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.

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 Sclerotinia Stem Rot of Chickpea Caused by Sclerotinia sclerotiorum in North Dakota and Washington.

Chickpea (Cicer arietinum L.) is cultivated as a rotational crop in the cereal-based production system in the U.S. Pacific Northwest (PNW) and its production is expanding to other northern tier states. During July 2005, symptoms of Sclerotinia stem rot were observed on chickpea cv. Dwelley and Dylan in fields near Spangle, WA and Carrington, ND, respectively, with disease incidence of approximately ≤1% in affected areas at both locations. Symptoms included stem whitening, wilting, and stem breakage. Occasionally, white fluffy mycelium was observed; however, production of sclerotia on infected plants was rarely observed. Sclerotinia sclerotiorum was isolated from diseased stems collected from both states. The isolates produced a ring of sclerotia near the edge of potato dextrose agar (PDA) plates in 7 days and produced neither conidia nor other fruiting bodies in culture after 30 days. PCR amplification of the rDNA internal transcribed spacer region from two representative isolates and subsequent digestion with restriction enzymes, Mbo I and Taq I, produced identical banding patterns to previously identified isolates of S. sclerotiorum from pea from the PNW (2). Chickpea cvs. Dwelley and Spanish White (eight plants of each) were inoculated by fastening mycelial agar plugs from an actively growing colony on PDA onto the stems with Parafilm. Symptoms of stem whitening were observed as early as 2 days after inoculation, and the lesions extended upward and downward from the inoculation site. Wilting and stem breakage were also observed. Control inoculations of four plants of each cultivar with PDA plugs without mycelium produced no visible symptoms. S. sclerotiorum was consistently reisolated from inoculated plants but not from control plants. Chickpea had been grown in the PNW for more than 20 years without any reported incidence of Sclerotinia stem rot although the disease has been reported from Arizona (3) and Asian countries (1). This is likely because of the upright growth habit of the chickpea plant coupled with relatively dry conditions late in the growing season. Previous chickpea cultivars were very susceptible to Ascochyta blight, an early-season disease of chickpea in the PNW that reduced chickpea stands and canopy coverage. Current cultivars possess much improved resistance to Ascochyta blight, allowing greater vegetative growth to occur and creating microenvironmental conditions conducive to Sclerotinia stem rot. In North Dakota, where humid conditions prevail late in the growing season, symptoms of Sclerotinia stem rot had been observed in previous years but had not been documented because of a recent history of chickpea cultivation there. To our knowledge, this is the first report of confirmed Sclerotinia stem rot of chickpea in North Dakota and Washington. References: (1) G. J. Boland and R. Hall. Can. J. Plant Pathol. 16:93, 1994. (2) I. Jimenez-Hidalgo et al. Phytopathology (Abstr.) 94(suppl.):S47, 2004. (3) M. E. Matheron and M. Porchas. Plant Dis. 84:1250, 2000.

Consistent Quantitative Trait Loci in Pea for Partial Resistance to Aphanomyces euteiches Isolates from the United States and France.

ABSTRACT Development of pea cultivars resistant to Aphanomyces root rot, the most destructive root disease of pea worldwide, is a major disease management objective. In a previous study of a mapping population of 127 recombinant inbred lines (RILs) derived from the cross 'Puget' (susceptible) x '90-2079' (partially resistant), we identified seven genomic regions, including a major quantitative trait locus (QTL), Aph1, associated with partial resistance to Aphanomyces root rot in U.S. fields (21). The objective of the present study was to evaluate, in the same mapping population, the specificity versus consistency of Aphanomyces resistance QTL under two screening conditions (greenhouse and field, by comparison with the previous study) and with two isolates of Aphanomyces euteiches originating from the United States and France. The 127 RILs were evaluated in the greenhouse for resistance to pure culture isolates SP7 (United States) and Ae106 (France). Using the genetic map previously described, a total of 10 QTL were identified for resistance in greenhouse conditions to the two isolates. Among these were Aph1, Aph2, and Aph3, previously detected for partial field resistance in the United States. Aph1 and Aph3 were detected with both isolates and Aph2 with only the French isolate. Seven additional QTL were specifically detected with one of the two isolates and were not identified for partial field resistance in the United States. The consistency of the detected resistance QTL over two screening environments and isolates is discussed with regard to pathogen variability, and disease assessment and QTL detection methods. This study suggests the usefulness of three consistent QTL, Aph1, Aph2, and Aph3, for marker-assisted selection.

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.

Construction of a HindIII Bacterial Artificial Chromosome library and its use in identification of clones associated with disease resistance in chickpea.

A chickpea ( Cicer arietinum L.) Bacterial Artificial Chromosome (BAC) library from germplasm line, FLIP 84-92C, was constructed to facilitate positional cloning of disease resistance genes and physical mapping of the genome. The BAC library has 23,780 colonies and was calculated to comprise approximately 3.8 haploid-genome equivalents. Studies on 120 randomly chosen clones revealed an average insert size of 100 kb and no empty clones. Colony hybridization using the RUBP carboxylase large subunit as a probe resulted in a very low percentage of chloroplast DNA contamination. Two clones with a combined insert size of 200 kb were isolated after the library was screened with a Sequence Tagged Microsatellite Site (STMS) marker, Ta96, which is tightly linked to a gene ( Foc3) for resistance to fusarium wilt caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris (Padwick) race 3 at a genetic distance of 1 cM. Also, these two clones were analyzed with several resistance gene analog (RGA) markers. End sequencing of these clones did not identify repetitive sequences. The development of the BAC library will facilitate isolation of Foc3 and allow us to perform physical mapping of this genomic region where additional R genes against other races of the wilt causing pathogen are positioned.

Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny

Molecular markers closely linked to fusarium resistance genes in chickpea show significant alignments to pathogenesis-related genes located on Arabidopsis chromosomes 1 and 5.

A population of 131 recombinant inbred lines from a wide cross between chickpea ( Cicer arietinum L., resistant parent) and Cicer reticulatum (susceptible parent) segregating for the closely linked resistances against Fusarium oxysporum f.sp. ciceri races 4 and 5 was used to develop DNA amplification fingerprinting markers linked to both resistance loci. Bulked segregant analysis revealed 19 new markers on linkage group 2 of the genetic map on which the resistance genes are located. Closest linkage (2.0 cM) was observed between marker R-2609-1 and the race 4 resistance locus. Seven other markers flanked this locus in a range from 4.1 to 9.0 cM. These are the most closely linked markers available for this locus up to date. The sequences of the linked markers were highly similar to genes encoding proteins involved in plant pathogen response, such as a PR-5 thaumatin-like protein and an important regulator of the phytoalexin pathway, anthranilate N-hydroxycinnamoyl-benzoyltransferase. Others showed significant alignments to genes encoding housekeeping enzymes such as the MutS2 DNA-mismatch repair protein. In the Arabidopsis genome, similar genes are located on short segments of chromosome 1 and 5, respectively, suggesting synteny between the fusarium resistance gene cluster of chickpea and the corresponding regions in the Arabidopsis genome. Three marker sequences were similar to retrotransposon-derived and/or satellite DNA sequences. The markers developed here provide a starting point for physical mapping and map-based cloning of the fusarium resistance genes and exploration of synteny in this highly interesting region of the chickpea genome.

Genomic location of the Fw gene for resistance to Fusarium wilt race 1 in peas.

Resistance to fusarium wilt in peas (Pisumsativum L.) caused by Fusarium oxysporum Schlect. f. sp. pisi race 1 (van Hall) Snyd. & Hans. is conferred by a single dominant gene, Fw. The gene was located in the pea genome by analyzing progenies from crosses involving genetic markers across all pea linkage groups. Phenotyping of the progenies for reaction to race 1 of the fusarium wilt pathogen was determined by field screening in a "wilt-sick" plot in Pullman, Washington. Fw was shown to be located on linkage group III, about 13 map units from Lap-1 and b and 14 map units from Td. The relatively large distances between these markers and Fw precludes the use of the linked markers in marker-assisted selection for wilt resistance. Additional markers in this region of the pea genome will be required if marker-assisted selection for Fw is to be successful.

Quantitative trait loci for partial resistance to Aphanomyces root rot in pea.

Aphanomyces root rot, caused by Aphanomyces euteiches Drechs, is the most-important disease of pea ( Pisum sativum L.) worldwide. No efficient chemicals are available to control the pathogen. To facilitate breeding for Aphanomyces root rot resistance and to better understand the inheritance of partial resistance, our goal was to identify QTLs associated with field partial resistance. A population of 127 RILs from the cross Puget (susceptible) x 90-2079 (partially resistant) was used. The lines were assessed for resistance to A. euteiches under field conditions at two locations in the United States (Pullman, Wash. and LeSueur, Minn.) in 1996 and 1998 for three criteria based on symptom intensity and disease effects on the whole plant. The RILs were genotyped using automated AFLPs, RAPDs, SSRs, ISSRs, STSs, isozymes and morphological markers. The resulting genetic map consisted of 324 linked markers distributed over 13 linkage groups covering 1,094 cM (Kosambi). Twenty seven markers were anchored to other published pea genetic maps. A total of seven genomic regions were associated with Aphanomyces root rot resistance. The first one, located on LG IVb and named Aph1, was considered as "major" since it was highly consistent over the years, locations and resistance criteria studied, and it explained up to 47% of the variation in the 1998 Minnesota trial. Two other year-specific QTLs, namely Aph2 and Aph3, were revealed from different scoring criteria on LG V and Ia, respectively. Aph2 and Aph3 mapped near the r (wrinkled/round seeds) and af (normal/afila leaves) genes, and accounted for up to 32% and 11% of the variation, respectively. Four other "minor" QTLs, identified on LG Ib, VII and B, were specific to one environment and one resistance criterion. The resistance alleles of Aph3 and the two "minor" QTLs on LG Ib were derived from the susceptible parent. Flanking markers for the major Aphanomyces resistance QTL, Aph1, have been identified for use in marker-assisted selection to improve breeding efficiency.

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome.

A size-selected genomic library comprising 280,000 colonies and representing approximately 18% of the chickpea genome, was screened for (GA)n, (GAA)n and (TAA)n microsatellite-containing clones, of which 389 were sequenced. The majority (approximately 75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%-9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P > or = 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.

First Report of Ascochyta Blight of Cicer montbretii, a Wild Perennial Chickpea in Bulgaria.

In the Stranja Mountains of southeastern Bulgaria, native populations of Cicer montbretii Jaub. & Spach were found on the edge of a road in an oak forest near the village of Gramatikova (42°1'38″N; 27°36'49″E) at an elevation of about 125 m. C. montbretii, a perennial species, is the only wild Cicer sp. native to Bulgaria. At the time of collection, necrotic lesions were observed on the stems, leaflets, and pods of several plants, and these lesions were reminiscent of those induced by Ascochyta rabiei (Pass.) Labrousse. The teleomorph (sexual stage) of A. rabiei, Didymella rabiei (Kovachevski) v. Arx (syn. Mycosphaerella rabiei Kovachevski), was discovered in 1936 on overwintered chickpea residue in southern Bulgaria. The fungus is heterothallic and requires the pairing of two compatible mating types for development of fertile pseudothecia. Both mating types of A. rabiei were isolated previously from naturally infected, cultivated chickpeas (C. arietinum L.) from northeastern and southern Bulgaria (1), and the teleomorph, Didymella rabiei (Kovachevski) v. Arx, developed on naturally infested chickpea debris from both regions when it was incubated at appropriate environmental conditions. Isolations were made from lesions on the leaflets, stems, pods, and seeds of C. montbretii by surface disinfecting tissue in 0.25% NaOCl for 5 min, drying on paper hand towels, and placing small pieces of tissue on 2% water agar and Difco potato dextrose agar. Plates were incubated at 22 to 24°C under fluorescent lights with a 12-h photoperiod. A. rabiei was isolated from all foliar tissues of the plant, including seeds. Koch's postulates were fulfilled by inoculating the foliage of chickpea PI 458870 and reisolating the fungus from lesions that developed on the leaflets and stems. Six Bulgarian isolates of A. rabiei from C. montbretii were paired with compatible mating type tester isolates of A. rabiei, MAT1-1 (ATCC 76501) and MAT 1-2 (ATCC 76502), following the procedure of Kaiser and Kusmenoglu (2). Both mating types were found among the six isolates. Two were MAT 1-1 and four MAT 1-2. The teleomorph did not develop on the small amount of naturally infested chickpea residue tested. Therefore, in Bulgaria, both cultivated and wild chickpeas are infected naturally by A. rabiei and both mating types have been isolated from these hosts. D. rabiei will likely be found in native stands of C. montbretii in Bulgaria as more samples of overwintered infested debris are examined for the teleomorph. This is the first report of A. rabiei causing blight of a wild Cicer sp. References: (1) W. J. Kaiser. Can. J. Plant Pathol. 19:215, 1997. (2) W. J. Kaiser and I. Kusmenoglu. Plant Dis. 81:1284, 1997.

First Report of Natural Infection of Pisum sativum subsp. elatius by Mycosphaerella pinodes in Bulgaria.

Pisum sativum L. subsp. elatius (Steven ex M. Bieb.) Asch. & Graebn. is a wild pea species that is native to Bulgaria. It readily crosses to the cultivated pea species P. sativum subsp. sativum. Field pea is an important component in the crop rotation system of the northeast region of Bulgaria. Little is known or published on the diseases of wild Pisum subspecies. In June 1997, brown to reddish brown, irregularly shaped lesions 5 to 10 mm in diameter were found on the leaves and stems of P. sativum subsp. elatius growing under native conditions in the low growing vegetation in a mixed forest habitat on the Black Sea coast at Albena, Bulgaria (43°22'26″N; 28°05'02″E) at an elevation of about 50 m. Black pycnidia were observed within lesions and contained hyaline, primarily two-celled conidia that measured 7 to 17 × 3 to 5 µm. On artificially inoculated pea stem pieces incubated on 2% water agar (WA) at 22 to 24°C for 28 days, pseudothecia developed with hyaline, two-celled ascospores constricted at the septum and measuring 12 to 17 × 4 to 7 µm. Black chlamydospores produced singly or in chains also formed in infected foliar tissues and on potato dextrose agar (PDA) and WA. Isolations were made from the lesions on pea tissue onto WA and PDA after disinfesting in 0.25% NaOCl for 5 min. Koch's postulates were fulfilled by inoculating the foliage of P. sativum subsp. sativum cvs. Dark Skin Perfection and Sounder and P. sativum subsp. elatius (W6-20047), and reisolating the fungus from lesions that developed on the inoculated leaves and stems. The wild Pisum fungus was identified as Mycosphaerella pinodes (Berk. & Blox.) Vestergr. based on cultural and morphological characteristics (2), pathogenicity tests, and by comparing random amplified polymorphic DNA (RAPD) markers with those of American Type Culture Collection (ATCC) isolates 201628 to 201633 of M. pinodes. The fungus was identified as a pathogen of cultivated peas in Bulgaria by Kovachevsky and Hristov (1) in 1949. This is the first report of M. pinodes infecting P. sativum subsp. elatius in Bulgaria and other countries where P. sativum subsp. elatius is a native plant species. References: (1) I. H. Kovachevsky and A. Hristov. 1949. Bulgarian Acad. Sci., Scientific-Popular Ser. 10. (2) E. Punithalingam and P. Holliday. 1972. CMI Descript. of Pathog. Fungi and Bacteria, no. 340. Commonwealth Mycol. Institute, Kew, England.

First Report of Anthracnose of Lentil Incited by Colletotrichum truncatum in Bulgaria.

In June 1992 and 1995, anthracnose of lentil (Lens culinaris Medik.) incited by Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore was widespread in field trials at the Institute for Wheat and Sunflower 'Dobroudja' near General Toshevo in northeastern Bulgaria. Lesions on the leaves, stems, and pods were usually white to grayish on younger plants, often turning brown as plants matured. Severe infection usually resulted in dieback and/or death of plants. Acervuli containing spores and dark setae were observed within lesions, and conidia from the acervuli produced pure cultures of C. truncatum. Conidia were hyaline, onecelled, falcate to nearly straight with a prominent clear area in the center of highly granular cytoplasm, and measured 17.6 to 19.8 × 4.4 µm. C. truncatum was seed-borne in naturally infected lentil cv. Tadjikskaya 95 at low frequencies (<2%). Koch's postulates were fulfilled by inoculating the foliage of lentil cvs. Brewer and Pardina and reisolating the fungus from stem and petiole lesions. In pathogenicity tests, three isolates of C. truncatum from the foliage and seeds of lentil caused severe symptoms on inoculated lentil cvs. Brewer and Pardina, similar to those observed on diseased lentils in Bulgaria. The fungus also caused moderate symptoms on inoculated faba bean (Vicia faba L.) and pea (Pisum sativum L.), and light symptoms on inoculated chickpea (Cicer arietinum L.). In 1995, 258 USDA Plant Introduction (PI) accessions from the USDA lentil core collection were screened in replicated trials in northeastern Bulgaria and disease symptoms were observed in >90% of the lines. Anthracnose severity ranged from light to severe. A few accessions appeared to have acceptable levels of resistance to the disease. These included accessions from Iran (PI 431714 and 431717) and Spain (PI 533693). Also that year, C. truncatum was isolated from stem lesions of naturally infected bitter vetch (Vicia ervilia (L.) Willd.) at the Institute for Wheat and Sunflower 'Dobroudja'. The disease in Bulgaria appears to be identical to one causing anthracnose of lentil in Canada (1) and the United States (2). This is the first report of C. truncatum causing anthracnose of lentil in Bulgaria. References: (1) R. A. A. Morrall. Plant Dis. 72:994, 1988. (2) J. R. Venette et al. Plant Dis. 78:1216, 1994.

Inheritance of inter-simple-sequence-repeat polymorphisms and linkage with a fusarium wilt resistance gene in chickpea.

The inheritance of an inter-simple-sequence-repeat (ISSR) polymorphism was studied in a cross of cultivated chickpea (Cicer arietinum L.) and a closely related wild species (C. reticulatum Lad.) using primers that anneal to a simple repeat of various lengths, sequences and non-repetitive motifs. Dinucleotides were the majority of those tested, and provided all of the useful banding patterns. The ISSR loci showed virtually complete agreement with expected Mendelian ratios. Twenty two primers were used for analysis and yielded a total of 31 segregating loci. Primers based on (GA)n repeats were the most abundant while primers with a (TG)n repeat gave the largest number of polymorphic loci. Nucleotides at the 5' and 3' end of the primers played an important role in detecting polymorphism. All the markers showed dominance. We found an ISSR marker linked to the gene for resistance to fusarium wilt race 4. The marker concerned, UBC-855500, was found to be linked in repulsion with the fusarium wilt resistance gene at a distance of 5.2 cM. It co-segregated with CS-27700, a RAPD marker previously shown to be linked to the gene for resistance to fusarium wilt race 1, and was mapped to linkage group 6 of the Cicer genome. This indicated that genes for resistance to fusarium wilt races 1 and 4 are closely linked. The marker UBC-855500 is located 0.6 cM from CS-27700 and is present on the same side of the wilt resistance gene. To our knowledge this is the first report of the utility of an ISSR marker in gene tagging. These markers may provide valuable information for the development of sequence-tagged microsatellite sites (STMS) at a desired locus.