Steven Y. Newell

Fungal farming in a snail

Mutualisms between fungi and fungus-growing animals are model systems for studying coevolution and complex interactions between species. Fungal growing behavior has enabled cultivating animals to rise to major ecological importance, but evolution of farming symbioses is thought to be restricted to three terrestrial insect lineages. Surveys along 2,000 km of North Americas Atlantic coast documented that the marine snail Littoraria irrorata grazes fungus-infected wounds on live marsh grass throughout its range. Field experiments demonstrate a facultative, farming mutualism between Littoraria and intertidal fungi. Snails graze live grass primarily not to feed but to prepare substrate for fungal growth and consume invasive fungi. Fungal removal experiments show that snails and fungi act synergistically to suppress marsh grass production. These results provide a case of fungus farming in the marine environment and outside the class Insecta and reveal a previously undemonstrated ecological mechanism (i.e., facilitation of fungal invasion) by which grazers can exert top-down control of marine plant production.

Established and potential impacts of eukaryotic mycelial decomposers in marine/terrestrial ecotones

Abstract Marine mycelial decomposers (eumycotes, members of Kingdom Fungi, and oomycotes, zoosporic members of the Kingdom Protoctista) are highly adapted for capture of solid substrate by pervasion and digestion from within. Thus they exert their influence in areas of large input of litter of vascular plants, especially at some types of terrestrial/marine ecosystemic interfaces (ecotones). Unavailability of methods easily used by general microbial ecologists has hampered progress in the study of marine mycelial decomposers, and there are still pockets of difficulty in this regard (especially for oomycotes). Recently published or refined methods for measuring fungal mass and productivity have begun to allow us to realize the impacts of fungi in marine ecotones. For example, it is now clear that the older paradigm reflecting negligible contribution of microbial mass to litter nitrogen content is false for the standing-decay system of saltmarsh grasses — in these decay systems, fungal mass can account for virtually all of the nitrogen present at some point(s) in the standing-decay period. Another generally held belief about marine fungi has also been reversed — ascomycetes (Fungi) of a saltmarsh grass (smooth cordgrass) clearly do digest lignocellulose under natural-decay circumstances. Much more work is needed to clarify the situation, but at present it appears that major types of marine ecotones (e.g., saltmarshes and mangroves) differ sharply in the balance among major groups of decomposers (eumycotes, oomycotes, and bacteria) with regard to their utilization of vascular-plant litter. In saltmarshes, microbial production in standing grass litter is strongly dominated by fungi, and oomycotes do not show evidence of a substantial role in decomposition. In mangroves, submerged fallen leaves appear to support minor fungal occupancy, but ubiquitous and rapid occupancy by oomycotes (especially Halophytophthora vesicula ). Many exciting areas of research are now more open than ever before to marine microbial ecologists interested in working with mycelial decomposers.

Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

ABSTRACT Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α-Proteobacteria and one with the Cytophagales) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate “4clt”).

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

SummaryDead 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.

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.

Decomposition of Shoots of a Salt-Marsh Grass

Researchers interested in accurately describing natural microbial participation in the decay of portions of vascular plants must try to avoid altering genuine conditions of decay via their methods (Swift et al., 1979; Boulton and Boon, 1991; Newell, 1994). This is an old refrain (e.g., Park, 1974, on the elusive balance between particle retention and shredder admittance using litterbags), but one that continues to go unheeded (e.g., a paper published in 1992 in a leading aquatic-science journal; methods: green shoots of grass cut, dried, and placed in litterbags on or buried in salt-marsh sediment). “The challenge of ecology immediately reveals the correlated dangers, for it is singularly easy to fall into error through a failure to describe accurately the various parts of the system and to appreciate their possible significance. An error at this stage may lead to the development of inapposite experimental techniques, so that the final synthesis must inevitably fail. A constant temptation besetting the ecologist is that of loose thinking to enable him to gloss over intractable parts of his study” (Griffin, 1972).

Misting and nitrogen fertilization of shoots of a saltmarsh grass: effects upon fungal decay of leaf blades

We conducted a 12-week field manipulation experiment in which we raised the nitrogen availability (ammonium sulfate fertilization to roots) and/or water potential (freshwater misting) of decaying leaf blades of a saltmarsh grass (smooth cordgrass, Spartina alterniflora) in triplicate 11-m2 plots, and compared the manipulated plots to unmanipulated, control plots. The ascomycetous fungi that dominate cordgrass leaf decomposition processes under natural conditions exhibited a boosting (>2-fold) of living standing crop (ergosterol content) by misting at the 1 st week after tagging of senescent leaves, but afterwards, living-fungal standing crop on misted blades was equivalent to that on control blades, confirming prior evidence that Spartina fungi are well adapted to natural, irregular wetting. Misting also caused 2-fold sharper temporal declines than control in instantaneous rates of fungal production (ergosterol synthesis), 5-fold declines in density of sexual reproductive structures that were not shown by controls, and 2-fold higher rates of loss of plant organic mass. Extra nitrogen gave a long-term boost to living-fungal standing crop (about 2-fold at 12 weeks), which was also reflected in rates of fungal production at 4 weeks, suggesting that saltmarsh fungal production is nitrogen-limited. Although bacterial and green-microalgal crops were boosted by manipulations of nitrogen and/or water, their maximal crops remained ≤0.3 or 2% (bacteria or green microalgae, respectively) of contemporaneous living-fungal crop. The fungal carbon-productivity values obtained, in conjunction with rates of loss of plant carbon, hinted that fungal yield can be high (>50%), and that it is boosted by high availability of nitrogen. We speculate that one partial cause of high fungal yield could be subsidy of fungal growth by dissolved organic carbon from outside decomposing leaves.

Direct-count Estimates of Fungal and Bacterial Biovolume in Dead Leaves of Smooth Cordgrass (Spartina alterniflora Loisel.)

Two types of hyphal-extraction, direct-count methods of estimating fungal biovalume in standing-dead, autumn leaves of Spartina alterniflora were compared with a clearing+staining method which does not require homogenization. Bacterial biovolume also was estimated, by an acridine-orange direct-count method. Type of homogenization had little effect on measured fungal volume, but counts made using water-soluble-aniline-blue epifluorescence were consistently lower than those made using phase-contrast (by 6–10x). Clearing+staining could not be used to estimate hyphal lengths, but was of use in estimating total ascocarp volume (=0.06 mm3 per mm3 of leaf). Estimated fungal hyphal volume was approximately 0.27 mm3 per mm3 of leaf. Bacterial volume was <3% of fungal volume.

Experimental studies of particle ingestion by the sand fiddler crab Uca pugilator (Bosc)

Abstract Particle ingestion by the sand fiddler crab Uca pugilator (Bosc) was investigated using clean sand sediment inoculated with glass and plastic beads. For all bead types, the number ingested was positively correlated with the initial sediment bead concentration. Ingestion efficiency, however, was inversely correlated with bead diameter, weight, and initial sediment bead concentration. The latter result suggests that the particle sorting mechanism can be saturated at high substratum particle concentrations. Crab size was directly related to the efficiency at which large beads were ingested, but had no effect on the ingestion of small beads. Ciliates, unbound bacteria, and bacteria attached to sand grains were ingested with high efficiency, but the crabs did not preferentially ingest live over inert particles. Other results suggest that maximization of food intake by sand fiddlers occurs at the habitat patch level rather than at the level of particle ingestion. Supporting this view is the observation that inert particles which were ingested with high efficiencies did not stimulate crab feeding. Sediment harvesting rates were inversely related to substratum organic and silt concentrations suggesting that sand fiddlers can control ingestion rates by regulating sediment harvesting rates

Microbial Secondary Production from Salt Marsh-Grass Shoots, and Its Known and Potential Fates

Several lines of evidence (direct microscopy, index biochemicals) point to predominance of eukaryotic decomposers in natural decay of dead shoots of smooth cordgrass (Spartina alterniflora). Recent research shows that this is also true for black needlerush (Juncus roemerianus). Ascomycetous fungi are the major initial secondary producers based on the dead shoots. There is no overlap between the species of the cordgrass (e.g., Phaeosphaeria spartinicola) and needlerush (e.g., Loratospora aestuarii) fungal-decay communities. Even when conditions in the marsh are manipulated in directions that would be expected to favor prokaryotes (extra water and nitrogen), the ascomycetes accumulate maximum organic masses in standing-decaying shoots hundreds of times larger than prokaryotic masses. Rates of fungal production are not increased by raising duration of high water availability, probably due to fine-tuned fungal adaptation to periodic dryness, but nitrogen does limit fungal productivity in decaying cordgrass. Content of living-fungal mass can be 10 to 20% of totalsystem (= microbes + remaining plant) mass, depending on nitrogen availability, rates of invertebrate mycophagy, and probably several further factors yet to be determined. Standing crops of living fungi in cordgrass marshes in Georgia (per-m2) basis) have been calculated to be equal to 3% (summer) to 28% (winter) of living-cordgrass standing crop. This is calculated to be about 50 to 100% of total (non-cyano) bacterial crop; the great bulk of bacterial crop is sedimentary. Fungal productivity per m2 standing-decaying-cordgrass marsh has been provisionally found to be 10 times greater in winter than in summer (3652 mg per m2 per day; μ=0.07 day−1). Total bacterial productivity per m2 was calculated to be about x2 fungal in summer, and x0.07 fungal in winter. High yields of fungi (on the order of 50%) from cordgrass shoots may be part of the explanation for high rates of animal secondary production in saltmarsh ecosystems. Cordgrass-fungal standing crops and productivities (per unit leaf mass) do not show pronounced variation (in autumn) along a south-north latitudinal gradient from 30° to 44°N. One major known fate of saltmarsh-fungal secondary production is output to shredder gastropods (periwinkles, Littoraria irrorata). Other potential substantial fluxes are to amphipods (especially Uhlorchestia spartinophila) and other gastropods (especially Melampus bidentatus), and fluxes as sexual propagules (ascospores) and as remnant hyphal wall/sheath mass in fallen, decayed fragments. Key opportunities for saltmarsh-ecological research lie: in learning the details of the life histories of the more important saltmarsh-fungal producers; in determining the biotic and abiotic controls on saltmarsh-fungal productivity; and in investigations of impacts of fungal activities, such as the probable role that saltmarsh ascomycetes have in release of dimethylsulfide to the atmosphere.