Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Butyl succinate-mediated control of Bacillus velezensis ce 100 for apple anthracnose caused by Colletotrichum gloeosporioides.

AIMS: Microbial biocontrol agents have become an effective option to mitigate the harmfulness of chemical pesticides in recent years. This study demonstrates the control efficacy of Bacillus velezensis CE 100 on the anthracnose causal agent, Colletotrichum gloeosporioides. METHODS AND RESULTS: In vitro antifungal assays revealed that the culture filtrate and volatile organic compounds of B. velezensis CE 100 strongly restricted the mycelial development of C. gloeosporioides. Moreover, a bioactive compound, butyl succinate, was isolated from the n-butanol crude extract of B. velezensis CE 100 (bce), and identified by liquid chromatography-electrospray ionization hybrid ion-trap and time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR). Treatment with purified butyl succinate at a concentration of 300 µg mL-1 strongly controlled conidial germination of C. gloeosporioides with an inhibition rate of 98.66%, whereas butyl succinate at a concentration of 400 µg mL-1 showed weak antifungal action on the mycelial growth of C. gloeosporioides with an inhibition rate of 31.25%. Scanning electron microscopy revealed that the morphologies of butyl succinate-treated hyphae and conidia of C. gloeosporioides were severely deformed with shriveled and wrinkled surfaces. Furthermore, butyl succinate was able to control carbendazim-resistant C. gloeosporioides, demonstrating that it could be a promising agent for the suppression of other carbendazim-resistant fungal pathogens. An in vivo biocontrol assay demonstrated that the strain ce 100 broth culture and butyl succinate showed higher control efficacy on apple anthracnose than bce. CONCLUSIONS: Our findings provide insight into the antifungal potential of B. velezensis  ce 100 and its butyl succinate for efficient control of phytopathogenic fungi, such as C. gloeosporiodes, in plant disease protection. This is the first study to demonstrate the antifungal potential of bacteria-derived butyl succinate for control of C. gloeosporioides.

Antifungal Potential of Bacillus velezensis CE 100 for the Control of Different Colletotrichum Species through Isolation of Active Dipeptide, Cyclo-(D-phenylalanyl-D-prolyl).

Colletotrichum species are important fungal pathogens causing anthracnose of tropical and subtropical fruit and vegetable crops. Dual culture assay indicated that Bacillus velezensis CE 100 was a strong antagonist against C. acutatum, C. coccodes, C. dematium, and C. gloeosporioides. The volatile organic compounds produced by B. velezensis CE 100 affected mycelial growth of Colletotrichum species tested in our study and caused twisted hyphal structures of all these fungal species. Chloroform crude compounds of B. velezensis CE 100 inhibited four Colletotrichum species in a concentration-dependent manner and induced severe damage in hyphal morphology of these fungal pathogens, including swelling, bulging, and multiple branching. Moreover, the active cyclic dipeptide, cyclo-(D-phenylalanyl-D-prolyl), was isolated from chloroform crude extract and identified by nuclear magnetic resonance (NMR) and mass spectrometry. The inhibitory effect of cyclo-(D-phenylalanyl-D-prolyl) on conidial germination of C. gloeosporioides occurred in a concentration-dependent manner. The conidial germination rate was completely inhibited by a concentration of 3 mg/mL of cyclo-(D-phenylalanyl-D-prolyl). Scanning electron micrographs revealed that the exposure to cyclic dipeptide resulted in seriously deformed hyphae and conidia with shriveled surfaces in dipeptide-treated C. gloeosporioides. Therefore, active dipeptide-producing B. velezensis CE 100 is a promising biocontrol agent for Colletotrichum species causing anthracnose.