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. 2024 Nov 28:15:1429646.
doi: 10.3389/fmicb.2024.1429646. eCollection 2024.

Evaluation of Streptomyces sporoverrucosus B-1662 for biological control of red pepper anthracnose and apple bitter rot diseases in Korea

DaYoung Kim  1 , Jungyeon Kim  1 , Younmi Lee  1 , Kotnala Balaraju  2 , Ye-Ji Hwang  3 , Mi-Hwa Lee  4 , Wonsu Cheon  5 , Hye Yeon Mun  5 , Chang Soo Lee  5 , Yongho Jeon  1
Affiliations

Evaluation of Streptomyces sporoverrucosus B-1662 for biological control of red pepper anthracnose and apple bitter rot diseases in Korea

DaYoung Kim et al. Front Microbiol. .

Abstract

Fungi are the prominent phytopathogens that have significant impact on the productivity of agriculture worldwide. Streptomyces species have been extensively studied for the production of various bioactive metabolites. These metabolites have been used as biocontrol agents for the management of diseases caused by phytopathogenic fungi. The purpose of this investigation is to assess the efficacy of Streptomyces sporoverrucosus B-1662, an antagonistic agent in the control of red pepper anthracnose caused by Colletotrichum acutatum KACC 42403 and apple anthracnose caused by Colletotrichum siamense CGCP6 (GYUN-10348). On the basis of the morphological, and molecular characterization using 16S rRNA, the strain B-1662 was determined to be S. sporoverrucosus. The strain B-1662 exhibited antagonistic activity against seven fungal phytopathogens, including C. acutatum KACC 42403 and C. siamense CGCP6. The culture filtrates (CF) from B-1662 showed antifungal activity against all seven fungal pathogens with greater inhibition rate (%) in comparison with a control. The bacterial suspensions of B-1662 showed an excellent biological control effect on the red pepper anthracnose and apple bitter rot using an in planta assay. The anthracnose disease rate (%) was controlled by over 90% with B-1662 cell suspensions at 105 to 107 CFU/mL. Compared to a control, the strain B-1662 played a more effective role in controlling the anthracnose disease in field conditions in both years 2022 and 2023. From the effective solvent fractions, the effect compound (dibutoxybutane) has been isolated exhibiting with antifungal effect. The genetic base underlying the biocontrol traits of B-1662 was characterized using the whole-genome sequence of B-1662, which was compared with closely related strains. Consequently, these results collectively suggest that S. sporoverrucosus B-1662 can aid in the management of red-pepper anthracnose.

Keywords: Colletotrichum disease; Streptomyces sporoverrucosus B-1662; apple; biocontrol agent; pepper; whole-genome sequencing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Scanning electron micrographs of the spore morphology of Streptomyces sp. B-1662 on IPS 3 medium after incubation at 28°C for 3 days, which showed the aerial hyphae, non-fragmented, and most spores were rod-shaped with a fragmented spore chain and smooth surface.
FIGURE 2
FIGURE 2
Neighbor-joining phylogenetic tree based on 16s rRNA gene sequence comparing Streptomyces B-1662 strain with other Streptomyces species collected from NCBI-BLAST. The numbers on the branches indicate the percentage bootstrap values of 1000 replicates. The Neighbor-joining phylogenetic tree was generated by the MEGA-X program. The scale bar indicates 0.02 Substitutions per nucleotide position.
FIGURE 3
FIGURE 3
In vitro antagonistic activity of Streptomyces sp. B-1662 against fungal phytopathogens using a dual culture plate assay. The mycelial plug of a pathogenic fungus from a 5-day-old PDA plate was inoculated onto PDK medium, while B-1662 was streaked on the opposite side of the same plate. The plates inoculated with only mycelial discs alone served as the control group. (A) The inhibition zones of various fungal pathogens, such as Colletotrichum acutatum KACC 42403, C. coccodes KACC 48737, Fusarium oxysporum f.sp. lycopercisi KACC 40043, C. siamense CGCP6, Alternaria alternata MGFJ029, Botryosphaeria dothidea USFJ2, and C. fructicola ANBA7 were measured 16, 10, 11, 15, 15, 5, and 13 days, respectively, after incubation at 25°C; the left and right colonies on the plates are phytopathogenic fungus and B-1662, respectively. (B) The percentage of mycelial growth inhibition rate was calculated from the inhibition zone. The experiment was performed twice in triplicate. Bars with the same letters do not differ from each other according to the least significant difference (LSD) (P < 0.05).
FIGURE 4
FIGURE 4
Inhibitory effect of culture filtrate (CF) of antagonistic Streptomyces sp. (A) B-1662 on growths of various phytopathogenic fungi under in vitro conditions. Paper disks impregnated with TSB were used as a non-treated control. The diameter of mycelial growths of fungal pathogens on PDK plates was recorded 12 d after incubation at 25°C. (B) The percentage of mycelial growth inhibition rate was calculated from the inhibition zone. The experiment was performed twice in triplicate. Bars with the same letters do not differ from each other according to the least significant difference (LSD) (P < 0.05).
FIGURE 5
FIGURE 5
Effect of bacterial suspensions of Streptomyces sporoverrucosus B-1662 treatment on conidia germination rate (%) of C. acutatum and microscopic observation: (A) Conidial germination rate (%) was suppressed by bacterial suspensions, while the germination rate (%) was increased in the non-treated control; and (B) appressorium formation rate (%) at different time intervals were recorded. (C) Microscopic observations of C. acutatum spore germination after treatment with B-1662 cell suspensions at various concentrations (105, 106, and 107 CFU/mL) during the incubation period from 0 to 48 h. The germination counting was carried out using a hemocytometer. The percentage at a given time was recorded by observing at least 200 conidia for each treatment. Bar = 10 μm. The experiment was performed two times in triplicate producing similar results.
FIGURE 6
FIGURE 6
Effect of various solvent fractions of B-1662 against the mycelial growth of Colletotrichum siamense CGCP6 in vitro. Mycelial growth inhibition rate (%) of Colletotrichum siamense CGCP6 on PDAA medium was recorded 9 d after incubation at 25°C in comparison with a non-treated control. The experiment was performed two times in triplicates. Bars with the same letters do not differ from each other according to the least significant difference (LSD) (P < 0.05). HX, Hexane; CX, Chloroform; EX, Ethyl acetate; BX1, Butanol layer 1; BX2, Butanol layer 2; BX4, Butanol layer 4.
FIGURE 7
FIGURE 7
Effect of S. sporoverrucosus B-1662 cell suspensions on suppression of disease rate (%) of anthracnose caused by C. acutatum on red-pepper fruits in vivo. (A) Wounded red-pepper fruits were treated with B-1662 culture suspensions at three different concentrations (105, 106, and 107 CFU/mL), followed by inoculation with C. acutatum ANUP-HJ conidia suspensions (5 × 105 conidia/mL). Red-pepper fruits treated with TSB served as a control. (B) The results were compared with a non-treated control 9 days after incubation at 25°C. The experiment was performed two times with three replicates. Bars with the same letters do not differ from each other according to the least significant difference (LSD) (P < 0.05).
FIGURE 8
FIGURE 8
Effect of S. sporoverrucosus B-1662 cell suspensions and its culture filtrate (CF) on control of apple bitter rot caused by C. siamense in wounded apples. (A) B-1662 cell suspensions (106 CFU/mL) and CFs (obtained from 5 days after incubation) were dropped on the detached apple fruits, followed by inoculation with C. siamense conidia suspensions (5.3 × 105 conidia/mL) after 1 day. (B) The results were compared with a non-treated control 11 days after incubation at 25°C. The experiment was performed two times with three replicates. Bars with the same letters do not differ from each other according to the least significant difference (LSD; p < 0.05).
FIGURE 9
FIGURE 9
Effect of B-1662 treatment on suppression of anthracnose disease caused by C. acutatum under field conditions in 2022 (A) and 2023 (B). (A) In 2022, after transplanting two-month-old red pepper seedlings in the field, the plants were treated with B-1662 cell suspensions by foliar spray or foliar spray + soil drench or water (control), chemical control, and pyraclostrobin (negative control) for seven times in 60 days. (B) In 2023, the plants were treated with B-1662 cell suspensions by foliar spray or cross-spraying or Mixed spraying or water (control), chemical control, and pyraclostrobin (negative control) for seven times in 60 days. Disease rate (%) was recorded from disease-infected red pepper fruits after 10 days of the last treatment, and compared with chemical and non-treated controls. For all the treatments, three different plots with twenty replicates (plants) were used. Differences in letters on bars indicate statistically significant between the treated and the control according to the least significant difference (LSD) (P < 0.05). FS, foliar spray, FS+SD: foliar spray+soil drench, CS, Cross spray; MS, Mixed spray.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Korea Environment Industry and Technology Institute (KEITI) through a project to make multi-ministerial national biological research resources a more advanced program funded by the Korea Ministry of Environment (MOE) (2021003420002).