Isolation of bacteria from ectomycorrhizae of Tuber aestivum Vittad.

Fifteen different cultivation media were used to isolate bacteria with the idea to obtain taxa specifically associated with ectomycorrhizae of Tuber aestivum . Ectomycorrhizae were collected at the sampling points previously analyzed for bacterial molecular diversity. We isolated 183 bacterial strains and identified them on the basis of the partial sequence of 16S rDNA. Out of these isolates, only 4 corresponded to operational taxonomic units significantly associated with T. aestivum ectomycorrhizae in previous molecular study. Preliminary study of the effect of 12 selected isolates on growth of T. aestivum mycelium showed no stimulation and one isolate induced the damage of hyphae. Different isolation strategy has to be developed to increase the probability of cultivation of potentially important components of T. aestivum mycorrhizosphere.


INTRODUCTION
For centuries, truffles have been highly appreciated as a special culinary ingredient, being highly priced on the markets.Although some of the truffle species have successfully been produced in plantations, attempts to culture other species have repeatedly failed and they can only be collected from their primary habitats.Life strategy of different truffle species is understood insufficiently and mainly the interactions between truffles and mycorrhizae-and mycelium-associated microflora merits attention.
Truffles are ectomycorrhizal Ascomycota deriving significant portion, if not all, of their carbon nutrition from a host tree via specialized communication organs, ectomycorrhizae.The soil space directly affected by ectomycorrhizae is called mycorrhizosphere (Linderman 1988) and is characterized by high biological activity (Chalot, Brun 1998).Presence of some bacteria may enhance the formation of ectomycorrhizal structures on the roots and was shown to change gene expression of the ectomycorrhizal fungal hyphae (Deveau et al. 2007).Some of these microbes referred to as "mycorrhization helper bacteria" (Garbaye 1994) have received a special attention because they can stimulate mycorrhizal colonization of the host roots by the truffles with economic consequences Of the few available studies directed to microbial associates of truffles, some have focused on bacteria (Citterio et al. 2001;Bedini et al. 1999;Sbrana et al. 2002) and yeasts (Zacchi et al. 2003) associated with the ascocarp or ectomycorrhizae.The information on the microflora of the soil inhabited by truffles is also infrequent and has been published for yeasts by Zacchi et al. (2003) and, for saprotrophic fungi by Luppi-Mosca (1973, in Napoli et al. 2008) and for bacteria by Sbrana et al. (2002).The most striking result of these studies is a strong association of yeasts Cryptococcus spp. with ectomycorrhizae of the truffle ground reported by Zacchi et al. (2003).Some novel information is gradually becoming available using cultivation-independent molecular methods such as high-throughput PCR and DNA sequencing.These approaches allowed insights into mycorrhizal community of the productive spots of T. magnatum -showing that the mycorrhizae of this fungus are generally rare and most roots of the host trees are occupied by other mycorrhizal fungi (Murat et al 2005).Molecular analysis of bacterial and fungal communitites associated with the productive spots of T. magnatum failed to identify any specific fungi associated with this truffle, whereas they suggested a bacterium Moraxella osloensis (a gamma-Proteobacterium) to be preferentially associated with the productive spots of this truffle (Mello et al. 2010).Our previous study (Gryndler et al. 2012) revealed a specific association of some bacteria, including four genera of actinobacterial suborder Pseudonocardineae, with ectomycorrhizae of Tuber aestivum Vittad.
In this study, we aimed at isolation of bacteria from the communities associating to the T. aestivum ectomycorrhizae.T. aestivum (incl.forma uncinatum) has been recently rediscovered as a valuable alimentary product in many European countries and is currently considered to be the most common European truffle with gradually increasing commercial value.Knowledge of the interactions of this truffle species with accompanying soil microflora might be of practical importance, for example in formulation of complex truffle inocula used in artificial host seedlings inoculations, containing beneficial (e.g., mycorrhization-helper) bacteria.
Ten replicate plates were established per medium.Growing bacteria were subcultured on medium B with the same composition as the above medium A, except that CaCO 3 was omitted and the concentration of anhydrous CaCl 2 was increased to 1.27 g per liter.Bacterial colonies of different morphological properties were chosen for subcultivation in order to obtain as many as possible different bacterial taxa.
Bacterial isolates were then inoculated to 10 ml of the liquid medium B (without agar) and bacterial biomass was pelleted by centrifugation.DNA was extracted from the pellet using Nucleo-Spin Soil DNA kit (Macherey-Nagel GmBH & Co., Germany) and a fragment of 16S rDNA was amplified in PCR with forward primer eub530F (5´-gtg cca gcm gcn gcg g-3´) and reverse primer eub1100aR (5´-ggg ttn cgn tcg ttg cg-3´).The primers were modified from Dowd et al. (2008).Cycling conditions were 94 ºC for 5 min, followed by 35 cycles of 94 ºC for 1 min, 62 ºC for 50 s, and 72 ºC for 30 s, and concluded by incubation at followed by 72 ºC for 10 min.
PCR products were then sequenced and the identity of isolates was estimated using comparison of their partial 16S rDNA sequence with GenBank database.
Selected isolates were tested for their possible effects on the growth of culture of T. aestivum, strain Tae5 (maintained in the Laboratory of Fungal Biology, Institute of Microbiology ASCR, Prague, Czech Republic).The truffle mycelium was first pre-cultured on the medium PEX (Pebeyre S.A., Cahors, France) for 6 weeks and the bacterium was then applied as one 4-cm line per dish, whose middle was 5 mm apart from the edge of the mycelial colony.The growth of Tuber mycelium in the proximity of the bacterial colony was observed after 7 days of co-cultivation.
The order Pseudomonadales was represented by the genera Pseudomonas (possibly P. putida, the only organism willing to grow on the medium containing oak root extract) and Moraxella.
Escherichia (possibly E.coli, 1 isolate) was the only representative of the order Enterobacteriales and the order Xanthomonadales was represented by the genera Lysobacter (possibly L. antibioticus, 3 isolates) and Dyella (1 isolate).
The identification of the isolates is provisional and may be refined in future, if a particular isolate will prove to have beneficial effects on mycorrhizal colonization of truffle-inoculated tree seedlings.

DISCUSSION
In spite of extensive effort during the isolation of the bacteria from ectomycorrhizae of T. aestivum, we were able to cultivate the members of 6 bacterial orders.This is very low number in comparison with the results of molecular analysis of the same material (Gryndler et al. 2012), which detected operational taxonomic units that can be grouped into a total of 79 bacterial orders.This discrepancy confirms the hypothesis that cultivation-based studies reveal just negligible portion of the microbial diversity of mycorrhizosphere.
Our recently reported effort is the continuation of the past works (Gryndler et al. 2012) led by the intention to cultivate mainly the bacterial taxa that are specifically associated with ectomycorrhizae of T. aestivum.This effort was, however, only partially successful and the isolates of Lysobacter sp. and Ensifer sp.remain the only bacteria that were significantly positively associated with T. aestivum ectomycorrhizae.In particular, we repeatedly failed to isolate the members of the actinobacterial genera Actinosynnema, Allokutzneria, Kibdelosporangium and Lentzea, the members of the suborder Pseudonocardineae which proved to positively correlate with the presence of T. aestivum in ectomycorrrhizae.In spite of the fact that Pseudonocardineae members are generally considered rare organisms in the nature (Jarerat et al. 2002), our molecular data predict that Pseudonocardineae members represent significant portion of biomass of the root-associated microbial community.However, they either do not produce sufficient amounts of colony forming units necessary for isolation or cannot be cultivated using our methodology.At the same time, their colony forming units may be hidden on dilution plates by high numbers of massively sporulating members of Actinomycetales.
There is no information on interactions of the members of this suborder with fungi or plants available in the literature.Some members of this group possess the ability to degrade poly-L-lactate (a kind of plastic, also used as a cultivation medium component in our work), which was originally considered as unique among the actinobacteria (Jarerat et al. 2002).Even though some other actinobacterial degraders of this material were described later (Sukkum et al. 2009), the suborder Pseudonocardineae remains important pool of organisms possessing this activity.This may indicate that the members of this suborder may possess exotic metabolic capabilities, perhaps connected with very specific nutritional demands which were not met during our isolation approach.
Further work and probably different isolation strategy will be needed to obtain the cultures of the bacteria specifically associated with ectomycorrhizae of T. aestivum.We hope, this work will be worth of effort as these bacteria may have potentially interesting effects on truffle mycelium and mycorrhiza functioning.

Fig. 1 .
Fig. 1.Interaction of in vitro-cultivated mycelium of T. aestivum with Phyllobacterium sp.(A, C) or Rhizobium cf.leguminosarum (B, D).During the 7-day co-cultivation, the bacteria (dark mass in the figure) became to close contact with hyphae (A, B).Whereas hyphae in the proximity of Phylobacterium sp.colony had normal morphology (C), the proximity of Rhizobium cf.leguminosarum caused extensive vacuolization.Scale bar = 100 μm.