Mycorrhiza DOI 10.1007/s00572-014-0611-6
ORIGINAL PAPER
Development of growth media for solid substrate propagation of ectomycorrhizal fungi for inoculation of Norway spruce (Picea abies) seedlings Irmeli Vuorinen & Leena Hamberg & Michael Müller & Pekka Seiskari & Taina Pennanen
Received: 19 March 2014 / Accepted: 7 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A silica-based propagation medium was developed for large-scale production of ectomycorrhizal (ECM) fungal inoculum by solid state fermentation. Development of the medium was started by screening for an optimal growth medium among six different semisynthetic agar media traditionally used in cultivation of ECM fungi. The majority (65 %) of the twenty tested ECM fungal strains that typically colonize Norway spruce (Picea abies) seedlings grew best on modified Melin-Norkrans (MMN) medium with reduced sugar content (½MMN). In order to develop a nutritionally similar medium for large-scale cultivation of the ECM fungi, we chose silica to form a solid matrix and light brewery malt extract to provide nutrients. The medium was supplemented with a commercial humic acid product that was shown to boost fungal growth. The optimal concentration of the constituents was screened for in two assays by determining the growth rates of seven potential inoculant ECM fungal strains (Amphinema sp., Cenococcum geophilum, Hebeloma sp., Meliniomyces bicolor, Paxillus involutus, Piloderma byssinum, and Tylospora asterophora). As a result, we composed a silica-based mass propagation medium (pH 5.8) containing 2.5 % brewery malt extract and 0.5 g/l humic acid product Lignohumate AM. This medium is easily produced and supported good growth of even the slowly growing and rarely studied Athelioid ECM strains. Furthermore, root systems of Norway spruce nursery seedlings were colonized by the tested ECM fungi by using solid inoculum formulated from the silica medium. I. Vuorinen (*) : L. Hamberg : M. Müller : T. Pennanen Finnish Forest Research Institute—Metla, Jokiniemenkuja 1, Box 18, 01301 Vantaa, Finland e-mail: [email protected] P. Seiskari Verdera Ltd., Kurjenkellontie 5 B, Box 5, 02270 Espoo, Finland
Keywords Ectomycorrhizal fungi . Norway spruce . Picea abies . Fungal inoculum . Solid state fermentation . Malt extract . Humic acid
Introduction Benefits of mycorrhizal symbiosis to host plant growth are well known. Ectomycorrhizal (ECM) fungi can improve the quality of coniferous tree seedlings, increase survival in the field (Quoreshi and Timmer 2000; Menkis et al. 2007), accelerate their growth (Selosse et al. 2000), and facilitate health of the host tree (Duchesne 1994; Hyder et al. 2013). Controlled synthesis of the symbiosis by using fungal inocula has therefore been the target of past research (Moser 1958; Theodorou and Bowen 1973; Marx et al. 1982). Commercially available arbuscular mycorrhizal inocula are currently produced in large scale by several companies (Gianinazzi and Vosátka 2004). ECM fungal inocula have been used experimentally in forestry (Lapeyrie and Bruchet 1985; Stenström and Ek 1990; Parlade 1993; Rincón et al. 2001, 2005) and several different ECM fungus inocula have been produced and marketed commercially (Marx et al. 1982, 2002; Rossi et al. 2007). Sphagnum peat substrate is widely used in Finnish nurseries. However, the use of this substrate in combination with high levels of mineral fertilizers and biocides can lead to poor ECM colonization level in Norway spruce (Picea abies (L.) H. Karst.) seedlings (Flykt et al. 2008; Vaario et al. 2009). Because commercially raised Norway spruce seedlings used for reforestation in Finland are produced in fertilized peat, new approaches to cultivate and inoculate ECM fungi onto Norway spruce seedlings are urgently needed. For commercial purposes fungal inoculum, biomass needs to be produced economically in great quantities and the product should be applicable at the nursery with little additional effort. Inocula based on fungal spores have proven to retain
Mycorrhiza
their viability and efficacy for a long time (Castellano and Molina 1989), but collecting spores can be laborious and susceptible to contaminations with other organisms. Therefore, the use of vegetative mycelia from pure ECM fungus cultures is considered the more suitable inoculation method especially for larger scale production (Marx et al. 1982; Rossi et al. 2007). The use of mycelial cultures has, however, been severely hindered by the inability to produce large quantities of viable and economical inoculum (Marx et al. 2002). Fortunately, fungal strains that promote host tree growth can be maintained for years in laboratory conditions. For inoculum production, the fungal mycelium may be cultivated by means of solid state fermentation or submerged liquid fermentation, of which the former has been more commonly used for ECM fungi (Rossi et al. 2007). In their recent study, Rossi and Oliveira (2011) optimized submerged cultivation conditions for efficient large-scale production of Pisolithus microcarpus and concluded that the best results were obtained using Pridham-Gottlieb medium where yeast extract was substituted with peptone. Smith (1982) had earlier screened for optimal submerged cultivation conditions for P. tinctorius and ended up using modified Melin-Norkrans (MMN) agar medium, where the ratio of carbohydrate to ammonium was similar as in one-half strength MMN. However, submerged cultivations are susceptible to contaminations and have a high energy demand. Solid state fermentation is a more economical, energy efficient, and environmentally friendly process (Virtanen et al. 2008). Peat and vermiculite supplemented with nutrient solution has been the preferred medium used in solid state fermentation (Marx et al. 1982; Castellano and Molina 1989; Duponnois and Garbaye 1991; Rincón et al. 2001) with production focused on a few fungal species such as Pisolithus tinctorius and Laccaria bicolor. One of the aims of our work was to develop a suitable growth medium that can be used in solid state cultivation of ECM fungal strains such as Athelioid ECM fungi (e.g. Amphinema, Piloderma, and Tylospora species) that commonly colonize wild Norway spruce seedlings (Erland et al. 1999; Taylor et al. 2000; Korkama et al. 2006). However, we poorly understand the growing demands of Athelioid ECM fungi. In order to develop a solid state ECM fungal inoculum for nursery grown Norway spruce seedlings in Finland, we conducted four interdependent experiments to address the following three objectives. First, determine which nutrient agar media commonly used for laboratory cultivation of ECM fungi provided the best mycelial growth for a variety of ECM fungi. Secondly, based on the nutrient agar study, optimize the most effective carbon source with humic acid additions and silica as the solid carrier agent in order to find a medium suitable for large-scale production of ECM fungal inoculum. Thirdly, test the efficacy of the developed silicabased solid state ECM fungus inoculum when applied to Norway spruce seedlings in the nursery.
Material and methods Fungal strains The ectomycorrhizal fungal strains used in this study are deposited in the culture collection of the Finnish Forest Research Institute. They were isolated from ECM root tips of young Norway spruce (Picea abies) seedlings growing in natural habitats or from fruiting bodies (Table 1). The strains have been identified by sequencing and compared with known sequences in GenBank and UNITE (Kõljalg et al. 2005). Twenty fungal strains were selected for a large preliminary growth experiment on common nutrient media (Growth assay 1) and seven strains for further experiments with silica-based media (Growth assays 2 and 3). All of the strains have proven to form ectomycorrhizas with spruce seedlings, and the seven strains chosen for Growth assays 2 and 3 are known to enhance seedling growth (Pennanen et al., data not shown). Most of the selected seven fungal taxa are known to commonly associate with Norway spruce seedlings in natural environments (Korkama et al. 2006; Tedersoo et al. 2008). Several strains per species or genera were included in the first experiment in order to assess the variation within species and genera level, but in the latter experiments, all isolates represented different species or genera in order to maintain the phylogenetical representativeness of the results. Growth assay 1—commonly used laboratory scale nutrient media Five different semisynthetic nutrient agar media commonly used to cultivate ECM fungi were tested with twenty ECM fungus strains: (1) Hagem’s agar medium (modified from Modess 1941), (2) Pachlewski agar medium (modified from Pachlewski and Pachlewska 1974), (3) Pridham-Gottlieb agar medium (PGM; slightly modified from Litchfield and Arthur 1983), (4) commercial potato dextrose agar medium (Difco), (5) modified Melin-Norkrans agar medium (MMN; Marx 1969; Molina and Palmer 1982), and (6) MMN agar medium with reduced sugar content (½MMN). Three different pH levels, 4.5, 5.5, and 6.1 (adjusted with HCl/NaOH), were tested for ½MMN medium resulting in altogether eight different media in this experiment. All the media contained 15-g agar per liter. Hagem’s agar medium was modified to contain 5.0-g glucose, 5.0-g malt extract, 0.5-g NH4Cl, 0.5-g KH2PO4, 0,5-g MgSO4°7H2O, and 11.3-mg Fe-EDTA per one liter water, and its pH value was set at 4.5 with NaOH-HCl. Pachlewski agar medium was modified to contain 20-g glucose, 5.0-g maltose, 0.5-g (NH4)2tartrate, 0.5-g KH2PO4, 0.5-g MgSO4°7H2O, and 1mg Thiamine-HCl per one liter water, and its pH value was set at 5.0. Pridham-Gottlieb agar medium was modified to contain 30-g glucose, 10-g peptone, 3.0-g NH4NO3, 2.38-g
Mycorrhiza Table 1 Fungal strains selected for the growth experiments on common semisynthetic nutrient media (Growth assay 1) and for the commercial propagation experiments (Growth assays 2, 3, and seedling inoculation experiment 4) Species Ascomycetes 1 Cenococcum geophilum 2 Cenococcum geophilum 3 Meliniomyces bicolor 4 Meliniomyces bicolor Basidiomycetes 5 Amanita muscaria 6 Amphinema byssoides 7 Amphinema sp. 8 Amphinema sp. 9 Atheliaceae 10 Cortinarius sp. 11 Hebeloma sp. 12 Hebeloma sp. 13 14 15 16 17 18 19 20 21 22 23
Hebeloma sp. Laccaria sp. Paxillus involutus Paxillus involutus Piloderma byssinum Piloderma fallax Tylospora asterophora Tylospora asterophora Tylospora asterophora Tylospora asterophora Tylospora asterophora
Straina
Growth assay
R-FC01 R-FC03 R-FC06 R-MF01
1 2, 3, 4 1 1, 2, 3, 4
F-SS01 R-AR03 R-AR01 R-SP03 R-RS06 F-SS11 F-NB01 F-RS01
1 1 1 2, 3, 4 1 1 1, 2, 3, 4 1
R-RS01 F-NC01 F-SS02 F-CY01 R-FC07 R-SP02 R-MF02 R-NC01 R-RS03 R-RS05 R-SP01
1 1 1 2, 3, 4 1, 2, 3, 4 1 1 1 1 1 1, 2, 3, 4
a
Strains including R as the first letter were isolated from ECM root tips of young Norway spruce (Picea abies) seedlings growing in natural habitats and those including F were isolated from fruiting bodies. Other letters in the strain codes indicate the biotope where the strain has been isolated in southern Finland: FC in the codes refer to forest clear cut, MF to Myrtillus type mature forest soil, SS to sapling stand, AR to afforested arable soil, SP to sand pit, RS to road side, NC to containerized nursery, and CY to courtyard
KH2PO4, 5.65-g KH2PO4, 1.0-g MgSO4°7H2O, 6.4-mg CuSO4°5H2O, 1.1-mg FeSO4°7H2O, 1.9-mg MnCl2°4H2O, and 1.5-mg ZnSO4°7H2O per one liter water, and its pH value was set at 6.5. Potato dextrose agar medium was prepared according to manufacturer’s instruction (Difco, BD Company, New Jersey, USA) to contain 20-g glucose per one liter water, and its pH was set at 5.6. MMN agar medium was modified to contain 2.5-g glucose, 10-g malt extract, 0.25-g (NH4)2HPO4, 0.5-g KH2PO4, 0.15-g MgSO4°7H2O, 0.05-g CaCl2°2H2O, 0.025-g NaCl, 20.16-mg Fe-EDTA, and 10-mg ThiamineHCl per one liter water, and its pH value was set at 5.5. ½MMN agar medium was further modified to contain only half the amount of glucose and malt extract, 1.25 g and 5 g per
liter, respectively, while the amount of other nutrients was not reduced. The media were autoclaved in +121 °C for 20 min. The agar plates were inoculated with round agar plugs of 0.5-cm diameter taken from the peripheral growth zone of an actively growing fungal colony grown for approximately 4 weeks after last transfer. The Petri dishes (diameter 8.9 cm) were sealed with Parafilm foil, and the fungi were allowed to grow in dark at +25 °C temperature. Five replicative plates per each fungal strain and nutrient medium were inoculated with one agar plug each. Three isolates of the T. asterophora strain R-SP01 were tested separately and thus n=120.
Growth assay 2—development of a solid matrix cultivation medium We designed the second assay aiming to find a universal growth medium for commercial cultivation. Seven selected ECM fungus strains (Table 1) were grown on a variety of silica-based nutrient media containing light brewery malt extract and humic substances in different ratios. The results of Growth assay 1, which are in accordance with the summation by Marx and Kenney (1982), showed that malt extract containing ½MMN agar medium is the best choice for a common nutrient medium supporting growth of the tested ECM fungal strains. Thus, we aimed to develop a nutritionally similar medium that is based on less costly bulk ingredients. According to the manufacturer’s information, light brewery malt extract (Maltax 10; Senson Oy, Lahti, Finland) contains most important macro- and micronutrients (N, K, Na, Ca, Mg, Zn, Fe, Cu, Cl) and even thiamin in roughly the same ratios as ½MMN medium and was thus selected to be the main constituent of the commercial silica-based medium. In this experiment, we investigated different brewery malt extract concentrations, 0.1, 2.5, 5, 7.5, and 10 % (w/V), in medium. The ½MMN medium was included in this experiment to represent traditionally used media. We also tested the effects of two different humic acid products, Humistar (Tradecorp International, Belgium) and Lignohumate AM (later referred to as Lignohumate; Amagro, Prague, Czech Republic), as humic acid-like substances have been shown to promote plant growth (Adani et al. 1998; Arancon et al. 2004, 2005; David et al. 1994) and are widely used in agriand horticulture. Furthermore, humic acid has been shown to stimulate growth of ECM fungi in vitro (Hršelová et al. 2007; Soukupová et al. 2008). Different malt extract concentrations were tested with the following humic acid additions: no added humic acid, Humistar 0.2, 0.6, and 1.2 g/l and Lignohumate 0.6 g/l resulting in 25 different malt extract combinations. For comparison, the same humic acid additions were also tested in ½MMN medium. Furthermore, pH of the growth medium was adjusted to 5.8 with HCl/NaOH.
Mycorrhiza
Finely ground silica powder Sipernat 22S (Evonic industries, Essen, Germany) was used as the solid carrier agent in the media. In order to enable monitoring of colony growth in the same way as on traditional agar media, we developed a laboratory scale version of the silica medium used in solid state fermentation (300 g silica per 700 g liquid, i.e., 1:2.3 w/w); by adding relatively more liquid (1:4 w/w), the medium was formulated into a thick paste that could be spread into the Petri dishes as an even layer. Two replicative plates of each of the 25 media combinations were inoculated with each of the seven fungal strains chosen for Growth assay 2. Inoculation procedure and the growth conditions were similar as in the Growth assay 1.
Growth assay 3—optimization of a solid matrix cultivation medium On the basis of the obtained results from the Growth assays 1 and 2, we continued screening for the optimal nutrient combination in silica-based media for the seven selected fungal strains (Table 1). We further tested media on Petri dishes with 2.5 and 5 % (w/V) brewery malt extract concentrations in combination with four different humic acid treatments (Lignohumate 0, 0.2, 0.5, and 0.8 g/l). Different Lignohumate concentrations were tested here as Lignohumate had clear positive effect on the growth of Meliniomyces bicolor whereas Humistar had only negative or indifferent effects on the growth of the investigated ECM fungi. We also tested the potential growth enhancing effect of phosphate addition, as the used brewery malt extract has a remarkably low P content (315 mg/kg; Senson Oy, Lahti, Finland). In the 2.5 % (w/V) malt extract medium, the P concentration is only 7.9 mg/l compared to 173.7 mg/l in ½MMN medium. Phosphate buffer is also used as a nutrient source in Pridham-Gottlieb medium for fungi (Pridham and Gottlieb 1948; Litchfield and Arthur 1983). Phosphate buffer system (K2HPO4/KH2PO4, pH 5.8) was added until the total P content of the medium reached the same level as in ½MMN medium. In all media, finally, pH was adjusted to 5.8 using HCl/NaOH. The silica-based medium was formulated similarly as in the Growth assay 2. We prepared two replicates for each combination of the two different malt extract concentrations and the four different humic acid treatments with and without the addition of phosphate resulting in 16 different media combinations. Inoculation procedure and the growth conditions were similar as in the Growth assay 1. In a separate growth test, we compared ground dolomite lime (Nordkalk) and HCl/NaOH as pH regulative agent in media containing 2.5 % (w/w) malt extract and 0.6 g/l Lignohumate as dolomite lime would be a practical pH regulative agent in large-scale media (Virtanen et al. 2008). The target pH 5.8 was reached by adding 0.4-g dolomite lime/l into the medium. Two replicative
plates of both treatments were inoculated with each of the seven selected ECM strains listed in Table 1. Monitoring fungal growth In Growth assay 1, the growth of the fungi was monitored for 40–90 days. In the Growth assay 2, all the cultures that remained uncontaminated were allowed to grow up to 80– 100 days, and in the Growth assay 3, up to 150 days. The growth was linear during this time period. The growth of the fungi in all the experiments was monitored weekly to every fortnight. The radius of the colony was measured in two opposing directions with the accuracy of 0.5 mm from the edge of the inoculum to the outermost hyphae of the peripheral growth zone. The average growth from these measurements was plotted against incubation time, and the growth rates were calculated using the last value in the linear growth phase, as radial growth of fungal colonies on agar has been shown to be linear as long as the conditions remain constant (Trinci 1971). On agar, the growing hyphae of the peripheral growth zone of a colony always proceed into fresh, unused medium, which enables them to maintain their maximum specific growth rate (Trinci 1971). The growth rates were only compared within a strain in order to find the optimal nutrient media promoting the best fungal growth for each strain. Seedling inoculation—testing the efficacy of the solid state inoculum in the nursery In order to test the efficacy of the silica carrier inoculum in vivo, we prepared solid ECM fungal cultures similar to the potential commercial product. Fungal cultures on silicabased media may be produced in large scale by solid substrate fermentation in aerated fermenters or plastic bags (Virtanen et al. 2008). The same fungal isolates as in the Growth assay 3 (Table 1) were first grown for two weeks in ½MMN liquid culture, homogenized, mixed with Omni-mixer (Sorvall), and pipetted into sterile, aerated, transparent plastic bags filled with solid silica nutrient medium. Forty ml of homogenized liquid culture was used per kg of nutrient medium. The prepared nutrient medium contained 2.5 % (w/V) light brewery malt extract (Maltax 10) and 0.5 g/l humic acid product Lignohumate AM, which was determined to be the best substrate in Growth Assay 3. The amount of silica powder was 300 g per 700-ml liquid. Moreover, pH was adjusted to 5.8 using 0.4-g dolomite lime/l. The fungi were grown from 2 (M. bicolor R-MF01 and C. geophilum R-FC03) to 4 weeks (all the other strains) in dark at 20 °C until the silica medium was thoroughly occupied by fungal vegetative mycelia. P. involutus F-CY01 did not grow in the system at all and was therefore excluded from the nursery inoculations.
Mycorrhiza
Unfertilized white nursery peat PP03 (Kekkilä Oy) was supplemented with 10 % (V/V) fully grown silica inoculum, and PL81F seedling containers (81 × cells of 85 cm3, BCC Ab) were filled with the peat-fungal inoculum mixture. Norway spruce seeds (seed-orchard seed R01-89-1002, Sv. 109) were sown on top of the peat–inoculum mixture. Altogether, 45 seedlings were grown with Amphinema sp. R-SP03 inoculum, and with the other inocula, 81 seedlings per ECM strain were grown. The seedlings were grown for 6 months in greenhouse at Haapastensyrjä forest nursery of the Finnish Forest Research Institute. The seedlings were fertilized from June to August by Superex-irrigation fertilization N-P-K (125-27) program (Kekkilä Oy) modified to keep the peat conductivity as low as 0.5–1 mS cm−1. Fertilization rate was approximately 5 ml/seedling/week. In the autumn, height of the seedlings was measured. Root systems of five replicate seedlings per fungal strain were subjected to gross morphotyping in order to assess the colonization of the roots by the inoculated fungi. The roots of the seedlings were washed and cut into 1–2 cm pieces, which were placed on water-filled Petri dishes. Root pieces were randomly selected until about 250 short roots were analyzed under a dissecting microscope (Euromex). The degree of colonization of the inoculated fungus per total number of short roots was determined as well as that of other fungi and the proportion of bare root tips. Statistical analyses The effects of different agar media on the growth rates of mycorrhizal species (Growth assay 1, Table 1) were tested using linear models in statistical program R (R Development Core Team 2011). The response variable in the models was the growth rate of a fungal strain per 30 days, and as an explanatory variable, we had different media as a factor with eight levels (i.e., MMN pH 5.5, ½MMN pH 4.5, ½MMN pH 5.5, ½MMN pH 6.1, Hagem’s pH 4.5, Pachlewski pH 5.0, PGM pH 6.5, and potato dextrose pH 5.6). In the models, the media with the highest growth rate was compared to the growth rates in the other media. Other pairwise comparisons were not performed. The effects of increasing brewery malt extract and humic acid (Humistar and Lignohumate) concentrations on the growth rates of different mycorrhizal species (Growth assay 2, Table 1) were analyzed using linear mixed models with lmer function in R (Pinheiro et al. 2011; R Development Core team 2011). As a response variable, we had the growth rate of each species (mm/30 d), and as explanatory variables, we had (1) a variable describing different malt extract concentrations (Maltax 2.5, 5, 7.5, and 10 %), (2) a variable describing different Humistar concentrations (HS 0, 0.2, 0.6, and 1.2 g/ l), and (3) a variable describing different Lignohumate concentrations (LH 0 and 0.6 g/l). The growth in the lowest malt
extract level, 0.1 %, was very poor, and therefore, this concentration was removed from the statistical analyses. We added two interaction terms to the models, i.e., between malt extract and Humistar concentrations and between malt extract and Lignohumate concentrations to investigate whether the effects of humic acids are different when the malt extract concentration increases. The interaction terms were removed if both of them were not statistically significant. Thus, we kept the simplest model with statistically significant effects for each species. Furthermore, the models included one random factor taking into account pseudoreplicates (the same fungus strain was always placed on two similar plates in each medium combination, and these observations are always correlated with each other because they are similar, i.e., the growth of a fungus has been measured twice on exactly similar plates). If data is not fully random, i.e., correlated observations exist, random factors should be included in the models (see e.g., Bolker et al. 2008). The effect of increasing brewery malt extract, Lignohumate, and phosphorus concentrations on the growth rate of different mycorrhizal species (Growth assay 3, Table 1) was analyzed using linear mixed models with lmer function in R (Pinheiro et al. 2011; R Development Core team 2011). The growth rate of each species (mm/30 d) was as a response variable in the models, and as explanatory variables, we had (1) a variable describing different malt extract concentrations (2.5 and 5 %), (2) a variable describing different Lignohumate concentrations (0, 0.2, 0.5, and 0.8 g/l), and (3) a variable describing the effect of phosphorus (as a factor 0 = phosphorus not added, 1 = phosphorus added). We added interaction terms between the explanatory variables 1 and 2, 1 and 3, 2 and 3, and between the variables 1, 2, and 3 to the models. We removed interaction terms from the models if they were not significant. However, non-significant interaction terms between two explanatory variables were removed only if a non-significant interaction between the explanatory variables 1, 2, and 3 was removed first. A random factor describing pseudoreplicates (two replicate plates per medium) was included in the models. Differences between the heights of spruce seedlings inoculated with different mycorrhizal fungi were investigated with linear model and ANOVA test (R Core Team 2011) which revealed whether any differences between the treatments exist.
Results Growth assay 1—commonly used laboratory scale nutrient media Investigated mycorrhizal species grew especially well on ½MMN media pH 5.5 and 6.1 (65 % of the strains, Table 2,
Mycorrhiza Table 2 The growth rates of 20 ECM fungal strains (Growth assay 1) in semisynthetic nutrient agar media Fungal strain
n
MMNa pH 5.5
½MMNb pH 4.5
½MMN pH 5.5
½MMN pH 6.1
Hagem’ s pH 4.5
Pachlewski pH 5.0
PGMc pH 6.5
PDd pH 5.6
Amanita muscaria F-SS01
40
Amphinema byssoides R-AR03
38
Amphinema sp. R-AR01
40
Atheliaceae R-RS06
40
−8.657± 0.337 −2.748± 0.179 −5.357± 0.489 −1.024± 0.556 −5.464± 0.272 −5.143± 0.458 −7.007± 0.477 −4.167± 0.279 −7.948± 0.146 −3.846± 0.958 −2.271± 0.371 −3.129± 0.303 −11.897± 1.209
−9.257± 0.337 −3.857± 0.168 −6.429± 0.489 −1.262± 0.556 −11.036± 0.272 −6.471± 0.458 −7.693± 0.477 −6.286± 0.279 −10.071± 0.146 −7.680± 0.958 −3.793± 0.371 −4.050± 0.303 35.224± 0.855
−9.279± 0.337 −1.479± 0.168 −0.750± 0.489 6.429± 0.393 −3.964± 0.272 −4.414± 0.458 −7.457± 0.477 −3.881± 0.279 −7.422± 0.146 −0.430± 0.958 −2.229± 0.371 −2.314± 0.303 −4.552± 1.209
−9.000± 0.337 9.686± 0.119 11.250± 0.345 −0.976± 0.556 −0.429± 0.272 −4.800± 0.458 −3.429± 0.477 −1.071± 0.279 −0.838± 0.146 29.638± 0.677 −4.939± 0.371 −1.521± 0.303 −5.224± 1.209
−8.743± 0.337 −6.257± 0.168 −6.279± 0.489 −2.762± 0.556 −11.491± 0.272 −9.021± 0.458 −8.046± 0.477 −5.833± 0.279 −9.857± 0.146 −17.013± 0.958 −7.479± 0.371 −4.864± 0.303 −9.879± 1.209
−9.086± 0.337 −1.543± 0.168 −2.400± 0.489 −2.071± 0.556 18.777± 0.192 −5.743± 0.458 −4.564± 0.477 −3.024± 0.279 −7.461± 0.146 −1.888± 0.958 15.386± 0.262 13.650± 0.214 −18.155± 1.209
−11.250± 0.337 −8.507± 0.168 −10.436± 0.489 −5.000± 0.556 −18.777± 0.272 −11.486± 0.458 −10.843± 0.477 −5.119± 0.279 −12.701± 0.146 −29.638± 0.958 −3.236± 0.371 −2.057± 0.303 −34.035± 1.209
12.214± 0.239 −3.846± 0.179 −4.136± 0.489 −1.786± 0.556 −2.277± 0.272 11.914± 0.324 10.843± 0.338 7.691± 0.197 12.701± 0.103 −21.846± 0.958 −7.286± 0.371 −4.693± 0.303 −26.638± 1.209
−1.136± 0.315 −2.293± 0.516 −3.150± 0.527 −2.191± 0.240 −2.500± 0.229 −1.805± 0.530 −1.145± 0.220
7.800± 0.223 −1.050± 0.516 10.393± 0.373 4.191± 0.170 −1.214± 0.229 −0.136± 0.530 5.364± 0.155
−4.532± 0.334 9.129± 0.365 −0.407± 0.527 −0.452± 0.240 6.143± 0.162 −0.115± 0.530 −1.567± 0.220
−2.229± 0.315 −5.250± 0.516 −4.221± 0.527 −1.857± 0.240 −2.691± 0.229 −2.502± 0.530 −1.054± 0.220
−0.836± 0.315 −3.064± 0.516 −4.114± 0.527 −1.548± 0.240 −1.333± 0.229 −2.058± 0.530 −2.812± 0.220
−7.800± 0.315 −9.129± 0.516 −10.393± 0.527 −4.191± 0.240 −6.143± 0.229 −6.896± 0.530 −5.364± 0.220
−4.864± 0.315 −5.314± 0.516 −3.921± 0.527 −0.214± 0.240 −1.708± 0.243 −0.833± 0.530 −0.659± 0.220
Cenococcum geophilum R-FC01 40 Cortinarius sp. F-SS11
40
Hebeloma sp. F-NB01
40
Hebeloma sp. R-RS01
40
Hebeloma sp. F-RS01
40
Laccaria sp. F-NC01
40
Meliniomyces bicolor R-FC06
40
Meliniomyces bicolor R-MF01
40
Paxillus involutus F-SS02
40
−0.043± 0.315 Piloderma fallax R-SP02 40 −1.307± 0.516 Tylospora asterophora R-MF02 40 −6.150± 0.527 Tylospora asterophora R-NC01 40 −1.167± 0.240 Tylospora asterophora R-RS03 40 −1.429± 0.229 Tylospora asterophora R-RS05 40 6.896± 0.375 Tylospora asterophora R-SP01 120 −1.391± 0.220 Piloderma byssinum R-FC07
39
The underlined value in each line indicates the growth rate (±standard error) of a fungus strain (mm/30 d) in the best medium. The growth rate in this medium has been compared with the others in the same line. Media which are as good as the best one are in bold (no statistically significant differences between the media, p≥0.05, linear models). Values not underlined indicate how much the growth rate (mm/30 d) differs from the underlined medium (the best one), see also Fig. 1 a
Melin-Norkrans medium
b
Melin-Norkrans medium with reduced sugar content
c
Pridham-Gottlieb medium
d
Potato dextrose medium
Fig. 1). Also, potato dextrose, Pachlewski, and MMN media were suitable for some of the fungal strains (preferred by 35, 20, and 15 % of the strains, respectively), but fungal growth in
Hagem’s and Pridham-Gottlieb media was quite poor. The 20 fungal strains showed species and genus specific preferences for the tested semisynthetic nutrient agar media (Table 2,
Mycorrhiza
16
12
8
PD 5.6
*
*
*
*
*
growth rate (mm/30 d)
20
*
*
12
*
8
*
4
0
24
Pach 1/2 MMN PD 5.0 6.1 5.6
* 1/2 MMN MMN 1/2 MMN Hagem PGM 5.5
5.5
4.5
4.5
12
4
0
24
*
*
*
* *
PD 1/2 MMN Pach 5.6 6.1 5.0
16
12
4
0
1/2 MMN MMN 5.5 5.5
*
*
*
*
Pach 1/2 MMN Hagem1/2 MMN PD 5.6 4.5 5.0 4.5 6.1
*
* PGM 6.5
1/2 MMN Pach 1/2 MMN MMN 1/2 MMN PD 5.6 5.5 6.1 5.5 4.5 5.0
*
*
Hagem PGM 4.5 6.5
16
12
*
8
*
*
*
*
4
PD 1/2 MMN 1/2 MMN MMN 5.5 5.6 5.5 6.1
*
*
Pach 1/2 MMN Hagem PGM 5.0 4.5 4.5 6.5
Meliniomyces bicolor R-FC06, n=40
20
16
*
*
12
*
*
* *
8
*
4
Pach 1/2 MMN MMN 5.0 5.5 5.5
24
20
*
*
Cortinarius sp. F-SS11, n=40
0
MMN1/2 MMN 1/2 MMN Hagem PGM 5.5 5.5 4.5 4.5 6.5
Piloderma byssinum R-FC07, n=39
8
*
20
24
16
*
*
4
0
Hebeloma sp. F-NB01, n=40
*
*
8
6.5
20
8
12
24
Cenococcum geophilum R-FC01, n=40
*
16
0
MMN Hagem 1/2 MMN Pach 1/2 MMN1/2 MMN PGM 5.5 6.5 5.5 4.5 6.1 5.0 4.5
16
Amphinema byssoides R-AR03, n=38
20
*
growth rate (mm/30 d)
growth rate (mm/30 d)
*
4
24
growth rate (mm/30 d)
growth rate (mm/30 d)
20
0
growth rate (mm/30 d)
24
Amanita muscaria F-SS01, n=40
growth rate (mm/30 d)
growth rate (mm/30 d)
24
PGM 1/2 MMN 1/2 MMN PD 6.1 6.5 5.6 4.5
Hagem 4.5
Tylospora asterophora R-SP01, n=120
20
16
12
8
4
0
1/2 MMN 5.5
* PD 5.6
*
*
*
*
*
Hagem 1/2 MMN MMN 1/2 MMN Pach 4.5 5.5 6.1 4.5 5.0
*
PGM 6.5
Fig. 1 Growth rates and standard errors of eight ECM fungal strains (mm/30 d) on different semisynthetic nutrient agar media in Growth assay 1: Hagem’s medium (Hagem), Melin-Norkrans medium (MMN), MelinNorkrans medium with reduced sugar content (½MMN), Pachlewski medium (Pach), Potato dextrose agar medium (PD), and Pridham-
Gottlieb medium (PGM). pH values of the media are given below each medium. Growth rates differing significantly (p
ORIGINAL PAPER
Development of growth media for solid substrate propagation of ectomycorrhizal fungi for inoculation of Norway spruce (Picea abies) seedlings Irmeli Vuorinen & Leena Hamberg & Michael Müller & Pekka Seiskari & Taina Pennanen
Received: 19 March 2014 / Accepted: 7 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A silica-based propagation medium was developed for large-scale production of ectomycorrhizal (ECM) fungal inoculum by solid state fermentation. Development of the medium was started by screening for an optimal growth medium among six different semisynthetic agar media traditionally used in cultivation of ECM fungi. The majority (65 %) of the twenty tested ECM fungal strains that typically colonize Norway spruce (Picea abies) seedlings grew best on modified Melin-Norkrans (MMN) medium with reduced sugar content (½MMN). In order to develop a nutritionally similar medium for large-scale cultivation of the ECM fungi, we chose silica to form a solid matrix and light brewery malt extract to provide nutrients. The medium was supplemented with a commercial humic acid product that was shown to boost fungal growth. The optimal concentration of the constituents was screened for in two assays by determining the growth rates of seven potential inoculant ECM fungal strains (Amphinema sp., Cenococcum geophilum, Hebeloma sp., Meliniomyces bicolor, Paxillus involutus, Piloderma byssinum, and Tylospora asterophora). As a result, we composed a silica-based mass propagation medium (pH 5.8) containing 2.5 % brewery malt extract and 0.5 g/l humic acid product Lignohumate AM. This medium is easily produced and supported good growth of even the slowly growing and rarely studied Athelioid ECM strains. Furthermore, root systems of Norway spruce nursery seedlings were colonized by the tested ECM fungi by using solid inoculum formulated from the silica medium. I. Vuorinen (*) : L. Hamberg : M. Müller : T. Pennanen Finnish Forest Research Institute—Metla, Jokiniemenkuja 1, Box 18, 01301 Vantaa, Finland e-mail: [email protected] P. Seiskari Verdera Ltd., Kurjenkellontie 5 B, Box 5, 02270 Espoo, Finland
Keywords Ectomycorrhizal fungi . Norway spruce . Picea abies . Fungal inoculum . Solid state fermentation . Malt extract . Humic acid
Introduction Benefits of mycorrhizal symbiosis to host plant growth are well known. Ectomycorrhizal (ECM) fungi can improve the quality of coniferous tree seedlings, increase survival in the field (Quoreshi and Timmer 2000; Menkis et al. 2007), accelerate their growth (Selosse et al. 2000), and facilitate health of the host tree (Duchesne 1994; Hyder et al. 2013). Controlled synthesis of the symbiosis by using fungal inocula has therefore been the target of past research (Moser 1958; Theodorou and Bowen 1973; Marx et al. 1982). Commercially available arbuscular mycorrhizal inocula are currently produced in large scale by several companies (Gianinazzi and Vosátka 2004). ECM fungal inocula have been used experimentally in forestry (Lapeyrie and Bruchet 1985; Stenström and Ek 1990; Parlade 1993; Rincón et al. 2001, 2005) and several different ECM fungus inocula have been produced and marketed commercially (Marx et al. 1982, 2002; Rossi et al. 2007). Sphagnum peat substrate is widely used in Finnish nurseries. However, the use of this substrate in combination with high levels of mineral fertilizers and biocides can lead to poor ECM colonization level in Norway spruce (Picea abies (L.) H. Karst.) seedlings (Flykt et al. 2008; Vaario et al. 2009). Because commercially raised Norway spruce seedlings used for reforestation in Finland are produced in fertilized peat, new approaches to cultivate and inoculate ECM fungi onto Norway spruce seedlings are urgently needed. For commercial purposes fungal inoculum, biomass needs to be produced economically in great quantities and the product should be applicable at the nursery with little additional effort. Inocula based on fungal spores have proven to retain
Mycorrhiza
their viability and efficacy for a long time (Castellano and Molina 1989), but collecting spores can be laborious and susceptible to contaminations with other organisms. Therefore, the use of vegetative mycelia from pure ECM fungus cultures is considered the more suitable inoculation method especially for larger scale production (Marx et al. 1982; Rossi et al. 2007). The use of mycelial cultures has, however, been severely hindered by the inability to produce large quantities of viable and economical inoculum (Marx et al. 2002). Fortunately, fungal strains that promote host tree growth can be maintained for years in laboratory conditions. For inoculum production, the fungal mycelium may be cultivated by means of solid state fermentation or submerged liquid fermentation, of which the former has been more commonly used for ECM fungi (Rossi et al. 2007). In their recent study, Rossi and Oliveira (2011) optimized submerged cultivation conditions for efficient large-scale production of Pisolithus microcarpus and concluded that the best results were obtained using Pridham-Gottlieb medium where yeast extract was substituted with peptone. Smith (1982) had earlier screened for optimal submerged cultivation conditions for P. tinctorius and ended up using modified Melin-Norkrans (MMN) agar medium, where the ratio of carbohydrate to ammonium was similar as in one-half strength MMN. However, submerged cultivations are susceptible to contaminations and have a high energy demand. Solid state fermentation is a more economical, energy efficient, and environmentally friendly process (Virtanen et al. 2008). Peat and vermiculite supplemented with nutrient solution has been the preferred medium used in solid state fermentation (Marx et al. 1982; Castellano and Molina 1989; Duponnois and Garbaye 1991; Rincón et al. 2001) with production focused on a few fungal species such as Pisolithus tinctorius and Laccaria bicolor. One of the aims of our work was to develop a suitable growth medium that can be used in solid state cultivation of ECM fungal strains such as Athelioid ECM fungi (e.g. Amphinema, Piloderma, and Tylospora species) that commonly colonize wild Norway spruce seedlings (Erland et al. 1999; Taylor et al. 2000; Korkama et al. 2006). However, we poorly understand the growing demands of Athelioid ECM fungi. In order to develop a solid state ECM fungal inoculum for nursery grown Norway spruce seedlings in Finland, we conducted four interdependent experiments to address the following three objectives. First, determine which nutrient agar media commonly used for laboratory cultivation of ECM fungi provided the best mycelial growth for a variety of ECM fungi. Secondly, based on the nutrient agar study, optimize the most effective carbon source with humic acid additions and silica as the solid carrier agent in order to find a medium suitable for large-scale production of ECM fungal inoculum. Thirdly, test the efficacy of the developed silicabased solid state ECM fungus inoculum when applied to Norway spruce seedlings in the nursery.
Material and methods Fungal strains The ectomycorrhizal fungal strains used in this study are deposited in the culture collection of the Finnish Forest Research Institute. They were isolated from ECM root tips of young Norway spruce (Picea abies) seedlings growing in natural habitats or from fruiting bodies (Table 1). The strains have been identified by sequencing and compared with known sequences in GenBank and UNITE (Kõljalg et al. 2005). Twenty fungal strains were selected for a large preliminary growth experiment on common nutrient media (Growth assay 1) and seven strains for further experiments with silica-based media (Growth assays 2 and 3). All of the strains have proven to form ectomycorrhizas with spruce seedlings, and the seven strains chosen for Growth assays 2 and 3 are known to enhance seedling growth (Pennanen et al., data not shown). Most of the selected seven fungal taxa are known to commonly associate with Norway spruce seedlings in natural environments (Korkama et al. 2006; Tedersoo et al. 2008). Several strains per species or genera were included in the first experiment in order to assess the variation within species and genera level, but in the latter experiments, all isolates represented different species or genera in order to maintain the phylogenetical representativeness of the results. Growth assay 1—commonly used laboratory scale nutrient media Five different semisynthetic nutrient agar media commonly used to cultivate ECM fungi were tested with twenty ECM fungus strains: (1) Hagem’s agar medium (modified from Modess 1941), (2) Pachlewski agar medium (modified from Pachlewski and Pachlewska 1974), (3) Pridham-Gottlieb agar medium (PGM; slightly modified from Litchfield and Arthur 1983), (4) commercial potato dextrose agar medium (Difco), (5) modified Melin-Norkrans agar medium (MMN; Marx 1969; Molina and Palmer 1982), and (6) MMN agar medium with reduced sugar content (½MMN). Three different pH levels, 4.5, 5.5, and 6.1 (adjusted with HCl/NaOH), were tested for ½MMN medium resulting in altogether eight different media in this experiment. All the media contained 15-g agar per liter. Hagem’s agar medium was modified to contain 5.0-g glucose, 5.0-g malt extract, 0.5-g NH4Cl, 0.5-g KH2PO4, 0,5-g MgSO4°7H2O, and 11.3-mg Fe-EDTA per one liter water, and its pH value was set at 4.5 with NaOH-HCl. Pachlewski agar medium was modified to contain 20-g glucose, 5.0-g maltose, 0.5-g (NH4)2tartrate, 0.5-g KH2PO4, 0.5-g MgSO4°7H2O, and 1mg Thiamine-HCl per one liter water, and its pH value was set at 5.0. Pridham-Gottlieb agar medium was modified to contain 30-g glucose, 10-g peptone, 3.0-g NH4NO3, 2.38-g
Mycorrhiza Table 1 Fungal strains selected for the growth experiments on common semisynthetic nutrient media (Growth assay 1) and for the commercial propagation experiments (Growth assays 2, 3, and seedling inoculation experiment 4) Species Ascomycetes 1 Cenococcum geophilum 2 Cenococcum geophilum 3 Meliniomyces bicolor 4 Meliniomyces bicolor Basidiomycetes 5 Amanita muscaria 6 Amphinema byssoides 7 Amphinema sp. 8 Amphinema sp. 9 Atheliaceae 10 Cortinarius sp. 11 Hebeloma sp. 12 Hebeloma sp. 13 14 15 16 17 18 19 20 21 22 23
Hebeloma sp. Laccaria sp. Paxillus involutus Paxillus involutus Piloderma byssinum Piloderma fallax Tylospora asterophora Tylospora asterophora Tylospora asterophora Tylospora asterophora Tylospora asterophora
Straina
Growth assay
R-FC01 R-FC03 R-FC06 R-MF01
1 2, 3, 4 1 1, 2, 3, 4
F-SS01 R-AR03 R-AR01 R-SP03 R-RS06 F-SS11 F-NB01 F-RS01
1 1 1 2, 3, 4 1 1 1, 2, 3, 4 1
R-RS01 F-NC01 F-SS02 F-CY01 R-FC07 R-SP02 R-MF02 R-NC01 R-RS03 R-RS05 R-SP01
1 1 1 2, 3, 4 1, 2, 3, 4 1 1 1 1 1 1, 2, 3, 4
a
Strains including R as the first letter were isolated from ECM root tips of young Norway spruce (Picea abies) seedlings growing in natural habitats and those including F were isolated from fruiting bodies. Other letters in the strain codes indicate the biotope where the strain has been isolated in southern Finland: FC in the codes refer to forest clear cut, MF to Myrtillus type mature forest soil, SS to sapling stand, AR to afforested arable soil, SP to sand pit, RS to road side, NC to containerized nursery, and CY to courtyard
KH2PO4, 5.65-g KH2PO4, 1.0-g MgSO4°7H2O, 6.4-mg CuSO4°5H2O, 1.1-mg FeSO4°7H2O, 1.9-mg MnCl2°4H2O, and 1.5-mg ZnSO4°7H2O per one liter water, and its pH value was set at 6.5. Potato dextrose agar medium was prepared according to manufacturer’s instruction (Difco, BD Company, New Jersey, USA) to contain 20-g glucose per one liter water, and its pH was set at 5.6. MMN agar medium was modified to contain 2.5-g glucose, 10-g malt extract, 0.25-g (NH4)2HPO4, 0.5-g KH2PO4, 0.15-g MgSO4°7H2O, 0.05-g CaCl2°2H2O, 0.025-g NaCl, 20.16-mg Fe-EDTA, and 10-mg ThiamineHCl per one liter water, and its pH value was set at 5.5. ½MMN agar medium was further modified to contain only half the amount of glucose and malt extract, 1.25 g and 5 g per
liter, respectively, while the amount of other nutrients was not reduced. The media were autoclaved in +121 °C for 20 min. The agar plates were inoculated with round agar plugs of 0.5-cm diameter taken from the peripheral growth zone of an actively growing fungal colony grown for approximately 4 weeks after last transfer. The Petri dishes (diameter 8.9 cm) were sealed with Parafilm foil, and the fungi were allowed to grow in dark at +25 °C temperature. Five replicative plates per each fungal strain and nutrient medium were inoculated with one agar plug each. Three isolates of the T. asterophora strain R-SP01 were tested separately and thus n=120.
Growth assay 2—development of a solid matrix cultivation medium We designed the second assay aiming to find a universal growth medium for commercial cultivation. Seven selected ECM fungus strains (Table 1) were grown on a variety of silica-based nutrient media containing light brewery malt extract and humic substances in different ratios. The results of Growth assay 1, which are in accordance with the summation by Marx and Kenney (1982), showed that malt extract containing ½MMN agar medium is the best choice for a common nutrient medium supporting growth of the tested ECM fungal strains. Thus, we aimed to develop a nutritionally similar medium that is based on less costly bulk ingredients. According to the manufacturer’s information, light brewery malt extract (Maltax 10; Senson Oy, Lahti, Finland) contains most important macro- and micronutrients (N, K, Na, Ca, Mg, Zn, Fe, Cu, Cl) and even thiamin in roughly the same ratios as ½MMN medium and was thus selected to be the main constituent of the commercial silica-based medium. In this experiment, we investigated different brewery malt extract concentrations, 0.1, 2.5, 5, 7.5, and 10 % (w/V), in medium. The ½MMN medium was included in this experiment to represent traditionally used media. We also tested the effects of two different humic acid products, Humistar (Tradecorp International, Belgium) and Lignohumate AM (later referred to as Lignohumate; Amagro, Prague, Czech Republic), as humic acid-like substances have been shown to promote plant growth (Adani et al. 1998; Arancon et al. 2004, 2005; David et al. 1994) and are widely used in agriand horticulture. Furthermore, humic acid has been shown to stimulate growth of ECM fungi in vitro (Hršelová et al. 2007; Soukupová et al. 2008). Different malt extract concentrations were tested with the following humic acid additions: no added humic acid, Humistar 0.2, 0.6, and 1.2 g/l and Lignohumate 0.6 g/l resulting in 25 different malt extract combinations. For comparison, the same humic acid additions were also tested in ½MMN medium. Furthermore, pH of the growth medium was adjusted to 5.8 with HCl/NaOH.
Mycorrhiza
Finely ground silica powder Sipernat 22S (Evonic industries, Essen, Germany) was used as the solid carrier agent in the media. In order to enable monitoring of colony growth in the same way as on traditional agar media, we developed a laboratory scale version of the silica medium used in solid state fermentation (300 g silica per 700 g liquid, i.e., 1:2.3 w/w); by adding relatively more liquid (1:4 w/w), the medium was formulated into a thick paste that could be spread into the Petri dishes as an even layer. Two replicative plates of each of the 25 media combinations were inoculated with each of the seven fungal strains chosen for Growth assay 2. Inoculation procedure and the growth conditions were similar as in the Growth assay 1.
Growth assay 3—optimization of a solid matrix cultivation medium On the basis of the obtained results from the Growth assays 1 and 2, we continued screening for the optimal nutrient combination in silica-based media for the seven selected fungal strains (Table 1). We further tested media on Petri dishes with 2.5 and 5 % (w/V) brewery malt extract concentrations in combination with four different humic acid treatments (Lignohumate 0, 0.2, 0.5, and 0.8 g/l). Different Lignohumate concentrations were tested here as Lignohumate had clear positive effect on the growth of Meliniomyces bicolor whereas Humistar had only negative or indifferent effects on the growth of the investigated ECM fungi. We also tested the potential growth enhancing effect of phosphate addition, as the used brewery malt extract has a remarkably low P content (315 mg/kg; Senson Oy, Lahti, Finland). In the 2.5 % (w/V) malt extract medium, the P concentration is only 7.9 mg/l compared to 173.7 mg/l in ½MMN medium. Phosphate buffer is also used as a nutrient source in Pridham-Gottlieb medium for fungi (Pridham and Gottlieb 1948; Litchfield and Arthur 1983). Phosphate buffer system (K2HPO4/KH2PO4, pH 5.8) was added until the total P content of the medium reached the same level as in ½MMN medium. In all media, finally, pH was adjusted to 5.8 using HCl/NaOH. The silica-based medium was formulated similarly as in the Growth assay 2. We prepared two replicates for each combination of the two different malt extract concentrations and the four different humic acid treatments with and without the addition of phosphate resulting in 16 different media combinations. Inoculation procedure and the growth conditions were similar as in the Growth assay 1. In a separate growth test, we compared ground dolomite lime (Nordkalk) and HCl/NaOH as pH regulative agent in media containing 2.5 % (w/w) malt extract and 0.6 g/l Lignohumate as dolomite lime would be a practical pH regulative agent in large-scale media (Virtanen et al. 2008). The target pH 5.8 was reached by adding 0.4-g dolomite lime/l into the medium. Two replicative
plates of both treatments were inoculated with each of the seven selected ECM strains listed in Table 1. Monitoring fungal growth In Growth assay 1, the growth of the fungi was monitored for 40–90 days. In the Growth assay 2, all the cultures that remained uncontaminated were allowed to grow up to 80– 100 days, and in the Growth assay 3, up to 150 days. The growth was linear during this time period. The growth of the fungi in all the experiments was monitored weekly to every fortnight. The radius of the colony was measured in two opposing directions with the accuracy of 0.5 mm from the edge of the inoculum to the outermost hyphae of the peripheral growth zone. The average growth from these measurements was plotted against incubation time, and the growth rates were calculated using the last value in the linear growth phase, as radial growth of fungal colonies on agar has been shown to be linear as long as the conditions remain constant (Trinci 1971). On agar, the growing hyphae of the peripheral growth zone of a colony always proceed into fresh, unused medium, which enables them to maintain their maximum specific growth rate (Trinci 1971). The growth rates were only compared within a strain in order to find the optimal nutrient media promoting the best fungal growth for each strain. Seedling inoculation—testing the efficacy of the solid state inoculum in the nursery In order to test the efficacy of the silica carrier inoculum in vivo, we prepared solid ECM fungal cultures similar to the potential commercial product. Fungal cultures on silicabased media may be produced in large scale by solid substrate fermentation in aerated fermenters or plastic bags (Virtanen et al. 2008). The same fungal isolates as in the Growth assay 3 (Table 1) were first grown for two weeks in ½MMN liquid culture, homogenized, mixed with Omni-mixer (Sorvall), and pipetted into sterile, aerated, transparent plastic bags filled with solid silica nutrient medium. Forty ml of homogenized liquid culture was used per kg of nutrient medium. The prepared nutrient medium contained 2.5 % (w/V) light brewery malt extract (Maltax 10) and 0.5 g/l humic acid product Lignohumate AM, which was determined to be the best substrate in Growth Assay 3. The amount of silica powder was 300 g per 700-ml liquid. Moreover, pH was adjusted to 5.8 using 0.4-g dolomite lime/l. The fungi were grown from 2 (M. bicolor R-MF01 and C. geophilum R-FC03) to 4 weeks (all the other strains) in dark at 20 °C until the silica medium was thoroughly occupied by fungal vegetative mycelia. P. involutus F-CY01 did not grow in the system at all and was therefore excluded from the nursery inoculations.
Mycorrhiza
Unfertilized white nursery peat PP03 (Kekkilä Oy) was supplemented with 10 % (V/V) fully grown silica inoculum, and PL81F seedling containers (81 × cells of 85 cm3, BCC Ab) were filled with the peat-fungal inoculum mixture. Norway spruce seeds (seed-orchard seed R01-89-1002, Sv. 109) were sown on top of the peat–inoculum mixture. Altogether, 45 seedlings were grown with Amphinema sp. R-SP03 inoculum, and with the other inocula, 81 seedlings per ECM strain were grown. The seedlings were grown for 6 months in greenhouse at Haapastensyrjä forest nursery of the Finnish Forest Research Institute. The seedlings were fertilized from June to August by Superex-irrigation fertilization N-P-K (125-27) program (Kekkilä Oy) modified to keep the peat conductivity as low as 0.5–1 mS cm−1. Fertilization rate was approximately 5 ml/seedling/week. In the autumn, height of the seedlings was measured. Root systems of five replicate seedlings per fungal strain were subjected to gross morphotyping in order to assess the colonization of the roots by the inoculated fungi. The roots of the seedlings were washed and cut into 1–2 cm pieces, which were placed on water-filled Petri dishes. Root pieces were randomly selected until about 250 short roots were analyzed under a dissecting microscope (Euromex). The degree of colonization of the inoculated fungus per total number of short roots was determined as well as that of other fungi and the proportion of bare root tips. Statistical analyses The effects of different agar media on the growth rates of mycorrhizal species (Growth assay 1, Table 1) were tested using linear models in statistical program R (R Development Core Team 2011). The response variable in the models was the growth rate of a fungal strain per 30 days, and as an explanatory variable, we had different media as a factor with eight levels (i.e., MMN pH 5.5, ½MMN pH 4.5, ½MMN pH 5.5, ½MMN pH 6.1, Hagem’s pH 4.5, Pachlewski pH 5.0, PGM pH 6.5, and potato dextrose pH 5.6). In the models, the media with the highest growth rate was compared to the growth rates in the other media. Other pairwise comparisons were not performed. The effects of increasing brewery malt extract and humic acid (Humistar and Lignohumate) concentrations on the growth rates of different mycorrhizal species (Growth assay 2, Table 1) were analyzed using linear mixed models with lmer function in R (Pinheiro et al. 2011; R Development Core team 2011). As a response variable, we had the growth rate of each species (mm/30 d), and as explanatory variables, we had (1) a variable describing different malt extract concentrations (Maltax 2.5, 5, 7.5, and 10 %), (2) a variable describing different Humistar concentrations (HS 0, 0.2, 0.6, and 1.2 g/ l), and (3) a variable describing different Lignohumate concentrations (LH 0 and 0.6 g/l). The growth in the lowest malt
extract level, 0.1 %, was very poor, and therefore, this concentration was removed from the statistical analyses. We added two interaction terms to the models, i.e., between malt extract and Humistar concentrations and between malt extract and Lignohumate concentrations to investigate whether the effects of humic acids are different when the malt extract concentration increases. The interaction terms were removed if both of them were not statistically significant. Thus, we kept the simplest model with statistically significant effects for each species. Furthermore, the models included one random factor taking into account pseudoreplicates (the same fungus strain was always placed on two similar plates in each medium combination, and these observations are always correlated with each other because they are similar, i.e., the growth of a fungus has been measured twice on exactly similar plates). If data is not fully random, i.e., correlated observations exist, random factors should be included in the models (see e.g., Bolker et al. 2008). The effect of increasing brewery malt extract, Lignohumate, and phosphorus concentrations on the growth rate of different mycorrhizal species (Growth assay 3, Table 1) was analyzed using linear mixed models with lmer function in R (Pinheiro et al. 2011; R Development Core team 2011). The growth rate of each species (mm/30 d) was as a response variable in the models, and as explanatory variables, we had (1) a variable describing different malt extract concentrations (2.5 and 5 %), (2) a variable describing different Lignohumate concentrations (0, 0.2, 0.5, and 0.8 g/l), and (3) a variable describing the effect of phosphorus (as a factor 0 = phosphorus not added, 1 = phosphorus added). We added interaction terms between the explanatory variables 1 and 2, 1 and 3, 2 and 3, and between the variables 1, 2, and 3 to the models. We removed interaction terms from the models if they were not significant. However, non-significant interaction terms between two explanatory variables were removed only if a non-significant interaction between the explanatory variables 1, 2, and 3 was removed first. A random factor describing pseudoreplicates (two replicate plates per medium) was included in the models. Differences between the heights of spruce seedlings inoculated with different mycorrhizal fungi were investigated with linear model and ANOVA test (R Core Team 2011) which revealed whether any differences between the treatments exist.
Results Growth assay 1—commonly used laboratory scale nutrient media Investigated mycorrhizal species grew especially well on ½MMN media pH 5.5 and 6.1 (65 % of the strains, Table 2,
Mycorrhiza Table 2 The growth rates of 20 ECM fungal strains (Growth assay 1) in semisynthetic nutrient agar media Fungal strain
n
MMNa pH 5.5
½MMNb pH 4.5
½MMN pH 5.5
½MMN pH 6.1
Hagem’ s pH 4.5
Pachlewski pH 5.0
PGMc pH 6.5
PDd pH 5.6
Amanita muscaria F-SS01
40
Amphinema byssoides R-AR03
38
Amphinema sp. R-AR01
40
Atheliaceae R-RS06
40
−8.657± 0.337 −2.748± 0.179 −5.357± 0.489 −1.024± 0.556 −5.464± 0.272 −5.143± 0.458 −7.007± 0.477 −4.167± 0.279 −7.948± 0.146 −3.846± 0.958 −2.271± 0.371 −3.129± 0.303 −11.897± 1.209
−9.257± 0.337 −3.857± 0.168 −6.429± 0.489 −1.262± 0.556 −11.036± 0.272 −6.471± 0.458 −7.693± 0.477 −6.286± 0.279 −10.071± 0.146 −7.680± 0.958 −3.793± 0.371 −4.050± 0.303 35.224± 0.855
−9.279± 0.337 −1.479± 0.168 −0.750± 0.489 6.429± 0.393 −3.964± 0.272 −4.414± 0.458 −7.457± 0.477 −3.881± 0.279 −7.422± 0.146 −0.430± 0.958 −2.229± 0.371 −2.314± 0.303 −4.552± 1.209
−9.000± 0.337 9.686± 0.119 11.250± 0.345 −0.976± 0.556 −0.429± 0.272 −4.800± 0.458 −3.429± 0.477 −1.071± 0.279 −0.838± 0.146 29.638± 0.677 −4.939± 0.371 −1.521± 0.303 −5.224± 1.209
−8.743± 0.337 −6.257± 0.168 −6.279± 0.489 −2.762± 0.556 −11.491± 0.272 −9.021± 0.458 −8.046± 0.477 −5.833± 0.279 −9.857± 0.146 −17.013± 0.958 −7.479± 0.371 −4.864± 0.303 −9.879± 1.209
−9.086± 0.337 −1.543± 0.168 −2.400± 0.489 −2.071± 0.556 18.777± 0.192 −5.743± 0.458 −4.564± 0.477 −3.024± 0.279 −7.461± 0.146 −1.888± 0.958 15.386± 0.262 13.650± 0.214 −18.155± 1.209
−11.250± 0.337 −8.507± 0.168 −10.436± 0.489 −5.000± 0.556 −18.777± 0.272 −11.486± 0.458 −10.843± 0.477 −5.119± 0.279 −12.701± 0.146 −29.638± 0.958 −3.236± 0.371 −2.057± 0.303 −34.035± 1.209
12.214± 0.239 −3.846± 0.179 −4.136± 0.489 −1.786± 0.556 −2.277± 0.272 11.914± 0.324 10.843± 0.338 7.691± 0.197 12.701± 0.103 −21.846± 0.958 −7.286± 0.371 −4.693± 0.303 −26.638± 1.209
−1.136± 0.315 −2.293± 0.516 −3.150± 0.527 −2.191± 0.240 −2.500± 0.229 −1.805± 0.530 −1.145± 0.220
7.800± 0.223 −1.050± 0.516 10.393± 0.373 4.191± 0.170 −1.214± 0.229 −0.136± 0.530 5.364± 0.155
−4.532± 0.334 9.129± 0.365 −0.407± 0.527 −0.452± 0.240 6.143± 0.162 −0.115± 0.530 −1.567± 0.220
−2.229± 0.315 −5.250± 0.516 −4.221± 0.527 −1.857± 0.240 −2.691± 0.229 −2.502± 0.530 −1.054± 0.220
−0.836± 0.315 −3.064± 0.516 −4.114± 0.527 −1.548± 0.240 −1.333± 0.229 −2.058± 0.530 −2.812± 0.220
−7.800± 0.315 −9.129± 0.516 −10.393± 0.527 −4.191± 0.240 −6.143± 0.229 −6.896± 0.530 −5.364± 0.220
−4.864± 0.315 −5.314± 0.516 −3.921± 0.527 −0.214± 0.240 −1.708± 0.243 −0.833± 0.530 −0.659± 0.220
Cenococcum geophilum R-FC01 40 Cortinarius sp. F-SS11
40
Hebeloma sp. F-NB01
40
Hebeloma sp. R-RS01
40
Hebeloma sp. F-RS01
40
Laccaria sp. F-NC01
40
Meliniomyces bicolor R-FC06
40
Meliniomyces bicolor R-MF01
40
Paxillus involutus F-SS02
40
−0.043± 0.315 Piloderma fallax R-SP02 40 −1.307± 0.516 Tylospora asterophora R-MF02 40 −6.150± 0.527 Tylospora asterophora R-NC01 40 −1.167± 0.240 Tylospora asterophora R-RS03 40 −1.429± 0.229 Tylospora asterophora R-RS05 40 6.896± 0.375 Tylospora asterophora R-SP01 120 −1.391± 0.220 Piloderma byssinum R-FC07
39
The underlined value in each line indicates the growth rate (±standard error) of a fungus strain (mm/30 d) in the best medium. The growth rate in this medium has been compared with the others in the same line. Media which are as good as the best one are in bold (no statistically significant differences between the media, p≥0.05, linear models). Values not underlined indicate how much the growth rate (mm/30 d) differs from the underlined medium (the best one), see also Fig. 1 a
Melin-Norkrans medium
b
Melin-Norkrans medium with reduced sugar content
c
Pridham-Gottlieb medium
d
Potato dextrose medium
Fig. 1). Also, potato dextrose, Pachlewski, and MMN media were suitable for some of the fungal strains (preferred by 35, 20, and 15 % of the strains, respectively), but fungal growth in
Hagem’s and Pridham-Gottlieb media was quite poor. The 20 fungal strains showed species and genus specific preferences for the tested semisynthetic nutrient agar media (Table 2,
Mycorrhiza
16
12
8
PD 5.6
*
*
*
*
*
growth rate (mm/30 d)
20
*
*
12
*
8
*
4
0
24
Pach 1/2 MMN PD 5.0 6.1 5.6
* 1/2 MMN MMN 1/2 MMN Hagem PGM 5.5
5.5
4.5
4.5
12
4
0
24
*
*
*
* *
PD 1/2 MMN Pach 5.6 6.1 5.0
16
12
4
0
1/2 MMN MMN 5.5 5.5
*
*
*
*
Pach 1/2 MMN Hagem1/2 MMN PD 5.6 4.5 5.0 4.5 6.1
*
* PGM 6.5
1/2 MMN Pach 1/2 MMN MMN 1/2 MMN PD 5.6 5.5 6.1 5.5 4.5 5.0
*
*
Hagem PGM 4.5 6.5
16
12
*
8
*
*
*
*
4
PD 1/2 MMN 1/2 MMN MMN 5.5 5.6 5.5 6.1
*
*
Pach 1/2 MMN Hagem PGM 5.0 4.5 4.5 6.5
Meliniomyces bicolor R-FC06, n=40
20
16
*
*
12
*
*
* *
8
*
4
Pach 1/2 MMN MMN 5.0 5.5 5.5
24
20
*
*
Cortinarius sp. F-SS11, n=40
0
MMN1/2 MMN 1/2 MMN Hagem PGM 5.5 5.5 4.5 4.5 6.5
Piloderma byssinum R-FC07, n=39
8
*
20
24
16
*
*
4
0
Hebeloma sp. F-NB01, n=40
*
*
8
6.5
20
8
12
24
Cenococcum geophilum R-FC01, n=40
*
16
0
MMN Hagem 1/2 MMN Pach 1/2 MMN1/2 MMN PGM 5.5 6.5 5.5 4.5 6.1 5.0 4.5
16
Amphinema byssoides R-AR03, n=38
20
*
growth rate (mm/30 d)
growth rate (mm/30 d)
*
4
24
growth rate (mm/30 d)
growth rate (mm/30 d)
20
0
growth rate (mm/30 d)
24
Amanita muscaria F-SS01, n=40
growth rate (mm/30 d)
growth rate (mm/30 d)
24
PGM 1/2 MMN 1/2 MMN PD 6.1 6.5 5.6 4.5
Hagem 4.5
Tylospora asterophora R-SP01, n=120
20
16
12
8
4
0
1/2 MMN 5.5
* PD 5.6
*
*
*
*
*
Hagem 1/2 MMN MMN 1/2 MMN Pach 4.5 5.5 6.1 4.5 5.0
*
PGM 6.5
Fig. 1 Growth rates and standard errors of eight ECM fungal strains (mm/30 d) on different semisynthetic nutrient agar media in Growth assay 1: Hagem’s medium (Hagem), Melin-Norkrans medium (MMN), MelinNorkrans medium with reduced sugar content (½MMN), Pachlewski medium (Pach), Potato dextrose agar medium (PD), and Pridham-
Gottlieb medium (PGM). pH values of the media are given below each medium. Growth rates differing significantly (p
