Identification and distribution of Armillaria species associated with an oak decline event in the Arkansas Ozarks

Forests in the Ozark Mountains of northern Arkansas recently experienced a widespread oak decline event. Armillaria, a root rot fungus, has been associated with other oak decline events and may have been an important contributing factor to tree mortality in this event. Although Armillaria has been identified from the Ozark Mountains in Missouri, it has never been investigated in the Arkansas Ozarks. Molecular diagnostic techniques were used in this study to identify species of Armillaria present on roots removed from dead trees of two common oak species, northern red oak, Quercus rubra L., and white oak, Q. alba L., from three geographic areas and on three topographic positions – ridges, south‐ and west‐facing benches. Armillaria(A. mellea, A. gallica or A. tabescens) was identified from 31% of root samples taken from 102 trees in seven of nine sample plots. Armillaria mellea, occurred most often (20 samples, both oak species on seven plots) followed by A. gallica (10 samples, northern red oak only on four plots), and A. tabescens occurred twice (on northern red oak in a single plot). Thus, all three Armillaria species occurred on northern red oaks while A. mellea was the only species recovered from white oaks. Results varied by topographic position with samples from tree roots on ridges having the fewest positive identifications, one of 29. West‐facing benches had the highest positive samples with 20 of 41 testing positive and trees on south‐facing benches were intermediate with 11 of 32 samples from infected trees. This study documents the occurrence of three species of Armillaria in the Arkansas Ozarks and their association with oak mortality resulting from an oak decline event coupled with a red oak borer, Enaphalodes rufulus, outbreak. Further, it documents some potential variation in host/pathogen combinations and forest site conditions.     

Ecology and distribution of Armillaria species in Norway

The occurence of Armillaria species was assessed in Norway, enabling the northern‐most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand.     

Armillaria species distribution on symptomatic hosts in northern hardwood and mixed oak forests in western Massachusetts

Armillaria spp. are some of the most important forest pathogens in mixed hardwood forests of southern New England, yet their role as prominent disturbance agents is still not fully appreciated. We investigated the distribution of Armillaria species across eight separate stands of northern hardwood and mixed oak forests in western Massachusetts. We were specifically interested in the Armillaria species distribution from live, symptomatic hosts and not in determining overall incidence in the forest. From 32 plots (16 within each forest type), 320 isolates were collected. Armillaria was routinely encountered causing disease of live trees. In total, 89% (286/320) of all isolations came from live hosts exhibiting symptoms of root and butt rot. Overall, A. gallica was the dominant species in each forest type, making up 88/160 (55%) isolates from northern hardwood and 153/160 (96%) of all isolations from mixed oak stands. However, northern hardwood forests showed much greater species diversity, as A. calvescens, A. gemina, A. ostoyae, and A. sinapina were all found. At one site, a northern hardwood forest surrounding a high elevation spruce-fir forest, A. ostoyae was the most abundant species encountered. All five Armillaria species were found causing disease of live hosts, including A. gemina, a species considered by some as weakly virulent. Armillaria gallica was found on 22/23 tree species’ sampled, and was found most often causing butt rot.     

Phylogeography and host range of Armillaria gallica in riparian forests of the northern Great Plains,USA

Root disease pathogens, including Armillaria, are a leading cause of growth loss and tree mortality in forest ecosystems of North America. Armillaria spp. have a wide host range and can cause significant reductions in tree growth that may lead to mortality. DNA sequence comparisons and phylogenetic studies have allowed a better understanding of Armillaria spp. taxonomic diversity. Genetic sequencing has facilitated the mapping of species distributions and host associations, providing insights into Armillaria ecology. These studies can help to inform forest management and are essential in the development of disease risk maps, leading to more effective management strategies for Armillaria root disease. Armillaria surveys were conducted on publicly owned lands in North Dakota, South Dakota, and Nebraska, U.S.A. Surveyed stands consisted of riparian forests ≥0.4 hectares in area. Armillaria was found at 78 of 101 sites. A total of 57 Armillaria isolates—associated with 12 host tree species—were used for DNA sequencing of the translation elongation factor‐1 alpha (tef1) gene. Armillaria gallica was the only species identified within the study sites. Results suggest that A. gallica is a common root pathogen of hardwood trees in riparian forests of the northern Great Plains with a wider host range and geographic distribution than previously recognized.     

Ecology of Armillaria species in managed forests and plantations in Serbia

The distribution of Armillaria species was investigated in Serbian forest ecosystems, in relation to the main host species attacked, forest‐types, geography and altitude. In total, 388 isolates were identified from 36 host species in 47 sites. Armillaria gallica was the most commonly observed species with the widest distribution and with an altitudinal range of 70–1450 m, it was the dominating Armillaria species in lowland alluvial forests and in Quercus and Fagus forests at higher elevations. Armillaria mellea occurred in Quercus spp. – dominated forests in the north and central regions at 70–1050 m. Sixty‐eight per cent of the A. mellea isolates were collected from living hosts, most commonly in declining conifer plantations. Armillaria ostoyae was distributed in the cooler coniferous forest types and plantations in the Dinaric Alps in the south of Serbia, at 850–1820 m. Armillaria cepistipes was found in the eastern and southern hilly and mountainous regions of the country, at 600–1900 m. Most isolates were obtained from conifers and rhizomorphs in the soil around decaying stumps. Armillaria tabescens was found only on dead oak material in the northern and eastern regions of the country at altitudes lower than 600 m.     

Development of species‐specific PCR primers on rDNA for the identification of European Armillaria species

Attempts to design species‐specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa.     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

Phylogenetic analysis of Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA

To determine the phylogenetic positions of two new species, Armillaria jezoensis and Armillaria singula, and one new subspecies, Armillaria mellea suhsp. nipponica, the nucleotide sequences of the intergenic spacers (IGS) of their ribosomal DNA were investigated, and compared with those of tour other Armillaria species from Japan, and those of nine Armillaria species from Europe and North America. We conclude that Armillaria jezoensis, and Armillaria singula belong to the Armillaria gallica cluster as Armillaria cepistipes, Armillaria gallica and Armillaria sinapina from Japan. Two isolates of Armillaria ostoyae from Japan were placed within the Armillaria ostoyae cluster. Armillaria mellea subsp. nipponica had an IGS sequence as long as the IGS of Armillaria mellea from Europe and North America. However, the IGS sequences of Armillaria mellea subsp. nipponica, whose basidium base lacks a clamp connection could not be satisfactorily aligned with the IGS sequences of other species possessing this morphological feature.     

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.     

Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Ecology of Armillaria species on silver fir (Abies alba) in the Spanish Pyrenees

? We describe the distribution and the ecology of three Armillaria species observed in silver fir (Abies alba) forests of the Pyrenees.

? We surveyed the presence and abundance of Armillaria above and belowground in 29 stands. Isolates were identified by the PCR-RFLP pattern of the IGS-1 region of their ribosomal DNA. We measured several ecological and management parameters of each stand in order to describe Armillaria infected sites.

? Armillaria cepistipes was the most abundant of three species observed. Armillaria gallica was dominant in soils with a higher pH and at lower elevations. Armillaria ostoyae seemed to be more frequent in stands where A. alba recently increased its dominance relative to other forest tree species. Thinning activities correlated with an increased abundance of Armillaria belowground. In 83% of the stands the same Armillaria species was observed above and belowground.

? It seems that in a conifer forest, A. cepistipes can be more frequent than A. ostoyae, a virulent conifer pathogen. Since logging is related to a higher abundance of Armillaria in the soil, the particular Armillaria species present in a given stand could be considered an additional site factor when making management decisions.

     

Incidence and phylogenetic analyses of Armillaria spp. associated with root disease in peach orchards in the State of Mexico,Mexico

Incidence of peach [Prunus persica (L.) Batsch] tree mortality attributed to Armillaria root disease was assessed from 2009 to 2011 in 15 orchards in the State of Mexico, Mexico. Incidence increased gradually every year of assessment, reaching average values of 9.7, 15.3 and 20.3% tree mortality and 23.2, 24.7 and 28.3% disease‐impacted area of the orchards during 2009, 2010 and 2011, respectively. The cultivars ‘Nemaguard’ and ‘Criollo of La Goleta’, a local rootstock used in the region, were both susceptible to the disease. To identify species of Armillaria isolated from infected peach trees, two nuclear rDNA regions (partial 5.8S‐ITS2‐LSU D‐domains and partial 3′ LSU‐IGS1) and the translation elongation factor‐1α (tef‐1α) gene were sequenced and compared with sequences of known Armillaria species. DNA sequence analysis from 49 Armillaria isolates revealed that five isolates (10.2%) were Armillaria mellea and eight isolates (16.3%) were Armillaria gallica. DNA sequences from the remaining 36 isolates (73.5%) showed no close similarity to Armillaria sequences in GenBank, and apparently represent an undescribed Armillaria species. This undescribed species was the most widely distributed in the region of study. Separate phylogenetic analyses of the LSU region (D1–D3 domains concatenated with the partial 3′ end) and the tef‐1α region show that the undescribed species is quite distinct from other Armillaria spp. reported in North America.     

Distribution,ecology and host range of Armillaria species in Albania

Species of Armillaria were identified from 645 isolates obtained in a nation‐wide survey in Albania. The material was collected from ca. 250 permanent plots, established for monitoring forest health, and from forests and orchards attacked by Armillaria. Armillaria mellea s.s. occurred on several coniferous and broadleaved trees in most areas examined, although it was absent above 1100–1200 m in northern Albania. This species damaged Abies and Quercus spp. and, to a lesser extent, other forest trees. Armillaria mellea was also commonly recorded causing damage in orchards and vineyards. Armillaria gallica was a common saprophyte or weak pathogen in coniferous and deciduous forests at altitudes from 600 to 1600 m, and less commonly on oaks at lower altitudes. Armillaria ostoyae was rare in central and southern Albania, but common in northern Albania, causing significant damage to pine and other conifers, mostly at altitudes from 600 to 1800 m. Armillaria cepistipes was recorded at altitudes from 800 to 1800 m as a saprophyte or weak pathogen on conifers and deciduous trees, mostly in beech and silver fir forests. Armillaria tabescens was found in oak forests at altitudes from sea level to 900 m. In orchards, A. tabescens occasionally attacked almond and pear trees. Armillaria borealis was found in a few locations in northern Albania, at altitudes from 800 to 1800 m.     

Initial characterization of an unidentified Armillaria isolate from Serbia using LSU‐IGS1 and TEF‐1‐α genes

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer‐1 regions of rDNA (LSU‐IGS1) and the translation elongation factor‐1 alpha gene (tef‐1α) genes, and phylogenetic analyses. Despite previously obtained LSU‐IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU‐IGS1 and tef‐1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU‐IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well‐supported clade (99%). Based on tef‐1α, the Serbian isolate appeared as a separate, well‐supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high‐level sequence heterogeneity, represented by multiple two‐nucleotide codes, within tef‐1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia.     

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.     

Geographical distribution of the Armillaria species in The Netherlands in relation to soil type and hosts

The geographical distribution of the annulate Armillaria species was studied in The Netherlands during the period 1983–1992. Armillaria gallica (incl. A. cepistipes), A. mellea and A. ostoyae appear to be widespread, the first two species being rather common on broad-leaved hosts growing on clay and loess soils, and the third species common on both broad-leaved and coniferous hosts on acid sandy soils. The distribution of the Armillaria species encountered was primarily determined by soil type. From a silvicultural point of view, A. ostoyae is the most important species, being pathogenic and occurring most frequently on sandy soils, the soil type predominantly used for forestry in The Netherlands.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.     

Oak decline in the Boston Mountains,Arkansas, USA: Spatial and temporal patterns under two fire regimes

A spatially explicit forest succession and disturbance model is used to delineate the extent and dispersion of oak decline under two fire regimes over a 150-year period. The objectives of this study are to delineate potential current and future oak decline areas using species composition and age structure data in combination with ecological land types, and to investigate how relatively frequent simulated fires and fire suppression affect the dynamics of oak decline. We parameterized LANDIS, a spatially explicit forest succession and disturbance model, for areas in the Boston Mountains of Arkansas, USA. Land type distribution and initial species/age class were parameterized into LANDIS using existing forest data. Tree species were parameterized as five functional groups including white oak (Quercus alba L., Quercus stellata Wangenh., Quercus muehlenbergii Engelm.), red oak (Qurecus rubra L., Quercus marilandica Muenchh., Quercus falcata Michx., Quercus coccinea Muenchh.), black oak (Quercus velutina Lam.), shortleaf pine (Pinus echinata Mill), and maple (Acer rubrum L., Acer saccharum Marsh.) groups. Two fire regimes were also parameterized: current fire regime with a fire return interval of 300 years and a historic fire regime with an overall average fire return interval of 50 years. The 150-year simulation suggests that white oak and shortleaf pine abundance would increase under the historic fire regime and that the red oak group abundance increases under the current fire regime. The black oak group also shows a strong increasing trend under the current fire regime, and only the maple group remains relatively unchanged under both scenarios. At present, 45% of the sites in the study area are classified as potential oak decline sites (sites where red and black oak are >70 years old). After 150 simulation years, 30% of the sites are classified as potential oak decline sites under the current fire regime whereas 20% of the sites are potential oak decline sites under the historic fire regime. This analysis delineates potential oak decline sites and establishes risk ratings for these areas. This is a further step toward precision management and planning.     

Effects of metals and pH on in vitro growth of Armillaria ostoyae and other root and butt rot fungi of red spruce

Armillaria ostoyae, Perenniporia subacida, Resinicium bicolor and Scytinostroma galactinum, root and butt rot fungi found on red spruce, Picea rubens, were tested, in vitro, for their sensitivity to metals typically found in high elevation forest soils where red spruce grows. Rhizomorph production by A. ostoyae from woody inocula in soils from red spruce stands at three elevations at each of five mountainous sites in the eastern United States was inhibited completely in the mineral soil from all elevations at all sites, and was also reduced significantly in the organic horizon from the upper two elevations at three of the sites. Inhibition was correlated with concentrations of metal ions in the soil. Growth of rhizomorphs into an agar medium containing lead and other heavy metals was inhibited for isolates of A. ostoyae from red spruce, but not for an isolate of Armillaria gallica from sugar maple; aluminium inhibited rhizomorph growth of isolates of both species. Mycelial growth of all four root and butt rot fungi was inhibited by lead, aluminium and other heavy metals depending on the solubility and concentration of metal and pH of the medium; growth inhibition was usually greater at an initial pH of 3.5 than at pH 4.5. Metal ions inhibited radial growth of Armillaria species more than that of the other three fungi. Rhizomorph growth of Armillaria was inhibited more than radial growth. Because local spread of A. ostoyae occurs frequently by means of rhizomorph growth between near roots, increases in lead, aluminium and other metals in the forest floor may contribute to this fungus' scarcity in high elevation soils and reduced incidence of infection at these sites in the eastern United States.