The Prospect of Hydrolytic Enzymes from Bacillus Species in the Biological Control of Pests and Diseases in Forest and Fruit Tree Production.

Plant diseases and insect pest damage cause tremendous losses in forestry and fruit tree production. Even though chemical pesticides have been effective in the control of plant diseases and insect pests for several decades, they are increasingly becoming undesirable due to their toxic residues that affect human life, animals, and the environment, as well as the growing challenge of pesticide resistance. In this study, we review the potential of hydrolytic enzymes from Bacillus species such as chitinases, ß-1,3-glucanases, proteases, lipases, amylases, and cellulases in the biological control of phytopathogens and insect pests, which could be a more sustainable alternative to chemical pesticides. This study highlights the application potential of the hydrolytic enzymes from different Bacillus sp. as effective biocontrol alternatives against phytopathogens/insect pests through the degradation of cell wall/insect cuticles, which are mainly composed of structural polysaccharides like chitins, ß-glucans, glycoproteins, and lipids. This study demonstrates the prospects for applying hydrolytic enzymes from Bacillus sp. as effective biopesticides in forest and fruit tree production, their mode of biocidal activity and dual antimicrobial/insecticidal potential, which indicates a great prospect for the simultaneous biocontrol of pests/diseases. Further research should focus on optimizing the production of hydrolytic enzymes, and the antimicrobial/insecticidal synergism of different Bacillus sp. which could facilitate the simultaneous biocontrol of pests and diseases in forest and fruit tree production.

Entomopathogenic Potential of Bacillus velezensis CE 100 for the Biological Control of Termite Damage in Wooden Architectural Buildings of Korean Cultural Heritage.

Biocontrol strategies are gaining tremendous attention in insect pest management, such as controlling termite damage, due to the growing awareness of the irreparable harm caused by the continuous use of synthetic pesticides. This study examines the proteolytic and chitinolytic activities of Bacillus velezensis CE 100 and its termiticidal effect through cuticle degradation. The proteolytic and chitinolytic activities of B. velezensis CE 100 systematically increased with cell growth to the respective peaks of 68.3 and 128.3 units/mL after seven days of inoculation, corresponding with the highest cell growth of 16 × 107 colony-forming units (CFU)/mL. The in vitro termiticidal assay showed that B. velezensis CE 100 caused a rapid and high rate of termite mortality, with a median lethal time (LT50) of >1 h and the highest mortality rates of 91.1% and 92.2% recorded at 11 h and 12 h in the bacterial broth culture and crude enzyme fraction, respectively. In addition to broken setae and deformed sockets, termites treated with the bacterial broth culture exhibited degraded epicuticles, while the crude enzyme fraction caused severe disintegration of both the epicuticle and endocuticle. These results indicate the tremendously higher potential of B. velezensis CE 100 in the biological control of subterranean termites compared to the previously used entomopathogenic bacteria.

Control of the bacterial soft rot pathogen, Pectobacterium carotovorum by Bacillus velezensis CE 100 in cucumber.

Pectobacterium carotovorum is a problematic bacterial pathogen causing soft rot in different vegetable crops, resulting in yield losses during pre- and post-harvest periods. In this study, Bacillus velezensis CE 100 showed antibacterial activity against P. carotovorum. Co-inoculation experiment indicated that B. velezensis CE 100 reduced the proliferation rate of P. carotovorum at the early incubation period and that a long incubation time induced a loss of viability of the bacterial pathogen. Agar well diffusion assay revealed that the culture filtrate of strain CE 100 affected the growth of P. carotovorum in a dose-dependent pattern. In time-kill assay, inoculation of P. carotovorum with 50% culture filtrate of strain CE 100 resulted in a complete loss of survival at 4 h incubation period. An antibacterial compound isolated from chloroform extract of B. velezensis CE 100 was identified as macrolactin A based on results of 1H and 13C NMR and mass spectrometry. However, time-kill assay showed that purified macrolactin A at a concentration of 200 µgmL-1 was not highly effective to control the growth of P. carotovorum although reduction in cell number of P. carotovorum was observed. Moreover, in vivo assay revealed that B. velezensis CE 100 effectively controlled bacterial soft rot. As a consequence, it significantly improved cucumber growth. Therefore, B. velezensis CE 100 could be used as an eco-friendly bioagent for effective control of bacterial soft rot to minimize global economic losses in crop production.

Antifungal Activity of Bacillus velezensis CE 100 against Anthracnose Disease (Colletotrichum gloeosporioides) and Growth Promotion of Walnut (Juglans regia L.) Trees.

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 ß-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.

Antifungal Activity of Cyclic Tetrapeptide from Bacillus velezensis CE 100 against Plant Pathogen Colletotrichum gloeosporioides.

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.