Joan Pera

Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi

Abstract. Containerized Pinus pinea L. seedlings are commonly used for reforestation in the Mediterranean area. While there is an increasing knowledge of the potential ectomycorrhizal fungi associated with Pinus pinea, few studies exist of inoculation techniques with selected ectomycorrhizal fungi. We tested seven ectomycorrhizal fungi for their effectiveness with containerized Pinus pinea seedlings. Hebelomacrustuliniforme, Laccarialaccata and Pisolithustinctorius were applied as vegetative inocula while Melanogasterambiguus, Pisolithus tinctorius, Rhizopogonluteolus, Rhizopogonroseolus and Sclerodermaverrucosum were tested as spore inocula. The inoculum of each fungus was tested at several application rates. Among the fungi tested as vegetative inocula, the highest percentages of ectomycorrhizas were obtained with H. crustuliniforme at all rates tested. The ectomycorrhizas formed by L. laccata varied from 11% to 40% depending on the inoculum rate applied. Vegetative inoculum of Pisolithus tinctorius was only effective at the highest inoculum rates and gave mycorrhization percentages around 60%. Pisolithus tinctorius applied as a spore inoculum formed ectomycorrhizas at a frequency of about 50% at the effective inoculum rates. The rest of the fungi applied as spore inocula produced more than 50% of ectomycorrhizas at the effective spore concentrations. These included the highest percentages of ectomycorrhizas (>80%) obtained with both Rhizopogon species. Differences in growth due to inoculation with the different fungi were not detected and in some cases inoculation even reduced the total biomass accumulated by seedlings. All seedlings reached a size suitable for transplantation.

Inoculation of containerized Pseudotsuga menziesii and Pinus pinaster seedlings with spores of five species of ectomycorrhizal fungi

Abstract Container-grown Pseudotsuga menziesii and Pinus pinaster seedlings were inoculated with water suspensions of spores of five ectomycorrhizal fungi commonly found in northeastern Spain. Pseudotsuga menziesii seedlings were inoculated with basidiospores of Melanogaster ambiguus, or Rhizopogon subareolatus, or with ascospores of Tuber maculatum. Pinus pinaster seedlings were inoculated with basidiospores of Melanogaster ambiguus, Rhizopogon roseolus or Scleroderma citrinum. The spore concentrations were 102–107 spores per seedling for Melanogaster ambiguus (in Pseudotsuga menziesii) and Rhizopogon subareolatus, 103–107 for Melanogaster ambiguus (in Pinus pinaster), Rhizopogon roseolus, and Scleroderma citrinum, and 102–104 for Tuber maculatum. Melanogaster ambiguus colonized more short roots in a larger proportion of plants at 107 spores per seedling than at any other rate. The highest colonization by Rhizopogon subareolatus was obtained at 104 spores per seedling and higher, and all inoculated plants became infected at 106 spores per seedling and higher. Tuber maculatum colonized a high percentage of short roots at all rates tested; the proportion of infected plants was over 80% at 103–104 spores per plant, decreasing to 50% at 102 spores per plant. Rhizopogon roseolus colonized the highest number of short roots on nearly all the inoculated plants when applied at 105 spores per seedling and higher. Scleroderma citrinum colonized a high percentage of short roots on all inoculated plants when applied at 105 spores per seedling and higher. The abundance of sporocarps of Melanogaster ambiguus, Rhizopogon subareolatus, Rhizopogon roseolus and Scleroderma citrinum and their colonization ability at relatively low rates allows these spores to be used as ectomycorrhizal inocula on a large scale.

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.

Ectomycorrhizal fungi of Pinus pinaster

A study was undertaken to determine the ability to form ectomycorrhizae with Pinus pinaster Ait. in pure culture syntheses of 98 isolates of putative mycorrhizal fungi, mainly collected in northern Spain. A total of 35 species in 16 genera — Amanita, Cenococcum, Collybia, Cortinarius, Hebeloma, Laccaria, Lactarius, Lyophyllum, Melanogaster, Paxillus, Pisolithus, Rhizopogon, Scleroderma, Suillus, Thelephora and Xerocomus — formed ectomycorrhizae. Many of these fungal species were not previously reported as symbiotic with Pinus pinaster. Results obtained increase the range of potential fungal candidates for inoculation of nursery seedlings.

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.

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

Abstract Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P.pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery.

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.

Coinoculation of containerized Douglas-fir (Pseudotsuga menziesii) and maritime pine (Pinus pinaster) seedlings with the ectomycorrhizal fungi Laccaria bicolor and Rhizopogon spp.

Abstract Coinoculations with mycelium of Laccaria bicolor and spores of Rhizopogon spp. included in alginate gel have been carried out to determine: (1) the ability of the mixed inoculum to produce dual-colonized containerized Douglas-fir and maritime pine planting stocks and (2) the colonization pattern of the two fungi in individual root systems. For both tree species, the maximal proportion of dual-colonized seedlings obtained almost never exceeded 50%. The rest of the seedlings remained colonized by a single fungus or were non-colonized. In Douglas-fir inoculations, the relationship between the dual-colonized seedlings obtained and the initial dose of the two fungi was highly significant. The highest proportion of dual-colonized seedlings was obtained when the highest dose of R. subareolatus was used (106 spores/seedling), regardless of the dose of L. bicolor. Among the treatments producing 25% or more dual-colonized seedlings, differences in the proportion of Laccaria/Rhizopogon mycorrhizas and total root colonization percentages were not clearly related to the initial combination of doses. The proportion of dual-colonized maritime pine seedlings was not significantly related to the initial inoculation doses of the two fungi. The proportion of Laccaria/Rhizopogon mycorrhizas was not significantly different among treatments with 25% or more dual-colonized seedlings, whereas total colonization percentages ranged from 37% with the combination 0.08/104 (g L. bicolor / spores R. roseolus per seedling) to 74% with the combination 0.08/106, this difference being statistically significant.

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.