Paola Giorni

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.

Penicillium populations in dry-cured ham manufacturing plants

Seven ham manufacturing plants were sampled for 1 year to assess the mycoflora present in the air and on hams, with special attention given to potential mycotoxin producers. Temperature and relative humidity were recorded in the ripening rooms. Maturing rooms held hams from 2 to 3 through 6 to 7 ripening months, and aging rooms held hams for the following 6 to 7 months, until the 14-month ripening point, when they were ready for the market. Mean temperatures and relative humidities registered during the study were 14.9 degrees C and 62.4%, respectively, in maturing rooms and 16.3 degrees C and 57.6% in aging rooms. Aspergilli and penicillia, potential mycotoxin producers, were isolated in all the plants from the air and the ham. Aspergilli represented 5% of the isolates, while penicillia were largely dominant, with Penicillium nalgiovense being the most represented species (around 60% of the penicillia), followed by Penicillium nordicum, with 10 and 26% of the penicillia isolated, respectively, from the air or the ham. Ochratoxin A production ability, checked in vitro at 250C, was observed in 50% of the P. nordicum isolates obtained both from the air and the ham. Air and ham surface contamination by penicillia was greater in the ripening rooms, where higher temperatures were registered. A certain correlation was also observed between air and ham surface contamination. On the basis of this study, P. nordicum, the ochratoxin A producer that is notable on proteinaceous substrates, is normally present in ham manufacturing plants in Italy, even though not a dominant species. Further studies are necessary to clarify and ensure if dry-curing conditions minimize the potential risk of ochratoxin A formation in the product.

Environmental factors modify carbon nutritional patterns and niche overlap between Aspergillus flavus and Fusarium verticillioides strains from maize.

This study examined the utilization patterns of key carbon sources (CS, 24: including key sugars, amino acids and fatty acids) in maize by strains of Aspergillus flavus and Fusarium verticillioides under different water activity (a(w), 0.87-0.98 a(w)) and temperature (20-35 degrees C) values and compared the niche overlap indices (NOI) that estimate the in vitro CS utilization profiles [Wilson, M., Lindow, S.E., 1994. Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Applied and Environmental Microbiology 60, 4468-4477.]. The ability to grow in these key CS in minimal media was studied for 120 h in 12 h steps. The NOI was calculated for inter-species (F. verticillioides-A. flavus) and for intra-species (A. flavus-A. flavus) using CS utilization patterns over the range of interacting environmental conditions. 30 degrees C, over the whole a(w) range examined, was found to be optimal for utilization of the maximum number of CS by A. flavus. In contrast, for F. verticillioides this was more so at 20 degrees C; 25 degrees C allowed a suboptimal usage of CS for both species. NOIs confirmed the nutritional dominance of A. flavus at 30 degrees C, especially at lower a(w) levels and that of F. verticillioides at 20 degrees C, mainly at 0.95 a(w). In other conditions of a(w), based on CS utilization patterns, the data indicated that A. flavus and F. verticillioides occupied different ecological niches. The variability in nutritional sources utilization between A. flavus strains was not related to their ability to produce aflatoxins (AFs). This type of data helps to explain the nutritional dominance of fungal species and strains under different environmental conditions. This could be useful in trying to find appropriate natural biocontrol microorganisms to compete with these mycotoxigenic species.

Field control of Fusarium ear rot, Ostrinia nubilalis (Hübner), and fumonisins in maize kernels

BACKGROUND A 6 year study was conducted to evaluate the possible support of pesticides in mitigating mycotoxin contamination in maize grown in northern Italy. Different pesticides to control Ostrinia nubilalis (Hübner) (ECB), Fusarium verticillioides (Sacc.) Nirenb. and Aspergillus flavus Link were considered to check their efficacy and the best schedule to reduce fumonisin and aflatoxin contamination. RESULTS Fumonisin B1 (FB1) contamination at harvest has been reduced by ECB control, while the addition of ingredients targeted to F. verticillioides gave a limited advantage, mainly with low FB1 contamination; nevertheless, fusaria control is relevant in high-risk conditions, when FB1 is likely to exceed the legal limit. The combination of fungal and pest control in a single spray at silk browning stage (BBCH 67) is the best way of application. These results are robust because they were obtained in a 6 year trial, with relevant differences in meteorological conditions, ECB and fungal presence and mycotoxin contamination. CONCLUSION Insecticides have been shown to give advantages in their application, while the low FB1 contamination in the final years of the study did not lead to conclusive data on the advantage of adding a fungicide. Chemicals applied did not modify A. flavus incidence or aflatoxin contamination (only detected in trace amounts).

Comparison of temperature and moisture requirements for sporulation of Aspergillus flavus sclerotia on natural and artificial substrates.

A key step in the infection cycle by Aspergillus flavus in maize is sporulation of sclerotia present in soil or in crop debris. However, little information is available on this critical and important phase. This study included experiments on artificial (Czapek Dox Agar (CZ)) and natural (maize stalks) substrates under different conditions of temperature (T; from 5 to 45 °C) and water activity (a(w); from 0.50 to 0.99) levels to quantify sporulation from sclerotia. The mean numbers of spores were higher on defined nutritional medium in vitro on CZ agar than on maize stalks (4.5×10(6) spores/sclerotium versus 4.2×10(4) spores/sclerotium) with production initiated after 6 and 24h, respectively. Surprisingly, the optimal temperature was found at 30-35 °C for CZ agar (9.23×10(6) spores/sclerotium) and to be 20-25 °C for maize stalks (6.26×10(4) spores/sclerotium). Water stress imposition only reduced sporulation at ≤0.90 a(w.) With more available water no significant differences were found between 0.90 and 0.99 a(w). This type of data is critical in the development of a mechanistic model to predict the infection cycle of A. flavus in maize in relation to meteorological conditions.

Growth and aflatoxin production of an Italian strain of Aspergillus flavus: influence of ecological factors and nutritional substrates

The aim of this study was to define quantitative relationships between temperature and water activity (aw), fungal growth and aflatoxin B1 (AFB1) production. A strain of Aspergillus flavus isolated from maize in north Italy, and previously tested and found positive for AFB1 production, was used for these experiments. The optimum temperature for AFB1 production was at 25 °C, slightly lower with respect to results obtained in other countries. 0.83 aw was the limit for growth of this strain of A. flavus after 60 days incubation at the optimum temperature. The solutes used to modify aw, glycerol and NaCl, influenced both growth and secondary metabolite production. Media modified with glycerol resulted in more AFB1 production when compared to the non-ionic solute NaCl added media. Maize based media, prepared with flour obtained from kernels at different ripening stages, only slightly influenced growth rates of A. flavus. The quantitative relationship obtained between fungal growth and AFB1 production in divers...

LDS1-produced oxylipins are negative regulators of growth, conidiation and fumonisin synthesis in the fungal maize pathogen Fusarium verticillioides

Oxylipins are fatty acid-derived signaling compounds produced by all eukaryotes so far investigated; in mycotoxigenic fungi, they modulate toxin production and interactions with the host plants. Among the many enzymes responsible for oxylipin generation, Linoleate Diol Synthase 1 (LDS1) produces mainly 8-hydroperoxyoctadecenoic acid and subsequently different di-hydroxyoctadecenoic acids. In this study, we inactivated a copy of the putative LDS1 ortholog (acc. N. FVEG_09294.3) of Fusarium verticillioides, with the aim to investigate its influence on the oxylipin profile of the fungus, on its development, secondary metabolism and virulence. LC-MS/MS oxylipin profiling carried out on the selected mutant strain revealed significant quali-quantitative differences for several oxylipins when compared to the WT strain. The Fvlds1-deleted mutant grew better, produced more conidia, synthesized more fumonisins and infected maize cobs faster than the WT strain. We hypothesize that oxylipins may act as regulators of gene expression in the toxigenic plant pathogen F. verticillioides, in turn causing notable changes in its phenotype. These changes could relate to the ability of oxylipins to re-shape the transcriptional profile of F. verticillioides by inducing chromatin modifications and exerting a direct control on the transcription of secondary metabolism in fungi.

Structure of an Aspergillus flavus population from maize kernels in northern Italy.

In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy between 2003 and 2010, were unable to produce detectable levels of aflatoxins. The genetic diversity of the population was assessed by analysis of vegetative compatibility groups (VCGs) and presence or absence of several aflatoxin biosynthesis genes. Forty-eight VCGs were identified through complementation between nitrate non-utilizing mutants. Twenty-five VCGs contained only atoxigenic isolates, and the remaining 23 only aflatoxin producers. Members of the largest atoxigenic VCG (IT6) were found in 4 of the 5 districts sampled. Six deletion patterns of genes in the aflatoxin biosynthesis gene cluster were detected. No deletions in the cluster were detected for twelve atoxigenic isolates and 10 had the entire cluster deleted. One isolate had a deletion pattern only seen once before in Nigeria. The basis for initial selection of endemic atoxigenic strains of A. flavus for biological control of aflatoxin contamination of maize in Italy is provided.

Fusarium verticillioides and maize interaction in vitro: relationship between oxylipin cross-talk and fumonisin synthesis

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the path...

Maize lipids play a pivotal role in the fumonisin accumulation

The role of lipids in maize – Fusarium verticillioides interaction and fumonisin production in natural field conditions were investigated. In 2010, ten maize hybrids were grown in fields located in 3 districts in Northern Italy and sampled at 4 growing stages, from early dough to full ripe. Chemical composition, fungal incidence and free and hidden fumonisin contamination were determined in all grain samples. All the hybrids considered within this study showed a strong fungal incidence, with Fusarium section Liseola as prevalent, already at the early dough maturity and along the ripening period. Fumonisins accumulated over the growing season and reached the maximum level at the full ripe stage; hidden fumonisins were found significant in all the considered samples (~57% of the free form at harvest). Hybrid H9 showed more than 50% of kernels infected by Aspergillus flavus and no hidden fumonisins were detected. This finding stresses the relevance of monitoring both free and total fumonisins for a comprehen...