Rhizoctonia solani is a destructive soil-borne pathogen causing substantial yield losses in diverse crops worldwide. To develop sustainable alternatives to synthetic pesticides, a thermally resistant Bacillus amyloliquefaciens strain, KW21, was isolated from agricultural and forest soils in Korea. KW21 was selected for its antifungal activity against R. solani and its ability to retain this activity after high-temperature exposure. 16S rRNA gene sequencing and phylogenetic analysis confirmed > 99% similarity to B. amyloliquefaciens. In vitro assays demonstrated consistent inhibition of R. solani growth. Thermal resistance testing revealed D-values of 47.6 h at 110°C, 8.3 h at 120°C, and 1.13 h at 130°C, with Z-values of 13.2°C and 11.5°C.
Research Article
Thermal Resistance and Antifungal Properties of Bacillus amyloliquefaciens KW21
https://doi.org/10.21203/rs.3.rs-7595067/v1
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Antifungal Activity
Bacillus amyloliquefaciens
Rhizoctonia solani
Thermal Resistance
D-values
Z-values
Rhizoctonia solani is a major plant pathogenic fungus that causes severe damage to crops worldwide [1]. As the detrimental effects of synthetic pesticide use on soil ecosystems and human health have become increasingly evident, the development of microbial biocontrol agents has emerged as an important alternative [2]. Among these, Bacillus species are considered promising candidates due to their ability to form endospores, produce antimicrobial compounds, and promote plant growth [3, 4]. However, most microorganisms with antifungal activity are sensitive to heat, which limits their practical application in distribution, storage, and especially high-temperature treatment processes [5]. To overcome this limitation, recent studies have explored microbial resources from diverse and extreme environments to identify heat-resistant strains [6, 7]. Nevertheless, reports on microorganisms capable of producing heat-stable antifungal substances, particularly those active above 100°C, remain scarce [8]. Therefore, this study reports the isolation of a heat-stable, antifungal strain from agricultural and forest soils in Korea and evaluates its thermal resistance characteristics.
Isolation of candidate Strains Soil samples were collected from potato fields, ginseng fields, and forest soils in Korea. For strain isolation, a minimal medium containing 0.9% complex fertilizer (N:19, P:10, K:8, Mg:1, B:0.1), 0.4% colloidal chitin, and 0.1% peptone was used. Eight candidate strains were selected based on preliminary antifungal activity against R. solani.
Antifungal activity and thermal resistance Assay Each strain was cultured in LB (Lennox L) broth at 28°C for 72 h under static conditions. The culture broth was filtered, and a 50 µL aliquot of the filtrate was pipetted onto a sterilized paper disc (Ø 8 mm). The loaded disc was then dry-heat treated at 100°C for 15 min and placed 20 mm away from a mycelial plug (Ø 8 mm; taken with a No. 3 cork borer) excised from the actively growing margin of an R. solani culture on Potato Dextrose Agar (PDA). The inoculated plates were incubated at 28°C for 96 h. The strain with antifungal activity, as indicated by its inhibition zone, was further treated at 120°C for 1 h to select the final strain. Thermal resistance of this strain was quantified by determining D-values at 110°C, 120°C, and 130°C in triplicate experiments, and Z-values were calculated from the slopes of the thermal death time curves.
Strain identification The 16S rRNA gene of the selected strain was amplified and sequenced (SolGent Co., Ltd., located in Daejeon, Korea), and the obtained sequence was compared with those in the NCBI GenBank database using BLASTn for species identification [9].
Strain identification The 16S rRNA gene sequence analysis revealed that Bacillus strain KW21 exhibited over 99% sequence similarity with Bacillus amyloliquefaciens when searched against the NCBI BLASTn database [9]. Key similar strains identified included B. amyloliquefaciens strain NBRC 15535, B. vallismortis strain KCTC 13913 and B. siamensis strain KCTC 13613. Phylogenetic analysis positioned KW21 within the B. amyloliquefaciens clade (Table 1, Fig. 1), supporting its taxonomic assignment.
The tree was constructed using the neighbor-joining method based on 16S rRNA gene sequences. Bootstrap values (> 50%) from 1,000 replicates are shown at branch nodes. Bacillus subtilis strain IAM 12118 was used as an outgroup. The scale bar indicates 0.005 substitutions per nucleotide position.
Thermal resistance KW21, which retained antifungal activity after heat treatment, exhibited D-values of 47.6 h at 110 °C, 8.3 h at 120 °C, and 1.13 h at 130 °C (triplicate experiments). Thermal death time curves for each temperature are shown in Fig. 2, where the slopes of the survival curves were used to calculate D-values.
The Z-value was calculated from the slope of the regression line obtained by plotting log D values against temperature (Fig. 3), which showed that increasing temperature led to a decrease in D-values, indicating accelerated microbial inactivation [10]. The Z-value between 110°C and 120°C was 13.2°C, and between 120°C and 130°C was 11.5°C. A regression-based overall Z-value across 110–130°C was 12.31°C (R² = 0.999), as shown in Fig. 3, indicating strong linearity [10].
This study isolated Bacillus KW21 with antifungal activity and unique thermal resistance from agricultural soils in Korea. The strain exhibited high D-values and Z-values, attributed to heat-resistant endospore formation. This distinctive thermal resistance addresses a key limitation of existing microbial biocontrol agents and supports KW21’s potential as a novel candidate for sustainable agricultural applications, particularly in heat-intensive environments.
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