S OF PRESENTATIONS AT T H E 27TH C O N G R E S S O F T H E I S R A E L I P H Y T O P A T H O L O G I C A L S O C I E T Y February 27-28, 2006 ARO, The Volcani Center, Bet Dagan, Israel A: PATHOGEN CHARACTERIZATION AND PROCESSES IN PATHOGENICITY Occurrence ofAspergillus carbonarius, the Producer of Ochratoxin A, in the Vineyards of Israel and in Table Grapes after Harvest A. Lichter, 1,* L. Guzev, 1 A. Ovadia, 2 Tirza Zahavi, a S. Ziv 1 and N. Paster 4 1Dept. of Postharvest Science of Fresh Produce [*e-mail: vtlicht@volcani.agri.gov.il] and 4Dept. of Food Science, ARO, The Volcani Center, Bet Dagan; 2Agronomia Ltd., Gedera; and 3Extension Service, Ministry of Agriculture and Rural Development, Qiryat Shemona, Israel Aspergillus carbonarius belongs to the A. niger group. Reports on its ability to produce the nephrotoxic and carcinogenic mycotoxin Ochratoxin A (OTA) raised the concern that its occurrence in vineyards might account for the presence of OTA detected in red wines in Europe. To address this issue, vineyards in several countries around the Mediterranean Basin were sampled during 3 years. In Israel, bunches without decay symptoms were sampled in ten vineyards of wine and table grapes at different stages of bunch development. The Aspergillus isolates on the surfaces of the berries were characterized morphologically, for production of OTA in vitro, and by molecular taxonomy for representative isolates. The following were the major findings: (i) The number of Aspergillus isolates present on the berries increased during the season. (ii) All the vineyards contained A. carbonarius. (iii) A large percentage of A. carbonarius isolates produced high levels of OTA. (iv) No OTA was identified in grape samples without decay symptoms. (v) More than one-third of the bunches that exhibited decay typical of black Aspergillus were infected with A. carbonarius. (vi) The infected part of the bunch contained OTA that did not translocate to the healthy section of the bunch. (vii) Application of fungicides in the vineyard did not achieve sufficient control of decay or fungal contamination of the berries. (viii) The composition of isolates on the berries did not change significantly after storage at 20~ or cold storage. (ix) Disinfecting the berries with ethanol was not effective in reducing the Aspergillus inoculum. (x) A correct dose of SO2 during cold storage decontaminated the berries from Aspergillus isolates. Many of these findings related to wine grapes were also reported by other countries participating in this research and they clearly indicate that there is an actual risk of contamination of grapes and their products with OTA. Recently, EU regulations concerning the presence of OTA in wine have been implemented. Therefore, there is an urgent need to develop useful means to minimize the contamination of grapes and their products with A. carbonarius in order to eliminate OTA from grapes and their products. [L] Expression of siRNA Targeted against T Y L C V C P Transcripts Leads to Silencing of C P Gene Expression and Resistance to the Virus A. Zrachya, 1'2 U. Ramakrishnan, 3 P.P. Kurnar, 3 Yael Levy, 1 A. Loyter, 2 T. Arazi, 4 M. Lapidot I and Y. Gafni 1'* L = lecture sessions; P = poster (market place) sessions.

The effects of nisin and propionic acid (PA) on aflatoxin production and on mycelial growth and spore germination of the mycotoxigenic fungi Aspergillus parasiticus, A. ochraceus, and Fusarium moniliforme were investigated. The growth of A. ochraceus was completely inhibited on media containing PA with nisin in concentrations of 0.05% PA with 1,000 ppm nisin, and 0.1% PA with 500 or 1,000 ppm nisin. The growth of both F. moniliforme and A. parasiticus was completely inhibited by PA with nisin at a concentration of 0.1% PA with 1,000 ppm nisin. Nisin alone caused a significant increase in mycelial growth when applied to A. ochraceus at 500 or 1,000 ppm and when applied to A. parasiticus at 1,000 ppm. Spore germination of A. ochraceus was completely inhibited on media containing 0.1% PA with 500 or 1,000 ppm nisin. Spores of F. moniliforme failed to germinate in 0.05% PA with 500 or 1,000 ppm nisin, whereas spores of A. parasiticus did not germinate on media containing 0.1% PA with 1,000 ppm nisin. For all three fungi tested, the inhibitory effect on mycelial growth was found to be fungistatic rather than fungicidal. The combined treatment of PA with nisin produced better fungistatic activity than treatment involving either material alone. Nisin, applied alone, did not stimulate aflatoxin production (expressed by microg toxin/mg mycelium), but the combined treatment at certain concentrations was inhibitory to aflatoxin B1 or G1. The production of aflatoxin G1, but not of B1, was stimulated in 0.05% PA with 1,000 ppm nisin and on media containing 0.1% PA with 100 ppm nisin. Nisin is currently applied in foods to prevent spoilage induced by bacteria but not by mold. The results of the present study indicate that a combined treatment of nisin in small concentrations of PA might be useful in preventing mold damage in certain foods and stored grain.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and floods. Three genes, ver-1, omt-1, and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis, respectively, have been identified, and their DNA sequences have been published. In the present study, three primer pairs, each complementing the coding portion of one of the genes, were generated. DNA extracted from mycelia of five Aspergillus species, four Penicillium species, and two Fusarium species was used as PCR template for each of the primer pairs. DNA extracted from peanut, corn, and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds Aspergillus parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined by using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only after a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10(2) spores per g). No DNA spores per g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive, and specific detection system, using PCR, for aflatoxigenic strains in grains and foods.

It was shown that the ability of three different strains of Penicillium expansum (NRRL 2034, NRRL 6069, and CBS 481.84) to grow and to produce patulin in pears (cv. Spadona) and apples (cv. Starking) varied under the different temperatures tested (0, 3, 6, 17 and 25 degrees C). Strain NRRL 2034 did not produce patulin at 0 or 25 degrees C while the two other strains produced the toxin at all temperatures, the maximum production occurring at 25 degrees C (for pears) and 17 degrees C (for apples). No significant differences in pathogenicity, as determined by lesion diameter, was recorded among the strains. Patulin production was totally inhibited when the fungi were grown in apples stored under 3% CO2/2% O2 (25 degrees C). The weight of infected tissue in apples contaminated with any of the strains and stored at that modified atmosphere was 70% that of the control. Under 3% CO2/10% O2 or 3% CO2/20% O2, strain NRRL 2034 did not produce patulin while the two other strains produced the toxin in different amounts.

Essential oils from oregano and thyme were applied for 24 h as fumigants against the mycelia and spores of Aspergillus flavus , Aspergillus niger and Aspergillus ochraceus , as well as against natural microflora of wheat grains. The minimal inhibitory concentration (MIC) of oregano oil needed to inhibit the mycelial growth of the fungi was 2.0 μl/L, while spores were eradicated following exposure to 2.0 to 2.5 μl/L. The thyme essential oil was less efficient in controlling mycelia and growth was observed even following exposure to 4.0 μl/L. However, the thyme essential oil was fungitoxic to spores (MIC = 3.0 μl/L). In another set of trials the efficacy of the oils and two of their constituents (carvacrol and thymol) in controlling natural microflora of surface-sterilized wheat grain was studied. Of the four materials investigated, only oregano essential oil exhibited fungicidal activity and, following 24 h exposure to 20 μl/L, a significant reduction in the percent of infested grain was observed even after 5 days of incubation on potato dextrose agar. A reduction in the germinability of the grains was evident following exposure to the materials tested. When the fungicidal activity of oregano essential oil was evaluated using grains with different moisture contents (MC), data revealed that the better inhibitory effect was achieved in grain with a high MC. The findings emphasize the toxicity of oregano and thyme essential oils as fumigants against fungi attacking stored grain and strengthen the possibility of using them as an alternative to chemicals for preserving stored grains.

Moulding and mycotoxin production have been studied during storage of rice at 85 and 90% relative humidity (r.h.) following treatment with cinnamon and clove oils. The clove oil-treated rice was artificially inoculated with Aspergillus flavus. Aflatoxin B1 and ochratoxin A were determined by monoclonal antibody-based enzyme-linked immunosorbent assay. Both moulding and mycotoxin were inhibited by 9 μl cinnamon oil g−1 but, with smaller doses, fungi could be isolated during the early part of storage, but not subsequently. Thus, following treatment with 3 or 6 μl cinnamon oil g−1, fungi were isolated from direct plated grains after 30 days but not after 45 days. Fungi were also isolated from washings of grain up to 30 days after treatment with 3 or 6 μl g−1, although fewer than from untreated grain. Propagules could also be detected up to 15 days after treatment with 9 μl g−1. Clove oil was much less effective than cinnamon oil in preventing moulding. After treatment with 8 μl g−1, the proportion of seeds infected with fungi and numbers of propagules in washings decreased only after 60 days' storage and with 4 μ g−1 after 90 days at both relative humidities. However, aflatoxin contamination could not be detected 90, 60 and 60 days after treatment with, respectively, 2, 4 and 8 μl g−1 at 85% r.h., although ochratoxin A was present in all treatments. At 90% r.h. aflatoxin was absent only after 90 days from treatment with 4 and 8 μl g−1. No T2 toxin was detected.