Identification and Mechanism Characterization of Antagonistic Actinomyces SD-64 Against Pepper Southern Blight

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

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (1): 175-189.doi: 10.16420/j.issn.0513-353x.2023-0058

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Identification and Mechanism Characterization of Antagonistic Actinomyces SD-64 Against Pepper Southern Blight

LIU Shida, QIU Zelan, LI Xiaogang, ZHONG Jie, ZHU Junzi^*()

College of Plant Protection，Hunan Agricultural University，Changsha 410128，China

Received:2023-04-23 Revised:2023-09-19 Online:2024-01-25 Published:2024-01-16
Contact: * （E-mail：zhujunzi20@163.com）

Abstract

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

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.

Figures/Tables 15

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

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

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

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

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.

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	+++

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

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.

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

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

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

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.

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

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

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

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Identification and Mechanism Characterization of Antagonistic Actinomyces SD-64 Against Pepper Southern Blight

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