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.

SUMMARYA survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3'-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population.

Lavandin (Lavandula × intermedia Emeric ex Loiseleur) is cultivated on a large scale in some South European countries for the extraction of essential oils or as an ornamental plant for gardens and landscapes. In May of 2012, virus-like symptoms including bright yellow calico mosaic, leaf distortion, and growth reduction were observed on 15% of lavandin plants in a commercial nursery in Banovo Brdo locality, Baranja County, Republic of Croatia. Leaves from 15 symptomatic lavandin plants were collected and examined by double-antibody sandwich (DAS)-ELISA using commercial antisera (Bioreba AG, Reinach, Switzerland) against two viruses known to infect Lavandula spp.: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) (2,3). Commercial positive and negative controls and extracts from healthy lavandin leaves were included in each ELISA. Only AMV was detected serologically in all 15 tested samples. Five plants each of Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (70-12) using 0.01 M phosphate buffer (pH 7). Local chlorotic spots accompanied by systemic mosaic on both Chenopodium species and bright yellow mosaic on N. benthamiana were observed 6 and 12 days post-inoculation, respectively. Test plants were assayed by DAS-ELISA and all inoculated plants of each species tested positive for AMV. The presence of AMV in all symptomatic lavandin plants was further confirmed by reverse transcription (RT)-PCR assay. Total nucleic acid was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using AMV specific primer pair CP AMV1 (5′-TCCATCATGAGTTCTTCAC-3′) and CP AMV2 (5′-AGGACTTCATACCTTGACC-3′) (1). Total RNAs obtained from the Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and healthy L. × intermedia plant served as the positive and negative control, respectively. The 751-bp amplicons, covering the partial coat protein (CP) gene and 3′-UTR, were obtained from all 15 samples that were serologically positive to AMV as well as from positive control. No amplification product was observed when extract from healthy L. × intermedia plant was used as template in the RT-PCR assay. The RT-PCR product derived from isolate 70-12 was directly sequenced in both directions using the same primer pair as in RT-PCR and deposited in GenBank (JX996119). Multiple sequence alignment of the CP open reading frame was performed by MEGA5 software (4) and revealed that the isolate 70-12 showed the highest nucleotide identity of 99.4% (99.5% amino acid identity) with Serbian AMV isolate from tobacco (FJ527749). To our knowledge, this is the first report of AMV on L. × intermedia in Croatia. Because lavandin is an aromatic plant traditionally and widely grown in Croatia, the presence of AMV could be a limiting factor for its successful production. References: (1) M. M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (2) T. Kobylko et al. Plant Dis. 92:978, 2008. (3) L. Martínez-Priego et al. Plant Dis. 88:908, 2004. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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 functioning of soil ecosystems greatly depends on the interactions occurring between soil biota communities. It is well known that earthworms are an important soil component that substantially affects its function, including their meaningful impact on the development of different phytopathogenic soil fungi. Phytopathogenic fungi are responsible for crop disease and cause great economic damage. It has previously been established that earthworms’ coelomic fluid can suppress the growth of phytopathogenic fungi, but the exact molecular mechanism is unknown. The present study aimed at broadening the proof of this observed phenomenon by investigating the effects of the coelomic fluid extract of three different earthworm species (Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica) on the growth of six different phytopathogenic fungi species (Berkeleyomyces basicola, Fusarium culmorum, Globisporangium irregulare, Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). Coelomic fluid extract was obtained by electrostimulation or usage of extraction buffer (only in case of A. chlorotica) and prepared in three different concentrations by diluting the obtained coelomic fluid with physiological saline. The coelomic fluid extract of the three investigated earthworm species had an inhibitory effect on the growth of all six phytopathogenic fungi species. The greatest inhibitory effect was achieved with the E. andrei coelomic fluid extract reducing the growth of R. solani fungi. The findings of this research confirm the antifungal activity of coelomic fluid obtained from earthworm species belonging to different ecological categories and may be of potential use in crop protection against phytopathogenic fungi.