Themis J. Michailides

Ochratoxin Production by the Aspergillus ochraceus Group and Aspergillus alliaceus

ABSTRACT Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

Morphological, pathogenic, and molecular characterization of Alternaria isolates associated with alternaria late blight of pistachio

ABSTRACT Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arborescens species-groups were pathogenic to pistachio and no significant differences in pathogenicity were observed. Isolates from the infectoria species-group were only weakly pathogenic to pistachio. Based on cluster analysis of RAPD and PCR-RFLP data, three distinct clusters were evident; the infectoria cluster, the arborescens cluster, and a combined alternata/tenuissima cluster. Based on analysis of ITS sequence data, the infectoria species-group was phylogenetically distinct from the other species-groups. Isolates of the alternata, tenuissima, and arborescens species-groups comprised a monophyletic clade in which the three species-groups could not be further resolved.

Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

Effects of wounding, inoculum density, and biological control agents on postharvest brown rot of stone fruits

The effects of wounding, inoculum density, and three isolates (New, Ta291, and 23-E-6) of Trichoderma spp. and one isolate (BI-54) of Rhodotorula sp. on postharvest brown rot of stone fruits were determined at 20°C and 95% relative humidity (RH). Brown rot was observed frequently on wounded nectarine, peach, and plum fruits inoculated with two spores of Monilinia fructicola per wound, and occasionally on unwounded nectarine and peach fruits inoculated with the same spore load. Brown rot was observed on wounded plums only. A substantial increase in lesion diameter of brown rot was also recorded on wounded nectarines and peaches inoculated with suspensions of ≤20 spores and ≤200 spores per wound, respectively, compared with unwounded fruit. At concentrations of 107 and 108 spores per ml, all Trichoderma isolates substantially reduced brown rot on peaches (63 to 98%) and plums (67 to 100%) when fruits were inoculated with M. fructicola following the application of a biological control agent. Similarly, at 108 spores per ml, the yeast BI-54 also suppressed brown rot on peaches completely and on plums by 54%. Significant brown rot reduction was also achieved with the isolate New at a concentration of 108 spores per ml, even when the biocontrol agent was applied 12 h after inoculation with M. fructicola and under continuous conditions of 95% RH. The isolates Ta291 and 23-E-6 also reduced brown rot significantly under drier (50% RH) incubation conditions. These isolates provided the best control of brown rot on plums when they were applied 12 h earlier than inoculation with M. fructicola. Satisfactory control of brown rot on plums inoculated with M. fructicola at 8 × 104 spores per ml was achieved with New at 106 spores per ml and with Ta291 at 107 spores per ml. Measures taken to avoid injuring fruit will greatly reduce brown rot of stone fruit at any spore load for plum, but only at ≤50 spores per mm2 for peach, and at ≤5 spores per mm2 for nectarine. This study identifies two isolates (Ta291 and New) of Trichoderma atroviride, one isolate (23-E-6) of T. viride, and one of Rhodotorula sp. that show potential for further development as biocontrol agents of postharvest brown rot of stone fruits.

Aspergillus species and mycotoxins in figs from California orchards.

Although 23 different Aspergillus spp. decayed figs in California orchards, only A. niger occurred in more than 0.2% of the figs. The black-spored Aspergillus isolates that caused the disease fig smut were classified as A. niger var. niger, A. niger var. awamori, A. japonicus, and A. carbonarius. Different fungi differed in their association with Aspergillus Section Nigri (causal agents of fig smut) infections in figs. For example, most figs infected with Aspergillus Section Flavi (potential aflatoxin producers) also had infections by Section Nigri. For other fungi, there was either no significant relationship between fig infections by these fungi and Section Nigri or simultaneous infections by Section Nigri were fewer than expected. Insect damage to the fig fruit, predominantly by navel orangeworm (Amyelois transitella), did not significantly increase the colonization of figs by Aspergillus spp. The incidences of infection by Aspergillus (Sections Nigri, Aspergillus, Flavi, and Circumdati) in figs differed little for different harvests. Figs naturally infected with A. alliaceus, A. melleus, A. ochraceus, and A. sclerotiorum of Aspergillus Section Circumdati contained ochratoxin up to 9,600 ng/g, although only 40% of the figs with these fungi had more than a trace amount of ochratoxin. Aflatoxin contamination in figs naturally infected with Aspergillus Section Flavi varied according to the species involved. No aflatoxins were detected in all figs infected with A. tamarii and in most figs infected with A. flavus. High levels of aflatoxin (>100 ng/g) were detected in 83% of the figs infected by A. parasiticus, but in only 32% of the figs infected by A. flavus. Section Flavi isolates from fig orchard soils were tested for their ability to produce the mycotoxins aflatoxin and cyclopiazonic acid. Aspergillus parasiticus isolates always produced aflatoxin but never cyclopiazonic acid; A. flavus strain S (producers of small sclerotia) isolates always produced both aflatoxin and cyclopiazonic acid, but strain L (producers of large sclerotia) isolates frequently did not produce aflatoxin or cyclopiazonic acid; and A. tamarii isolates never produced aflatoxin but always produced cyclopiazonic acid. Aspergillus flavus was recovered from the soil, at fewer than 6 CFU/g of dry soil of every fig orchard assayed in 1992 and 1993. Although A. parasiticus was rarer in fig fruit than was A. flavus for each year, in orchard soil A. parasiticus was more frequent than A. flavus. Isolates of A. flavus strain L were much more common in the orchard soil and fig fruit than those of strain S. Figs in commercial orchards seem to be a favorable substrate for infection by and growth of Aspergillus spp.

Resistance to Boscalid Fungicide in Alternaria alternata Isolates from Pistachio in California

Boscalid is a new carboxamide fungicide recently introduced in a mixture with pyraclostrobin in the product Pristine for the control of Alternaria late blight of pistachio. In all, 108 isolates of Alternaria alternata were collected from pistachio orchards with (59 isolates) and without (49 isolates) prior exposure to boscalid. The sensitivity to boscalid was determined in conidial germination assays. The majority of isolates from two orchards without a prior history of boscalid usage had effective fungicide concentration to inhibit 50% of spore germination (EC50) values ranging from 0.089 to 3.435 μg/ml, and the mean EC50 was 1.515 μg/ml. Out of 59 isolates collected from an orchard with a history of boscalid usage, 52 isolates had EC50 values ranging from 0.055 to 4.222 μg/ml, and the mean EC50 was 1.214 μg/ml. However, in vitro tests for conidial germination and mycelial growth also revealed that seven A. alternata isolates, originating from the orchard exposed to boscalid were highly resistant (EC50 > 100 μg/ml) to this fungicide. Furthermore, in vitro tests showed no significant differences between wild-type and boscalid-resistant mutants in some fitness parameters such as spore germination, hyphal growth, sporulation, or virulence on pistachio leaves. Experiments on the stability of the boscalid-resistant phenotype showed no reduction of the resistance after the mutants were grown on fungicide-free medium. Preventative applications of a commercial formulation of boscalid (Endura) at a concentration which is effective against naturally sensitive isolates failed to control disease caused by the boscalid-resistant isolates in laboratory tests. To our knowledge, this is first report of field isolates of fungi resistant to boscalid.

Epidemiology of Botrytis cinerea in Orchard and Vine Crops

Substantial economic crop losses occur worldwide in tree fruits, nut crops, vines and small fruits as a result of infection by Botrytis cinerea. Fungicide-based management, once an accepted practice, is becoming increasingly restricted, a trend likely to continue in the future. Greater emphasis on alternative, non-chemical control will require improved knowledge of B. cinerea ecology and epidemiology in affected crops. Epidemics are often initiated in the spring from conidial inoculum produced on over-wintering structures on a very wide range of plant species. From floral infection in the spring, several infection pathways to fruit infection and crop loss at harvest are described. The majority of these pathways include a degree of symptomless latency, or quiescence, in the host tissue. In some crops (e.g. grapes) multiple pathways are described, each one dependant upon many complex host, pathogen and environmental factors. In other crops (e.g. berry fruits), a single dominant pathway is described. Latency, once poorly understood, has become the focus of research in the last decade. Several host defence mechanisms are described which may account for this period of enforced dormancy. Once pathogenic growth resumes and typical B. cinerea symptoms appear, many factors affect the subsequent rate of B. cinerea epidemics and we describe some of these in detail. The growth of organic production in the last decade has high-lighted the need for a greater understanding of the complexities of epidemic development in order to develop durable and sustainable disease control strategies.

Significance of thinned fruit as a source of the secondary inoculum of Monilinia fructicola in California nectarine orchards

The significance of thinned fruit as a source of secondary inoculum in the spread of brown rot, caused by Monilinia fructicola, under semi-arid weather conditions of the San Joaquin Valley in California, was investigated in seven nectarine orchards in 1995 and 1996. Between 6 and 60% (depending on the orchard) of thinned fruit showed sporulation by M. fructicola. Brown rot was significantly less severe at preharvest (five orchards) and postharvest (one orchard) on fruit harvested from trees in plots from which thinned fruit were completely removed than on those in plots from which thinned fruit were not removed. M. fructicola sporulated more frequently on thinned fruit placed into irrigation trenches than on those left on the dry berms in tree rows. The incidence of preharvest fruit brown rot increased exponentially as the density of thinned fruit increased on the orchard floor. These results suggest that thinned fruit left on the floor of nectarine orchards can be a significant inoculum source of secondary infections. Removal or destruction of thinned fruit should reduce brown rot in nectarine and possibly other stone fruit orchards under semi-arid California conditions.

Characterization of Mutations in the Iron-Sulphur Subunit of Succinate Dehydrogenase Correlating with Boscalid Resistance in Alternaria alternata from California Pistachio

Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.

Genetic Structure of Botrytis cinerea Populations from Different Host Plants in California

The population structure of Botrytis cinerea was investigated by using transposable elements, DNA fingerprinting generated by microsatellite primed-polymerase chain reaction (MP-PCR), and sensitivity to the hydroxyanilide fungicide, fenhexamid, for 234 isolates collected from fig, grape, kiwifruit, pea, and squash in California. Among 234 isolates tested, 195 had two transposable elements, Boty and Flipper (transposa type), 38 had only the Boty element (Boty type), and one had neither of these elements (vacuma type). Four of these 234 isolates, which belonged to the Boty type, were resistant to fenhexamid. A phenogram generated based on MP-PCR markers showed that the isolates were not clustered based on their source hosts or the presence of transposable elements. Analysis of molecular variance (AMOVA) showed that there were no significant genetic differentiations among isolates collected from grape, kiwifruit, pea, and squash at the Kearney Agricultural Center. A more detailed analysis based on AMOVA partition of the total genetic variance indicated that 96% of the variation occurred within populations. The parsimony tree length permutation (PTLPT) and index of association ( IA) analyses of MP-PCR data set were consistent with absence of sexual recombination in sampled populations of this pathogen.