Diversity of ascomycete laccase gene sequences in a southeastern US salt marsh.

The diversity of ascomycete laccase sequences was surveyed in a southeastern US salt marsh using a degenerate primer set designed around copper binding sites conserved in fungal laccases. This gene was targeted for diversity analysis because of its potential function in lignin degradation in the salt marsh ecosystem and because few studies have assessed functional gene diversity in natural fungal communities. Laccase sequences were amplified from genomic DNA extracted from 24 isolates (representing 10 ascomycete species) cultured from decaying blades of Spartina alterniflora, and from DNA extracted directly from the decaying blades. Among the ascomycete isolates, 21 yielded a PCR product of expected size (900 bp) that was tentatively identified as laccase based on sequence similarities to previously published laccase sequences from related organisms. Overall, 13 distinct sequence types, containing 39 distinct sequences, were identified among the isolates, with several species yielding multiple distinct laccase types. PCR amplifications from early and late decay blades of S. alterniflora yielded seven laccase types. Of these, five were composed of sequences >96% similar at the amino acid level to sequences from three cultured ascomycetes previously found to be dominant members of the fungal communities on decaying S. alterniflora blades. Two of the laccase types from the natural-decay clone library were novel and did not match any of the sequences obtained from the cultured ascomycetes. The 39 distinct sequences and 15 distinct laccase sequence types retrieved from the S. alterniflora decay system demonstrate high sequence diversity of this functional gene in a natural fungal community.

Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh.

The ascomycete community colonizing decaying Spartina alterniflora blades in a southeastern U.S. salt marsh was characterized by analysis of internal transcribed spacer (ITS) regions of fungal rRNA genes. ITS sequences were amplified with ascomycete-specific primers from DNA extracted from S. alterniflora blades at two stages of decay (early and late) and were identified based on sequence analysis of a companion ascomycete culture collection. The S. alterniflora ITS libraries were dominated by clones from three species of ascomycetes: Mycosphaerella sp. 2, Phaeosphaeria spartinicola, and Phaeosphaeria halima. ITS sequences from five other less abundant ascomycete species were also found in the clone libraries, only two of which could be identified based on the culture collection, Hydropisphaera erubescens and a new species nicknamed '4clt'. Ascospore expulsion assays indicated dominance by the same three species as the ITS analysis, although this non-molecular approach differed from the molecular method in relative ranking of the dominant species and in characterization of minor species. Analysis of ITS amplicons from three replicate plots by terminal restriction fragment length polymorphism (T-RFLP) analysis showed significant spatial homogeneity in ascomycete community composition for both early- and late-stage decay. ITS sequence analysis identified morphologically cryptic subgroups for two of the three dominant salt marsh ascomycetes.

The effect of mercury and PCBs on organisms from lower trophic levels of a Georgia salt marsh.

We examined several indicators of salt marsh function, focusing on primary producers, microbes, and grass shrimp, at a Superfund site (LCP) contaminated with mercury and polychlorinated biphenyls (PCBs) and a reference site (Cross-River) in Georgia. Primary production of Spartina alterniflora was assessed by measuring peroxidase activity (POD), glutathione concentration (tGSH), photosynthesis (A(net)), and transpiration (E). Microbial populations were assessed by measuring living-fungal standing crop (as ergosterol) and Microtox(R). Grass shrimp (Palaemonetes pugio) reproductive potential was determined by measuring individual egg mass, average egg area, brood size, and brood mass of gravid females. Comparison of the sites suggested that P. pugio reproduction was affected at the LCP site, but we were unable to document clear negative effects on other organisms we investigated. Due to natural environmental gradients, the Cross-River site may not have been a perfect control for the LCP site. Therefore, data from just the LCP site were reanalyzed using multiple regression. Fungal biomass was related to methylmercury concentrations, but the direction of the relationship differed between wholly dead shoots (positive) and partially dead shoots (negative). S. alterniflora POD was positively related to methylmercury concentrations. S. alterniflora A(net) and E were negatively related to elevation and salinity, respectively. Despite high levels of contamination at the LCP site, our results provided only suggestive evidence for impacts on organisms at lower trophic levels.

Fungal biomass in saltmarsh grass blades at two contaminated sites.

Ascomycetous fungi are the principal drivers of the decomposition of shoots of smooth cordgrass (Spartina alterniflora). Shoots of smooth cordgrass move into the saltmarsh food web via the decomposition system. Therefore, influences on saltmarsh ascomycetes by pollutants of saltmarshes could have far-reaching impacts. Earlier examination of impacts of severe contamination of a Georgia saltmarsh by mercury and polychlorinated biphenyls (PCBs) revealed little or no influence of the toxicants on living standing crops or sexual productivities of cordgrass ascomycetes. Extension of the examination of saltmarsh-ascomycete response to sites containing other toxic pollutants (the chlorinated organocyclic insecticide toxaphene; chromium, copper, and lead; and polycyclic aromatic hydrocarbons [PAHs]) has shown that none of the additional toxicants engendered saltmarsh-fungal responses in the form of reduced living standing crops or sexual productivities. Thus the ascomycetes of the cordgrass-decay system appear to be as resistant to anthropogenic-pollutant poisoning as smooth cordgrass itself. Unless the fungal and plant resistance mechanisms involve degradation of the toxicants, this may imply that saltmarshes are especially dangerous as receiving sites for toxic waste because they may have the potential to readily move toxicants into the food web.

Autumnal biomass and potential productivity of salt marsh fungi from 29 degrees to 43 degrees north latitude along the United States Atlantic Coast.

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20 degrees C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = -0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine.

Release of dimethylsulfide from dimethylsulfoniopropionate by plant-associated salt marsh fungi.

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium [M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106-111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora and Pythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer's ability to catabolize DMSP via the DMSP lyase pathway and the host plant's production of DMSP as a secondary product.

Lignocellulolysis by ascomycetes (fungi) of a saltmarsh grass (smooth cordgrass).

Lignocellulose (LC) makes up greater than 70% of the mature shoots of the prodigiously photosynthetically productive saltmarsh grass Spartina alterniflora. Naturally decaying shoots of this cordgrass were examined by transmission electron microscopy (after high-pressure freezing and freeze-substitution) as a means of directly detecting lysis of the LC-rich tissues. Portions of the cordgrass were selected that contained ascomata (sexual reproductive structures) of only one of each of four species of fungi (Kingdom Fungi; Subdivision Ascomycotina): Phaeosphaeria spartinicola and Buergenerula spartinae from leaf blades, Phaeosphaeria spartinae from leaf sheaths, and Passeriniella obiones from naked stems. All four of the ascomycetes were LC-lytic. Phaeosphaeria spartinicola caused both thinning of LC-rich secondary walls of fiber cells from cell lumina outwards (type 2 soft rot, akin to white rot) and digestion extending from hyphae within longitudinal cavities in the secondary walls (type 1 soft rot). The other three species caused either one or the other type of soft rot. Bacterial erosion of cordgrass cells was found only in the samples of naked stems. Ascomycetous decomposers of standing-dead grasses may have potential for biotechnological applications involving alterations of lignocellulose or toxic polyphenolic substances.

Minimizing Ergosterol Loss during Preanalytical Handling and Shipping of Samples of Plant Litter.

Common preliminary treatments of samples of decaying material can involve changes in water content (e.g., via storage in relatively dry air or rinsing) that could conceivably result in loss or gain of fungal membranes and, consequently, ergosterol. A related problem is that collecting of ergosterol content data from widely distributed locales by shipment of samples ideally requires an inexpensive, safe alternative to submerging the samples in methanol for prevention of ergosterol loss. Experimental testing showed that fungal occupants of decaying salt marsh grass leaves did not exhibit loss or gain of ergosterol during air drying (to a water potential of <-8 MPa) or rewetting (to -0.8 MPa). Wet leaves of one grass species (Juncus roemerianus, black needlerush) could be fixed and dried for shipment by microwaving, or by fully drying after alcoholic or pentane fixation, without ergosterol loss, but those of smooth cordgrass (Spartina alterniflora) lost about 40% of their ergosterol content by all three of these drying methods. Ergosterol content of wet leaves of cordgrass could be maintained by alcoholic fixation and subsequent drying down to a thin film of alcohol.

Total and free ergosterol in mycelia of saltmarsh ascomycetes with access to whole leaves or aqueous extracts of leaves.

Three species of saltmarsh ascomycetes were grown in the presence of all of the constituents of their natural substrate (leaves of cordgrass) or were presented only with aqueous extracts of the leaves. These two growth-condition treatments had no significant effect on total ergosterol content of the fungal mycelia, contrary to an earlier hypothesis that availability of plant lipids would lower fungal ergosterol contents. Mycelial content of free ergosterol was about twice as variable as that for total (free plus esterified) ergosterol. Total ergosterol (data pooled for all species) was strongly correlated to organic mycelial mass (r = 0.43, P < 0.00001, and slope = 4.59 mug of ergosterol mg of organic mass).

Ecomethodology for organoosmotrophs: Prokaryotic unicellular versus eukaryotic mycelial.

Although they are very unlikely to play large direct roles in water-column microbial loops, eukaryotic mycelial decomposers (the mycelial true fungi, eumycotes, and zoosporic "fungi," oomycotes) have the potential to be important secondary producers in decaying plant material in shallow aquatic systems. Their secondary productivity may lead to important exchanges of material with microbial loops: output of ascospores, conidia, zoosporic flagellates, leaked lysates, and particles of decayed plants containing mycelium; input of dissolved organics and inorganic nutrients. Development of methods for ecological study of the aquatic mycelial eukaryotic decomposers has not advanced as rapidly as that for the prokaryotes of microbial loops, probably because (1) there are fewer aquatic microbial ecologists with mycological training and inclination than with prokaryotic leanings; and (2) the mycelial decomposers are difficult to work with, because they produce their mycelial mass virtually entirely within opaque solid substrates. Direct microscopic methods have emerged as prime tools for the measurement of prokaryotic mass, whereas an index-chemical assay (ergosterol) is currently the most efficient way to measure the mass of eumycotes. For measuring productivity of prokaryotes of microbial loops, microbial ecologists may choose from several (>10) published and field-tested methods, involving direct microscopy or monitoring of radiotracers. Extensive reviews of distribution and dynamics of aquatic bacterial mass and productivity have appeared. For measuring productivity of eukaryotic mycelial decomposers, one has only two published methods from which to choose, a direct-microscopic and a radiotracer method, neither of which has had adequate field testing. We are, furthermore, much less well equipped to obtain mass and productivity information for the poorly known mycelial oomycotes than we are for the eumycotes. Application of productivity techniques and nucleic-acid technology, may within the next decade allow knowledge of ecology of aquatic eukaryotic mycelial decomposers to advance to levels approaching that for the prokaryotes of microbial loops.

Digestive enzymes of the saltmarsh periwinkleLittorina irrorata (Mollusca: Gastropoda).

The saltmarsh periwinkleLittorina irrorata is well adapted for the digestion of a wide range of polysaccharides. Enzyme extracts attacked cellulose, pectin, xylan, bean gum and mannan (common in cell walls of higher plants), as well as starch and laminarin (representative of major polysaccharide classes in fungal, algal, and animal tissues). Activities were generally highes at a ph of 5 or 6. There was no evidence that chitin was digested, but 19 other enzymes, active toward various carbohydrates, lipids and peptides, were demonstrated. Enzymatic activity toward Azocoll, a general substrate for proteinase activity, was weak compared to that of other aquatic detritivores. The maximum was reached at a pH of 8. Enzymatic activities were generally measured with extracts of the entire visceral hump. Separate stomach or intestine extracts also gave strong activities. The stomach was the most acidic section of the digestive system with an average pH of 5.8; the intestine had an average pH of 7.3.

Fundamental procedures for determining ergosterol content of decaying plant material by liquid chromatography.

Portions of published procedures for measurement of ergosterol content of decomposing plants were examined for their influence upon ergosterol yield. Common methods of treatment of plant samples prior to sterol extraction (e.g., oven drying, freezing, lyophilization) led to reduced recoveries of ergosterol (ca. 20 to 80%). The least destructive method was direct placement and storage in methanol. Photoconversion of ergosterol is not likely to cause losses during analysis, but losses are likely if there is insufficient mixing during neutral-lipid partitioning from base-hydrolysis reagents. Homogenization (two times for 2 min) and refluxing (2 h) in methanol were equally effective in extracting ergosterol. Direct extraction in base-hydrolysis reagents was less effective (by ca. 40%).

Rapid and pervasive occupation of fallen mangrove leaves by a marine zoosporic fungus.

Samples of leaves of red mangrove (Rhizophora mangle) were incubated on an agar medium selective for pythiaceous oomycetes. Leaves on trees above the water did not contain oomycetes. Marine oomycetes, principally Phytophthora vesicula, had colonized leaves within 2 h of leaf submergence, probably finding them by chemotaxis. The frequency of occurrence of P. vesicula in submerged leaves reached 100% within 30 h of submergence. By 43 h most, if not all, parts of leaves were occupied, and surface treatment with a biocide indicated that leaves were occupied internally. Frequencies of P. vesicula remained near 100% through about 2 weeks of submergence and then declined to about 60% in older (>/=4 weeks) leaves. Leaves of white mangrove (Laguncularia racemosa) were also extensively occupied by P. vesicula after falling into the water column, but decaying leaves of turtlegrass (Thalassia testudinum) were not colonized by oomycetes. Ergosterol analysis indicated that the standing crop of living, non-oomycete (ergosterol-containing) fungal mass in submerged red-mangrove leaves did not rise above that which had been present in senescent leaves on the tree; decaying turtlegrass leaves had an ergosterol content that was only about 2% of the maximum concentration detected for red-mangrove leaves. These results suggest that oomycetes are the predominant mycelial eucaryotic saprotrophs of mangrove leaves that fall into the water column and that for turtlegrass leaves which live, die, and decompose under submerged conditions, mycelial eucaryotes make no substantial contribution to decomposition.

Thymidine Incorporation by the Microbial Community of Standing Dead Spartina alterniflora.

Thymidine incorporation by the microbial community on standing dead leaves of Spartina alterniflora did not obey many of the assumptions inherent in the use of the technique in planktonic systems. Incorporation rates of [methly-H]thymidine were nonsaturable over a wide concentration range (10 to 10 nM). Owing to metabolism by both fungi and bacteria, a major fraction of the radiolabel (mean, 48%) appeared in protein. Extraction of the radiolabeled macromolecules was inefficient, averaging 8.8%. Based on an empirically derived conversion factor, 4 x 10 cells . mol of thymidine, doubling times ranged from 4 to 69 h for the epiphytic bacterial assemblage.

Influence of rain, tidal wetting and relative humidity on release of carbon dioxide by standing-dead salt-marsh plants.

Dead parts of salt-marsh plants form a considerable fraction of their annual average standing crop. A microbial assemblage living on and in the standing-dead leaves and stems of Spartina alterniflora and Juncus roemerianus responds to saltwater, freshwater or water-vapor wetting by immediately beginning to release CO2. Water-saturated, standing-dead leaves and culms of S. alterniflora release CO2 at steady rates of as much as about 200 and 140 µg CO2-C·g-1 dry·h-1, respectively, at temperatures of 25-30°C, after an initial burst of higher rates. These CO2-release rates are within the range of maximal rates reported for decaying terrestrial litter, and are as high as most rates reported for S. alterniflora decaying under continuously wetted or submerged conditions.

Relative contributions of bacteria and fungi to rates of degradation of lignocellulosic detritus in salt-marsh sediments.

Specifically radiolabeled [C-lignin]lignocellulose and [C-polysaccharide]lignocellulose from the salt-marsh cordgrass Spartina alterniflora were incubated with an intact salt-marsh sediment microbial assemblage, with a mixed (size-fractionated) bacterial assemblage, and with each of three marine fungi, Buergenerula spartinae, Phaeosphaeria typharum, and Leptosphaeria obiones, isolated from decaying S. alterniflora. The bacterial assemblage alone mineralized the lignin and polysaccharide components of S. alterniflora lignocellulose at approximately the same rate as did intact salt-marsh sediment inocula. The polysaccharide component was mineralized twice as fast as the lignin component; after 23 days of incubation, ca. 10% of the lignin component and 20% of the polysaccharide component of S. alterniflora lignocellulose were mineralized. Relative to the total sediment and bacterial inocula, the three species of fungi mediated only very slow mineralization of the lignin and polysaccharide components of S. alterniflora lignocellulose. Experiments with uniformly C-labeled S. alterniflora material indicated that the three fungi and the bacterial assemblage were capable of degrading the non-lignocellulosic fraction of S. alterniflora material, but only the bacterial assemblage significantly degraded the lignocellulosic fraction. Our results suggest that bacteria are the predominant degraders of lignocellulosic detritus in salt-marsh sediments.

Modification of the gelatin-matrix method for enumeration of respiring bacterial cells for use with salt-marsh water samples.

In this report I describe a modification of the gelatin-matrix method for determining the percentages of bacterial cells having active electron transport systems. This modification is one-step fluorochroming followed by use of an antioxidant solution instead of the secondary gelatin layer. The modified technique is more successfully applicable to water samples containing high densities of flocculant particulate matter, owing to the brighter green fluorescence of the cells, which contrasts with the red-orange fluorescence of the floc.

An improved gas chromatographic method for measuring glucosamine and muramic acid concentrations.

A method of simultaneously measuring glucosamine and muramic acid concentrations in marsh grass litter was developed. Spartina alterniflora samples were preextracted with acetone to remove lipids containing amino sugars and then hydrolyzed in 6 N HCl (100 degrees C, 4.5 h). Amino sugars in the hydrolysates were isolated by ion-exchange chromatography, which gave good recoveries (greater than 90%) and reproducibility (CV less than 5%). Isolated amino sugars were converted to O-methyloxime acetates. beta-Phenylglucose and N-methylglucamine were added as internal standards. Sample derivatives were quantified by capillary column gas chromatography. OV-101 and SE-54 capillary columns completely separated glucosamine and muramic acid from other amino sugars. The detection limit of glucosamine and muramic acid during gas chromatographic analysis was below 30 pmol using splitless-mode injection (SE-54 column). Filamentous fungal and procaryotic biomasses may be estimated simultaneously by using glucosamine and muramic acid biomass conversion factors in conjunction with this method.

Comparison of methods for measurement of bacterial growth rates in mixed batch cultures.

Eight methods of assessing growth rate constants of bacteria were compared in batch cultures of 3-micrometers-filtered estuarine water from the Skidaway River in Ga. Mixed assemblages of bacteria were grown under four nutrient regimes of added yeast extract ranging from 0 to 100 mg/liter. Linear and exponential growth rate constants were computed from changes in cell densities, biovolumes, and ATP concentrations. Exponential growth rate constants were obtained from the frequency of dividing cells and RNA synthesis as measured by [3H]adenine uptake. Rate constants obtained during lag, exponential, and stationary growth phases depended largely on the method used. Constants calculated from changes in cell densities, frequency of dividing cells, and adenine uptake correlated most closely with each other, whereas constants calculated from changes in ATP concentrations and biovolumes correlated best with each other. Estimates of in situ bacterial productivity and growth vary depending on the method used and the assumptions made regarding the growth state of bacteria.

Bacterial productivity in the water column and sediments of the Georgia (USA) coastal zone: Estimates via direct counting and parallel measurement of thymidine incorporation.

Three methods of estimating bacterial productivity were compared using parallel samples of Atlantic Ocean water (within 0.25-15 km of the Georgia coast). The frequency-of-dividing cells (FDC) method and the [(3)H]thymidine incorporation method gave results which were strongly correlated (r=0.97), but the FDC estimates were always higher (X2 to X7) than the [(3)H]thymidine estimates. Estimates of bacterial productivity ranged from 2-4×10(8) cells·l(-1)·h(-1) at 0.25 km from shore to 1-9×10(7)cells·l(-1)·h(-1) at 15 km. A method involving incubation of 3-µm filtrates and direct counting gave results that could not be easily translated into estimates of bacterial productivity. Application of the FDC method to sediment samples gave high productivity estimates, which could be not reconciled with productivity estimates based on sediment oxygen uptake.