How Does Botrytis cinerea Infect Red Raspberry?

Botrytis cinerea, causal agent of gray mold, is one of the most important pathogens affecting raspberry in the U.S. Pacific Northwest and worldwide. Fungicides are currently applied to control the disease starting from 5 to 10% bloom and continuing on a calendar basis throughout the season rather than according to inoculum level or infection risk primarily because the disease cycle on red raspberry is poorly understood. Botrytis cinerea was isolated from raspberry flowers and fruit sampled at seven developmental stages during each of 2015 and 2016 in a northwestern Washington raspberry field untreated with fungicides. Incidence of colonization of flowers was low (15% of total sampled flowers), but increased as fruit developed, and peaked in mature fruit (67% of total sampled fruit). In the early stages of flower development, B. cinerea recovery was greatest from the carpel (80% of carpels colonized) compared with other floral organs. As fruit matured, additional floral parts were colonized by B. cinerea, possibly facilitating secondary internal or external infections of mature fruit. Average weekly minimum air temperature, average weekly night air temperature, cumulative rain, average weekly leaf wetness percentage, and duration of leaf wetness >90% were significantly positively correlated with B. cinerea colonization of raspberry in NW Washington during two seasons of this study. Our data does not support the hypothesis that the bloom period is the critical window for B. cinerea colonization of red raspberry and suggest that later colonization of developing fruit may be more important for gray mold development on raspberry. The outcomes of this research provide useful information for improvement of gray mold disease management strategies for red raspberry in NW Washington and elsewhere.

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata.

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including ß-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, ß-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.

ACTTS3 encoding a polyketide synthase is essential for the biosynthesis of ACT-toxin and pathogenicity in the tangerine pathotype of Alternaria alternata.

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerine and tangerine hybrids. Sequence analysis of a genomic BAC clone identified part of the ACT-toxin TOX (ACTT) gene cluster, and knockout experiments have implicated several open reading frames (ORF) contained within the cluster in the biosynthesis of ACT-toxin. One of the ORF, designated ACTTS3, encoding a putative polyketide synthase, was isolated by rapid amplification of cDNA ends and genomic/reverse transcription-polymerase chain reactions using the specific primers designed from the BAC sequences. The 7,374-bp ORF encodes a polyketide synthase with putative beta-ketoacyl synthase, acyltransferase, methyltransferase, beta-ketoacyl reductase, and phosphopantetheine attachment site domains. Genomic Southern blots demonstrated that ACTTS3 is present on the smallest chromosome in the tangerine pathotype of A. alternata, and the presence of ACTTS3 is highly correlated with ACT-toxin production and pathogenicity. Targeted gene disruption of two copies of ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS3 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

Population structure of Potebniamyces pyri in the U.S. Pacific Northwest and evidence of outcrossing inferred with sequence-characterized amplified region markers.

Potebniamyces pyri is the cause of Phacidiopycnis rot of d'Anjou pear, which is grown primarily in Washington and Oregon. To estimate the population structure of P. pyri, 146 single-spore isolates were sampled from five major pear-production areas and scored for variation at eight sequence-characterized amplified region (SCAR) loci. Significant genetic differentiation was detected among five subpopulations and a total of 54 multilocus genotypes were identified, with significant genotypic diversity in each subpopulation. No genotype was shared among more than three subpopulations. To estimate the relationship between phenotype and multilocus SCAR genotype, four to five representative isolates of each dominant SCAR genotype in each subpopulation were assayed for growth rate on oatmeal agar and virulence on d'Anjou pear fruit. Significant differences in daily growth rates and virulence were detected among genotypes; however, genotype was not predictive of virulence. To assess the mating system of the pathogen, 10 ascospores were sampled from each of 20 apothecia from a commercial orchard and scored for five SCAR markers. Segregation of alleles at one or more SCAR loci was detected among 18 of 20 ascospore progeny sets, indicating that P. pyri is likely a heterothallic fungus with a predominantly outcrossing mating system.

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.

Function of genes encoding acyl-CoA synthetase and enoyl-CoA hydratase for host-selective act-toxin biosynthesis in the tangerine pathotype of Alternaria alternata.

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.

First Report of Chickpea Wilt Caused by Clonostachys rhizophaga in Syria.

In 2007 and 2008, disease symptoms were observed on four cultivars of chickpea (Cicer arietinum L.), including two of the most popular cultivars grown in Syria (Ghab 3 and Ghab 4), in a replicated on-farm trial conducted in the fertile Al Ghab Plains. Affected plants exhibited chlorosis of the foliage, vascular discoloration, and death. In both years, plant mortality reached 100% in plots of cvs. ICC 12004, Ghab 3, and Ghab 4, but only 60% in plots of cv. ILC 97-706. Five monosporic isolates obtained from surface-disinfested stems and roots were identified morphologically. All micromorphological characteristics indicated that the isolated fungi fit the description of Clonostachys rhizophaga Schroers (1). Wilting of chickpea was widespread in the area, and fungal isolations from a random sample of diseased plants in neighboring farmers' fields revealed the presence of C. rhizophaga. In culture, isolates formed dimorphic, Verticillium-like (primary) or penicillate (secondary) conidiophores and ovoidal to elongate, slightly curved or asymmetrical, 5 to 9 µm long and 2.5 to 3.5 µm wide conidia showing a slightly laterally displaced hilum. The identification of the five isolates as C. rhizophaga was supported by sequencing approximately 600 bp of the ß-tubulin gene (tub2). Two representative sequences have been deposited under GenBank, Accession No. FJ593882 for strain CBS 124507 and No. FJ593883 for CBS 124511. Both were 100% similar to the sequence of C. rhizophaga strain CBS 361.77 (GenBank Accession No. AF358158) but differed by a deletion of 2 nucleotides relative to the ex-type strain of C. rhizophaga, CBS 202.37 (GenBank Accession No. AF358156). Two methods were used to inoculate plants and complete Koch's postulates. Method 1 used a 10-mm-diameter mycelial plug to inoculate healthy 3-day-old seedlings grown on 40 ml of Hoagland nutrient agar medium in a glass tube (one seedling per tube). The plug was placed mycelial-side down on the surface of the medium, and the fungus subsequently colonized the medium and penetrated the plant roots. Method 2 involved mixing autoclaved seed that had been colonized by each isolate with sterilized soil (1:12 vol/vol) prior to transplanting healthy seedlings into the soil mix. Thirty plants of each cultivar were tested per isolate per method, and controls received sterile agar plugs or autoclaved chickpea seed only. Irrespective of inoculation method, all five isolates caused wilt and plant death of all cultivars within 15 days (method 1) or 2 months (method 2) postinoculation. Symptoms were similar to those originally observed in the field and controls remained healthy. C. rhizophaga was recovered from all affected plants. To our knowledge, this is the first report of C. rhizophaga as a pathogen of chickpea. In an earlier report, C. rhizophaga (as Verticillium rhizophagum Tehon & Jacobs, nom. invalid.) was identified as the causal agent of a disastrous disease of Ulmus americana in Ohio (2). C. rhizophaga has been reported from Chile, Ecuador, the United States, and Switzerland (1). References: (1) H.-J. Schroers. Stud. Mycol. 46:85, 2001. (2) L.-R. Tehon and H. L. Jacobs. Bull. Davey Tree Expert Company, Kent, OH. 6:3, 1936.

Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata using RNA silencing.

Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a serious disease of commercially important tangerines and their hybrids. The pathogen produces host-selective ACT toxin, and several genes (named ACTT) responsible for ACT-toxin biosynthesis have been identified. These genes have many paralogs, which are clustered on a small, conditionally dispensable chromosome, making it difficult to disrupt entire functional copies of ACTT genes using homologous recombination-mediated gene disruption. To overcome this problem, we attempted to use RNA silencing, which has never been employed in Alternaria spp., to knock down the functional copies of one ACTT gene with a single silencing event. ACTT2, which encodes a putative hydrolase and is present in multiple copies in the genome, was silenced by transforming the fungus with a plasmid construct expressing hairpin ACTT2 RNAs. The ACTT2 RNA-silenced transformant (S-7-24-2) completely lost ACTT2 transcripts and ACT-toxin production as well as pathogenicity. These results indicated that RNA silencing may be a useful technique for studying the role of ACTT genes responsible for host-selective toxin biosynthesis in A. alternata. Further, this technique may be broadly applicable to the analysis of many genes present in multiple copies in fungal genomes that are difficult to analyze using recombination-mediated knockdowns.

Postbloom fruit drop of citrus and key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum.

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (São Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.

First Report of Ascochyta Blight Outbreak of Pea Caused by Ascochyta pisi in Spain.

Characteristic Ascochyta blight lesions were observed on leaves and stems of pea (Pisum sativum L.) 'Dove' grown at two sites in the province of Burgos (northern Spain) during May and June of 2005 and 2006. Mean disease severity of affected tissue reached 47% in 2005 and 72% in 2006. Dark brown, circular, necrotic lesions were sometimes covered with pycnidia. Fungal isolations were made from small pieces of infected tissue by surface disinfecting in 1% NaOCl for 1 min and then washing in deionized, sterile water for 2 min. Tissue pieces were placed on potato dextrose agar (PDA) for 7 days at 20 to 24°C under fluorescents lights with a 12-h photoperiod to induce sporulation. Single-spore isolations were made by streaking conidia from PDA cultures on 2% water agar and picking germinated conidia after 18 h. Fungal colonies grown on PDA and conidia from these cultures were similar to that of Ascochyta pisi Lib., and no chlamydospores or pseudothecia were observed, eliminating the possibility that the isolated fungi were A. pinodes or A. pinodella (3), the other fungi associated with the "Ascochyta complex" of pea. Conidial suspensions (5 × 105 conidia/ml) of two single-spore isolates (Spain-47 and Spain-48) were spray inoculated to runoff on 3-week-old plants of bean (Phaseolus vulgaris L. 'Contender'), chickpea (Cicer arietinum L. 'Blanco lechoso'), lentil (Lens culinaris Medik. 'Pardinar'), pea ('Lincoln'), and faba bean (Vicia faba L. 'Alameda') with 10 replicate plants per isolate. Plants were incubated in a growth chamber at 20 to 24°C and 100% relative humidity (RH) for 48 h and then incubated at the same temperature and 50 to 80% RH for 3 weeks. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation on leaves and stems of pea. No disease symptoms were observed on the other inoculated plants. DNA was extracted from both isolates (Spain-47 and Spain-48) and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD) was amplified with gpd-1 and gpd-2 primers (2). Amplicons were direct sequenced on both strands and consensus sequences were aligned. Spain-47 and Spain-48 had identical sequences. A BLAST search of the NCBI nucleotide database with the consensus sequence revealed A. pisi G3PD Accession No. DQ383963 (isolate ATCC 201617, Bulgaria) as the closest match in the database with 100% sequence similarity. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. Although infections by A. pinodes or by unidentified Ascochyta spp. are well known in pea crops in Spain (1), to our knowledge, this is the first report of an outbreak of Ascochyta blight of pea caused by A. pisi under field conditions in Spain. References: (1) M. F. Andrés et al. Patógenos de Plantas Descritos en España. MEC, Madrid, 1998. (2) M. L. Berbee et al. Mycologia 91:964, 1999. (3) E. Punithalingam and P. Holliday. No 334 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1972.

First Report of Spring Black Stem and Leaf Spot of Alfalfa in Washington State Caused by Phoma medicaginis.

Lesions were observed on leaves and stems of alfalfa (Medicago sativa L.) growing as weeds in Pullman, Washington in June of 2001. Lesions appeared similar to those described for spring black stem and leaf spot caused by Phoma medicaginis Malbr. & Roum. in Roum. var. medicaginis Boerema (synonyms Phoma herbarum Westend. var. medicaginis Fckl. and Ascochyta imperfecta Peck). Sporulation was induced by placing surface-disinfested pieces of infected tissue on 3% water agar (WA) for 24 h under fluorescent light with a 12-h photoperiod. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia after 18 h. Isolates had cultural and conidial morphology similar to descriptions of P. medicaginis and isolate ATCC52798 when grown on V8 agar and PDA at room temperature (3). Distinction between P. medicaginis var. medicaginis and P. medicaginis var. macrospora was not attempted. Conidial suspensions (1 × 106 conidia/ml) of isolates AS1, AS2, AS3, and AS4 were spray inoculated to runoff onto 3-week-old plants. PI lines 536535 and 536534 of M. sativa subsp. sativa (4-trifolate stage) and PI lines 442896 and 577609 of M. truncatula (5- to 7-trifolate stage) from the USDA Western Region Plant Introduction Station, Pullman, Washington were inoculated, with at least two replicate plants inoculated per isolate. Plants were incubated in a dew chamber at 20°C in the dark for 24 h to promote infection and then transferred to a growth chamber at 18°C with a 12-h photoperiod. Lesions were apparent on M. sativa subsp. sativa plants 4 days postinoculation (dpi) and 7 dpi on M. truncatula plants. At 12 dpi, many dark brown lesions with chlorotic halos were noted on leaves of M. sativa subsp. sativa, occasionally killing the entire trifoliate leaf and progressing approximately 1 cm down the stem. According to the previously published 1-to-5 visual rating scale for this disease (4), disease scores on both genotypes of M. sativa subsp. sativa were 4 (susceptible), while disease ratings on M. truncatula were 1-2 (resistant) with a few dark brown lesions noted on leaves and stems generally restricted to less than 2 mm in diameter. DNA was extracted from isolates AS1 and AS4, and PCR was performed using gpd-1 and gpd-2 primers for the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) (1), and EF1-728F and EF1-986R primers for the translation elongation factor 1-alpha gene (EF) (2), resulting in amplification of an approximately 600-bp fragment from each primer set. Amplicons were direct-sequenced on both strands, and BLAST searches of the NCBI nucleotide database were conducted with consensus G3PD and EF sequences of both isolates AS1 and AS4. Closest matches obtained for the G3PD and EF sequences were P. medicaginis isolate ATCC52798 (Accession No. DQ525740) and P. medicaginis var. medicaginis CBS316.90 (Accession No. AY831548), respectively. The G3PD and EF sequences for these isolates have been deposited in GenBank database (Accession Nos. EU394712-EU394715). To our knowledge, this is the first confirmed report of spring black stem and leaf spot of alfalfa in Washington State supported by Koch's postulates, cultural morphology, and multigene sequencing. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (3) G. C. Kinsey. No. 1503 in: IMI Descriptions of Fungi and Bacteria. CABI Bioscience, Surrey, UK, 2002. (4) R. M. Salter and K. L. Leath. Spring blackstem and leafspot resistance. Online publication. North American Alfalfa Improvement Conference, Beltsville, MD, 1992.

Evolutionary relationships among Ascochyta species infecting wild and cultivated hosts in the legume tribes Cicereae and Vicieae.

Evolutionary relationships were inferred among a worldwide sample of Ascochyta fungi from wild and cultivated legume hosts based on phylogenetic analyses of DNA sequences from the ribosomal internal transcribed spacer regions (ITS), as well as portions of three protein-coding genes: glyceraldehyde-3-phosphate-dehydrogenase (G3PD), translation elongation factor 1-alpha (EF) and chitin synthase 1 (CHS). All legume-associated Ascochyta species had nearly identical ITS sequences and clustered with other Ascochyta, Phoma and Didymella species from legume and nonlegume hosts. Ascochyta pinodes (teleomorph: Mycosphaerella pinodes [Berk. & Blox.] Vestergen) clustered with Didymella species and not with well characterized Mycosphaerella species from other hosts and we propose that the name Didymella pinodes (Berk. & Blox.) Petrak (anamorph: Ascochyta pinodes L.K. Jones) be used to describe this fungus. Analysis of G3PD revealed two major clades among legume-associated Ascochyta fungi with members of both clades infecting pea ("Ascochyta complex"). Analysis of the combined CHS, EF and G3PD datasets revealed that isolates from cultivated pea (P. sativum), lentil (Lens culinaris), faba bean (Vicia faba) and chickpea (Cicer arietinum) from diverse geographic locations each had identical or similar sequences at all loci. Isolates from these hosts clustered in well supported clades specific for each host, suggesting a co-evolutionary history between pathogen and cultivated host. A. pisi, A. lentis, A. fabae and A. rabiei represent phylogenetic species infecting pea, lentil, faba bean and chickpea, respectively. Ascochyta spp. from wild relatives of pea and chickpea clustered with isolates from related cultivated hosts. Isolates sampled from big-flower vetch (Vicia grandiflora) were polyphyletic suggesting that either this host is colonized by phylogenetically distinct lineages of Ascochyta or that the hosts are polyphyletic and infected by distinct evolutionary lineages of the pathogen. Phylogenetic species identified among legume-associated Ascochyta spp. were fully concordant with previously described morphological and biological species.

First Report of Ascochyta Blight of Pisum elatius (Wild Pea) in the Republic of Georgia Caused by Ascochyta pisi.

Characteristic Ascochyta blight lesions were observed on leaves and pods of wild pea (Pisum elatius Steven. ex M. Bieb.) growing at three sites in the Republic of Georgia during June and July of 2004. Site characteristics were 41°36.11'N, 44°31.34'E (elevation 919 m), 41°54.221'N, 44°05.667'E (elevation 744 m), and 41°44.907'N, 43°12.263'E (elevation 884 m). Lesions appeared similar to those induced by Ascochyta pisi Lib. on cultivated pea (P. sativum L.). Fungi were isolated by surface disinfesting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then in deionized sterile H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia 18 h later. Three fungi (isolates Georgia-6, -7, and -12) had colony morphology similar to that of A. pisi on V8 juice agar. Conidial suspensions (1 × 105 conidia/ml) of each isolate above were spray inoculated to runoff on three genotypes of 2-week-old P. elatius plants. Plants inoculated included PI lines 560055 and 513252 and W6 line 15006 from the USDA Western Region Plant Introduction Station, Pullman, WA with 11 replicate plants inoculated per isolate. Plants were incubated in a growth chamber for 48 h at 18°C and covered with a plastic cup to maintain high humidity. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation. DNA was extracted from each isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of chitin synthase 1, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands, and BLAST searches of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences of isolates Georgia-6, -7, and -12 were performed. The closest match obtained for the G3PD sequences was A. pisi isolate ATCC 201617 (Accession No. DQ383963). G3PD sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ383966 [Georgia-6 and -7] and DQ383963 [A. pisi isolate AP1 and Georgia-12]). Closest matches to CHS and EF sequences were A. pisi isolate ATCC 201618 (EF Accession No. DQ386494) and Didymella fabae isolate ATCC 96418 (CHS Accession No. DQ386481, EFAccession No. DQ386492), respectively. CHS sequences for Georgia-6, -7, and -12 were identical to each other and to A. fabae isolate AF1 and were deposited in GenBank (Accession No. DQ386481. EF sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ386494 [Georgia-6 and A. pisi isolate AP2], DQ386495, and DQ386496, respectively. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. To our knowledge, this is the first report of Ascochyta blight of P. elatius in the Republic of Georgia. References: (1) M. L. Berbee et al. Mycologia 91:964. 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.

Spread of Heterobasidion annosum in Christmas Tree Plantations of the United States Pacific Northwest.

ABSTRACT The population structure of Heterobasidion annosum in the Pacific Northwest (PNW) Christmas tree plantations was estimated at two spatial scales to assess the relative importance of primary and secondary infection, colonization, and spread of the pathogen. Ninety-three isolates from single trees in 27 discrete mortality pockets and 104 isolates from 12 individual root systems of noble and Fraser fir trees were sampled near Mossyrock, Washington. Isolates were genotyped using somatic compatibility assays and microsatellite markers to determine the spatial scale at which dispersal of single genotypes (genets) was occurring. All isolates sampled from different trees in discrete mortality pockets had distinct genotypes, whereas the root systems of single trees were dominated by one or two genotypes. These results suggest that infection of PNW Christmas trees results from frequent primary infection events of adjacent stumps and localized secondary spread within root systems rather than clonal spread of the pathogen between adjacent trees. We hypothesize that mortality pockets may be due to availability of infection courts and/or variation in inoculum levels during selective harvesting of patches of mature trees.

First Report of Ascochyta Blight of Vicia hirsuta (Hairy Tare) in the Republic of Georgia Caused by Ascochyta sp.

Tan lesions with dark margins containing concentric rings of black pycnidia were observed on leaves and pods of hairy tare (Vicia hirsuta L.) growing near Ateni, GA (41°54.631'N, 44°05.586'E, elev. 730 m) on 1 July 2004. Lesions were reminiscent of those induced by Ascochyta rabiei (Pass.) Labrousse on chickpea (Cicer arietinum L.). At the time of collection, necrotic lesions were observed on the stems, leaflets, and pods of several plants. The fungus was isolated by surface-disinfecting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then deionized H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking cirrhi on 3% WA and picking germinated single conidia. After 14 days of growth, the isolated fungus had colony morphology similar to that of A. rabiei on V8 juice agar. A conidial suspension of the fungus (1 × 105 conidia/ml) was spray-inoculated onto 2-week-old plants including PI lines 628303, 628304, 420171, and 422499 of V. hirsuta and C. arietinum cv. Burpee. Plants were obtained from the USDA Western Region Plant Introduction Station, Pullman, WA, and 20 replicate plants of each genotype were inoculated. Inoculated plants were covered with a plastic cup to maintain high humidity and incubated in a growth chamber for 48 h at 18°C. Following removal of the cups, characteristic Ascochyta blight lesions were apparent 14 days after inoculation on both plant species. DNA was extracted from the isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of the chitin synthase 1 gene, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands and a BLAST search of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences revealed the chickpea pathogen Didymella rabiei (anamorph Ascochyta rabiei) accessions DQ383958, DQ386480, and DQ386488 as the closest matches in the databases with 95, 95, and 88% sequence similarity, respectively. These results, coupled with the morphological identification and the inoculation results, confirm the identity of the fungus as Ascochyta sp. Further research needs to be performed to determine if this represents a new species of Ascochyta. The identification of this fungus is part of a larger project to develop a phylogeny for Ascochyta spp. infecting cultivated legumes and their wild relatives that will provide a framework for the study of the evolution of host specificity and speciation of plant-pathogenic fungi. This is the second report of an Ascochyta species on V. hirsuta, and to our knowledge, the first report of Ascochyta blight of this host in the Republic of Georgia. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.

First Report of Alternaria Brown Spot of Citrus Caused by Alternaria alternata in Peru.

Alternaria brown spot, caused by Alternaria alternata (Fr.) Keissler, causes leaf, twig, and fruit lesions and reduces yield and fruit quality of many tangerines (Citrus reticulata Blanco) and their hybrids (3). In 2003, characteristic symptoms of brown spot were observed on young leaves and fruit of 'Minneola' tangelo in the Satipo Province of Peru. In 2004, the disease was discovered in the provinces of Chanchamayo, Leoncio Prado, and La Convención in the Junin, Huanuco, and Cusco regions, respectively, as well as in the Apurimac and the Ene valleys. In 2005, it was confirmed in the province of Oxapampa in the Pasco Region. Brown-to-black lesions surrounded by yellow halos and veinal necrosis were observed on young leaves, often causing abscission of young shoots and twig dieback. Light brown, circular lesions were observed on fruit, and when severe, resulted in premature abscission. Isolations from infected leaves and twigs were made on potato dextrose agar (PDA) with 10 µg/ml of benomyl. Colonies that developed after 5 days at 27°C were olive brown-to-black and produced small, muriform, pigmented conidia typical of A. alternata. On PDA without benomyl, gray colonies with conidia typical of Colletotrichum gloeosporioides were recovered frequently. Inoculation of three detached young shoots of 'Minneola' by spraying with a suspension of 105 conidia/ml of A. alternata produced leaf and twig symptoms characteristic of the disease after 48 h and confirmed pathogenicity of three isolates. Symptoms were not observed on control leaves sprayed with water nor on an equal number of leaves inoculated with a suspension of 105 conidia/ml of C. gloeosporioides. Reisolation of A. alternata from diseased tissue fulfilled Koch's postulates. DNA was extracted from 17 isolates and a partial endopolygalacturonase gene was amplified and sequenced (2). Sequences of all 17 isolates were identical, and in BLAST searches of the NCBI database, the closest matches were A. alternata accession nos. AY295023.1, AY295022.1, and AY295021.1 with 100, 99.8, and 99.8% sequence similarity, respectively. Phylogenetic analyses revealed that all isolates from Peru clustered with brown spot isolates from Israel, Turkey, South Africa, and Australia (1). These results, along with morphological characterization and pathogenicity tests, confirm the identity of the fungus as the tangerine pathotype of A. alternata. The disease has significantly reduced yield and the commercial value of fruit and may be a limiting factor for the production of susceptible cultivars in those areas of Peru. References: (1) T. L. Peever et al. Phytopathology 92:794, 2002. (2) T. L. Peever et al. Mycologia 96:119, 2004, (3) L.W. Timmer et al. Pages 19-21 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al eds. The American Phytopathological Society, St. Paul, MN, 2000.

Host Specificity of Ascochyta spp. Infecting Legumes of the Viciae and Cicerae Tribes and Pathogenicity of an Interspecific Hybrid.

ABSTRACT Ascochyta spp. (teleomorphs: Didymella spp.) infect a number of legumes, including many economically important species, and the diseases they cause represent serious limitations of legume production worldwide. Ascochyta rabiei, A. fabae, A. pisi, A. lentis, and A. viciae-villosae are pathogens of chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), lentil (Lens culinaris), and hairy vetch (V. villosa), respectively. Inoculations in the greenhouse and in growth chambers demonstrated that A. fabae, A. lentis, A. pisi, A. rabiei, and A. viciae-villosae were host specific. Isolates caused no visible disease symptoms on "nonhost" plants (plants other than the hosts they were originally isolated from) but were recovered consistently from inoculated, surface-disinfested, nonhost tissues. Interspecific crosses of A. pisi x A. fabae and A. viciae-villosae x A. lentis produced pseudothecia with viable ascospores, and the hybrid status of the ascospore progeny was verified by the segregation of mating type and amplified fragment length polymorphism (AFLP) markers. Interspecific progeny were morphologically normal in culture but exhibited more phenotypic variation compared with progeny from intraspecific crosses. Mating type and the majority of AFLP markers segregated in Mendelian 1:1 ratios in both intraspecific and interspecific crosses. A total of 11 and 7% of AFLP markers showed segregation distortion among progeny from interspecific crosses and intraspecific crosses, respectively; however, this difference was not significant (P = 0.90). Only 30 of 114 progeny isolates from the A. fabae x A. pisi cross inoculated in the greenhouse caused lesions on pea and only 4 caused disease on faba bean. In all, 15 of 110 progeny isolates were pathogenic to pea and none were pathogenic to faba bean under growth chamber conditions. Although no obvious postzygotic, intrinsic isolating barriers were identified in any of the interspecific crosses, it appears that host specialization may act as both a prezygotic, ecological isolating barrier and a postzygotic, extrinsic, ecological isolating barrier in these fungi. Host specificity, coupled with low pathogenic fitness of hybrids, may be an important speciation mechanism contributing to the maintenance of hostspecific, phylogenetic lineages of these fungi.

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins.

ABSTRACT Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.

Citrus Black Rot is Caused by Phylogenetically Distinct Lineages of Alternaria alternata.

ABSTRACT Phylogenetic analysis revealed that isolates of Alternaria alternata causing black rot of citrus were associated with six well-supported evolutionary lineages. Isolates recovered from brown spot lesions on Minneola tangelo, leaf spot lesions on rough lemon, and healthy citrus tissue and noncitrus hosts were related closely to isolates from black-rotted fruit. Phylogenies estimated independently from DNA sequence data from an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1-3 and OPA2-1) had similar topologies, and phylogenetic analysis was performed on the combined data set. In the combined phylogeny, isolates from diverse ecological niches on citrus and noncitrus hosts were distributed in eight clades. Isolates from all lineages, regardless of ecological or host association, caused black rot in fruit inoculation assays, demonstrating that small-spored Alternaria isolates associated with different ecological niches on citrus and other plant hosts are potential black rot pathogens. These data also indicated that the fungi associated with black-rotted fruit do not form a natural evolutionary group distinct from other Alternaria pathogens and saprophytes associated with citrus. The use of the name A. citri to describe fungi associated with citrus black rot is not justified and it is proposed that citrus black rot fungi be referred to as A. alternata.

Historical and contemporary multilocus population structure of Ascochyta rabiei (teleomorph: Didymella rabiei) in the Pacific Northwest of the United States.

The historical and contemporary population genetic structure of the chickpea Ascochyta blight pathogen, Ascochyta rabiei (teleomorph: Didymella rabiei), was determined in the US Pacific Northwest (PNW) using 17 putative AFLP loci, four genetically characterized, sequence-tagged microsatellite loci (STMS) and the mating type locus (MAT). A single multilocus genotype of A. rabiei (MAT1-1) was detected in 1983, which represented the first recorded appearance of Ascochyta blight of chickpea in the PNW. During the following year many additional alleles, including the other mating type allele (MAT1-2), were detected. By 1987, all alleles currently found in the PNW had been introduced. Highly significant genetic differentiation was detected among contemporary subpopulations from different hosts and geographical locations indicating restricted gene flow and/or genetic drift occurring within and among subpopulations and possible selection by host cultivar. Two distinct populations were inferred with high posterior probability which correlated to host of origin and date of sample using Bayesian model-based population structure analyses of multilocus genotypes. Allele frequencies, genotype distributions and population assignment probabilities were significantly different between the historical and contemporary samples of isolates and between isolates sampled from a resistance screening nursery and those sampled from commercial chickpea fields. A random mating model could not be rejected in any subpopulation, indicating the importance of the sexual stage of the fungus both as a source of primary inoculum for Ascochyta blight epidemics and potentially adaptive genotypic diversity.