John H. Andrews

THE ECOLOGY AND BIOGEOGRAPHY OF MICROORGANISMS ON PLANT SURFACES.

The vast surface of the plant axis, stretching from root tips occasionally buried deeply in anoxic sediment, to apical meristems held far aloft, provides an extraordinarily diverse habitat for microorganisms. Each zone has to a greater or lesser extent its own cohort of microorganisms, in aggregate comprising representatives from all three primary domains of life-Bacteria, Archaea, and Eucarya. While the plant sets the stage for its microbial inhabitants, they, in turn, have established varied relationships with their large partner. These associations range from relatively inconsequential (transient epiphytic saprophytes) to substantial (epiphytic commensals, mutualistic symbionts, endophytes, or pathogens). Through recent technological breakthroughs, a much better perspective is beginning to emerge on the nature of these relationships, but still relatively little is known about the role of epiphytic microbial associations in the life of the plant.

r - and K -Selection and Microbial Ecology

The essence of the concept of r- and K-selection is that organisms strive to maximize their fitness for survival in either uncrowded (r-selection) or crowded (K-selection) environments. Fitness is defined following ecological convention as the proportion of genes left in the population gene pool (Pianka, 1983, p. 10). The terms r and K refer, respectively, to the maximum specific rate of increase (maximum specific growth rate minus minimum specific death rate) of an organism and to the density of individuals that a given environment can support at the population equilibrium. Since both r and K can vary within a species and are subject to modification, the division of natural selection into r- and K-selection is of considerable basic interest in evolutionary ecology.

THE PATHOLOGY OF MARINE ALGAE

I11 . Non-infectious diseases . . . . . . . . . . . . . (I) Diseases caused by deleterious changes of the natural environment (2) Diseases caused by man’s activities: pollution damage . . . . . . . . . . (a) Oil and dispersants . . . . . . . . . . . . (b) Pesticides and related compounds (c) Heavy metals and radio-isotopes (d) Sewage and industrial effluents (e) Thermal pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

Putative mechanism and dynamics of inhibition of the apple scab pathogen Venturia inaequalis by compost extracts

Abstract Clarified water extracts of slurries of spent mushroom substrate (SMS) inhibited in vitro germination of conidia of the apple scab pathogen Venturia inaequalis by up to 98% relative to germination in water controls. Inhibition of conidial germination increased with incubation time of slurries over 5 to 7 d and persisted for at least 14 d, at which time experiments were terminated. Compost slurries became anaerobic within 1 h when incubated without aeration. Aeration of slurries decreased efficacy of the resulting extracts compared to non-aerated controls. When aerated slurries were allowed to incubate without further aeration, extracts regained efficacy, becoming not significantly different from non-aerated controls. There was no difference in efficacy between filtered (0.1μm) and untreated extracts. Passage of filtrate through microconcentrators with molecular cut-off limits ranging from 100 to 3 kDa did not diminish activity of the extracts. Autoclaved extracts were less effective than mutreated extracts but retained most of their efficacy. Extracts produced from sterile SMS were virtually ineffective compared with those from non-sterile SMS. When small volumes of slurry from raw SMS were added to slurries of sterile SMS and incubated for an additional period, efficacy of the inoculated SMS was enhanced compared to uninoculated controls. We conclude that a major inhibitory principle of the SMS extract is a low molecular weight, heat-stable, non-protein metabolite produced by anaerobic microorganisms in the compost.

Phylogenetic placement ofAthelia bombacina, Aureobasidium pullulans, andColletotrichum gloeosporioides inferred from sequence comparisons of small-subunit ribosomal RNAs

Complete nucleotide sequences of the small-subunit ribosomal RNA (16 S-like rRNA) coding regions from Athelia bombacina (Basidiomycotina, Corticiaceae) (1809 nucleotides), Aureobasidium pullulans (Deuteromycotina, Dematiaceae (1800 nucleotides), and Colletotrichum gloeosporioides (Deuteromycotina, Melanconiaceae) (1795 nucleotides) were determined. The coding regions were amplified from total genomic DNA preparations in polymerase chain reactions using oligodeoxynucleotide primers complementary to the 5′ and 3′ termini of the rRNA cistron. Distance matrix methods based on nucleotide sequence comparisons for the small-subunit rRNA were used to infer a phylogeny where the basidiomycete A. bombacina diverged after the separation of chytrids from the ascomycetes. A. pullulans is affiliated with the ascomycetes rather than the basidiomycetes, which implies that if a teleomorph is identified, then it will probably be an ascomycete. A. pullulans is related more closely to Neurospora crassa and Podospora anserina than it is to the ascosporogenous yeast, Saccharomyces cerevisiae , or to the asporogenous yeast Candida albicans. C. gloeosporioides is more closely related to the Neurospora/Podospora cluster than it is to either Aureobasidium or the Candida/Saccharomyces lineage.

Fungi, leaves, and the theory of island biogeography.

Species dynamics of fungi (filamentous fungi and yeasts) on apple leaves were studied within the framework of the theory of island biogeography by following “immigration” and “extinction” patterns on individual apple leaf “islands” over time. Total fungi were censused on unmanipulated leaves collected throughout two seasons; filamentous fungi only were monitored additionally for several weeks in one season on newly created, axenic, model (seedling) islands introduced to the orchard, and on surface-sterilized, preexisting leaves. Analyses based on both the natural and the surface-sterilized systems showed that an equilibrium in species number was reached and turnover in species composition occurred in both. Immigration and extinction events were strongly related to number of species present on each island. The balance between immigration and extinction implies that species number on leaves and “real” (oceanic) islands is determined by a common mechanism, and emphasizes the need to regard leaf microbial communities as dynamic.

The Synergistic Effects of Stochasticity and Dispersal on Population Densities

Using laboratory experiments, simulation models, and analytical techniques, we examined the impact of dispersal on the mean densities of patchily distributed populations. Even when dispersal leads to no net additions or removals of individuals from a population, it may nonetheless increase mean population densities if the net immigration rate is positive when populations are growing and negative when they are declining. As a model system for exploring this phenomenon, we used the yeastlike fungus Aureobasidium pullulans. In a laboratory experiment, we showed that dispersal can both ensure persistence and increase mean population densities even when dispersal among populations causes no direct addition or loss of fungal cells. From the laboratory data, we constructed a plausible model of A. pullulans dynamics among apple leaves within an orchard. This simulation model demonstrated that the effect of dispersal on mean densities is enhanced by three factors: weak density dependence of the dynamics within populations, high environmental variability affecting population growth rates, and lack of synchrony among the fluctuations of populations. Using an analytical model, we showed that the underlying mechanisms for this phenomenon are general, suggesting that a large effect of dispersal on mean population densities is possible in many natural systems.

Positional variation in phylloplane microbial populations within an apple tree canopy

Variation in density of epiphytic yeasts, filamentous fungi, and bacteria on apple leaves collected from eight trees at nine dates for two seasons was determined with respect to three positional factors: height, compass direction from the center of the tree, and lateral proximity to the canopy periphery. Univariate analyses of variance were performed on each of the microbial classes for each date according to a model that excluded tree effect but accounted for the positional factors with interactions. The assumption of no tree effect was explored by residual analysis and examination of the seasonal pattern of microbial densities for each tree. No persuasive evidence was obtained to invalidate this assumption. For filamentous fungi and yeasts, height and lateral position were the most significant factors withp<0.05 for yeasts at several periods. The two factors appeared to be of equal importance. Trends were less clear for bacteria, but all three positional factors and some two-way interactions seemed of some importance. For filamentous fungi and bacteria, frequently no factors were significant at a level of 0.10, but at almost all sampling dates certain positional factors and interactions were significant at a level of 0.25. Inspection of partial correlation coefficients indicated no apparent linear association between densities of most pairs of microbial classes. Implications of these results for experimental design and for the microbial ecology of the phylloplane community are discussed.

Fungal immigration dynamics and community development on apple leaves

Fungal immigration dynamics and community development were followed over time on sets of surface-disinfested apple leaves in the field. Immigration was defined as the arrival of viable propagules on the leaf surface. In three separate experiments (May, June, July), total numbers of fungal immigrants, numbers of filamentous fungal immigrants, and numbers of yeast immigrants per leaf were estimated for successive 12-hour immigration periods. Communities developing over 2–14 immigration periods (1–7 days) were compared with the corresponding estimates of cumulative immigration. There were significant differences among both experiments and immigration periods within each experiment in mean numbers of immigrants per leaf. Leaf area was often significantly correlated with numbers of immigrants. Developing communities supported progressively fewer individuals than the corresponding sums of immigrants, suggesting that losses due to emigration and/or death play a critical role in shaping these communities.

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) ofAureobasidium pullulans

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.