Epidemiology of Toxin-Producing Fungi and Ochratoxin a Occurrence in Grape

Fungi responsible for ochratoxin A (OTA) production have been studied especially on cereals, where Penicillium verrucosum and Aspergillus ochraceus are to be considered the main producers. Until 1998, these fungi were also believed to be responsible for the production of the toxin in grape, but OTA-producing A. carbonarius and A. niger were identified in dried vine fruits in 1999. Further studies pointed out that mycoflora potentially responsible for the presence of OTA in grapes are present in the field. Aspergilli are dominant to Penicillia, and among these Aspergilli section Nigri. A. carbonarius probably plays an important role because of the high percentage of positive strains and the amount of OTA produced. Aspergilli section Nigri are present on grape bunches early in the season and their frequency increases during later growth stages. At early veraison and ripening, the incidence of colonised berries is more related to the year than to the growth stage, but not to visible symptoms, since it is normal to isolate fungi from intact berries. Differences in ochratoxin content of berries have been detected between years, when the same vineyards, managed in the same way, showed high levels (1999) or the absence (2000) of the toxin. The results suggest that meteorological differences between years and grape-growing areas are responsible for differences in OTA levels, but the data are at present insufficient to draw firm conclusions.     

PCR Assay for Identification of Aspergillus Carbonarius and Aspergillus Japonicus

Black Aspergilli, and in particular Aspergillus carbonarius, are the main causes of contamination of grapes and their by-products by ochratoxin A. A PCR-based method was developed to detect DNA of A. carbonarius and A. japonicus. Two pairs of primers (CARBO1/2 and JAPO1/2) designed from the calmodulin gene, produced PCR products of 371 and 583 bp for A. carbonarius and A. japonicus, respectively. Primer specificity was tested with DNA of 107 strains belonging to Aspergillus section Nigri isolated mostly from grapes in Europe. The sensitivity of primers CARBO1/2 and JAPO1/2 was 12.5 pg when using pure total genomic DNA of the two species. The developed primers provide a powerful tool for detection of the main ochratoxigenic producing Aspergillus species in grapes.     

Ochratoxin A-producing fungi in Spanish wine grapes and their relationship with meteorological conditions

Forty vineyards from four wine making regions of Spain were sampled at three different growth stages in 2002 and 2003. The aim was to study the fungi associated with grapes and their ability to produce ochratoxin A (OTA) on synthetic media. Among the total mycoflora, 464 (7.7%) and 648 (10.8%) Aspergillus section Nigri (black aspergilli) strains were isolated in 2002 and 2003, respectively, and were classified into three groups: isolates with uniseriate heads, A. niger aggregate and A. carbonarius. The latter presented the highest percentage of OTA-positive strains (82% in 2002 and 76% in 2003) and produced the highest levels of toxin (2.5–25 μg g⁻¹). The sampling year, sampling date, the region and their interactions presented significant differences in the number of black aspergilli isolated. Most black aspergilli were found in 2003 and at harvest. A positive correlation between the number of black aspergilli found in grapes and the temperature in the field was found. Grapes from 2003, the warmest year, and from Costers del Segre, the warmest region, were significantly the most contaminated. No significant correlation between black aspergilli presence and other meteorological factors such as relative humidity or rainfall could be established. Musts from all the vineyards were also analysed in both years, although no OTA was found in either year.     

Strains of Aureobasidium pullulans can lower ochratoxin A contamination in wine grapes

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.     

Molecular characterisation and pathogenicity of <Emphasis Type="Italic">Aspergillus</Emphasis> Sect. <Emphasis Type="Italic">Nigri</Emphasis> causing Aspergillus vine canker of table grapes in Italy

Thirty-two isolates belonging to black aspergilli (Aspergillus section Nigri) associated to vine canker disease of grapevine were collected in seven vineyards located in southeastern Sicily (Italy). Molecular analysis was performed to identify the isolates by multilocus sequence analysis. Amplification of part of the β-tubulin gene (benA) and partial calmodulin (CaM) gene were performed using the Bt2a, Bt2b and CL1, CL2A primers, respectively. Molecular characterisation showed a high distribution of Aspergillus niger “aggregate” species on grapes in Sicily and in particular of A. niger (21 isolates), A. tubingensis (9 isolates), and A. carbonarius (2 isolates). The 21 isolates of A. niger found to belong within the newly described cryptic species A. awamori. Six isolates (3 of A. tubingensis, 2 of A. carbonarius, and 1 of A. niger) were used in pathogenicity studies on mature canes of cv. Italia grape. All species caused Aspergillus vine canker equally well, with no differences in virulence.     

Epidemiology of Toxigenic Fungi and their Associated Mycotoxins for Some Mediterranean Crops

Recent data on the epidemiology of the common mycotoxigenic species of Fusarium, Alternaria, Aspergillus and Penicillium in infected or colonized plants, and in stored or processed plant products from the Mediterranean area are reviewed. Emphasis is placed on the toxigenicity of the causal fungal species and the natural occurrence of well known mycotoxins (aflatoxins, ochratoxins, fumonisins, trichothecenes, zearalenone, patulin, Alternaria-toxins and moniliformin), as well as some more recently described compounds (fusaproliferin, beauvericin) whose toxigenic potential is not yet well understood. Several Fusarium species reported from throughout the Mediterranean area are responsible of the formation of mycotoxins in infected plants and in plant products, including: Fusarium graminearum, F. culmorum, F. cerealis, F. avenaceum, F. sporotrichioides and F. poae, which produce deoxynivalenol, nivalenol, fusarenone, zearalenone, moniliformin, and T-2 toxin derivatives in wheat and other small grains affected by head blight or scab, and in maize affected by red ear rot. Moreover, strains of F. verticillioides, F. proliferatum, and F. subglutinans, that form fumonisins, beauvericin, fusaproliferin, and moniliformin, are commonly associated with maize affected by ear rot. Fumonisins, were also associated with Fusarium crown and root rot of asparagus and Fusarium endosepsis of figs, caused primarily by F. proliferatum. Toxigenic A. alternata strains and associated tenuazonic acid and alternariols were commonly found in black mould of tomato, black rot of olive and citrus, black point of small cereals, and black mould of several vegetables. Toxigenic strains of A. carbonarius and ochratoxin A were often found associated with black rot of grapes, whereas toxigenic strains of A. flavus and/or P. verrucosum, forming aflatoxins and ochratoxin A, respectively, were found in moulded plant products from small cereals, peanuts, figs, pea, oilseed rape, sunflower seeds, sesame seeds, pistachios, and almonds. Finally, toxigenic strains of P. expansum and patulin were frequently found in apple, pear and other fresh fruits affected by blue mould rot, as well as in derived juices and jams.     

Identification of <Emphasis Type="Italic">Pestalotiopsis bicilita,Diplodia seriata</Emphasis> and <Emphasis Type="Italic">Diaporthe eres</Emphasis> causing fruit rot in withered grapes in Italy

Pestalotiopsis biciliata, Diplodia seriata and Diaporthe eres were recovered from severely diseased bunches of withered grapes for Amarone wine production. To our knowledge this post-harvest disease has never been seen before in withered grapes. Fruit rot occurred with bunches containing decayed berries partially or totally covered by whitish mycelium. Morphological and phylogenetic analyses of fungal isolates from symptomatic fruits enabled species delimitation. Pathogenicity tests revealed that the three species are able to infect grape berries confirming that they are casual agents of fruit rot. This study provides preliminary insights on this post-harvest disease that might have important implications in grape quality.     

Ochratoxin a in Cereals, Foodstuffs and Human Plasma

Aspergillus and Penicillium fungi contaminate cereals and foodstuffs, and can thus introduce ochratoxin A (OTA) into the food chain. In this work, five new isolates of Aspergillus: A. albertensis, A. auricomus, A. wentii, A. fumigatus and A. versicolor, were found to produce ochratoxin A. Data on the occurrence and the concentration levels of ochratoxin A in European food of vegetable and animal origin are reported. Furthermore, data on the concentration of ochratoxin A in blood of citizens of Western Europe are compared with those of some areas where Balkan endemic nephropathy is endemic. The results of the studies of Stoev and co-workers are reviewed and the possibility that OTA alone cannot be the cause of the Balkan endemic nephropathy is discussed.     

澧县‘红地球’葡萄表面黑色组曲霉种群结构及产毒情况研究

葡萄和葡萄制品面临着真菌毒素的威胁, 这些真菌毒素主要来自于黑色组曲霉Aspergillus section Nigri?本研究对采自湖南省澧县‘红地球’葡萄上的黑色组曲霉进行了分离鉴定及种群结构分析, 并采用超高效液相色谱串联质谱技术(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)对12株代表菌株产赭曲霉毒素(ochratoxin A, OTA)和伏马毒素B2(fumonisin B2, FB2)的能力进行了测定?结果表明, 澧县地区‘红地球’葡萄带菌率为92%?基于钙调素序列, 分离得到的131株黑色组曲霉分属于5个种, 其中A.aculeatinus为优势种?12株代表菌株均不产生OTA; A.welwitschiae(4/5)及A.niger(2/2)可产生FB2, 平均产毒量分别为3.36 ng/g及741.37 ng/g?上述结果为评估国内葡萄表面黑色组曲霉及其毒素的污染风险提供参考?     

Stilbene oligomer phytoalexins in grape as a response to Aspergillus carbonarius infection

Stilbenes are grapevine phytoalexins elicited by biotic and abiotic agents; Aspergillus carbonarius is a widespread ochratoxin A producing fungus present in warm conditions, such as in Southern Italy. To increase the knowledge on biosynthesis of stilbene oligomers induced by A. carbonarius infection, grape berries of the Southern Italian grape cv. Negro Amaro were inoculated. Significant increase of trans-resveratrol and resveratrol dimers and oligomers, such as caraphenol, E-ε-viniferin, ω-viniferin, δ-viniferin, α-viniferin, E-miyabenol C, and two tetramers, was observed, and concomitant decrease of glycoside derivatives. These findings improve the knowledge on the phytoalexin production as response against this pathogen.     

Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa

Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan™) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10⁵ cells per reaction in water and grape extracts and 10–10⁵ cells in insect DNA. Regression curves were similar, with correlation coefficients of r ² > 0.97. In quantitative PCR, C_t values ranged between 20 and 36 cycles for 5–10⁵ bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.     

Relationship between azinphos-methyl usage and residues on grapes and in wine in Australia

Azinphos-methyl was applied to Shiraz winegrapes by commercial high-volume and hand-held sprayers during seasons 1993/94 and 1994/95. Residue levels in grapes resulting from treatments applied by commercial sprayer were below the maximum residue level (MRL) of 2 mg kg^-1 for grapes in Australia, whereas residues resulting from treatments applied by hand-held sprayer still exceeded the MRL five weeks after final application. There was a strong correlation for most treatments between treatment concentration of azinphos-methyl and residue level in grapes, and in wine made from treated grapes. Applied at the recommended rate (1·2 g litre^-1 wettable powder (WP) and 2·4 ml litre^-1 suspension concentrate (SC)) by commercial high-volume sprayer, azinphos-methyl residue levels in wine were well below the MRL, and below the MRLs of most importing countries, except Denmark and Sweden. When applied by hand-held sprayer, residue levels in wine were 5·9–29·6 fold higher than those previously obtained by commercial application of insecticide. Since wines are often blends from different grape blocks and grape-growing districts, in practice, this is unlikely to be of concern. Wine made from grapes treated by commercial sprayer showed no detectable residues of azinphos-methyl after one year of storage. In both years, residue levels in grapes of both formulations of azinphos-methyl fluctuated during the five-week post-treatment period, although there was an overall downward trend. Previously unrecorded systemicity in azinphos-methyl was demonstrated in laboratory studies with barley seedlings, and this may explain these fluctuating data in grapes. The reduction of azinphos-methyl residues in grapes over time appears to be a complex phenomenon involving translocation of active ingredient combined with an increase in the size and weight of berries, producing fluctuating residue levels. © 1998 SCI     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。     

Mycotoxin Genetics and Gene Clusters

Fungi produce low molecular weight secondary metabolites such as antibiotics and mycotoxins. Antibiotics cure diseases whereas mycotoxins cause diseases in plants, animals and human beings. Species such as Aspergillus, Fusarium, Penicillium and Stachybotrys are known to produce mycotoxins that accumulate in processed foods and feeds, although the incidence of infection occurs before processing, during the active growth of the organism. Among the mycotoxins, aflatoxins produced by Aspergillus flavus and A. parasiticus have been extensively studied at the molecular level. A complex biosynthetic pathway involving sixteen steps is mediated by individual major genes. These fungi have eight linkage groups, but the aflatoxin/sterigmatocystin (AF/ST) metabolic pathway genes have been mapped to only three linkage groups; ten of them belong to linkage group VII, and one of each to linkage group II and VIII. These genes are involved in both the regulatory and biosynthetic pathways and are clustered on the respective chromosomes. Clustering of genes in fungi indicates an evolutionary trend among genes that orchestrate gene function. Being linked together they segregate as a unit, thereby conferring a selective advantage to the organism. The evolution of gene clusters takes place through vertical or horizontal gene transfer. In fungi, horizontal gene transfer is most effective. Functionally, the mechanism of evolution of mycotoxin gene clusters in fungi seems to be similar to the evolution of a super-gene. The possible implications of evolutionary parallelism of gene clusters and super-genes is briefly explored.     

Ozone for control of post-harvest decay of table grapes caused byRhizopus stolonifer

The effect of ozone on post-harvest decay of table grapes was studied both with regard to its effectiveness and its possible mode of action. Ozone concentrations fell rapidly upon contact with organic matter and the amount which reacted with grape berries and the microflora on their surface was about 0.1mgg^-1, when supplied at a rate of 8mgmin^-1for 20min. The dose applied could be increased by longer periods of exposure, but symptoms of toxicity were observed on certain cultivars. The number of colony forming units (cfu) of fungi, yeasts and bacteria naturally present on the berry surface was considerably reduced by a 20min exposure to ozone. Ozone treatments significantly reduced the extent of berry decay caused by fungi following cold storage and increased shelf-life. A significant decrease in decay was observed in berries that were treated with ozone either before or after being inoculated withRhizopus stolonifer.This finding indicates that, in addition to its sterilizing effect, ozone also induced resistance to post-harvest decay development. The phytoalexins resveratrol and pterostilbene were elicited by ozone treatments, at levels similar to those produced by uv-c irradiation. Resveratrol accumulated in greater quantities than pterostilbene. Inoculation withR. stoloniferin addition to ozone treatment, raised the levels of both stilbenes even more. Exposing berries to ozone was almost as effective as SO₂fumigation for the control of storage decay caused byR. stoloniferand no deleterious effects were observed on the appearance of the grape cluster. Ozone treatments can therefore be considered as a possible substitute for SO₂fumigation for the control of post-harvest fungal decay.     

Use of nitrate non‐utilizing (nit) mutants to determine phenological stages at which Botrytis cinerea infects wine grapes causing botrytis bunch rot

Botrytis bunch rot (BBR), caused by Botrytis cinerea, degrades wine grapes during ripening, even though infection can occur as early as flowering. Effective BBR management requires knowledge of whether some stages of fruit development are more important than others in relation to infection and BBR severity at harvest. Bunches of Vitis vinifera ‘Sauvignon blanc’ and/or ‘Pinot noir’ were inoculated in two vineyard trials and one glasshouse trial with nitrate non‐utilizing (nit) mutant strains at three phenological stages: early flowering, pre‐bunch closure (PBC) and veraison. Isolates recovered from symptomless berries at veraison and from bunches with symptoms at harvest were screened to measure the incidence of the nit strains used in the inoculations. It was found that latent infections, which resulted in BBR at harvest, could become established at all three phenological stages and no single stage was associated with greater latent incidence or harvest severity than any other stage. It was concluded that a proportion of BBR at harvest resulted from the expression of latent infections that had accumulated throughout the season. However, the time between infection and BBR symptom expression in near‐ripe grape berries was sufficiently short for polycyclic secondary infection to also contribute to epidemic development.     

Toxigenic Fusarium Species of Liseola Section in Pre-harvest Maize Ear Rot, and Associated Mycotoxins in Slovakia

The occurrence of Fusarium species of Liseola section and related toxins was investigated for two years (1996 and 1998) on maize ear rot samples collected in the most important areas for maize growing in Slovakia. The species most frequently isolated was F. verticillioides, followed by F. proliferatum in 1996 and F. subglutinans in 1998. Most of the strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusarium graminearum was also frequently recovered in both the years of investigations. Toxin analysis of maize ears showed that most of the samples (21 out of 22) were contaminated with at least one toxin. In particular, the concentration of fumonisin B₁, and fumonisin ₂ was up to 26.9 and 5.1gg^-1, respectively in 1996, and up to 12.1 and 6.3gg^-1, respectively in 1998. Beauvericin was detected only in one sample in 1996. Seven samples in 1996 were contaminated by fusaproliferin up to 8.2gg^-1, but just traces of the toxin were found in one sample in 1998. All 29 strains of F. verticillioides, two of three strains of F. proliferatum and none of eight F. subglutinans strains isolated from samples produced fumonisin B₁ in culture on whole maize kernels (0.1–5646 and 940–1200ugg^-1, respectively). Two strains of F. subglutinans and two of F. proliferatum produced beauvericin (up to 65 and 70gg^-1, respectively). Ten strains of F. verticillioides produced beauvericin: 9 strains produced a low amount (up to 3gg^-1), while only one of them produced a high level of toxin (375gg^-1). Fusaproliferin was produced by two F. proliferatum strains (220 and 370gg^-1), by seven F. subglutinans (20–1335gg^-1) and by three F. verticillioides (10–35gg^-1). This is the first report on fusaproliferin production by F. verticillioides, although at low level.     

渭北旱原酿酒葡萄与葡萄酒质量的研究

选取陕西省耀州区3个不同海拔高度(A点1 152 m,B点1 096 m,C点818 m)的葡萄园,对其葡萄与葡萄酒进行了成分分析和感官鉴定.结果表明,各点葡萄浆果含糖量以B点最高,A点最低;含酸量山地高于平地,山地上随海拔的升高而增加,糖酸比山地小于平地,在山地上随海拔的升高而递减;单宁含量以B点最高,C点最低.葡萄酒酒精发酵进程无明显差异,发酵趋势基本一致,发酵彻底,残糖均在2 g/L以下.葡萄酒的酒精度、总酸含量及单宁含量的变化趋势分别与浆果含糖量、含酸量和单宁含量相一致.山地葡萄酒的感官质量优于平地,在山地上又以海拔较低处优于海拔较高处.     

Studies on insects and mites causing galls on the leaves of purple willow Salix purpurea L. in the Ojców National Park in Poland

Analytical studies were conducted on the species composition of galls occurring on the leaves of Salix purpurea L. in the Ojców National Park in 1999–2000. The species composition of galls was identical in two consecutive years. Galls were caused by 5species (a total of 480galls). The most numerous species in both years was Pontania viminalis (L.). Index of occurrence frequency and Agrell's index of species co-existence have been calculated. This index reached the highest values in case of Pontania dolichura (Thoms.) and P. viminalis (L.) in 1999 as well as Phyllocolpa leucaspis (Tischb.) and P. viminalis (L.) in 2000.