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. Hebeloma crustuliniforme, Laccaria laccata and Pisolithus tinctorius were applied as vegetative inocula while Melanogaster ambiguus, Pisolithus tinctorius, Rhizopogon luteolus, Rhizopogon roseolus and Scleroderma verrucosum 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.

Effects of ectomycorrhizal inoculation and the type of substrate on mycorrhization, growth and nutrition of containerised Pinus pinea L. seedlings produced in a commercial nursery

“…programmes for the production of mycorrhizal P. pinea seedlings with similar results [27,29,32]. Both, the percentages of mycorrhizal seedlings and the colonisation level obtained with all the fungi tested in this study, except with S. verrucosum, were significantly reduced when seedlings were grown in the substrate containing peat and composted pine bark (p/pb).…”

“…Low mycorrhization rates were obtained with M. ambiguus, with only half of seedlings being mycorrhizal and percentages of ectomycorrhizas (ECM) under 20%. The quantity of spores applied was probably not high enough, since ECM percentages over 80% have been previously obtained with this fungus for P. pinea with application of spore rates over 10 7 spores per seedling [32]. In the case of P. tinctorius, even if all plants became mycorrhizal, the percentages of ECM did not reach 50%, the minimal limit established for this fungus to ensure an effect on plant development after transplanting [23,24].…”

“…The minimal level of ECM necessary to ensure seedling performance in the field has been poorly studied, and probably depends on environmental characteristics of the site of transplantation and on the fungus-tree combination used [4,34]. The spore inoculum of P. tinctorius has been successfully used in previous experiments to produce containerised Pinus species, including P. pinea [15,28,32].…”

“…Inoculation with these fungi was carried out by mixing the vermiculite containing the spores with the potting substrate to reach a final concentration of 10 7 spores per seedling in both cases. For all fungi tested, the choice of the spore concentration was based on previous results [32].…”

“…Seedling roots were washed free of substrate and ectomycorrhizas were identified according to morphological criteria derived from previous results obtained in synthesis and inoculum dosage trials [31,32]. Each seedling root was cut into 2-3 cm segments, and the percentage of ectomycorrhizal short roots was assessed by counting at least 200 randomly selected short roots under the stereomicroscope.…”