Yeast infections such as otitis externa and seborrheic dermatitis in dogs and cats are frequently associated with Malassezia pachydermatis secondary infection. It is part of the normal cutaneous microflora of most warm-blooded vertebrates, however, under certain conditions, it can become a causative agent of infection that needs to be treated pharmacologically. Azole derivatives are the drugs of the first choice. An interesting trend in developing resistance is the use of natural substances, which include manuka honey with confirmed antimicrobial properties. The main intention of this research was to evaluate the mutual effect of manuka honey in combination with four conventional azole antifungals – clotrimazole, fluconazole, itraconazole, and miconazole – on 14 Malassezia pachydermatis isolates obtained from dogs and 1 reference strain. A slightly modified M27–A3 method (CLSI 2008) and the checkerboard test (Nikolić et al. 2017) were used for this purpose. Our results show an additive effect of all 4 antifungals with manuka honey concurrent use. Based on the determined values of fractional inhibitory concentration index (FICI – 0.74±0.03 when manuka honey combined with clotrimazole, 0.96±0.08 with fluconazole, 1.0±0 with miconazole and 1.16±0.26 with itraconazole), it was found in all cases that the effect of substances used is more pronounced in mutual combination than when used separately.

The aim of this study was to investigate interactions between conventional antifungal drug and essential oils against isolates of Malassezia pachydermatis. Antifungal activity of Cinnamomum cassia, Melaleuca alternifolia, Mentha piperita, Origanum vulgare and Syzygium aromaticum essential oils were tested against 19 strains of M. pachydermatis isolated from healthy dogs and reference strain M. pachydermatis CBS 1879. The checkerboard assay was used to search for in- teractions. Synergism was observed for the combination of clotrimazole with Melaleuca alternifolia essential oil, Mentha piperita and Origanum vulgare. The combinations of Cinnamomum cassia and Syzygium aromaticum essential oils with clotrimazole showed indifferent effect. Additive antimicrobial activity was observed for the combination of clotrimazole with Syzygium aromaticum and Melaleuca alternifolia essential oils against reference strain. The obtained results showed synergistic interactions between essential oils and clotrimazole which could improve effectiveness of this antifungal drug.

In a previous study, the endophytic Bacillus velezensis NC318 was isolated from the rhizosphere of date palm and showed strong antifungal activity against the soil-borne plant pathogenic fungus, Sclerotium rolfsii Sacc, the causal agent of Southern blight. The potential of the Bacillus genus in the inhibition of plant pathogens is mainly due to the production of certain bioactive compounds. In the present study, secondary metabolites extracted from the cell-free supernatant of strain NC318 showed strong antifungal activity on the mycelial growth and germination of S. rolfsii sclerotia in vitro. With 50 μl of bioactive compounds crude extracts, the mycelial growth inhibition rate was 97% and any germination of sclerotia was reported. Chemical analysis of the secondary metabolite crude extracts performed by high performance liquid chromatography coupled with mass spectrometry (HPLC/MS), revealed that the secreted bioactive compounds belonged to the family of lipopeptides (iturin, fengycin, surfactin), polyketides (bacillaene, macrolactin, difficidin and bacilysin) and siderophores (bacillibactin). These results provide a better understanding of the biocontrol mechanism of the bacteria strain B. velezensis NC318 against the soil fungal pathogens, especially S. rolfsii root rot.

Actinomycetes are considered to be the biggest producer of bioactive compounds which are expected to have antifungal activity for controlling many fungi such as Rhizoctonia solani. The objective of this study was to obtain potential soybean rhizosphere actinomycetes as a biocontrol agent for R. solani which cause damping-off disease both in vitro and in vivo, including their ability to produce siderophore, chitinase, and HCN. Out of 26 isolates, 18 (56%) showed diverse antifungal activities against R. solani with percentages of inhibition radial growth (PIRG) from 18.9 to 64.8%, as evaluated by a dual culture method. Ten isolates with the strongest antifungal activity were numbered for further characterization. All the tested isolates were not antagonistic towards Bradyrhizobium japonicum. These isolates were able to suppress damping-off disease caused by R. solani in the greenhouse experiment. Isolate ASR53 showed the highest disease suppression, 68% and 91% in sterile and non-sterile soil, respectively. Based on 16S rRNA sequence analysis this isolate belonged to Streptomyces violaceorubidus LMG 20319 (similarity 98.8%) according to GenBank data base available at www.ncbi.nlm.gov.nih. Furthermore, isolate ASR53 had significantly longer roots and shoots, as well as greater fresh and dry weights of seedlings than the control. Crude extract derived from ASR53 isolates contained 10 dominant compounds that were biologically active against fungal pathogens. Thus, this study suggests that the application of potential actinomycetes of the soybean rhizosphere can act as a promising biocontrol agent against damping-off disease caused by R. solani.

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is a worrisome destructive foliar disease of wheat-growing areas around the world. Streptomyces spp. have been investigated as biocontrol agents because they beneficially interact with host plants and produce important bioactive substances that can act in the suppression of diseases in plants. In the present study, antifungal activity and plant growth-promoting of Streptomyces spp. strains 6(4), R18(6), and their consortium, were evaluated through in vitro and greenhouse assays. The Ultra High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-QTOF MS) technique was used to analyze the crude extract of each strain. The results of the in vitro tests showed that the 6(4) metabolites caused several abnormalities in the conidial germination of Ptr. This strain also produced indole acetic acid (IAA) and siderophores. Strain R18(6) did not alter conidial germination of Ptr, and produced IAA and phosphate solubilizers. In the greenhouse, the treatment ‘seed inoculation plus foliar spray’ with streptomycetes propagules and metabolites contributed to biomass gain, with no statistical difference between the strains ( p < 0.05). Treatments with 6(4) ‘seed inoculation’, ‘seed inoculation plus foliar spray’, and consortium ‘seed inoculation’ showed the lowest percentage of injured area compared to other treatments ( p < 0.05). UHPLC-QTOF MS data showed that erucamide is present in the culture of 6(4), but not in the culture of R18(6). Therefore, this substance is one of those involved in Ptr hyphal abnormalities, and R18(6) use indirect mechanisms of action to control Ptr. We concluded that these Streptomyces spp. and their metabolites have a promising potential for biological control of Ptr to protect wheat plants from tan spot damage.

In the current study the antifungal activity of inorganic reagents was tested against Cryphonectria parasitica in vitro in a mycelial growth inhibition test. Three reagents, each consisting of chloride silver (AgCl) in combination with (1) aluminum oxide − Al2O3, (2) zinc oxide − ZnO, and (3) Al2O3 and titanium dioxide – TiO2, were tested. Significant differences of the tested reagents on the growth of C. parasitica were recorded. The study demonstrated that silver in mixture with ZnO had an antifungal effect and significantly reduced the mycelial growth of C. parasitica in vitro. The mixture of AgCl with the other two combinations of inorganic metal oxides had no inhibition effect on the growth of the pathogen. It was confirmed that ZnO (applied in a single compound test) is responsible for inhibition of C. parasitica mycelium growth. A preliminary in planta assay was performed but statistically significant differences were not recorded in the average increment of canker length.

This study illustrates the antifungal activity of green biosynthesis of a silver nanoparticle solution using one of Sinai’s natural plant extracts, namely Zygophyllum album which was used as a stabilizer and reducing agent to reduce Ag+ to metallic silver. In this study the plant extract was prepared by boiling in water for 10 min., 70% ethanol and wet autoclaving for 5 min. AgNPs were prepared using these three different extract methods. Transmission electron microscope (TEM) and zeta potential techniques were employed to characterize the synthesis of nanoparticles. The size of particles ranged from 6.28 nm to 28.89 nm at x100 and the zeta potential had one peak at –16.6 mean (mV) at area 100% for green synthesized AgNPs from Z. album prepared from boiling in water for 10 min. The size of particles ranged from 6.64 nm to 54.82nm at 100x and the zeta potential had one peak at – 12.9 mean (mV) at 100% area for green synthesized AgNPs from the plant ethanol extract. The size of particles ranged from 9.39 nm to 31.93 nm at 100x and the zeta potential had one peak – 19.8 mean (mV) at 100% area for green synthesized AgNPs from the wet autoclaved plant extract of Z. album for 5 min. All treatments of plant extract and AgNPs solutions, prepared from these plant extracts of Zygophyllum album, were compared with the positive control and Tachigaren – 30% W/P was conducted on the radial growth of F. oxysporium and caused antifungal activity with a high inhibition percent. There was a highly significant difference between the various extraction techniques. Increasing the concentration of treatments was accompanied with a significant effect on Fusarium wilt. Thus, this study may provide a good alternative approach to control Fusarium wilt disease in the field and under storage conditions of vegetables. Our study suggests that silver nanoparticles of plant extracts can be used for controlling Fusarium wilt.

The aim of this study was to compare various methods of anti fungal activity determination in model experiments. For the investigations Bacillus coagulans (No. 6) isolate with the high anti fungal activity, isolated from lupine composts was used. Anti fungal activity of the examined isolate was estimated against five pathogenic species of fungi: Rhizoctonia so/ani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Trichothecium roseum and Fusarium oxysporum. Determination of fungal growth was carried out by three methods: ergosterol assessment, counting colony-forming units (CFU) and agar plate diffusion assay. Statistically significant Spearman's rank coefficients were recorded between ergosterol assessment and two other methods. On the basis of this data the assessment of ergosterol is the best way ofdetermination of antifungal activity and may be used as a reference procedure.