基于高通量测序的杨树人工林根际土壤真菌群落结构

研究不同根序细根根际土壤微生物群落组成结构对深入了解根系-微生物互作关系具有重要意义.本研究采用Illumina MiSeq测序平台,对杨树人工林非根际土壤和不同根序细根根际土壤的真菌群落结构进行分析.物种注释结果显示: 杨树1～2级根(R1)、3级根(R2)和4～5级(R3)根际及非根际土壤(NR)中分别包含128、124、130和101个真菌属,表明杨树根际存在对真菌群落构建的选择性机制.不同根序根际土壤中相对丰度>1%的真菌属有7个,木霉属在1～2级根根际土壤中丰度较高,毛孢子菌属和曲霉属分别是3级根和4～5级根根际土壤中丰度最高的真菌属.α多样性指数表明: 根际土壤真菌的多样性在不同根序间存在显著差异,低级根显著高于高级根(P<0.05).β多样性指数表明: 真菌群落随着序级的升高差异性不断上升,相似性不断降低.不同根序细根根际真菌群落的趋异化组成和结构与细根功能具有密切关系.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities

Fungal communities play a key role in ecosystem functioning. However, only little is known about their composition in plant roots and the soil of biomass plantations. The goal of this study was to analyze fungal biodiversity in their belowground habitats and to gain information on the strategies by which ectomycorrhizal (ECM) fungi form colonies. In a 2-year-old plantation, fungal communities in the soil and roots of three different poplar genotypes (Populus × canescens, wildtype and two transgenic lines with suppressed cinnamyl alcohol dehydrogenase activity) were analyzed by 454 pyrosequencing targeting the rDNA internal transcribed spacer 1 (ITS) region. The results were compared with the dynamics of the root-associated ECM community studied by morphotyping/Sanger sequencing in two subsequent years. Fungal species and family richness in the soil were surprisingly high in this simple plantation ecosystem, with 5944 operational taxonomic units (OTUs) and 186 described fungal families. These findings indicate the importance that fungal species are already available for colonization of plant roots (2399 OTUs and 115 families). The transgenic modification of poplar plants had no influence on fungal root or soil communities. Fungal families and OTUs were more evenly distributed in the soil than in roots, probably as a result of soil plowing before the establishment of the plantation. Saprophytic, pathogenic, and endophytic fungi were the dominating groups in soil, whereas ECMs were dominant in roots (87%). Arbuscular mycorrhizal diversity was higher in soil than in roots. Species richness of the root-associated ECM community, which was low compared with ECM fungi detected by 454 analyses, increased after 1 year. This increase was mainly caused by ECM fungal species already traced in the preceding year in roots. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localized ECM fungi.     

广西喀斯特地区毛唇芋兰根内与根际土壤真菌群落组成分析

为探索兰科(Orchidaceae)植物毛唇芋兰(Nervilia fordii)根内和根际土壤真菌群落多样性，该研究采用Illumina MiSeq高通量测序技术，分析了大新(DX)和龙州(LZ)两个样地(简称两地)毛唇芋兰根内和根际土壤的真菌组成。结果表明：(1)两地的毛唇芋兰根内和根际土壤真菌多样性很丰富，根际土壤真菌多样性均高于根内，主根的真菌多样性高于走茎。(2)通过测序共获得有效序列118 040条，207个可操作分类单元(OTUs)涉及8门19纲42目86科123属。(3)担子菌门(Basidiomycota)真菌是两地毛唇芋兰根内真菌的共同优势菌群，涉及胶膜菌科(Tulasnellaceae)、Trimorphomycetaceae、角担菌科(Ceratobasidiaceae)、马拉色菌科(Malasseziaceae)和小皮伞科(Marasmiaceae)等，其中优势科和优势属分别是胶膜菌科(75%)和瘤菌根菌属(Epulorhiza)(56%),而土壤中的优势菌属则是镰刀菌属(Fusarium)。综上认为，毛唇芋兰根内与根际土壤中的优势菌群既差异显著也存在一些共同...     

Root Colonization by Bipolaris sorokiniana in Different Cereals and Relations to Lesion Development and Natural Root Cortical Cell Death

A GUS-transformed strain of B. sorokiniana was used to study the relationship between fungal growth, lesion development and natural root cortical cell death (RCD) in roots of different cereals. Roots of 10-day-old seedlings, grown on filter paper, were inoculated with the fungus and at different time intervals lesion size and GUS-activity in the roots were determined. A significant, positive correlation was found between GUS-activity and ergosterol content in barley roots infected with this transformed strain and these results indicate that GUS can be used as a marker to study fungal growth in plant tissue. The fungus had a slower growth rate in resistant barley varieties, i.e., those producing smaller lesions, than in more susceptible varieties.
In wheat and triticale, the fungal growth was faster than in barley, despite the smaller and lighter coloured lesions in these species. This may be explained by the fact that wheat and triticale have a faster rate of root cortical senescence than barley. Presumably, dying cortex cells cannot respond to the fungal invasion by producing phenolic compounds that cause browning of the tissue. Among seven investigated cereal species, there was a positive correlation between the degree of RCD and fungal biomass increase in roots after inoculation.     

Co-existing grass species have distinctive arbuscular mycorrhizal communities

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing the majority of land plants, and are of major importance in plant nutrient supply. Their diversity is suggested to be an important determinant of plant community structure, but the influence of host-plant and environmental factors on AM fungal community in plant roots is poorly documented. Using the terminal restriction fragment length polymorphism (T-RFLP) strategy, the diversity of AM fungi was assessed in 89 roots of three grass species (Agrostis capillaris, Festuca rubra, Poa pratensis) that co-occurred in the same plots of a field experiment. The impact of different soil amendments (nitrogen, lime, nitrogen and lime) and insecticide application on AM fungal community was also studied. The level of diversity found in AM fungal communities using the T-RFLP strategy was consistent with previous studies based on clone libraries. Our results clearly confirm that an AM fungal host-plant preference exists, even between different grass species. AM communities colonizing A. capillaris were statistically different from the others (P < 0.05). Although grass species evenness changed in amended soils, AM fungal community composition in roots of a given grass species remained stable. Conversely, in plots where insecticide was applied, we found higher AM fungal diversity and, in F. rubra roots, a statistically different AM fungal community.     

Arbuscular mycorrhizal community composition associated with two plant species in a grassland ecosystem

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing about two-thirds of land plant species and found in all ecosystems. They are of major importance in plant nutrient supply and their diversity is suggested to be an important determinant of plant community composition. The diversity of the AM fungal community composition in the roots of two plant species (Agrostis capillaris and Trifolium repens) that co-occurred in the same grassland ecosystem was characterized using molecular techniques. We analysed the small subunit (SSU) ribosomal RNA gene amplified from a total root DNA extract using AM fungal-specific primers. A total of 2001 cloned fragments from 47 root samples obtained on four dates were analysed by restriction fragment length polymorphism, and 121 of them were sequenced. The diversity found was high: a total of 24 different phylotypes (groups of phylogenetically related sequences) colonized the roots of the two host species. Phylogenetic analyses demonstrate that 19 of these phylotypes belonged to the Glomaceae, three to the Acaulosporaceae and two to the Gigasporaceae. Our study reveals clearly that the AM fungal community colonizing T. repens differed from that colonizing A. capillaris, providing evidence for AM fungal host preference. In addition, our results reveal dynamic changes in the AM fungal community through time.     

Effects of Co-Inoculation with Arbuscular Mycorrhizal Fungi and Rhizobia on Fungal Occupancy in Chickpea Root and Nodule Determined by Real-Time PCR

Legume roots in nature are usually colonized with rhizobia and different arbuscular mycorrhizal fungi (AMF) species. Light microscopy that visualizes the presence of AMF in roots is not able to differentiate the ratio of each AMF species in the root and nodule tissues in mixed fungal inoculation. The purpose of this study was to characterize the dominant species of mycorrhiza in roots and nodules of plants co-inoculated with mycorrhizal fungi and rhizobial strains. Glomus intraradices (GI), Glomus mosseae (GM), their mix (GI + GM), and six Mesorhizobium ciceri strains were used to inoculate chickpea. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess occupancy of these fungal species in roots and nodules. Results showed that GI molecular ratio and relative density were higher than GM in both roots and nodules. These differences in molecular ratio and density between GI and GM in nodules were three folds higher than roots. The results suggested that M. ciceri strains have different effects on nodulation and mycorrhizal colonization pattern. Plants with bacterial S3 and S1 strains produced the highest root nodulation and higher fungal density in both the roots and nodules.     

Changes in mycorrhizal colonization and overall fungal communities across upland and wetland <Emphasis Type="Italic">Acer</Emphasis> forests of the Virginia Coastal Plain

Arbuscular mycorrhiza (AM) are common in the roots of most plants and provide benefits such as enhanced uptake of soil nutrients and water when the environment limits these resources. Two primary factors proposed in the literature as limiting the extent of mycorrhizal colonization of wetland plants are: (1) the low oxygen environment; and (2) the level of available phosphorus. The present study evaluated AM colonization of maple (Acer) roots and the fungal community structure in paired upland/wetland locations in the Virginia Coastal Plain to determine if wetland conditions affected the root fungal community. The range of observed AM colonization, based on 60 samples collected on 11 dates covering 3 years, was from 4 to 55%. Overall, soil redox potential and available phosphorus concentration were not significant in explaining the difference in AM colonization of Acer roots. In fact, the site with the greatest percent colonization in this study was a wetland site. A comparison of fungal diversity between the pooled wetland site communities and the pooled upland site communities was conducted and the difference was significant (p = 0.003), but the upland sites exhibited the lower diversity. Communities from all sites were dissimilar. Geographic location exerted a greater effect over community structure than did upland versus wetland status. This study concludes that the forested wetland environments studied here neither limited AM colonization nor reduced the overall fungal community and in fact may present a more favorable environment for fungal diversity.     

Various forms of organic and inorganic P fertilizers did not negatively affect soil- and root-inhabiting AM fungi in a maize–soybean rotation system

Arbuscular mycorrhizal (AM) fungi are key components of most agricultural ecosystems. Therefore, understanding the impact of agricultural practices on their community structure is essential to improve nutrient mobilization and reduce plant stress in the field. The effects of five different organic or mineral sources of phosphorus (P) for a maize–soybean rotation system on AM fungal diversity in roots and soil were assessed over a 3-year period. Total DNA was extracted from root and soil samples collected at three different plant growth stages. An 18S rRNA gene fragment was amplified and taxa were detected and identified using denaturing gradient gel electrophoresis followed by sequencing. AM fungal biomass was estimated by fatty acid methyl ester analysis. Soil P fertility parameters were also monitored and analyzed for possible changes related with fertilization or growth stages. Seven AM fungal ribotypes were detected. Fertilization significantly modified soil P flux, but had barely any effect on AM fungi community structure or biomass. There was no difference in the AM fungal community between plant growth stages. Specific ribotypes could not be significantly associated to P treatment. Ribotypes were associated with root or soil samples with variable detection frequencies between seasons. AM fungal biomass remained stable throughout the growing seasons. This study demonstrated that roots and soil host distinct AM fungal communities and that these are very temporally stable. The influence of contrasting forms of P fertilizers was not significant over 3 years of crop rotation.     

Vertical Transmission of Endobacteria in the Arbuscular Mycorrhizal Fungus Gigaspora margarita through Generation of Vegetative Spores

Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences, these endobacteria have recently been identified as a new taxon, Candidatus Glomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungal reproduction (asexual), a system based on transformed carrot roots and single-spore inocula of Gigaspora margarita was used. Under these in vitro sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored, and it was shown, by using primers designed for both 16S and 23S ribosomal DNAs, to occur through several vegetative spore generations (SG0 to SG4). A method of confocal microscopy for quantifying the density of endobacteria in spore cytoplasm was designed and applied; endobacteria were consistently found in all of the spore generations, although their number rapidly decreased from SG0 to SG4. The study demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus, and suggests that these bacteria are obligate endocellular components of their AM fungal host.     

Arbuscular mycorrhizal colonization of giant sequoia (Sequoiadendron giganteum) in response to restoration practices

Interactions with soil microbiota determine the success of restoring plants to their native habitats. The goal of our study was to understand the effects of restoration practices on interactions of giant sequoia Sequoiadendron giganteum with arbuscular mycorrhizal (AM) fungi (Glomeromycota). Natural regeneration of Sequoiadendron is threatened by the absence of severe fires that create forest canopy gaps. Generating artificial canopy gaps offers an alternative tool for giant sequoia restoration. We investigated the effect of regeneration practices, including (i) sapling location within gaps, (ii) gap size and (iii) soil substrate, on AM fungal colonization of giant sequoia sapling roots in a native giant sequoia grove of the Sierra Nevada, California. We found that the extent of AM fungal root colonization was positively correlated with sapling height and light availability, which were related to the location of the sapling within the gap and the gap size. While colonization frequency by arbuscules in saplings on ash substrate was higher relative to saplings in mineral soil, the total AM fungal root colonization was similar between the substrates. A negative correlation between root colonization by Glomeromycota and non-AM fungal species indicated antagonistic interactions between different classes of root-associated fungi. Using DNA genotyping, we identified six AM fungal taxa representing genera Glomus and Ambispora present in Sequoiadendron roots. Overall, we found that AM fungal colonization of giant sequoia roots was associated with availability of plant-assimilated carbon to the fungus rather than with the AM fungal supply of mineral nutrients to the roots. We conclude that restoration practices affecting light availability and carbon assimilation alter feedbacks between sapling growth and activity of AM fungi in the roots.     

Resistance and resilience of root fungal communities to water limitation in a temperate agroecosystem

Understanding crop resilience to environmental stress is critical in predicting the consequences of global climate change for agricultural systems worldwide, but to date studies addressing crop resiliency have focused primarily on plant physiological and molecular responses. Arbuscular mycorrhizal fungi (AMF) form mutualisms with many crop species, and these relationships are key in mitigating the effects of abiotic stress in many agricultural systems. However, to date there is little research examining whether (1) fungal community structure in agroecosystems is resistant to changing environmental conditions, specifically water limitation and (2) resilience of fungal community structure is moderated by agricultural management systems, namely the integration of trees into cropping systems. Here, we address these uncertainties through a rainfall reduction field experiment that manipulated short‐term water availability in a soybean‐based (Glycine max L. Merr.) agroforest in Southern Ontario, Canada. We employed terminal restriction fragment length polymorphism analysis to determine the molecular diversity of both general fungal and AMF communities in soybean roots under no stress, stress (rainfall shelters added), and poststress (rainfall shelters removed). We found that general fungal and AMF communities sampled from soybean roots were resistant to rainfall reduction in a monoculture, but not in an agroforest. While AMF communities were unchanged after stress removal, general fungal communities were significantly different poststress in the agroforest, indicating a capacity for resiliency. Our study indicates that generalist fungi and AMF are responsive to changes in environmental conditions and that agroecosystem management plays a key role in the resistance and resilience of fungal communities to water limitation.     

Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forest

We have used molecular techniques to investigate the diversity and distribution of the arbuscular mycorrhizal (AM) fungi colonizing tree seedling roots in the tropical forest on Barro Colorado Island (BCI), Republic of Panama. In the first year, we sampled newly emergent seedlings of the understory treelet Faramea occidentalis and the canopy emergent Tetragastris panamensis, from mixed seedling carpets at each of two sites. The following year we sampled surviving seedlings from these cohorts. The roots of 48 plants were analysed using AM fungal-specific primers to amplify and clone partial small subunit (SSU) ribosomal RNA gene sequences. Over 1300 clones were screened for random fragment length polymorphism (RFLP) variation and 7% of these were sequenced. Compared with AM fungal communities sampled from temperate habitats using the same method, the overall diversity was high, with a total of 30 AM fungal types identified. Seventeen of these types have not been recorded previously, with the remainder being similar to types reported from temperate habitats. The tropical mycorrhizal population showed significant spatial heterogeneity and nonrandom associations with the different hosts. Moreover there was a strong shift in the mycorrhizal communities over time. AM fungal types that were dominant in the newly germinated seedlings were almost entirely replaced by previously rare types in the surviving seedlings the following year. The high diversity and huge variation detected across time points, sites and hosts, implies that the AM fungal types are ecologically distinct and thus may have the potential to influence recruitment and host composition in tropical forests.     

Mycorrhizal features and fungal partners of four mycoheterotrophic Monotropoideae (Ericaceae) species from Yunnan, China

We provide a preliminary report of the mycobionts found within four Monotropoideae (Ericaceae) species from China: Monotropa uniflora, Hypopitys monotropa, Monotropastrum humile and Monotropastrum sciaphilum (a rare endemic species never previously studied for mycorrhizae). Such achlorophyllous Monotropoideae plants obtain their carbohydrates from mycorrhizal fungi linking them to surrounding trees, on which these fungi form ectomycorrhizae. Since Monotropoideae were rarely studied in continental Asia, the root systems of the four species sampled in Yunnan were examined using morphological and molecular methods. All the roots of these four species exhibit a typical monotropoid mycorrhizal morphology, including a fungal mantle, a Hartig net and hyphal pegs. In M. uniflora and M. humile mycorrhizae, cystidia typical of Russula symbionts covered the fungal mantle. ITS barcoding revealed that Russulales were the most frequent colonizers in all species, but Hypopitys monotropa displayed various additional mycorrhizal taxa. Moreover, a few additional ectomycorrhizal and saprotrophic Basidiomycota taxa were identified in the three other species, challenging that these four Monotropoideae species are as strictly fungal specific as the other Monotropoideae species hitherto studied. Moreover, a comparison with accompanying fungus sporocarps revealed that the fruiting fungal community significantly differed from that associated with the Monotropoideae roots, so that a clear fungal preference was evident. Finally, four fungal species were found on more than one Monotropoideae species: this contrasted with previous reports of sympatrically growing mycoheterotrophic plants, which did not reveal any overlap. This again challenges the idea of strict fungal specificity.     

Distribution and diversity of arbuscular mycorrhizal fungi in grapevines from production vineyards along the eastern Adriatic coast

The colonisation and diversity of arbuscular mycorrhizal fungi (AMF) on roots of grapevines were investigated in production vineyards located along a 500-km-long stretch of karst along the coast of the Adriatic Sea. AMF communities on roots of grapevines were analysed using temporal temperature gel electrophoresis and sequencing of the 18S and internal transcribed spacer segments of the rDNA operon. The AMF colonisation of these grapevines roots was consistent along the whole of this east Adriatic karst region, at 64 to 82 % of fine roots. The comparison of the AMF communities on the roots of these grapevines showed that the fungal community associated with grapevine roots seems to be relatively stable, with inter-vineyard variability comparable to intra-vineyard variability. Some of the changes in the fungal communities were attributed to environmental factors (plant-available P) and location of the vineyard, although the latter could also have been influenced by an unmeasured environmental factor. A total of 27 taxa of fungi were identified, including taxa from Glomus group B, based on the sequencing of 18S rDNA. Sequencing of the internal transcribed spacer rDNA yielded 30 different fungal taxa, which comprised eight different Glomeromycota taxa, including Glomus sinuosum and Glomus indicum. To our knowledge, this is the first report of grapevine colonisation by G. indicum.     

Does the presence of arbuscular mycorrhizal fungi influence growth and nutrient uptake of a wild-type tomato cultivar and a mycorrhiza-defective mutant, cultivated with roots sharing the same soil volume?

We investigated the growth and nutrient uptake of the Lycopersicon esculentum symbiosis mycorrhiza-defective plant mutant rmc, challenged with arbuscular mycorrhiza (AM) fungal propagules, in the presence or absence of roots of the commercial wild-type tomato cv. Golden Queen (GQ). Two plants shared the middle (combi) compartment of a horizontal three-compartment split-root pot with one part of their root system; the other part was grown separately in an outer (solo) pot. Combinations of rmc and GQ plants were grown together in soil that was either mycorrhiza-free (-M) or prepared with AM fungal inoculum (+M). Surface colonization of rmc roots was strongly increased in the presence of (+M) GQ roots. AM fungal inoculation increased phosphorus uptake of GQ plants, but decreased growth and P uptake of rmc plants. Growth and P uptake of (+M) GQ plants were reduced when plants were grown in combination with rmc rather than another GQ plant. AM fungi in the (combi) compartment may have preferentially formed hyphae spreading infection rather than functioning in P uptake in (+M) GQ plants grown in combination with rmc. Surface colonization of (+M) rmc roots, in the presence of GQ roots, was probably established at the expense of carbohydrates from associated GQ plants. Possible reasons for a decreased P uptake of rmc plants in response to AM fungal inoculation are proposed.     

Impact of Collimonas bacteria on community composition of soil fungi

The genus Collimonas consists of soil bacteria that have the potential to grow at the expense of living fungal hyphae. However, the consequences of this mycophagous ability for soil fungi are unknown. Here we report on the development of fungal communities after introduction of collimonads in a soil that had a low abundance of indigenous collimonads. Development of fungal communities was stimulated by addition of cellulose or by introducing plants ( Plantago lanceolata ). Community composition of total fungi in soil and rhizosphere and of arbuscular mycorrhizal fungi in roots was examined by PCR-DGGE. The introduction of collimonads altered the composition of all fungal communities studied but had no effects on fungal biomass increase, cellulose degrading activity or plant performance. The most likely explanation for these results is that differences in sensitivity of fungal species to the presence of collimonads result in competitive replacement of species. The lab and greenhouse experiments were complemented with a field experiment. Mesh bags containing sterile sand with or without collimonads were buried in an ex-arable field and a forest. The presence of collimonads had an effect on the composition of fungi invading these bags in the ex-arable site but not in the forest site.