Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Grapevine trunk diseases associated with fungi from the Diaporthaceae family in Croatian vineyards.

Grapevine trunk diseases (GTD) have a variety of symptoms and causes. The latter include fungal species from the family Diaporthaceae. The aim of our study was to determine Diaporthaceae species present in the woody parts of grapevines sampled from 12 vine-growing coastal and continental areas of Croatia. The fungi were isolated from diseased wood, and cultures analysed for phenotype (morphology and pathogenicity) and DNA sequence (ITS1, 5.8S, ITS2). Most isolates were identified as Phomopsis viticola, followed by Diaporthe neotheicola and Diaporthe eres. This is the first report of Diaporthe eres as a pathogen on grapevine in the world, while for Diaporthe neotheicola this is the first report in Croatia. Pathogenicity trials confirmed Phomopsis viticola as a strong and Diaporthe neotheicola as a weak pathogen. Diaporthe eres turned out to be a moderate pathogen, which implies that the species could have a more important role in the aetiology of GTD.

Ochratoxin A contamination of food from Croatia.

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, genotoxic and carcinogenic properties produced by Penicillium and Aspergillus moulds under different climatic conditions. Humans and animals are exposed to this compound mainly via ingestion of contaminated food. In Croatia, research on mycotoxins focused on OTA when the mycotoxin theory of endemic nephropathy (EN) was postulated. Ochratoxin A was more frequent and at higher concentration in foods from EN than those from the control regions. Subsequently, OTA concentrations were determined in some commodities intended for human consumption such as maize, wheat, beans and wine. Samples from all parts of Croatia were analyzed and OTA was found in all types of commodities. It was frequently found together with other mycotoxins (fumonisin B(1), fumonisin B(2) and zearalenone). In general, OTA concentration in foods from Croatia is low, but the frequency of positive samples shows considerable variations from year to year depending also on sampling location. Although low levels of OTA were found in a large proportion of analyzed food samples, its persistent co-occurrence with other significant mycotoxins should raise serious public health concerns as there interactions may be synergistic or additive in causing toxicity in humans and animals. There is need to establish control measures through which such contaminations in foods can be managed.

Fusarium spp. contamination of wheat, maize, soybean, and pea grain in Croatia.

From 2002 to 2008, 203 samples of wheat, maize, soybean, and pea were analysed for the presence of Fusarium species. Contamination with Fusarium spp., expressed as the percentage of seeds with Fusarium colonies, ranged from 5 % to 69 % for wheat, from 25 % to 100 % for maize, from 4 % to 17 % for soybean, and from 3 % to 17 % for pea. 187 isolates were collected and the following 19 species determined: F. graminearum, F. poae, F. avenaceum, F. verticillioides, F. sporotrichioides, F. heterosporum, F. crookwellense, F. tricinctum, F. semitectum, F. oxysporum, F. proliferatum, F. solani, F. equiseti, F. pseudograminearum, F. chlamydosporum, F. sambucinum, F. compactum, F. scirpi, and F. culmorum. Dominant species were F. graminearum on wheat (27 % of isolates), F. verticillioides on maize (83 % of isolates), F. sporotrichioides on soybean (34 % of isolates), and F. proliferatum on pea (29 % of isolates). Among species identified, F. heterosporum, F. crookwellense, F. pseudograminearum, F. sambucinum, and F. compactum have been reported for the first time in Croatia.

Mould contamination and co-occurrence of mycotoxins in maize grain in Croatia.

Maize grain samples (n=15) collected during the autumn of 2002 were analyzed for the presence of moulds and mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA), and ochratoxin A (OTA). Mycological analysis showed that all samples were contaminated with Fusarium spp. and Penicillium spp., while Aspergillus spp. were found in 5 samples. F. proliferatum and F. verticilloides, the producers of fumonisins, were found in 14 and 8 samples, respectively, while F. graminearum, the producer of ZEA, was present in all samples. The most frequent mycotoxins were FB1 (15/15) and ZEA (12/15), followed by OTA (7/15), while FB2 was found in only two samples. Seven samples were contaminated with two mycotoxins, seven with three, and one sample with only one mycotoxin. The concentrations (mean+/-SD) of FB1, ZEA, and OTA in positive samples were 459.5+/-314.6, 1.70+/-0.80, and 1.40+/-0.55 microg kg-1, respectively, and the concentrations of FB2 in two samples were 68.4 and 3084.0 microg kg-1. In general, such low mycotoxin concentrations are not a significant source of exposure to humans, but they may contribute to exposure from other commodities. A few samples with extreme values indicate that strict control is needed.

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia.

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB1(100%), followed by ZEA (84%) and OTA (39%), while FB2 was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean +/- SD) of FB1, ZEA and OTA in positive samples were 459.8 +/- 310.7, 3.84 +/- 6.68 and 1.47 +/- 0.38 microg kg(-1), respectively, and the concentrations of FB2 in three positive samples were 68.4, 109.2 and 3084.0 microg kg(-1). Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.

Prevention of exposure to mycotoxins from food and feed.

Mycotoxins are metabolites of moulds that may be found in food and feed of plant and animal origin. This paper gives a short review of the agronomical methods and food and feed storage recommendations for the prevention of mould contamination. It describes the practical methods of feed decontamination and the use of feed additives where mycotoxin contamination prevention has failed. However, these methods should be avoided as much as possible because they may increase the cost of production, reduce the nutritional value of feed, and leave residues of mycotoxins or their toxic metabolites. Since there is no universal and reliable method of feed decontamination for all mycotoxins, the paper stresses the importance of preventive measures.