Mycorrhiza (2016) 26:161–168 DOI 10.1007/s00572-015-0657-0 ORIGINAL ARTICLE Mycorrhization between Cistus ladanifer L. and Boletus edulis Bull is enhanced by the mycorrhiza helper bacteria Pseudomonas fluorescens Migula Olaya Mediavilla1,2 & Jaime Olaizola2 & Luis Santos-del-Blanco1,3 & Juan Andrés Oria-de-Rueda1 & Pablo Martín-Pinto1 Received: 30 April 2015 /Accepted: 16 July 2015 /Published online: 26 July 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Boletus edulis Bull. is one of the most economically work was to optimize an in vitro protocol for the mycorrhizal and gastronomically valuable fungi worldwide. Sporocarp synthesis of B. edulis with C. ladanifer by testing the effects of production normally occurs when symbiotically associated fungal culture time and coinoculation with the helper bacteria with a number of tree species in stands over 40 years old, Pseudomonas fluorescens Migula. The results confirmed suc- but it has also been reported in 3-year-old Cistus ladanifer cessful mycorrhizal synthesis between C. ladanifer and L. shrubs. Efforts toward the domestication of B. edulis have B. edulis. Coinoculation of B. edulis with P. fluorescens dou- thus focused on successfully generating C. ladanifer seedlings bled within-plant mycorrhization levels although it did not associated with B. edulis under controlled conditions. Micro- result in an increased number of seedlings colonized with organisms have an important role mediating mycorrhizal sym- B. edulis mycorrhizae. B. edulis mycelium culture time also biosis, such as some bacteria species which enhance mycor- increased mycorrhization levels but not the presence of my- rhiza formation (mycorrhiza helper bacteria). Thus, in this corrhizae. These findings bring us closer to controlled B. edulis study, we explored the effect that mycorrhiza helper bacteria sporocarp production in plantations. have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this Keywords MHB . Mycelium culture . Mycorrhizal plants . Sporocarps . Ectomycorrhizal fungi cultivation * Pablo Martín-Pinto [email protected] Introduction Olaya Mediavilla [email protected] The species belonging to the Boletus edulis Bull. complex, Jaime Olaizola commonly known as porcini, are currently among the most [email protected] appreciated fungi worldwide (Hall et al. 1998). The different Luis Santos-del-Blanco species in the complex occur frequently, and their combined [email protected] distribution range spans both hemispheres, from Scandinavia Juan Andrés Oria-de-Rueda to Southern Africa and Australia (Dentinger et al. 2010). As a [email protected] result, global marketed productions are high (Boa 2004)with their annual world market value exceeding $250 million (Catcheside and Catcheside 2012). In Spain alone, annual 1 Sustainable Forest Management Research Institute, Fire and Applied Mycology Laboratory, Departments of Agroforestry Sciences and harvested B. gr. edulis production widely ranges between Vegetal Production and Natural Resources, University of Valladolid 2000 and 20,000 Tm, depending on the year (Oria-de-Rueda (Palencia), Avda, Madrid 44, 34071 Palencia, Spain et al. 2008). However, a drastic decrease in the presence and 2 IDForest-Biotecnología Forestal Aplicada, Calle Tren Ter, s/n. productivity of B. gr. edulis has been reported in several parts Parcela 238, 34200 Venta de Baños, Palencia, Spain of Europe (Salerni and Perini 2004). Coupled by an ever- 3 Department of Ecology and Evolution, Univeristy of Lausanne, increasing demand of edible fungi (Sitta and Floriani 2008) CH-1015 Lausanne, Switzerland and considering that currently B. gr. edulis are only collected 162 Mycorrhiza (2016) 26:161–168 from the wild (Cannon and Kirk 2007), there is a high interest melanosporum Vitt. under the presence of P. fluorescens in achieving a controlled production of these fungi, similar to (Dominguez et al. 2012). that of the mycorrhizal fungus Tuber melanosporum Vitt. Also relevant to the feasibility of large-scale mycorrhized (Bonet et al. 2009). plant production is our ability to produce a large number of Most of the porcini only produce fruitbodies in association sterile plants ready to host ECM fungi (Diez et al. 2000). The with Pinus, Quercus,orCastanea (Olivier et al. 1997), micropropagation of plants is a fast method of propagation, reaching high productions in relatively mature forests (40+ which allows us to improve the quality of the plant by years old) (Díaz-Balteiro et al. 2003), thus limiting the feasi- selecting optimum plant material and provide genetic unifor- bility of their domestication. Interestingly, B. edulis species mity (Diez et al. 2000). Despite the high cost of preparing have recently been reported to occur in Cistus ladanifer L. vitroplants as the first step, it pays off with a higher number shrublands in Northwest Spain (Oria-de-Rueda et al. 2005; of mycorrhized plants. Once the in vitro production protocol Martín-Pinto et al. 2006). This finding is very relevant be- with a suitable plant genotype(s) for mycorrhization is cause sporocarp production occurs as early as in 3-year-old achieved, the cost per plant may be reduced, thus creating a C. ladanifer shrubs, and high yields have been reported in 8- profitable inoculation system. The positive results achieved year-old plants (Oria-de-Rueda et al. 2008). with MHB and ECM fungus coinoculations, paired with im- The first steps toward C. ladanifer controlled provements in plant propagation methods, open new expecta- mycorrhization with B. edulis have already been taken tions toward the domestication of valuable ECM fungus spe- (Águeda et al. 2008). However, we poorly understand how cies. Thus, in this study, we aimed to optimize a protocol for to efficiently produce a large number of plants that harbor the mycorrhizal synthesis of B. edulis with C. ladanifer the targeted ectomycorrhizal (ECM) fungi in their root system. vitroplants by assessing the qualitative and quantitative effects Several factors are known to influence the success of man- of coinoculation with MHB P. fluorescens. Our specific aims mediated ectomycorrhizal symbiosis, including fungal inocu- were (i) to assess the influence of P. fluorescens on the my- lum preparation, choice, and preparation of substrates, choice corrhizal presence (i.e., on the number of pots containing of nutrient solutions (Honrubia et al. 1994; Parladé et al. 2004; mycorrhized plants) and mycorrhizal colonization level of Pera and Parladé 2005), and the presence of other microor- C. ladanifer vitroplants inoculated with B. edulis and (ii) to ganisms in the rhizosphere (Walder et al. 2012). assess the influence of the culture time of fungal inoculum on Bacteria can enhance the ectomycorrhizal symbiosis in two mycorrhizal presence and colonization level alone and in in- ways (Frey-Klett et al. 2007), by helping the ectomycorrhizal teraction with P. fluorescens. symbiosis to take place, known as mycorrhization helper bac- teria, or by enhancing within-plant mycorrhization rate, known as mycorrhiza helper bacteria. In either case, they are Material and methods referred to as MHB (Garbaye 1994). The mechanisms by which bacteria interact with fungi involve the stimulation of Mycorrhizal synthesis protocol mycelial growth, the increase of contact points between roots and fungi, and the reduction of environmental stress on the The mycorrhizal synthesis protocol consisted of a preliminary mycelium (Brulé et al. 2001; Kurth et al. 2013). Nonetheless, step for fungal strain isolation and growth, in parallel with the activity of bacteria can also be detrimental to the mycor- propagation of C. ladanifer vitroplants. This step was follow- rhizal symbiosis in some cases (Kataoka et al. 2009). ed by the preparation of bacterial inoculum and finally Many different bacterial groups and genera have been re- coinoculation of vitroplants with MHB into a substrate colo- ported as MHB in both endomycorrhizal and ectomycorrhizal nized by B. edulis. Below, we provide a detailed description of symbiosis (Duponnois and Plenchette 2003). Among strains the different steps. isolated from ectomycorrhizae, Pseudomonas fluorescens Migula is among the most frequent (Garbaye and Bowen Fungal inoculum 1989; Garbaye et al. 1990 ). In addition, various studies have confirmed that P. fl uo re sc en s generally improves the symbi- Sporocarps of B. edulis were collected from pure C. ladanifer otic relationship of ectomycorrhizae by increasing the within- shrublands located in northwestern Spain. Immediately after plant ratio of mycorrhization, i.e., the percentage of mycorrhi- collection, sporocarps were stored in closed polyethylene bags zal short roots to total short roots (Duponnois and Garbaye until their processing in the laboratory within 24 h after col- 1991;Duponnois2006), and stimulating the ectomycorrhizal lection (Honrubia et al. 1994). Strain isolation was carried out fungus growth under experimental conditions (Frey-Klett under aseptic conditions in order to avoid contamination. A et al. 2007). Interestingly, in the context of ECM fungus do- small piece of inner flesh from the cap was placed on MMN mestication, a recent study reported a significant increase in nutritive medium plates (Modified Melin-Norkrans) (Marx colonization of Pinus halepensis Mill. roots by Tuber 1969)atpH5.5andkeptat22–24 °C in dark (Honrubia Mycorrhiza (2016) 26:161–168 163 et al. 1994). Mycelium was transferred to fresh MMN plates Tween 20® detergent.