Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.     

Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community▿

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.     

Ectomycorrhizal fungal communities in late-successional Swedish boreal forests, and their composition following wildfire

This study was conducted to evaluate the effects of wildfires on ectomycorrhizal (EM) fungal communities in Scots pine ( Pinus sylvestris ) stands. Below- and above-ground communities were analysed in terms of species richness and evenness by examining mycorrhizas and sporocarps in a chronosequence of burned stands in comparison with adjacent unburned late-successional stands. The internal transcribed spacer (ITS)-region (rDNA) of mycobionts from single mycorrhizas was digested with three restriction enzymes and compared with an ITS–restriction fragment length polymorphism (RFLP) reference database of EM sporocarps. Spatial variation seemed to be more prominent than the effects of fire on the EM fungal species composition. Most of the common species tended to be found in all sites, suggesting that EM fungal communities show a high degree of continuity following low-intensity wildfires. Species richness was not affected by fire, whereas the evenness of species distributions of mycorrhizas was lower in the burned stands. The diversity of EM fungi was relatively high considering that there were only three EM tree species present in the stands. In total, 135 EM taxa were identified on the basis of their RFLP patterns; 66 species were recorded as sporocarps, but only 11 of these were also recorded as mycorrhizas. The species composition of the below-ground community of EM fungi did not reflect that of the sporocarps produced. EM fungal species present in our ITS–RFLP reference database accounted for 54–99% of the total sporocarp production in the stands, but only 0–32% of the mycorrhizal abundance.     

Dynamics of bacterial and fungal communities on decaying salt marsh grass

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

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

Ectomycorrhizal community structure in a xeric Quercus woodland based on rDNA sequence analysis of sporocarps and pooled roots

Quercus woodlands are key components of California's wild landscapes, yet little is known about ectomycorrhizal (EM) fungi in these ecosystems. We examined the EM community associated with Quercus douglasii using sporocarp surveys and by pooling EM roots and subjecting them to DNA extraction, polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP) screening and DNA sequencing. Ectomycorrhizal root symbionts were sampled four times in 2003-04. During this time, the below-ground community structure was relatively stable; we found no evidence of taxa adapted to winter or spring conditions and only one species varied widely in occurrence between years. The EM community from sporocarps and roots was diverse (161 species), rich in Ascomycota (46 species), and dominated by fungi with cryptic sporocarps. This included a large number of resupinate and hypogeous taxa, many of which were detected both above- and below-ground.     

毛竹入侵阔叶林对土壤真菌群落的影响

为揭示天目山毛竹入侵原始阔叶林后土壤真菌群落特征的变化,采用T-RFLP以及荧光定量PCR技术,分析毛竹纯林、竹阔混交林及原始阔叶林土壤真菌群落结构和数量特征.结果表明： 土壤真菌群落结构差异在毛竹纯林和阔叶林之间最为明显,其次为竹阔混交林和阔叶林;竹阔混交林土壤具有最高的真菌Shannon指数、均匀度指数及最低的Simpson指数.硝态氮含量和pH显著影响了真菌群落结构的变异,毛竹林土壤真菌群落结构受pH和铵态氮影响较大,而阔叶林主要受硝态氮影响.阔叶林土壤真菌数量显著高于毛竹纯林和竹阔混交林,真菌数量分别与土壤pH和硝态氮呈现显著负相关和正相关.表明真菌在阔叶林土壤中介导了异养硝化作用,毛竹入侵可能对此过程产生了显著影响.     

Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

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

Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium

In order to clarify the functional role of individual ectomycorrhizal (EcM) fungal species in the field, we need to relate their abundance and distribution as mycorrhizas to their abundance and distribution as extramatrical mycelium (EMM). We divided each of four 20 cm x 20 cm x 2 cm slices of pine forest soil into 100 cubes of 2 cm x 2 cm. For each cube, ectomycorrhizas were identified and the presence of EMM of the EcM fungi recorded as ectomycorrhizas was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA. Ectomycorrhizas and EMM of seven EcM species were mapped. Spatial segregation of mycorrhizas and EMM was evident and some species produced their EMM in different soil layers from their mycorrhizas. The spatial relationship between mycorrhizas and their EMM generally conformed to their reported exploration types, but EMM of smooth types (e.g. Lactarius rufus) was more frequent than expected. Different EcM fungi foraged at different spatial scales.     

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.     

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.     

Identical genotypes of an ericoid mycorrhiza-forming fungus occur in roots of Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) in an Australian sclerophyll forest

Assemblages of fungi associated with roots of cooccurring Epacris pulchella ( Ericaceae ) and Leptospermum polygalifolium ( Myrtaceae ) seedlings at a sclerophyll forest site in New South Wales, Australia, were investigated by direct DNA extraction and analysis of rRNA gene internal transcribed spacer (ITS) products by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses. While ordination of the DGGE data suggested that the assemblages did not differ significantly between the two plant taxa, T-RFLP data provided marginal statistical support for the presence of different assemblages. Fungi isolated from roots of both plants were identified by ITS sequence comparisons largely as ascomycetes, several of which had close sequence identity to Helotiales ericoid mycorrhizal (ERM) fungi. One isolate morphotype from E. pulchella had close sequence similarity to ectomycorrhizal fungi in the Cenococcum geophilum complex, and neighbour-joining analysis grouped this strongly with other Australian C. geophilum- like sequences. Distribution of genotypes of an ERM Helotiales ascomycete in root systems of the two plant taxa was also investigated using inter-simple sequence repeat (ISSR)-PCR. Nineteen ISSR genotypes were identified, two of which were present in roots of both plant taxa. The results are discussed in the context of potential mycelial connections between Ericaceae and non- Ericaceae plants.     

Soil chemical properties,rather than landscape context,influence woodland fungal communities along an urban‐rural gradient

With the expansion of cities around the world there is a growing interest in the factors that influence biodiversity and ecosystem processes in urban areas. Fungi are exceptionally diverse and play key roles in ecosystem function, yet despite predictions of negative impacts due to urbanization, fungi have been generally overlooked in urban ecological studies. We surveyed fungi in 16 remnant river red gum (Eucalyptus camaldulensis: Myrtaceae) woodlands along a gradient of 4–35 km from the city of Melbourne (south‐east Australia). Using both sporocarp surveys and terminal restriction fragment length polymorphism (T‐RFLP; primer pair ITS1‐F‐ITS4), we examined relationships between fungal community composition, landscape context (i.e. urbanization) and soil physicochemical properties. Community compositions from sporocarp data were significantly correlated with those from T‐RFLP data, largely because of correlations with ectomycorrhizal sporocarps (Spearman rank correlation coefficients ρ 0.31–0.42) rather than saprotrophic fungi (ρ 0.18–0.21). Principal components analysis of soil properties and non‐metric multidimensional scaling ordinations of fungal community composition showed no clear separation of sites according to urbanization, and there were no significant correlations between fungal community composition and urbanization. However, fungal community composition was significantly correlated with soil chemical properties (ρ 0.41–0.55). These data suggest that site‐scale soil properties, and associated effects of past and current land management activities, were more important in determining fungal community composition than the landscape‐level influences of urbanization.     

Small mammal mycophagy in hemiboreal forest communities of Lithuania

The diets of small mammals in different hemiboreal spruce-dominated, oak-dominated and mixed forests in western part of Lithuania were studied by examination of fungal spores in fresh fecal pellets of caught animals. In the diets of mice (Apodemus spp.), bank voles (Myodes glareolus), and common and pygmy shrews (Sorex araneus and S. minutus), 22 different fungal taxa were identified, 15 of which were hypogeous fungi. The sporocarp abundance and the spores in fecal samples of Elaphomyces fungi prevailed in study area during this investigation. Although most of the captured individuals consumed fungi, the consumption varied among small mammal species. The data show that the fungi were more frequent and taxonomically diverse in Myodes glareolus than in Apodemus spp. diets. The study provided evidence that the fungal component in the diets of insectivorous Sorex species is more diverse than previously known. The availability of sporocarps and the fungal component in the diets of small mammals showed seasonal effects. Annual hypogeous and epigeous sporocarp abundances did not vary significantly across forest types. The significant difference in mycophagy was observed across all forest cover types, with the greatest fungal diversity in fecal samples collected in mixed coniferous-deciduous tree stands.     

Allocasuarina tree hosts determine the spatial distribution of hypogeous fungal sporocarps in three tropical Australian sclerophyll forests

Across three tropical Australian sclerophyll forest types, site-specific environmental variables could explain the distribution of both quantity (abundance and biomass) and richness (genus and species) of hypogeous fungi sporocarps. Quantity was significantly higher in the Allocasuarina forest sites that had high soil nitrogen but low phosphorous. Three genera of hypogeous fungi were found exclusively in Allocasuarina forest sites including Gummiglobus, Labyrinthomyces and Octaviania, as were some species of Castoreum, Chondrogaster, Endogone, Hysterangium and Russula. However, the forest types did not all group according to site-scale variables and subsequently the taxonomic assemblages were not significantly different between the three forest types. At site scale, significant negative relationships were found between phosphorous concentration and the quantity of hypogeous fungi sporocarps. Using a multivariate information theoretic approach, there were other more plausible models to explain the patterns of sporocarp richness. Both the mean number of fungal genera and species increased with the number of Allocasuarina stems, at the same time decreasing with the number of Eucalyptus stems. The optimal conditions for promoting hypogeous fungi sporocarp quantity and sporocarp richness appear to be related to the presence and abundance of Allocasuarina (Casuarinaceae) host trees. Allocasuarina tree species may have a higher host receptivity for ectomycorrhizal hypogeous fungi species that provide an important food resource for Australian mycophagous animals.     

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.     

Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests?

A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiss and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata.     

Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities

We have investigated colonization strategies by comparing the abundance and frequency of ectomycorrhizal fungal species on roots in a mature Pinus muricata forest with those present as resistant propagules colonizing potted seedlings grown in the same soil samples. Thirty-seven fungal species were distinguished by internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs); most were identified to species level by sporocarp RFLP matches or to genus/family level by using sequence databases for the mitochondrial and nuclear large-subunit rRNA genes. The below-ground fungal community found in the mature forest contrasted markedly with the resistant propagule community, as only four species were found in both communities. The dominant species in the mature forest were members of the Russulaceae, Thelephorales and Amanitaceae. In contrast, the resistant propagule community was dominated by Rhizopogon species and by species of the Ascomycota. Only one species, Tomentella sublilacina (Thelephorales), was common in both communities. The spatial distribution of mycorrhizae on mature roots and propagules in the soil differed among the dominant species. For example, T. sublilacina mycorrhizae exhibited a unique bias toward the organic horizons, Russula brevipes mycorrhizae were denser and more clumped than those of other species and Cenococcum propagules were localized, whereas R. subcaerulescens propagules were evenly distributed. We suggest that species differences in resource preferences and colonization strategies, such as those documented here, contribute to the maintenance of species richness in the ectomycorrhizal community.     

The annual invasive plant Impatiens glandulifera reduces hyphal biomass of soil fungi in deciduous forests

Soil fungi play a crucial role in ecosystem functioning and there is increasing evidence that exotic plants invading forests can affect soil fungal communities. We examined potential effects of the invasive plant Impatiens glandulifera on hyphal biomass of ectomycorrhizal fungi, their genetic diversity and the diversity of other soil fungi in deciduous forests in Switzerland. We compared invaded patches with patches where I. glandulifera had been removed, by establishing pairs of 3-m long transect lines at the edge of seven areas of either type. Along the transects we assessed the length of ectomycorrhizal fungal hyphae using the ‘ingrowth mesh bag method’, and used terminal restriction fragment length polymorphism (T-RFLP) analysis to examine fungal genetic diversity. The invasive plant reduced fungal hyphal biomass by 30–80%: the reduction was largest in the centre of the patch. I. glandulifera did not alter fungal richness, but affected the composition of fungal communities. This is probably the result of a decrease of mycorrhizal fungi, coupled with an increase of saprotrophic fungi. Our findings demonstrate the adverse impacts of an annual invasive plant species on both fungal hyphal biomass and the composition of soil fungal communities. This may negatively affect forest nutrient and carbon cycling, soil stability and the functionality of the fungal community, with major consequences for forest ecosystem functioning.     

云南松(Pinus yunnanensis)林外生菌根真菌的时空分布

2000年至2005年,调查了滇中及其附近云南松林下外生菌根真菌的生态分布,共采集、鉴定标本834号,计有27科39属211种（含变种、变型）。结果表明,红菇属（Russula）、牛肝菌属（Boletus）、乳菇属（Lactarius）、乳牛肝菌属（Suillus）、口蘑属（Tricholoma）、鸡油菌属（Cantharellus）和革菌属（Thelephora）等为云南松林下的主要外生菌根菌类群。它们的发生与分布受到气候（如：气温和降水）、植被（如：林龄、林地郁闭度和草本植被）、地形特征（如：海拔、坡向和坡度）、土壤条件（如：pH值、地表腐殖质和枯枝落叶层等）和人为干扰（比如：商业化采集、林木采伐、火烧和地表物清理）诸多因素的影响。总结为如下：（1）5a的调查结果显示,云南松外生菌根菌的分布表现出季节性变化的规律;其中以每年1、2、3月份的物种多样性为最低,雨季期间急剧增加,至中夏和秋末达到顶峰,种类最为繁多。（2）在海拔1500-2100m,云南松外生菌根菌种类随着海拔的升高而逐渐增加,至顶峰后,又呈缓慢下降趋势。海拔因素不但对其物种多样性,而且对于类群的组成也具有重要的影响。特定的类群往往发生在特定海拔范围。（3）随着云南松林龄的增加,外生菌根菌呈现由少至多的演替过程。外生菌根菌多样性随云南松林生长而逐渐增加的演替方式,可能与宿主光合作用产物、根部分泌物和土壤条件的逐渐变化有关。（4）人类干扰是云南松外生菌根菌物种多样性和类群组成的主要负影响因子。大规模的商业化采集可破坏或枯竭地下菌丝体,打破各物种之间的竞争平衡,减少孢子释放影响资源再生能力,进而直接影响到子实体的产生。外生菌根菌物种多样性的减少趋势会随林木砍伐和火烧强度的增加而加剧。地表枯枝落叶层与杂草密度也会影响子实体的产生,其中枯枝落叶层的厚度与云南松外生菌根菌子实体的发生呈负相关性,而被紫茎泽兰覆盖的云南松林地内也很少会发现相应的子实体。