Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo

After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B₁ and fumonisin B₂. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.     

Advances in molecular diagnosis of toxigenic Fusarium species: A review

The development of advanced molecular diagnosis for the critical toxigenic Fusarium species is considered in this review. The specific topics discussed are (1) isolation of mating type genes of Gibberella complex, (2) molecular detection of Fusarium-producing fumonisins, (3) molecular detection of Fusarium-producing trichothecenes and enniatins. Particular attention is given to the development of PCR assays for genes involved in the toxin biosynthesis that would permit the early detection of Fusarium species-producing toxins and potentially even reveal which particular toxin may be present within a food or feed product. Most of these data have been obtained within the 'De-Tox Fungi' project supported by the European Commission (QLK1-CT-1998-01380).     

Associations of planting date,drought stress,and insects with Fusarium ear rot and fumonisin B1 contamination in California maize

Fusarium ear rot, caused by Fusarium verticillioides, is one of the most common diseases of maize, causing yield and quality reductions and contamination of grain by fumonisins and other mycotoxins. Drought stress and various insects have been implicated as factors affecting disease severity. Field studies were conducted to evaluate the interactions and relative influences of drought stress, insect infestation, and planting date upon Fusarium ear rot severity and fumonisin B₁ contamination. Three hybrids varying in partial resistance to Fusarium ear rot were sown on three planting dates and subjected to four irrigation regimes to induce differing levels of drought stress. A foliar-spray insecticide treatment was imposed to induce differing levels of insect injury. Populations of thrips (Frankliniella spp.), damage by corn earworm (Helicoverpa zeae), Fusarium ear rot symptoms, and fumonisin B₁ levels were assessed. There were significant effects of hybrid, planting date, insecticide treatment, and drought stress on Fusarium ear rot symptoms and fumonisin B₁ contamination, and these factors also had significant interacting effects. The most influential factors were hybrid and insecticide treatment, but their effects were influenced by planting date and drought stress. The more resistant hybrids and the insecticide-treated plots consistently had lower Fusarium ear rot severity and fumonisin B₁ contamination. Later planting dates typically had higher thrips populations, more Fusarium ear rot, and higher levels of fumonisin B₁. Insect activity was significantly correlated with disease severity and fumonisin contamination, and the correlations were strongest for thrips. The results of this study confirm the influence of thrips on Fusarium ear rot severity in California, USA, and also establish a strong association between thrips and fumonisin B₁ levels.     

Biodiversity of complexes of mycotoxigenic fungal species associated with Fusarium ear rot of maize and Aspergillus rot of grape

Fusarium ear rot of maize and Aspergillus rot of grape are two examples of important plant diseases caused by complexes of species of mycotoxigenic fungi. These complexes of species tend to be closely related, produce different classes of mycotoxins, and can induce disease under different environmental conditions. The infection of maize and grape with multiple fungal species and the resulting production of large classes of mycotoxins is an example of mutual aggressiveness of microorganisms toward host species as well as to humans and animals that eat feed or food derived from the infected and contaminated plants. Infection of crop plant with a complex of microbial species certainly represents a greater threat to a crop plant and to human and animal health than infection of the plant with a single fungal species.     

Reaction of winter wheat (Triticum aestivum L.) cultivars to infection with Fusarium spp.: mycotoxin contamination in grain and chaff

This study compares the susceptibility of winter wheat (Triticum aestivum L.) cultivars to Fusarium head blight (FHB) and accumulation of mycotoxins in kernels and chaff under different climatic conditions in two locations–Cerekwica near Poznan (Central West Poland) and Sitaniec, near Zamosc, Lublin region (South East Poland). Very high variations were found in the concentrations of mycotoxins (zearalenone, ZEA; nivalenol, NIV; deoxynivalenol, DON; moniliformin, MON) in examined fractions: Fusarium-damaged kernels (FDK) and healthy looking kernels (HLK) and in chaff for individual cultivars in both locations. In most cases, significantly higher concentrations of investigated toxins were recorded in wheat from the area of Lublin than from Poznan (p < 0.05). The highest Fusarium infection rates and mycotoxin biosynthesis levels were observed in the Lublin location, with the percentage of the FDK fraction ranging 8.1–81.6. In this region, ZEA concentration (µg g^?1) after inoculation with F. culmorum and F. graminearum ranged from 0.02–0.48 and 0.32–1.04, respectively. In the Poznan area, the toxin concentrations were considerably lower, ranging 0.01–0.10 and 0.03–0.13 µg g^?1 for both isolates, respectively. The concentration of DON was significantly higher than ZEA or NIV levels. The levels of MON accumulation (µg g^?1) in the FDK fraction were between 0.14 and 1.73 (Poznan area) and ND (not detected) to 2.51 (Lublin area). F. avenaceum infection rate ranged 7–35% in samples where the toxin was detected.     

Reduction of Fusarium head blight and deoxynivalenol in wheat with early fungicide applications of prothioconazole

Numerous studies have identified the benefit of fungicides applied at flowering (Zadoks Growth Stage (GS) 59–69) in the reduction of Fusarium head blight and the reduction of deoxynivalenol (DON) in harvested wheat grain. Two experiments were performed to identify the ability of prothioconazole (Proline®) at three timings to reduce Fusarium head blight and resulting DON in harvested grain of wheat. Prothioconazole (150 g ha^?1) was applied to plots of wheat at GS31, GS39, and GS65 in a full-factorial design. Plots were inoculated with Fusarium-infected oat grain at GS30 and mist-irrigated at GS65 to encourage head blight development. Plots were assessed for head blight symptoms at GS77 and harvested grain was analysed for yield, specific weight, thousand grain weight, and DON. Factorial analysis of variance (ANOVA) identified prothioconazole applications at each timing that resulted in significant reductions in Fusarium head blight and DON. The control achieved with combinations of spray timings was additive with no significant interactions. The control of Fusarium head blight at GS31, GS39, and GS65 was 50, 58 and 83%, respectively. The reduction in Fusarium head blight achieved by all three timings combined was 97% compared to the fully untreated control plots. The reduction of DON after application of prothioconazole at GS31, GS39, and GS65 was 27%, 49%, and 57%, respectively. The application of prothioconazole at all three timings achieved 83% reduction of DON compared with the fully untreated control plots. These experiments have determined, for the first time, significant additional head blight disease control and mycotoxin reduction with applications of a fungicide before flowering.     

Analysis of deoxynivalenol and deoxynivalenol-3-glucosides content in Canadian spring wheat cultivars inoculated with Fusarium graminearum

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.     

Contamination of barley seeds with Fusarium species and their toxins in Spain: an integrated approach

Fusarium is a globally distributed fungal genus that includes different species pathogenic to cereals among others crops. Some of these Fusarium species can also produce toxic compounds towards animals and humans. In this work, the presence of the most important Fusarium toxins was determined in barley seeds from Spain, sampled according to European Union requirements. The results obtained were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction protocols. Fumonisins B₁ and B₂, zearalenone, trichothecenes type A (T-2 and HT-2) and trichothecenes type B (deoxynivalenol and nivalenol) were analysed by using high-performance liquid chromatography. Deoxynivalenol and zearalenone were detected in 72% and 38% of the barley samples, respectively, at levels below European Union limits in all cases. However, the co-occurrence of both toxins in 34% of the samples suggested that synergistic activity of these two mycotoxins should be evaluated. Nivalenol and HT-2/T-2 were detected at low levels in 17% and 10% of the samples, respectively. Fumonisins occurred in 34% of the samples at levels up to 300?µg/kg. This suggested that they might represent a risk in Spanish barley, and to our knowledge, this is the first report on the presence of fumonisins in barley in this country. The species-specific polymerase chain reaction assays to detect mycotoxin-producing Fusarium species showed a very consistent correlation between F. verticillioides detection and fumonisin contamination as well as F. graminearum presence and zearalenone, deoxynivalenol and nivalenol contamination in barley samples. The approach used in this study provided information of mycotoxin contamination of barley together with the identification of the fungal species responsible for their production. Detection of the species with the current polymerase chain reaction assay strategy may be considered predictive of the potential mycotoxin risk in this matrix.     

DNA arrays, electronic noses and tongues, biosensors and receptors for rapid detection of toxigenic fungi and mycotoxins: A review

This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out-of-laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission-sponsored project Good-Food (FP6-IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on-site generation of data that can aid the safe production of high-quality foodstuffs.     

Natural deoxynivalenol occurrence and genotype and chemotype determination of a field population of the Fusarium graminearum complex associated with soybean in Argentina

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLP_s markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.     

A systematic review of plant-conjugated masked mycotoxins: Occurrence,toxicology, and metabolism

Abstract

Masked mycotoxins are biologically modified phase II metabolites formed by plant defense mechanisms through glucosylation catalyzed by uridine diphosphate -glucosyltransferases. Most of the current reports focus on the occurrence of masked mycotoxins in Europe, America, Africa, and cover other geographic regions, e.g. China and Japan. High proportions of masked mycotoxins co-occurring with their parent forms in various cereal-based food and feedstuff could clearly increase total exposures and pose additional health risks to humans and animals. In contrast to the parent mycotoxins, the data on the toxicity of masked mycotoxins are still scarce, however, the poor existing information showed that masked mycotoxins generally exhibit significant in vitro and in vivo toxicities lower than those of their parent forms, especially for deoxynivalenol-3-glucoside, which is the only thoroughly investigated masked mycotoxin. Although the lower toxicity level of masked mycotoxins, these are probably hydrolyzed into their free forms by intestinal microorganisms in the digestive tract of mammals and thus contribute to unpredicted toxicity. The metabolic characteristics of reported masked mycotoxins are species-specific. The most relevant animal model of human sensitivity, the pig, is most sensitive to masked mycotoxins. This review focuses on updates in the current knowledge on country-specific natural-occurrence data in global surveys, as well as in vitro and in vivo toxicology and metabolic investigations of masked mycotoxins.     

Blood,breast milk and urine: potential biomarkers of exposure and estimated daily intake of ochratoxin A: a review

The purposes of this review are to study potential biomarkers of exposure for ochratoxin A (OTA) in biological fluids (blood, urine and breast milk) for the period 2005–14, calculate the estimated daily intake (EDI) of OTA by using database consumption for the Spanish population, and, finally, to correlate OTA levels detected in blood and EDI values calculated from food products. The values of OTA detected in potential biomarkers of exposure for blood, breast milk and urine ranged from 0.15 to 18.0, from 0.002 to 13.1, and from 0.013 to 0.2 ng ml^–1, respectively. The calculated EDI for OTA in plasma ranged from 0.15 to 26 ng kg^–1 bw day^–1, higher than that obtained in urine (0.017–0.4 ng kg^–1 bw day^–1). All these values are correlated with the range of EDI for OTA calculated from food products: 0.0001–25.2 ng kg^–1 bw day^–1.     

A survey of pre-harvest ear rot diseases of maize and associated mycotoxins in south and central Zambia

Maize ear rots reduce grain yield and quality with implication on food security and health. Some of the pathogenic fungi produce mycotoxins in maize grain posing a health risk to humans and livestock. Unfortunately, the levels of ear rot and mycotoxin infection in grain produced by subsistence farmers in sub-Saharan countries are not known. A survey was thus conducted to determine the prevalence of the ear rot problem and levels of mycotoxins in maize grain. A total of 114 farmsteads were randomly sampled from 11 districts in Lusaka and southern provinces in Zambia during 2006. Ten randomly picked cobs were examined per farmstead and the ear rot disease incidence and severity were estimated on site. This was followed by the standard seed health testing procedures for fungal isolation in the laboratory. Results indicated that the dominant ear rots were caused by Fusarium and Stenocarpella. Incidence of Fusarium verticillioides ranged from 2 to 21%, whereas that of Stenocarpella maydis reached 37% on ear rot diseased maize grain. In addition, 2-7% F. verticillioides, and 3-18% Aspergillusflavus, respectively, were recovered from seemingly healthy maize grain. The mean rank of fungal species, from highest to lowest, was F. verticillioides, S. maydis, A.flavus, Fusarium graminearum, Aspergillus niger, Penicillium spp., Botrydiplodia spp., and Cladosporium spp. The direct competitive ELISA-test indicated higher levels of fumonisins than aflatoxins in pre-harvest maize grain samples. The concentration of fumonisins from six districts, and aflatoxin from two districts, was 10-fold higher than 2 ppm and far higher than 2 ppb maximum daily intake recommended by the FAO/WHO. The study therefore suggested that subsistence farmers and consumers in this part of Zambia, and maybe also in similar environments in sub-Saharan Africa, might be exposed to dangerous levels of mycotoxins due to the high levels of ear rot infections in maize grain.     

Identification,mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia,Italy

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1α gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg^?1; 30 strains produced beauvericin (BEA) up to 190 mg kg^?1; 60 strains produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) up to 1575 mg kg^?1 of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg^?1; all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg^?1; one strain produced FB₁ and FB₂, 1100 and 470 mg kg^?1, respectively; and one strain produced FUP, 820 mg kg^?1; F. solani (30 strains) produced FA, 13 strains up to 215 mg kg^?1. Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB₁/FB₂ by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.     

Mycotoxin food and feed regulation and the specific case of ochratoxin A: a review of the worldwide status

Mycotoxins are naturally occurring contaminants whose presence in food- and feedstuffs cannot be completely avoided. The safety and economic impact arising from the commercialization of mycotoxin-contaminated food and feed supply chain is considerable and acknowledged. To protect consumers from mycotoxin-derived health risks, many countries have adopted regulations or guidelines to limit exposure. Similar to regulation for other mycotoxins, the regulatory status of ochratoxin A (OTA) lacks consensus. Although this was one of the first fungal toxins to be subjected to compliance control due to its toxic properties, the conflict between the prime objective of consumer health safeguard and the economic interests of producers and traders remains. One of the key challenges facing policymakers is to balance these conflicting demands and reach consensual regulatory actions or limits. The noteworthy transboundary implications are recognized, both in the case of absence as the unsubstantiated tightening of regulatory limits. This paper scrutinizes the rationales and implications of mycotoxin regulation, with special attention devoted to OTA. In view of the ongoing debate concerning this complex issue, a review of the arguments and suggested strategies proposed by different parties is also made. The specific case of OTA regulation in food and feed is updated and analysed at a European Union and global level.     

Chemometrics coupled with ultraviolet spectroscopy: a tool for the analysis of variety,adulteration, quality and ageing of apple juices

This study demonstrates the use of UV spectroscopy (UV) in combination with chemometrics as a simple and feasible approach for analysis of variety, adulteration, quality and ageing of apple juice. The results show that PCA‐UV is adequate to differentiate apple juice varieties and adulteration. The percentage of the adulterant can be detected by PLSR‐UV with RMSE < 0.7783% and R² > 0.9980. For the evaluation of juice quality, PLSR‐UV (RMSE = 0.2555–2.3448; R² = 0.7276–0.9816) is recommended for the prediction of soluble solids, ascorbic acid, total flavonoids, total sugar and reducing sugar, whilst PCR‐UV (RMSE = 0.0000–2.7426; R² = 0.7073–1.0000) is adequate for the prediction of pH and antioxidant activity. In addition, PLSR‐UV may be used to predict the storage time with RMSE = 0.4681 day and R² = 0.9832. Therefore, UV coupled with chemometrics has potential to be developed as a portable tool for the detection of variety, adulteration, quality and ageing of not only apple juices, but also other fruit and vegetable juices.     

Mold spoilage of bread and its biopreservation: A review of current strategies for bread shelf life extension

Microbial spoilage of bread and the consequent waste problem causes large economic losses for both the bakery industry and the consumer. Furthermore the presence of mycotoxins due to fungal contamination in cereals and cereal products remains a significant issue. The use of conventional chemical preservatives has several drawbacks, necessitating the development of clean-label alternatives.

In this review, we describe current research aiming to extend the shelf life of bread through the use of more consumer friendly and ecologically sustainable preservation techniques as alternatives to chemical additives. Studies on the in situ-production/-expression of antifungal compounds are presented, with special attention given to recent developments over the past decade. Sourdough fermented with antifungal strains of lactic acid bacteria (LAB) is an area of increasing focus and serves as a high-potential biological ingredient to produce gluten-containing and gluten-free breads with improved nutritional value, quality and safety due to shelf-life extension, and is in-line with consumer's demands for more products containing less additives. Other alternative biopreservation techniques include the utilization of antifungal peptides, ethanol and plant extracts. These can be added to bread formulations or incorporated in antimicrobial films for active packaging (AP) of bread. This review outlines recent progress that has been made in the area of bread biopreservation and future perspectives in this important area.     

Invited review: Antimicrobial resistance in bovine mastitis pathogens: A review of genetic determinants and prevalence of resistance in European countries

Antimicrobial resistance is an urgent and growing problem worldwide, both for human and animal health. In the animal health sector actions have been taken as concerns grow regarding the development and spread of antimicrobial resistance. Mastitis is the most common infection in dairy cattle. We aimed to summarize the genetic determinants found in staphylococci, streptococci, and Enterobacteriaceae isolated from mastitic milk samples and provide a comparison of percentage resistance to a variety of antimicrobials in European countries.     

白腐菌与酶生物预处理对麦草NaOH-AQ制浆性能的影响

研究了麦草白腐菌预处理条件的优选以及白腐菌、木聚糖酶、漆酶生物预处理对麦草蒸煮性能的影响。结果表明：白腐菌预处理半个月优于一个月，漆酶生物预处理脱木素的选择性优于白腐菌、木聚糖酶。麦草经生物预处理后，均能改善后续蒸煮性能，改善程度漆酶好于木聚糖酶、白腐菌。在提高蒸煮得率、浆料强度方面漆酶优于木聚糖酶、白腐菌，与未处理浆和白腐菌生物化学浆相比，实际得率分别提高4．；59、5．49个百分点；裂断长分别提高55．59％、16．05％；,耐破指数分别提高42．86％、19．48％；撕裂指数分别提高4；5．91％、9．57％。木聚糖酶预处理对浆料白度的改善程度优于漆酶、白腐菌，与禾处理浆和漆酶生物化学浆相比白度分别提高4．2和2．0SBD。