Identification and Pathogenicity of Fusarium Isolated from Soybean in Poland.

Fungi belonging to the Fusarium genus are commonly isolated from soybean plants and seeds but not all of them are pathogenic. The aim of this study was to compare the pathogenicity among different Fusarium isolates obtained from soybean plants with disease symptoms originating from an experimental field located in the southeast of Poland. Nineteen fungal isolates were selected for the pathogenicity assay, including eight isolates of F. oxysporum, six isolates of F. graminearum, four isolates of F. culmorum and one isolate of F. redolens. Species identification of these isolates was carried out using microscopic methods and sequencing of two genes: translation elongation factor 1-alpha (TEF1) and RNA polymerase second largest subunit (RPB2). To our knowledge, this is the first report of F. redolens being isolated from soybean in Europe. The pathogenicity test was set up by fungal inoculation of healthy soybean seeds of three cultivars: Abelina, Atlanta and Mavka. Symptoms were assessed seven days after inoculation. Disease area percentage of Fusarium inoculated seeds was significantly higher compared to uninoculated control. Nineteen isolates differed in their aggressiveness as the median disease area percentage ranged between 5.0 and 88.0% depending on isolate. The obtained isolates of four Fusarium species may be used in the future screening of soybean cultivars for resistance to these pathogens.

First report of Sclerotinia sclerotiorum causing Sclerotinia wilt of hop (Humulus lupulus) in Poland.

The common hop (Humulus lupulus L., Cannabaceae) is a perennial plant cultivated in the temperate climate zone and used in the brewing and pharmaceutical industry. In June 2021, symptoms of wilting and subsequent drying of shoots were observed on hop plants (cv. Lubelski) in Lubelskie Province, in Poland (50°55'30.5"N, 22°10'35.4"E). Wilted shoots showed no symptoms of chlorosis. Usually both healthy and wilted shoots were present on the same plant. Approx. 20% of the plants in the 2 hectare hop garden showed these symptoms. An inspection of the underground parts of the infected shoots revealed a brown necrosis of the tissues partly covered by white mycelium with black sclerotia. These symptoms were characteristic for Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006), but this pathogen has never been detected in hop gardens in Poland before. In order to confirm the preliminary diagnosis, shoots from five different plants with symptoms of necrosis were collected, disinfected with 1% sodium hypochlorite for 1 min, rinsed with sterile water and dried. Twelve fragments from partially necrotized tissue were placed on potato dextrose agar (PDA) amended with chlortetracycline hydrochloride. After three days of incubation at room temperature and daylight, 75% of the explants had white or light grey mycelia present. Pure cultures were obtained by transplanting mycelium to fresh PDA. Within 4 days, the mycelium has reached the diameter of Petri dishes (90 mm). On PDA at room temperature and daylight, black spherical or cylindrical sclerotia formed after 6-14 days. Sclerotial size was 1.0 to 10.0 x 1.0 to 5.0 mm (average 2.8 x 2.0 mm; n = 57 from 3 plates). Five isolates were subjected to DNA extraction. Then ITS region was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). The sequences obtained from the five fungal isolates were identical with each other and BLAST analysis revealed also 100% identity with the GenBank records of S. sclerotiorum (e.g. KT224645, Baturo-Ciesniewska et al. 2017). One representative sequence of isolate Ss5HL was deposited to GenBank (OQ998981). Pathogenicity of the isolate Ss5HL was confirmed in inoculation tests on approx. 30 cm high, young shoots of hop plants (cv. Lubelski) grown from rootstock for 4 weeks in the greenhouse. Five plants were inoculated by placing mycelial discs from a 7-day-old culture on shoots and covering them with wet sterilized cotton pads and aluminum foil. Two control plants were treated in the same way but instead of mycelial discs pure agar discs were placed on the shoots. Then plants were kept in a climate chamber (80% relative humidity, 15-h light at 22°C, 9-h dark at 18°C). All fungal inoculated shoots wilted within 3-4 days, then necrosis developed and spread from the place of inoculation up the shoot. No tissue necrosis was observed below the place of inoculation. The control plants remained asymptomatic. The inoculation test was carried out twice with the same result. Fungal cultures reisolated from the inoculated shoots were morphologically identical with the culture used as inoculum. Isolate Ss5HL was deposited in the collection of the Westerdijk Fungal Biodiversity Institute (CBS 150077).To our knowledge, this is the first case of Sclerotinia wilt of hops in Poland and also in Europe, with the exception of one, nearly 100 years old report from the United Kingdom (Salmon and Ware 1936). S. sclerotiorum rarely occurs on hop but it can cause severe yield reduction if pathogen accumulates in the soil.

Effects of Silybum marianum L. Seed Extracts on Multi Drug Resistant (MDR) Bacteria.

Wound infections became a great challenge, especially after the emergence of bacterial resistance to commonly used antibiotics. Medicinal plants can be the source of alternative antibacterial agents effective against multi drug resistant (MDR) bacteria. This research aimed to evaluate the effectiveness of different Silybum marianum seed extracts in fighting MDR bacteria that infect wounds. First, thirty purified bacterial cultures obtained from superficial, infected wounds were subjected to antibiotic sensitivity tests. The selected MDR isolates were then used to test the antimicrobial effects of different S. marianum seed extracts. The most potent extract was evaluated for its impact on the ultrastructure of the cells of sensitive bacterial isolates using transmission electron microscopy (TEM). The bioactive ingredients of this extract were analyzed by means of gas chromatography-mass spectroscopy (GC-MS). Then, in-silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties were predicted for the main components. The results indicated that four out of 30 bacterial isolates were considered MDR bacteria. Primary morphological features of colonies, secondary (automatic) identification using the Biomerieux Vitek 2 System, and 16S rRNA sequencing of the four isolates confirmed that they represent Staphylococcus aureus, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Escherichia coli. Among different extracts of S. marianum seeds, ethanol extract showed the strongest inhibitory effect on both Gram-positive and Gram-negative bacteria, with minimum inhibitory concentration (MIC) values between 9.375 and 1.172 mg/mL. However, at concentrations four times higher, this extract was unable to kill bacterial cells, indicating that it had a bacteriostatic effect on the tested MDR strains. TEM revealed denaturation and distorted cell ultrastructure in S. aureus and S. maltophilia after exposure to ethanol extract. In addition, GC-MS analysis of the ethanol extract identified nine compounds known to have important biological activities, and ADMET analysis showed good drug-likeness for two of these compounds. Consequently, S. marianum seeds could be a good source of alternative bacteriostatic agents effective against MDR bacterial strains that cause wound infections.

The Use of Carbohydrate Biopolymers in Plant Protection against Pathogenic Fungi.

Fungal pathogens cause significant yield losses of many important crops worldwide. They are commonly controlled with fungicides which may have negative impact on human health and the environment. A more sustainable plant protection can be based on carbohydrate biopolymers because they are biodegradable and may act as antifungal compounds, effective elicitors or carriers of active ingredients. We reviewed recent applications of three common polysaccharides (chitosan, alginate and cellulose) to crop protection against pathogenic fungi. We distinguished treatments dedicated for seed sowing material, field applications and coating of harvested fruits and vegetables. All reviewed biopolymers were used in the three types of treatments, therefore they proved to be versatile resources for development of plant protection products. Antifungal activity of the obtained polymer formulations and coatings is often enhanced by addition of biocontrol microorganisms, preservatives, plant extracts and essential oils. Carbohydrate polymers can also be used for controlled-release of pesticides. Rapid development of nanotechnology resulted in creating new promising methods of crop protection using nanoparticles, nano-/micro-carriers and electrospun nanofibers. To summarize this review we outline advantages and disadvantages of using carbohydrate biopolymers in plant protection.

Resistance vs. tolerance to Potato virus Y in tobacco-comparing effectiveness using virus isolates from Central Europe.

Growing resistant cultivars is the best method of protecting the crops against Potato virus Y (PVY). There are a few sources of PVY resistance/tolerance in tobacco acquired through mass selection, X-ray induced mutagenesis and introgressions from wild Nicotiana species. Here, we compare major sources of PVY resistance/tolerance in inoculation tests using ten PVY isolates collected in Central Europe (Poland and Germany) and differing with their virulence. The diversity of collected isolates was confirmed by DAS-ELISA tests and two PCR assays targeting the most common recombination sites in the PVY genome. We used these isolates in inoculation tests on five resistant cultivars 'V.SCR', 'PBD6', 'TN86', 'VAM', 'Wislica', a tolerant breeding line 'BPA' and four susceptible cultivars 'BP-210', 'K326', 'NC95', 'Samsun H'. None of the tested cultivars/breeding lines showed universal resistance against all ten isolates. However, 'VAM' and 'Wislica' appeared to be the most effective sources, as they showed no symptoms and gave negative DAS-ELISA tests for four out of ten tested PVY isolates. In contrast, tolerance of the breeding line 'BPA' was effective against all tested isolates, because inoculation did not lead to development of full disease symptoms in that breeding line.