Grey mould is a destructive disease in a broad range of economically important crops, and Botrytis cinerea is the main fungal species causing grey mould in many plants. Pepper plant and fruit production declined due to B. cinerea infection, and fruit decay by grey mould should also be managed during the storage period after harvest (Polat et al., 2018; Wang et al., 2022b). Various disease control methods have been suggested as eco-friendly alternatives to chemical pesticides. Amendment with macro- and micro-nutrients changed grey mould severity in pepper plants. Internal calcium and nitrate levels of pepper plants were changed by treatment with calcium and nitrate, which are correlated with the decreased grey mould in pepper stems (Yoon et al., 2010). Zinc supply during seedling growth decreased grey mould in pepper leaves via enhancing immunity (Kuvelja et al., 2024). Reduced pepper grey mould through enhanced plant immunity was also possible by chemical plant defence activators (salicylic acid, melatonin) and biocontrol agents (Bacillus pumilus, B. safensis, B. siamensis, and B. thuringiensis) (Nasiri et al., 2024; Poveda et al., 2022).

In our previous studies, rhizobacteria B. siamensis H30-3 and its volatiles showed in vitro antifungal activity against diverse phytopathogenic fungi (Hong et al., 2024; Lee et al., 2018; Park et al., 2021). The unidentified antifungal compounds diffused on the media during dual culture with B. siamensis H30-3 restricted the mycelial growth of phytopathogenic fungi such as Alternaria brassicicola, Colletotrichum fructicola, C. higginsianum, Lasiodiplodia theobromae, Neopestalotiopsis clavispora, Phytophthora cactorum and Sclerotinia sclerotiorum (Hong et al., 2024; Lee et al., 2018; Park et al., 2021). Volatiles from B. siamensis H30-3 exerted their antifungal activity against the mycelial growth of C. fructicola, L. theobromae, N. clavispora, P. cactorum and S. sclerotiorum (Hong et al., 2024; Park et al., 2021). B. siamensis H30-3 and its volatiles were applied to B. cinerea and B. cinerea-inoculated pepper plants to see whether pepper grey mould can be controlled by B. siamensis H30-3.

In this study, B. cinerea mycelial growth was significantly restricted by co-culture with B. siamensis H30-3 on the 1/2 potato dextrose agar (PDA) media for 6 days (Fig. 1). Presumably, diffusible compounds from B. siamensis H30-3 also contain antifungal metabolites against B. cinerea, and the bacterial strain can be applied to control grey mould occurring in diverse crops. Further investigation is needed to determine whether the significant antifungal metabolites active against B. cinerea are also the most effective against other fungal species.

Bacterial motility and proliferation may influence the antifungal activity of B. siamensis H30-3 against B. cinerea, so two different agar concentrations were applied to the bacterial growth media. B. siamensis H30-3 bacterial suspension (10⁸ cfu/ml) (5 µl each) was dropped on the tryptic soy agar (TSA; 1.5%, 1.2%, 0.9%, and 0.6% agar) media (18 drops per medium) in the square dish (100×100×13 mm) and cultured at 30°C for 1 day. The colony morphology of B. siamensis H30-3 on the TSA media was changed depending on agar concentration, and bacterial colonies formed more diffusely on the TSA media of lower agar concentration (Fig. 2A). All bacterial colonies were separated on TSA containing 1.5% agar, and each bacterial colony diffused and contacted other bacterial colonies by reducing agar concentrations. The higher agar concentration in the media made the bacterial colony density look dense. Bacterial cells on the TSA media containing different agar concentrations were collected using distilled water, and optical densities of the bacterial suspensions were measured by a spectrophotometer at 600 nm. No significant difference was found among the numbers of bacterial cells derived from different growth media (data not shown). It indicates that B. siamensis H30-3 may move faster on the surface harbouring high water contents without high multiplication, and that highly diffused bacterial colony can be associated with metabolic activation for elevated production of antifungal compounds.

It was evaluated whether B. siamensis H30-3 volatiles could limit the mycelial growth of B. cinerea, and whether the changed colony formation by different agar concentrations led to the altered antifungal activity. B. siamensis H30-3 bacterial suspension (10⁸ cfu/ml) (5 µl each) was dropped on the TSA media (9 drops per medium) in the Petri dish (85 mm in diameter) and cultured at 30°C for 1 day. The mycelial disc (5 mm in diameter) of B. cinerea was inoculated at the centre in the 1/2-PDA media (20 ml) in a Petri dish (85 mm in diameter), and then exposed to the B. siamensis H30-3 colonies preformed on the TSA at 20°C for 3 days. B. siamensis H30-3 volatiles exerted antifungal activity against the mycelial growth of B. cinerea shown by reduced colony diameter (Fig. 2B, C). The limited fungal mycelial growth by B. siamensis H30-3 volatiles has been found in the other phytopathogenic fungi in our previous studies, but increased antifungal efficacy of the volatiles from B. siamensis H30-3 grown on TSA media containing a low agar concentration (0.6%) was first demonstrated on B. cinerea in the present study. It seems that the mycelial growth of phytopathogenic fungi investigated previously could also be more delayed by volatiles of B. siamensis H30-3 grown on TSA media containing a low agar concentration. Antifungal activity against B. cinerea has been found in volatiles from many Bacillus spp., and some volatile metabolites have been chemically identified as antifungal compounds (Calvo et al., 2020; Essghaier et al., 2009; Wang et al., 2022a). However, it is worth noting that changing bacterial environments, such as water potential, can enhance their capacity for antifungal metabolite production in the current study. Finding other environmental factors to increase the antifungal activity of B. siamensis H30-3 will be a promising way for efficient crop protection.

The enhanced antifungal activity of volatiles from B. siamensis H30-3 grown on TSA media containing a low agar concentration was also found during the in vitro conidial germination of B. cinerea (Fig. 3). Conidial germination of B. cinerea was induced on glass slides under humid conditions according to slightly modified methods (Hong et al., 2023). Two glass slides with four conidial suspension drop in the square dishes (100×100×13 mm) were covered with the same-sized square dishes, in which B. siamensis H30-3 colonies grown on TSA media 1 day above described. The conidia and germ tubes were stained with lactophenol-trypan blue at 4 hr after incubation at 20°C, and conidial germination was investigated under a light microscope. B. siamensis H30-3 volatiles efficiently decreased conidial germination of B. cinerea (Fig. 3A). Most conidia in the mock-treated control germinated and formed germ tubes at a similar level regardless of agar concentration in the TSA media, whilst conidial germination was distinctly reduced by B. siamensis H30-3 volatiles, in particular, by volatiles from B. siamensis H30-3 grown on the low-agar media. The differently reduced conidial germination by volatiles from B. siamensis H30-3 grown on the high-agar and low-agar media was quantified (Fig. 3B). The conidial germination in the mock-treated control was ca. 92.9% and 89.2% by the high-agar and low-agar media, respectively, without a significant difference. The conidia germination was reduced to ca. 65.7% by volatiles from B. siamensis H30-3 on the high-agar media but to ca. 3.0% by volatiles from B. siamensis H30-3 on the low-agar media. These suggest that volatiles from B. siamensis H30-3 on the low-agar media are more effective in suppressing B. cinerea during conidial germination and mycelial growth simultaneously.

The volatiles from B. siamensis H30-3 grown on the two different agar media were applied to the detached pepper leaves to investigate the crop protection efficacies against grey mould. Pepper seeds (cv. Nockwang) were sown, and the seedlings were grown until the eight-leaf stage under controlled environments (Jo et al., 2020). Primary and secondary leaves from the bottom were detached and placed on the sterile water-saturated gauze in square Petri dishes (125×125×20 mm). Two drops (10 µl each) of conidial suspension (5×10⁴ conidia/ml) were applied to the detached leaves and incubated in the moist boxes at 20°C for 3 days to develop necrotic lesions with or without B. siamensis H30-3 volatiles. Reduced grey mould lesions were found in the pepper leaves by volatiles from B. siamensis H30-3 grown on the high agar and low agar media (Fig. 4A), and statistically analysed (Fig. 4B). The B. siamensis H30-3 volatiles-mediated disease control was not different in the primary and secondary leaves, regardless of agar concentration in the growth media. Compared to the mock treatment, the reductions in lesion diameters were ca. 22.3% and ca. 32.5% in the primary and secondary leaves by B. siamensis H30-3 grown on the high-agar media, respectively. By contrast, the reductions were ca. 54.7% and ca. 61.8% in the primary and secondary leaves by B. siamensis H30-3 grown on the low agar media. Disease control efficacies by B. siamensis H30-3 grown on the low agar media were much higher than those on the high agar media. The volatiles from B. siamensis H30-3 grown on the less solid media could decrease grey mould on the detached leaves more efficiently. The arrested lesion enlargement on the pepper leaves may be attributed to limited conidial germination and mycelial growth of B. cinerea by B. siamensis H30-3 volatiles shown in Figs. 2, 3. These suggest that increasing wetness conditions on the plant leaf surface may enhance the disease protection efficacy of B. siamensis H30-3 volatiles in crop fields. The grey mould in postharvest blueberry and raspberry fruits was suppressed by volatiles from B. siamensis strains YJ15 and G-3, respectively (Wang et al., 2022a; Zhang et al., 2020). Recently, efficient control of grey mould was demonstrated in pepper fruits by B. siamensis SCFB 2-2 and SCFB 3-4, but it was hard to show the effect of antifungal volatiles from B. siamensis strains on disease reduction because B. cinerea was inoculated on pepper fruits at the same site where B. siamensis strains were treated (Poveda et al., 2022). The decreased grey mould in pepper leaves by B. siamensis H30-3 volatiles in the current study may be the first report on the pepper grey mould control by B. siamensis volatiles. We could not exclude the possibility of enhanced plant immunity of pepper leaves by volatiles of B. siamensis H30-3, because microbial volatiles-mediated activation of plant immunity has been continuously suggested (Kwon et al., 2010; Naznin et al., 2014; Ryu et al., 2004; Thankappan et al., 2022). More recently, treatment with B. siamensis B30 on faba bean plants mitigated disease severity caused by bean yellow mosaic virus infection via enhancing systemic resistance (Abdelkhalek et al., 2025). It will be worthy to investigate B. siamensis H30-3 volatiles-triggered defence-associated biochemical changes in pepper plants.

B. velezensis 5YN8 and DSN012 volatiles exerted in vitro antifungal activity against B. cinerea, but the suppressed grey mould in the pepper seedlings mediated by the volatiles could not be confirmed using the two bacterial application methods (Jiang et al., 2018). The B. velezensis 5YN8 and DSN012 volatile compounds can be diffused to the ambient space through the root-sprinkling and leaf-spraying bacterial suspension onto the pot seedlings before B. cinerea challenge-inoculation. In our study, B. cinerea conidial suspension (5×10⁴ conidia/ml) was sprayed on the two eight-leaf staged pepper seedlings in a plastic box (35×24.5×19 cm), and volatiles from B. siamensis H30-3 grown on the two low agar media were applied simultaneously in the box. Eight pepper seedlings were used for mock and volatile treatments, respectively. The plastic boxes were tightly closed to keep the bacterial volatiles and moisture and placed at 20°C for 48 hr. After transfer to the plant growth room, the grey mould severity in the pepper seedlings was evaluated at 3 days and 5 days after the fungal inoculation (Fig. 4C). Pepper seedlings were highly damaged by B. cinerea infection in the absence of B. siamensis H30-3 volatiles. The fungal infection caused severe blight on most leaves, with some lower leaves undergoing defoliation, and only newly developed leaves showed slightly lower damage. B. siamensis H30-3 volatiles attenuated grey mould symptom development in the pepper seedlings. The 1st to 3rd lower leaves were blighted, as shown in the inoculated pepper seedlings without B. siamensis H30-3 volatiles. However, the 4th to 5th upper leaves from the bottom did not exhibit severe necrosis, and scattered spots appeared on the leaves. The disease severity index was followed by a scale of 0-10 based on the number of severely necrotic leaves. Significantly different disease severities were recorded on the B. cinerea-inoculated pepper seedlings with or without B. siamensis H30-3 volatiles (Fig. 4D). A distinct reduction in the disease severity was found in the inoculated seedlings at 3 days and 5 days in the presence of B. siamensis H30-3 volatiles. These results show that B. siamensis H30-3 volatiles can be applied to the pepper seedlings grown under greenhouse conditions to control grey mould.

In conclusion, B. siamensis H30-3 and its volatiles efficiently suppressed conidial germination and mycelial growth of B. cinerea, and the antifungal activity was enough to reduce grey mould in detached pepper leaves. A drastic reduction in the fungal growth and grey mould lesion was uncovered by volatiles from B. siamensis H30-3 grown on the low agar media. These results support B. siamensis H30-3 volatiles as a bio-fungicide to manage grey mould in pepper seedlings. For credible disease control under the greenhouse, it is necessary to determine further how much B. siamensis H30-3 volatiles should be introduced depending on greenhouse size and how long the seedlings should be exposed to the bacterial volatiles.