Influence of the Culture Media and the Organic Matter in the Growth of Paxillus ammoniavirescens (Contu & Dessi).

The genus Paxillus is characterized by the difficulty of species identification, which results in reproducibility problems, as well as the need for large quantities of fungal inoculum. In particular, studies of Paxillus ammoniavirescens have reported divergent results in the in vitro growth while little is known of its capacity to degrade organic matter. For all the above, and assuming that this variability could be due to an inappropriate culture media, the aim of this study was to analyse growth in different culture media (MMN, MS, and 1/2 MS) and in the case of MMN in presence/absence of two types of organic matter (fresh litter and senescence litter) to probe the saprophytic ability of P. ammoniavirescens . We also evaluated the effects of pH changes in the culture media. Growth kinetics was assessed by weekly quantification of the area of growth in solid culture media over 5 wk, calculating the growth curves and inflection points of each culture media. In addition, final biomass after 5 wk in the different culture media was calculated. Results showed that best culture media are MS and 1/2 MS. Moreover, an improvement in growth in culture media containing decomposing fall litter was observed, leading to confirm differences in the culture media of this species with others of the same genus. Further, we established that all growth media suffered a significant acidification after fungal growth.

The genus Paxillus Fr. (Agaricomycota, Boletales, Paxillaceae) is widespread in the Northern Hemisphere and forms ectomycorrhizal associations with various host plant species, including hardwood and coniferous trees [12], and is common an array of ecosystems including degraded or contaminated habitats. The species Paxillus involutus is one of the best studied and a model species for ecological and physiological studies. Several authors have reported a large variability in the morphology and physiology between different isolates of this fungus, which suggests that P. involutus encompasses multiple species [3457].

Phylogenetic analyses of nuclear DNA performed by different authors [289] confirmed the presence of five different species in the P. involutus complex: P. involutus, P. ammoniavirescens, P. cuprinus, P. obscurosporus, and P. vernalis; among which it is extremely difficult to differentiate based on morphological characters or differential ammonia reactions. Misidentification can prompt numerous reproducibility problems and difficulties optimizing culture conditions, as demonstrated by the study of Jargeat et al. [2], who observed conflicting behaviours in the same culture medium for P. ammoniavirescens, P. cuprinus, and P. involutus.

Traditionally the most employed culture medium is Melin-Norkrans agar medium (MMN) [10], but numerous species present culturing issues in this medium. Molina and Palmer [11] argue these problems might be due to an inappropriate culture media, as demonstrated with the recalcitrant fungus Cantharellus cibarius that only grew in modified fries medium (MFM) [12], a complex and highly defined medium. In the case of P. ammoniavirescens very little is known about its growth kinetics, and only two authors [25] analyzed the growth of this species with varying outcomes. Additionally, it has been demonstrated that P. involutus in symbiosis with Pinus sylvestris seedlings have only a reduced ability to decompose fresh beech litter (Fagus sylvatica) [13]. Unfortunately, there is a lack of reporting on the saprotrophic abilities of P. ammoniavirescens.

Assuming that the observed variability could be caused by not using a culture media that provide the essential substances necessary for the optimal growth of P. ammoniavirescens, and with the main objective of using P. ammoniavirescens in reforestation programs, we studied in detail the growth kinetics of this species in different culture media with/without different types of organic matter, aiming to increase mycelium production. Additionally, we determined the pH variation caused by the fungi growth in different culture media.

MATERIALS AND METHODS

Insolates and culture conditions

Several pure mycelia cultures were obtained from fruit bodies collected in a polluted site of Asturias (North Spain), under Betula celtiberica [14]. Basidiome fragments were placed on solid MMN medium in Petri dishes (90 × 15 mm) containing 15 mL medium, and incubated at 23℃ in the dark. Cultures are available at the culture collection of the Area of Plant Physiology, Department of Biology of Organism and Systems, University of Oviedo.

PCR and DNA sequencing

DNA was extracted from culture (pool of five isolates) using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR was performed following Hedh et al. [8] using the primers ITS1 and ITS4 [15] and a further clean up of PCR products (NucleoSpin Gel and PCR Clean-Up kit; Macherey-Nagel, Düren, Germany) was performed before being sequenced using Big Dye Terminator 3 and an ABI 3730xl DNA Analyzer (both from Applied Biosystems, Foster City, CA, USA). The consensus sequence was determined by overlapping sequence data from both DNA strands, using the software Geneious Pro 4.8.2. Sequence is available from the GenBank database under the accession number KP241048.

Alignment and phylogenetic analyses

For species determination, the generated internal transcribed spacer (ITS) sequence was compared with 14 sequences from GenBank (Table 1). Sequences were aligned using MUSCLE (v3.7) [16], and gaps were scored as missing data. The best-fit evolutionary model was determined using the software jModelTest-2.1.4 [17] comparing different nucleotide substitution models, and selected based on the Akaike Information Criterion [18]. Bayesian analyses were performed using MrBayes v3.2.2 [19], with four Markov Chain Monte Carlo tests running simultaneously for 1,000,000 generations under General Time Reversible evolutionary model [20] with the proportion of invariable sites (I). Evolutionary trees were saved every 1,000 generations and the first 25% were discarded as burn-in. For the remaining trees, a majority rule consensus tree was computed to obtain estimations for Bayesian posterior probabilities (PPs). The phylogenetic tree was visualized and edited with FigTree v1.4.1.

Table 1

ITS sequences of the genus Paxillus included in the studied

All sequences are identified with the ID GenBank, the location and the host. Type specimens in bold.

ITS, internal transcribed spacer; ND, not determined.

aJargeat et al. (2014) [2].

bVellinga et al. (2012) [9].

cHedh et al. (2008) [8].

Maximum likelihood (ML) genealogies were constructed using MEGA v5.2 [21], under the same evolutionary model used for Bayesian analyses. The bootstrap consensus tree was inferred from 10,000 replicates. In both analyses Paxillus rubicundulus was used as outgroup.

Effects of culture media and organic matter

For the growth experiments in nine nutritive media, a 10 mm diameter mycelium plug was taken with a cork borer from actively growing mycelia (2 wk on MMN at 23℃). This plug was placed into Petri dishes (90 × 15 mm) containing 20 mL of each medium. The culture media lacking organic matter were MMN, Murashige and Skoog medium (MS) [22], and a modification of the previous media called 1/2 MS, wherein calcium and other macronutrients are halved. To test the effect of organic matter on fungi growth, we used an MMN medium, to which we added two types of organic matter at different concentrations. The first consisting of green leaves collected directly from the tree (green organic matter [G]); and a second, consisting of fallen leaves (brown organic matter [B]). In both cases, leaves were washed and dried at 70℃ for 24 hr. Later, they were ground to reach a size lower than 1 mm. In both cases three concentrations were assayed: 0.25, 0.5, and 1 g per plate. For a homogeneous distribution of the organic matter in the culture media, it was autoclaved separately and added in the solidifying media.

Growth was quantified weekly over the course of 5 wk by measuring the area occupied by fungal colonies in the different culture media, using the image analysis program Image-Pro Plus v4.5 [23]. At the end of this period, biomass was measured using dry colony weight. For this purpose, we eliminated the agar and traces of organic matter by heating the colonies in a warm water bath. Thereafter, the fungal material was dryed at 75℃ until it reached a constant weight. Ten samples per treatment were evaluated.

pH variation

To determine the pH variations caused by fungal growth in the nine culture media used, the pH was initially adjusted to 5.6 in all media before autoclaving at 120℃ for 20 min and 20 mL of each medium was poured into 10 Petri dishes (90 × 15 mm). Once solidified, the pH was measured on two plates (three points per plate, called Control 1) per media to determine how the autoclaving process affected it. In six plates, a 10 mm diameter fungal plug was taken with a cork borer from actively growing mycelia (2 wk on MMN at 23℃), and incubated at 23℃ in the dark until the colony had achieved a diameter of 5 cm. pH was measured at three zones on each plate: inside the mycelium, at the border of the mycelium and in an area without growth. In each zone four points were measured. After 5 wk, two more plates of each medium without inoculum (Control 2) were evaluated for changes to the pH.

Statistical and mathematical analysis

All data were analysed by analysis of variance (ANOVA) and post hoc Fisher's least significant difference (LSD) tests using SPSS software (SPSS Inc., Chicago, IL, USA). The level of significance was 0.05 for all purposes. The inflection point, which represents the moment when the exponential growth begins to decelerate, was calculated as the second derivative of the linear equation of the growth curves of each medium.

RESULTS AND DISCUSSION

Phylogenetic analyses

Sequence analyses of the ITS-rDNA from the examined samples produced identical topology and relationships among clades using ML (66% bootstrap) and Bayesian (90% PPs) tree analyses (Fig. 1), placing our isolate clearly inside the P. ammoniavirescens clade, in line with the results obtained by other authors [289].

Fig. 1

>Maximum likelihood (ML) consensus tree derived from the internal transcribed spacer data set. On each branch, the percentages (%) of 10,000 bootstrapping replicates supported by ML are shown, so are the Bayesian posterior probabilities (PPs) (in bold). The sequence of our isolates is shown in bold. The tree was rooted using a sequence from Paxillus rubicundulus. The scale shows the expected number of changes per nucleotide. PPs and bootstrap values under 50 not included.

We observed significant differences among the various culture media, with MS and 1/2 MS showing higher growth, while 1 g G MMN medium shows the worst growth index (Fig. 2).

Fig. 2

Growth curves of the colonies of Paxillus ammoniavirescens in the nine culture media analyzed. The ordinates axis represents the growth area expressed in cm2 and the abscissa axis the amount of time in weeks spent in culture. IP indicates the inflection point. Different letters within a single column indicate significant differences between different cultures media weekly for 5 wk (p ≤ 0.05 ANOVA and post hoc least significant difference tests). MMN, Melin-Norkrans agar medium; MS, Murashige and Skoog medium; G, green organic matter; B, brown organic matter plus MMN.

Growth kinetics of filamentous fungi is difficult to characterize, especially in solid medium. Erroneously, there is a tendency to interpret their growth in the same manner as with bacteria and yeast. However, growth in these fungi only takes place at the tip of the hypha and not in each individual cell. French and Hebert [24] consider that when a constant and uniform progress of the fungi in solid medium is observed, the fungus has a defined growth, whereas when the growth is not uniform the fungus has an undefined growth, which varies in response to different environmental factors. Analyzing the growth curves, we can observe that in all culture media where the fungi grew, a lag phase took place in the first week, except in 1/2 MS medium where it started growing exponentially from the beginning. Aside of this fact, in solid medium P. ammoniavirescens seems to have a defined growth, without constant growth which decelerated between the second and fourth week in all the culture media, as was reflected by the inflection points.

Our results demonstrate that MS and 1/2 MS were the best culture media for P. ammoniavirescens, given that whitin 3 wk all isolates covered the entire medium; with the lowest inflection points (MS 2.91 and 1/2 MS 2.07) and were also the ones which generated the highest biomass after 5 wk. Both media were very rich in microelements and vitamins, improving on the growth descriptions for malt extract peptone (MP) medium, which seemed to be key for increasing the growth rates on this species. It is also notable that when comparing the results obtained in MMN and MMN supplemented with organic matter, an improvement of growth rates is observed in all media with brown litter, as well as in the 0.25 g G MMN. The other two culture media with fresher litter showed either a decrease (0.5 g G MMN) or an inhibition (1 g G MMN) of growth.

The highest fungi dry weight was recorded in MS and 1/2 MS as culture mediums (Fig. 3). There were no significant differences between MMN and MMN with brown organic matter, although in the 0.5 g B MMN and 1 g B MMN the biomass increased and did not show a significant difference with 1/2 MS, which clearly supports that addition of G organic matter caused a significant decrease of dry weight.

Fig. 3

Means ± standard error of dry weight (mg) of the colonies of Paxillus ammoniavirescens after 5 wk of growth in the different culture media. Different letters above bars indicate significant differences between culture media (p ≤ 0.05 ANOVA and post hoc least significant difference tests). MMN, Melin-Norkrans agar medium; MS, Murashige and Skoog medium; G, green organic matter; B, brown organic matter plus MMN.

Only two authors [25] have studied the growth of P. ammoniavirescens with conflicting results; while Hahn and Agerer [5] observed slow growth (1 cm per month) in yeast malt agar, Jargeat et al. [2] reported a faster growth of all isolates on MP medium. This last author hypothesized that this discordance was due to insufficient cultures under analysis. However, the composition of the culture media used was also very different, the MP medium used by Jargeat et al. [2] is more complex than the medium used by Hanh and Agerer [5], with numerous microelements and macronutrients of MMN media, which could also explain those divergences in growth rates. Moreover, previous results from our group [23] showed that in a similar medium to that used by Hahn and Agerer [5], the culture medium MEPA [6], a significantly lower growth is observed as compared with other culture media (MMN, BAF Biotin-aneurin-folic acid agar [25], and MFM). In sight of these results, we hypothesise that the differences observed in this species are not only due to genetic variability but could be potentially caused by an inappropriate culture media.

Data for Control 1 showed that autoclaving slightly modified the initial pH, as seen by the acidification of the MMN, MS, and 1/2 MS media, which is not attributable to the addition of organic matter (Table 2). Additionally, a significant pH increase is observed after 5 wk in plates without fungus in all culture media with green organic matter and in 1 g B MMN (Control 2).

Table 2

pH of Paxillus ammoniavirescens colonies in the different culture media

Values are presented as mean ± standard error.

Different notation within a single row indicate significant differences within a single medium.

Control 1, pH of culture media after autoclaving; Control 2, pH after 5 wk (p ≤ 0.05 ANOVA and post hoc least significant difference tests); MMN, Melin-Norkrans agar medium; MS, Murashige and Skoog medium; G, green organic matter; B, brown organic matter plus MMN.

Our results showed that the addition of green organic matter caused an initial acidification greater than that exhibited by all other culture media. Moreover, in absence of fungi, the pH increased in all culture media with organic matter, with significant differences found in all those containing G organic matter and in 1 g B MMN. Fungal growth produced significant acidification in all media, moreover there were also significant differences among the various areas recorded in all media with B organic matter.

In conclusion, assuming that environmental conditions are stable, pH variations in the culture media, the depletion of some elements or essential substances or even the accumulation of toxic metabolites [24] can affect the growth of P. ammoniavirescens. Further, the results above allow us to conclude that culture media MS and 1/2 MS are the most suitable for the growth of P. ammoniavirescens. Moreover, P. ammoniavirescens was shown to be capable of decomposing fallen leaf litter but not fresh litter. The use of MS and organic matter could potentially help to improve the culture of recalcitrant fungal species.