Comparison of Alternaria spp. collected in Italy from apple with A. mali and other AM-toxin producing strains.

Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in northern Italy resulting in necrotic spots on leaves and on fruit. Forty-four single-spored isolates were obtained from diseased plant materials to investigate the diversity of this fungus in Italy and to compare these isolates to isolates of Alternaria associated with apple disease in previous studies, including A. mali, causal agent of apple blotch. All isolates, including the reference strains, were tested for pathogenicity utilizing in vitro bioassays on detached leaf or on fruit ('Golden Delicious'). In addition, morphological characterizations were conducted describing both the three-dimensional sporulation pattern and the colony morphology of each isolate. In order to assess the genetic diversity within the Italian Alternaria population, sequence characterization of specific loci and anonymous regions (endoPG, OPA1-3, OPA2-1, and OPA10-2) and genetic fingerprinting based on amplified fragment length polymorphism and inter simple sequence repeat markers were performed. The single spore isolates exhibited differential pathogenicity, which did not correlate with the morphological groupings or to groupings defined by molecular approaches. Moreover, 10 pathogenic isolates out of the 44 single-spored tested were positive for the host-specific AM-toxin gene based upon polymerase chain reaction amplification using specific primers for the AM-toxin gene. This suggests that the production of the AM-toxin may be involved in pathogenesis by some of the Italian isolates of A. alternata from apple. However, this research also suggests that a number of different Alternaria genotypes and morphotypes may be responsible for the apple disease in Italy and that a single taxon cannot be defined as the sole causal agent.

Effect of Sclerotium Density and Irrigation on Disease Incidence and on Efficacy of Coniothyrium minitans in Suppressing Lettuce Drop Caused by Sclerotinia sclerotiorum.

Field experiments were conducted over 2 years in Yuma, AZ, and Holtville, CA, to establish the relationship between soil sclerotium density of Sclerotinia sclerotiorum and the incidence of lettuce drop on different lettuce (Lactuca sativa) types under different irrigation systems, and to determine the efficacy of the biocontrol agent Coniothyrium minitans (Contans) against S. sclerotiorum on crisphead lettuce at varied sclerotium densities under different irrigation systems. There was no significant interaction of irrigation (overhead sprinkler versus furrow) with either sclerotium density or with biocontrol treatment. Lettuce drop incidence was lowest in romaine lettuce compared with crisphead or leaf lettuce at all soil sclerotium densities. There was a significant positive correlation between the sclerotial density and the percent disease incidence. Disease incidence in plots infested with 2 sclerotia/m2 of bed was not significantly higher than in control plots regardless of lettuce type. However, plots infested with 40 or 100 sclerotia/m2 of bed revealed a significantly higher disease incidence over the control in all lettuce types. A single application of Contans at planting significantly reduced the incidence of lettuce drop in all lettuce types even under high disease pressure. There were no significant differences between recommended (2.2 kg/ha) and high (4.4 kg/ha) application rates of Contans or between one or two applications of the product.

An expanded multilocus phylogeny does not resolve morphological species within the small-spored Altemrnaria species complex.

Small-spored Alternaria species are a taxonomically challenging group of fungi with few morphological or molecular characters that allow unambiguous discrimination among taxa. The protein-coding genes most commonly employed in fungal systematics are invariant among these taxa, so noncoding, anonymous regions of the genome were developed to assess evolutionary relationships among these organisms. Nineteen sequence-characterized amplified regions (SCAR) were screened for phylogenetic utility by comparing sequences among reference isolates of small-spored Alternaria species. Five of nineteen loci were consistently amplifiable and had sufficient phylogenetic signal. Phylogenetic analyses were performed with 150 small-spored Alternaria isolates using sequence data from an endopolygalacturonase gene and two anonymous loci. Associations among phylogenetic lineage, morphological classification, geography and host were evaluated for use as practical taxonomic characters. Samples included isolates from citrus in Florida, pistachio in California, desert plants in Arizona, walnuts in France/Italy and apples in South Africa. No associations were found between host or geographic associations and phylogenetic lineage, indicating that these characters were not useful for cladistic classification of small-spored Alternaria. Similarly strict congruence between morphology and phylogenetic lineage was not found among isolates grouped morphologically with A. alternata or A. tenuissima. In contrast 34 isolates grouped morphologically with A. arborescens fell into discrete clades for all datasets. Although 5-9 well supported clades were evident among isolates, it is currently unclear if these clades should be considered phylogenetic species or emerging evolutionary lineages within the phylogenetically defined alternata species-group.

Biocontrol of Lettuce Drop Caused by Sclerotinia sclerotiorum and S. minor in Desert Agroecosystems.

Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to determine the efficacy of several biocontrol agents for the management of lettuce drop caused by Sclerotinia spp. Commercial formulations of Trichoderma harzianum (Plantshield, Supersivit), Gliocladium virens (Soilgard), Coniothyrium minitans (Contans), and Bacillus subtilis (Companion) were evaluated and compared with the chemical fungicide iprodione (Rovral) against Sclerotinia sclerotiorum and S. minor. A single application of biocontrol products or of Rovral did not reduce lettuce drop caused by either Sclerotinia species. However, two applications of Contans, one at planting and one at post-thinning, significantly reduced the incidence of lettuce drop caused by S. sclerotiorum and increased yield but had no effect on S. minor at both locations in both years. Two applications of other biocontrol products did not significantly reduce disease incidence despite medium to high recovery following application. In contrast, Contans was only sporadically recovered following application. In vitro fungicide sensitivity evaluation revealed that both Trichoderma and Gliocladium species were tolerant to iprodione, dicloran (Botran), and vinclozolin (Ronilan) up to 1,000 ppm a.i., whereas both Sclerotinia spp. and C. minitans were sensitive to all three fungicides above 1 ppm. In summary, Contans was the most effective treatment for the control of lettuce drop caused by S. sclerotiorum, but no treatment was effective against S. minor in the desert lettuce production systems.

First Report of Seedling Damping-Off of Fennel Caused by Alternaria petroselini in the Netherlands.

In April 2005, serious seedling damping-off was noted on fennel (Foeniculum vulgare Mill. cv. Rondo) in a transplant greenhouse facility in Maasdijk, the Netherlands. Symptoms appeared 3 to 4 weeks after sowing and included black, sunken lesions aboveground on the stem and belowground on the hypocotyls. Mortality of seedlings was 6 to 10% (10 to 15 seedlings per 150-plant tray). Following removal of diseased seedlings, further transplant mortality in the field was not evident. Samples of diseased tissue were collected, surface disinfested, and placed in petri dishes containing water agar. After 7 to 10 days of incubation at 25°C under fluorescent lights, an Alternaria sp. was growing from each sample. Single conidium cultures were obtained from representative colonies and cultured on potato dextrose agar (PDA) and potato carrot agar (PCA) for morphological examination. On PDA, colonies were dark olive brown, cottony, subsurface microsclerotia production abundant, and no production of pigments in the medium. On PCA, conidia were darkly pigmented, broadly ellipsoidal to subsphaerical, and produced singly. Mature conidia were 28 to 45 × 20 to 25 µm with two to four transepta and one to three longisepta. Characteristics were consistent with those of Alternaria petroselini (2,3). In subsequent freezer-blotter assays (ISTA blotter method; www.seedtest.org ) of seed lot used in the original planting, the same fungus was recovered at an infestation level of 30%, confirming that it was seedborne. To confirm pathogenicity on fennel, pathogenicity tests were conducted on a common fennel cultivar (Floro F1) in the greenhouse and on fennel stalks in the laboratory. Fennel seeds were soaked in a conidia suspension (106/ml in sterile H2O) for 10 min. Control seeds were soaked in sterile H2O. Seeds were dried on paper, sown in soil plugs, and grown in the greenhouse at 16 to 20°C. After 4 weeks, black lesions were observed on the fennel stems and symptoms were similar to those observed on the original infected material. Control plants remained healthy. A fungus was reisolated from the lesions of symptomatic plants and was identical to the fungus isolated from the original infected material. For the fennel stalk assay, two surface-sterilized fennel stalks were sliced longitudinally and three 4-mm plugs from a 10-day-old culture of each isolate were placed along the fennel stalks. Sterile agar plugs were used as negative controls. After 7 to 10 days of incubation at 25°C in plastic boxes, test isolates grew extensively from agar plugs and resulted in extensive black necrosis of the fennel stalks. No necrosis resulted from control plugs. DNA was extracted from field isolates, and the nuclear internal transcribed spacer region was sequenced using protocol previously described (1). A representative sequence was deposited in GenBank (Accession No. EF636901). A BLAST search of the NCBI database revealed A. petroselini Accession No. AY154685 as the closest match (total score = 1,014, 100% coverage, 99% sequence identity). The next closest match was A. radicina Accession No. DQ394074 (total score 987, 100% coverage, 98% sequence identity). To our knowledge, this is the first report of A. petroselini causing disease of fennel and the fungus being seedborne on fennel seed. An isolate has been deposited at the Centraalbureau voor Schimmelcultures (Accession No. 118228). References: (1) B. M. Pryor and D. M. Bigelow. Mycologia 95:1141, 2003. (2) B. M. Pryor and R. L. Gilbertson. Mycologia 94:49, 2002. (3) E. G. Simmons. Mycotaxon 55:55, 1995.

First Report of Alternaria Species Groups Involved in Disease Complexes of Hazelnut and Walnut Fruit.

During the last 5 years, two new diseases, brown apical necrosis (BAN) and gray necrosis (GN), were observed on English walnut (Juglans regia) and hazelnut (Corylus avellana), respectively (2,3). Both diseases caused severe fruit drop resulting in yield loss often exceeding 30%. Previous work demonstrated that BAN and GN are disease complexes caused by several fungi (Alternaria spp., Fusarium spp., and a Phomopsis sp.) (2,3). In both diseases, preliminary identification of Alternaria spp. revealed they were a complex of small-spored catenulate taxa related to A. alternata. To further characterize these taxa, additional pathogenicity tests and morphological examinations were conducted with isolates obtained from each host. Single-spored isolates were prescreened for pathogenicity by inoculating detached, surface-disinfested hazelnut leaves or walnut leaflets (1). Only isolates that produced foliar lesions after 5 days were used in subsequent fruit inoculations. From this screening, 35 isolates were selected (19 from walnut and 16 from hazelnut). For each isolate, attached fruit of respective hosts were inoculated at bloom by placing 10 µl of a conidial suspension (1 × 106 conidia per ml of H2O + 0.26% agar) onto the stigmas (150 fruit per isolate). Controls (150 fruit) were treated with agar solution only. After 15 days, fruit were examined for development of disease symptoms, and examination continued until fruit maturation (late July). Approximately 20 to 50% of the inoculated fruit displayed discoloration or necrosis of internal tissue, particularly the pericarp and the embryo, although symptoms were more limited than those typically seen in fully expressed BAN and GN. No differences in symptoms were evident among the isolates tested. The controls showed no symptom development initially, although 5% began to develop discoloration at fruit maturity. Fungal isolates used as inoculum were reisolated from all symptomatic fruit by surface disinfesting tissue from the margins of necrotic lesions. For each isolate, the conidial characteristics were described from cultures grown under defined conditions (4). Three distinct groups of isolates were identified. Alternata sp. group isolates produced conidial chains (8 to 20 spores) with numerous secondary and occasionally tertiary chains branching from apical and median cells. Conidia were typically ovate and often possessed a one-celled apical extension. Tenuissima sp. group isolates developed conidial chains (10 to 22 spores) with occasional branching forming secondary chains from apical and median cells. Conidia were ovate to obclavate, often with long apical extensions (10 to 35 µm). Arborescens sp. group isolates developed conidial chains (5 to 12 spores) with numerous secondary, tertiary, and quaternary short chains branching from apical cells. Conidia were typically ovate with minimal apical extensions. Of the walnut isolates, 12, 4, and 3 were from the arborescens, alternata, and tenuissima sp. groups, respectively. Of the hazelnut isolates, 7, 6, and 3 were from the arborescens, alternata, and tenuissima sp. groups, respectively. The finding that Alternaria from several distinct sp. groups can cause similar disease on a single host is consistent with previous work on pistachio, almond, and pear (4). References: (1) A. Belisario et al. Plant Dis. 83:696, 1999. (2) A. Belisario et al. Plant Dis. 86:599, 2002. (3) A. Belisario et al. Inf. Agrario 59:71, 2003. (4) B. M. Pryor et al. Phytopathology 92:406, 2002.

Survival and Persistence of Alternaria dauci in Carrot Cropping Systems.

Alternaria dauci was recovered in California from carrot crop residue and from volunteer carrot plants in fallow carrot fields. The fungus was not recovered from common weeds surrounding fallow fields. To evaluate further the survival of A. dauci on carrot crop residue, infected carrot leaf tissue was placed in fields or in soil in greenhouse pots, and recovered over time. In California, A. dauci was recovered from infected leaf tissue in both fallow and irrigated fields for as long as 1 year. In Florida, A. dauci was recovered from infected leaf tissue in fallow fields for up to 30 weeks. In greenhouse experiments, A. dauci was recovered from infected leaf tissue for as long as 1 year in dry soil, but only up to 30 weeks in soil that was watered weekly. To determine the infectivity of A. dauci borne on carrot crop residue, infected carrot crops were incorporated into organic and mineral field soils, and soil samples were collected over time. Carrot seed were planted in collected soil, and seedling infection by A. dauci was recorded. Seedling infection was detected up to 13 and 14 weeks after crop incorporation in organic and mineral soil, respectively. Seedling infection was detected for up to 5 weeks in soil that remained dry compared with 3 weeks in flooded soil.

A PCR-based Assay for Detection of Alternaria radicina on Carrot Seed.

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Detection of Soilborne Alternaria radicina and Its Occurrence in California Carrot Fields.

Alternaria radicina, causal agent of black rot disease of carrot, was recovered from soil by plating dilutions on a semi-selective medium, A. radicina semi-selective agar. The efficiency of this soil assay was 93% based on recovery of the fungus from non-infested field soil amended with A. radicina conidia. Soilborne A. radicina was recovered from five of six carrot-growing areas in California, but was only commonly found in the Cuyama Valley, where the fungus was detected in 83% of sampled fields. Over a 3-year period of sampling, A. radicina soil populations in Cuyama Valley fields prior to carrot planting ranged from 0 to 317 CFU/g. There was a positive correlation between A. radicina soil populations in these fields and the incidence of black rot disease at harvest. A. radicina was recovered from dry soil after 4 years of storage, and the fungus survived in this soil as solitary conidia or as conidia associated with organic debris.