Nursery mycorrhization of Quercus ilex with Tuber melanosporum using different substrates.

Authors: Benito-Matas, L.F.1; lvarez Lafuente, A.1; Suz, L.M.2,3,4; Honrubia, M.4; Peuelas, J.L.1. 1 CNRGF El Serranillo. Ministerio de Medio Ambiente y Medio Rural y Marino. Direccin General de Medio Rural y Poltica Forestal. Crta Fontanar, s/n. 19080, Guadalajara, SPAIN. e-mail: lusfbm@gmail.com 2 Imperial College London, London SW7 2AZ, UK Royal Botanic Gardens, Kew TW9 3DS, UK. 3 Departamento de Biologa Vegetal (Botnica), Facultad de Biologa, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain INTRODUCTION. Inoculation with edible ectomycorrhizal fungi and production of forest plant species in nurseries have experienced a very important development during the last few years. Truffles (Tuber spp.) are cultivated in plantations to offset the declining of wild production. Although methods for truffle plantation management are currently known, published protocols for production of truffle-colonized seedlings for commercial use are diverse (Reyna, 2000). The sporal inoculum method is the most used for Tuber species, and a high diversity of substrates have been used. Soil from wild truffle grounds is the most common substrate used in truffle seedling production. In a greenhouse and field study we analyzed the effect of four non-sterilized substrates used in nursery production on the Tuber melanosporum mycorrhization rate and growth of Quercus ilex seedlings at the end of cultivation and on seedling outplanting survival. MATERIALS AND METHODS. Plastic 400 mL containers (ForestPot400, Nuevos Sistemas de Cultivo, S.L., Gerona, Spain) previously esterilized in water and bleach (1:1), were filled with 5 types of substrate: 1. SOIL: A mixture of soil-vermiculite (1:1 v/v), pH=8,5. Sterilized in a vacuum 3 times per week at 121 C for 20 min. Esto cambiar como 5 si dejis la descripcin de los tratamientos en la introduccin 2. FPM: Fertilized (0,8-1 kg/m3 N) peat moss (Kekkila F6, pH=4,5). 3. FPMV: Mixture fertilized peat moss with vermiculite 1:1. 4. NFPM: Unfertilized peat moss (Kekkila F0, pH=4,5). 5. NFPMV: Unfertilized peat moss with vermiculite 1:1. All subtrates containing peat moss were not sterilized and were ammended with CaCO3 to adjust pH=8,5. Quercus ilex acorns were obtained from provenance Alcarria (2007, Central Spain). The seeds were rinsed in tap water and surface-sterilized with H2O2 (30%) for 20 min. After sterilization, the seeds were pregerminated in peat moss sterilized with himexazol (0,072%) in a germination chamber at 20 C in February 2008. Germination occurred

21days after; acorns with at least 1 cm of radicle were sown in a greenhouse in the CRGF El Serranillo (Ministerio de Medio Ambiente) in the vicinities of Guadalajara (Central Spain). The seedlings were well irrigated and kept indoors till the end of the experiment. Truffle inoculum was obtained from Soria, Barracas-Valencia, Sarrin-Teruel and Huesca (Spain). Ascosporal inoculum was made by cleaning the peridium of each truffle, coarsely chopping and resuspending the gleba (fertile inner part) in distilled water, and blending the gleba to produce a suspension of nearly uniform particle size. The aqueous sporal suspension was incorporated thoroughly by hand into the substrate at a rate of 1 g of fresh truffle/seedling in May 2008. Fertilization of 52 mg N/seedling, 15 mg P/seedling and 58 mg K/seedling was applied by hand on the first year, accordingly with the initial fertilization of each substrate, excepting to sterilized soil, that was not fertilized. During 2009, second year of experiment, all plants were fertilized with a final amount of 95 mg N, 29 mg P and 110 mg K (Hakaphos Amarillo, NPK 17-5-19, BASF). Each treatment was replicated 4 times for a total of 152 inoculated seedlings (38 wells per container), and other 152 non-inoculated. Previous to the field establishment of the plants, in January 2009, a mycorrhizal control was done to check for the presence of Tuber melanosporum mycorrhizas in the roots of the seedlings. Twenty inoculated and 30 non-inoculated seedlings were planted in March 2009 in Abanades, Guadalajara, (1050 m). The experimental design consisted in replication blocks of seedlings by substrate, each block had 5 plants of the same treatment. Four blocks of inoculated seedlings and six blocks of non-inoculated plants were planted at 1m intervals. Field survival was measured in February 2010. At the end of experiment, in February 2010, five seedlings from each treatment were extracted from the field and T. melanosporum mycorrhization rate was evaluated following the methology proposed by Fisher & Colinas (1997). Two to five mycorrhizas previously identified as T. melanosporum by their morphological and anatomical features were selected per treatment and their DNA was extracted. The internal transcribed spacer (ITS) if the r-DNA region was amplified and sequenced. BLAST searches in GenBank were carried out to identify the obtained sequences. To characterize morphologically the plants we measured the height and stem diameter at the root collar after the first and second year of cultivation. RESULTS. Table I shows the quantitative and qualitative evaluation of seedlings obtained from each treatment; mean black truffle mycorrhizal and other fungal contaminants rates are detailed. No differences in Tuber melanosporum mycorrhizal rate and other competing ectomycorrhizas were found. The identification of the mycorrhizas as T. melanosporum was confirmed by their ITS-rDNA sequences. After 10 months in the greenhouse, NFPMV seedlings were significantly shorter and smaller in diameter (P < 0,05) compared to the seedlings from the other four treatments

(Table 1). At the end of the experiment only NFPMV and NFPM seedlings were shorter and smaller (P<0,05) than the rest of seedlings from other substrates. Thus, fertilization improved growth of seedlings.
Table I. Characteristics of Tuber melanosporum-inoculated Quercus ilex seedlings evaluated. Sterilized soil (SOIL), fertilized peat moss (FPM), fertilized peat moss-vermiculite (FPMV), unfertilized peat moss (NFPM) and unfertilized peat moss with vermiculite (NFPMV).Variables: Height (cm) first and second year; stem diameter (mm) measured the root collar first and second year; Tuber melanosporum mycorrhizae rate and other competing ectomycorrhizae (%). Within each variable, different letters represent significantly different means ( = 0,05). Height (cm) 1s year n=100 9,80 ab 11,35 a 11,03 ab 11,15 ab 9,68 b Diameter (mm) 1s year n=100 3,241 a 2,887 ab 2,817 ab 3,099 ab 2,793 b Height (cm) 2nd year n=12 17,86 abc 18,79 ab 20,65 a 13,60 c 14,90 bc Diameter (mm) 2nd year n=12 5,478 a 6,38 a 5,79 a 5,87 a 5,84 a Others Competing ECM n=5 0,55 a 0,0 a 0,0 a 0,0 a 0,0 a

Substrate

Mycorrhization rate (%) n=5 37,1 a 32,4 a 35,9 a 35,9 a 36 a

Survival (%) n=100 65 a 65 a 65 a 70 a 75 a

SOIL FPM FPMV NFPM NFPMV

However, after one year of plantation in the field there were no significant differences in survival.
DISCUSSION

Substrates did not seem to affect mycorrhization rates. All substrates presented enough ventilation, drainage and nutrients to allow the germination of Tuber melanosporum spores. The non sterilization of the substrates did not enhance other competing fungi colonization of the seedlings. Fertilization did no difficult T. melanosporum mycorrhization. Although fertilization has not been recommended in several publications about mycorrhizal seedlings, we consider that it is necessary for the correct development of the plant.. In Mediterranean areas, fertilization is linked to best quality of plants and greater survival in the field (Villar Salvador et al., 2004). The mycorrhization with truffles improved seedling of Quercus ilex growth, especially that of the shoots, and encouraged water uptake of the seedlings during the summer drough (Dominguez Nuez et al., 2006). In 2009, annual drought in Guadalajara was long and hard, and this could explain the higher survival rate of seedlings. We can conclude that the utilization of non sterilized peat moss substrate and fertilize during growing season provides good conditions to produce well mycorrhized and high quality plants, and at a lower cost than other media traditionally used for growing plants inoculated with Tuber melanosporum. BIBLIOGRAFA. DOMNGUEZ NUEZ J.A., SELVA SERRANO J., RODRGUEZ BARREAL J.A., SAIZ DE OMEACA GONZLEZ J. A. 2006. The influence of mycorrhization with Tuber melanosporum in the afforestation of a Mediterranean site with Quercus ilex and Quercus faginea. Forest Ecology and Management 231: 226-233

FISHER & COLINAS C. 1997 REYNA DOMENECH S., 2000. Trufa, truficultura y selvicultura trufera. Mundiprensa, S. A. Madrid. 229 pp. VILLAR SALVADOR P., PLANELLES R., ENRQUEZ E., PEUELAS J., 2004. Nursery cultivation regimes, plant functional attributes and field perfromance relantioships in the Mediterranean oak Quercus ilex L. Forest Ecology and Management 196, 257-266.