First Report of Syzygites megalocarpus (Mucorales) Web Mold on the Commercial Portabella Button Mushroom Agaricus bisporus in North America.

Agaricus bisporus (Lange) Imbach mushrooms, which are cultivated commercially under environmentally controlled conditions, are the most valuable crop in Pennsylvania. In August 2011, we first observed a mucoraceous mold colonizing primordia and mature basidiocarps of a new brown portabella strain of A. bisporus at two commercial mushroom farms in Chester County, PA. This strain is a hybrid between a commercial strain producing white basidiocarps and a brown wild type isolate of A. bisporus. Mushrooms mature in weekly "flushes". By third flush, 25% of the production surface at both farms was colonized by a fast growing mycelium that was initially white, subsequently yellow to golden brown, and finally grayish. Mushrooms colonized by the mold showed pitting, discoloration, and necrosis. Two pure cultures of the mold were obtained by the hyphal tip method from mature, necrotic basidiocarps at each farm. These isolates were accessioned in the ARS Culture Collection (NRRL, Peoria, IL) as NRRL 54814 to 54815 and 54818 to 54819. The cultures produced abundant aerial sporangiophores that branched dichotomously on potato dextrose agar. Light microscopic examination revealed that each branch terminated in a globose, multispored sporangium with a conspicuous columella. Individual cultures of NRRL 54818 and 54819 produced large (175 to 250 × 200 to 250 µm), barrel-shaped, dark brown to black zygosporangia between opposed suspensors, indicating they were homothallic. Morphological and cultural characteristics of the mold matched the description of Syzygites megalocarpus (3), a member of the Mucorales reported to colonize diverse, mostly fleshy basidiomycetes (2), including cultivated matsutake (Tricholoma matsutake) in Korea (1). Molecular phylogenetic confirmation of the morphological identification was obtained by PCR amplifying and sequencing domains D1 and D2 at the 5' end of the nuclear ribosomal large subunit (LSU rDNA). The four isolates shared an identical LSU rDNA allele. A search of the NCBI nucleotide database, using a partial LSU rDNA sequence from NRRL 54814 as the BLAST query, revealed that it shared 99.5% identity with AF157216.1, a reference isolate of S. megalocarpus NRRL 6288 (3). To assess whether cultures of S. megalocarpus could induce the disease, caps of portabella and white button mushrooms were inoculated with 3.7 × 106 sporangiospores. When incubated in moist chambers at 21 to 22°C with a 12-h photoperiod, disease symptoms developed in 2 to 3 days on portabella that included discoloration and pitting at the site of inoculation. S. megalocarpus was reisolated from the symptomatic mushrooms and produced a colony identical to the original. By comparison, white button mushrooms inoculated with S. megalocarpus, using the same method, only showed minor pitting and discoloration. Disease symptoms were not observed on mushrooms inoculated with water as a negative control. Although development of new commercial varieties derived using "wild" genetically diverse stocks is an effective way to introduce desirable traits into cultivated mushrooms, it carries the risk of introducing new diseases into the industry. References: (1) K.-H. Ka et al. Korean J. Mycology 27:345, 1999. (2) R. L. Kovacs and W. J. Sundberg. Trans. Il. State Acad. Sci. 92:181, 1999. (3) K. O'Donnell. Zygomycetes in culture. Palfrey Contributions in Botany. No. 2. Department of Botany, University of Georgia, Athens, 1979.

Autologous Hematopoietic Stem Cell Transplantation for Adults With Acute Myeloid Leukemia.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) remains the most efficacious therapy in patients with acute leukemia. For older patients and those lacking a related HLA-compatible donor, autologous transplantation (auto-HSCT) is a valid alternative therapeutic option. METHODS: From 1997 until 2014 in the Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Poland, 29 auto-HSCT were performed in patients with acute myeloid leukemia (AML; 15 men and 14 women; median age, 52.2 years). The following FAB types of AML were diagnosed: M0, 3; M1, 4; M2, 6; M4, 10; and M5, 6. Patients with AML were classified into 3 cytogenetic prognostic groups: high risk, 9; intermediate risk, 16; and low risk, 4. Twenty-five were in first complete remission and 4 in second complete remission. The peripheral HSCs mobilized after chemotherapy (mainly second course of consolidation) and granulocyte colony-stimulating factor were the source of the stem cells in all cases. The median number of infused CD34+ cells was 3.58 × 10(6)/kg. The conditioning regimen was busulfan and cyclophosphamide in all patients with AML. The intravenous form of busulfan was applied in the last 15 patients. RESULTS: The median time for absolute neutrophil count recovery >0.5 × 10(9)/L and for platelet count >20.0 × 10(9)/L was 12 and 16.5 days, respectively. Treatment-related mortality rate in the whole group was 3.4% (1 patient with sepsis in the aplastic period). The median follow-up time of survivors was 21.9 months (range, 11.7-142.4). The 3-year projected disease-free survival and overall survival rates were 60% and 68%, respectively. CONCLUSIONS: Our data confirm that auto-HSCT is a valuable therapeutic option for patients with AML, especially older patients and those lacking related HLA-compatible donors.

Addition of cladribine to induction/consolidation regimen does not impair peripheral blood stem cell mobilization and bone marrow harvest for autotransplantation in acute myeloid leukemia patients.

BACKGROUND: The previous study by the Polish Adult Leukemia Group has demonstrated that addition of cladribine to standard DNR+AraC induction potentiates the antileukemic activity. The goal of this study was to compare the efficacy of bone marrow or peripheral blood hematopoietic cell collection in patients who obtained remission after daunorubicine plus cytarabine induction with cladribine (DAC-7) or without addition of cladribine (DA-7) in preparation for autotransplantation. PATIENTS AND METHODS: Sixty-six patients aged 41 years (range, 17-58 years) were included in this study: 33 cases in the DAC-7 and 33 in the DA-7 arm. Hematopoietic cells were collected from the bone marrow (ABMT, n = 29) or from the peripheral blood (ABCT, n = 37) using cytopheresis after administration of AraC (2 x 2 g/m2) on days 1, 3, 5 and subsequent G-CSF (10 microg/kg) from day 7 as mobilization therapy. RESULTS: The numbers of harvested CD34+ cells were similar in the DAC-7 and DA-7 pretreated patients both after harvesting from peripheral blood (2.55 x 10(6)/kg vs 2.5 x 10(6)/kg) and from bone marrow (1.62 x 10(6)/kg vs 1.55 x 10(6)/kg), respectively. The proportion of patients with sufficient material for autologous bone marrow transplantation was higher in the DAC-7 compared with the DA-7 arm. All patients engrafted; hematopoietic recovery was similar in both subgroups. CONCLUSION: Addition of cladribine to a standard DA induction does not impair the harvesting of hematopoietic cells and their engraftment after autotransplantation.

High-dose chemotherapy with autologous stem cell transplantation is an effective treatment of primary refractory Hodgkin's disease. Retrospective study of the Polish Lymphoma Research Group.

We analysed the treatment outcome of primary refractory HD patients managed with high-dose chemotherapy and haematopoietic cell transplantation. Data of 65 adult patients who underwent HDC/ASCT in nine Polish centres for primary resistant Hodgkin's disease between June 1991 and July 2000 were collected retrospectively. Response rate to HDC/ASC: CR, 54%; PR, 20%; less than PR, 15%; early deaths, 11%. Actuarial 3-year OS and PFS were 55% and 36%, respectively. In multivariate analysis, lack of bulky lymph nodes and use of immunotherapy were favourable factors for both OS and PFS. IPF <3 at the time of transplantation was predictive for PFS. However, the prognostic impact of immunotherapy should be interpreted with caution since this group included more patients who achieved CR after HDC/ASCT. The results of HDC/ASCT are encouraging and confirm earlier findings. The role of immunotherapy should be further investigated in prospective trials.

Attempted reconstruction of the immune system using low doses of interleukin 2 in chronic lymphocytic leukemia patients treated with 2-chlorodeoxyadenosine: results of a pilot study.

This study was designed to investigate the immunostimulatory effect of low dose Il-2 treatment in B-CLL patients previously treated with 2-chlorodeoxyadenosine (2CdA) in whom severe depletion of T lymphocyte subsets was observed. Four patients enrolled into the study had previously been treated with 3-6 courses of 2CdA. All patients suffered from recurrent infections and showed CD4+ and CD8+ immunosuppression. Recombinant Il-2 was given subcutaneously at a dose of 100 micrograms (1.8 x 10(6)IU) daily for 6 weeks. The drug was administered between 2CdA courses. These preliminary studies showed a marked increase in T cell subsets after Il-2 treatment. All patients displayed an increase of NK cells and there was increased expression of Il-2 receptors (CD 25 and CD 122) on lymphocytes. It is possible that the combination of cytotoxic therapy with 2CdA and low dose rIl-2 could stimulate the T-cell immune system and may be a promising regimen in patients with B-CLL with severe depletion in T-cell subsets.

[Results of the pilot treatment of 9 patients with chronic lymphocytic leukemia or CLL lymphoplasmacytoid lymphoma by the CHOP protocol]. / Pilotowe wyniki leczenia ukladem CHOP 9 chorych z przewlekla bialaczka limfatyczna lub limfoplazmocytoidalna.

In a group of 9 patients with chronic lymphocytic or lymphoplasmocytic leukaemia in clinical stage from 2 to 4 (classification of Rai et al.) 8 various CHOP programmes (cyclophosphamide, hydroxyldaunomycin, oncovin, prednisone) were used. In 6 cases (67%) partial remission was obtained, with normalization of peripheral blood and bone marrow patterns, with statistically significant decrease of the proportion of cells forming rosettes with murine erythrocytes, and with reduction or full normalization of the size of previously enlarged lymph nodes. In one case the control examination of a lymph node failed to demonstrate the previously present clone of cells with chromosomal aberration, although in histological examination the diagnosis of lymphoplasmocytic lymphoma was maintained. In the remaining 3 cases no partial remission was noted, and in one case progression was recognized. We think similarly as the French haematologists studying chronic lymphatic leukaemia, that the CHOP programme is effective in the treatment of chronic lymphatic or lymphoplasmocytic leukaemia.

[Immunologic phenotype of lymphocytes in chronic B-cell lymphocytic leukemia. I. B- and T-lymphocytes in chronic lymphocytic leukemia]. / Fenotyp immunologiczny limfocytów przewleklej bialaczki limfatycznej B-komórkowej. Czesc I. Limfocyty B i T w przewleklej bialaczce limfatycznej.

This article contains the review and the critical discussion of studies of T and B lymphocytes in chronic B-cell lymphocytic leukaemia. It is not explicitly known now, which stage of differentiation of lympho-haemopoietic cells undergoes malignant transformation in CLL-B. Basing on VDJ recombination activity it is supposed that some cases derive from the stem cell, others--from B lineage cells.

[Phenotypic changes in peripheral blood lymphocytes of patients with chronic lymphoma and B-cell leukemia after treatment]. / Zmiany fenotypowe limfocytów krwi obwodowej chorych z przewleklymi chloniakami/bialaczkami B komórkowymi pod wplywem leczenia.

The purpose of the study was evaluation of the effect of treatment on the immunological phenotype of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia or in leukaemic forms of non-Hodgkin lymphomas, with low or medium grade malignancy. The study was carried out in 13 patient. In those responding to treatment a tendency was observed for normalization of the immunological phenotype of peripheral blood lymphocytes (increased proportion of cells forming rosettes with sheep erythrocytes, reduced proportion of cells forming rosettes with murine erythrocytes), and a tendency for polyclonal pattern of B-cells with immunoglobulins on their surface (although this feature was not a rule). In non-responders these tendencies were much less pronounced or absent. Similarly, in non-responders the tendency for polyclonal B-cell pattern may be only apparent, and may be connected with proliferation of a new lymphocyte population with another type of light chain.

[In vitro studies directed at inducing differentiation of leukemic B-lymphocytes]. / Doswiadczenia in vitro w kierunku indukcji róznicowania limfocytów B-bialaczkowych.

Lipopolysaccharide (LPS) and phorbol esters (TPA) stimulate lymphocytes proliferation in two different ways. While LPS primary function is specific receptor binding, TPA directly activate cellular protein kinase C. The stimulation of human leukaemic lymphocytes (from chronic lymphocytic leukaemia patients) with LPS and TPA results in two different types of response: to both stimulators, and to LPS only. Therefore the supposed defect of cellular receptors can not explain all the observed effects. The existence of TPA independent second messengers and changes in signal transduction pathways downstream of PKC can be considered.

[Immunologic phenotype of lymphocytes in chronic B-cell lymphocytic leukemia. II. Studies of immunologic phenotype of peripheral blood and lymph node lymphocytes in patients with chronic lymphocytic leukemia or leukemic forms of lymphoplasmacytoid lymphoma]. / Fenotyp immunologiczny limfocytów przewleklej bialaczki limfatycznej B-komórkowej. Czesc II. Badania wlasne fenotypu immunologicznego limfocytów krwi obwodowej i wezla chlonnego u chorych na przewlekla bialaczke limfatyczna, lub bialaczkowa postac chloniaka limfoplazmocytoidalnego.

UNLABELLED: In a group of 16 patients with chronic lymphocytic leukaemia and leukaemic forms of the lymphoma lymphoplasmocytoides immunophenotypes of peripheral blood and lymph node lymphocytes were studied. CONCLUSIONS: 1) immunophenotype heterogeneity observed in a minority of patients in lymph-nodes or peripheral blood seems to be connected with the co-existence of leukaemic and normal reactive B-cells, 2) SIgG+ cells seem to represent activated B lymphocytes producing and secreting autoantibodies, 3) circulating peripheral T lymphocytes do not reflect the distribution of T cell subpopulations in lymph-nodes.

[Myelodysplastic syndrome associated with clonal proliferation of B lymphocytes]. / Zespól mielodysplastyczny przebiegajacy z klonalna proliferacja limfocytów B.

We present an unusual case of the myelodysplastic syndrome (subtype refractory anemia with the excess of blasts in transformation--RAEB-t) associated with significant increase of IgG (4,700 mg/dl), lambda (160 U/dl) in blood serum and circulating clone of B lymphocytes SIgG, lambda, manifesting clonal rearrangement of JH domain. Peripheral blood cells of the patient showed two different chromosomal abnormalities: 47,XY, + del/8/p? and 47,XY, +22, +14, -19. We suppose that two independent neoplastic clones are developed in the described case, i.e. a population displaying markers of myeloblasts and monoblasts, and a clone of B lymphocytes.

Biosimilar granulocyte colony-stimulating factor is effective in reducing the duration of neutropenia after autologous peripheral blood stem cell transplantation.

BACKGROUND: Autologous peripheral blood stem cell transplantation (APBSCT) is the standard of therapy for patients with multiple myeloma and refractory Hodgkin's and non-Hodgkin's lymphomas. Granulocyte colony-stimulating factor (G-CSF) is widely used to accelerate hematopoietic recovery after transplantation and to reduce the morbidity and mortality associated with prolonged neutropenia. Biosimilar G-CSF is approved for the same indications as the originator G-CSF. This is one of the first reported uses of a biosimilar G-CSF for neutrophil recovery after APBSCT. METHODS: A total of 23 consecutive patients with hematological malignancy (multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, and acute myelogenous leukemia) were recruited at the Department of Haematooncology and Bone Marrow Transplantation at the Medical University of Lublin. Patients (12 men and 11 women; median age, 47 ± 13 years) received biosimilar G-CSF (Zarzio, Sandoz Biopharmaceuticals) after myeloablative chemotherapy (primarily BiCnU, etoposide, cytarabine, and melphalan or melphalan 140/200 mg/m(2)) followed by PBSCT. The median number of transplanted CD34+ cells was 4.2 ± 0.8 × 10(6)/kg body wt. G-CSF therapy was started when absolute neutrophil count (ANC) was <0.5 × 10(9)/L and was continued until ANC reached >1.5 × 10(9)/L for 3 consecutive days. Hematopoietic recovery parameters were compared with those in the control group, which consisted of 23 consecutive patients transplanted in the period before the biosimilar G-CSF group and receiving originator G-CSF (Neupogen, Amgen). RESULTS: The mean duration of treatment with biosimilar and originator G-CSF was 14.4 ± 5.1 and 18.6 ± 11.5 days, respectively (P = .43). The adverse event profile was comparable between the biosimilar G-CSF and originator G-CSF groups, with similar occurrence of neutropenic fever (5 versus 6 patients) and bone pain (7 patients in each group). One patient in the biosimilar group had neutropenic enterocolitis and sepsis. There was no case of death in either group. Granulocyte recovery in the study group was as follows: mean days to ANC >0.5 × 10(9)/L was 13.0 ± 4.0 days; to ANC >1.5 × 10(9)/L, 13.6 ± 4.5 days; and to ANC >1.5 × 10(9)/L, 14.0 ± 4.7 days. Mean duration until platelet recovery >20 × 10(9)/L was 16.1 ± 4.4 days. There were no statistically significant differences between the biosimilar and originator G-CSF groups in hematopoietic recovery parameters. CONCLUSIONS: Biosimilar G-CSF is safe and effective in reducing the duration of neutropenia in patients undergoing myeloablative therapy followed by APBSCT and probably in cost savings in transplantation budgets.

A multicenter, open, noncomparative, phase II study of the combination of cladribine (2-chlorodeoxyadenosine), cytarabine, granulocyte colony-stimulating factor and mitoxantrone as induction therapy in refractory acute myeloid leukemia: a report of the Polish Adult Leukemia Group.

Purine nucleoside analogues, cladribine (2-chlorodeoxyadenosine, 2-CdA) and fludarabine (FAMP) are active agents in acute myeloid leukemias (AMLs). Synergistic interaction between FAMP or 2-CdA with cytarabine (cytosine arabinoside, Ara-C) has been demonstrated in preclinical and clinical studies. The current multicenter phase II study was initiated to evaluate the efficacy and toxicity of induction treatment consisting of 2-CdA (5 mg/m2), Ara-C (2 g/m2), mitoxantrone (MIT, 10 mg/m2) and granulocyte colony-stimulating factor (G-CSF) (CLAG-M) in refractory AML. In case of partial remission, a second CLAG-M was administered. Patients in complete remission (CR) received consolidation courses based on high-dose Ara-C and MIT with or without 2-CdA. Forty-three patients from five centers were registered: 25 primary resistant and 18 relapsed. CR was achieved in 21 (49%) patients, 20 (47%) were refractory and 2 (5%) died early. Hematologic toxicity was the most prominent toxicity of this regimen. The overall survival (OS; 1 year) for the 42 patients as a whole and the 20 patients in CR were 43% and 73%, respectively. Disease-free survival (1 year) was 68.6%. None of the analyzed prognostic factors influenced the CR and OS probability significantly. We conclude that CLAG-M regimen has significant antileukemia activity in refractory AML, which seems to be better than the activity of many other regimens. The toxicity of the treatment is acceptable.

[Studies of immunoglobulin surface expression in lymphocytes of peripheral blood, bone marrow and lymph nodes in patients with B-cell chronic lymphocytic leukemia]. / Badania powierzchniowych immunoglobulin limfocytów krwi obwodowej, szpiku i wezla chlonnego chorych z przewlekla bialaczka limfatyczna B komórkowa.

The expression of surface immunoglobulins (SIg) on peripheral blood, bone marrow, and lymph node lymphocytes was studied in a group of 10 patients with B cell chronic lymphocytic leukaemia. In 4 patients differences in the SIg phenotypes were found when cells from blood, bone marrow and lymph nodes were examined. Peripheral blood lymphocytes were characterized as a monoclonal B cell population whereas in the marrow or lymph node lymphocytes a tendency toward poly-clonality was found. We suggest that these differences reflect the origin of the initial leukaemic transformation: intra versus extra-medullar. In case of intramedullary origin of leukaemia the transformed clone infiltrates subsequently lymph nodes or other lymphoid structures. In leukaemia of extra-medullar origin the bone marrow is infiltrated later and therefore the tendency toward polyclonal SIg picture of the bone marrow lymphocytes contrasts with the monoclonality of the peripheral blood cells.

[Intravascular malignant lymphoma (angiotrophic large-cell lymphoma)]. / Chloniak wewnatrznaczyniowy (angiotropowy chloniak wielkokomórkowy).

Intravascular lymphoma (IVL) is characterized by proliferation of large malignant lymphoid cells within the lumen of small vessels. Sites usually affected include the central nervous system and skin although involvement of multiple organ symptoms have been described. IVL is very rare and aggressive type of lymphoma. Based on review of the literature we present clinicopathological, immunohistochemical and molecular features of the IVL. The etiological possibilities are discussed.

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.

[Unusual cases of non-Hodgkin's lymphomas--case reports]. / Niezwykle przypadki chloniaków zlosliwych--opis przypadków.

We describe 4 cases of non-Hodkin's lymphomas that were interesting because of their curiosal clinical courses and spontaneous complete remissions during the course of high malignancy lymphoma. We present three of them for the first time in Poland. Case 1: a 61-year old woman was admitted to the hospital because of the headache, lasting for 4 months before hospitalization and right hemiparesis. CT scans revealed the presence of tumor in the temporo-occipital region. The diagnosis of B-cell lymphoma was established at histopathological examination of the postoperative material. Co60--therapy of these region was applied after the operation with good response. Case 2: a 38-year woman was admitted to the hospital because of L5-S1 spondylolisthesis to operate it. During the hospitalization haemolytic anaemia of unknown origin, thrombocytopoenia, splenomegaly, fever and rising acute insufficiency of kidneys, heart, liver and CNS were occurred. The patient died, despite applying corticosteroidotherapy. The diagnosis of intravascular lymphoma was established at postmortem examination. Case 3: a 51-year old woman was admitted to the hospital with diagnosis: anaplastic non-Hodgkin lymphoma B-cell type high malignancy established after the double histopathological examination of lymph nodes and biopsy of the lung. At the admission to the Department of Haematology we stated absolute regression of these changes. The patient had been only observed in the Outpatient Department over 1 year. She died after 6 months since the beginning of the relapse despite intensive chemotherapy. Case 4: a 43-year old man was admitted to the hospital because of great hyperleukocytosis, hepatosplenomegaly and neurological symptoms. The diagnosis: chronic prolymphocytic leukaemia was established. The cerebrospinal fluid examination showed presence of mononuclears which infiltrated CNS. CT scans of the brain revealed leucaemic infiltrations of the hemispheres and cerebellum. The patient died despite intensive therapy due to rising progressive multiorgan failure.

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus.

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg(2+) and the four nucleoside triphosphates and was insensitive to actinomycin D, alpha-amanitin, and rifampin. The (3)H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 x 10(6) and 1.4 x 10(6); they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs(2)SO(4) equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 x 10(6) and 1.4 x 10(6). The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity.

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non-ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N-methylated cyclic dipeptides such as thaxtomins. Based on high-performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies.

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.