In Vitro Evaluation of Some Endophytic Bacillus to Potentially Inhibit Grape and Grapevine Fungal Pathogens.

Romania has a long history of grapevine culturing and winemaking. However, like any agricultural sector, viticulture faces devastating biological threats. Fungi responsible for grapevine trunk diseases (GTDs) and grape spoilage lead to considerable yield losses and a decline in grapevine quality. In the actual context, many countries, including Romania, have reoriented their approaches to minimize chemical inputs, which have been proven to be toxic and to have negative impacts on the environment, and to replace them with sustainable biocontrol strategies for the wine-growing sector. Within biocontrol strategies, Bacillus spp. is a well-known plant-protective bacteria with antifungal properties. Within this paper, six endophytic bacteria from various plant sources were studied. The bacterial strains were identified as B. pumilus, B. subtilis, and B. velezensis by sequencing their 16S rDNA region. Regardless of the in vitro test methods (using living bacterial cells, bacterial-cell-free supernatant (CFS), and volatile active compounds (VOCs)), B. velezensis strains revealed strong and broad antifungal activity against grape and grapevine fungal pathogens such as Aspergillus spp., Botrytis cinerea, Penicillium expansum, Diplodia seriata, Eutypa lata, Fusarium spp., Clonostachys rosea, Neofusicoccum parvum, and Stereum hirsutum. The functional antifungal genes encoding for difficidin, fengycin, iturins, macrolactin, and mycosubtilin were molecularly detected, which could support the proven antifungal activity of the endophytic strains. Lytic enzymes involved in fungal growth inhibition, such as chitinase, cellulase, and proteases, were also revealed to be produced by some of these bacterial strains. Various other in vitro tests, such as phosphate and phytate solubilization, phytohormone synthesis, the production of enzymes involved in the polyamine biosynthetic pathway, and pH as well as temperature tolerance tests were carried out to reveal the plant-beneficial potential of these bacterial strains. These results revealed that the B. velezensis strains, especially BAHs1, are the most suitable endophytes for grapevine biologic control, which could lead to the future development of sustainable management strategies.

Genetic Polymorphism at 15 Codons of the Prion Protein Gene in 156 Goats from Romania.

BACKGROUND: The variability of prion protein gene (PRNP) codons and the frequency of alleles (K222, D146, and S146) that appear to confer genetic resistance to classical scrapie are still unknown in several goat populations/breeds prevalent in Romania. This work aims to assess the genetic polymorphism at 15 PRNP codons in Romanian goat populations to inform the development of goat breeding programs for scrapie resistance. METHODS: Whole blood and hair follicles from Carpathian (50), French Alpine (53), and Banat's White (53) breed goats were sampled to extract genomic DNA for genetic analyses and Sanger sequencing. In the targeted goat groups, one classical scrapie-positive Banat's White goat was included. RESULTS: The codons without polymorphisms were G37G, W102W, N146N, R151R, S173S, and I218I. The following non-synonymous polymorphisms of PRNP were recorded: P110P, P110S, P110T, T110T, G127G, G127S, I142I, I142M, T142I, H143H, P143P, R143R, R154R, H154R, P168P, Q168Q, Q211Q, Q211R, Q222Q, H222Q, K222K, S240S, P240P, P240S, and S240P. CONCLUSIONS: PRNP polymorphism was recorded in 60% (9/15) of codons. The scrapie-positive Banat's White goat had G37G, W102W, T110T, G127G, I142I, H143H, N146N, R151R, R154R, P168P, S173S, R211R, I218I, Q222Q, and S240S. The K222 allele had a frequency of 6% (3/50) in Carpathian, 9.43% (5/53) in Banat's White, and 15.09% (8/53) in French Alpine. Therefore, the polymorphisms detected in this sample of Romanian goat breeds are too rare to design a breeding program at the current time.

The Biotechnological Potential of Pediococcus spp. Isolated from Kombucha Microbial Consortium.

In the past decade, the probiotic market has grown rapidly, both for foods and supplements intended to enhance wellness in healthy individuals. Different lactic acid bacteria (LAB), especially Lactobacillus spp., of different origins have already been used to develop commercial probiotic products. Nowadays, LAB new alternative sources, such as non-dairy fermented food products, are being exploited. One such source is Kombucha, a fermented low-alcohol beverage made of tea leaves. In this regard, we tested seven Pediococcus spp. strains isolated from a local industrial Kombucha for their biotechnological potential. Two, out of the seven isolates, identified as Pediococcus pentosaceus (L3) and Pediococcus acidiliactici (L5), were selected as successful candidates for the food industry, due to their probiotic and technological properties. In regard to their resistance in the gastro-intestinal tract, both selected strains were tolerant to a pH of 3.5, presence of 0.3% pepsin, and 0.5% bile salt concentration. On the antagonistic side, the fresh suspension of selected isolates had high inhibitory activity against pathogenic bacteria, such as Salmonella enterica Typhimurium, Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Proteus hauseri, and methicillin resistant Staphylococcus aureus. In addition, moderate to high inhibitory activity was noticed against foodborne molds (e.g., Penicillium expansum and Penicillium digitatum). These safety issues were supported by their negative hemolytic activity and good antioxidant potential (56-58%). Selected isolates were sensitive to ampicillin, penicillin, erythromycin, and lincomycin, while a broad range of other antibiotics were not effective inhibitors. On the technological side, both strains tolerated 5% NaCl and, during the freeze-drying process, had a good survival rate (86-92%). The selected Pediococcus strains have proven properties to be used for further development of functional products.

Bee Collected Pollen with Enhanced Health Benefits, Produced by Fermentation with a Kombucha Consortium.

The bioavailability of pollen bioactive compounds for humans is limited. In this study, our aim was to enhance the health-related benefits of pollen by fermentation with a Kombucha/SCOBY (symbiotic culture of bacteria and yeasts) consortium. We performed the fermentation of pollen suspended from the beginning with SCOBY on sweetened green tea or on Kombucha vinegar, by adding pollen after 20 days of Kombucha fermentation. We analyzed: formation of bioactive compounds (anti-oxidant polyphenols, soluble silicon, hydroxy-acids, short chain fatty acids-SCFA); parameters related to Kombucha fermentation (dynamics of lactic acid bacteria-LAB, formation of organic acids, soluble sugar evolution on Kombucha vinegar); the influence of Kombucha fermentation on pollen morphology and ultrastructure; in vitro cytotoxic and antitumoral effects of the Kombucha fermented pollen. The pollen addition increases LAB proportion in the total number of SCOBY microbial strains. SEM images highlight the adhesion of the SCOBY bacteria to pollen. Ultrastructural analysis reveals the release of the pollen content. The content of bioactive compounds (polyphenols, soluble silicon species and SCFA) is higher in the fermented pollen and the product shows a moderate antitumoral effect on Caco-2 cells. The health benefits of pollen are enhanced by fermentation with a Kombucha consortium.