Johann N. Bruhn

Contrasting patterns of genetic diversity and population structure of Armillaria mellea sensu stricto in the eastern and western United States.

ABSTRACT Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (theta(ST) = -0.002, P = 0.5 ) or western subpopulations (theta(ST) = 0.004, P = 0.3 ) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States.

Temporal dynamics of ectomycorrhizal community composition on root systems of oak seedlings infected with Burgundy truffle.

Truffles, the hypogeous ascocarps of the ectomycorrhizal genus Tuber, are currently cultivated in orchards to partially offset declining wild production in Europe. Truffle cultivation begins with inoculation of seedling root systems in the greenhouse. Once the mycorrhizal relationship is well established, colonized seedlings are transplanted to a suitable field site. In the USA, little is known about the effect of indigenous ectomycorrhizal species on colonization of host trees by the European Burgundy truffle fungus (T. aestivum). Here we identify the ectomycorrhizal community composition on inoculated seedlings grown in the greenhouse in three types of potting media. We subsequently monitored the ectomycorrhizal community composition for two years after seedlings were transplanted into a field site that had been prepared for truffle cultivation by lime applications. We found that the infection rates of contaminant ectomycorrhizal species present in the greenhouse declined to low levels in this field site. We also found that after two years in the field, both T. aestivum colonization levels and indigenous ectomycorrhizal species richness and abundance increased, indicating that indigenous species, in the short term, did not displace T. aestivum.

Fractal geometry of diffuse mycelia and rhizomorphs of Armillaria species

Fractal geometry was used to quantify the variation in branching patterns of sibling basidiospore-derived diffuse mycelia of Armillaria gallica and rhizomorph systems of A. gallica and A. ostoyae in laboratory culture. The fractal dimension (D) of 11 A. gallica diffuse mycelia ranged from 1·05 to 1·20 at 74 h after basidiospore germination, and from 1·43 to 1·57 at 116 h after germination. The coefficient of variation (CV) for the regression coefficients of D on time was 14·8%, whereas the CV for D of all 11 mycelia at the fourth measured time step was only 2·9%, indicating significant convergence towards a given branching pattern for a particular set of environmental conditions. The rhizomorph systems of diploid A. gallica thalli branched more profusely than those of diploid A. ostoyae . D values among replicate rhizomorph systems for strains representing three A. gallica genets were similar, whereas rhizomorph systems of eight of the 12 A. ostoyae thalli, also representing three genets, either failed to develop or did not branch sufficiently to permit calculation of D. D values of rhizomorph systems were generally temporally invariate for both species. The CV of D for replicate rhizomorph systems at the fourth measured time step was between 1·6% and 3·9% for A. gallica genets and 2·4% for the A. ostoyae genet; D was significantly lower for the A. ostoyae genet than for the three A. gallica genets. A. gallica is only weakly pathogenic and can colonize a taxonomically broad range of food bases saprotrophically. A. ostoyae is more pathogenic and colonizes a narrower range of substrata. The profuse rhizomorph branching pattern of A. gallica is consistent with a foraging strategy in which acceptable food bases may be encountered at any distance, and which favours broad and divisive distribution of potential inoculum. The sparse branching pattern of A. ostoyae rhizomorph systems is less divisive, consistent with the conservation of nutrients necessary to maintain rhizomorph inoculum potential more efficiently with increasing distance from a foodbase.

Foraging behaviour of Armillaria rhizomorph systems

The foraging behaviour of Armillaria rhizomorph systems is poorly understood owing to their cryptic position within the soil. We investigated foraging in a homogeneous environment (i.e. agar), finding that rhizomorph systems of the more parasitic species, A. mellea, A. ostoyae, and A. tabescens, lacked melanin and the approximately cylindrical cord-like form observed in the field. In contrast, rhizomorph systems of the more saprotrophic species, A. calvescens, A. gallica, and A. sinapina, developed radially resembling those in the field. For the three saprotrophic Armillaria species, the number of rhizomorph tips, total rhizomorph length and total rhizomorph surface area were significantly positively correlated with increasing rhizomorph system diameter and elapsed time in two developmental tests. However, the fractal dimension (D), used as a measure of foraging intensity, was temporally invariable, suggesting that one component of foraging behaviour is innate. In a heterogeneous environment (i.e. sand) and in the absence of a potential nutrient source, we observed that rhizomorph systems of A. gallica most often developed asymmetrically. While rhizomorph foraging was unresponsive to the lateral placement of an uncolonised stem segment, we were able to demonstrate directional growth toward an uncolonised Quercus velutina stem segment placed above or below the colonised source stem segment. When neighboring rhizomorph systems were conspecific genets of A. gallica, we observed that the growth of one rhizomorph system was directed toward zones unoccupied by its neighbour. However, the foraging intensity of the neighbouring genets, as measured by fractal dimension (D), was unaffected by the proximity of a neighbour. When neighbouring rhizomorph systems represented different species (A. gallica and A. mellea), A. gallica rhizomorph systems produced more total length and more foraging tips but concentrated their rhizomorph production away from the neighbouring A. mellea genet. In contrast, A. mellea rhizomorph systems produced significantly more foraging tips per unit length, both overall and in the zone of confrontation with the neighbouring A. gallica genet. Our observations are consistent with field observations of territoriality among Armillaria genets, and provide evidence that rhizomorph systems of more parasitic Armillaria spp. are able to compete effectively with the larger rhizomorph systems of more saprotrophic Armillaria species.

The effects of moisture and oxygen availability on rhizomorph generation by Armillaria tabescens in comparison with A. gallica and A. mellea

Compared with other Armillaria species, natural melanized rhizomorphs of A. tabescens are rarely observed. Growing in autoclaved Vitis stem segments, A. tabescens isolates from the Ozark Mountains in the central USA formed fully melanized rhizomorphs, thinner and shorter than those observed for other Armillaria species under field conditions, and only under conditions of both high oxygen availability, ζ2 ug cm −2 min −1 and moisture near saturation. Conducive conditions were used to compare the rhizomorph generation capacities of A. tabescens, A. gallica , and A. mellea , which have overlapping host and geographic ranges in central North America. While the rhizomorphs of A. tabescens were significantly shorter than those of the other two species, A. gallica and A. tabescens produced similar numbers of rhizomorph initials. Finally, we demonstrated the ability of melanized A. tabescens rhizomorphs to span woody food bases and thereby establish viable infections by penetration of intact bark of Vitis stem segments. We hypothesize that A. tabescens rhizomorphs form under conditions of periodic saturation which promote rapid water movement through naturally occurring lacunae in the soil. Thus, the role of A. tabescens rhizomorphs in the spread of the fungus should be re-evaluated.

The fractal dimension of young colonies of Macrophomina phaseolina produced from microsclerotia

Concepts of fractal geometry have been used to describe the branching patterns of a variety of or? ganisms ranging from microorganisms to trees. The fractal dimension is a measure of branching density. The fractal dimension was used to investigate the hy? phal branching patterns of young colonies of the soil- borne phytopathogen Macrophomina phaseolina. The temporal development of branching density was ex? amined by assembling composite photomicrographs of 17 entire thalli (representing three test isolates) at 2-hour time intervals. These photomicrographic im? ages were digitized, and the fractal dimension was de? termined using a box-count algorithm. Analysis of 90 images revealed patterns of thallus development which were remarkably radially asymmetric. The fractal di? mension ranged from 1.21 to 1.84, indicating a wide range of branching density. The temporal develop? ment of branching density was investigated by regres? sion of fractal dimension on time. Branching density increased with time at rates characteristic of each iso? late. Further, the magnitude of fractal dimension to? gether with the rate of temporal increase of the fractal dimension permitted clear distinction among the three test isolates of M. phaseolina. These results suggest that quantitative description of branching density using the fractal dimension might become a novel supplement to the morphological criteria traditionally used in fun? gal taxonomy.

Colonization of Pedunculate oak by the Burgundy truffle fungus is greater with natural than with pelletized lime

European black truffles can be profitable agroforestry crops outside their native ranges. Truffle fungi grow symbiotically as ectomycorrhizae on the roots of host trees, notably hazels and oaks. Conditions in the central USA appear conducive to cultivation of the Burgundy truffle (Tuber aestivum Vitt. syn. T. uncinatum Chatin), but research is needed to determine effects of management practices on truffle establishment and fruiting. In a greenhouse study we tested the effect of lime type, inoculation technique, and two truffle sources on Pedunculate oak (Quercus robur L.) growth and mycorrhizal colonization. We found that the type of lime used to raise potting mix pH can differentially affect the growth rate of root systems inoculated with different selections of Burgundy truffle inoculum. Seedlings inoculated with one selection of the truffle and grown in potting mixes amended with natural crushed dolomitic limestone developed larger root systems with more truffle mycorrhizae compared with potting mix amended with high-calcium pelletized quick-release lime. Seedlings inoculated with a second truffle selection were not affected by lime source and developed root systems as large as those developed with the first truffle source grown with natural lime. Supplemental root dip inoculation did not improve levels of colonization beyond those accomplished by potting mix infestation with truffle ascospores. Use of a hygroscopic polymer to maintain ascospore suspension in the inoculum slurry used to infest the potting mix had no effect on root system development or mycorrhiza formation.

Evidence of natural hybridization among homothallic members of the basidiomycete Armillaria mellea sensu stricto

Populations of Armillaria mellea (Basidiomycota, Agaricales) across much of its range are heterothallic; homothallic populations occur only in Africa (A. mellea ssp. africana), China (China Biological Species CBS G), and Japan (A. mellea ssp. nipponica). Monosporous isolates of heterothallic A. mellea are haploid and their mating behaviour is consistent with the requirement of two different alleles at two mating-type loci (tetrapolar mating system) to create a diploid individual. In contrast, monosporous isolates of homothallic A. mellea are putatively diploid; they bypass the haploid phase by undergoing karyogamy in the basidium (a unique type of secondary homothallism/pseudohomothallism). In order to determine the genetic origin of this homothallism, we analyzed genetic variation of 47 heterothallic isolates from China, Europe, and North America, and 14 homothallic isolates from Africa, China, and Japan. Gene trees and mutational networks were constructed for partial mitochondrial gene ATP synthase subunit 6 (ATP6) and for the following nuclear genes: actin (ACTIN), elongation factor subunit 1-alpha (EFA), glyceraldehyde 3-phosphate dehydrogenase (GPD), and the RNA polymerase subunit II (RPB2). Homothallic isolates from Africa and Japan shared a common mitochondrial ATP6 haplotype with homothallic isolates from China, and are likely introductions. Homothallic isolates from China that shared a common mitochondrial haplotype with all European isolates did not share European nuclear haplotypes, as revealed by median-joining networks, but instead clustered with haplotypes from China or were intermediate between those of China and Europe. Such mitochondrial-nuclear discordance in homothallic isolates from China is indicative of hybridization between lineages originating from China and Europe.

Opportunistically Pathogenic Root Rot Fungi: Armillaria Species

Numerous gap-model studies have attempted to predict responses of tree populations to climate change. Although these models incorporate various disturbance factors, the multifaceted influences of structural-root disease activity on gap dynamics have not been included [e.g., (2000)]. The fact that apparently similar models yield very different projections for the same forest [e.g., (1997)] suggests that most of these models are incomplete and/or may be inappropriately formulated (Bachelet and Neilson 2000). The existence of belowground root gaps corresponding to aboveground canopy gaps and their influence on plant competition and establishment have also received little attention [see (1997)]. Yet, Armillaria root disease and other structural-root diseases have been associated with gap dynamics [e.g., (1988); (1993); (1993), 1995); (1997); (2000)].

Forest farming of wine-cap Stropharia mushrooms

Cultivation of the wine-cap Stropharia mushroom (Stropharia rugoso-annulata Farlow ex. Murrill) has potential as a forest farming practice in agroforestry. Two outdoor cultivation experiments were established in central Missouri during 2006. We examined Stropharia production in response to two substrate/casing systems: (1) an uncased mixture of lightly composted poplar wood chips and soil, without casing; and (2) leached and partially fermented wheat straw cased with the same chip/soil mixture. We also examined the effect of soil contact (presence/absence of water-permeable fabric), and inoculation season (mid versus late summer) over two field seasons. Mushroom production during both seasons (2006 and 2007) was greater from the straw/chips system than from the wood chip system. The use of ground-cover fabric did not enhance mushroom production. Further research is needed to determine the most productive dimensions of cultivation beds, to evaluate alternative casing methods, and to explore the possibility of cultivation bed renewal with fresh substrate.