Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.     

A sheathing mycorrhiza between the tropical bolete Phlebopus spongiosus and Citrus maxima

Phlebopus (Ph.) spongiosus was recently described from several pomelo orchards (Citrus maxima) in southern Vietnam. This fungus was suspected to associate with pomelo plants as an ectomycorrhiza, although members of the genus Phlebopus have previously been presumed saprotrophic. To clarify this association, pomelo roots collected from the orchard (in situ roots), and those cultured with Ph. spongiosus (in vitro roots) in test tubes for 12 wk, were examined for ectomycorrhizal colonization. Both in vitro and in situ roots were analyzed for colonization using fungal LSU nuclear ribosomal DNA sequencing. The in situ roots exhibited the anatomical features of ectomycorrhizae: a thick fungal mantle, Hartig net, and extramatrical hyphae. The Hartig net, however, was very rare and showed discontinuous development. The in vitro association between Ph. spongiosus and C. maxima showed ectomycorrhiza-like structures, i.e., mantles and rhizomorphs in the plant roots, but no Hartig net development in the roots. Continuous hyphal penetration was restricted to the exodermis in both in situ and in vitro roots. Although the association between Ph. spongiosus and C. maxima could be considered ectomycorrhizal, its anatomy matches the unique feature known as sheathing mycorrhiza.     

Distinct Microbial Communities within the Endosphere and Rhizosphere of Populus deltoides Roots across Contrasting Soil Types

The root-rhizosphere interface of Populus is the nexus of a variety of associations between bacteria, fungi, and the host plant and an ideal model for studying interactions between plants and microorganisms. However, such studies have generally been confined to greenhouse and plantation systems. Here we analyze microbial communities from the root endophytic and rhizospheric habitats of Populus deltoides in mature natural trees from both upland and bottomland sites in central Tennessee. Community profiling utilized 454 pyrosequencing with separate primers targeting the V4 region for bacterial 16S rRNA and the D1/D2 region for fungal 28S rRNA genes. Rhizosphere bacteria were dominated by Acidobacteria (31%) and Alphaproteobacteria (30%), whereas most endophytes were from the Gammaproteobacteria (54%) as well as Alphaproteobacteria (23%). A single Pseudomonas-like operational taxonomic unit (OTU) accounted for 34% of endophytic bacterial sequences. Endophytic bacterial richness was also highly variable and 10-fold lower than in rhizosphere samples originating from the same roots. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Pezizomycotina (40%), while the Agaricomycotina were more abundant in the rhizosphere (34%) than endosphere (17%). Both fungal and bacterial rhizosphere samples were highly clustered compared to the more variable endophyte samples in a UniFrac principal coordinates analysis, regardless of upland or bottomland site origin. Hierarchical clustering of OTU relative abundance patterns also showed that the most abundant bacterial and fungal OTUs tended to be dominant in either the endophyte or rhizosphere samples but not both. Together, these findings demonstrate that root endophytic communities are distinct assemblages rather than opportunistic subsets of the rhizosphere.     

Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes

 We describe and document the in vitro synthesis of ectomycorrhiza between roots of wild type and transgenic aspen (Populus tremula × P. tremuloides), expressing Agrobacterium tumefaciens T-DNA indoleacetic acid (IAA)-biosynthetic genes, and Amanita muscaria. Plantlets were raised from tissue culture. The root system of approximately 4-week-old plantlets was transferred to Petri dishes and incubated together with fungal mycelia under sterile conditions. Ectomycorrhiza showing both a well developed hyphal mantle and Hartig net were established within 3 to 4 weeks. Formation and morphology of ectomycorrhiza were not affected by the transformation of aspen, expressing the IAA biosynthetic genes in roots. As both hybrid aspen and fungal cells can be genetically engineered, this system offers a new approach to the study of mycorrhizal symbioses. Received: 19 January 1996 / Accepted: 23 January 1996     

Ability of native ectomycorrhizal fungi from northern Spain to colonize Douglas-fir and other introduced conifers

 Thirty-six isolates from 27 species of native ectomycorrhizal fungi collected in northern Spain were tested for ectomycorrhiza formation with Pseudotsuga menziesii seedlings in pure culture syntheses. Thirteen of those species were also tested for ectomycorrhiza formation with six other species of conifers (two native and four introduced) to compare their colonization potential. Twenty-three fungal isolates from 18 species formed ectomycorrhizas with Pseudotsuga menziesii. The colonization level of the root system varied markedly among the different fungal species. Eight fungi colonized over 50% of the short roots. Nine fungi did not form ectomycorrhizas even though some of them were collected in pure stands of Pseudotsuga menziesii. Laccaria laccata, Lyophyllum decastes, Pisolithus tinctorius, and Scleroderma citrinum formed abundant ectomycorrhizas on all the conifers tested. Lactarius deliciosus, Rhizopogon spp., and Suillus luteus showed the greatest host specificity. The success in the introduction of some exotic conifers for reforestation in northern Spain is discussed in relation to their compatibility with native ectomycorrhizal fungi. Accepted: 28 August 1995     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza-forming Ceratobasidiaceae fungi

? We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. ? We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. ? In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. ? This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.     

Tubulin and actin protein patterns in Scots pine (Pinus sylvestris) roots and developing ectomycorrhiza with Suillus bovinus

The role of tubulin and actin in the development of Scots pine ( Pinus sylvestris ) roots and in the formation of the ectomycorrhiza with the basidiomycete Suillus bovinus was studied by immunoblotting of 2D-gels with anti-tubulin and anti-actin antibodies. In the short roots the α-tubulin pattern was different from that in the other root types due to the more acidic pI of the two α-tubulins. During the formation of the ectomycorrhiza, two new α-tubulins were detected in the acidic α-tubulin cluster. No such variation occurred in the plant β-tubulin patterns. The fungal tubulins dominated in the ectomycorrhiza, but no changes in tubulin polypeptide patterns from those in the S. bovinus mycelium were observed. Contrary to the tubulins, plant actin dominated in the mycorrhiza. The specific α-tubulin patterns of uninfected and infected short roots indicate that α-tubulin is involved in the morphogenesis of Pinus sylvestris short roots. The high level of plant actin at early stage of the mycorrhiza formation suggests a significant role of this protein in the interaction between plant cells and fungal hyphae.     

Dichotomization of mycorrhizal and NPA-treated short roots in Pinus sylvestris

Conifers like Scots pine (Pinus sylvestris) have a complicated root system consisting of morphologically and anatomically different root types, of which the short roots have a very limited ability to elongate. Short roots have an important role in nature since they are able to establish ectomycorrhizal symbiosis, in which the growth of fungal mycelium between the epidermal cells and in the intercellular space between cortical cells leads to formation of dichotomous short roots, which may, through further splitting of the meristem, form coralloid root structures. Dichotomous short roots have been suggested to result from changes in either auxin or ethylene concentrations due to the fungal growth inside the root. NPA, the inhibitor of polar auxin transport, enhances the dichotomization of P. sylvestris short root tips similarly to the fungal growth in the root, thus confirming that auxin plays a role in short root morphogenesis. Ethylene is also known to have an important role in the regulation of root morphogenesis. In future the research dealing with the root system and ectomycorrhiza development in P. sylvestris must take into account that both auxin and ethylene are involved and that there is no contradiction in obtaining the same phenotype with both hormones. The expression analysis of PIN proteins, auxin efflux carriers, could give valuable information about the role of auxin transport in regulating the root growth and morphogenesis of coniferous root system and mycorrhiza.Key words: auxin transport, dichotomization, ethylene, mycorrhiza, open meristem, PIN, Pinus sylvestris, short root     

赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用

本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不定根和愈伤组织均起源于无菌苗的下胚轴,接种2周后,菌丝体开始包围不定根。接种3周后,菌丝体出现在不定根皮层细胞间,哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中,细胞间也能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成外生菌根的报道。