Walnut anthracnose caused by Colletotrichum gloeosporioides is a deleterious disease that severely affects the production of walnut (Juglans regia L.). The aim of this study was to assess the antifungal and growth promotion activities of Bacillus velezensis CE 100 as an alternative to chemical use in walnut production. The crude enzyme from B. velezensis CE 100 exhibited chitinase, protease, and β-l,3-glucanase activity and degraded the cell wall of C. gloeosporioides, causing the inhibition of spore germination and mycelial growth by 99.3% and 33.6% at 100 µL/mL, respectively. The field application of B. velezensis CE 100 culture broth resulted in a 1.3-fold and 6.9-fold decrease in anthracnose disease severity compared to the conventional and control groups, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (up to 1.4 µg/mL) and exhibited the potential for ammonium production and phosphate solubilization to enhance the availability of essential nutrients. Thus, field inoculation of B. velezensis CE 100 improved walnut root development, increased nutrient uptake, enhanced chlorophyll content, and consequently improved total biomass by 1.5-fold and 2.0-fold compared to the conventional and control groups, respectively. These results demonstrate that B. velezensis CE 100 is an effective biocontrol agent against anthracnose disease and a potential plant growth-promoting bacteria in walnut tree production.

This study investigated the control of Fusarium root rot and development of coastal pine (Pinus thunbergii) seedlings in a container nursery by using Bacillus licheniformis MH48. High-quality seedlings without infectious diseases cause vigorous growth. Fusarium root rot caused by Fusarium oxysporum is responsible for serious damage to coastal pine seedlings in nurseries. B. licheniformis MH48 produced enzymes that degraded the fungal cell walls, such as chitinase and β-1,3-glucanase. These lytic enzymes exhibited destructive activity toward F. oxysporum hyphae, which were found to play key roles in the suppression of root rot. In addition, B. licheniformis MH48 increased the nitrogen and phosphorus in soils via fixed atmospheric nitrogen and solubilized inorganic phosphate. B. licheniformis MH48 produced the phytohormone auxin, which stimulated seedling root development, resulting in increased nutrient uptake in seedlings. Both the bacterial inoculation and the chemical fertilizer treatments significantly increased seedling growth and biomass, and the bacterial inoculation had a greater effect on seedling development. Based on the results from this study, B. licheniformis MH48 showed potential as a biological agent against Fusarium root rot and as a promoter of growth and development of Pinus thunbergii seedlings.

The aim of this study was to investigate the antifungal activity of a cyclic tetrapeptide from Bacillus velezensis CE 100 against anthracnose-causing fungal pathogen Colletotrichum gloeosporioides. Antifungal compound produced by B. velezensis CE 100 was isolated and purified from ethyl acetate extract of B. velezensis CE 100 culture broth using octadecylsilane column chromatography. The purified compound was identified as cyclo-(prolyl-valyl-alanyl-isoleucyl) based on mass spectrometer and nuclear magnetic resonance analyses. This is the first report of the isolation of a cyclic tetrapeptide from B. velezensis CE 100 culture filtrate. Cyclic tetrapeptide displayed strong antifungal activity at concentration of 1000 µg/mL against C. gloeosporioides mycelial growth and spore germination. Our results demonstrate that the antifungal cyclic tetrapeptide from B. velezensis CE 100 has potential in bioprotection against anthracnose disease of plants caused by C. gloeosporioides.

Colletotrichum gloeosporioides is the most prevalent phytopathogen, causing anthracnose disease that severely affects the production of various fruit trees, including walnut and jujube. In this study, the volatile organic compounds (VOCs) from Bacillus velezensis CE 100 disrupted the cell membrane integrity of C. gloeosporioides and reduced the spore germination by 36.4% and mycelial growth by 20.0% at a bacterial broth concentration of 10%, while the control group showed no antifungal effect. Based on the headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis, seven VOCs were identified from the headspace of B. velezensis CE 100. Out of the seven VOCs, 5-nonylamine and 3-methylbutanoic acid were only detected in the headspace of B. velezensis CE 100 but not in the control group. Both 5-nonylamine and 3-methylbutanoic acid showed significant antifungal activity against the spore germination and mycelial growth of C. gloeosporioides. Treatment with 100 µL/mL of 5-nonylamine and 3-methylbutanoic acid suppressed the spore germination of C. gloeosporioides by 10.9% and 30.4% and reduced mycelial growth by 14.0% and 22.6%, respectively. Therefore, 5-nonylamine and 3-methylbutanoic acid are the potential antifungal VOCs emitted by B. velezensis CE 100, and this is the first report about the antifungal activity of 5-nonylamine against C. gloeosporioides.

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Subterranean termites of the species Reticulitermes speratus kyushuensis Morimoto (Isoptera: Rhinotermitidae) are notoriously destructive soil-dwelling pests that feed on the cellulosic wood biomass. This leads to tremendous losses of forest trees such as Pinus densiflora Siebold and Zucc. (Pinales: Pinaceae) and precious wooden structures of cultural heritage. This study investigated the efficacy of chitinase and protease produced by Bacillus licheniformis PR2 as cuticle-degrading enzymes for the biocontrol of worker termites. Bacillus licheniformis PR2 produced a strong chitinase and protease activity up to a maximum of 82.3 unit/mL and 35.9 unit/mL, respectively, and caused a lethal effect on termites under laboratory conditions. Treatment of termites with the bacterial broth culture and the crude enzyme fraction of B. licheniformis PR2 resulted in a maximum mortality rate (with a median lethal time (ET50)) of 83.3% (3 h, 36 min) and 88.9% (2 h, 59 min), respectively. The termites treated with B. licheniformis PR2 exhibited loss of setae, disintegration of epicuticle, rupturing of procuticle, and swelling at sockets. The degradation of cuticular chitin and glycoprotein polymers in the termite cuticle by chitinase and protease enzymes produced by B. licheniformis PR2 represents an effective eco-friendly strategy for controlling termite damage in Korean cultural heritage sites and forests.