Identification of Internal Transcribed Spacer Sequence Motifs in Truffles: a First Step toward Their DNA Bar Coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

Reevaluation of the Life Cycle of Tuber magnatum

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.     

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.     

Selection of a set of specific primers for the identification of Tuber rufum: a truffle species with high genetic variability

Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.     

Effects of drought stress and arbuscular mycorrhiza on the growth of Gliricidia sepium (Jacq). Walp, and Leucaena leucocephala (Lam.) de Wit. in simulated eroded soil conditions

A greenhouse investigation was conducted to determine the effect of arbuscular mycorrhiza and drought on the growth of two tropical hedgerow legume trees (Gliricidia sepium and Leucaena leucocephala) under simulated eroded soil conditions. It was a factorial design with two levels of watering regime (adequate watering and drought), inoculation with Glomus deserticola (with and without), and two soil types (0-30 cm topsoil and 30-60 cm subsoil). Each treatment was replicated 3 times. After ten drought cycles, the growth of Gliricidia sepium in the subsoil was enhanced by mycorrhizal inoculation under both watering regimes whereas there was no significant contribution of mycorrhizal inoculation to the growth of L. leucocephala in both soil types under the two watering regimes. Drought stress significantly reduced most growth parameters for the two tree species in both soils with or without fungal inoculation. The N-fixing activity of Gliricidia sepium benefited from Glomus deserticola inoculation while that of L. leucocephala was not significantly affected in the topsoil. Mycorrhizal colonization was reduced for both tree species in the subsoil compared to the topsoil while it was significantly increased for both species in the subsoil when compared to the uninoculated subsoil counterpart. In the subsoil, inoculation of Gliricidia sepium with the mycorrhizal fungus increased root colonization by 89% and 73% under adequate watering and drought, respectively, whereas L. leucocephala had only a 38% and 42% increase in root colonization under comparative conditions in the subsoil. Thus Glomus deserticola inoculation may be beneficial to the growth of Gliricidia sepium in a badly eroded site where topsoil is missing.     

Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

The green-fluorescent protein (GFP) gene from the Pacific Northwest jellyfish, Aequorea victoria, was used as a screenable marker in the production of transgenic barley plants. Isolated barley microspore culture was biolistically transformed with two synthetic forms of GFP, sgfp and pgfp. Thirty-seven fluorescing multicellular structures were isolated using epifluorescent microscopy. Sixteen structures developed shoots, but only five regenerated into green plants. Three events had been co-bombarded with #-glucuronidase (gus) and assayed positive for gus expression in the leaves, and all five events were positive for gfp expression. The expected transgene band size was PCR-amplified from all five plants, and Southern blots performed on three plants revealed unique patterns of gfp transgene integration. Fluorescent in situ hybridization also revealed the transgenic status and hemizygous nature of all the events. GFP-based visual screening provides a viable alternative method to chemical selection of transgenic plants from barley microspore culture.     

Molecular identification of Tuber magnatum ectomycorrhizae in the field

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).     

Intraspecific invariability of the internal transcribed spacer region of rDNA of the truffleTerfezia terfezioides in Europe

ITS regions (internal transcribed spacers—ITS1 andITS2—with the 5.8S gene of the nuclear rDNA) of 25 fruit body samples ofTerfezia terfezioides, originating from Hungary and Italy, were compared. The amplification and sequencing of the ITS region was successful with both theITS1-ITS4 andITSIF-ITS4 primer pairs. No differences of the restriction fragment length polymorphism profiles were detected among 19 samples collected in one place at the same time. The sequences of the ITS region of 9 samples collected in different localities were highly invariable, differing in only two bases. Thus the intraspecific homogeneity of the ITS region seems to be an important species-specific characteristic ofT. terfezioides in contrast to otherTerfezia species. As the samples of the species were collected from different and distant localities of Europe, the ITS sequence ofT. terfezioides can be considered a very conservative, reliable molecular marker of the fungus. *** DIRECT SUPPORT *** A00EN076 00008     

Fossil ectomycorrhizae from the Middle Eocene

Fossil ectomycorrhizae were found recently among permineralized plant remains in the middle Eocene Princeton chert of British Columbia. The ectomycorrhizae are associated with roots of Pinus and have a Hartig net that extends to the endodermis, a pseudoparenchymatous mantle, and contiguous extramatrical hyphae that are simple-septate. The mycorrhizal rootlets lack root hairs and dichotomize repeatedly to form large, coralloid clusters. Reproductive structures are absent. Based on the morphological characteristics, and the identity of the host, the closely related basidiomycete genera Rhizopogon and Suillus are suggested as comparable extant mycorrhizal fungi. These exquisitely preserved specimens represent the first unequivocal occurrence of fossil ectomycorrhizae and demonstrate that such associations were well-established at least 50 million years ago.     

The early-stage ectomycorrhizal Thelephoroid fungal sp. is competitive and effective on<Emphasis Type="Italic"> Afzelia africana</Emphasis> Sm. in nursery conditions in Senegal

This study was conducted to evaluate the competitiveness and effectiveness of Thelephoroid fungal sp. ORS.XM002 against native ectomycorrhizal fungal species colonizing potted Afzelia africana seedlings during 3 months of growth in different forest soils collected from under mature trees. Using morphotyping and restriction fragment length polymorphism (RFLP) analysis of the nuclear rDNA internal transcribed spacer (ITS), we were able to distinguish the introduced Thelephoroid fungal sp. ORS.XM002 among native ectomycorrhizal fungal species that form ectomycorrhizae in A. africana seedlings. The morphotype (MT) of the introduced fungus showed some color variation, with a shift from light- to dark-brown observed from younger to older mycorrhizal tips. We were able to differentiate the ITS type xm002 of the introduced fungus from the 14 ITS-RFLP types characterizing the 9 native MT that occurred in forest soils. The frequency of ITS type xm002 ranged from 40% to 49% depending on the forest soil used, and was always higher than those of ITS types from native dark-brown MT that occurred in inoculated seedlings 3 months after inoculation. We considered Thelephoroid fungal sp. ORS.XM002 to be responsible for stimulation of mycorrhizal colonization of inoculated A. africana seedlings when compared with control seedlings in forest soils. This fungus appeared to be more effective in increasing the root dry weight of A. africana seedlings. To identify the unknown introduced fungal species and native MT, we sequenced the ML5/ML6 region of the mitochondrial large subunit rRNA. Sequence analysis showed that these fungi belong to three ML5/ML6 groups closely related to the Cortinarioid, Thelephoroid, and Sclerodermataceous taxa. The molecular evidence for the persistence of Thelephoroid fungal sp. ORS.XM002 despite competition from native fungi argues in favor of using this fungus with A. africana in nursery soil conditions in Senegal.     

Seasonal variations of symbiotic ultrastructure and relationships of two natural ectomycorrhizae of beech (Fagus sylvatica/Lactarius blennius var. viridis and Fagus sylvatica/Lactarius subdulcis)

This study concerned two perennial ectomycorrhizae of beech: Fagus sylvatica L./Lactarius blennius var. viridis and Fagus sylvatica L./Lactarius subdulcis, collected in a natural forest site. Nutritional exchanges existing between beech roots and fungi were evaluated over a seasonal cycle by studying the variations of ultrastructure, polyphosphate and nitrogenous granules and glycogen content (periodic acid thiocarbohydrazide silver test). The acid phosphatase activity, which is an indicator of the intensity of the physiological activity of symbiosis partners, was also revealed by transmission electron microscopy. The occurrence of the two ectomycorrhizae in the soil was not related to the climatic conditions. In winter, the number of polyphosphate and nitrogenous granules was high in the fungi, and glycogen was abundant in the cytoplasm of hyphae. In late winter, an intense acid phosphatase activity was revealed on plasmalemmas of live hyphae. It could indicate the beginning of fungal reserve mobilization, which progressively disappeared as spring progressed. In summer, glycogen was quasi non-existent and only few polyphosphate and nitrogenous granules were observed. At this period, the acid phosphatase activity was weak. Finally, in autumn and early winter, glycogen and granules were progressively restored in fungal structures. Relationships between changes of granules and glycogen content and the physiological states of each symbiont throughout the year are discussed.     

Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe²⁺, BO₄^3-, Ca²⁺, Cu²⁺ and Zn²⁺, very low in PO₄^3- and lacking MoO₄^2-, NH₄⁺ and Co²⁺), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.     

Arbuscular mycorrhiza increased the activity of a biotrophic leaf pathogen – is a compensation possible?

Arbuscular mycorrhizal barley-plants were more susceptible to the obligate biotrophic shoot pathogen Erysiphe graminis f. sp. hordei. In experiments under greenhouse and open-air conditions on leaves of mycorrhizal plants, the sporulation rate of the mildew fungus was more than twice that on control plants. However, mycorrhizal plants suffered less than non-mycorrhizal plants in terms of grain number, ear yield and thousand-grain weight. Disease-yield-relationship analysis showed that the symbiosis neutralised the positive correlation between disease severity and yield loss (up to 25% infected leaf area tested). After mildew infection, nitrogen in ears of non-mycorrhizal barley was higher because of an impaired starch accumulation during grain filling. In mycorrhizal plants, leaf disease did not impair either the quantity or quality of grain yield. This improved compensation in mycorrhizal plants was related to maintained photosynthetic capacity and a delay in pathogen-induced senescence. Thus filling of long-term storage pools (fructans in internodes) and consequently reallocation of these reserves during grain filling was improved. The results suggest that higher availability of energy and material during grain formation, together with longer physiological activity, were the basis of yield maintenance and, therefore, expression of mycorrhiza-induced tolerance towards the pathogen.     

Assessing the quality of different ant species as partners of a myrmecophilous butterfly

We assessed the quality of different ant species as partners of the facultatively myrmecophilous lycaenid butterfly Glaucopsyche lygdamus. We compared disappearance and parasitism rates of G. lygdamus larvae in the field, and development of non-feeding prepupae in the laboratory, when individuals were untended or tended by one of four ant species. Formica podzolica was the only ant species to provide a clear benefit to G. lygdamus, in the form of reduced larval parasitism relative to untended larvae. F. 'neogagates' (F. neogagates + F. lasioides) and Tapinoma sessile were essentially neutral partners, providing no significant cost or benefit for any of the parameters measured. Relative to untended individuals, association with F. obscuripes significantly increased larval disappearance and significantly decreased pupal mass. Thus, F. obscuripes may act as a parasite of the general association between G. lygdamus and ants under certain conditions. Ant species also differed in their persistence as tenders of G. lygdamus larvae once an interaction was established. Over the lifetime of a larva, F. podzolica and F. obscuripes usually remained as the attendant ant species on plants over consecutive census dates, while F. 'neogagates' and T. sessile were frequently replaced, most commonly by F. obscuripes. It remains to be determined if disappearance and developmental outcomes reported here reflect true fitness costs (i.e. reduced survivorship and lower reproductive success) for G. lygdamus. The potential and limitations for specialization in association between G. lygdamus and high quality ant partners are discussed.     

Soil phosphorus heterogeneity and mycorrhizal symbiosis regulate plant intra-specific competition and size distribution

We investigated the interactive effects of soil phosphorus (P) heterogeneity, plant density and mycorrhizal symbiosis on plant growth and size variability of Trifolium subterraneum. We set up mesocosms (trays 49Ꮉ cm and 12 cm deep) with the same amount of available P, but distributed either homogeneously or heterogeneously, in randomly arranged cells (7ǻ cm each) with high or low available P. The trays were planted with either 1 or 4 seedlings of T. subterraneum per cell. Half of the trays were inoculated with spores of the mycorrhizal fungus Gigaspora margarita. We harvested the plants when leaves just started to overlap, 8 weeks after planting. Plants growing in high P cells had the lowest percentage infection, but the highest mean shoot and root biomass and root length. The mean size of the plants in each cell was determined mainly by local P concentration. However, in plants growing in high density, low P cells, ca. 20% of the variability in plant biomass was explained by the number of adjacent cells with high P. Patchy trays had the highest total shoot biomass, independently of mycorrhizal infection or plant density. Inoculated trays (M) had higher total shoot biomass and relative competition intensity (measured as reduction in plant biomass due to increased density) than non-inoculated trays (NM). Plant density reduced the plant response to mycorrhizal infection, and its effect was independent of P distribution. All populations growing in patchy trays, and low density mycorrhizal ones, had the highest plant-size inequality, presumably because patchy distribution of P and mycorrhizal infection increased competitive asymmetry. We conclude that mycorrhizal symbiosis has the potential to strongly influence plant population structure when soil nutrient distribution is heterogeneous because it promotes pre-emption of limiting resources.     

Arbuscular mycorrhizal fungi influence life history traits of a lepidopteran herbivore

Results from pot and microcosm studies in the greenhouse have shown that plant growth and foliar chemistry is altered by the presence and species composition of arbuscular mycorrhizal fungi (AMF). The growth and survival of herbivores which feed on plants could, as a consequence, also be affected by these mutualistic soil fungi. Consequently, interactions between AMF, plants and herbivores could occur. To test this, larvae of the common blue butterfly, Polyommatus icarus (Lycaenidae), were fed with sprigs of Lotus corniculatus (Fabaceae) plants which were inoculated with one of two different AMF species, with a mixture of these AMF species or with sprigs of plants which were not inoculated with AMF. Survival and larval weight of third instar larvae fed with plants colonised by AMF were greater than those of larvae fed with non-mycorrhizal plants. Survival of larvae feeding on non-mycorrhizal plants was 1.6 times lower than that of larvae feeding on plants inoculated with a mixture of AMF species and 3.8 times lower than that of larvae feeding on plants inoculated with single AMF species. Furthermore, larvae fed with non-mycorrhizal plants attained only about half the weight of larvae fed with mycorrhizal plants after 11 days of growth. These differences in larval performance might be explained by differences in leaf chemistry, since mycorrhizal plants had a 3 times higher leaf P concentration and a higher C/N-ratio. Our results, thus, show that the presence of belowground mutualistic soil fungi influences the performance of aboveground herbivores by altering their food quality. Larval consumption, larval food use and adult lipid concentrations of the common blue butterfly differed between larvae which were fed with plants inoculated with different AMF species. This suggests that the performance of herbivores is not only influenced by the presence of AMF but also depends on the identity of the AMF species colonising the host plants. Moreover, a significant interaction term between AMF species and maternal identity of the larvae occurred for adult dry weight, indicating that the performance of offspring from different females was differently influenced by AMF species composition. To our knowledge, these results show for the first time that the species composition of AMF communities can influence life-history traits of butterfly larvae and possibly herbivores in general.     

Identification of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis Makino (Triuridaceae)

The identity of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis was investigated by DNA analysis and examination of the morphology of the association. The morphological features of the mycorrhizal fungus, i. e. aseptate hyphal coils, vesicles, arbuscule-like branching, and degenerate coils were similar to those previously reported for other achlorophyllous plants. Spore-like propagules identified asa glomalean fungus were propagated from root pieces of S. tosaensis in pot culture using alfalfa as the host trap plant. A PCR product was obtained from colonized root of S. tosaensis using the taxon-specific primers, VANS1 and VAGLO. Sequence analysis of the DNA fragment showed it to be almost identical to other Glomus species. Although it has been reported many times that the morphology of mycorrhizal fungi in achlorophyllous plants is quite similar to that of arbuscular mycorrhizal fungi, this is the first report of the isolation and identification of such a fungus itself.