Diversity and aggressiveness of the Diaporthe species complex on sunflower in Serbia.

This study aimed to investigate the Diaporthe species associated with Phomopsis stem canker of sunflower (Helianthus annuus L.) in Serbia. The significant increase in sunflower and soybean (Glycine max (L.) Merr.) cultivation may have created the bridge favorable conditions for the distribution of Diaporthe species in this region. The present study identified five Diaporthe species on sunflower: D. gulyae, D. helianthi, D. pseudolongicolla, D. stewartii, and the newly identified D. riccionae based on morphological, molecular, and pathogenic characteristics. The research emphasizes the importance of effective inoculation methods and evaluates the aggressiveness of isolates. Sunflower plants were inoculated using the stem wound method, while seeds of sunflower and soybean were inoculated using the standard seed method. Most of the tested isolates demonstrated high aggressiveness, resulting in over 80% premature wilting of sunflower plants. Additionally, this research examined the aggressiveness of Diaporthe species on sunflower seeds, highlighting D. stewartii and D. pseudolongicolla as common pathogens of both sunflower and soybean. The most aggressive species on seeds was D. stewartii, causing seed decay of up to 100% in sunflower and 97% in soybean. The findings suggest the development of resilient sunflower genotypes through breeding programs and the implementation of strategies to manage cross-contamination risks between sunflower and soybean crops. Furthermore, this study provides insights into the interactions between Diaporthe species and the seeds of sunflower and soybean. Future research will enhance our understanding of the impact of Diaporthe species on sunflower and soybean.

First report of brown spot on stored apple fruits caused by Stemphylium vesicarium in Serbia.

Apple is one of the most economically important fruit crops worldwide, and fungal postharvest diseases can cause significant losses during storage (Petres et al. 2020). Apple fruits (cultivar Fuji) with necrosis symptoms were collected during the fall of 2022 from the cold storage facility (ULO - Ultra Low Oxygen) in Titel, Serbia. The fruits originated from the apple orchard in Titel, Serbia (45°12'47.1"N, 20°15'23.6"E). The pathogens were isolated from collected fruit samples using standard phytopathological techniques. Fruits were surface-sterilized, rinsed with sterile water, aseptically cut in half, and small fragments collected from the border of healthy and diseased tissue were placed into Petri dishes on Potato Dextrose Agar medium (PDA) and incubated at 25±1 °C in dark for seven days. The obtained 11 isolates were identified to the genus level as Alternaria (incidence 46%), Penicillium (36%), Fusarium (9%) and Stemphylium (9%) based on morphological characteristics. Pathogenicity of all isolates was confirmed on apple fruits of cultivars Fuji and Golden Delicious. The fruits were surface-sterilized, sprayed with 5 ml conidial suspension (1×105 conidia/ml) and incubated at room temperature for 21 days. Symptoms developed on inoculated fruits were the same as symptoms observed on apple fruit samples collected from cold storage. Reisolation from artificially inoculated fruits resulted in colonies that morphologically corresponded with the colonies used for inoculation. Stemphylium isolate was the only one included in further research. Initial symptoms and symptoms on artificially inoculated apple fruits caused by Stemphylium sp. occurred as circular dark brown necrosis located near the calyx, without visible sporulation on the fruit surface. The isolate and reisolate formed aerial, white to light brown mycelia. The pigmentation of the culture medium was pale to dark brown. Conidia were singular, cylindrical and multicellular, brown to dark brown, 22-35.1 long and 12.6-18.9 µm wide. Based on morphological properties, isolate and reisolate were identified as Stemphylium vesicarium which is in line with the description reported by Sharifi et al. (2021) and Gilardi et al. (2022). The identification of S. vesicarium isolate was confirmed by polymerase chain reaction (PCR) by amplifying and sequencing three regions using following primer pairs: Bt2a (5'- GGT AAC CAA ATC GGT GCT GCT TTC -3') and Bt2b (5'-ACC CTC AGT GTA GTG ACC CTT GGC-3') for ß-tubulin region (Nasri et al. 2015), ITS1 (5'-TCC GTA GGT GAA CCT GCG G - 3') and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC-3') for ITS region (White et al. 1990), and EF1 (5' - ATG GGT AAG GAG GAC AAG AC - 3') and EF2 (5'- GGA AGT ACC AGT GAT CAT GTT - 3') for TEF-1α region (O'Donnell et al. 1998). PCR products were separated by horizontal gel electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and visualization under UV light revealed amplified fragments of the expected size of 500 bp for Bt2a/ Bt2b primer pair, 600 bp for ITS1/ITS4 primer pair, and 700 bp for EF1/EF2 primer pair. The obtained amplicons were Sanger sequenced (Macrogen Europe BV) in both directions. BLASTn analysis showed the identity of amplified fragments of the isolates with sequences of S. vesicarium present in the GenBank of 100% (MT881940.1 and JQ671944.1) for the ß-tubulin region, 99.40% (MT520589.1 and OR256793.1) for the ITS region, and 99.49% (DQ471090.2 and MT394642.1) for the TEF-1α region. The sequences were deposited to NCBI GenBank (Accession No. OQ653540 for the ß-tubulin region, OQ678016 for the ITS region, and OR232710 for the TEF-1α region). To our knowledge, this is the first finding of S. vesicarium on apple fruits in the Republic of Serbia, and the finding of a new causal agent of postharvest apple fruit rot.

Biological Control of Aflatoxin in Maize Grown in Serbia.

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51-83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains.

A New Concept to Secure Food Safety Standards against Fusarium Species and Aspergillus Flavus and Their Toxins in Maize.

Commercial maize hybrids are exposed to different degrees of ear infection by toxigenic fungal species and toxin contamination. Their resistance to different fungi and toxin relationships are largely unknown. Without this knowledge, screening and breeding are not possible for these pathogens. Seven- to tenfold differences were found in resistance to Fusarium spp., and there was a five-fold difference in ear coverage (%) in response to A. flavus. Three hybrids of the twenty entries had lower infection severity compared with the general means for toxigenic species. Three were highly susceptible to each, and 14 hybrids reacted differently to the different fungi. Differences were also observed in the toxin content. Again, three hybrids had lower toxin content in response to all toxigenic species, one had higher values for all, and 16 had variable resistance levels. Correlations between infection severity and deoxynivalenol (DON) content were 0.95 and 0.82 (p = 0.001) for F. graminearum and F. culmorum, respectively. For fumonisin and F. verticillioides ear rot, the Pearson correlation coefficient (r) was 0.45 (p = 0.05). Two independent isolates with different aggressiveness were used, and their mean X values better described the resistance levels. This increased the reliability of the data. With the introduction of this methodological concept (testing the resistance levels separately for different fungi and with two isolates independently), highly significant resistance differences were found. The resistance to different fungal species correlated only in certain cases; thus, each should be tested separately. This is very useful in registration tests and post-registration screening and breeding. This would allow a rapid increase in food and feed safety.