Use of chitinase to assess ruminal fungi associated with plant residues in vitro.

Treatment of fibrous residue from in vitro digestion trials with chitinase was evaluated gravimetrically and microscopically to determine the associated fungal biomass. The percent dry weight removed by chitinase with time paralleled changes in the number of sporangia associated with leaves. The fungal biomass contributed about 12% dry matter to the residue of leaves incubated with ruminal fluid plus streptomycin and penicillin.     

Mass loss,fungal colonisation and nutrient dynamics of Phragmites australis leaves during senescence and early aerial decay

This study examined the mass loss, fungal biomass, and nutrient dynamics of standing Phragmites australis leaf blades during senescence and early decay in littoral reed stands of two hardwater lakes. Green living leaves were tagged at defined canopy heights in early autumn (late August or early September) and periodically collected until all leaf blades had fallen off the parent shoot. Samples were analysed for leaf dry mass remaining, fungal biomass associated with leaves (ergosterol concentrations), and nitrogen and phosphorus concentrations. Considerable mass loss of leaves occurred in the standing position (up to 28%). Nitrogen and phosphorus concentrations of leaves decreased substantially with time (by 39–77%), indicating that a major portion of these nutrients was translocated to the rhizome during senescence. Fungal biomass associated with leaves increased during the study period, reaching an estimated maximum of about 40 mg g⁻¹ of leaf dry mass. Fungal biomass was negatively correlated with leaf N and P concentrations. The observed patterns of leaf mass loss, nutrient dynamics, and fungal biomass were consistent with the successive senescence and death of leaves from the shoot base to its tip. The results of this study point to a notable mass loss of P. australis leaf blades in the standing position, which appears to be mediated by both plant and microbial processes. Nutrient dynamics, in contrast, appear to be largely governed by plant processes.     

Influence of fungal-bacterial interactions on bacterial conjugation in the residuesphere

Conjugal gene transfer among bacteria in the residuesphere (area between decaying plant material and soil) of leaves of barley straw was studied. The residuesphere was shown to be a hot-spot for conjugal gene transfer compared to conjugation in sterile sand and non-sterile bulk soil. Impact of fungal colonisation of the residuesphere on bacterial colonisation and conjugation was also investigated. The inhibition of fungal colonisation, due to the application of an eukaryotic inhibitor, increased bacterial colonisation of the residuesphere in soil microcosms compared to non-treated leaves. This treatment also had a transient, positive effect on conjugation. Bacterial conjugation in the residuesphere of leaves subjected to 17 days of fungal colonisation was significantly lower than in the residuesphere of non-colonised leaves. Fungal biomass, as measured by chitinase activity, was inversely related to the conjugation efficiency.     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.     

Comparison of ATP and Ergosterol as Indicators of Fungal Biomass Associated with Decomposing Leaves in Streams

ATP and ergosterol were compared as indicators of fungal biomass associated with leaves decomposing in laboratory microcosms and streams. In all studies, the sporulation rates of the fungi colonizing leaves were also determined to compare patterns of fungal reproductive activity with patterns of mycelial growth. During leaf degradation, ATP concentrations exhibited significant, positive correlations with ergosterol concentrations in the laboratory and when leaves had been air dried prior to being submerged in a stream. However, when freshly shed leaves were submerged in a stream, concentrations of ATP and ergosterol were negatively correlated during degradation. This appeared to be due to the persistence of leaf-derived ATP in freshly shed leaves during the first 1 to 2 weeks in the stream. Estimates of fungal biomass from ergosterol concentrations of leaf litter were one to three times those calculated from ATP concentrations. ATP, ergosterol, and sporulation data generally provided similar information about the fungi associated with decomposing leaves in streams during periods when fungi were growing. Ergosterol concentrations provide a more accurate indication of fungal biomass in situations in which other organisms make significant contributions to ATP pools.     

Cell Surfaces in Plant-Microorganism Interactions : VI. Elicitors of Ethylene from Colletotrichum lagenarium Trigger Chitinase Activity in Melon Plants

Treatment of melon leaves or seedlings with elicitors of Colletotrichum lagenarium, a fungal pathogen of melon, increases chitinase activity. In treated leaves, chitinase is enhanced within the first 6 hours and becomes 2 to 10 times higher than in control leaves after 24 hours. Ethylene is increased simultaneously and is correlated with chitinase elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor-induced ethylene and elicitor-induced chitinase are inhibited. This inhibition is overcome by added exogenous ethylene. On the other hand, 1-aminocyclopropane-1-carboxylic acid the direct precursor of ethylene, triggers chitinase activity. Chitinase elicitation is thought to be a protein synthesis dependent process, as it does not occur in the presence of cycloheximide.     

Placing the Effects of Leaf Litter Diversity on Saprotrophic Microorganisms in the Context of Leaf Type and Habitat

Because of conflicting results in previous studies, it is unclear whether litter diversity has a predictable impact on microbial communities or ecosystem processes. We examined whether effects of litter diversity depend on factors that could confound comparisons among previous studies, including leaf type, habitat type, identity of other leaves in the mixture, and spatial covariance at two scales within habitats. We also examined how litter diversity affects the saprotrophic microbial community using terminal restriction fragment length polymorphism to profile bacterial and fungal community composition, direct microscopy to quantify bacterial biomass, and ergosterol extraction to quantify fungal biomass. We found that leaf mixture diversity was rarely significant as a main effect (only for fungal biomass), but was often significant as an interaction with leaf type (for ash-free dry mass recovered, carbon-to-nitrogen ratio, fungal biomass, and bacterial community composition). Leaf type and habitat were significant as main effects for all response variables. The majority of variance in leaf ash-free dry mass and C/N ratio was explained after accounting for treatment effects and spatial covariation at the meter (block) and centimeter (litterbag) scales. However, a substantial amount of variability in microbial communities was left unexplained and must be driven by factors at other spatial scales or more complex spatiotemporal dynamics. We conclude that litter diversity effects are primarily dependent on leaf type, rather than habitat type or identity of surrounding leaves, which can guide the search for mechanisms underlying effects of litter diversity on ecosystem processes.     

Fungal biomass and decomposition in Spartina maritima leaves in the Mondego salt marsh (Portugal)

Spartina maritima (Curtis) Fernald is a dominant species in the Mondego salt marsh on the western coast of Portugal, and it plays a significant role in estuarine productivity. In this work, leaf litter production dynamics and fungal importance for leaf decomposition processes in Spartina maritima were studied. Leaf fall was highly seasonal, being significantly higher during dry months. It ranged from 42 g m^-2 in June to less than 6 g m^-2 during the winter. Fungal biomass, measured as ergosterol content, did not differ significantly between standing-decaying leaves and naturally detached leaves. Fungal biomass increased in wet months, with a maximum of 614 g g^-1 of ergosterol in January in standing-decaying leaves, and 1077 g g^-1 in December, in naturally detached leaves, decreasing greatly in summer. Seasonal pattern of fungal colonization was similar in leaves placed in litterbags on the marsh-sediment surface. However, ergosterol concentrations associated with standing-decaying and naturally detached leaves were always much higher than in litterbagged leaves, suggesting that fungal activity was more important before leaf fall. Dry mass of litterbagged leaves declined rapidly after 1 month (about 50%), mostly due to leaching of soluble organic compounds. After 13 months, Spartina leaves had lost 88% of their original dry weight. The decomposition rate constant (k) for Spartina maritima leaves was 0.151 month^-1.     

Fungal biomass associated with the phyllosphere of bryophytes and vascular plants

Little is known about the amount of fungal biomass in the phyllosphere of bryophytes compared to higher plants. In this study, fungal biomass associated with the phyllosphere of three bryophytes (Hylocomium splendens, Pleurozium schreberi, Polytrichum commune) and three vascular plants (Avenella flexuosa, Gymnocarpium dryopteris, Vaccinium myrtillus) was investigated using ergosterol content as a proxy for fungal biomass. Phyllosphere fungi accounted for 0.2-4.0 % of the dry mass of moss gametophytes, representing the first estimation of fungal biomass associated with bryophytes. Significantly more fungal biomass was associated with the phyllosphere of bryophytes than co-occurring vascular plants. The ergosterol present in moss gametophytic tissues differed significantly between species, while the ergosterol present in vascular plant leaf tissues did not. The photosynthetic tissues of mosses had less associated fungal biomass than their senescent tissues, and the magnitude of this difference varied in a species-specific manner. The fungal biomass associated with the vascular plants studied varied significantly between localities, while that of mosses did not. The observed differences in phyllosphere community biomass suggest their size could be affected by host anatomical and physiological attributes, including micro-niche availability and chemical host defenses, in addition to abiotic factors like moisture and nutrient availability.     

The role of chitosan in protection of soybean from sudden death syndrome caused by Fusarium solani f. sp. glycines

The in vitro antifungal properties of chitosan and its role in protection of soybean from a sudden death syndrome (SDS) were evaluated. Chitosan inhibited the radial and submerged growth of F. solani f. sp. glycines with a marked effect at concentrations up to 1mg/ml indicating antifungal property and at 3mg/ml was able to delay SDS symptoms expression on soybean leaves for over three days after fungal inoculation when applied preventively. Chitosan was able to induce the level of chitinase activity in soybean resulting in the retardation of SDS development in soybean leaves. However, the SDS symptoms gradually appeared and were associated with the reduction of chitinase activity level after five days of infection period. These results suggested the role of chitosan in partially protecting soybeans from F. solani f. sp. glycines infection.     

Use of a Monoclonal Antibody-Based Immunoassay for the Detection and Quantification of Heliscus lugdunensis Colonizing Alder Leaves and Roots

A genus-specific monoclonal antibody, NG-CF10, raised in a previous study to the fungal pathogen Nectria galligena, was found to recognize the aquatic hyphomycete Heliscus lugdunensis (anamorph) and its teleomorph Nectria lugdunensis. Using this MAb in a plate trapped antigen- ELISA we could detect and determine the biomass of Heliscus lugdunensis in mixed assemblages in both naturally occurring and artificially inoculated leaves and roots of Alnus glutinosa trees. Initial studies indicate that the biomass associated with naturally occurring leaf material is significantly lower than that recorded with laboratory inoculated leaves, suggesting that biomass production is limited in the natural environment. Significantly lower biomass was associated with roots when compared with leaf material, which supports the proposition that rather than a major substrate for the growth of aquatic hyphomycetes, roots act as a refugium for fungal growth.     

Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.     

A Cross-System Comparison of Bacterial and Fungal Biomass in Detritus Pools of Headwater Streams

The absolute amount of microbial biomass and relative contribution of fungi and bacteria are expected to vary among types of organic matter (OM) within a stream and will vary among streams because of differences in organic matter quality and quantity. Common types of benthic detritus [leaves, small wood, and fine benthic organic matter (FBOM)] were sampled in 9 small (1st-3rd order) streams selected to represent a range of important controlling factors such as surrounding vegetation, detritus standing stocks, and water chemistry. Direct counts of bacteria and measurements of ergosterol (a fungal sterol) were used to describe variation in bacterial and fungal biomass. There were significant differences in bacterial abundance among types of organic matter with higher densities per unit mass of organic matter on fine particles relative to either leaves or wood surfaces. In contrast, ergosterol concentrations were significantly greater on leaves and wood, confirming the predominance of fungal biomass in these larger size classes. In general, bacterial abundance per unit organic matter was less variable than fungal biomass, suggesting bacteria will be a more predictable component of stream microbial communities. For 7 of the 9 streams, the standing stock of fine benthic organic matter was large enough that habitat-weighted reach-scale bacterial biomass was equal to or greater than fungal biomass. The quantities of leaves and small wood varied among streams such that the relative contribution of reach-scale fungal biomass ranged from 10% to as much as 90% of microbial biomass. Ergosterol concentrations were positively associated with substrate C:N ratio while bacterial abundance was negatively correlated with C:N. Both these relationships are confounded by particle size, i.e., leaves and wood had higher C:N than fine benthic organic matter. There was a weak positive relationship between bacterial abundance and streamwater soluble reactive phosphorus concentration, but no apparent pattern between either bacteria or fungi and streamwater dissolved inorganic nitrogen. The variation in microbial biomass per unit organic matter and the relative abundance of different types of organic matter contributed equally to driving differences in total microbial biomass at the reach scale.     

Isolation and Characterization of a Barley Chitinase Genomic Clone—Expression in Powdery Mildew Infected Barley

Chitinases (E.C.3.2.1.14) are thought to play an important role in the defense of plants against fungal invasion. By screening a barley genomic library with a previously identified chitinase eDNA clone (clone 10), a genomic clone was isolated and characterized by DNA sequencing of the chitinase coding region and flanking sequences. This clone contains an open reading frame capable of coding for a 34 kD chitinase. Comparison of the amino acid sequence of the encoded protein with other barley chitinases suggests that the genomic clone encodes chitinase T, which has been characterized extensively by protein sequencing. Treatment of barley leaves and aleurone protoplasts with N-acetyl glucosamine oligomers which act as elicitors in other plants, did not lead to the elevation of the levels of the chitinases. However, infection of barley seedlings with the powdery mildew fungus, Erysiphe graminis, resulted in the induction of several isoforms of chitinase. The level and number of chitinase isozymes was correlated with the severity of infection. The infection-related chitinases found in barley leaves are different from those found in seeds.     

<Emphasis Type="Italic">Encentrum (Parencentrum) walterkostei</Emphasis> n. sp., a new dicranophorid rotifer (Rotatoria: Monogononta) from the high alpine zone of the Central Alps (Austria)

Leaf litter processing rates and fungal biomass on leaf detritus were compared in four streams of different water chemistry. The streams drained catchments underlain by different bedrock types and varied in mean pH from 4.3 to 7.5 and in mean alkalinity from 0.0 to 35.8 mg CaCO₃ l^–1. Processing rates were fastest in WS3 and WS4, which had a pH of 6.0; slowest in SFR, which had a pH of 4.3; and intermediate in HSR which had a pH of 7.5. Fungal biomass as measured by the fungal sterol, ergosterol, was similar in WS3, WS4, and HSR but was much lower in SFR. These results suggest that reduced processing rates in SFR were associated in part with reduced fungal biomass on the leaves, whereas reduced processing rates in HSR were not related to differences in fungal biomass on the leaves.The Unit is jointly sponsored by the U.S. Fish and Wildlife Service, the West Virginia Division of Natural Resources, West Virginia University, and the Wildlife Management Institute.     

Fungal growth, production, and sporulation during leaf decomposition in two streams.

I examined the activity of fungi associated with yellow poplar (Liriodendron tulipifera) and white oak (Quercus alba) leaves in two streams that differed in pH and alkalinity (a hard water stream [pH 8.0] and a soft water stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 microg liter(-1)) and phosphorus (<3 microg liter(-1)). The leaves of each species decomposed faster in the hard water stream (decomposition rates, 0.010 and 0.007 day(-1) for yellow poplar and oak, respectively) than in the soft water stream (decomposition rates, 0.005 and 0.004 day(-1) for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [(14)C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hard water stream but not in the soft water stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hard water stream (5.8 mg g(-1) day(-1) on yellow poplar leaves and 3.1 mg g(-1) day(-1) on oak leaves) than in the soft water stream (1.6 mg g(-1) day(-1) on yellow poplar leaves and 0.9 mg g(-1) day(-1) on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the soft water stream (12.8% of detrital mass) than in the hard water stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the soft water stream.     

The Effect of the Herbicide Mecoprop on Heliscus lugdunensis and Its Influence on the Preferential Feeding of Gammarus Pseudolimnaeus

Abstract Exposure of the aquatic hyphomycete Heliscus lugdunensis to the herbicide Mecoprop did not significantly affect production of the antigen recognized by the specific monoclonal antibody NG-CF10. Therefore, an ELISA method, developed in a previous study, could be used to quantify the biomass of H. lugdunensis colonizing leaves exposed to this herbicide. Exposure to Mecoprop significantly reduced the mycelial biomass associated with alder leaves. This was shown to be a threshold response rather than a dose response, with higher biomass recorded on control leaves. No significant differences were found over the range of Mecoprop concentrations used. In laboratory experiments, Gammarus pseudolimnaeus was offered a choice of alder leaves exposed to a range of Mecoprop concentrations. The animals were able to discriminate between the exposed and control leaves, and between inoculated and sterile leaves. Presence of the fungus resulted in increased leaf consumption, but no interaction between the Mecoprop concentrations and fungal colonization was observed. The major factor affecting food choice was the concentration of Mecoprop that the leaves were exposed to—not the Mecoprop-mediated effects on fungal biomass. Received: 10 February 1997; Accepted: 8 May 1997     

Quantification of Fungal Hyphae in Leaves of Deciduous Trees by Automated Image Analysis

An optical method to quantify the fungal hyphae within decomposing leaves of deciduous trees was developed. The plant matrix was partially destroyed under hydrolytic conditions, and fungal hyphae and cellulose residues within the leaves were stained with Calcofluor M2R. Cellulose residues were subsequently depolymerized by cellulase, and fungal hyphae were separated from the remaining plant matrix with a pressurized air-water mixture. An image analysis program to quantify the fungal hyphae was written. The program included the recognition of fungal hyphae, the elimination of stomata from the images, and the measuring of lengths of fungal hyphae. The optical method was verified by a chemical method relying on glucosamine as an indicator of fungal biomass. The fungal biomass in leaves of Fagus silvatica and Quercus petraea at early states of decomposition was 0.2 to 0.4% of the leaf weight. The biomass reached a maximum within 2 to 4 weeks (optical method, 0.5 to 0.7%; chemical method, 1 to 1.4% of the initial leaf weight) and decreased thereafter.     

A comparison of bacterial and fungal biomass in several cultivated soils

Bacterial and fungal biomass was estimated in incubated samples of three cultivated soils, the influence of glucose, ammonium nitrate and cattle slurry on its formation being studied. The microbial biomass was determined in stained microscopic preparations of soil suspension. Bacterial biomass in the control samples was from 0.17 to 0.66 mg dry wt per 1 g dry soil and independently of the applied supplements was on the average two times larger in muck soils than in sand. Fungal biomass in the control soils ranged from 0.013 to 0.161 mg dry wt per 1 g dry soil, no relationship being found between its size and the soil type. As a result, the ratio of the size of fungal to bacterial biomass was dependent on the soil type; in sand the fungal biomass corresponded to 1/3 of the bacterial biomass, and in muck soils--only to 1/7.     

The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.