Vegetation and Soil Environment Influence the Spatial Distribution of Root-Associated Fungi in a Mature Beech-Maple Forest

Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.In temperate forests, most trees form relationships with ectomycorrhizal (ECM) fungi, and the diversity of this fungal group alone can approach 100 species within a forest stand (17, 20, 60). The ECM mutualism may be necessary for the success of some native plant species, as approximately 90% of roots of some tree species are colonized by ECM fungi (65). Nevertheless, we still know surprisingly little about what controls the community structure and distribution of root-associated fungi in forest systems (44, 46). The occurrence of root-associated fungi may broadly reflect soil environmental conditions and the presence of preferred plant hosts (28, 61), but how these factors interact to influence the diversity, distribution, and community structure of these fungi within forest habitat patches at a local scale is uncertain.The distribution of root-associated fungi may be primarily a species response to local soil environmental conditions. For example, both the quality (i.e., nutrient content) and the quantity of soil organic matter are known to influence the diversity of ECM communities (18, 20, 32). ECM fungi also vary in drought tolerance (14, 36), resistance to fire (61, 65), and tolerance to soil acidity (19) and temperature (56). Changes in soil chemistry, especially as they relate to pH and the availability of nitrogen (N) and phosphorous (P), might favor selection of fungi most capable of tolerating environmental extremes (2, 28, 29).Plant distribution and identity may, however, play the strongest role in structuring the below-ground diversity of root-associated fungi. Many ECM fungi can colonize a wide range of plant species, and plant species can be host to a large number of ECM fungi (63), especially those in the families Russulaceae and Thelephoraceae (34, 35, 62). Moreover, some ECM fungi are also specific to certain tree species (e.g., Suillus and Rhizopogon species are specific to species in the family Pinaceae [38, 39]). At the local scale, fungal distribution and richness might be influenced by differences in root growth and architecture (30, 42), by the distance to the bole of the tree (11, 42, 49), or by the presence of neighboring trees (29, 64). Temporal changes in ECM communities could be associated with seasonal changes in plant physiology and phenology (3, 8, 17).An often overlooked factor influencing root-associated fungi of tree roots is the occurrence of herbaceous plant species within forest stands. Many species of parasitic, achlorophyllous angiosperms obtain carbon (C) from ECM fungi that colonize tree roots (43), and some autotrophic plants could also obtain C from ECM fungi during certain times of the year (58). Herbaceous plants also influence the cycling of nutrients, including N, P, and K (potassium) (31, 50), within forests, which could affect the distribution of root-associated fungi. Herbaceous plants can also produce secondary compounds that inhibit colonization of tree roots (68).In this study, we examine the effect of soil environment and plant distribution on root-associated fungi of tree roots in a mature beech-maple forest at two points in the growing season. We predict that plant distribution, both the distribution of host trees and that of herbaceous plants, influences fungi associated with tree roots in terms of both community structure and diversity. Molecular typing protocols, including a site-specific database of fungal sequences and fingerprints, were used to identify fungi on tree roots (i.e., beech or maple trees) to the species level.     

Impact of Endochitinase-Transformed White Spruce on Soil Fungal Biomass and Ectendomycorrhizal Symbiosis

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.White spruce [Picea glauca (Moench) Voss] is a tree species with an extensive distribution in boreal and subboreal forests and with significant ecological roles (37, 38). It is also an important commercial species for production of pulpwood and construction-grade lumber. However, in nurseries and plantations, white spruce is sensitive to multiple fungal diseases (23, 29, 42, 62, 76). Climate change scenarios suggest that diseases could result in increased mortality in conifer forests (22, 48). Genetic engineering offers a potential means to mitigate biotic and abiotic stresses.During the last 2 decades, chitinase genes isolated from plants, fungi, or bacteria have been studied and used to transform crops or trees in order to increase their resistance to plant-pathogenic fungi. One potential goal is improving white spruce tolerance to fungal infection through insertion of a chitinase gene. Chitin is a biopolymer of β-(1-4)-linked molecules of N-acetylglucosamine (NAG), a derivative of glucose, and is the primary constituent of the fungal cell wall and arthropod exoskeleton (3, 51). Chitinases are plant defense pathogenesis-related proteins (6, 11) that break down the chitin chain either by cleavage of internal glycoside bonds (endochitinases), by hydrolysis of the nonreducing end of the chitin chain (exochitinases), or by hydrolysis of NAG oligomers and trimers into NAG monomers (chitobiases). Endo- and exochitinase genes have been well characterized using sugar beet (Beta vulgaris) (44) and the filamentous fungal genus Trichoderma (14, 24, 69). Chitinolytic genes have been inserted into the genomes of cultivated plants and trees in an attempt to boost plant chitinase activity. Among the different genes involved in the production of chitinolytic enzymes, the ech42 endochitinase gene from Trichoderma harzianum has been inserted into plant genomes to enhance their resistance against phytopathogenic fungi. In McIntosh apple cultivars transformed with the ech42 gene there was limited attack by the apple scab fungus Venturia inaequalis (5). Transgenic black spruce (Picea mariana) expressing the ech42 gene was more resistant to the root rot pathogen Cylindrocladium floridanum (45).However, field deployment of crops and trees genetically transformed to improve nonspecific resistance against phytopathogenic fungi has raised concerns about the impact on nontarget fungi, including potentially beneficial symbionts. This is particularly worrisome when nonspecific constitutive promoters control expression of the resistance gene and the gene is expressed in all tissues from roots to leaves. As a consequence, the natural colonization of such transformed plants by endophytic or mycorrhizal fungi can be altered.Mycorrhizal fungi play a key role in plant nutrition (55) by mobilizing and transferring nutrients to the host through an intimate and highly organized association with plant roots (52, 63). Furthermore, their involvement in soil nutrient recycling (56) makes mycorrhizal symbiosis a major ecological process that is important for the health of soil and forest ecosystems. Crops, fruits, and forest trees exhibit mycorrhizal colonization by arbuscular mycorrhizae, ectomycorrhizae, and ectendomycorrhizae (EEM). While numerous studies have addressed the impact of transgenic plants on arbuscular mycorrhizae (10, 26, 64, 68, 72, 73) and ectomycorrhizae (32, 43, 50, 60), no previous study focused on EEM.Ectendomycorrhizal fungi can be distinguished from ectomycorrhizae by the presence of a thin or fragmented mantle and intracellular penetration into root cortical cells. All EEM fungi identified so far belong to the Ascomycetes, and these fungi are represented by several genera of Helotiales and Pezizales (77). EEM fungi are prevalent in conifer and deciduous tree nurseries (27, 39, 40, 70) and are also very common on seedling root tips at disturbed sites (15, 16, 19). The prevalence of EEM fungi on seedling roots, from which the genus Wilcoxina is frequently recovered (16, 67), suggests that they can play a significant role in establishment and growth of seedlings (77) and provide protection against root diseases (31, 61). Consequently, the potentially negative effects of chitinase-transformed trees on ectendomycorrhizal fungi could be detrimental to plant health.The present study addressed the potential impact of ech42-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis. It was hypothesized that (i) the soil fungal biomass in a transgenic white spruce rhizosphere is less than the soil fungal biomass in a control tree rhizosphere and (ii) the development of Wilcoxina spp. on root tips of transgenic white spruce is less important than the development of Wilcoxina spp. on root tips of control trees. To test these hypotheses, 5-year-old white spruce trees transformed with the 35S promoter-ech42 construct were analyzed by performing a greenhouse trial. The amount of soil fungal biomass was estimated using measurements of ergosterol in soil. A real-time PCR method was developed to detect changes in the quantity of ectendomycorrhizal hyphae involved in colonization of transgenic white spruce root tips.     

Identity,Diversity, and Molecular Phylogeny of the Endophytic Mycobiota in the Roots of Rare Wild Rice (Oryza granulate) from a Nature Reserve in Yunnan,China

Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.The majority of terrestrial plant roots are intimately associated with mycorrhizal fungi, and many aspects of the ecological roles played by these mycorrhizal fungi are well understood. In recent years, however, endophytic fungi have been gaining increasing interest. There is accumulating evidence that plant roots usually harbor mycorrhizal as well as endophytic fungi (29, 30, 34, 39, 52, 63). Dark septate endophytes (DSEs), which are characterized by dark pigmented hyphae and sclerotium-like structures, are believed to represent primary nonmycorrhizal root-inhabiting fungi (23). In some cases, DSEs are even more frequent than mycorrhizal fungi (68).Endophytic fungi have frequently been reported to be associated with crop plants, including wheat (Triticum aestivum), wild barley (Hordeum brevisubulatum and Hordeum bogdanii), soya bean (Glycine max), and maize (Zea mays) (6, 9, 11, 13, 21, 26, 27, 33, 36, 67). Some of the endophytic fungi in these crops conferred resistance of the plant to insect or fungal pathogens (55).Domesticated from the wild grass Oryza rufipogon 10,000 to 14,000 years ago, rice is today the main staple for more than 3 billion people (i.e., half of the world''s population). Its consumption exceeds 100 kg per capita annually in many Asian countries, and it is the principal food for most of the world''s poorest people, particularly in Asia. The association of arbuscular mycorrhizal fungi and endophytic bacteria with rice plants has been well documented (15, 32, 35, 44, 53, 56, 60). Less, however, is known about its fungal endophytes. Fungal endophytes have been detected in cultivated rice (Oryza sativa L.) (12, 14, 37, 61, 70), and antagonistic or plant growth-stimulating properties have been claimed for some of these isolates. For example, endophytic Fusarium spp. from cultivated rice roots proved to be effective in biocontrol of a root-knot nematode (28). The occurrence of mycorrhizal and endophytic fungi in a variety of rice cultivars has also recently been reported (63).Nondomesticated, wild plant species may live in symbiosis with a unique and rich mycoflora that may have been lost during breeding of the cultivars used in agriculture (20, 59). The purpose of this research was to characterize the endophytic fungal community of the roots of rare (nearly extinct) wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Our results showed that arbuscular mycorrhizal fungi were apparently absent from wild rice roots. This finding was confirmed by standard root staining techniques and molecular detection using the arbuscular mycorrhizal (AM)-specific primer pairs (69). The characterization of root endophytes in wild rice as reported in this study will improve our knowledge concerning the ecology and evolution of mutualistic plant-fungus interactions.     

Diverse Bacteria Inhabit Living Hyphae of Phylogenetically Diverse Fungal Endophytes

Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.Traits related to the establishment and outcome of plant-fungus symbioses can reflect not only abiotic conditions and the unique interactions of particular fungal and plant genotypes (49, 50, 56, 59, 62, 67) but also additional microbes that interact intimately with fungal mycelia (4, 12, 42). For example, mycorrhizosphere-associated actinomycetes release volatile compounds that influence spore germination in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita (Glomeromycota) (14). Levy et al. (34) describe Burkholderia spp. that colonize spores and hyphae of the AM fungus Gigaspora decipiens and are associated with decreased spore germination. Diverse “helper” bacteria have been implicated in promoting hyphal growth and the establishment of ectomycorrhizal symbioses (23, 26, 57, 70). Minerdi et al. (43) found that a consortium of ectosymbiotic bacteria limited the ability of the pathogen Fusarium oxysporum to infect and cause vascular wilts in lettuce, with virulence restored to the pathogen when ectosymbionts were removed.In addition to interacting with environmental and ectosymbiotic bacteria, some plant-associated fungi harbor bacteria within their hyphae (first noted as “bacteria-like organisms” of unknown function) (38). These bacteria, best known from living hyphae of several species of the Glomeromycota and Mucoromycotina, can alter fungal interactions with host plants in diverse ways (see references 12, 31, and 51). For example, the vertically transmitted bacterium “Candidatus Glomeribacter gigasporarum” colonizes spores and hyphae of the AM fungus Gigaspora gigasporarum (9, 10). Removal of the bacterial partner from the fungal spores suppresses fungal growth and development, altering the morphology of the fungal cell wall, vacuoles, and lipid bodies (37). In turn, the discovery of phosphate-solubilizing bacteria within Glomus mossae spores (44), coupled with the recovery of a P-transporter operon in Burkholderia sp. from Gigaspora margarita (54), suggests a competitive role in phosphate acquisition and transport by these bacteria within the AM symbiosis. Within the Mucoromycotina, Partida-Martinez and Hertweck (51) reported that a soilborne plant pathogen, Rhizopus microsporus, harbors endosymbiotic Burkholderia that produces a phytotoxin (rhizoxin) responsible for the pathogenicity of the fungus.These examples, coupled with the discovery of bacteria within hyphae of the ectomycorrhizal Dikarya (Tuber borchii; Ascomycota; Laccaria bicolor and Piriformospora indica; Basidiomycota) (5-8, 58), suggest that the capacity to harbor endohyphal bacteria is widespread among fungi. To date, however, endocellular bacteria have been recovered only from fungi that occur in the soil and rhizosphere (12, 31). Here we report for the first time that phylogenetically diverse bacteria occur within living hyphae of foliar endophytic fungi, including members of four classes of filamentous Ascomycota. We use a combination of light and fluorescence microscopy to visualize bacterial infections within living hyphae of representative strains. Then, drawing from surveys of endophytes from asymptomatic foliage of cupressaceous trees in five biogeographic provinces, we provide a first characterization of the phylogenetic relationships, host associations, and geographic distributions of endohyphal bacteria associated with focal fungal endophytes.     

Calcium Oxalate Biomineralization by Piloderma fallax in Response to Various Levels of Calcium and Phosphorus

Piloderma fallax is an ectomycorrhizal fungus commonly associated with several conifer and hardwood species. We examined the formation of calcium oxalate crystals by P. fallax in response to calcium (0.0, 0.1, 0.5, 1, and 5 mM) and phosphorus (0.1 and 6 mM) additions in modified Melin-Norkrans agar medium. Both calcium and phosphorus supplementation significantly affected the amount of calcium oxalate formed. More calcium oxalate was formed at high P levels. Concentrations of soluble oxalate in the fungus and medium were higher at low P levels. There was a strong positive linear relationship between Ca level and calcium oxalate but only under conditions of phosphorus limitation. Calcium oxalate crystals were identified as the monohydrate form (calcium oxalate monohydrate [COM] whewellite) by X-ray diffraction analysis. Prismatic, styloid, and raphide forms of the crystals, characteristic COM, were observed on the surface of fungal hyphae by scanning electron microscopy. P. fallax may be capable of dissolving hyphal calcium oxalate under conditions of limited Ca. The biomineralization of calcium oxalate by fungi may be an important step in the translocation and cycling of Ca and P in soil.Many fungi from forest litter, including ectomycorrhizal fungi, exhibit calcium oxalate (CaOx) crystals on their hyphae. The ubiquity of CaOx crystals on fungal hyphae suggests that their formation may provide a selective advantage to the organism (4). CaOx formation is hypothesized to regulate intracellular pH and levels of oxalate and Ca and, hence, serves as a major sink for toxic amounts of Ca in soil and other environments (52, 53, 61). In plants, CaOx crystals have also been proposed to serve as a calcium source under conditions of calcium limitation (14, 18, 41), but such a process has yet to be established among fungi.CaOx on fungal hyphae is formed from soil-derived calcium and biologically synthesized oxalate. Oxalate released by ectomycorrhizae has been correlated with increased phosphorus bioavailability in the rhizosphere (V. Casarin, cited by Hinsinger in reference 25). The ability of oxalate to chelate metal ions makes it important in the solubilization and transport of metals in soil, the weathering and diagenesis of rocks and soil minerals (9, 23, 31, 57), and, consequently, the transport of nutrients. It is generally presumed that CaOx crystals form on the surface of fungal hyphae as a result of precipitation when released oxalic acid interacts with calcium cations (23, 43). However, the regularity of the CaOx crystals suggests that their formation is regulated and that they may be formed within the fungal hyphae at specific sites of origin (3, 5, 7).CaOx crystals vary in morphology, ranging from plates to raphides, druses, tetragonal bipyramids, and prisms. This variation in morphology can be seen among fungal genera and species (4). The crystals also usually occur either as CaOx monohydrate (COM; whewellite) (29) or CaOx dihydrate (weddellite) (3, 5, 28, 35, 60). Either crystal form or both may be present on fungal hyphae at the same time.In earlier studies (8, 9), we reported that Piloderma fallax is one of the major species of ectomycorrhizal fungi in subboreal forests. In addition, Piloderma sp. is found in temperate forest soils in association with conifer and hardwood species (34). Piloderma influences nutrient uptake and modifies mineral transformation in rock and soil systems (3, 33). In this study, we chose P. fallax because of (i) its ability to produce oxalate and form CaOx crystals (8, 56), (ii) its presence in many types of forest ecosystems, and (iii) its significant role in the breakdown and formation of soil minerals (9).The objective of this study was to quantify and characterize the formation of CaOx by P. fallax in response to various P and Ca levels in agar medium. We tested the hypothesis that P limitation will induce the production of oxalate and that increased concentrations of Ca will result in greater CaOx formation. This study also examined the dissolution of CaOx on P. fallax when it is grown on Ca-deficient medium and determined whether CaOx can serve as temporary Ca storage. Our study was conducted to add to knowledge of the ecological significance of CaOx, especially of its influence in biogeochemical cycling of P and Ca in soils.     

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.The poplar has become a model tree species in genetic engineering as it can easily be transformed and clonally propagated and has a small genome size (7, 77, 80). Tree growth, agronomic traits, and timber quality can be improved through genetic engineering (61), thereby avoiding the long reproductive cycles of conventional breeding (47, 59, 83). However, concerns have arisen about the potential impact of genetically modified (GM) trees on the environment (10). The potential environmental hazards linked to GM trees differ from those associated with transgenic crop plants at both spatial and temporal scales (84) because trees are long-lived perennials, unlike annual crop plants. They display several biotic interactions with soil microbial communities such as bacteria and fungi. Interactions between GM trees and these communities could result in exposure to the expression of new traits over several decades, a period longer than those for GM crop plants.Impact studies of GM plants on nontarget organisms usually focus on the potential risk linked to transgene expression (expected effects) that confers a genetic advantage to the transformed plant rather than on unforeseen (pleiotropic) effects from transgene insertion or the expression of other transgene components such as selection markers or reporter genes. The nptII gene, encoding neomycin phosphotransferase II (EC 2.7.1.95), and the GUS gene, encoding β-glucuronidase (GUS; EC 3.2.1.31), are frequently used for genetic selection of transformed cells and for monitoring transgene presence and expression during transgenic plant lifetime (76). The products of the nptII and GUS genes have been subjected to safety assessment studies and were shown to be nondeleterious to human and animal health (21, 23, 27, 51). Nevertheless, pleiotropic effects in crop plants transformed with the nptII and GUS genes have been observed (2, 15, 17, 43). Pleiotropic effects from GM trees coexpressing such selectable markers have also been recorded. For example, Pasonen et al. (56) showed a significant decrease in the number of root tips colonized by Paxillus involutus associated with a line of chitinase-transformed silver birch in vitro. Similar results have been observed in vivo with P. involutus associated with a line of lignin-modified silver birches (72).Many trees in temperate, boreal, tropical, and subtropical forests establish mutualistic interactions with ectomycorrhizal (EM) fungi (42, 66, 67, 68). EM fungi are a polyphyletic group comprising over 5,000 species (49) that play key roles in biogeochemical soil processes and plant health. They represent one-third of the total microbial biomass in the soil of boreal forests (32). Fine roots colonized by EM fungi, also called EM root tips or ectomycorrhizae, display a fungal mantle from which extends the extraradical mycelium to prospect the soil for nutrient uptake. These two anatomical parts can be sampled for EM fungus molecular identification, but some studies have highlighted dissimilarities between the EM fungal diversity recorded in root tip sampling and that recorded in extraradical mycelium sampling (26, 37, 39).Given the potential cumulative effects caused by the presence and stable constitutive expression of transgenes over years on GM tree fitness and on the environment, impact studies of GM trees require long-term field trials (5, 72, 84). In this study, we investigated the potential long-term impact on the EM fungal community of hybrid poplars transformed with the binary vector containing the selectable nptII marker and GUS reporter genes, field deployed for 8 years. This plantation was part of the first confined field trial of transgenic trees in Canada. Hybrid poplars constitutively expressed the nptII gene for kanamycin resistance driven by the NOS promoter (30). The activity of the NOS promoter has been shown to increase in the lower part of transgenic tobacco plants (4). Such a vertical gradient has also been observed in transgenic hybrid poplars, where the NOS promoter activity was 2.4-fold higher in roots than in leaves (87).As no direct negative impact of nptII or GUS gene expression on fungal organisms has been reported in the literature, we first tested the null hypothesis (H₀) that the EM fungal community recorded from transgenic poplars was similar to that from untransformed poplars. Second, since the EM fungal diversity picture can be influenced by the sampling method, we contrasted the EM fungal community recovered from root tips with that recorded in soil cloning analyses. Internal transcribed spacer (ITS) sequences from the nuclear rRNA were produced from both EM root tips and extraradical mycelia to compare the EM fungal communities associated with three control and three transgenic poplars. EM fungal communities were characterized by measuring the usual qualitative and quantitative EM species diversity within each community (α-diversity) and then estimating the nucleotide diversity between EM communities in relation to EM phylotype relative abundances (quantitative β-diversity).     

Girdling Affects Ectomycorrhizal Fungal (EMF) Diversity and Reveals Functional Differences in EMF Community Composition in a Beech Forest

The relationships between plant carbon resources, soil carbon and nitrogen content, and ectomycorrhizal fungal (EMF) diversity in a monospecific, old-growth beech (Fagus sylvatica) forest were investigated by manipulating carbon flux by girdling. We hypothesized that disruption of the carbon supply would not affect diversity and EMF species numbers if EM fungi can be supplied by plant internal carbohydrate resources or would result in selective disappearance of EMF taxa because of differences in carbon demand of different fungi. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Girdling did not affect root colonization but decreased EMF species richness of an estimated 79 to 90 taxa to about 40 taxa. Cenococcum geophilum, Lactarius blennius, and Tomentella lapida were dominant, colonizing about 70% of the root tips, and remained unaffected by girdling. Mainly cryptic EMF species disappeared. Therefore, the Shannon-Wiener index (H′) decreased but evenness was unaffected. H′ was positively correlated with glucose, fructose, and starch concentrations of fine roots and also with the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON), suggesting that both H′ and DOC/DON were governed by changes in belowground carbon allocation. Our results suggest that beech maintains numerous rare EMF species by recent photosynthate. These EM fungi may constitute biological insurance for adaptation to changing environmental conditions. The preservation of taxa previously not known to colonize beech may, thus, form an important reservoir for future forest development.In temperate and boreal forest ecosystems, most tree species form ectomycorrhizal fungal (EMF) associations. EM fungi ensheathe the root tip, forming characteristic mantlelike structures (1). The presence and lengths of hyphae emanating from the mantle are characteristic of different EMF species and establish different soil exploration types (2). It has been assumed that EMF communities are adapted specifically to mobilize sparse soil nutrient resources in boreal and temperate forests (11, 50). Current estimates indicate that about 80% of all nitrogen and phosphorus present in plants has been taken up via mycorrhizas (30, 41, 63).Unlike free-living soil microbes, EM fungi have direct access to reduced carbon from their host plants. More than 50 years ago, Melin and Nilsson (46) showed that ¹⁴C applied to leaves was recovered within one day in EM fungi, suggesting a strong dependence of fungal metabolism on host photosynthesis. Subsequent isotopic studies corroborated tight connections between current photosynthate and EM fungi (28, 42). EMF hyphae constitute the main path of plant-derived carbon into the soil (24, 29). Furthermore, EMF hyphae contribute substantially to soil respiration (25% from hyphae and 15% from roots) (27). As hyphal respiration decreases strongly in response to girdling of trees, a tight metabolic link between extramatrical mycelia and host photosynthetic activity must exist (5, 9, 32). In addition, fruiting body formation of EMF species was strongly dependent on host photosynthetic capacity (32, 40). In contrast, the significance of the current assimilate supply for EMF colonization of root tips and for community composition is not yet well understood. Since trees contain substantial stores of carbohydrates in the roots and stem (7), it may be expected that EM fungi can be maintained if this carbon resource is available. For example, defoliation experiments with conifers, which restricted but did not eliminate current photosynthate transfer to roots, showed no effects on root EMF colonization. Massive defoliation that negatively affected aboveground biomass production suppressed morphotypes with thick mantles compared to those with thin mantles, suggesting a shift to less-carbon-demanding EMF species (14, 40, 44, 54, 56). Earlier studies also reported decreased EMF colonization of root tips (21, 52).In a common garden experiment with young beech trees, strong shading over several years, which severely limited plant growth, suppressed EMF colonization and resulted in low EMF diversity (20). EMF community composition was affected strongly by shading and slightly by short-term girdling, suggesting that EMF taxa are sensitive to changes in plant internal carbohydrate resources (20). However, the overall EMF diversity was low, probably because the young trees were grown in nutrient-rich compost soil (20). The significance of photoassimilates for EMF abundance, diversity, and community composition, therefore, remains to be shown for adult forest trees, which usually have high EMF diversity and low nitrogen availability (10, 26, 53, 61).The aim of this work was to test the hypothesis that EMF abundance and diversity are independent of the current photoassimilate supply and can be maintained by internal resources. To investigate this concept, old-growth beech trees (Fagus sylvatica L.) were girdled to suppress carbon allocation to roots. Since disruption of the current assimilate flux affects the carbohydrate source strength, we hypothesized that changes in EMF taxon composition would occur if EMF species had different carbon demands. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Since girdling also affects carbon release into and probably nutrient uptake from soil, the influence of possible feedback by changes in the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON) in the soil on EMF diversity was also assessed.     

Migratory Response of Soil Bacteria to Lyophyllum sp. Strain Karsten in Soil Microcosms

In this study, the selection of bacteria on the basis of their migration via fungal hyphae in soil was investigated in microcosm experiments containing Lyophyllum sp. strain Karsten (DSM2979). One week following inoculation with a bacterial community obtained from soil, selection of a few specific bacterial types was noticed at 30 mm in the growth direction of Lyophyllum sp. strain Karsten in sterile soil. Cultivation-based analyses showed that the migration-proficient types encompassed 10 bacterial groups, as evidenced by (GTG)₅ genomic fingerprinting as well as 16S rRNA gene sequencing. These were (>97% similarity) Burkholderia terrae BS001, Burkholderia sordidicola BS026, Burkholderia sediminicola BS010, and Burkholderia phenazinium BS028; Dyella japonica BS013, BS018, and BS021; “Sphingoterrabacterium pocheensis” BS024; Sphingobacterium daejeonense BS025; and Ralstonia basilensis BS017. Migration as single species was subsequently found for B. terrae BS001, D. japonica BS018 and BS021, and R. basilensis BS017. Typically, migration occurred only when these organisms were introduced at the fungal growth front and only in the direction of hyphal growth. Migration proficiency showed a one-sided correlation with the presence of the hrcR gene, used as a marker for the type III secretion system (TTSS), as all single-strain migrators were equipped with this system and most non-single-strain migrators were not. The presence of the TTSS stood in contrast to the low prevalence of TTSSs within the bacterial community used as an inoculum (<3%). Microscopic examination of B. terrae BS001 in contact with Lyophyllum sp. strain Karsten hyphae revealed the development of a biofilm surrounding the hyphae. Migration-proficient bacteria interacting with Lyophyllum sp. strain Karsten may show complex behavior (biofilm formation) at the fungal tip, leading to their translocation and growth in novel microhabitats in soil.Bacterial-fungal interactions are common in a wide variety of habitats like decaying wood, human bodies, and marine and soil environments (7, 12, 13, 15, 18). Especially in soil, interactions are likely to occur frequently, as members of both kingdoms abound in this system and depend on strategies that allow them to utilize the sparse carbonaceous nutrients that are available (6, 22, 26, 27). Interactions may be deleterious, neutral, or even beneficial for either or both of the partners. In particular, the putative beneficial effects exerted by soil fungi on associated bacteria may enhance bacterial fitness and thus provide a selective force on these (4, 5, 11, 14, 29). A range of different mechanisms is thought to play a role in the putative bacterial selection, in which particular fungus-released compounds may exert key effects in this selection (1, 10, 14, 28). In addition, changes in the structure of the local (soil) habitat effected by either of the partners (2) and/or production of antibacterial substances by the fungal partner (7, 9) may play roles.The capacity of soil bacteria to use fungal hyphae as a means to reach and colonize novel microhabitats in soil has been proposed as a mechanism for pollutant-degrading bacteria to become efficient in polluted soil (16). However, the study addressed only bacterial migration with fungi via so-called fungal highways in non-soil systems like agar plates and glass bead systems. Clearly, such fungal highways might be used by bacteria to cross air gaps (23) during growth and movement in soil, but evidence for this is lacking. Movement of the bacterial partner was probably driven by motility of the bacterial cells in the water film surrounding the fungal hyphae. The observation of bacteria moving along fungal highways was supported by an earlier study that addressed bacterial motility via dead hyphae of an oomycete in soil (32). Together, these studies suggest that bacteria can utilize the mycosphere (here defined as the fungal hyphal network) in soil to reach and colonize novel microhabitats. However, these studies do not allow an in-depth assessment of which bacteria get selected by growing fungi and how they mechanistically make use of fungal highways.In the current study, we assessed the putative selection of organisms from a soil bacterial community that was able to migrate in the mycosphere of Lyophyllum sp. strain Karsten, a close saprotrophic relative of the ectomycorrhizal fungus Laccaria proxima. We initially assessed the selection of particular bacterial species by L. proxima (29), which was an abundant ectomycorrhizal species with hazel trees. Thus, we developed a microcosm system composed of three compartments, which allowed the outgrowth of fungal hyphae from a nutrient source into sterile soil. Different aspects of bacterial migration along with the fungal front were studied. Based on these findings, a mechanism for bacterial migration in which biofilm formation plays a role is proposed.     

ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A₁ or a protonophore, suggesting that ATP may not energize the reaction through H⁺-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that form symbiotic associations with most land plants (29). These fungi promote the growth of host plants via enhanced uptake of phosphate (P_i) and thus play important roles in the terrestrial phosphorus cycle. In the symbiotic phase, AM fungi take up P_i from soil through an extensive network of extraradical hyphae and rapidly accumulate inorganic polyphosphate (polyP). This accumulation was as rapid as that for a polyP-hyperaccumulating bacterium found in activated sludge (6). PolyP is a linear polymer of three to hundreds of molecules of P_i linked by high-energy phosphoanhydride bonds and has been found across all classes of organisms (19). Although polyP is considered to play a central role in long-distance translocation of P_i in AM fungal associations (4, 10, 30, 31), the translocation mechanism, metabolism, and dynamics in the fungi have not been elucidated due to the difficulty in obtaining sufficient fungal material for analysis.Many enzymes/genes involved in polyP synthesis/metabolism have been identified and characterized in prokaryotes (19). For instance, exopolyphosphatase hydrolyzes the terminal high-energy bonds of polyP, and polyphosphate glucokinase (PPGK) transfers the terminal P_i residue to glucose. Polyphosphate kinase 1 (PPK1) is responsible both for polyP synthesis, using ATP as a phosphoryl donor, and for the reverse ATP-generating reaction. This enzyme is bound to the plasma membrane (18) and has been found in a wide range of bacteria (17). Unlike the case for prokaryotes, knowledge of polyP synthesis/metabolism in eukaryotes remains limited. The first eukaryotic PPK genes, DdPPK1 (32) and DdPPK2 (14), were identified from the social slime mold Dictyostelium discoideum. The products of these genes, as known for bacterial PPK1s, are responsible both for polyP synthesis and for the ATP-generating reaction and have been suggested to be associated with vacuoles or small vesicles (14, 32). Although several homologues of bacterial PPK1 genes have now been found in the genomes of eukaryotic microorganisms (17), yeast Candida humicola is the only organism apart from D. discoideum for which PPK-like activity has been confirmed (22). The model organism Saccharomyces cerevisiae is known to accumulate polyP, to up to 10% of its dry weight (19). A unique polyP synthetic pathway different from those of PPK1 has been proposed for S. cerevisiae based on the observation that vacuolar-type H⁺-ATPase (V-ATPase)-defective mutants could not accumulate polyP (23). In this hypothetical pathway, P_i would be polymerized by an analogous system (enzyme) of mitochondrial F₁-ATPase on the vacuolar membrane, using the proton motive force created by V-ATPase (23). On the other hand, Hothorn et al. (16) demonstrated very recently that vacuolar transporter chaperone 4 (VTC4), a small transmembrane protein associated with the membrane, polymerizes P_i by using the γ-P_i residue of ATP as a phosphoryl donor in S. cerevisiae.More than 2 decades ago, Capaccio and Callow (3) reported the presence of polyP-hydrolyzing, -metabolizing (PPGK), and -synthesizing (PPK-like) activities in the soluble (cytosolic) fractions of the hyphae of the AM fungus Glomus mosseae. Recently, polyP-hydrolyzing activity was found in both the cytosolic and insoluble (membrane) fractions and then characterized (8). PPGK activity has also been confirmed in the cytosolic fraction, although the activity was quite low and hexokinase (ATP-hexose phosphotransferase) activity appeared to dominate in the glucose phosphorylation process (9). PPK-like activity, however, could not be detected in the same fraction (10), and this seems likely because all other prokaryotic (reviewed in reference 17) and eukaryotic (14, 16, 22, 32) polyP-synthesizing enzymes, so far, are associated with membranes. These observations suggest that AM fungi possess a polyP-synthesizing enzyme that is probably associated with membranes and that ATP may be essential in the synthesis as a phosphoryl donor or via H⁺-ATPase, as suggested by Ogawa et al. (23). In this study, a cell fractionation technique was applied to demonstrate polyP-synthesizing activity in an AM fungus, and then the role of ATP in the synthesis was investigated.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Differential Utilization of Carbon Substrates by Bacteria and Fungi in Tundra Soil

Little is known about the contribution of bacteria and fungi to decomposition of different carbon compounds in arctic soils, which are an important carbon store and possibly vulnerable to climate warming. Soil samples from a subarctic tundra heath were incubated with ¹³C-labeled glucose, acetic acid, glycine, starch, and vanillin, and the incorporation of ¹³C into different phospholipid fatty acids (PLFA; indicative of growth) and neutral lipid fatty acids (NLFA; indicative of fungal storage) was measured after 1 and 7 days. The use of ¹³C-labeled substrates allowed the addition of substrates at concentrations low enough not to affect the total amount of PLFA. The label of glucose and acetic acid was rapidly incorporated into the PLFA in a pattern largely corresponding to the fatty acid concentration profile, while glycine and especially starch were mainly taken up by bacteria and not fungi, showing that different groups of the microbial community were responsible for substrate utilization. The ¹³C-incorporation from the complex substrates (starch and vanillin) increased over time. There was significant allocation of ¹³C into the fungal NLFA, except for starch. For glucose, acetic acid, and glycine, the allocation decreased over time, indicating use of the storage products, whereas for vanillin incorporation into fungal NLFA increased during the incubation. In addition to providing information on functioning of the microbial communities in an arctic soil, our study showed that the combination of PLFA and NLFA analyses yields additional information on the dynamics of substrate degradation.Bacteria and fungi comprise more than 90% of the soil microbial biomass and are the main agents for decomposition of organic matter in soil. Until recently it was thought that these two organism groups could be lumped together in this respect, and total microbial biomass or total activity (respiration) was often the only variable included in soil microbiology studies of decomposition and soil organic matter turnover (39). However, there is increasing evidence suggesting that whether decomposition is performed by bacteria or fungi, thereby channeling energy through the bacterial or the fungal food web, has profound effects on the ecosystem. Such effects can have direct influence on the higher trophic levels in the food web (30) or indirect effects on nutrient mineralization rates (14) and nutrient transfer (19, 20), and they can even determine the extent of carbon sequestration in the soil (37). The situation becomes even more complex when the impact of changes in climate, nitrogen availability, and litter input on the balance between bacteria and fungi is taken into account. The Arctic region has been identified as an area that will be especially vulnerable to these changes (3).Little is known about the contribution of bacteria and fungi to the utilization of plant-derived carbon substrates in arctic soils. Differentiation of the bacterial and fungal contributions to decomposition has hitherto relied to a large extent on changes in bacterial and fungal biomasses, for example, by analysis of patterns of phospholipid fatty acids (PLFA) (40). PLFA are components of the cell membrane, and some of the PLFA extracted from the soil are characteristic for a certain microbial group in the environment. However, for changes in PLFA concentrations after the addition of substrates to be detected, substrates often have to be added at unrealistically large amounts. Even then only small changes in the PLFA concentrations will often be detected (35).One way of overcoming these problems is to follow the incorporation of ¹³C label from added substrates into specific fatty acids (8, 17). This approach adds a new dimension—metabolic function—to the study of soil microbial communities without the need of cultivation. It also increases the sensitivity in tracing responses of organism groups to different substrates as the addition of substrates at low and more realistic concentrations with high specific ¹³C label will induce large changes in the ¹³C concentration of the PLFA without changing the total amount of PLFA.Carbon-13 labeling has been used to follow uptake of recent photosynthates (11, 13, 27), pure substrates (10, 12, 32, 33, 41), and complex labeled plant material (28, 41, 43, 44) into PLFA although seldom in arctic soils. However, microorganisms incorporate carbon not only into phospholipids (indicating growth) but also into storage products, for example, when a nutrient other than carbon is limiting growth or under growth-restricting conditions. Thus, with excess carbon both bacteria and fungi will store carbon for later need, for example, as polyhydroxyalkanoate or glycogen (bacteria) and triacylglycerols (fungi). Thus, neutral lipid fatty acids (NLFA) of fungal origin can be used to indicate storage in fungi (4). Degraded PLFA, resulting in diacylglycerols, will also end up in the corresponding NLFA fraction, and NLFA has thus been suggested as an indicator of recently dead bacterial biomass (42). Therefore, the NLFA/PLFA ratio serves two purposes: for fungal lipids a higher NLFA/PLFA ratio would indicate allocation of lipids to energy storage while for bacterial lipids it would indicate turnover of this bacterial group. However, the latter will probably be of minor importance during short incubations. As far as we know, no studies on soil microorganisms have used incorporation of ¹³C from substrates to indicate both effects on growth (incorporation into PLFA) and storage (incorporation into NLFA).We assessed the uptake of ¹³C-labeled substrates into lipid biomarkers of different microbial groups in a laboratory incubation experiment using soil from an arctic tundra heath. The selected substrates represented carbon sources present in soil. Glucose, acetic acid, and glycine are simple compounds common in plant root exudates, and glycine is also a nitrogen source. Starch is a very common polysaccharide in plant residues. Vanillin is a common product of lignin depolymerization (18) containing a phenol ring and is often used as a model substance to indicate lignin degradation. Starch and vanillin are therefore examples of more complex substrates and are supposedly more difficult to decompose. We followed the incorporation of the label into different PLFA and NLFA over time. We hypothesized that ¹³C from the simple compounds would be more rapidly incorporated into microbial PLFA than ¹³C from the more complex substrates (more rapid growth), and thus we expected ¹³C emanating from the complex substrates to increase in concentration in the PLFA and NLFA over time. We also hypothesized that bacteria would be better than fungi in utilizing simple compounds while the label from the more complex substrates would preferentially be incorporated into PLFA, indicating fungi (6, 29). We also expected ¹³C from the C-rich substrates to be incorporated into NLFA (fungal storage) to a larger extent than C from glycine, which also serves as a nitrogen source (4). However, with time the carbon in storage structures would decrease as it would be used for growth or maintenance energy.     

Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers

Researchers agree that climate change factors such as rising atmospheric [CO₂] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO₂ concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO₂] but decreased in warmed plots under ambient atmospheric [CO₂]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of Proteobacteria and Acidobacteria, where Acidobacteria decreased with a concomitant increase in the Proteobacteria in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.Soil microbial communities are responsible for the cycling of carbon (C) and nutrients in ecosystems, and their activities are regulated by biotic and abiotic factors such as the quantity and quality of litter inputs, temperature, and moisture. Atmospheric and climatic changes will impact both abiotic and biotic drivers in ecosystems and the response of ecosystems to these changes. Feedbacks from ecosystem to the atmosphere may also be regulated by soil microbial communities (3). Although microbial communities regulate important ecosystem processes, it is often unclear how the abundance and composition of microbial communities correlate with climatic perturbations and interact to effect ecosystem processes. As such, much of the ecosystem climate change research conducted to date has focused on macroscale responses to climatic change such as changes in plant growth (43, 44), plant community composition (2, 37), and coarse scale soil processes (14, 18, 21, 26), many of which may also indirectly interact to effect microbial processes. Studies that have addressed the role of microbial communities and processes have most often targeted gross parameters, such as microbial biomass, enzymatic activity, or basic microbial community profiles in response to single climate change factors (22, 28, 29, 33, 61, 63).Climate change factors such as atmospheric CO₂ concentrations, warming, and altered precipitation regimes can potentially have both direct and indirect impacts on soil microbial communities. However, the direction and magnitude of these responses is uncertain. For example, the response of soil microbial communities to changes in atmospheric CO₂ concentrations can be positive or negative, and consistent overall trends between sites and studies have not been observed (1, 28, 34-36). Further, depending on what limits ecosystem productivity, precipitation and soil moisture changes may increase or decrease the ratio of bacteria and fungi, as well as shift their community composition (8, 50, 58). Increasing temperatures can increase in microbial activity, processing, and turnover, causing the microbial community to shift in favor of representatives adapted to higher temperatures and faster growth rates (7, 46, 60, 64, 65). Atmospheric and climatic changes are happening in concert with one another so that ecosystems are experiencing higher levels of atmospheric CO₂, warming, and changes in precipitation regimes simultaneously. Although the many single factor climate change studies described above have enabled a better understanding of how microbial communities may respond to any one factor, understanding how multiple climate change factors interact with each other to influence microbial community responses is poorly understood. For example, elevated atmospheric [CO₂] and precipitation changes might increase soil moisture in an ecosystem, but this increase may be counteracted by warming (10). Similarly, warming may increase microbial activity in an ecosystem, but this increase may be eliminated if changes in precipitation lead to a drier soil condition or reduced litter quantity, quality, and turnover. Such interactive effects of climate factors in a multifactorial context have been less commonly studied even in plant communities (45), and detailed studies are rarer still in soil microbial communities (25). Clearly, understanding how microbial communities will respond to these atmospheric and climate change drivers is important to make accurate predications of how ecosystems may respond to future climate scenarios.To address how multiple climate change drivers will interact to shape soil microbial communities, we took advantage of a multifactor climatic change experiment that manipulated atmospheric CO₂ (+300 ppm, ambient), warming (+3°C, ambient) and precipitation (wet and dry) in a constructed old-field ecosystem that had been ongoing for 3.5 years at the time of sampling. Previous work on this project has demonstrated direct and interactive effects of the treatments on plant community composition and biomass (15, 30), soil respiration (56), microbial activity (30), nitrogen fixation (21), and soil carbon stocks (20). These results led us to investigations of how the soil bacterial and fungal communities, important regulators of some of these processes, were responding using culture-independent molecular approaches. Our research addresses two overarching questions. (i) Do climatic change factors and their interactions alter bacterial and fungal abundance and diversity? (ii) Do climatic change factors and their interactions alter bacterial or fungal community composition?     

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.The vast majority of soil bacteria (1, 7, 27) and fungi (13, 29) cannot be cultured via traditional laboratory techniques and must be identified using molecular methods. Successful characterization of microbial communities is therefore often dependent on DNA that is extracted from the environment. However, extraction of high-quality DNA from soil can be problematic (8, 11, 22, 26). Commercial DNA extraction kits are now commonly used in the assessment of taxonomic and functional diversity, community composition, and population abundance (e.g., references 19, 21, 23, 25, and 31). Studies comparing various kits (18, 32) or comparing commercial kits to other methods (2, 10, 24) have shown that DNA yield and purity vary depending on methodology and soil type. While these comparative studies are valuable, it is still unclear to what extent these protocols yield genomic DNA representative of the microbial community found within soil.Our objective in this study was to optimize and assess the bias of a widely used commercial soil DNA extraction kit. We hypothesized that cell lysis would be enhanced and DNA would be removed from adsorption sites by conducting multiple extractions on a single sample, thereby increasing genomic DNA yield and obtaining a more complete survey of microbial taxa. This hypothesis was tested by (i) varying the extraction protocol and measuring DNA yield for three soils with differing characteristics and (ii) examining extraction bias in the genomic DNA obtained from successive extractions by using an improved method. Analytical replicates rather than biological replicates were used in order to focus strictly on variation and bias introduced through methodology, although multiple soil types were analyzed to determine whether biases detected were consistent.     

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6, 21, 22, 24, 37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7, 21, 26, 34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7, 10, 20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment—clay that contains humic substances—and enumerating a specific target sequence from the crude extract.     

Negative Fitness Consequences and Transmission Dynamics of a Heritable Fungal Symbiont of a Parasitic Wasp

Heritable bacterial symbionts are widespread in insects and can have many important effects on host ecology and fitness. Fungal symbionts are also important in shaping their hosts'' behavior, interactions, and evolution, but they have been largely overlooked. Experimental tests to determine the relevance of fungal symbionts to their insect hosts are currently extremely rare, and to our knowledge, there have been no such tests for strictly predacious insects. We investigated the fitness consequences for a parasitic wasp (Comperia merceti) of an inherited fungal symbiont in the Saccharomycotina (Ascomycota) that was long presumed to be a mutualist. In comparisons of wasp lines with and without this symbiont, we found no evidence of mutualism. Instead, there were significant fitness costs to the wasps in the presence of the yeast; infected wasps attacked fewer hosts and had longer development times. We also examined the relative competitive abilities of the larval progeny of infected and uninfected mothers, as well as horizontal transmission of the fungal symbiont among larval wasps that shared a single host cockroach egg case. We found no difference in larval competitive ability when larvae whose infection status differed shared a single host. We did find high rates of horizontal transmission of the fungus, and we suggest that this transmission is likely responsible for the maintenance of this infection in wasp populations.The majority of heritable bacterial symbionts associated with insects either provide nutritional benefits for hosts that feed on nutrient-poor diets, such as blood (e.g., Wigglesworthia sp. [1]) or sap (e.g., Buchnera spp. [33]), or manipulate the hosts'' reproduction to benefit their own transmission (e.g., Wolbachia spp. [38] or Cardinium sp. [40]). Thanks in part to these examples, research efforts have become more diverse, leading to the discovery of additional benefits, such as heat tolerance (29) and protection from parasitism (26).Despite growing interest in the cryptic roles of insect associates, fungal symbionts have largely been overlooked, and their prevalence, ecological importance, and evolutionary implications for hosts are still poorly understood. Yet we have reason to suspect that fungal symbionts may be as diverse and functionally important as bacteria in insects. Buchner''s (5) foundational work on arthropod-microbe symbioses included many fungi, and anecdotal reports of such symbioses are scattered throughout the literature (e.g., fire ants [3]; stingless bees [28]; earwigs, scale insects, flies, andrenid bees, and ants [39]; and leafhoppers [30]). Recent surveys of insects for fungi have resulted in an astonishing diversity, including fungi in beetles (35), a cockroach and five other neuropteran families (24), sap-feeding beetles, and flies and bees (15), and it has been suggested that the majority of unicellular fungal diversity may be in insects (35). It is often suggested that such associations are mutualistic, with the fungus presumably providing enzymes, essential amino acids, vitamins, or sterols (37) and the insect vectoring and providing a habitat for the fungus. Fitness consequences of these associations have been assessed in only a few cases, including associations in planthoppers (31), anobiid beetles (23, 32), and scolytid beetles (2). In most instances the significance of the relationship is not clear, especially in the many cases where the fungi are not obligate associates.In 1985, LeBeck (18) reported a unicellular fungal symbiont in Comperia merceti (Compere) (Hymenoptera: Encyrtidae), a gregarious endoparasitoid wasp that specializes on the egg cases of brown-banded cockroaches [Supella longipalpa (Serville) (Blattaria: Blattellidae)]. The fungus is found throughout the hemocoel in juvenile wasps, in adult males, and in the venom gland of adult females (18). In addition, the fungus is vertically transmitted from mother to offspring via the external surface of wasp eggs during oviposition into cockroach egg cases. Vertical transmission via the egg surface is a common method in other fungal symbiont systems (e.g., planthoppers [19]; lacewings [10]; and wood wasps, anobiid beetles, and cerambycid beetles [5]). LeBeck (18) characterized the fungus as a Candida sp. and suggested that it might alter the nutritional value of the host cockroach egg case for the benefit of the developing wasp larvae. However, this claim has never been tested. Further, the predacious diet of immature parasitic wasps would make them unusual candidates for nutritional symbionts; parasitic wasps consume other insects and do not ordinarily require the complementary nutrients that many fungal and bacterial symbionts provide to insects with unbalanced diets. To our knowledge, our study is the first to specifically test the role of an inherited fungus in an insect with a strictly predacious diet.C. merceti wasps house a single known fungal symbiont belonging to the Ascomycota (Saccharomycotina) and no detectable bacterial symbionts (9). Further, these wasps do not become infected with any of their host cockroaches'' symbionts (9). In in vitro trials of the C. merceti wasp fungus with other microbes there was no evidence of inhibition or any type of interaction (C. M. Gibson, unpublished). The current research tests the hypothesis that the wasps'' fungal symbiont is a mutualist and explores alternative means by which this fungus could be maintained in wasp populations.     

Roles of the Glyoxylate and Methylcitrate Cycles in Sexual Development and Virulence in the Cereal Pathogen Gibberella zeae

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.During the infection process, pathogenic fungi usually encounter nutrient deprivation in the host before gaining access to sufficient nutrients for successful colonization of the living tissue. To cope with a nutrient-limited environment, fungal pathogens seem to rely mostly on fatty acid metabolism for both energy supply and biosynthesis of essential molecules (29). The ability of fungi to use fatty acids as a carbon source for growth is based on the glyoxylate cycle. Fungal pathogens have been proposed to employ the glyoxylate bypass for the use of acetyl coenzyme A (CoA) units produced by the β-oxidation of even-chain-length fatty acids, probably available from host cell membranes or the lipid reservoir inside the fungal spore (7, 12, 20, 27, 28, 41, 44, 46). Recent studies suggest that the glyoxylate pathway plays an important role in fungal virulence toward both plant and animal hosts (12, 20, 27, 44, 46). The key enzymes of the glyoxylate pathway, such as isocitrate lyase (ICL), which catalyzes the cleavage of isocitrate to glyoxylate and succinate, and malate synthase, which mediates the condensation of acetyl-CoA and glyoxylate into malate, are strongly induced within the host (16, 27, 41, 44). Moreover, disruption of genes encoding either of these enzymes causes severely reduced virulence of fungal phytopathogens, including Leptosphaeria maculans (20), Magnaporthe grisea (46), Stagonospora nodorum (44), and Colletotrichum lagenarium (2), and the animal pathogen Candida albicans (27). In contrast, these glyoxylate cycle enzymes have been known to be dispensable in invasive aspergillosis caused by Aspergillus fumigatus (38, 43).During fatty acid and amino acid catabolism by fungi, propionyl-CoA can be generated along with acetyl-CoA, particularly from the breakdown of odd-chain-length fatty acids or of the amino acids valine, isoleucine, and methionine (14). Therefore, fungal pathogens may need to use or remove propionyl-CoA during the infection process because it is toxic to fungi. In fungi, propionyl-CoA is metabolized via the methylcitrate cycle, in which propionyl-CoA is oxidized to pyruvate in four enzymatic steps (4, 5, 6, 19, 30, 31, 40, 49, 50). Recently, the importance of the methylcitrate cycle in fungal virulence was demonstrated in A. fumigatus: a mutant defective in methylcitrate synthase, the first enzyme of this cycle, displayed attenuated virulence in mice and insects (19, 31). However, the role of methylisocitrate lyase (MCL), which catalyzes the last reaction in the methylcitrate cycle (i.e., the cleavage of methylisocitrate into pyruvate and succinate) in fungal virulence, has not been determined, although deletion of the MCL gene inhibits hyphal growth and conidiation in Aspergillus nidulans (4). The protein sequences of several fungal MCLs show high similarity to fungal ICLs of the glyoxylate cycle (4, 30). In the pathogenic bacterium Mycobacterium tuberculosis, the methylcitrate cycle, only when working together with the glyoxylate cycle, is involved in virulence as well as fatty acid metabolism and intracellular growth (34, 35).Here, we focused on the roles of these two cycles during disease development caused by the devastating cereal pathogen Gibberella zeae (anamorph: Fusarium graminearum). G. zeae is a ubiquitously distributed ascomycete fungus that causes major disease in cereal crops such as corn, wheat, barley, and rice (33). Severe epidemics of these diseases result in serious economic consequences due to yield losses and contamination by fungal mycotoxins (32, 33). Wind-disseminated sexual spores (ascospores), which are produced in perithecia formed on plant debris, can infect plant spikes during anthesis (13, 39, 45). Detailed studies of the G. zeae infection process on wheat and barley heads have shown that fungal hyphae on the inner surfaces of the spike penetrate epicarp cells through pits or pores and grow into the caryopses through the pericarp (21). Thus, the glyoxylate cycle, either alone or in conjunction with the methylcitrate cycle, is likely employed by G. zeae during the infection process, as in other fungus-plant interactions (20, 46). G. zeae genome searches have identified orthologs of fungal ICL and MCL genes, designated GzICL1 and GzMCL1, respectively. Here, we performed functional analyses of these genes to provide new insight into their importance in lipid metabolism during the G. zeae infection process in host plants.     

Horizontal Chromosome Transfer,a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.Fungi produce a huge variety of secondary metabolites. Some plant-pathogenic fungi, especially necrotrophic pathogens that kill plant cells during invasion, produce phytotoxic metabolites to impair host tissue functions (20, 30, 42, 47). Phytotoxins produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites that exert toxic effects on host plants. Among these phytotoxins, host-specific toxins (HSTs) are critical determinants of pathogenicity or virulence in several plant-pathogen interactions (13, 30, 33, 40, 42, 47, 49).Recent advances in molecular biological techniques for fungi have led to the identification of fungal genes involved in pathogenesis, as exemplified by those used in the biosynthesis of toxic secondary metabolites, such as HSTs. Genes involved in the biosynthesis of secondary metabolites are typically clustered in filamentous fungi, including plant pathogens (20, 24, 44). The origins and evolutionary processes of these gene clusters, however, are largely unknown. Analysis of the arrangement and sequences of genes in the clusters would shed light on how the clusters themselves and their ability to produce toxic secondary metabolites evolved (20, 24, 44).The involvement of horizontal gene transfer (HGT) in the evolution of fungal secondary-metabolite gene clusters has been discussed (34, 44). HGT events are well known in prokaryotes (21, 29), and the genomic regions that have undergone HGT are referred to as pathogenicity or genomic islands (7). In prokaryotes, the mechanisms of HGT are also associated with conjugation, transformation, and transduction (21, 29). Although these transfer mechanisms are generally unknown in eukaryotes such as fungi, interspecific transfer of a virulence gene encoding the production of a critical toxin has been reported in Pyrenophora tritici-repentis (14). There is also clear evidence of recent lateral gene transfer of the ToxA gene from Stagonospora nodorum to P. tritici-repentis (14, 30).In Alternaria alternata plant pathogens (37), we have shown that all strains of the A. alternata pathotypes harbor small extra chromosomes of less than 1.7 Mb, whereas nonpathogenic isolates do not have these small chromosomes (5). A cyclic peptide synthetase gene, AMT, which is involved in host-specific AM toxin biosynthesis of the apple pathotype of A. alternata, was located on a small chromosome of 1.1 to 1.7 Mb, depending on the strain (22, 23). The AF toxin biosynthesis gene cluster was also present on a single small chromosome of 1.05 Mb in the strawberry pathotype of A. alternata (18). Based on biological and pathological observations, those small chromosomes were regarded as supernumerary chromosomes, or conditionally dispensable chromosomes (CDCs) (10, 18, 22). Fungal supernumerary chromosomes, which are not important for normal growth but confer advantages for colonizing an ecological niche, such as infecting host plants, are regarded as CDCs (21). The functions and pathological roles of CDCs have been studied in the pea pathogen Nectria haematococca (11, 17, 25, 32, 43, 46).The origin and evolution of CDCs have been intriguing issues in the study of plant-microbe interactions. The supernumerary chromosomes of certain strains of N. haematococca have been suggested to have a different evolutionary history than essential chromosomes (ECs) in the same genome, and they might have been introduced into the genome by horizontal transfer from another strain (10, 12, 36). In Colletotrichum gloeosporioides, the 2-Mb supernumerary chromosome was transferred from a biotype A strain to a vegetative incompatible biotype B strain (19, 31). Transfer of the chromosome, however, did not affect the pathogenicity of the recipient fungus, perhaps because it did not harbor pathogenicity genes (19, 31). These results suggest that supernumerary chromosomes of fungi might have the capacity for horizontal transfer across an incompatibility barrier between two distinct strains.AAL toxins are HSTs produced by the tomato pathotype of A. alternata (synonym A. alternata f. sp. lycopersici, synonym Alternaria arborescens), the causal agent of Alternaria stem canker disease in tomatoes, which causes severe necrosis of susceptible tomato cultivars (15, 26, 35). AAL toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and termed sphinganine-analogue mycotoxins. AAL toxins and fumonisins are sphinganine-analogue mycotoxins, which are toxic to some plant species and mammalian cells (16, 48). They cause apoptosis in susceptible tomato cells and mammalian cells by inhibiting ceramide biosynthesis (9, 41, 45). In the tomato pathotype of A. alternata-tomato interactions, a major factor in pathogenicity is the production of host-specific AAL toxins capable of inducing cell death only in susceptible cultivars (3, 9, 48).In this study, we describe evidence showing that the ability to produce the host-specific AAL toxin and to infect host tomato plants could potentially be distributed among a population of strains of the A. alternata tomato pathotype by horizontal transfer of an entire pathogenicity chromosome of the pathogen.     

Treatment of Fungal Bioaerosols by a High-Temperature,Short-Time Process in a Continuous-Flow System

Airborne fungi, termed fungal bioaerosols, have received attention due to the association with public health problems and the effects on living organisms in nature. There are growing concerns that fungal bioaerosols are relevant to the occurrence of allergies, opportunistic diseases in hospitals, and outbreaks of plant diseases. The search for ways of preventing and curing the harmful effects of fungal bioaerosols has created a high demand for the study and development of an efficient method of controlling bioaerosols. However, almost all modern microbiological studies and theories have focused on microorganisms in liquid and solid phases. We investigated the thermal heating effects on fungal bioaerosols in a continuous-flow environment. Although the thermal heating process has long been a traditional method of controlling microorganisms, the effect of a continuous high-temperature, short-time (HTST) process on airborne microorganisms has not been quantitatively investigated in terms of various aerosol properties. Our experimental results show that the geometric mean diameter of the tested fungal bioaerosols decreased when they were exposed to increases in the surrounding temperature. The HTST process produced a significant decline in the (1→3)-β-d-glucan concentration of fungal bioaerosols. More than 99% of the Aspergillus versicolor and Cladosporium cladosporioides bioaerosols lost their culturability in about 0.2 s when the surrounding temperature exceeded 350°C and 400°C, respectively. The instantaneous exposure to high temperature significantly changed the surface morphology of the fungal bioaerosols.Fungi are omnipresent in indoor and outdoor environments (2, 28, 39). Most fungi are dispersed through the release of spores into the air, a phenomenon known to be driven by two kinds of energy (17): the energy provided by the fungus itself and the energy provided by external sources, such as air currents, rain, gravity, or changes in temperature and nutritional sources. Of these various mechanisms of fungal particle release, dispersal by air currents is the most prevalent mechanism for indoor fungal particles (19, 31). These airborne fungal spores, termed fungal bioaerosols, are resistant to environmental stresses and are adapted to airborne transport.Fungal bioaerosols constitute the major component of ambient airborne microorganisms (23, 50, 51). Several studies have reported that the concentration of fungal bioaerosols is relevant to the occurrence of human diseases and public health problems associated with acute toxic effects, allergies (3, 18), and asthma (4, 5, 13, 48). Fungal bioaerosols are of particular concern in healthcare facilities, where they can cause major infectious complications as opportunistic pathogens in patients with an immunodeficiency (9). For instance, invasive mycoses can affect patients undergoing high-dose chemotherapy for hematological malignancies associated with a prolonged period of neutropenia; they can also affect solid-organ transplant recipients. Despite all diagnostic and therapeutic efforts, the outcome of an invasive fungal infection is often fatal (with a mortality rate of around 50% for aspergillosis) (37). The main fungal genera responsible for these infections are as follows: Aspergillus spp., Fusarium spp., Scedosporium spp., and Mucorales spp. (10, 12, 20). However, virtually any filamentous fungus can be a pathogen (22, 41). In the hospital environment, possible sources of airborne nosocomial infection include ventilation or air-conditioning systems, decaying organic material, dust, water, food, ornamental plants, and building materials in and around hospitals (1).One of the major bioaerosols of concern is (1→3)-β-d-glucans, which comprises up to 60% of the cell wall of most fungal organisms. The (1→3)-β-d-glucans are glucose polymers with a variable molecular weight and a degree of branching (49). The results of several studies about the exposure of subjects to airborne (1→3)-β-d-glucans suggest that these agents play a role in bioaerosol-induced inflammatory responses and resulting respiratory symptoms, such as a dry cough, phlegmy cough, hoarseness, and atopy (11, 44). In addition, given that many epidemiological studies have reported that (1→3)-β-d-glucan has strong immuno-modulating effects (42, 47), (1→3)-β-d-glucan is an important parameter for exposure assessment by itself and as a surrogate component for fungi (16).To prevent the adverse health effects of fungal bioaerosols, we must ensure that control methods for airborne fungal spores are studied and developed. However, despite the necessity of controlling fungal bioaerosols, few studies have focused on such control mechanisms. The most common control methods are UV irradiation and electric ion emission. Given that UV irradiation is known to have a germicidal effect, several studies have examined how UV irradiation affects the viability of bioaerosols (35, 42). However, although UV irradiation can be easily applied by simply installing and turning on a UV lamp, the 254-nm-wavelength UV light produces ozone and radicals, which cause harmful effects to surrounding humans. Electric ion emission has also been studied as a means of controlling bioaerosols (21, 27). When the efficiency of the filter is increased, the efficacy of respiratory protection devices against bioaerosols can be enhanced. Although electric ions decrease the viability of airborne bacteria (25), the generation of the ions produces ozone, a pollutant, and also causes electric charges to accumulate on surrounding surfaces.Recently, heat treatment of indoor air using thermal processes has been considered a safe, effective, and environment-friendly method; it does not produce ozone or use ion or filter media. A thermal heating process has long been considered a suitable and reliable method for controlling microorganisms. Two types of heat are generally used, moist heat and dry heat. Moist heat utilizes steam under pressure, whereas dry heat involves high-temperature exposure without additional moisture. Several types of heat treatment are currently used for killing microorganisms. The treatments include incineration, Tyndallization, pasteurization, and autoclaving (32). However, most of these technologies were originally limited to controlling microorganisms in liquid or on material surfaces. In addition, they may not be adequate for controlling bioaerosols because the continuous surrounding environment of bioaerosols is significantly different from the conditions in liquid and on solid surfaces. Therefore, it is necessary to find adequate and practical conditions for controlling bioaerosols. Thus far, several investigations regarding the use of thermal processes against bioaerosols have been reported. Some of these studies have targeted airborne bacteria spores widely used as surrogates for biological warfare agents (8, 34), while others have focused on environmental parameters for the culture and survival of various vegetative cells (14, 29, 46). However, in these studies novel techniques for aerosols, such as measuring and analyzing aerosol particle size, distributions, and concentrations, were not utilized. In addition, to the best of our knowledge, there has been no study on the use of a thermal process for controlling fungal bioaerosols in continuous airflow. Fungal bioaerosols were found to be very resistant to a thermal environment in previous studies.In this study, we investigated the thermal heating effects on the physical, chemical, and biological properties of fungal bioaerosols using a high-temperature, short-time (HTST) sterilization process. The HTST process, a type of thermal heating process, is based on high-temperature stresses for very short periods. Although this thermal process has been used for the microbial decontamination of seeds and dried, powdered products, such as pharmaceuticals and heat-sensitive drink and food, it can be also applied to the control of an airborne microorganism in a continuous-flow system, such as a heating, ventilation, and air-conditioning system (15, 33, 38). When the fungal bioaerosol was passed through a thermal electric heating system, the fungal spores were exposed to various temperatures for short periods. Then, we examined the bioaerosol and aerosol characteristics, including aerosol size distribution, culturability, (1→3)-β-d-glucan production, and surface morphology, using a novel technique for sampling and measuring aerosols.