First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp in Croatia.

The production of industrial hemp (Cannabis sativa) has had a sharp increase in the past five years in Croatia (Mihelcic 2017). Production has been in constant increase, from 1560 ha in 2015 to 2476 ha in 2019 (PAAFRD). In August 2019, numerous (~1.5%) premature wilted hemp plants (cultivar Fibranova) were observed in commercial fields in Vladislavci (45.4646950° N, 18.5674770° E), around Osijek, Croatia. Diseased plants showed symptoms of chlorosis, rapidly wilting, necrosis and prematurely dying. The stalk of diseased plants was completely desiccated, while internal tissues were spongy and fluffy. Near the soil line, discoloration of the stalk with small spherical to oblong black microsclerotia was detected. Roots were necrotic with brown-gray areas. Twenty root and crown segments of the infected plants (2- to 3-mm long pieces) were surface sterilized with 2% NaOCl for 5 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA, pH 6.2) media, containing 250 mg per liter of chloramphenicol to inhibit bacterial growth (Zveibil and Freeman 2005). The culture plates were incubated at 28 ± 2°C for 7 days in the dark and purified to obtain a pure culture that produces numerous, dark, hard, ovoidal-shaped sclerotia, averaging 140 x 52 µm (n=50). The single sclerotium isolate produced flat light to dark gray colonies with entire margins. Based on field symptoms, colony and microsclerotia morphology, the fungus was identified as Macrophomina phaseolina (Tassi) Goid (Marquez et al. 2021). Total DNA from the isolate was extracted with Extract-N-Amp Plant PCR Kit (Sigma-Aldrich Co., USA). To confirm morphological identification, part of the TEF 1-alpha gene region was amplified using EF1-728F (Carbone and Kohn 1999) and EF2 (O'Donell et al. 1998). The sequence of the isolate MP1 (212 bp - GenBank accession no. OQ389757), showed 100% nucleotide sequence identity to the reference sequence of M. phaseolina GenBank sequence MG434668 (Casano et al. 2018). Eighteen hemp plants (cv. Fibranova) were sown in six plastic pots (three hemp plants per pot) for the pathogenicity test. Ten-day-old M. phaseolina culture (isolate MP1) was used for inoculum preparation. Each pot of one-week-old plant was irrigated with 100 ml of a microsclerotia suspension (105 microsclerotia/ml)(Abied et al. 2018). Plants were held at 28°C and 70% relative humidity in a growth chamber (aralab, Fitoclima 10.000 HP) with a 16-hour photoperiod. Pots with control plants were irrigated with the same amount of sterile distilled water. Ten weeks after inoculation percentage of wilted plants was 77.78%. In the control variant all plants were healthy. M. phaseolina was reisolated from inoculated plants and morphologically identified. With the liberalization of the law, hemp production in Croatia is increasing, which could result in general disease problems and the disease caused by M. phaseolina. Charcoal rot will be expressed in years with dry and warm summers (Lodha and Mawar 2020), and relatively short, cool, rainy winters (Nevo et al. 2012), which has become common in the last decade in Croatia. To our knowledge, this is the first report of M. phaseolina on hemp in Croatia. The authors declare no conflict of interest.

Antifungal Activities of Fluorinated Pyrazole Aldehydes on Phytopathogenic Fungi, and Their Effect on Entomopathogenic Nematodes, and Soil-Beneficial Bacteria.

Fluoro-substituted pyrazoles have a wide range of biological activities, such as antibacterial, antiviral, and antifungal activities. The aim of this study was to evaluate the antifungal activities of fluorinated 4,5-dihydro-1H-pyrazole derivatives on four phytopathogenic fungi: Sclerotinia sclerotiorum, Macrophomina phaseolina, Fusarium oxysporum f. sp. lycopersici, and F. culmorum. Moreover, they were tested on two soil beneficial bacteria-Bacillus mycoides and Bradyrhizobium japonicum-as well as two entomopathogenic nematodes (EPNs)-Heterorhabditis bacteriophora and Steinernema feltiae. The molecular docking was performed on the three enzymes responsible for fungal growth, the three plant cell wall-degrading enzymes, and acetylcholinesterase (AChE). The most active compounds against fungi S. sclerotiorum were 2-chlorophenyl derivative (H9) (43.07% of inhibition) and 2,5-dimethoxyphenyl derivative (H7) (42.23% of inhibition), as well as H9 against F. culmorum (46.75% of inhibition). Compounds were shown to be safe for beneficial soil bacteria and nematodes, except for compound H9 on EPN H. bacteriophora (18.75% mortality), which also showed the strongest inhibition against AChE (79.50% of inhibition). The molecular docking study revealed that antifungal activity is possible through the inhibition of proteinase K, and nematicidal activity is possible through the inhibition of AChE. The fluorinated pyrazole aldehydes are promising components of future plant protection products that could be environmentally and toxicologically acceptable.

Effects of Coumarinyl Schiff Bases against Phytopathogenic Fungi, the Soil-Beneficial Bacteria and Entomopathogenic Nematodes: Deeper Insight into the Mechanism of Action.

Coumarin derivatives have been reported as strong antifungal agents against various phytopathogenic fungi. In this study, inhibitory effects of nine coumarinyl Schiff bases were evaluated against the plant pathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiourum). The compounds were demonstrated to be efficient antifungal agents against Macrophomina phaseolina. The results of molecular docking on the six enzymes related to the antifungal activity suggested that the tested compounds act against plant pathogenic fungi, inhibiting plant cell-wall-degrading enzymes such as endoglucanase I and pectinase. Neither compound exhibited inhibitory effects against two beneficial bacteria (Bacillus mycoides and Bradyrhizobium japonicum) and two entomopathogenic nematodes. However, compound 9 was lethal (46.25%) for nematode Heterorhabditis bacteriophora and showed an inhibitory effect against acetylcholinesterase (AChE) (31.45%), confirming the relationship between these two activities. Calculated toxicity and the pesticide-likeness study showed that compound 9 was the least lipophilic compound with the highest aquatic toxicity. A molecular docking study showed that compounds 9 and 8 bind directly to the active site of AChE. Coumarinyl Schiff bases are promising active components of plant protection products, safe for the environment, human health, and nontarget organisms.

Biological Activities Related to Plant Protection and Environmental Effects of Coumarin Derivatives: QSAR and Molecular Docking Studies.

The aim was to study the inhibitory effects of coumarin derivatives on the plant pathogenic fungi, as well as beneficial bacteria and nematodes. The antifungal assay was performed on four cultures of phytopathogenic fungi by measuring the radial growth of the fungal colonies. Antibacterial activity was determined by the broth microdilution method performed on two beneficial soil organisms. Nematicidal activity was tested on two entomopathogenic nematodes. The quantitative structure-activity relationship (QSAR) model was generated by genetic algorithm, and toxicity was estimated by T.E.S.T. software. The mode of inhibition of enzymes related to the antifungal activity is elucidated by molecular docking. Coumarin derivatives were most effective against Macrophomina phaseolina and Sclerotinia sclerotiorum, but were not harmful against beneficial nematodes and bacteria. A predictive QSAR model was obtained for the activity against M. phaseolina (R2tr = 0.78; R2ext = 0.67; Q2loo = 0.67). A QSAR study showed that multiple electron-withdrawal groups, especially at position C-3, enhanced activities against M. phaseolina, while the hydrophobic benzoyl group at the pyrone ring, and -Br, -OH, -OCH3, at the benzene ring, may increase inhibition of S. sclerotiourum. Tested compounds possibly act inhibitory against plant wall-degrading enzymes, proteinase K. Coumarin derivatives are the potentially active ingredient of environmentally friendly plant-protection products.

An Improved Method for the Quaternization of Nicotinamide and Antifungal Activities of Its Derivatives.

The quaternization reactions of nicotinamide, with different electrophiles: methyl iodide and substituted 2-bromoacetophenones (4-Cl, 4-Br, 4-H, 4-CH3, 4-F, 4-OCH3, 4-Ph, 2-OCH3, 4-NO2) are reported. The preparations were carried out by conventional synthesis and under microwave irradiation in absolute ethanol and acetone. The synthesis performed by microwave dielectric heating significantly improved yield, up to 8 times, and shortened down the reaction time from ca. one day in conventional, to 10⁻20 min. The structures of the synthesized compounds were confirmed by IR, ¹H- and 13C-NMR spectroscopy, mass spectrometry and elemental analysis. The compounds have been screened for antifungal activities against Fusarium oxysporum, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum at concentrations of 10 µg/mL and 100 µg/mL. Six compounds showed the strong inhibition of mycelium growth at a concentration of 10 µg/mL. All tested compounds revealed the great inhibitory activities against S. sclerotiorum at a concentration of 100 µg/mL.

First Report of Olive Branch Dieback in Croatia Caused by Cytospora pruinosa Défago.

Olive (Olea europaea L.) is a very important crop grown in the Mediterranean part of Croatia. Olive branch and fruit dieback symptoms were observed in two olive orchards in Istria, Croatia. The samples from symptomatic trees were collected and brought to the laboratory for analysis. Based on their morphological characterization, isolated fungi were identified as Cytospora sp. Two representative isolates (one per orchard) were taken for molecular analysis, and based on DNA sequence data of the ITS and TUB gene regions, and phylogenetic analysis of the sequences, the isolates were identified as Cytospora pruinosa Défago. To determine pathogenicity, pathogenicity tests were conducted on detached olive branches and two-year-old olive trees in the greenhouse. This is the first report of C. pruinosa causing olive branch and fruit dieback in Croatia.

First Report of Hemp Fusarium Wilt Caused by Fusarium oxysporum in Croatia.

Wilted hemp (Cannabis sativa L.) plants were observed in August 2019 in commercial fields around Osijek, Croatia. Plants and roots with disease symptoms were collected. The single-spored isolates produced septate cottony white to light pink aerial mycelium and purple undersurface on potato dextrose agar (PDA). Smooth and hyaline hyphae were branched and septate. Macroconidia were fusiform to sickle-shaped with foot-shaped basal cells, elongated apical cells and three to five septa. Sequencing of the internal transcribed spacer and the partial elongation factor 1-α gene identified the species as Fusarium oxysporum. Artificial infection fulfills Koch's postulates, producing plants which show stunted growth and wilt symptoms similar to those observed in the commercial fields. Control seedlings remained symptomless and healthy. To the best of our knowledge, this is the first report of hemp Fusarium wilt causing F. oxysporum in Croatia. Considering that F. oxysporum has been reported in main field crops in Croatia, the presence of this pathogen could cause economically significant hemp production decreases, especially in humid and cold springs and susceptible varieties.

Influence of side-chain length on antifungal efficacy of N-alkyl nicotinamide-based compounds.

This article presents fungicidal properties of 9 synthesized nicotinamide-bromides with different alkyl side chain lengths toward Fusarium graminearum, Sclerotinia sclerotiorum, and Botrytis cinerea which were examined. The fungicidal properties were determined by the measurement of the radial growth of fungi, followed by the calculation of the antifungal index. The obtained results were correlated with the descriptors from DFT calculations to determine structural features that affect the fungicidal properties of nicotinamides. Based on the experimental and theoretical results, it was confirmed that F. graminearum is most resistant to the change of lipophilicity of compounds, while S. sclerotiorum is most sensitive. For all investigated compounds, the growth rate decreased with the increase of carbon atoms in the side chain until tetradecylnicotinamidium bromide, [C14Nic][Br], while the further prolongation of the alkyl side chain increased the growth rate of fungus. This behavior was explained by the distinguished hydrophobic and hydrophilic surfaces in [C14Nic][Br] due to interactions between keto oxygen and bromide anion absent in the case of nicotinamides with a longer chain.

First Report of Phaeoacremonium iranianum Causing Olive Twig and Branch Dieback.

In an olive orchard on the western part of Istria, Croatia, twig and branch dieback was observed on several olive trees. In total, seven samples from symptomatic trees were collected. Samples were analyzed, and four fungal isolates showed morphological similarities to the species Phaeoacremonium. One isolate, chosen as a representative, was taken for molecular identification and pathogenicity tests. Based on the DNA sequence data of the ITS, TUB, and EF1α gene regions, the isolate was identified as P. iranianum. Pathogenicity tests were conducted on detached olive branches and olive trees in the greenhouse. To the best of our knowledge, this is the first report of twig and branch dieback on olive caused by Phaeoacremonium iranianum.

Oxidative Status and Antioxidative Response to Fusarium Attack and Different Nitrogen Levels in Winter Wheat Varieties.

Abiotic and biotic stresses, such as mineral nutrition deficiency (especially nitrogen) and Fusarium attack, pose a global threat with devastating impact on wheat yield and quality losses worldwide. This preliminary study aimed to determine the effect of Fusarium inoculation and two different nitrogen levels on oxidative status and antioxidative response in nine wheat varieties. Level of lipid peroxidation, activities of antioxidant enzymes (catalase, ascorbate peroxidase, glutathione reductase), phenolics, and chloroplast pigments content were measured. In general, wheat variety, nitrogen, and Fusarium treatment had an impact on all tested parameters. The most significant effect had a low nitrogen level itself, which mostly decreased activities of all antioxidant enzymes and reduced the chloroplast pigment content. At low nitrogen level, Fusarium treatment increased activities of some antioxidative enzymes, while in a condition of high nitrogen levels, antioxidative enzyme activities were mostly decreased due to Fusarium treatment. The obtained results provided a better understanding on wheat defense mechanisms against F. culmorum, under different nitrogen treatments and can serve as an additional tool in assessing wheat tolerance to various environmental stress conditions.

Xanthium italicum, Xanthium strumarium and Arctium lappa as new hosts for Diaporthe helianthi.

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.

Antifungal activities of silver and selenium nanoparticles stabilized with different surface coating agents.

BACKGROUND: Extensive and growing use of different chemical pesticides that affect both the environment and human health raises a need for new and more suitable methods to deal with plant pathogens. Nanotechnology has enabled the use of materials at the nanoscale with exceptional functionality in different economic domains including agricultural production. This study aimed to evaluate antifungal potential of selenium nanoparticles (SeNPs) and silver nanoparticles (AgNPs) stabilized with different surface coatings and characterized by different surface charge on plant pathogenic fungi Macrophomina phaseolina, Sclerotinia sclerotiorum and Diaporthe longicolla. RESULTS: AgNPs were coated with three different stabilizing agents: mono citrate (MC-AgNPs), cetyltrimethyl ammonium bromide (CTAB-AgNPs) and polyvinylpyrrolidon (PVP-AgNPs). SeNPs were coated with poly-l-lysine (PLL-SeNPs), polyacrylic acid (PAA-SeNPs), and polyvinylpyrrolidon (PVP-SeNPs). Seven different concentrations (0.1, 0.5, 1, 5, 10, 50 and 100 mg L-1 ) of nanoparticles were applied. All AgNPs and SeNPs significantly inhibited the growth of the tested fungi. Among the tested NPs, PVP-AgNPs showed the best inhibitory effect on the tested plant pathogenic fungi, especially against S. sclerotiorum. The similar inhibition of the sclerotia formation was observed for S. sclerotiorum treated with PLL-SeNPs. CONCLUSION: Obtained results provides new insights on fungicide effect of AgNPs and SeNPs stabilized with different coating agents on different plant pathogens. Further work should focus on detailed risk/benefit ratio assessment of using SeNPs or AgNPs in agriculture taking into account whole agroecosystem. © 2020 Society of Chemical Industry.

Correlation between lipophilicity of newly synthesized ionic liquids and selected Fusarium genus growth rate.

The purpose of the present study was to examine the effectiveness of 23 different synthesized ionic liquids (ILs) on Fusarium culmorum and Fusarium oxysporum growth rate. The strategy of IL synthesis was a structural modification of ionic liquids through changing the polarity of imidazolium and pycolinium cations and replacing halide anions with well known antifungal anions (cinnamate, caffeate and mandelate). The findings clearly suggest that the type of alkyl chain on the cation is the most determining factor for IL toxicity. In order to examine how IL structure affects their toxicity towards Fusarium genus, lipophilic descriptor A log P is calculated from density functional theory and correlated with Fusarium growth rate. All these results demonstrate the high level of the interdependency of lipophilicity and toxicity for investigated ILs towards the Fusarium genus. The data collected in this research suggest that the inhibitory influence of ILs is more pronounced in the case of F. oxysporum.