4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.

Exploring the Diversity and Aromatic Hydrocarbon Degrading Potential of Epiphytic Fungi on Hornbeams from Chronically Polluted Areas

Plants can ‘catch’ and mitigate airborne pollutants and are assisted by fungi inhabiting their leaves. The structure and function of the fungal communities inhabiting the phyllosphere of hornbeam trees growing in two chronically polluted areas, the oilfield of Bóbrka and the city center of Warsaw, were compared to the ones growing in one nature reserve, the Białowieża National Park. Fungi were isolated and characterized both phylogenetically and functionally for their potential role in air pollution mitigation. Both culture-dependent (e.g., enzyme assays and tolerance tests) and culture-independent methods (e.g., ITS and shotgun sequencings) were used. Furthermore, the degradation potential of the fungi was assessed by gas chromatography mass spectrometry (GC-MS). Shotgun sequencing showed that the phyllosphere fungal communities were dominated by fungi belonging to the phylum Ascomycota. Aureobasidium was the only genus detected at the three locations with a relative abundance ≥1.0%. Among the cultivated epiphytic fungi from Bóbrka, Fusarium sporotrichioides AT11, Phoma herbarum AT15, and Lophiostoma sp. AT37 showed in vitro aromatic hydrocarbon degradation potential with laccase activities of 1.24, 3.62, and 7.2 μU L−1, respectively, and peroxidase enzymes with activities of 3.46, 2.28, and 7.49 μU L−1, respectively. Furthermore, Fusarium sporotrichioides AT11 and Phoma herbarum AT15 tolerated exposure to airborne naphthalene and benzene. Lophiostoma sp. AT37 was the most tolerant to exposure to these pollutants, in line with being the best potential aromatic hydrocarbon degrader isolated in this study.

BIODIVERSITY OF MOLD FUNGI IN THE AREAS OF THE CITY OF CHEBOKSARY

A study of 12 soil samples in the summer period in the districts of the city of Cheboksary was conducted. The aim of the research was to conduct a mycological analysis of the soil and isolate particularly dangerous micromycetes in the urban environment. Soil samples were taken from the Moskovsky, Kalininsky, Leninsky districts of Cheboksary and Marposadsky highway. The pH of the soil environment of the city of Cheboksary was: Embankment of the Moskovsky district – 7.67, Kirovsky – 7.23; Leninsky – 7.28; Marposadskoe highway – 7.51. In mycological analysis of soil samples on agarized media of Chapek, the highest concentration of micromycetes was recorded in soil samples from the Moskovsky district on the Embankment, where the concentration of fungi of the genus Aspergillus fumigates was 27 %, fungi of the genus Fusarium sporotrichioides and Fusarium graminearum was 15 %. The soil of the Leninsky district was dominated by fungi of the genus Penicillium, their concentration was 23 %, Aspergillus flavus – 18 %. Isolates of fungi of the genus Penicillium spp. were isolated in the soil of the Kalininsky district, and their concentration was – 7 %, Aspergillus flavus – 21 %. The soil from the Marpasad highway was dominated by fungi of the genus Mucor sp. Testing on Paramecium caudatum infusoria showed that the field isolate of Aspergillus fumigates had toxic properties, the evaluation criterion for biotesting was 38 %. Aspergillus flavus showed a 78 % survival rate when tested on infusoria.

First Report of Sea buckthorn Stem Wilt Caused by Fusarium sporotrichioides in Gansu, China

Sea buckthorn (Hippophae rhamnoides) is an important deciduous shrub for fruit and ecological restoration in arid and semi-arid regions of China. Twelve Chinese and Russian cultivars (cv. Shenqiuhong, eshi01, ... eshi11) were planted about 1.6 acre area in a seedling nursery, located in Qingyang City of Gansu province in northwest China, where high mortality (more than 70%) of sea buckthorn was observed in late July 2019. Symptoms consisted of massive chlorosis, drooping leaves and dried-up stems on 5-year-old trees. Pieces of tree roots and stems with irregular light-brown discoloration in the xylem vessels were selected. Small pieces of discolored tissue were surface disinfested (1 min in 1% sodium hypochlorite, followed by three rinses with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues. Colonies on PDA grew rapidly. Dense mycelia were pinky-white initially, and became carmine red color with age on the undersurface of the plate. Macroconidia were moderately curved, 3 to 5 marked septa, hyaline, thick walled, and measuring 27.8± 3.6 µm × 4.8 ± 0.5 µm (n = 30). Microconidia were abundant, pear-shaped, ellipsoid to fusoid, often with a papilla at the base, and 8.4 ± 2.2 µm ×3.1 ± 0.3 µm (n = 30). Genomic DNA was extracted for amplification and sequencing of the internal transcribed spacer region (ITS1 and ITS4 primers) (White et al. 1990) of the ribosomal DNA (Accession Nos. MN160235 to MN160238) and translation elongation factor-1 alpha (EF1 and EF2 primers, accession Nos. MN429075 to MN429078) (O’Donnell et al. 1998). The sequences revealed 99% similarity to the sequences of the ITS (AY188917), and 100% identity with EF1-α (JF740808) regions of Fusarium sporotrichioides. Based on morphological and molecular characteristics, the fungus was identified as F. sporotrichioides (Leslie and Summerell 2006). Koch’s postulates were fulfilled on healthy, potted 1-year-old sea buckthorn seedings using two isolates in a greenhouse at 25 °C, 90% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedings were inoculated with PDA plugs as controls. Seven to ten days after inoculation, typical symptoms of dark-brown necrotic lesions on chlorotic leaf margins were observed. About 2 weeks after inoculation, the inoculated stems were gradually dry up, accompanied by withering and fallen leaves. Control plants remained asymptomatic. Pathogens were successfully isolated from the inoculated stems again, exhibiting morphological characteristics identical to those of F. sporotrichioides. Previous papers reported F. sporotrichioides as a common pathogen caused lavender wilt (Cosic et al. 2012), foliar spots on forage corn (Moya-Elizondo et al. 2013) and maize ear rot (Wang et al. 2019). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. sporotrichioides on several Chinese and Russian cultivars in Gansu province of China. In Heilongjiang province, the same disease was reported in 2010 (Song et al. 2010), nearly 30 longitudes away from Gansu province. Therefore, this disease appears to be a serious risk for future sea buckthorn production.

Antagonistic activity of dominant strains of bacteria of spring barley rhizosphere against phytopathogenic micromycetes

The purpose of the work. The aim of the study was to conduct research on the manifestation of the dominant representatives of the bacterial biome of the rhizosphere of spring barley antagonistic activity against phytopathogenic micromycetes. Research methods. The standard diffusion method of double culture in Petri dishes was used to study the antagonistic properties of the dominant strains of rhizosphere bacteria. The level of antagonistic activity of microorganisms was assessed by the indicator (%) of inhibition of growth and development of the mycelium of micromycetes Fusarium sporotrichioides Sherb. 23.2, Alternaria alternata (Fr.) Keissl. 3.45, Nigrospora oryzae (Berk. & Broome) Petch. 18.77. Results and scope of their application. Studies have shown that Bacillus methylotrophicus 10 had an inhibitory effect on Fusarium sporotrichioides Sherb. 23.2 ‒ 77,4%, Alternaria alternata (Fr.) Keissl. 3.45 – 66,6% and Nigrospora oryzae (Berk. & Broome) Petch. 18.77 – 86,7%, while the bacteria Phyllobacterium ifriqiyense 1 showed activity against phytopathogens by 45,1%, 63,1% and 65,0%, respectively. Conclusions. Thus, both strains of rhizosphere dominant bacteria have high antagonistic activity against phytopathogenic micromycetes, which gives grounds for their further study.

Distribution of T-2 toxin and HT-2 toxin during experimental feeding of yellow mealworm (Tenebrio molitor)

Within the European Union (EU), edible insects need to be approved as “Novel Food” according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 μg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.

In vitro screening of fungicides and plant extracts against six pathogenic fungi isolated from cotton (Gossypium arboreum L.) seed

Six pathogenic fungi, namely Aspergillus flavus Link., A. niger van Tieghem (Type-I)., Curvularia lunata (Wakker) Boedijn, Fusarium moniliforme var. subglutinans Wr. & Reink., Fusarium sporotrichioides Sherb., Mem. and Rhizoctonia solani J.G. Kuhn were isolated from cotton seeds. Five fungicides viz., Acrobat MZ, Autostin 50 WDG, Capvit 50 WP, Nativo 75 WP and Thiovit 80 WG were selected to evaluate in vitro efficacy at 100, 200, 300, 400 and 500 ppm concentrations against pathogenic fungi following poisoned food technique. Out of 5 fungicides Nativo 75 WP showed the complete growth inhibition of above mentioned six pathogenic fungi at all the used concentrations. Autostin 50 WDG showed complete growth inhibition of all tested pathogenic fungi except Curvularia lunata. Leaf extract of five angiospermic plants viz., Adhatoda vasica, Aegle mermelos, Azadirachta indica, Datura metel and Psidium guajava were selected to evaluate in vitro fungitoxicity at 5, 10, 15 and 20% concentrations against the test pathogens. At 20% concentration, out of the 5 plants extracts A. indica was found to be most active to inhibit the growth of Aspergillus niger (Type-I) (65.56%) and Fusarium moniliforme var. subglutinans (75.00%), Psidium guajava were most active against A. flavus (81.29%) and Curvularia lunata (72.23%), and Datura metel was most active against Fusarium sporotrichioides (64.77%) and Rhizoctonia solani (42.44%).

Species Diversity of Micromycetes Associated with Epipactis helleborine and Epipactis purpurata (Orchidaceae, Neottieae) in Southwestern Poland

The Orchidaceae family is a diverse family of flowering plants that occur naturally in most parts of the world. However, fungal communities inhabiting different parts of orchids are not sufficiently described. The aim of the study was to conduct a mycological evaluation of Epipactis helleborine and E. purpurata (Orchidaceae), which grow naturally in Lower Silesia (SW Poland), by identifying the species composition of the culturable micromycetes fungi on the surfaces of the plants and from the inner layers of the tissues. Fungi were identified based on a phenotypic and genotypic analysis. To our knowledge, this is the first such analysis. This study showed that more species of micromycetes were cultured from E. helleborine compared with E. purpurata. The flowering plants of E. helleborine were inhabited by the largest number of culturable fungal species (13 species), and the fewest species were isolated from the flowering plants of E. purpurata (eight species). Some of these fungal species may be pathogens of the plants. The surface tissues of the orchids were mainly inhabited by Mucor moelleri and/or Penicillium biourgeianum. The inner layers of these plants were the most colonized by Alternaria tenuissima and/or Arthrinium arundinis and/or Fusarium sporotrichioides. The relative dominance of these fungal species depended mainly on the development phase of the plants.