Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins.

AIMS: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA-producing fungi from California raisin vineyard populations. METHODS AND RESULTS: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g⁻¹. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 µg g⁻¹. CONCLUSIONS: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that low-level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback.

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.

A Reassessment of the Species Concept in Eutypa lata, the Causal Agent of Eutypa Dieback of Grapevine.

ABSTRACT Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and beta-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.

The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus.

Aflatoxins are highly toxic and carcinogenic compounds produced by certain Aspergillus species on agricultural commodities. The presence of fatty acid hydroperoxides, which can form in plant material either preharvest under stress or postharvest under improper storage conditions, correlates with high levels of aflatoxin production. Effects on fungal growth and aflatoxin production are known for only a few of the numerous plant metabolites of fatty acid hydroperoxides. Jasmonic acid (JA), a plant growth regulator, is a metabolite of 13-hydroperoxylinolenic acid, derived from alpha-linolenic acid. The volatile methyl ester of JA, methyl jasmonate (MeJA), is also a plant growth regulator. In this study we report the effect of MeJA on aflatoxin production and growth of Aspergillus flavus. MeJA at concentrations of 10(-3)-10(-8) M in the growth medium inhibited aflatoxin production, by as much as 96%. Exposure of cultures to MeJA vapour similarly inhibited aflatoxin production. The amount of aflatoxin produced depended on the timing of the exposure. MeJA treatment also delayed spore germination and inhibited the production of a mycelial pigment. These fungal responses resemble plant jasmonate responses.

Inhibition of aflatoxin production by surfactants.

The effect of 12 surfactants on aflatoxin production, growth, and conidial germination by the fungus Aspergillus flavus is reported. Five nonionic surfactants, Triton X-100, Tergitol NP-7, Tergitol NP-10, polyoxyethylene (POE) 10 lauryl ether, and Latron AG-98, reduced aflatoxin production by 96 to 99% at 1% (wt/vol). Colony growth was restricted by the five nonionic surfactants at this concentration. Aflatoxin production was inhibited 31 to 53% by lower concentrations of Triton X-100 (0.001 to 0.0001%) at which colony growth was not affected. Triton X-301, a POE-derived anionic surfactant, had an effect on colony growth and aflatoxin production similar to that of the five POE-derived nonionic surfactants. Sodium dodecyl sulfate (SDS), an anionic surfactant, and dodecyltrimethylammonium bromide, a cationic surfactant, suppressed conidial germination at 1% (wt/vol). SDS had no effect on aflatoxin production or colony growth at 0.001%. The degree of aflatoxin inhibition by a surfactant appears to be a function of the length of the hydrophobic and hydrophilic chains of POE-derived surfactants.

Structure-activity relationship of flavones as growth inhibitors of the navel orangeworm.

A series of flavones with widely varying degrees of substitution was fed to neonate larvae of the navel orangeworm. Growth of navel orangeworm larvae is inhibited by 5-methoxy flavones and flavone itself; 5-hydroxy flavones do not inhibit growth. Host resistance of citrus fruit to attack by the navel orangeworm might be due to the 5-methoxy flavones that occur in the peels.