Imaging of long-distance alpha-aminoisobutyric acid translocation dynamics during resource capture by Serpula lacrymans

alpha-Aminoisobutyric acid (AIB) is a nonmetabolized amino acid analogue of alanine, which at low (muM) concentrations acts as a tracer for amino acid movements. At high concentrations (mM), it competitively inhibits membrane transport and metabolism of protein amino acids and acts as a systemic translocated inhibitor of mycelial extension in fungi. AIB can control mycelial spread of the basidiomycete Serpula lacrymans, the cause of brown rot of wood in buildings. However, it is not known how effectively the inhibitor is distributed throughout the mycelium. Realistically heterogeneous microcosms, in which the fungus grew across nutritionally inert sand to colonize discrete wood resources, were used to investigate patterns of inhibition and translocation following local application of AIB. At a 0.1 M concentration, locally applied AIB caused immediate arrest of extension throughout the whole mycelium, maintained for a 6-week experimental period. The dynamics of translocation of subtoxic amounts of [1-(14)C]AIB ([(14)C]AIB) were mapped by photon-counting scintillation imaging in conjunction with destructive harvest to establish the velocity, direction, and rate of translocation and the extent of [(14)C]AIB reallocation accompanying the invasion of fresh wood. Locally applied [(14)C]AIB was distributed throughout complex mycelial networks within 2 h of application, becoming localized in growing margins by 12 h. Encounter with a fresh wood resource triggered a widespread response, causing withdrawal of [(14)C]AIB from throughout the network, accompanied by accumulation in the newly colonized wood and associated mycelium. The results are discussed in the context of nutrient dynamics in wood decomposer fungi and the mechanism of the amino acid reallocation response.     

Behaviour of the fungus Rhizoctonia solani Kiihn in the soil

Rhizoctonia solani was found able to grow as a saprophyte through natural unsterilized soil. Its rate of growth under different soil conditions in glass tumblers was studied by the Rossi-Cholodny soil-plate method. Growth was most rapid at the lowest soil-moisture content tested, viz. 30 % saturation, and was accelerated by forced aeration of the soil. The maximum distance to which mycelial growth could be supported on the food reserves of the agar inoculum alone was some 5 cm., as shown by extent of growth through tubes of moist sand, but in 23 days the fungus grew 21–24 cm through tubes of soil. Removal of the agar disk 2 days after inoculation of the tubes reduced growth through sand by more than half, but through soil by only a small proportion. In soil, Rhizoctonia was able to cause 100% damping-off of radish seedlings planted at a radial distance of 4 cm. from the agar inoculum, and some 40 % damping-off at a distance of 9 cm. The depressing effect of additions of 1 % ground-wheat straw or dried grass to the soil upon growth of the fungus was attributed to (1) the negligible cellulose-decomposing ability of Rhizoctonia, (2) nitrogen starvation of the mycelium, through rapid utilization of the available soil nitrogen by the cellulose-decomposing micro-organisms multiplying upon the fresh organic material, (3) fungistatic action on Rhizoctonia of the respiratory carbon dioxide produced by the cellulose-decomposers.     

13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Acquisition of N by external hyphae of an arbuscular mycorrhizal fungus and its impact on physiological responses in maize under drought-stressed and well-watered conditions

This study examined the uptake of nitrogen by external hyphae of an arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck &; Smith) and its impact on physiological responses in maize plants subjected to well-watered or drought-stressed conditions. Plants were grown in compartmented boxes divided by a nylon mesh (40?μm) into a root compartment and a hyphal compartment. Maize plants (Zea mays cv. 'Tuxpeño sequia' selection cycle C0) were exposed to 2 weeks of drought 56 days after sowing. A ^[15]N tracer was applied as K^[15]NO_[3] to the hyphal compartment at a distance of 5?cm from the root compartment. Root and shoot samples were then analyzed for ^[15]N atom % excess (APE), glutamine synthetase (GS) activity, protein concentration and nutritional status. Evapotranspiration rate and stomatal resistance were monitored daily to determine the degree of drought stress. The APE values for AM shoots and roots were 32% and 33% higher than non-AM shoots and roots, respectively, under drought conditions. This provides clear evidence that the external mycelium of AM fungus transports considerable amounts of ^[15]NO_[3]^[– ]to the host plant under drought conditions. Drought-stressed AM roots had 28% higher GS activity, possibly as a consequence of higher hyphal acquisition of NO_[3]^[–] ions. Mycorrhizal colonization significantly increased the host plant P status regardless of soil moisture regime. In addition, the N status of drought-stressed AM shoots and roots was slightly higher than stressed non-AM shoots and roots. The improved nutritional status may assist AM plants to exploit available soil moisture more efficiently and to maintain higher leaf relative water content under moderate drought conditions.     

Suppression of the Root-knot Nematode Meloidogyne javanica by Alginate Pellets Containing the Nematophagous Fungi Hirsutella rhossiliensis , Monacrosporium cionopagum and M. ellipsosporum

The endoparasitic fungus Hirsutella rhossiliensis and the nematode-trapping fungi Monacrosporium cionopagum and M. ellipsosporum were formulated as hyphae in alginate pellets. In a soil microcosm experiment, dried pellets of all three fungi decreased the invasion of cabbage seedlings by the root-knot nematode Meloidogyne javanica when juvenile nematodes were placed 2 cm from roots; M. cionopagum was more effective than the other two fungi, reducing nematode invasion by 40-95% with 0.24-0.94 pellets cm - 3 of soil. In a field microplot experiment, in which neither H. rhossiliensis nor M. ellipsosporum suppressed nematodes, 0.5 pellets of M. cionopagum cm - 3 of soil suppressed M. javanica invasion of tomato seedlings by 73%. In a second microplot experiment with only M. cionopagum , again at 0.5 pellets cm - 3 of soil, the fungus suppressed the invasion of tomato seedlings whether the pellets were added 0, 5 or 14 days before planting; the population density of M. cionopagum increased to nearly 3000 propagules g - 1 of soil by day 8 and then declined to less than 300 by day 22. Enchytraeid worms were observed in and around damaged and apparently destroyed pellets in both microplot experiments. Whether enchytraeids consumed the fungi or otherwise affected biological control requires additional research.     

Mineralization of Polycyclic Aromatic Hydrocarbons by the White Rot Fungus Pleurotus ostreatus

The white rot fungus Pleurotus ostreatus was able to mineralize to (sup14)CO(inf2) 7.0% of [(sup14)C]catechol, 3.0% of [(sup14)C]phenanthrene, 0.4% of [(sup14)C]pyrene, and 0.19% of [(sup14)C]benzo[a]pyrene by day 11 of incubation. It also mineralized [(sup14)C]anthracene (0.6%) much more slowly (35 days) and [(sup14)C]fluorene (0.19%) within 15 days. P. ostreatus did not mineralize fluoranthene. The activities of the enzymes considered to be part of the ligninolytic system, laccase and manganese-inhibited peroxidase, were observed during fungal growth in the presence of the various polycyclic aromatic hydrocarbons. Although activity of both enzymes was observed, no distinct correlation to polycyclic aromatic hydrocarbon degradation was found.     

Use of α-aminoadipic acid for the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. l-alpha-Amino[6-(14)C]adipic acid has been prepared from the dl-amino acid by oxidation of the l-isomer with l-amino acid oxidase to alpha-oxo[6-(14)C]adipic acid and by transamination of the latter with l-glutamic acid in an extract of a Cephalosporium sp. prepared by ultrasonic treatment of the mycelium. 2. The optical configuration of small amounts of (14)C-labelled alpha-aminoadipic acid from the mycelium of the Cephalosporium sp. has been determined by treatment with l-amino acid oxidase and measurement of the proportion of radioactivity subsequently retained on a column of a strong cation-exchange resin. 3. alpha-Aminoadipic acid which had been labelled in the mycelium from [1-(14)C]acetate appeared to contain more than 99% of the l-isomer. 4. l-alpha-Amino[(14)C]adipic acid (sodium salt) was taken up much more rapidly than the d-isomer, or alpha-oxo[6-(14)C]adipic acid, by suspensions of washed mycelium of the Cephalosporium sp. in water. The pool of intracellular alpha-aminoadipic acid was expandable. 5. Intracellular products found to be labelled with (14)C from l-alpha-amino[(14)C]adipic acid were delta-aminovaleric acid, saccharopine, lysine, protein, compounds which behaved like penicillin N, cephalosporin C and deacetylcephalosporin C respectively on paper chromatography and electrophoresis, and a peptide whose amino acid residues include alpha-aminoadipic acid, cysteine and valine. 6. l-alpha-Amino[(14)C]adipic acid acted as a precursor of the delta-(d-alpha-aminoadipoyl) side chains of extracellular penicillin N and cephalosporin C. 7. (14)C from d-alpha-amino[(14)C]adipic acid was incorporated into penicillin N and cephalosporin C, but the incorporation was accompanied by a relatively high dilution of specific radioactivity and some l-alpha-amino[(14)C]adipic acid was found in the intracellular pool. 8. These findings are discussed in relation to the origin of the d- configuration of the alpha-aminoadipoyl side chain of the antibiotics.     

Biodegradation of Nigerian wood wastes by Pleurotus tuber-regium (Fries) Singer

Studies were carried out for 90days on the degradation of wood wastes of four economically important Nigerian trees; Terminalia superba, Mansonia altissima, Holoptelia grandis and Milicia excelsa by white rot fungus, Pleurotus tuber-regium a Nigerian edible mushroom. The pH of the wastes dropped to 4.0/4.2, 90days after incubation. On the contrary, amino nitrogen content of the wastes increased consistently during this period of solid-state fermentation. Lignin degradation also increased with the increase in incubation days. The greatest lignin reduction was observed in H. grandis followed by T. superba, M. altissima and M. excelsa. Digestibility of spent substrates by ruminants increased during fermentation as follows: M. excelsa>M. altissima>T. superba>H. grandis. These results are discussed in relation to the use of fermented wood wastes as feeds for ruminants.     

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus

Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.     

Studies on the biosynthesis of phenols in fungi. Conversion of [14C]orsellinic acid and [14C]orcinol into fumigatol by Aspergillus fumigatus I.M.I. 89353

1. Sodium [1-(14)C]acetate was incorporated into orsellinic acid and fumigatol by Aspergillus fumigatus. 2. [(14)C]Orsellinic acid was prepared biosynthetically. It was converted almost entirely into fumigatol and fumigatin within 2 days of supplementation of the medium. The apparent decrease in incorporation after a longer period of growth was due to decomposition of radioactive fumigatol and the production of relatively unlabelled material. The addition of orcinol to these cultures decreased the conversion of [(14)C]orsellinic acid into fumigatol. [(14)C]Orsellinic acid was incorporated into 3,4-dihydroxytoluquinol in both sets of cultures. 3. [(14)C]Orcinol was prepared from [(14)C]orsellinic acid after acid hydrolysis. It was also very effective as a precursor of fumigatol (60% incorporation). 4. The specific activity of fumigatin was lower than that of fumigatol at early stages of growth (4-5 days after inoculation) with all the labelled substrates that were tested. This indicated that fumigatin arose from fumigatol after oxidation in the medium. 5. The presence of orcinol in the medium greatly stimulated the incorporation of radioactivity (presumably derived from the (14)CO(2)H of orsellinic acid) into the isoprenoid compounds, ergosterol and ubiquinone, in the mycelium.     

Effects of chelators (EGTA, EDTA) and calcium ions on nematode capture by the nematode-trapping fungusArthrobotrys ellipsospora

The nematode-trapping fungusArthrobotrys ellipsospora developed an adhesive knob and trapped nematodes when cultured on a low-nutrient medium. It also trapped polystyrene beads in the same way. The adhesive knob produced mucus that was stained with alcian blue, while mycelium of the fungus was stained with periodic acid/Schiff (PAS). The amount of mucus increased with in days after culturing in the low-nutrient media. The fungus completely lost its ability to trap nematodes when treated with EDTA and EGTA, but it recovered the ability after incubation in the presence of a low concentration of Ca (10⁻⁶–10⁻⁷ M) for 1 h. Calmodulin inhibitor W-7 also inhibited the trapping ability of the fungus, and there was a significant (p<0.05) difference between the effects of W-7 and W-5. Ca-binding protein was also detected in the fungus.     

Spatiotemporal transfer of carbon-14-labelled photosynthate from ectomycorrhizal Pinus densiflora seedlings to extraradical mycelia

Seedlings of Pinus densiflora colonized by an unidentified ectomycorrhizal fungus (T01) were labelled photosynthetically with 14C. Movement of 14C-labelled photosynthates within the underground part of the seedlings was investigated by temporal autoradiography using an imaging plate. Within 1 day, 14C was transferred from the shoot to the underground part that included roots, mycorrhizae, and the extraradical mycelium; within 3 days, the 14C in the underground part reached its maximum density. Mycorrhizae and actively growing root tips were large C sinks. Three days after 14C labelling, counts of 14C radioactivity in the underground part of the mycorrhizal seedlings were 2.6 times those of nonmycorrhizal seedlings. The mycorrhizae of mycorrhizal plants accumulated 5.2 times the 14C counts in the short-root tips of nonmycorrhizal plants. 14C counts in various areas of the extraradical mycelium demonstrated that all 14C-photosynthate transfer from the host root to the extraradical mycelium occurred within 3 days after 14C labelling, and that there was only a short lag of < 1 day between 14C accumulation in the basal and distal parts of the mycelium. Although more 14C accumulated in the distal than in the basal parts, 14C counts per unit hyphal biomass were similar between the two. These results suggest that 14C spread rapidly throughout the entire mycelium. Thirteen days after 14C labelling, we estimated 14C allocation to extraradical mycelia by taking autoradiographs after removing host roots. About 24% of 14C counts in the underground part of the mycorrhizal seedlings had been allocated to extraradical mycelia in this system, indicating that the fugal mycelium is an important sink for photosynthates.     

Induction of Nematode-Trapping Organs in the Predacious Fungus Arthrobotrys oligospora (Hyphomycetales) by Infective Larvae of Ostertagia ostertagi (Trichostrongylidae)

Laboratory experiments were designed to study the influence of temperature, concentrations of nematodes, oxygen tension, light, and nutrient levels, on the induction of nematode-trapping hyphal nets in the predacious fungus Arthrobotrys oligospora. When induced by infective Ostertagia ostertagi larvae, a maximum number of nets was produced at 20°C, at which temperature nets in surplus were produced at larval concentrations up to 1,000 larvae per cm2. A. oligospora did not produce nets in an anaerobic atmosphere containing 21 % CO2 (v/v), and net induction was suppressed to a certain degree by exposure to light. The composition of the medium had an important influence on the saprophytic growth and the net-forming capability of A. oligospora as a maximum number of nets was induced at a relatively low concentration of corn meal supporting the relatively sparse mycelium. It was shown that a proportion of trapping nets in A. oligospora maintained their trapping potential for more than 7 weeks when the temperature was below 25°C. Induction of nematodetrapping organs in A. oligospora is discussed in relation to control of infective nematode parasite larvae in cow pats.     

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

Pattern of non-methanogenic and methanogenic degradation of cellulose in anoxic rice field soil

Rice field soils turn anoxic upon flooding. The complete mineralization of organic matter, e.g. cellulose, to gaseous products is then accomplished by the sequential reduction of nitrate, ferric iron, sulfate and finally by methanogenesis. Therefore, the anaerobic turnover of [U-(14)C]cellulose was investigated in fresh, non-methanogenic and in preincubated, methanogenic slurries of Italian rice field soil. In anoxic soil slurries freshly prepared from air-dried soil [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in a ratio of 3:1. In methanogenic soil slurries, on the other hand, which had been preincubated for 45 days under anaerobic conditions, [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in the ratio of 1:1. The turnover times (7-14 days) of cellulose degradation were not significantly different (P0.05) in fresh and methanogenic soil. Chloroform addition abolished CH(4) production, but only slightly (30%) inhibited cellulose degradation in both fresh and methanogenic soil. Under both soil conditions, [(14)C]acetate was the only labeled intermediate detected. A maximum of 24% of the applied radioactivity was transiently accumulated as [(14)C]acetate in both fresh and methanogenic soil slurries. However, when methanogenesis was inhibited by chloroform, 46% and 66% of the applied radioactivity were recovered as [(14)C]acetate in fresh and methanogenic soil, respectively. Only non-radioactive propionate accumulated during the incubation with [U-(14)C]cellulose, especially in the presence of chloroform, indicating that propionate was produced from substrates other than cellulose.     

Alphaproteobacteria dominate active 2-methyl-4-chlorophenoxyacetic acid herbicide degraders in agricultural soil and drilosphere

2-Methyl-4-chlorophenoxyacetic acid (MCPA) is a widely used phenoxyalkanoic acid herbicide and subject to aerobic microbial degradation. Earthworms stimulate both growth and activity of MCPA-degrading bacteria in soil. Thus, active MCPA degraders in soil and drilosphere (i.e. burrow walls, gut content and cast) were assessed by 16S rRNA stable isotope probing in soil columns under experimental conditions designed to minimize laboratory incubation biases. Agriculturally relevant concentrations of [(13) C]MCPA (20 μg g(dw) (-1)) were degraded in soil within 23 and 27 days in the presence and absence of earthworms respectively. Total 16S rRNA analysis revealed 73 operational taxonomic units indicative of active Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia in soil and drilosphere derived material. Seven operational taxonomic units indicative of Alpha-, Beta-, Gammaproteobacteria and Firmicutes consumed MCPA-[(13) C]. Dominant consumers of MCPA-[(13) C] were Alphaproteobacteria (Sphingomonadaceae and Bradyrhizobiaceae) in soil and drilosphere. Beta- (Comamonadaceae) and Gammaproteobacteria (Xanthomonadaceae) were also important MCPA-[(13) C] consumers in burrow walls only, indicating that earthworms favour betaproteobacterial MCPA degraders. In oxic microcosms with bulk soil, burrow walls and cast, 20 and 300-400 μg g(dw) (-1) [(13) C]MCPA were consumed within 24 h and 20 days respectively. Gut contents did not facilitate the degradation of [(13) C]MCPA. Sphingomonadaceae dominated MCPA-[(13) C] consumers in bulk soil and burrow wall microcosms, while Beta- and Gammaproteobacteria (Burkholderiacea, Comamonadaceae, Oxalobacteraceae and Xanthomonadaceae) dominated MCPA-[(13) C] consumers in microcosms of cast, indicating that the latter taxa are prone to respond to MCPA in cast. The collective data indicated that Alphaproteobacteria are major MCPA degraders in soil and drilosphere.     

Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 degrees C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid--histidine--serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.     

STUDIES OF WOOD-DESTROYING FUNGI I. POLYPOBUS HISPIDUS (FRIES)

The characteristics of the Polyporus Msjndus , when grown on artificial media, both solid and liquid, are described and compared with those given by Baxter.
Growth on wood of ash under laboratory conditions produces a rot which is indistinguishable from that occurring naturally. The distribution of the hyphae in the wood is described.
The mode of penetration of the cell-wall by the hyphae is figured. It is apparently by pits in the early stages of decay, but by bore-holes, formed entirely by enzyme activity, in-the more advanced rot.
The rate of growth of the fungus under controlled conditions has been measured, and shown to be 0–5 cm. per month.
Successful inoculation experiments have been carried out with young trees, confirming the results of Baxter, who states that the fungus can attack young, living sap wood.
An investigation of the enzymes produced by the mycelium has been carried out, and a method evolved for demonstrating the presence of a ligninase. This is a modification of Czapek's "hadromal" reaction.
The following enzymes are shown to be present in the mycelium: emulsin, diastase, invertase, ligninase, hemicellulase, oxidase, and catalase. The list is not intended to be exhaustive.
The writer desires to express his thanks to Mr W. R. Day, of the Imperial Forestry Institute, Oxford, and to Dr W. Brown, of the Royal College of Science, for helpful criticism and advice. Also to Mr R. S. Pearson, C.I.E., F.L.S., the Director of Forest Products Research, for permission to publish this paper.     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.