Leaf spot symptoms were spotted in two olive orchards in Istria and in Kvarner Gulf, Croatia. Fungal species from three representative isolates (P13 LECIII, R18 BI, JA20 NP) have been morphologically characterized based on the colony and conidial characteristics. Several techniques were performed for inducing the sporulation of the JA20 NP isolate. Only PDA + banana medium was successful. PCR was conducted for ITS, TUB, and EF1α gene regions. Phylogenetic analyses were performed using internal transcribed spacer, beta-tubulin, and translation elongation factor 1-alpha sequence data. Three types of tests were conducted: a pathogenicity test on detached leaves, on detached and scratched leaves, and on olive seedlings. Ultimately, from the morphological characterizations, DNA sequence analysis of ITS, TUB, and EF1α gene regions, and phylogenetic analysis, these species were identified as Nigrospora gorlenkoana Novobr., Nigrospora osmanthi Mei Wang & L. Cai, and Nigrospora philosophiae-doctoris M. Raza, Qian Chen & L. Cai. This is the first report of Nigrospora species causing leaf spot on olive trees and the first report of Nigrospora philosophiae-doctoris as a plant pathogen. Fungal leaf diseases in conditions that are favorable for infection and disease development can lead to a decrease in the yield and olive oil quality. Therefore, it is necessary to conduct further research and the monitoring of fungal leaf diseases.

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 (R²_tr = 0.78; R²_ext = 0.67; Q²_loo = 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.

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.

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.

The antifungal effectiveness of essential oils (EOs) and their predominant components were tested on 14 phytopathogenic fungi isolated from olive trees. Commercial EOs from holy basil (Ocimum tenuiflorum L.), Chinese cinnamon (Cinnamomum aromaticum Ness), lemon (Citrus × limon), peppermint (Mentha × piperita L.), oregano (Origanum compactum Benth), and thyme (Thymus vulgaris L.) and components eugenol, e-cinnamaldehyde, limonene, menthol, carvacrol, and thymol were used. Antifungal efficacy was tested on six species from the Botryosphaeriaceae family: Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not.; Diplodia mutila (Fr.) Fr.; D. seriata De Not.; Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves; Do. sarmentorum (Fr.) A.J.L. Phillips, Alves & Luque; and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Other tested species included Biscogniauxia mediterranea (De Not.) Kuntze, B. nummularia (Bull.) Kuntze; Cytospora pruinosa Défago; Nigrospora gorlenkoana Novobr.; N. osmanthi Mei Wang & L. Cai; N. philosophiae-doctoris M. Raza, Qian Chen & L. Cai; Phaeoacremonium iranianum L. Mostert, Grafenhan, W. Gams & Crous; and Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. The results show that Chinese cinnamon and oregano EOs, along with their components, completely inhibited the growth of all tested fungi, indicating their potential as biological control agents in sustainable agriculture. In contrast, the least effective treatments were the EOs derived from lemon and peppermint, as well as the components limonene, menthol, and thymol. Notably, the fungi Do. iberica and N. gorlenkoana were among the most sensitive to all the treatments applied.

A field investigation of olive trees in Istria, Croatia, revealed branch dieback and cracked bark. Samples of diseased branches were collected from eight different locations and analysed. Additionally, meteorological data from two locations were analysed to determine if there was a connection between climatic changes and the appearance of pathogens in the region. Pathogenicity tests were conducted on olive seedlings. This study provides a description of Biscogniauxia and Sordaria species’ morphology and elucidates their phylogeny based on the internal transcribed spacer (ITS), beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1- α) regions. This research represents the first documented occurrence of Biscogniauxia mediterranea causing charcoal disease in olive trees in Croatia. Additionally, it is the first report of Biscogniauxia nummularia (Bull.) Kuntze and Sordaria fimicola causing diseases in olive trees anywhere in the world. Furthermore, this study marks one of the initial forays into molecular investigations of these species isolated from olive trees. Considering the potential threat posed by the inherent aggressiveness of Biscogniauxia species, further research is deemed necessary to curb the development of charcoal disease.