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.

Evolution of four types of RNA editing in myxomycetes.

The myxomycete Physarum polycephalum requires extensive RNA editing to create functional mitochondrial transcripts. The cytochrome c oxidase subunit 1 (col) transcript exhibits a combination of editing forms not found together in any other eukaryotic RNA: 66 insertions of ribonucleotides (59 Cs, a single U, and three mixed dinucleotides) as well as base conversion of four Cs to Us (Gott et al., J Biol Chem, 1993, 268:25483-25486). Through a phylogenetic survey of col DNA genes and RNA transcripts in representative myxomycetes, we have decoupled the four types of editing in this lineage. Some myxomycetes share insertional editing with P. polycephalum, yet lack C--> U conversion, consistent with previous reports of separation of insertional and base conversion editing in P. polycephalum extracts (Visomirski-Robic & Gott, RNA, 1995, 3:821-837). Most remarkably, we detect unique evolutionary histories of the three different types of insertional editing, though these have been indistinguishable in vitro. For example, Clastoderma debaryanum exhibits insertions of Us, but not Cs or dinucleotides.

Mitochondrial RNAs of myxomycetes terminate with non-encoded 3' poly(U) tails.

We examined the 3' ends of edited RNAs from the myxomycetes Stemonitis flavogenita and Physarum polycephalum using a modified anchor PCR approach. Surprisingly, we found that poly(A) tails are missing from the cytochrome c oxidase subunit 1 mRNA (coI) from both species and the cytochrome c oxidase subunit 3 mRNA (cox3) from P. polycephalum. Instead, non-encoded poly(U) tails of varying length were discovered at the 3' ends of these transcripts. These are the first described examples of 3' poly(U) tails on mature mRNAs in any system.

Xenopus NK cells identified by novel monoclonal antibodies.

Early-thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monoclonal antibodies (mAb) which identify candidate NK cells at the amphibian level of evolution. Hybridomas, generated from mice immunized with splenocytes from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stained increased proportions of splenocytes from Tx compared with control frogs. These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor-rich medium, exhibited spontanous killing of MHC-deficient allotumor targets. mAb-defined splenocytes also rapidly induced apoptosis of such tumor targets. Dual color analysis confirmed that NK cells are neither T nor B cells. Cytospins of splenocytes isolated with anti-NK mAb revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated each anti-NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of approximately 70 - 85 kDa, whereas mAb 1F8 and 4D4 stained less intensely, but identified similar protein bands.

Natural cytotoxicity towards allogeneic tumour targets in Xenopus mediated by diverse splenocyte populations.

We have recently demonstrated NK-like activity in the spleen of the clawed frog, Xenopus laevis. This paper investigates the cellular basis of this natural cytotoxicity. Significant levels of cytotoxicity towards B3B7 allogeneic thymus tumour targets, that express neither class Ia nor class II MHC proteins, occurred after splenocytes from either control or early-thymectomized (Tx) year-old Xenopus were cultured for 48 hours. Killing by Tx cells required their culture in growth factor-rich medium (GFM) obtained from concanavalin A-stimulated cells. Immunomagnetic cell sorting revealed that cytotoxic effectors in both control and Tx frogs were found in the B cell-depleted population, but never in the B cell-enriched fraction. Splenocytes from control Xenopus, depleted of T cells by magnetic sorting and following culture in GFM, also developed natural cytotoxicity towards allotumour cells. Magnetic cell sorting also revealed that purified (CD5+) T cells cultured for 48 hours in GFM also became able to lyse the allogeneic tumour targets. Cytotoxicity mediated by T cells resided not only in the CD5+, CD8+ population, but also in the CD5+, CD8- (putative CD4+) T cell subset. Ontogenetic studies revealed that splenocytes from 6-7 week-old (stage 56-57) control larvae, even after 48 hr culture in GFM, were unable to spontaneously lyse the allotumour targets, whereas cultured splenocytes from 6 month old froglets were effective killers. Thymocytes from larvae or adults routinely failed to kill tumour cells. The work highlights the need to use Tx Xenopus to further explore non-T-cell-mediated, NK-like cytotoxicity at the amphibian level of evolution.

T-cell and natural killer cell development in thymectomized Xenopus.

The Xenopus early-thymectomy model system is used to investigate the extent to which the thymus controls T-cell development and to probe the evolution of natural killer (NK) cells. Loss of T-cell function following thymectomy, together with the paucity of cells expressing monoclonal antibody-defined T-cell surface markers, and greatly reduced expression of T-cell receptor beta transcripts in spleen, liver and intestine, indicate that T-cell development in minimal in the absence of the thymus. Our findings therefore mitigate against the idea that a substantial extrathymic pathway of T-cell development exists in early vertebrate evolution. Rather, they suggest that in this amphibian representative T cells are predominately thymus dependent. In vitro studies with control and thymectomized Xenopus splenocytes reveal that a non-T/non-B population and also two T-cell subsets all display natural cytotoxicity towards allogeneic thymus lymphoid tumour cells (which are deficient in MHC antigen expression). Since Xenopus thymectomized early in larval development are permanently deficient in T cells, they may provide a useful phylogenetic model for the study of NK cells.

NK-like activity against allogeneic tumour cells demonstrated in the spleen of control and thymectomized Xenopus.

This paper addresses the issue of natural killer (NK) cell evolution by searching for NK-like activity in an amphibian representative, the immunologically well-characterized clawed frog, Xenopus laevis. Using in vitro 6 h 51chromium release assays, we have shown that splenocyte effectors from early thymectomized (Tx) year-old frogs, but not from control siblings, are able to spontaneously lyse allogeneic thymus tumour cell lines that lack MHC antigen expression. Such lytic capacity can be readily induced in control Xenopus and elevated in Tx frogs by a single injection of tumour cells, with maximal splenocyte cytotoxicity occurring 3 days postinjection, the amount of 51Cr-release correlating directly with effector: target ratios. Splenocytes, even those from tumour-injected frogs, are unable to lyse allogeneic splenic lymphoblasts or erythrocyte targets, even when the latter are coated with IgY (the Xenopus IgG equivalent); moreover, we were unable to demonstrate any splenocyte-induced lysis of the human NK cell target K562. Lymphokine-activated killing (LAK) in Xenopus is suggested, since Tx splenocytes cultured in cytokine-rich medium (from concanavalin A-stimulated control splenocytes) display significantly elevated killing of allogeneic tumour targets. Flow cytometric analysis highlights the loss of T cell markers from the spleen of Tx frogs and reveals a variable staining pattern of both control and Tx splenocytes when treated with a mAb that binds to both fish non-specific cytotoxic cells and human NK cells. Prospects for identifying the cellular basis of NK-like activity in Xenopus are discussed in the light of these experiments.

Ontogeny and thymus-dependence of T cell surface antigens in Xenopus: flow cytometric studies on monoclonal antibody-stained thymus and spleen.

Recently generated anti-Xenopus T cell monoclonal antibodies (mAbs) to the 120 kDa XTLA-1 determinant and against the putative CD5 and CD8 homologues, together with anti-IgM and anti-MHC class II mAbs, are used in dual colour flow cytometric experiments to characterize cell surface antigenic expression on lymphocytes in thymus and spleen of Xenopus laevis during larval and early adult life and also in metamorphosis-inhibited animals. Histological confirmation of T cell emergence early in larval ontogeny is supplied by cryostat sections stained for CD8. Five-day thymectomy, i.e. prior to T-lineage cell differentiation in the thymus, abolishes T cell marker expression in the spleen for up to 1 year. Moreover, late larval (20 days) or early adult (3 months) thymectomy (i.e. removal after peripheralization of T cells has occurred) also leads to severe depletion of mAb-defined T cells in the spleen.

Site-dependent intestinal hydrolysis of valproate and morphine glucuronide in the developing rat.

A previous pharmacokinetic study in developing rats suggested that enterohepatic recirculation of valproic acid was absent prior to weaning. One explanation for this observation is that the rate, extent, and/or primary site of glucuronide hydrolysis in the gastrointestinal tract changes during postnatal development. To test this hypothesis, the hydrolysis of two model glucuronide conjugates, valproate glucuronide and morphine-3-beta,D-glucuronide, was examined in vitro in homogenates of small and large intestine obtained from rats at 5-60 days postpartum. Analysis of initial hydrolysis rates indicated that the principal hydrolytic site for both glucuronide conjugates shifted from the upper to lower intestine as the animals developed. The initial hydrolysis rate (nmol/min/g) for valproate glucuronide decreased from 38.1 +/- 10.2 to 8.25 +/- 2.42 in the small intestine, and increased from 14.2 +/- 2.3 to 105 +/- 22 in the large intestine, as rats developed from 5 to 60 days postpartum, respectively. Likewise, the intestinal hydrolysis rate for morphine-3-beta,D-glucuronide decreased from 3.70 +/- 0.46 to 0.646 +/- 0.165 in the small intestine, and increased from 3.50 +/- 0.48 to 115 +/- 30 in the large intestine, as rats developed from 5 to 60 days postpartum, respectively. If hydrolysis occurs immediately after excretion of conjugate into the intestine in neonatal rats, minimal temporal delay between excretion of conjugate and reabsorption of liberated parent may occur, therefore concealing the secondary increase in serum drug concentrations associated with enterohepatic recirculation. In contrast, the time required for conjugates to reach the primary hydrolytic site in adult animals is sufficient for appearance of secondary peaks in the serum drug concentration-time profile.

Incomplete tolerance induced in Xenopus by larval tissue allografting: evidence from immunohistology and mixed leucocyte culture.

Application of adult skin allografts to Xenopus larvae has been a favoured protocol for probing the development of self-tolerance. A more physiologic approach is presented here that examines the immunologic outcome of grafting semi- or fully allogeneic larval skin or spleen to age-matched, larval Xenopus (X. laevis/X. gilli clonal hybrids). Following such grafting at 2 or 4 weeks-of-age, young froglets (4-5-months-old) are generally unable to reject second-set skin transplants, but destroy third-party skin vigorously, the MHC class II-rich spleen proving especially effective at inducing this tolerance. In contrast, following larval grafting of semiallogeneic tissues, mixed leucocyte culture performed at the end of metamorphosis (6 weeks) and again at 6 months reveals splenocyte reactivity toward donor-strain stimulators. Immunohistological findings extend this observation of anti-donor reactivity (suggesting incomplete tolerance) to the graft site. Thus despite excellent health when viewed externally, apparently tolerated second-set skin transplants display localised infiltration (especially into the epidermis) by CD8+ T cells and increased numbers of MHC class I and II-expressing cells by 3 weeks post-grafting. The immunologic implications of these findings are discussed.

Skin xenograft rejection in Xenopus--immunohistology and effect of thymectomy.

Immunohistology, using the T-lineage-specific monoclonal antibody XT-1 and an anti-IgM mAb, illustrates differences in the cellular basis of skin allograft and xenograft destruction displayed by control froglets (X. laevis). Thus T cells predominate within allografts, whereas B-lineage cells accumulate under xenografts (from X. tropicalis). The possibility that T cells do not play a central role in mediating xenograft rejection is consistent with the finding that early thymectomy (at 7 days) has minimal effect on rejection end points of X. tropicalis transplants. However, rejection of skin from a "phylogenetically less distant" xenogeneic species (X. borealis) is shown here to be impaired in early thymectomized X. laevis. Differences in the extent to which the thymus has been shown to influence skin xenograft rejection in Xenopus are discussed.

Enterohepatic recirculation and renal metabolism of morphine in the rat.

Morphine (2.5 mg/kg) was administered iv to intact (I), bile duct-cannulated (BC), and bile duct-cannulated--renal-ligated (BC-RL) rats (n = 4 per group) to investigate the extent of enterohepatic recirculation and renal metabolism of the drug. A decrease in the serum area under the concentration-time curve (AUC) was observed for the BC in comparison with I rats. From these AUC values, it was determined that approximately 16% of the administered dose was subject to enterohepatic recirculation. In addition, a statistically significant (p less than 0.05) decrease in the systemic clearance of morphine was observed in the BC-RL rats compared with the BC animals (55.2 +/- 17.2 versus 31.4 +/- 8.5 mL/min/kg). This decrement in systemic clearance appeared to be the result of a significant decrease in the formation clearance of morphine glucuronide after ligation of the renal pedicles (23.2 +/- 4.8 versus 10.9 +/- 5.0 mL/min/kg). Renal metabolic clearance was calculated as 15.7 mL/min/kg, accounting for 28.5% of the systemic clearance of morphine. Hepatic clearance (31.4 +/- 8.5 mL/min/kg) accounted for 56.8% of total systemic clearance.

Splenocyte response to T cell-derived cytokines in thymectomized Xenopus.

A miniaturized, "hanging-drop" bioassay reveals that splenocytes from early-thymectomized (Tx) Xenopus can respond (by enhanced thymidine incorporation) to thymic-dependent "cytokines" generated in PHA- or alloantigen-stimulated cultures. Preliminary evidence, using fluorescence activated cell sorting, indicates that surface IgM- splenocytes, rather than sIgM+ cells, from Tx toads are sensitive to the crude, splenocyte-derived, active supernatants. Although these responsive cells display residual, but low, reactivity to PHA, their thymus independence is suggested by flow cytometric observations using the anti-T cell monoclonal antibody XT-1. The development of "T-like" cells in Tx Xenopus is discussed.

In vitro cytotoxicity in adult Xenopus generated against larval targets and minor histocompatibility antigens.

Our experiments reveal that application of several minor H antigen-disparate skin grafts to adult Xenopus over an 18-month period can lead to in vitro generation of CML reactivity toward these minor antigens. Furthermore, we demonstrate that, following MHC-disparate skin graft rejection, adult effectors can efficiently kill both adult and larval donor-strain targets; this killing is MHC-specific and requires MLC restimulation with cells syngeneic to the skin graft donor. The ability to kill larval lymphoblasts, which have been shown elsewhere to be MHC class I-negative but class II-positive, suggests the probable importance of class II-restricted killing in this species.

Attempts to break perimetamorphically induced skin graft tolerance by treatment of Xenopus with cyclophosphamide and interleukin-2.

The maintenance of skin allotolerance induced by perimetamorphic application of MHC-disparate skin to isogeneic Xenopus is investigated. Removal of the perimetamorphically applied first-set graft after 4 weeks did not, in general, completely break allotolerance; however, many second-set semi-allogeneic grafts, applied up to 14 weeks after first-set removal, were no longer maintained in perfect condition. Skin allografts tolerated for up to 42 weeks continued to express donor histocompatibility antigens, as indicated by their survival times when transplanted back to the original donor or recipient strain. Treatment with human recombinant IL-2 (rIL-2), shown elsewhere to be an effective immunoregulatory lymphokine for Xenopus in vivo, failed to cause long-term-tolerated 1st-set allografts, or newly-applied 2nd-set grafts, to be rejected. In contrast, cyclophosphamide (CyP) treatment led to acute (less than 4 weeks) destruction of both 1st- and 2nd-set allografts; breaking of tolerance was regularly seen when donor and host differed by two MHC haplotypes, but occurred infrequently in semiallogeneic combinations. The experiments suggest that skin-induced allotolerance is maintained by an immunosuppressive mechanism, that is CyP-sensitive but resistant to rIL-2 treatment.

Thymocyte/stromal cell chimaerism in allothymus-grafted Xenopus: developmental studies using the X. borealis fluorescence marker.

These experiments employ the X. borealis (quinacrine-fluorescence) cell marker to illustrate that froglet (normal or in vivo-irradiated) thymuses, alloimplanted to 4- to 6-week-old, 7-day-thymectomized hosts, become filled with host lymphoid cells, while a range of thymic stromal cell types (e.g. epithelial derivatives and reticuloendothelial cells) remain donor derived. A time-course study of 4 micron historesin-embedded sections reveals that for normal thymus implants, host cells begin to immigrate in good number only after metamorphosis. In contrast, 3000 rad-irradiated thymus implants begin to be repopulated with host lymphocytes within 2 weeks postimplantation, when hosts are still at a late larval stage of development. Despite rapid colonization by host lymphoid cells, irradiated thymuses remain small and often disappear in early adult life. Donor-derived lymphocytes frequent the blood and both the red pulp and perifollicular regions of the spleen following normal thymus implantation, whereas such thymic emigrants were not seen in the periphery of thymectomized hosts grafted with irradiated thymus glands.

Splenic involvement in amphibian transplantation immunity.

The spleen has been identified as a centre of alloimmune reactivity in control Xenopus. Levels of tritiated thymidine labelling and pyroninophilic cells are elevated in spleens of skin-allografted toadlets when compared with autografted and nongrafted animals. Second-set alloimmune reactivity can be transferred by implanting a spleen from a donor that has rejected one or two grafts into a nonsensitized host. Spleen donor and host in these experiments were mutually tolerant, following reciprocal transfer of embryonic tissue grafts. In contrast, studies on the uptake of tritiated thymidine and levels of pyroninophilia in animals thymectomized at 7 or 8 days of age suggest lack of splenic involvement in the chronic first-set allograft rejection that can still occur in the absence of the thymus. The lymphoid organ origin of "thymic-independent" alloimmunity still awaits clarification.