Genetic Characteristics and Enzymatic Activities of <i>Bacillus velezensis</i> KS04AU as a Stable Biocontrol Agent against Phytopathogens

Bacillus velezensis has a broad application in the agricultural and industrial sectors for its biocontrol properties and its potential active secondary metabolites. The defined phenotypic characteristics of a strain vary according to its ecosystem. We report the complete genomic analysis of B. velezensis KS04AU compared to four strains of B. velezensis (SRCM102752, ONU-553, FZB42, and JS25R) and two closely related Bacillus amyloliquefaciens (LL3 and IT-45). A total of 4771 protein coding genes comprises the KS04AU genome, in comparison with 3334 genes core genes found in the six other strains and the remaining 1437 shell genes. Average nucleotide identity of the target strain to the six other strains showed 99.65% to B. velezensis ONU-553, sharing 60 orthologous genes. Secondary metabolite gene cluster analysis of all strains showed that KS04AU has a mersacidin cluster gene, which is absent in the genome of the other strains. PHASTER analysis also showed KS04AU harboring two phages (Aeribacllus AP45 NC_048651 and Paenibacillus_Tripp NC_028930), which were also unique in comparison with the other strains. Analysis on anti-microbial resistance genes showed no difference in the genome of KS04AU to any of the other genomes, with the exception of B. amyloliquefaciens IT-45 which had one unique small multidrug-resistance antibiotic efflux-pump gene (qacJ). The CRISPR-Cas systems in the strains were also compared showing one CRISPR gene found only in KS04AU. Hydrolytic activity, antagonistic activity against phytopathogens (Fusarium oxysporum, Fusarium graminearum, Alternaria alternata and Pseudomonas syringae) and biocontrol against tomato foot and root rot experiments were carried out. B. velezensis KS04AU inhibits the growth of all phytopathogens tested, produces hydrolytic activity, and reduces Fusarium oxysporum f.sp. radicis-lycopersici (Forl) ZUM2407 lesions up to 46.02 ± 0.12%. The obtained results confirm B. velezensis KS04AU as a potential biocontrol strain for plant protection

Identification and Aggressiveness of <i>Fusarium</i> Species Associated with Onion Bulb (<i>Allium cepa</i> L.) during Storage

Plant pathogens present a major challenge to crop production, leading to decreased yield and quality during growth and storage. During long-term storage, healthy onions can develop diseases from latent pathogen infections. This poses a challenge for onion growers because infected bulbs without visible symptoms can lead to significant crop losses during the growing season. In this study, we aimed to isolate and identify Fusarium species from yellow onion bulbs (Allium cepa L.) that developed disease symptoms during storage. The aggressiveness of these strains against onion bulbs and seedlings was also evaluated. The isolated strains were further subjected to morphological and molecular differentiation. The results revealed that all 16 isolated strains belonged to the Fusarium complex species incarnatum-equiseti and Fusarium fujikuroi, namely, F. proliferatum (98%), F. oxysporum (1%), and Fusarium sp. (1%). Koch’s postulate analysis of isolated strains revealed varying aggressiveness on onion bulbs and plants depending on fungal species. Disease symptoms developed more slowly on plants than on onion bulb plants according to Koch’s postulates. Moreover, the results revealed that Fusarium strains that can infect onion plants were less pathogenic to onion bulbs and vice versa. In addition, three isolates were found to be non-pathogenic to onions. Furthermore, the in vitro control of Fusarium species through Bacillus velezensis KS04-AU and Streptomyces albidoflavus MGMM6 showed high potential for controlling the growth of these pathogenic fungi. These results may contribute to the development of environmentally friendly approaches for controlling onion spoilage caused by pathogens during storage

Persistence as a Constituent of a Biocontrol Mechanism (Competition for Nutrients and Niches) in <i>Pseudomonas putida</i> PCL1760

Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by Pseudomonas strains. Phenotypic and full genome analysis revealed Pseudomonas putida PCL1760 controlling tomato foot and root rot (TFRR) solely through CNN mechanism. Although the availability of nutrients and motility are the known conditions for CNN, persistence of bacteria through dormancy by ribosomal hibernation is a key phenomenon to evade both biotic and abiotic stress. To confirm this hypothesis, rsfS gene knockout mutant of PCL1760 (SB9) was first obtained through genetic constructions and compared with the wild type PCL1760. Primarily, relative expression of rsfS in PCL1760 was conducted on tomato seedlings which showed a higher expression at the apical part (1.02 ± 0.18) of the plant roots than the basal (0.41 ± 0.13). The growth curve and persistence in ceftriaxone after the induction of starvation with rifampicin were performed on both strains. Colonization on the tomato root by CFU and qPCR, including biocontrol ability against Fusarium, was also tested. The growth dynamics of both PCL1760 and SB9 in basal and rich medium statistically did not differ (p ≤ 0.05). There was a significant difference observed in persistence showing PCL1760 to be more persistent than its mutant SB9, while SB9 (pJeM2:rsfS) was 221.07 folds more than PCL1760. In colonization and biocontrol ability tests, PCL1760 was dominant over SB9 colonizing and controlling TFRR (in total, 3.044 × 104 to 6.95 × 103 fg/µL and 55.28% to 30.24%, respectively). The deletion of the rsfS gene in PCL1760 reduced the persistence and effectiveness of the strain, suggesting persistence as one important characteristic of the CNN