Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease.

The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

Intra-articular injection of tumor necrosis factor-alpha in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation.

OBJECTIVE: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. DESIGN: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. RESULTS: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. CONCLUSION: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.

Interactions Among a Soil Organic Amendment, Nematodes, and the Nematode-Trapping Fungus Dactylellina candidum.

ABSTRACT When alfalfa leaves (Medicago sativa) are added to soil, both the nematode-trapping fungus Dactylellina candidum and microbivorous nematodes increase. To determine whether the response of the fungus to alfalfa depends on consumption of bacterivorous and fungivorous nematodes, soil microcosm experiments were performed. D. candidum did not increase if alfalfa leaves were added to soil lacking nematodes, but did increase if nematodes were added to soil lacking alfalfa leaves. Although these results indicate that the response of D. candidum to alfalfa depends on nematodes, D. candidum and microbivorous nematodes did not exhibit classical predator-prey dynamics (i.e., D. candidum seldom reduced numbers of resident nematodes and, after initially increasing in alfalfa amended soil, numbers of D. candidum then decreased, whereas numbers of nematodes continued to increase). Fungivorous nematodes were abun dant in alfalfa-amended soil, and their potential to suppress trapping fungi requires more research.

Antiinflammatory 4,5-diarylpyrroles: synthesis and QSAR.

A series of 2-substituted- and 2,3-disubstituted-4-(4-fluorophenyl)-5-[4-(methylsulfonyl)phenyl]-1H- pyrroles was synthesized and found to be active in the rat adjuvant arthritis model of inflammation. The most active compounds were the 2-halo derivatives in the order of chloro > bromo > iodo. The same pattern of activity was observed for the 2,3-dihalopyrroles. Quantitative structure-activity relationship studies suggested that the activity could be correlated with the molar refractivity and the inductive field effect of the 2-substituent and the lipophilicity of the 3-substituent.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Minor antigen graft-versus-host reactions revealed in irradiated spleen and popliteal lymph node assays.

The graft-versus-hot (GVH) reaction across minor (non-H-2) histocompatibility barriers was studied in mice, in vivo. To increase GVH potential and to mimic clinical bone marrow transplantation protocols, we modified the popliteal lymph node (PLN) and the splenomegaly assays by irradiating the recipients before they received allogeneic lymphoid cell suspensions. In several combinations across major (H-2), minor (non-H-2) and multiple minor (non-H-2 plus minor lymphocyte stimulation) barriers, increased recipient organ weight (a measure of GVH activity) was seen with irradiated F1 recipients of parental cells. The irradiated splenomegaly (x-splenomegaly) assay was more sensitive than the (x-PLN) assay, but both correlated with in vivo GVH experiments of the P----F1 variety. The x-splenomegaly test indicated histoincompatibility in a system (B10.D2----BALB/c) in which the primary in vitro mixed leukocyte reactions is nonreactive, but in which systemic GVH can be induced. The x-splenomegaly test should be useful in analyzing complex reactions involving minor histocompatibility antigens in vivo.

Prolongation of murine skin allografts by prostaglandin E1.

The effects of 16,16-dimethyl prostaglandin E2 methyl ester (di-M-PGE2) and indomethacin (an inhibitor of endogenous prostaglandin biosynthesis) on mouse skin allograft survival were studies in B10.D2 female mice receiving skin allografts from (B10.BR X B10.D2)F1 mice. Control animals with and without i.p. diluent injections had a mean allograft survival of 13.8+/-0.6 and 13.5+/-0.5 days, respectively. Daily administration of di-M-PGE2 (200 microng/kg) prolonged mean allograft survival, both when administered alone, 16.7+/-0.6 days (P less than 0.001), or with indomethacin, 4 mg/kg thrice weekly, 16.0+/0.6 days (P less than 0.005). Increasing concentrations of indomethacin (4, 6, and 8 mg/kg thrice weekly) were inversely corrleated with allograft survival ((12.7+/-0.2, 11.8+/-0.2, and 10.9+/-0.4 days, respectively), coefficient of correlation=-0.6986; P less than 0.001. Mean plasma PGE levels at the time of total allograft rejection were 879+/-80 pg/ml in control, 717+/-59 pg/ml in 100 micron g of indomethacin-treated mice, and 654+/-59 pg/ml in 200 microng of indomethacin-treated mice (P less than 0.05). Exogenous di-M-PGE2 prolonged skin allograft survival in mice. Inhibition of endogenous prostaglandin biosynthesis by indomethacin chortened allograft survival, but this effect was completely abrogated by concurrent injection of di-M-PGE2.

Novel mechanisms of brequinar sodium immunosuppression on T cell activation.

Brequinar sodium (BQR) is a novel immunosuppressive agent that acts by inhibiting the activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine biosynthetic pathway. The activity of BQR as an immunosuppressive agent is believed to be inhibition of antigen-induced lymphocyte proliferation through inhibition of DNA and RNA synthesis. BQR, therefore, has a different mechanism of action than cyclosporine and may potentiate the immunosuppressive effects of cyclosporine. In this study, we determined the effect of BQR on peripheral blood mononuclear cell (PBMC) activation in a series of in vitro culture systems. In these studies, BQR inhibited PHA-stimulated activation in a dose-dependent fashion beginning at 10(-6) M. The immunosuppressive effect of BQR was similar in magnitude to cyclosporine. Proliferation assays suggested an additive immunosuppression by the combination of BQR and cyclosporine. Similar inhibition of CD2-stimulated or CD3-stimulated activation of PBMC was found. The mechanisms of action of BQR were complex. BQR inhibited interleukin 2 protein production in response to mitogen stimulation. Cell surface interleukin 2 receptor expression was inhibited by BQR. BQR inhibited cell cycle progression, preventing progression from G0/G1 into S and G2 + M phases. BQR had no effect on induction of transcripts for the interleukin 2 receptor, but markedly inhibited the production of transcripts for interleukin 2. Thus, our studies indicate that BQR exerts a potent immunosuppression on mitogen-induced PBMC activation through multiple mechanisms. Consequently, BQR may be an effective agent for immunosuppression in organ transplantation or inflammatory diseases.

The effect of a new immunosuppressive drug, brequinar sodium, on heart, liver, and kidney allograft rejection in the rat.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. BQR is capable of inhibiting immune responses in vitro and is effective in suppressing the development of contact sensitivity and adjuvant arthritis in rodent models. Based on the antiproliferative and immunosuppressive capacity of BQR, we have evaluated the efficacy of BQR in preventing allograft rejection utilizing experimental models of heterotopic heart and kidney and orthotopic liver transplantation in an MHC and non-MHC mismatched ACI----LEW rat strain combination. The immunosuppressive activity of BQR is illustrated by its ability to inhibit the development of delayed-type hypersensitivity to DNFB in mice. When BQR was administered orally throughout the sensitization and elicitation phases of the DNFB contact sensitivity response, it was found to be a potent immunosuppressant with an ED50 value of 0.5 mg/kg. This immunosuppressive activity is also seen in vitro, where BQR is capable of inhibiting the mixed lymphocyte response between allogeneic ACI and LEW rat strains with an IC50 of 150 ng/ml. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. Cardiac allografts are not rejected during the period of drug treatment at dosage levels of 12 to 24 mg/kg orally three times weekly. The grafts survive until the drug is discontinued (30 days posttransplantation), and the grafts are then rejected approximately 14 days later. Liver and kidney allografts are permanently accepted by approximately 50 to 90% of the recipient rats following 30 days of treatment with BQR at 12 mg/kg. The tolerance that is induced to the liver grafts extends in the majority of animals to greater than 250 days and is specific for the donor ACI strain. Challenge of long-term liver graft survivors with donor cardiac grafts is associated with permanent survival of donor, but not third-party, heart grafts. Combination therapy consisting of suboptimal doses of BQR and CsA demonstrates that the combination of these two immunosuppressive drugs results in an increased efficacy in prolonging graft survival. The results of these allograft experiments demonstrate that this new immunosuppressive agent is highly effective in preventing allograft rejection in the rat. The antiproliferative activity of BQR is effective for inhibiting T-lymphocyte-mediated immune responses, and Brequinar sodium should be an important addition to a polytherapeutic approach in the treatment of organ graft rejection.

The prolongation of concordant hamster-to-rat cardiac xenografts by brequinar sodium.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. In these experiments, we have tested the ability of BQR to prevent the rejection of concordant cardiac xenografts. LEW inbred rats transplanted with heterotopic hamster hearts were treated orally with brequinar sodium as a single agent. The survival of the cardiac xenografts was significantly prolonged with a variety of treatment regimens. The most effective treatment was the daily oral administration of BQR at 3 mg/kg. At this level, the median graft survival was approximately 25 days. Four animals had hamster heart xenografts that functioned for more than 90 days. The prolonged survival of the xenografts was associated with relatively constant plasma drug levels of approximately 1 to 3 micrograms/ml and a marked suppression of IgM production. At rejection, there was a significant rise in IgM levels compared with those of recipients with stable xenografts. In vitro MLR responses were effectively inhibited by BQR, with an IC50 of 0.08 microgram/ml. The results of these experiments demonstrate that BQR is a new immunosuppressive agent that is highly effective as a single agent in prolonging the survival of hamster-to-rat cardiac xenografts. The prolonged xenograft survival is associated with effective suppression of rat antihamster antibody production, suggesting that brequinar sodium may be an important addition to multidrug immunosuppressive regimes designed to prevent B and T lymphocyte-mediated immune responses.

Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium.

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.

Inhibition of the pyrimidine biosynthetic pathway with S-8660, an analogue of brequinar sodium, prolongs cardiac allograft survival in rats.

The compound S-8660 is a member of a family of antiproliferative drugs that act on de novo pyrimidine synthesis through selective inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase. S-8660 is highly effective in preventing the development of delayed-type hypersensitivity in mice and in suppressing human mixed-lymphocyte responses. We have tested its ability to prevent cardiac allograft rejection in the ACI (RT1a) to Lewis (RT1(1)) rat strain combination, based on the immunosuppressive activity of this compound and its similarity to another member of this group, brequinar sodium. Daily oral administration of the drug (5 to 20 mg/kg) was begun 2 days before transplantation and extended for periods of time up to 30 days after graft placement. Control grafts were promptly rejected (median survival time, 7.0 +/- 0.5 days). Administration of S-8660 was effective in extending graft survival in a dose-dependent fashion. The efficacy of S-8660 could be improved with a high initial concentration of the drug, followed by a reduction ("tapering") in the level of drug administration (median survival time, 32.0 +/- 4.6 days) or by use in combination with cyclosporine. The differences in the mode of action of S-8660, when compared to cyclosporine or FK 506, suggest that the disruption of de novo pyrimidine synthesis may be an effective and safe addition to a polytherapeutic approach for the prevention of allograft rejection in clinical transplantation.

Side effects of brequinar and brequinar analogues, in combination with cyclosporine, in the rat.

Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.

Regional lymph node x-radiation suppresses allograft rejection.

We studied the effect of a single dose of x-radiation given to the regional lymph nodes on allograft rejection. Our model was an A-strain tumor, transplanted to C57B1/6, DBA/2, and C57B1/6 x CBA hosts. Irradiation (1,000 rad) of the regional lymph nodes inhibited graft rejection, as judged by increased tumor growth, slower rejection, and, in the case of DBA/2 mice, by fewer regressions; irradiation of the contralateral lymph nodes did not have this effect. The mechanism of immunosuppression appeared to be B-cell independent, since immunosuppression could be induced by regional x-radiation as readily in B-cell deficient mice as in normal mice. The suppression was critically time-dependent, viz, irradiation before, or 6 or more days after, tumor graft was without significant effect. Treatment of the allogeneic host with cyclophosphamide several days before grafting, so as to reduce T-suppressor cells, attenuated the immunosuppression of regional x-radiation.

Suppression of nematophagous fungi by enchytraeid worms: a field exclosure experiment.

The feeding biology of Enchytraeus crypticus and other enchytraeids is poorly understood as is their effect on nematophagous fungi. Because enchytraeids had been associated with nematophagous fungi in the field and had suppressed these fungi in soil microcosms, we tested the hypothesis that exclusion of enchytraeids, largely E. crypticus, would improve establishment of certain nematophagous fungi in field plots. The fungi, Hirsutella rhossiliensis and Monacrosporium gephyropagum, are being studied as potential control agents of plant-parasitic nematodes and were formulated as hyphae in alginate pellets. The pellets were mixed into soil without enchytraeids and placed in cages (PVC pipe, 80 cm3 volume) with fine (20 µm) or coarse (480 µm) mesh; cages were buried 15 cm deep in field plots and then recovered after 6-52 days. When fine mesh was used, enchytraeids were excluded and the fungi increased to large numbers. When coarse mesh was used, enchytraeid numbers in cages increased rapidly and the fungi did poorly. Although mesh also affected other potential fungivores, including collembolans and large dorylaimid nematodes, we suspect that enchytraeids were more important because large numbers were consistently found in cages with coarse mesh soon after the cages were placed in soil. Organisms smaller than enchytraeids (bacteria, fungi, and protozoa) also appeared to be important because the fungi did better in heat-treated soil than in non-heat-treated soil, regardless of mesh size. The rapid increase in enchytraeid numbers in cages with hyphal pellets and coarse mesh was probably caused by movement of enchytraeids toward the pellets with hyphae: increase in enchytraeid numbers was minimal when movement into cages was blocked (or when cages contained pellets without hyphae). Overall, the data were consistent with the hypothesis that enchytraeids, or other meso- or macrofauna, contributed to suppression of nematophagous fungi in our field plots.

Augmentation of Soil with the Nematophagous Fungi Hirsutella rhossiliensis and Arthrobotrys haptotyla.

In previous studies, growth of Hirsutella rhossiliensis from pelletized assimilative hyphae was reduced by other soil organisms. In the current study, sensitivity to this biotic inhibition was compared when the fungus was added to soil as pelletized hyphae or as fungus-parasitized nematodes. The hypothesis was that the natural inoculum, the parasitized nematode, would be less sensitive than the artificial inoculum, pelletized hyphae. The soil was heated to 60 degrees C for 2 h to remove putative antagonists or was not heated. The soil was packed into vials (17 cm(3)) and kept at 20 degrees C in the laboratory or packed into cages (PVC pipe sealed at the ends with 480-mum pore mesh, 80 cm(3)) and buried 22 cm deep in a vineyard. After 2 or 4 weeks, assay nematodes were added to the vials or cages (recovered from the vineyard), respectively. The assay nematodes were extracted from soil after 2.0 or 2.5 days and examined for adhesive conidia of H. rhossiliensis. Consistent with the hypothesis, H. rhossiliensis was quite sensitive to biotic inhibition when formulated as pelletized hyphae but was insensitive to biotic inhibition when formulated as parasitized nematodes. These data suggest that the activity of the H. rhossiliensis pellet could be increased if the pellet better mimicked the natural inoculum. Similar experiments with the nematode-trapping fungus Arthrobotrys haptotyla, however, exhibited an opposite trend: A. haptotyla was more sensitive to biotic inhibition when added to soil as fungus-parasitized nematodes than as pelletized hyphae. Results from laboratory and field experiments were similar.

Correlations between most probable number and activity of nematode-trapping fungi.

ABSTRACT Soil cages were used to determine whether nematode-trapping fungi population density, as measured by most probable number (MPN) procedures, was correlated with the trapping of nematodes. Fungi studied (and trap type) were Arthrobotrys oligospora (adhesive networks), A. eudermata (adhesive networks), A. dactyloides (constricting rings), Dactylellina ellipsospora (adhesive knobs), and D. haptotyla (adhesive knobs). The fungi were formulated as assimilative hyphae in dried alginate pellets. Pellets were added to field soil, the soil was packed into 80-cm(3) cages (PVC pipe, 3.0 cm long and 3.9 cm in diameter), and the cages were buried in vineyards. After 14 to 61 days, the cages were recovered, and MPN data and trapping activity were determined. For all five fungi, MPN data were correlated with the number of pellets added. Regardless of fungus population density, A. oligospora and A. eudermata trapped few if any nematodes in soil, and consequently, trapping and fungus population density were not correlated. The correlation between population density and trapping was weak for A. dactyloides but relatively strong for D. ellipsospora and D. haptotyla. High levels of trapping by the latter two fungi required more than 10(2) fungus propagules per gram of soil.

Enchytraeids and nematophagous fungi in tomato fields and vineyards.

ABSTRACT I tested the hypothesis that exclusion of enchytraeids and microarthropods in agricultural fields improves establishment of two nematophagous fungi. Soil was collected from three tomato fields and two vineyards and either heat-treated (2 h at 60 degrees C) or not. Alginate pellets containing hyphae of the fungi Hirsutella rhossiliensis or Monacrosporium gephyropagum were added to the soil, which was packed into cages (PVC pipe, 80-cm(3) volume) sealed with fine (20 mum) or coarse (480 mum) mesh. Cages were buried 22 cm deep in the same fields from which the soil had been collected. After 7 to 50 days, the cages were recovered and fungi and fauna quantified. Fine mesh largely excluded enchytraeids, collembolans, and mites but rarely affected fungus numbers. In contrast, heat treatment of soil rarely affected enchytraeids, collembolans, or mites but frequently increased fungus numbers, regardless of mesh size. The data are inconsistent with the initial hypothesis but are consistent with the idea that organisms narrower than 20 mum interfere with fungal growth from the pellets.

Soil pH and the Activity of a Pelletized Nematophagous Fungus.

ABSTRACT When formulated as assimilative hyphae in alginate pellets, the nematophagous fungus Hirsutella rhossiliensis was more active (i.e., parasitized more assay nematodes) in an acidic vineyard soil than in a neutral vineyard soil. To determine whether soil pH explained the difference, fungus activity was measured in soil from the neutral site that had been acidified (by adding sulfuric acid) and in soil from the acidic site that had been neutralized (by adding calcium hydroxide). As hypothesized, the activity of pelletized Hirsutella rhossiliensis was negatively correlated with soil pH. Maximum activity occurred at pH(calcium chloride) 4.5, and activity gradually declined to near zero as the pH increased to 6.5 and rapidly declined to near zero as the pH dropped below 4.0. Assays performed on leached soil samples indicated that the effects of sulfuric acid and calcium hydroxide were largely due to pH rather than to specific ions or osmotic potential. The effect of pH, however, was indirect. Heating the neutral soil to 60 degrees C for 2 h did not alter soil pH or electrical conductivity but increased fungus activity to levels equivalent to those in acidified soil. We conclude that, in these two soils, heat treatment or low soil pH suppresses soil organisms that otherwise interfere with growth of Hirsutella rhossiliensis from alginate pellets.