Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.

Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.

Phytopathogenic fungal species of theDiaporthe/Phomopsiscomplex (DPC) are associated with three highly destructive diseases on soybean: seed decay, pod and stem blight, and stem canker. They are responsible for poor seed quality and significant yield reduction in most soybean-producing areas. Precise identification and classification of DPC species are important in understanding the epidemiology of disease and to develop effective control measures. Although cultural and morphological characteristics of DPC-associated pathogens have been described, establishing a more accurate taxonomic framework seems necessary for a revaluation of the taxonomy and phylogeny of DPC species. In this study, we focused on morphological and molecular analyses of species from DPC-damaged European soybean seeds obtained from several locations throughout Europe. Colony characteristics, conidia dimensions, existence of α- and β-conidia, and formation of perithecia were evaluated in order to assign the isolates to a species morphologically. Phylogenetic relationships were determined based on sequences from beta-tubulin (TUB), translation elongation factor 1-alpha (TEF1), and nuclear ribosomal DNA internal transcribed spacers (ITS). All isolates were tested for pathogenicity on soybean with positive results. In this study, we present updated taxonomic data by combining morphological observations and molecular tools which placed 32Diaportheisolates into four DPC species:D. longicolla,D. caulivora,D. eres, andD. novem, which are well-known soybean pathogens.

Diaporthe (anamorph: Phomopsis) species are endophytes or fungal pathogens for many different plant species. Soybean (Glycine max) can be infected by many different Diaporthe species; among them, D. caulivora and D. longicolla are responsible for the most significant damages. Diaporthe goulteri is a species that was only recently described and has so far been found on sunflower (Helianthus annuus) in Australia and an unknown host in Thailand. Here, we report isolation of D. goulteri from soybean in southern Germany, molecular species identification, and additional morphological description. We also show that D. goulteri can infect soybean and describe the symptoms we observed, both on the plant where the isolate came from and following artificial inoculation.

Two genetically engineered Bacillus subtilis strains, BMV9 and BsB6, were evaluated in terms of culture medium (effect of nutrients on surfactin yield) and potential biotechnological applications of surfactin in agriculture and the petrochemical industry. BMV9 (spo0A3; abrB*; ΔmanPA; sfp+) is, to date, the highest surfactin producer reported scientifically, and BsB6 is a sfp+ laboratory derivative strain that has also demonstrated considerable production potential. To assess their performance, fermentation experiments were conducted in shake flasks using two different culture media, a mineral salt medium and a complex medium, each supplemented with 2% (w/v) glucose. Lipopeptides (surfactin and fengycin) were extracted and quantified at multiple time points (up to 48 h) via high-performance thin-layer chromatography (HPTLC). Optical density, residual glucose, and pH were monitored throughout the cultivation. In parallel, microbial growth in both media were also validated in small-scale cultivation approaches. Antifungal activity of culture supernatants and lipopeptide extracts was tested against two Diaporthe species, key phytopathogens in soybean crops. Given the agricultural relevance of these pathogens, the biocontrol potential of lipopeptides represents a sustainable alternative to conventional chemical fungicides. Additionally, oil displacement tests were performed to evaluate the efficacy of surfactin in enhanced oil recovery (EOR), bioremediation, and related petrochemical processes. High-resolution LC-MS/MS analysis enabled structural characterization and relative quantification of the lipopeptides. Overall, these investigations provide a comprehensive comparison of strain production performance and the associated impact of cultivation media, aiming to define the optimal conditions for economically viable surfactin production and to explore its broader biotechnological applications in agriculture and the petrochemical industry.

Species of the genus Diaporthe have a mating-type system with the two mating types MAT1-1 and MAT1-2, like other ascomycetes. They can either be heterothallic, which means that any isolate only possesses one of the two mating types and needs a mating partner for sexual reproduction, or homothallic, which means that they possess both mating types and are self-fertile. For several Diaporthe species, no sexual reproduction has been observed so far. Using PCR with primers specific to the defining genes MAT1-1-1 and MAT1-2-1, we determined the mating types of 33 isolates of Diaporthe caulivora, D. eres, D. longicolla, and D. novem from central Europe. In addition, we partially sequenced the mating-type genes of 25 isolates. We found that different D. longicolla isolates either possess MAT1-1-1 or MAT1-2-1, making the species heterothallic, which is in contrast to previous studies and the general assumption that D. longicolla only reproduces asexually. D. eres and D. novem were also found to be heterothallic. Using genomic sequence information and re-sequencing of DNA and RNA, we identified the MAT1-1-1 gene in D. caulivora and present here the full sequence of the mating-type locus of this homothallic species. Finally, we used sequence information from MAT1-1-1 and MAT1-2-1, respectively, for improved phylogenetic resolution of our isolates.