Javier Parladé

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

Abstract Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccariabicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L.bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain.

Coinoculation of aseptically grown Douglas fir with pairs of ectomycorrhizal fungi

Douglas fir seedlings grown under aseptic conditions in a peat-vermiculite substrate were inoculated with four pairs of ectomycorrhizal fungi to assess the relative inoculum dosages needed to establish two mycorrhizal fungi simultaneously in the same root system. The dual fungal combinations tested were: Pisolithus arhizus + Rhizopogon subareolatus, P. arhizus + R. roseolus, Laccaria bicolor + P. arhizus and L. bicolor + R. subareolatus. A total of 12 ml of inocula per plant was applied at the rates: 0+12, 3+9, 6+6, 9+3, 12+0, and 0+0 (v+v) for each combination. After 3 months growth, the number of mycorrhizas and uninfected short roots as well as the total plant biomass produced were recorded. Inoculations were successful with the fungal combinations P. arhizus + R. subareolatus and L. bicolor + P. arhizus. Plants developed P. arhizus and R. subareolatus mycorrhizas only at the rate 9Pa + 3Rs; at other rates tested, only monospecific mycorrhizas were formed. Plants developed L. bicolor and P. arhizus mycorrhizas at the three rates containing both fungi. L. bicolor behaved as an aggressive root colonizer and its level of root colonization remained constant at increasing rates of P. arhizus inoculum. L. bicolor displaced R. subareolatus at all inocula rates. P. arhizus displaced R. roseolus except at the rate 3Pa + 9Rr, with only a low number of mycorrhizas formed by either fungus. Total plant biomass was significantly increased by the presence of any fungal combination up to four times the values for uninoculated controls. P. arhizus and R. subareolatus were more effective in promoting plant growth and stimulating short root formation than either L. bicolor or R. roseolus.

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

Abstract 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.

Influence of the fertilisation method in controlled ectomycorrhizal inoculation of two Mediterranean pines

The influence of the fertilisation method: soluble (SF) vs. slow-release fertiliser (SRF) and of inoculation with Laccaria laccata (Scop.) Fr., Pisolithus tinctorius (Pers.) Coker & Couch and Melanogaster ambiguus (Vittad.) Tul & C. Tul. on ectomycorrhizal colonization and growth of Pinus pinea L. and Pinus pinaster Ait. was evaluated. For both pines, mycorrhization with L. laccata was not affected by the fertilisation method. Percentages of ectomycorrhizas (ECM) formed by P. tinctorius were dependent on the fertilisation method, the inoculum type (vegetative or spores) and the pine species involved. ECM formed by M. ambiguus were increased with fertilisation in both pines. Inoculation significantly improved P. pinea biomass when seedlings were fertilised with SRF whereas no effect was found in non-fertilised ones. For non-fertilised P. pinaster, inoculation with L. laccata and both inocula of P. tinctorius increased seedling biomass whereas fertilisation neutralised the fungal effect. Fertilisation increased P. pinea and P. pinaster biomass, independently of the inoculation treatment.RésuméL’impact sur le degré de mycorhization et la croissance de jeunes plants de Pinus pinea L. et de Pinus pinaster Ait., de deux méthodes de fertilisation (fertilisant soluble (FS) et fertilisant à libération lente) et d’une inoculation contrôlée avec Laccaria laccata (Sco.) Fr., Pisolithus tinctorius (Pers.) Coker et Couch et Melanogaster ambiguus (Vittad.) Tul et C. Tul. Pour les deux pins, la mycorhization avec Laccaria laccata n’a pas été modifiée par la méthode de fertilisation. Le pourcentage d’ectomycorrhizes (ECM) formé by P. tinctorius dépendait de la méthode de fertilisation, su type d’inoculum (spores ou inoculum végétatif) et de l’espèce de pin. La fertilisation a augmenté les ECM produites par Melanogaster ambiguus chez les deux pins. L’inoculation a augmenté significativement la biomasse des semis de Pinus pinea lorsqu’ils ont été fertilisés avec SRF tandis qu’aucun effet n’a été trouvé pour les traitements non fertilisés. Pour les semis non fertilisés de Pinus pinaster, l’inoculation avec Laccaria laccata et avec les deux inoculums de Pisolithus tinctorius a augmenté la biomasse des semis tandis que la fertilisation a neutralisé l’effet de l’inoculation. La fertilisation a augmenté la biomasse de Pinus pinaster et de Pinus pinea indépendamment du traitement d’inoculation utilisé.

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan® real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical–chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4 °C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques

ABSTRACT Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species. IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems.

Tools to Trace Truffles in Soil

Different methodological tools have been developed to trace truffles in soil, from fruiting body collections and mycorrhiza morphotyping to recent high-sensitive, molecular techniques able to detect specific truffle DNA markers in the soil. Recent findings in disclosing the biological cycle of truffles based on molecular research have represented a significant step forward toward a rational cultivation. On the other hand, the relationships among the different phases of the symbiosis, mycorrhizas, extraradical mycelium, and fruiting body formation, remain unsolved. The detection and quantification of truffle mycelium in the soil along the truffle development might be the key to understand the processes leading to fertilization and fruiting body formation. Also, the knowledge of soil mycelium dynamics, at temporal and spatial scales, is necessary to evaluate the fungal responses to environmental changes and to develop appropriate management techniques to ensure fungal persistence and regular fruiting body production.

How Ectomycorrhizae Structures Boost the Root System

Mycorrhizae are classic examples to explain the mutualistic interaction between two different organisms in nature: the roots of a vascular plant and a fungus. Both species establish a permanent relationship, they live together in symbiosis, and that differentiates the nature of mycorrhizae from other plant–fungus interactions. Ectomycorrhizal associations increase the root exploration area in soil, boosting the potential for mineral nutrition, water availability, and mutual survival of plant and fungus. The diversity of ectomycorrhizal fungal communities in the roots of spermatophyte plants is impressively high and means a complex diversity of structures in the root system, including emanating hyphae and rhizomorphs, which enlarge its area of influence. The distribution of the ectomycorrhizae living in the root system in an ever-changing balance is conditioned by many factors. Some of them are related with the root morphology of the host tree and also with ectomycorrhizal morphology, but abiotic factors (such as soil properties) also play a role.