-64对辣椒白绢病菌菌核质量（A）和萌发率（B）的影响不同小写字母表示处理间差异显著，P < 0.05。下同。

Fig. 4 Effect of strain SD-64 on Sclerotium rolfsii weight (A) and germination rate (B) Different lowercase letters indicate significant differences between treatments，P < 0.05. The same below.

表2 菌株SD-64的生长和培养特征

Table 2 Growth and cultural characteristics of strain SD-64

培养基 Medium	气生菌丝 Aerial mycelium	基内菌丝 Substrate mycelium	可溶性色素 Soluble pigment	生长状况 Growth
ISP2	白色White	红色Red	无 None	+++
ISP3	白色White	白色White	无None	++
ISP4	白色White	淡黄色Light yellow	无None	+++
ISP5	淡红色Light red	淡黄色Light yellow	无None	+
ISP6	黄色Yellow	黄色Yellow	无None	+++
ISP7	白色White	黄色Yellow	黄色Yellow	+++

图5 菌株SD-64基于16S rDNA序列的系统进化树

Fig. 5 Phylogenetic tree of strain SD-64 based on 16S rDNA sequences

图6 菌株SD-64对辣椒白绢病菌防效试验 A、B为离体叶片接种；C为活体接种。Ⅰ~ Ⅳ分别为对照、SD-64菌悬液、SD-64发酵液、0.4%噻呋酰胺处理。

Fig. 6 Inoculation experiment of strain SD-64 on Sclerotium rolfsii A and B were in vitro leaves inoculation；C is in vivo inoculation. Ⅰ-Ⅳ were treated with control，SD-64 bacterial suspension，SD-64 fermentation broth and 0.4% Thiafuramide，respectively.

图7 菌株SD-64对辣椒白绢病菌菌丝形态的影响

Fig. 7 Effect of strain SD-64 on hyphal morphology of Sclerotium rolfsii

图8 用PI染色检测菌株SD-64对辣椒白绢病菌细胞膜完整性的影响

Fig. 8 Effect of strain SD-64 on cell membrane integrity of Sclerotium rolfsii observed by PI staining

图9 菌株SD-64对辣椒白绢病菌活性氧积累的影响

Fig. 9 Effect of strain SD-64 on the production of reactive oxygen species of Sclerotium rolfsii

图10 菌株SD-64的产胞外酶能力检测 A：产果胶酶培养基；B：产葡聚糖酶培养基；C：产纤维素酶培养基；D：产几丁质酶培养基。

Fig. 10 Results of extracellular enzyme production of strain SD-64 A：Pectinase production medium；B：Glucanase production medium；C：Cellulase production medium；D：Chitinase production medium.

图11 菌株SD-64发酵液粗提物的抑菌试验

Fig. 11 Antibacterial experiment of crude extract from fermentation broth of strain SD-64

表3 菌株SD-64发酵液粗提物对辣椒白绢病菌的抑菌率

Table 3 Antibacterial activity of crude extract from fermentation broth of strain SD-64 against Sclerotium rolfsii

浓度/（μg · mL^-1） Concentration	菌落直径/cm Colony diameter	抑菌率/% Inhibition rate
0（对照Control）	6.32 ± 0.04	—
30	5.21 ± 0.24 a	19.99 ± 0.04 f
60	4.05 ± 0.38 b	41.12 ± 0.07 e
90	3.47 ± 0.20 c	51.69 ± 0.04 d
120	2.22 ± 0.44 d	74.34 ± 0.08 c
150 180	1.25 ± 0.14 e 0.80 ± 0.00 f	91.85 ± 0.02 b 100.00 ± 0.00 a

图12 菌株SD-64发酵液粗提物主要化合物的化学结构

Fig. 12 Chemical structure of the main compounds in the crude extract of strain SD-64 fermentation broth

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