First Report of Diplodia Cane Dieback of Grapevine in Bolivia.

Grape (Vitis vinifera L.) is an important commercial crop in the temperate regions of Bolivia where it has been grown for hundreds of years. In October of 2001, diseased canes of grape (cv. Muscat of Alexandria) were collected in a vineyard in Yotala, Department of Chuquisaca in southern Bolivia. In this planting of more than 1,000 plants, more than 75% were exhibiting cane dieback symptoms and many were dead or dying. No disease was observed on grape berries. Symptoms of the disease were similar to those reported for Diplodia cane dieback (1). Cankers ranging from 2 to 10 cm long and 0.5 to 3 cm wide were observed. When diseased canes were placed in a moist chamber, conidia oozed from pycnidia in black cirri. Immature conidia were hyaline and one-celled, but mature conidia were dark brown (20 to 30 × 10 to 15 µm) with one median septum and longitudinal striations. The pathogen was tentatively identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (synonyms Diplodia natalensis Pole-Evans and Botryodiplodia theobromae Pat.), teleomorph Botryosphaeria rhodina (Cooke) Arx) (2). Fungi were isolated from cankers on diseased canes by surface disinfestation in 0.25% NaOCl for 5 min and placing small pieces of tissue on 2% water agar or potato dextrose agar (PDA). L. theobromae was isolated from these tissues. Koch's postulates were fulfilled by inoculating grape berries and canes with the pathogen. Five grape berries were surface disinfested and inoculated by wounding with a sterile scalpel and inserting a piece of fungal mycelium on PDA in the wounded sites. The same number of control berries was similarly treated with sterile PDA. Inoculated and control berries were placed in plastic, moist chambers in the laboratory at ambient temperature (15 to 28°C) in the dark. Five canes on two potted plants were inoculated with the same isolate of the pathogen in a similar manner as the berries. The inoculated and control sites on canes were wrapped with masking tape. Plants were placed in a moist chamber for 5 days. After 8 days, inoculated berries were rotting and the inoculated sites were covered with grayish mycelium. Within 12 days, cankers as much as 3 cm long developed on the inoculated canes, and in some lesions, black pycnidia were observed. No lesions developed in the wounded control canes. The pathogen was reisolated from inoculated berries and canes, but not from control berries or canes. The teleomorph was not observed on any naturally infected canes or on those inoculated with the anamorph. The pathogen was identified as L. theobromae based on symptoms (1), cultural and morphological characteristics (2), and pathogenicity tests. The disease poses a potential threat to the cultivation of grapevine in southern Bolivia. To our knowledge, this is the first report of Diplodia cane dieback of grapevine in Bolivia. References: R. C. Pearson and A. C. Goheen. Compendium of Grape Diseases. The American Phytopathological Society, St. Paul, MN, 1988. (2). E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, England, 1976.

Future innovations in anti-platelet therapies.

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

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.

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and hemostasis.

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SUMMARY: Objective Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells. Methods and Results The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47-deficient mice or following inhibition of HSP47. Conclusions Our study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen-mediated signalling and therefore thrombus formation and hemostasis.

Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM.

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183-10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.

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.

Anti- and pro-apoptotic activities of baculovirus and Drosophila IAPs in an insect cell line.

The anti-apoptotic activities of two baculovirus IAPs, OpIAP and CpIAP, were directly compared with that of two Drosophila IAPs, DIAP1 and DIAP2, in the same insect cell line, SF-21 cells. Like OpIAP and CpIAP, DIAP1 inhibited actinomycin D-induced apoptosis and apoptosis induced by Doom. Removal of the RING finger of DIAP1 reduced but did not eliminate its anti-apoptotic activity. DIAP2 was unable to inhibit actinomycin-D induced apoptosis but was able to partially inhibit Doom-induced apoptosis. The baculoviral BIR and RING finger regions, when separated, were unable to block apoptosis induced by actinomycin D or Doom. Instead, the BIR regions of OpIAP and CpIAP as well as the RING finger regions of CpIAP and DIAP1 induced apoptosis. Thus, there were significant differences in the manner in which the different domains of the viral and cellular homologues of IAPs interacted with the components of the pathways regulating apoptosis in SF-21 cells.

The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drICE.

Many members of the Inhibitor of Apoptosis (IAP) family inhibit cell death and existing data suggest at least two mechanisms of action. Drosophila IAPs (D-IAP1 and D-IAP2) and a baculovirus-derived IAP, Op-IAP, physically interact with and inhibit the anti-apoptotic activity of Reaper, HID, and Grim, three genetically defined inducers of apoptosis in Drosophila, while human IAPs, c-IAP1, c-IAP2, and X-IAP interact with a number of different proteins including specific members of the caspase family of cysteine proteases which are crucial in the execution of cell death. We have examined whether insect-active IAPs can inhibit apoptosis induced by selected caspases, Drosophila drICE, Sf-caspase-1, and mammalian caspase-3, in insect SF-21 cells. D-IAP1 inhibited apoptosis induced by the active forms of all three caspases tested and physically interacted with the active, but not the proform of drICE. MIHA, the mouse homolog of X-IAP and an effective inhibitor of caspase-3, also interacted with and blocked apoptosis induced by active drICE but was relatively ineffective in blocking Sf-caspase-1. Op-IAP and D-IAP2 were unable to inhibit effectively any of the active caspases tested and failed to interact with drICE. The Drosophila IAPs and Op-IAP, but not MIHA, blocked HID-initiated activation of pro-drICE. We conclude that D-IAP1 is capable of inhibiting the activation of drICE as well as inhibiting apoptosis induced by the active form of drICE. In contrast, D-IAP2 and Op-IAP are more limited in their inhibitory targets and may be limited to inhibiting the activation of caspases.

Distribution of Mating Types and the Teleomorph of Ascochyta rabiei on Chickpea in Turkey.

One hundred forty-five isolates of Ascochyta rabiei, the cause of Ascochyta blight of chickpea, were collected from chickpea (Cicer arietinum) in 23 provinces of Turkey. Each isolate was tested for mating type with compatible MAT1-1 and MAT1-2 tester isolates. Both mating types were found in 18 provinces. Of the isolates tested, 59% were MAT1-1 and 41% were MAT1-2. A great deal of variation in cultural characteristics was observed among the Turkish isolates in mycelial growth, sporulation, and colony appearance. Mature pseudothecia of Didymella rabiei, the teleomorph (sexual state) of A. rabiei, developed on naturally infested chickpea debris collected in 15 of 20 provinces when incubated under favorable conditions. This is a new geographic record for D. rabiei. The teleomorph may play an important role in long-distance dissemination of the pathogen and in increasing genetic diversity in the pathogen population in Turkey.

A Vascular Wilt of Turnsole Caused by Fusarium oxysporum.

Turnsole (Chrozophora tinctoria) is a common spring-summer weed in chickpea (Cicer arietinum) and other dry-land crop production areas in southern Spain. Under field conditions, this weed often develops a general wilt and eventual death associated with a vascular discoloration of stems and roots. Diseased turnsole plants frequently occurred together with chickpea plants affected by Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a major disease of chickpea crops in southern Spain (1). Isolations from roots, stems and leaf petioles of turnsole plants consistently yielded F. oxysporum, and it was morphologically similar to F. oxysporum f. sp. ciceris. To test the pathogenicity of this fungus, germinated and previously surface-sterilized seeds of turnsole and chickpea cultivar Blanco Lechoso were planted in a greenhouse soil mixture artificially infested with four isolates of F. oxysporum from turnsole and two isolates of F. oxysporum f. sp. ciceris, one of them inducing the wilt syndrome and the other causing yellowing (1). Pathogenicity tests were conducted following the standard inoculation method used for F. oxysporum in chickpea (1). Inoculated and control plants were maintained in a greenhouse at 15 to 30°C. Isolates of F. oxysporum from turnsole caused wilt symptoms and death of turnsole plants within 2 months, but chickpea isolates did not affect turnsole. Conversely, chickpea plants were affected only by the two isolates of F. oxysporum f. sp. ciceris from chickpea. All diseased chickpea and turnsole plants exhibited typical vascular discoloration. F. oxysporum was consistently reisolated from the vascular tissues of roots, stems, and leaf petioles of affected plants. Based on these results, the fungus causing wilt of turnsole was identified as a forma specialis of F. oxysporum different from the chickpea wilt pathogen. Since the Fusarium wilt diseases of turnsole and chickpea are caused by different pathogens, the occurrence of F. oxysporum causing wilt of turnsole in the field can not be used to forecast Fusarium wilt of chickpea, but it may be considered as a potential biocontrol agent of this weed under field conditions. This is the first report of F. oxysporum causing wilt of turnsole. Reference: (1) A. Trapero-Casas and R. M. Jiménez-Díaz. Phytopathology 75:1146, 1985.

Lupins, a New Host of Phytophthora erythroseptica.

Several lupin (Lupinus) species are native to southern Spain (2). The white lupin, Lupinus albus L., is the most important crop, and its seeds are used for human consumption and animal feed. Accessions of three indigenous species, L. albus, L. angustifolius L., and L. luteus L., and an introduced species from South America, L. mutabilis Sweet, were planted during October in replicated yield trials in acidic soils (pH 6.5) in the Sierra Morena Mountains (elevation 350 m) north of Córdoba. Root and crown rot disease was widespread and very serious on the indigenous lupins, particularly in several patches of white lupin cultivars. Infected plants were devoid of feeder rootlets, and the tap roots, crowns, and lower stems were necrotic and turned dark brown to black. Rotted roots were colonized heavily by fungal oospores. Many affected plants wilted and died before flowering. A Phytophthora sp. was isolated consistently from the necrotic roots and crowns of symptomatic white lupins. The same fungus also was isolated from the necrotic root tissues of the other indigenous lupin species. Isolates of the fungus from diseased white lupins were homothallic and produced oospores rapidly and abundantly on corn meal and V8 agars. Antheridia were amphigynous, and aplerotic oospores ranged from 22 to 32 µm (average 27 µm). Nonpapillate, ovoidobpyriform sporangia were produced only in water on simple sympodial sporangiophores. Cultures on V8 agar grew at 5 to 30°C (optimum ≈25°C). The species was identified as Phytophthora erythroseptica Pethybr. based on morphology of oospores, sporangia, and other cultural characteristics (1). Koch's postulates were fulfilled by planting seeds of white lupin cv. Multulupa in sterile potting soil infested with a blended culture on V8 agar from a white lupin isolate of P. erythroseptica and reisolating the fungus after 28 days from lesions that developed on the roots and crowns of inoculated plants incubated in a greenhouse at 16 to 26°C. The fungus was not isolated from white lupins seeded in potting soil inoculated with sterile V8 agar. In pathogenicity tests, two isolates of P. erythroseptica from white lupins caused severe symptoms on the roots and crowns of inoculated white lupin cv. Multulupa similar to those observed on white lupins naturally infected in field trials. These isolates also caused root and crown rots on inoculated L. luteus and L. angustifolius. The fungus did not infect the roots or crowns of tarwi (L. mutabilis cv. SCG 20), alfalfa (Medicago sativa cv. Moapa), bean (Phaseolus vulgaris cv. Contender), chickpea (Cicer arietinum cv. Blanco Lechoso), faba bean (Vicia faba cv. Arboleda), lentil (Lens culinaris cv. local), pea (Pisum sativum cv. Lancet), soybean (Glycine max cv. Akashi), or subterranean clover (Trifolium subterraneum cv. Seaton-park). The tests were repeated, and the results were similar. This is the first report of P. erythroseptica infecting Lupinus spp. References: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. (2) B. Valdés et al. 1987. Flora Vascular de Andalucía Occidental. Ketres, Barcelona, Spain.

First Report of Ascochyta Blight of Chickpea in Latin America.

Chickpea (Cicer arietinum L.) has been cultivated in different regions of Bolivia for hundreds of years. In the highlands (2,400 to 3,000 m above sea level) of the Department (state) of Chuquisaca in southern Bolivia, chickpea is an important cash crop for small farmers. During March through April 1999, a blight was observed infecting local chickpea landraces in Chamicle, Escana, Kullpa Ckasa, Presto, Q'ara Puncu, Santa Rosalia, Sucre, and Yotala in Chuquisaca, and its cause was tentatively identified as Ascochyta rabiei (Pass.) Labrousse (teleomorph Didymella rabiei (Kovachevski) v. Arx) based on disease symptomatology. Stems, leaflets, and pods of infected plants exhibited abundant necrotic lesions. Isolations were made from lesions on leaflets, stems, pods, and seeds of infected plants on 2% water agar and potato dextrose agar. The fungus was isolated from the foliar and reproductive tissues of infected plants. Koch's postulates were fulfilled by inoculating the foliage of 15-day-old seedlings of a local chickpea landrace with spore suspensions of three isolates of the pathogen from Escana, Santa Rosalia, and Sucre. Inoculated and control (sterile water) plants were incubated in moist chambers for 4 days in the laboratory at ambient temperatures and under natural daylight. The fungus was reisolated from lesions that developed on the leaflets, petioles, and stems of all inoculated seedlings but not from tissues on any of the noninoculated control plants. The fungus was identified as A. rabiei based on symptoms, cultural and morphological characteristics (2), and pathogenicity tests. Above average rainfall and cool weather during March and April favored development and spread of the disease in many chickpea-growing areas. Severe infection usually resulted in dieback and death of plants and reduced yields. Additionally, A. rabiei was isolated from chickpea seeds purchased in the markets of Sucre and Monteagudo and in seeds used by farmers in Escana to plant the 1999 crop (which had supplied the plants previously observed with blight). The teleomorph did not develop on naturally infested chickpea debris from five locations when incubated over the winter on the soil surface in Sucre. Based on farmers' reports, it appears that Ascochyta blight of chickpea has been present in the Department of Chuquisaca and possibly other Bolivian departments for many years. This is the first report of the disease in either Bolivia or other countries of Latin America (Mexico and Central and South America) (1). References: (1) CAB. 1991. CAB Distribution Maps of Plant Diseases: Ascochyta rabiei. Map No. 151. CAB International Mycological Institute, Wallingford, England. (2) E. Punithalingham and P. Holliday. 1972. Ascochyta rabiei. Descriptions of Pathogenic Fungi and Bacteria No. 337. Commonwealth Mycological Institute, Kew, England.

Ascochyta fabae and A. lentis: Host Specificity, Teleomorphs (Didymella), Hybrid Analysis, and Taxonomic Status.

Isolates of Ascochyta fabae from faba bean (Vicia faba) and A. lentis from lentil (Lens culinaris) collected from different countries were used in this study. The Didymella teleomorph (sexual state) of each fungus was induced to develop and mature on inoculated sterile lentil stems. Both fungi were heterothallic, with two mating types, designated MAT1-1 and MAT1-2. When certain isolates of A. fabae and A. lentis were crossed, hybrid pseudothecia developed. Growth, sporulation, colony appearance, morphology, and pathogenicity of the hybrid progeny frequently differed greatly from the parent isolates. Inoculations with single-ascospore progeny from matings among compatible isolates of A. fabae caused disease in faba bean but not in lentil; inoculations with single-ascospore progeny from matings among compatible isolates of A. lentis incited disease in lentil but not in faba bean. Inoculations with single-ascospore progeny from crosses between faba bean and lentil isolates did not induce disease in either host. Asci from crosses between A. fabae and A. lentis mostly contained fewer than eight ascospores that were, on average, larger than those from eight-spored asci. Matings among certain isolates of A. fabae resulted in production of pseudothecia with ascospores considerably larger than is typical for D. fabae. Random amplified polymorphic DNA (RAPD) banding patterns of Ascochyta isolates from faba bean and lentil are clearly different, and banding patterns from hybrid progeny from crosses between A. fabae and A. lentis confirmed hybridity. RAPD markers proved useful in supporting identifications of ascospore isolates from faba bean to known Ascochyta species. Dendrogram analysis indicated similarity between the two fungal species was low. The pathogenicity tests, morphological characteristics, and RAPD markers indicate that A. fabae and A. lentis represent distinct taxa. D. lentis, with its anamorph, A. lentis, is proposed as a new species that is distinct from D. fabae, with its anamorph, A. fabae.

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.

First Report of Botryosphaeria dothidea and B. obtusa on Apple in Bolivia.

Gala and Winter Banana apples are important commercial crops in Azurduy and Lima Bamba, which are located in the Department (state) of Chuquisaca, Bolivia. White or bot rot (causal agent Botryosphaeria dothidea (Moug.:Fr.) Ces. De. Not. [anamorph Fusicoccum aesculi Corda]) and black rot (causal agent B. obtusa (Schwein.) Shoemaker [anamorph Sphaeropsis malorum Berk.]) have not been reported previously from Bolivia. Both fungi were isolated from apple fruit and branch cankers in Azurduy, but only B. dothidea was isolated from rotted fruit and limb cankers in Lima Bamba. Both fungi also were isolated from rotted Gala and Winter Banana fruit purchased in the markets in Sucre, Bolivia. Symptoms on fruit consisted of light-to-dark brown lesions that ranged from 3- to 8-cm in diameter. Cankers on limbs were sunken and reddish brown and ranged from 2 to 25+ cm in length and 0.5 to 3 cm in diameter. Neither pathogen produced pycnidia in lesions on rotted fruit, but they often developed in branch cankers. Pseudothecia of B. dothidea and B. obtusa were not observed. Identification of both pathogens was based on descriptions of their anamorphic stages (1). To fulfill Koch's postulates, four healthy Gala apple fruit were inoculated with two isolates of each pathogen by wounding the opposite faces of surface-disinfected fruit with a 5-mm-diameter cork borer and inserting mycelial plugs of the pathogens. Plugs were obtained from the margins of cultures growing on potato dextrose agar (PDA). Wounds were made on the opposite sides of each fruit, a mycelial plug of one of the pathogens was inserted in one wound, and on the opposite side, a plug of sterile PDA was inserted as a control. Each plug containing fungal mycelium or sterile PDA was covered with a plug of trimmed apple tissue, and the apple fruit were incubated in a moist chamber at 17 to 20°C for 10 days. Six branches on two young apple trees growing outdoors in a nursery were inoculated in a similar manner with one isolate of each pathogen: bark was wounded with a 5-mm-diameter cork borer, and the wounded area was inoculated with a plug of PDA containing the pathogen or a plug of sterile PDA for the control. The inoculated sites were wrapped with masking tape to prevent dehydration. Within 10 days, all fruit wounds inoculated with isolates of each pathogen developed brown lesions up to 5 cm in diameter. Each pathogen was reisolated from tissues in which it had been inoculated, but not from any of the noninoculated control sites. Within 6 to 8 weeks, all but one wound on branches inoculated with each pathogen developed depressed canker lesions up to 2 cm in length. Each pathogen was reisolated from the canker produced by inoculation with that pathogen, but not from any of the control sites. Reference: (1) T. B. Sutton. White rot and black rot. Pages 16-20 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinckle, eds. The American Phytopathological Society, St. Paul, MN, 1991.

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca(2+) entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca(2+) entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca(2+)-sensing role of STIM1 is served by the protein in the ER.

Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi.

BACKGROUND: Integrin-linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes. METHODS: The impact of ILK inhibition on integrin αII b ß3 activation and degranulation was assessed with the ILK-specific inhibitor QLT0267, and a conditional ILK-deficient mouse model was used to assess the impact of ILK deficiency on in vivo platelet aggregation and thrombus formation. RESULTS: Inhibition of ILK reduced the rate of both fibrinogen binding and α-granule secretion, but was accompanied by only a moderate reduction in the maximum extent of platelet activation or aggregation in vitro. The reduction in the rate of fibrinogen binding occurred prior to degranulation or translocation of αII b ß3 to the platelet surface. The change in the rate of platelet activation in the absence of functional ILK led to a reduction in platelet aggregation in vivo, but did not change the size of thrombi formed following laser injury of the cremaster arteriole wall in ILK-deficient mice. It did, however, result in a marked decrease in the stability of thrombi formed in ILK-deficient mice. CONCLUSION: Taken together, the findings of this study indicate that, although ILK is not essential for platelet activation, it plays a critical role in facilitating rapid platelet activation, which is essential for stable thrombus formation.