Endobacteria in Some Ectomycorrhiza of Scots Pine (Pinus Sylvestris) Hironari Izumi1,2, Ian C

Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris) Hironari Izumi1,2, Ian C. Anderson1, Ian J. Alexander2, Ken Killham2 & Edward R.B. Moore1

1The Macaulay Institute, Craigiebuckler, Aberdeen, UK; and 2School of Biological Sciences, University of Aberdeen, Aberdeen, UK

Correspondence: Ian Anderson, The Abstract

Macaulay Institute, Craigiebuckler, Aberdeen Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 AB15 8QH, UK. Tel.: 144 0 1224 498200; The diversity of cultivable endobacteria associated with four different ectomycor- fax: 144 01224 498207; e-mail: rhizal morphotypes (Suillus flavidus, Suillus variegatus, Russula paludosa and [email protected] Russula sp.) of Scots pine (Pinus sylvestris) was analysed by restriction fragment length polymorphism profiling of PCR-amplified rDNA intergenic spacer regions Present address: Edward R.B. Moore, and by sequence analyses of 16S rRNA genes. Ectomycorrhizal root tip surface- Department of Clinical Bacteriology, sterilization methods were developed and assessed for their efficiencies. Bacterial Sahlgrenska University Hospital, Goteborg¨ communities from surface-sterilized ectomycorrhizal root tips were different from University, Guldhedsgatan 10 A, SE-413 46 those of ectomycorrhizal root tips without surface-sterilization for all the Goteborg,¨ Sweden. morphotypes studied. Endobacteria belonging to the genera Pseudomonas, Bur-

Received 23 May 2005; revised 23 September kholderia and Bacillus were isolated from more than one ectomycorrhizal 2005; accepted 25 September 2005. morphotype, whereas species of Rahnella, Janthinobacterium and Rhodococcus First published online 15 February 2006. were only isolated from the single morphotypes of S. variegatus, R. paludosa and Russula sp., respectively. Some of the isolated endobacteria utilized fungal sugars doi:10.1111/j.1574-6941.2005.00048.x more readily than typical plant sugars in carbon utilization assays.

Editor: Jim Prosser

Keywords endobacteria; ectomycorrhiza; ectomycorrhizal fungi; 16S rRNA genes.

was greater in the mycorrhizosphere of Douglas fir colo- Introduction nized by Laccaria bicolor than in the bulk soil, and bacteria Ectomycorrhizas are ubiquitous in temperate forest ecosys- in the mycorrhizosphere solubilize inorganic phosphorus in tems (Smith & Read, 1997). Ectomycorrhizal fungi are vitro more efficiently than those from the bulk soil (Frey- known to have beneficial effects on host plants by improving Klett et al., 2005). nutrient acquisition from soil through hyphae connected to Bacteria that colonize plant tissues internally without the roots, while host plants allocate 10–20% of their current causing disease symptoms are known as endophytic bacteria photosynthate to ectomycorrhizal fungi (Smith & Read, (Chanway, 1998). Endophytic bacteria have been observed 1997). The positive contribution of ectomycorrhizal fungi in a wide range of plant species and tissues, including Scots to host plants has been hypothesized to vary depending pine buds and spruce roots (Pirttila et al., 2000; Shishido upon the morphology of the ectomycorrhiza (Agerer, 2001). et al., 1999), and may play an important role in the According to this hypothesis, ectomycorrhizas with exten- improvement of growth of host plants (Bent & Chanway, sive external hyphae can reach nutrient sources in soil more 1998) or defence against pathogens (Barka et al., 2002). efficiently than those without them. Because the morpholo- Bacteria have also been found in association with ectomy- gies of ectomycorrhiza are primarily determined by ectomycorrhizas. For example, endobacteria belonging to the gen- corrhizal fungal species, different ectomycorrhizal fungi era Burkholderia and Paenibacillus were isolated from Pinus provide different contributions to the growth of host plants sylvestris – Lactarius rufus ectomycorrhizas following sur-

(Read & Perez-Moreno, 2003). face-sterilization with H2O2 (Poole et al., 2001). Endobac- Mycorrhizal fungi may also have an indirect influence on teria in this context are defined as those bacteria that exist host nutrition through their effects on bacterial commu- within the fungal or host compartments of the mycorrhiza, nities in the mycorrhizosphere (Johansson et al., 2004). For or conceivably within the cells of either of the symbionts. example, the diversity of Pseudomonas fluorescens genotypes There is some evidence that endobacterial communities may

c 2005 The Macaulay Land Use Research Institute FEMS Microbiol Ecol 56 (2006) 34–43 Published by Blackwell Publishing Ltd. All rights reserved Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris) 35 differ between ectomycorrhizas formed by different fungal surface. After washing, the root tips were sorted, classified species. For instance, Pseudomonas spp. were isolated more and grouped into morphotypes using the approach of Agerer frequently from Thelophora ectomycorrhizas of subalpine fir (1987–1993). In the first experiment, three distinct but than from those of other ectomycorrhizal fungi such as unidentified morphotypes were used. In the second experi- Cenococcum and E-strain after extensive washing (Khetmalas ment, four distinct morphotypes, two of which had a et al., 2002). Furthermore, endobacterial Pseudomonas spp. morphology characteristic of Suilloid fungi and two charac- and Bacillus spp. have been isolated from inside sporocarps teristic of Russulaceae, were selected for further study. Several of Tuber borchii and Suillus grevillei (Varese et al., 1996; hundred individual tips of these morphotypes were carefully Gazzanelli et al., 1999). Such lines of evidence suggest that cleaned of debris using fine forceps under a dissecting different ectomycorrhizal fungi may enrich different endo- microscope, and kept on moist filter paper, at 4 1C, until bacterial communities, although differences in isolation further processing. Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 methods may influence any comparisons made. Therefore, assessment of the diversity of endobacteria associated with Identification of ectomycorrhizal morphotypes ectomycorrhiza requires the analysis of more than a single DNA was extracted from several representative ectomycorrhi- species of ectomycorrhiza. Although the role of endobacteria zal root tips of each morphotype as described by Gardes & in ectomycorrhizas is not fully understood, there is some Bruns (1993). The fungal internal transcribed spacer (ITS) evidence to suggest that they may act as ‘mycorrhization region of rDNA was amplified using the primer pairs ITS1F helper bacteria’ (MHB) that promote mycorrhizal formation and ITS4B (Gardes & Bruns, 1993). PCR programmes con- (Garbaye, 1994). For example, Paenibacillus amylolyticus has sisted of one cycle of 94 1C for 10 min, followed by 35 cycles of been shown to enhance ectomycorrhizal colonization of 94 1C for 30 s, 55 1Cfor30sand741C for 30 s, and a final Scots pine roots (Poole et al., 2001). primer-extension of 74 1C for 10 min. The 25-mL reaction mix The aim of this study was to analyse the diversity of included 0.5 mL of extracted DNA, 25 pmol of primers, 1.25 U cultivable endobacterial communities associated with Scots of Taq DNA polymerase (Bioline, London, UK), 250 mMeach pine ectomycorrhiza. One of the problems in studying such dNTP, 1Â reaction buffer (Bioline) and 1 mM MgCl .Purified bacteria is being able to distinguish between those bacteria 2 fungal ITS PCR products were subjected to digestion with Taq that are genuinely endobacterial in the sheath or host tissues, I restriction enzyme in a final volume of 20 mL containing and those that reside on the surface of the ectomycorrhizas. 1Â reaction buffer with 10 U of endonuclease (Promega, Two approaches were taken to address this problem. In the Madison, MI), and 0.2 mgofbovineserumalbumin(BSA) first, the efficiencies of several surface-sterilization proce- (Promega). The reaction was incubated for 3 h at 65 1C, with a dures were compared by checking for cultivable bacteria in final inactivation step of 95 1C for 10 min. The entire volume the final rinse water after surface sterilization of the ectomy- of the reaction was analysed by agarose gel (3%) electrophor- corrhizal root tips. In the second approach, an efficient esis in Tris-borate-EDTA (TBE) running buffer, stained with sterilizing agent (30% H O ) was used. Restriction fragment 2 2 ethidium bromide. Hyperladder VI (Bioline) was used to length polymorphism (RFLP) patterns of PCR-amplified determine the size of the digested fragments. The RFLP 16S rRNA genes of mixed cultures obtained from unster- patterns were visualized and documented using the Gene ilized macerated ectomycorrhizas were compared with those Genius Bio Imaging System (Synoptics, Cambridge, UK). ITS obtained from mycorrhizas treated with the sterilizing agent PCR products were also sequenced using the primers ITS1F for various time periods. Sequences were obtained from and ITS4B and the Big Dye Terminator v.1.1 Cycle Sequencing individual endobacterial isolates, and the carbon utilization Kit on an ABI PRISM 310 Genetic Analyser (Applied Biosys- patterns of these were determined to examine whether these tems, Foster City, CA). The sequences obtained were aligned isolates were capable of utilizing both fungal- and plant- using Sequencher software version 3.0 (Gene Codes Corp., derived carbon sources. Ann Arbor, MI) and comparisons were made with reference sequences of the European Molecular Biology Laboratory Materials and methods (EMBL) Nucleotide Sequence Database (Kulikova et al., 2004), using the FASTA algorithm (Pearson & Lipman, Collection and morphotyping of ectomycorrhizal 1988). Sequences were obtained in a similar manner from roottips fruit bodies of ectomycorrhizal fungi growing at the same site. Ectomycorrhizal root tips were collected in April 2003 from Efficiency of surface-sterilization of the surface organic layer of a Pinus sylvestris stand at Red ectomycorrhizal roottips (Experiment1) Moss, Aberdeenshire, Scotland (NJ 705 176 GB). The ectomycorrhizas were washed by shaking with water in a 50 mL One per cent sodium hypochlorite and 15% and 30% H2O2 conical tube in order to remove soil particles from the (both from Fisher Scientific, Loughborough, UK) were com-

FEMS Microbiol Ecol 56 (2006) 34–43 c 2005 The Macaulay Land Use Research Institute Published by Blackwell Publishing Ltd. All rights reserved 36 H. Izumi et al. pared as sterilization reagents. Ten to 20 ectomycorrhizal form–isoamyl alcohol (25 : 24 : 1) (Sigma, Poole, UK). The root tips of each morphotype were immersed in respective CTAB extraction buffer was prepared by mixing 6 g of CTAB sterilant for time periods up to 5 min or were washed 10 times and 3 g of polyvinylpyrrolidone (Sigma) in 1.4 M NaCl and in 3 mL of 0.1% Tween 80. The sterilization treatment was 100 mM Tris Base (Promega) with 20 mM EDTA and 0.2% terminated by removing the sterilant using vacuum filtration b-mercaptoethanol (Sigma). The bacterial cell suspension followed by five rinses with 3 mL sterile water. The final rinse was lysed, using the FastPrep cell disruptor (Bio101, Vista, water was collected and inoculated onto culture media for CA) at the machine speed setting of 5.5 m sÀ1, for 30 s. The sterility assessments. Trypticase Soy Broth (TSB) and R2A aqueous phase was separated by centrifugation (20 817 g) for (both Oxoid, Basingstoke, UK) were used for isolating fast 5 min at 4 1C and residual phenol was removed by mixing growing and more oligotrophic bacteria, respectively. Inocu- with 600 mL of cold chloroform, followed by centrifugation lated culture media, containing 50 mg LÀ1 cycloheximide to (20 817 g) for 5 min at 4 1C. The final aqueous phase was Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 inhibit fungal growth, were incubated at 25 1Cfor5days. recovered and DNA was precipitated by adding two volumes Root tips were homogenized using a micro-pestle and of 2-propanol with 0.1 M sodium acetate (pH 7.0), followed inoculated into the two media after surface-sterilization. by centrifugation (20 817 g) for 30 min at 4 1C. Pelleted DNA was washed with 600 mL cold 70% ethanol and air dried Cultivation and isolation of endobacteria prior to resuspension in 50 mL of RNAse-free water (Fisher (Experiment 2) Scientific). Intergenic spacer (IS) regions between the bacterial 16S Ectomycorrhizas of the four different morphotypes (Table and 23S rRNA genes were amplified by PCR using the 1) were each divided into seven subgroups consisting of primers M16F1195 (5-AGGAAGGTGGGGATGACGTC-3) approximately 10 root tips and immersed in 3 mL of 30% and 23R458 (5-CCCCTTTCCCTCACGGTAC-3), hybridiz- H O for 0 and 10 s, or 1, 2, 5, 7 and 10 min. The 2 2 ing at 16S rRNA gene nucleotide positions 1176–1195 and sterilization treatment was terminated as described above. 23S rRNA gene nucleotide positions 458–476 (Escherichia coli The sterilized mycorrhizas were macerated using a micro- rRNA gene sequence numbering), respectively (based on the pestle, in 400 mL of TSB or R2A broth medium. The sequences in Hauben et al. (1997) and Guasp et al. (2000). homogenate (100 mL) was inoculated into TSB or R2A Bacterial rRNA IS sequences allow the assessment of bacterial medium containing 50 mg LÀ1 cycloheximide. After 4–5 diversity at the species level. Reactions were performed on a days of incubation at 25 1C, with shaking, bacterial suspen- DNA Engine PTC-200 Peltier Thermal Cycler (MJ Research, sions were inoculated onto TSA or R2A agar medium as Waltham, MA), using the following programme: one cycle of streak plates for isolation of individual colonies. The final 95 1Cfor5min,35cyclesof951C for 45 s, 55 1Cfor45sand rinse waters were also collected and inoculated into media 72 1C for 2 min, followed by a final extension at 72 1Cfor for validation of sterility. For molecular analyses of total 10 min. The 25 mL reaction mix contained 1 mLof100-fold cultivable populations, the bacterial cells were recovered diluted DNA extract, 25 pmol of primers, 0.6 U Taq DNA from the broth cultures produced using ectomycorrhizal polymerase (Qiagen, Crawley, UK), 250 mM of each dNTP homogenate by centrifugation (20 817 g, 10 min) and the (Bioline) and 1 mL BSA (Roche, Essex, UK) in a 1Â Qiagen cell pellets were stored at À 20 1C. PCR buffer with Q-Solution (Qiagen). After amplification, 10 mL of the PCR products were digested with Taq I restric- rRNA intergenic spacer RFLPanalysis of tion enzyme and analysed as described above for the ectomy- endobacteria corrhizal root tips. Bacterial DNA was extracted from cell pellets of bacterial cultures using a modified Griffiths’ method (Griffiths et al., 2000). The bacterial cell pellets were suspended in a mixture 16rRNA gene sequence analysis of 0.6 mL CTAB extraction buffer and 0.6 mL phenol–chloro- The bacteria were selected on the basis of colony morphologies on agar media cultures originating from mycorrhizas Table 1. Identity of Scots pine ectomycorrhizal morphotypes used in that had been surface sterilized for 2 min or longer. DNA was Experiment 2 extracted from single colonies as described above. Nearly Sequence complete (1466 nucleotides) 16S rRNA genes were amplified Closest species similarity Overlap by PCR as described above, using the forward primer Morphotype match (%) (bp) M16F28 (5-AGAGTTTGATCKTGGCTCAG-3) and reverse p10 (AJ971401) Suillus variegatus (AJ971399) 99.0 639 primer M16R1494 (5-TACGGYTACCTTGTTTACGAC-3), p11 (AJ971402) Russula paludosa (AJ971400) 100.0 647 hybridizing at 16S rRNA gene sequence nucleotide positions p12 (AJ971403) Suillus flavidus (AY641460) 99.6 544 (E. coli gene sequence numbering) 9–28 and 1494–1514, p14 (AJ971404) Russula paludosa (AY061703) 98.2 667 respectively. The PCR products were purified, using the

QIAquick PCR purification kit (Qiagen) and were se- Table 2. Presence of cultivable bacteria in final rinse water and surface- quenced as described above except that primers M16F355 sterilized homogenates of the ectomycorrhizal root tips before and after (5-ACTCCTACGGGAGGCAGC-3) and M16R1087 (5- treatment with different sterilizing reagents (Experiment 1) CTCGTTGCGGGACTTAACCC-3) (Hauben et al., 1997) TSB R2A were used. The sequences obtained were aligned and ana- Before After Before After lysed as described above for ectomycorrhizal root tips. Sterilization method WMWMWMWM Analysis of carbon-utilization by endobacteria None (0.1% Tween 80 only) 11111111 1% NaClO (5 min treatment) 11111111 Bacterial isolates obtained as detailed above were inoculated 15% H2O2 into Biolog GN2 plates, according to the manufacturer’s (1 min treatment) 11À 11 1 À 1 instructions (Biolog Inc., Hayward, CA). Before inoculation (2 min treatment) 11À 11 1 À 1 Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 of the Biolog plates, cell turbidity of the inoculum (15 mL) (5 min treatment) 11À 11 1 À 1 was standardized at a transmittance level of 55%. Inoculated 30% H2O2 Biolog plates were incubated at 25 1C for 3–4 days and (1 min treatment) 11À 11 1 À 1 (2 min treatment) 11À 11 1 À 1 utilization of carbon sources was determined by measuring (5 min treatment) 11ÀÀ11ÀÆ Redox dye colour change at 590 nm using the Biolog Micro Distilled water (negative control) À NNNÀ NNN station (Biolog Inc.). Plus and minus indicate presence and absence of cultivable bacteria, respectively. N, nonapplicable; W, final rinse water; M, homogenized Nucleotide sequence accession numbers ectomycorrhizal root tips. Distilled water of the negative control is the The bacterial 16S rRNA gene sequences were deposited in rinse water of sterile glassware before surface-sterilization was carried the EMBL nucleotide sequence database under accession out. numbers AJ971378–AJ971398. Ectomycorrhizal fungal sequences were also deposited under the accession numbers indicated in Table 1 or longer. Cultivable bacteria were recovered from homo-

genized root tips treated with 15% H2O2 for 1, 2 and 5 min, Results and from tips treated with 30% H2O2 for 1 and 2 min. Treatment with 30% H2O2 for 5 min eliminated all surface- Identification of ectomycorrhizal morphotypes attached bacteria and those in the ectomycorrhizas. The results were the same for all three morphotypes. PCR–RFLP (Taq I) patterns of fungal ITS from replicate ectomycorrhizal extractions of the same morphotype were identical, confirming that the morphotype collections were Cultivable endobacterial community structures genotypically uniform. Based on the sequence similarities among different ectomycorrhizal morphotypes between ectomycorrhizas and fruit bodies collected at the Cultivable surface bacteria were again removed from all site, morphotypes p10 and p11 were identified as Suillus ectomycorrhizal morphotypes as indicated by the absence variegatus and Russula paludosa, respectively. Morphotype of growth in final rinse waters after 2 min of treatment with p12 was identified as Suillus flavidus by comparison with a 30% H O . In the case of Suillus flavidus and Russula sp. fruit body sequence from another Scots pine site in north- 2 2 morphotypes, the surface bacteria appeared to be eliminated east Scotland (Table 1). No close matches to fruit bodies by even shorter periods (Fig. 1). Therefore, bacterial cultures were obtained for morphotype p14 and the closest database recovered from ectomycorrhizal root tips treated for more match was to Russula paludosa with 98.2% sequence simi- than 2 min were presumed to be endobacterial. larity. Given the amount of sequence variation that can Genotypic profiling of the total cultivable bacterial com- occur within Russula species and the fact that p14 was munities associated with the ectomycorrhizal morphotypes different from p11, which had a 100% match with Russula was carried out by RFLP, using amplified IS regions of paludosa, p14 was designated as ‘Russula sp’. bacterial rRNA digested with Taq I restriction enzyme (Fig. 1). For all morphotypes, IS RFLP profiles for bacteria Surface-sterilization of ectomycorrhizal roottips isolated from nonsurface-sterilized root tips were the same Bacteria grew in both TSA and R2A media inoculated with on TSB and R2A media (Fig. 1). Variation in bacterial IS the final rinse waters after the treatments with 0.1% RFLP banding patterns were observed only after prolonged detergent (Tween 80) or 1% sodium hypochlorite for 5 min exposure to H2O2, although the time period of exposure (Table 2), but bacteria could not be cultivated from the rinse before changes were observed differed for each morphotype. waters after treatments with 15% and 30% H2O2 for 1 min Changes in IS RFLP profiles for Suillus flavidus and Russula

TSB R2A

0sec. 10sec 1min 2min 5min 7min 0sec. 10sec 1min 2min 5min 7min MMM (a)

1000bp

400bp

200bp

100bp Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021

+ ++– – – +++–––

(b) 1000bp 400bp 200bp 100bp

+ – ++–– –––

400bp 200bp

100bp

+––– +––– (d) 1000bp Fig. 1. Taq I restriction fragment length polymorphism patterns of bacterial intergenic spacer regions 400bp over the course of surface sterilization (Experiment 200bp 2). Plus and minus below each lane indicate positive or negative bacterial growth in the ﬁnal rinse water 100bp after treatment. Blank lanes indicate no growth obtained in the sample. a, p10 (Suillus variegatus); b, p11 (Russula paludosa); c, p12 (Suillus ﬂavidus); ++–––– ++––– d, p14 (Russula sp.).

sp. were observed after 1 min exposure to H2O2, whereas 16S rRNA gene sequence analysis of endobacteria root tips of Russula paludosa and Suillus variegatus had to be exposed for 2 and 7 min, respectively, before different IS Endobacterial isolates with different colony morphologies RFLP profiles were observed (Fig. 1). Interestingly, after on culture plates exhibited different RFLP profiles (data not surface sterilization for these time periods, IS RFLP profiles shown). Partial sequencing of the 16S rRNA genes showed were extremely diverse among the four different morpho- that most of the isolates could be matched, with greater than types, and further changes in RFLP profiles were observed 99% similarity, to sequences in the EMBL database (Table with longer exposures to the sterilizing reagent in all cases 3). Sequence similarities ranged from 99.6% (Pseudomonas except Suillus variegatus (Fig. 1). For example, bacteria tolaasii in Suillus flavidus) to 93.4% (several Paenibacillus isolated from Russula sp. root tips in TSB produced identical spp. in Russula sp.). In general, sequences most closely

IS RFLP proﬁles after 0 and 10 sec exposure to H2O2, but related to those of Pseudomonas spp. had higher similarities were changed after 1, 2, 5 and 7 min exposure (Fig. 1). to the reference database sequences than those most closely

Table 3. Characterization of endobacterial isolates based on 16S rRNA gene sequence comparisons Bacterial Associated Most closely related Sequence EMBL accession Most closely related Sequence EMBL accession isolate ectomycorrhiza reference bacterium similarity (%) number type strain similarity (%) number tAp10 p10 Rahnella aquatilis 98.7 AY253921 Serratia grimesii 98.2 AJ233430 tDp10 p10 Pseudomonas spp. 99.2 AJ492829 Pseudomonas trivialis 99.2 AJ492831 Pseudomonas poae 99.2 AJ492829 rAp10 p10 Pseudomonas 98.5 AJ292381 Pseudomonas 98.5 AF100323 brassicacearum thivervalensis rHp10 p10 Burkholderia glathei 97.1 AY605695 Burkholderia sordidicola 95.2 AF512826 tDp11 p11 Pseudomonas spp. 99.4 AJ492829 Pseudomonas trivialis 99.4 AJ492831 Pseudomonas poae AJ492829

tKp11 p11 Bacillus 99.2 AB021195 Bacillus muralis 97.5 AJ628748 Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 psychrosaccharolyticus tMp11 p11 Paenibacillus spp. 94.5 AB073363 Paenibacillus wynnii 94.5 AJ633647 Paenibacillus kobensis 94.5 AB073363 rGp11 p11 Paenibacillus graminis 97.2 AJ223987 Paenibacillus graminis 97.2 AJ223987 tAp12 p12 Pseudomonas tolaasii 99.5 AF320989 Pseudomonas 98.9 AJ537603 proteolytica tJp12 p12 Paenibacillus odorifer 98.8 AJ223990 Paenibacillus odorifer 98.8 AJ223990 tKp12 p12 Burkholderia glathei 99.3 AY605695 Burkholderia 97.4 U9636 phenazinium 97.4 AF12826 Burkholderia sordidicola tMp12 p12 Pseudomonas ﬂuorescens 98.8 AF094730 Pseudomonas 98.7 AJ492827 cannabina 98.7 AJ492828 Pseudomonas congelans 98.7 AF320991 Pseudomonas gingeri rDp12 p12 Janthinobacterium 99.0 Y08845 Janthinobacterium 99.0 Y08845 agaricidamnosum agaricidamnosum tGp14 p14 Burkholderia glathei 98.8 AY154374 Burkholderia 97.4 U9636 phenazinium 97.4 AF12826 Burkholderia sordidicola tJp14 p14 Bacillus 99.7 AB021195 Bacillus muralis 97.5 AJ628748 psychrosaccharolyticus tMp14 p14 Bacillus arvi 99.4 AJ627211 Bacillus arvi. 99.4 AJ627211 tPp14 p14 Paenibacillus polymyxa 96.6 AY359629 Paenibacillus polymyxa 96.3 AJ320493 Paenibacillus taejonensis 96.3 AF391124 tSp14 p14 Rhodococcus opacus 98.3 AB032565 Rhodococcus opacus 98.2 X80631 Nocardia corynebacteroides 98.3 AY167850 rGp14 p14 Pseudomonas ﬂuorescens 99.2 AF094730 Pseudomonas congelans 99.1 AJ492828 rJp14 p14 Rhodococcus 98.7 X80617 Rhodococcus 98.7 X80617 marinonascens marinonascens rMp14 p14 Paenibacillus 93.9 AY598818 Paenibacillus 93.9 AY598818 phyllosphaerae phyllosphaera

EMBL, European Molecular Biology Laboratory. related to the sequences of other genera, such as Paeniba- damnosum, Bacillus arvi and Pseudomonas congelans, re- cillus. Twenty-two different isolates were obtained, distrib- spectively. uted within seven genera. Species of Pseudomonas, Burkholderia, Paenibacillus and Bacillus made up half the Carbon utilization patterns of endobacteria isolates. The most frequently isolated bacteria belonged to Pseudomonas (28%) and Paenibacillus (23%). As well as D-Glucose was utilized by most isolates, but sucrose was only comprising the most common isolates, Pseudomonas utilized by four isolates (Table 4). Whereas consumption of and Paenibacillus spp. were found in all four morpho- fructose was variable among the isolates, the fungal carbo- types. Rahnella and Janthinobacterium spp. were found hydrates D-trehalose and D-mannitol (Koide et al., 2000) only in single morphotypes. Three bacterial isolates, rDp12, were widely used. Additionally, individual isolates showed tMp14 and rGp14, had more than 99% sequence different utilization patterns. For example, isolate tAp10 similarity to the type strains of Janthinobacterium agarici- (most similar to Rahnella aquatilis) degraded all the carbon

Table 4. Carbon source utilization patterns of endobacteria from ectomycorrhiza

Isolate Water D-Mannitol Fructose Sucrose D-Trehalose D-Glucose L-Serine L-Aspartic acid tAp10 (Rahnella aquatilis) À 1111111 tDp10 (Pseudomonas spp.) À 1 b À 1111 rAp10 (Pseudomonas brassicacearum) À 1 b 1 bb11 rIp10 (Burkholderia glathei) À 1 b ÀÀ 1 À 1 rGp11 (Paenibacillus graminis) À 1 b À 1111 tAp12 (Pseudomonas tolaasii) À 11À 1111 tMp12 (Pseudomonas ﬂuorescens) À 11À 11À 1 rDp12 (Janthinobacterium agaricidamnosum) ÀÀ À bb b ÀÀ tJp12 (Paenibacillus odorifer) À b 111 1 ÀÀ

tPp14 (Paenibacillus polymyxa) À bb11 1 1 À Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 tSp14 (Rhodococcus opacus) ÀÀ À À b 1 ÀÀ rJp14 (Rhodococcus marinonascens) À 11ÀÀ 1 ÀÀ rGp14 (Pseudomonas ﬂuorescens) À 1 b ÀÀ 111

Bacterial cell suspensions in saline were inoculated to Biolog GN2 plates and were incubated at 25 1 C for 3–4 days. The closest species match in the European Molecular Biology Laboratory (EMBL) database of each isolate was indicated. B, borderline.

sources examined whereas isolate tSp14 (most similar to 2 min followed by three washes in sterile H2O is a simple and Rhodococcus opacus) only utilized D-glucose and weakly effective sterilization procedure for the removal of surface degraded D-trehalose. Interestingly, the isolates that utilized bacteria from ectomycorrhizal morphotypes p10 (Suillus sucrose also degraded D-trehalose, although the isolates such variegatus), p11 (Russula paludosa), p12 (Suillus flavidus) as tDp10 (Pseudomonas spp.), rGp11 (most similar to Paeni- and p14 (Russula sp.) from Scots pine. This is demonstrated bacillus graminis), tAp12 (most similar to Pseudomonas by the fact that Taq I RFLP profiles of bacteria from non tolaasii) and tMp12 (most similar to Pseudomonas fluores- surface sterilized ectomycorrhizal root tips are markedly cens), which were unable to utilize sucrose, were still able to different from those that were sterilized, suggesting that degrade D-trehalose and D-mannitol. Isolates rIp10 (most endobacterial communities of the ectomycorrhizas studied similar to Burkholderia glathei) and rGp14 (most similar to are different from the bacterial communities associated with Pseudomonas fluorescens) exhibited limited substrate utiliza- the ectomycorrhizal sheath surfaces. Although there are no tion, only utilizing D-mannitol and fructose weakly in addi- other published studies comparing bacterial communities tion to D-glucose. Most isolates utilized one or both of L- from the internal tissue and the surface of ectomycorrhizal serine and L-aspartic acid, but four isolates could not. The root tips, similar results have been reported recently for utilization patterns of these amino acids were also diverse. roots of field-grown canola and wheat (Germida et al., 1998) Isolate rGp14 was observed to consume these amino acids and of potato (Berg et al., 2005). Further changes in RFLP preferentially relative to sugars, whereas tJp12 (most similar profiles of endobacteria in ectomycorrhizas were observed to Paenibacillus odorifer) did not utilize them at all. after continued exposure to H2O2 (i.e. 42 min), probably due to further penetration of the sterilizing reagent into the ectomycorrhizal tissue, resulting in the elimination of more Discussion bacterial species. The observed differences between mor- Surface sterilization using chemical reagents is a common photypes may be explained by differences in their physical approach for the removal of bacteria from the surface of characteristics. For example, Suillus spp. (p10 and p12) form plant tissues (Miche & Balandreau, 2001), including ecto- distinctive tuberculate ectomycorrhizas consisting of a clus- mycorrhizal root tips (Poole et al., 2001). A number of ter of colonized root tips enveloped in a hydrophobic fungal different chemicals have been used previously, including the sheath (Bucking et al., 2002), which may provide more sequential use of sodium hypochlorite and H2O2 (Sturz & resistance to penetration of the sterilizing reagent than Kimpinski, 2004) or ethanol (Coombs & Franco, 2003) for Russula spp. (p11 and p14) ectomycorrhizas, which usually analysis of endophytic bacterial communities associated consist of a single colonized root tip and are more hydro- with roots. H2O2 has also been used for the removal of philic. Another possibility is that the observed variation surface bacteria from ectomycorrhizal root tips and is reflects the relative abundance of different cultivable bacter- commonly used for the isolation of the fungal symbiont ial populations inside ectomycorrhizas. In this case, the into axenic culture (Heinonen-Tanski & Holopanainen, most abundant species may require a longer time period to 1991). Our data demonstrate that immersion of ectomycor- be eliminated when all the isolated bacteria are equally rhizal root tips in 30% H2O2 for a period of longer than susceptible to H2O2.

Although the surface-sterilization procedure was effective rhizal endobacteria, in particular the pseudomonads, are at removing bacteria from the surface of the ectomycor- common rhizosphere (Espinosa-Urgel, 2004) and mycorrhizas included in this study, it is possibile that some rhizosphere (Timonen et al., 1998) bacteria. resistant species remained on the surface of the root tips. Carbon utilization analysis was carried out to characterize For example, Paenibacillus polymyxa is known to produce the ecophysiology of the endobacterial isolates. It was of endospores that are resistant to sodium hypochlorite (Bent particular interest to determine whether the bacteria showed & Chanway, 2002). Although it is possible the isolates preferences for either fungal-derived (D-trehalose) or plant- belonging to Paenibacillus detected in this study might be derived (D-sucrose) sugars, or if they were capable of from residual spores remaining on the ectomycorrhizal utilizing both. Interestingly, although the majority of the surface, this is unlikely given that sterilization with 30% isolates tested could utilize the fungal-derived sugar D-tre-

H2O2 was observed to be more efﬁcient at removing surface halose (with the exception of rIp10, rJp14 and rGp14), only Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 bacteria from ectomycorrhizas than sodium hypochlorite four isolates (tAp10, rAp10, tJp12 and tPp14) were capable

(Table 2). In addition, 5% H2O2 has been reported to kill of using the plant-derived sugar sucrose. This suggests that Bacillus subtilis and Bacillus megaterium spores by attacking the majority of endobacteria isolated in this study have a either proteins (Riesenman & Nicholson, 2000) or DNA preference for fungal-derived sugars as a carbon source. (Cortezzo & Setlow, 2005), respectively. Moreover, Paeniba- Heinonsalo et al. (2001) and Frey et al. (1997) reported that, cillus spp. have previously been identified as being impor- whereas mycorrhizosphere bacterial communities in Scots tant endobacteria of Suillus tomentosus ectomycorrhizas pine and Douglas fir stands were efficient at degrading D- (Paul, 2002) and have also been identified within fungal trehalose, bacteria in the humus and mineral soil could not hyphae of the basidiomycete Laccaria bicolor, with fluores- utilize this carbon source. Based on these observations, it is cence in situ hybridization (Bertaux et al., 2003). tempting to hypothesize that a significant proportion of the A wide range of bacterial genera have been identified as isolated endobacteria from ectomycorrhizas may be more endophytes in various plant tissues (Lodewyckx et al., 2002) closely associated with the fungal partner rather than the and some data suggest that specific associations exist plant in the symbiotic ectomycorrhizal tissue. Clearly, between certain endophytic bacteria and host plant species, further work is required to test this hypothesis. However, including Gluconobacter diazotrophicus with sugar cane and Bertaux et al. (2003) found, using in situ hybridization, that Rhizobium spp. with leguminous plants (Sprent & Sprent, Paenibacillus spp. were localized inside fungal hyphae in 1990; Chanway, 1998). The data presented here demonstrate Laccaria bicolor ectomycorrhizas. In addition, one of the that a diverse assemblage of endobacteria are associated with isolates in this study, rDp12, is closely related to the type four different Scots pine ectomycorrhizal morphotypes and strain of Jathinobacterium agaricidamnosum, which is it appears that some bacteria are associated with more than known to be a soft rot pathogen of Agaricus bisporus one Scots pine ectomycorrhizal species. In fact, 16S rRNA (Lincoln et al., 1999). gene sequence analysis of the endobacteria in this study Elucidating the functional relationships between endo- reveals that approximately 50% of the isolates belong to the bacteria and ectomycorrhizal fungi and/or host plant tissue genera Pseudomonas and Paenibacillus, suggesting that these is a key for understanding the biological significance of the two genera may be widely distributed across different Scots interaction between these organisms. An interesting obser- pine ectomycorrhizas. In accordance with these data, a vation is the detection of bacterial species in Scots pine previous study has also shown that these genera represent ectomycorrhizas that are known to be capable of fixing more than 30% of the bacteria isolated from Cenococcum, atmospheric nitrogen. Isolate rGp11 was found to be related Thelephora, Tomentella, Russulaceae and E-strain ectomy- closely (97.2% 16S rRNA gene sequence similarity) to the corrhizas of subalpine fir (Abies lasiocarpa) (Khetmalas type strain of Paenibacillus graminis, which is known to be et al., 2002). Additionally, some other bacteria detected in extremely efficient at fixing atmospheric nitrogen (Berge this study, such as Rhodococcus spp., have been reported et al., 2002). Previous studies have also reported the from Scots pine Lactarius rufus ectomycorrhizas (Poole presence of nitrogen-fixing Bacillus spp. in tuberculate et al., 2001). These data indicate that these bacteria, which ectomycorrhizas (Li et al., 1992). Additionally, Paul (2002) belong to a wide range of genera, may be able to associate has demonstrated, using field experiments, that Suillus with a broad range of host plant and fungal species. tomentosus tuberculate ectomycorrhizas in young and ma- Although the sampling design employed here does not allow ture stands of Pinus contorta produce significant amounts of any firm conclusions regarding specificities between endo- nitrogenase activity. Further studies targeting the expression bacteria and ectomycorrhizas to be drawn, a lack of specifi- of genes involved in fixation of atmospheric nitrogen (e.g. city has been observed and reported previously (Khetmalas nifH) in ectomycorrhizal root tips may provide a better et al., 2002). This is, perhaps, not surprising given the fact understanding of the potential role that endobacteria play in that the majority of bacterial species detected as ectomycor- forest nitrogen cycles.

Cultivation-based approaches similar to those employed Berg G, Krechel A, Ditz M, Sikora RA, Ulrich A & Hallmann J in this study have been commonly used for the isolation of (2005) Endophytic and ectophytic potato-associated bacterial bacteria from the natural environment, although it is now communities differ in structure and antagonistic function well accepted that only small proportions of the bacterial against plant pathogenic fungi. FEMS Microbiol Ecol 51: species in nature are readily cultivated (Amann et al., 1995). 215–229. This has been demonstrated through the use of cultivation- Berge O, Guinebretiere MH, Achouak W, Normand P & Heulin T independent molecular methods to reveal, for example, (2002) Paenibacillus graminis sp. nov. and Paenibacillus Cytophaga–Flexibacter–Bacteriodes phylogroup bacteria as- odorifer sp. nov., isolated from plant roots, soil and food. Int J sociated with ectomycorrhizal Tuber borchii that are not Syst Evol Microbiol 52: 607–616. detected through cultivation-based approaches (Barbieri Bertaux J, Schmid M, Prevost-Boure NC, Churin JL, Hartmann et al., 2000). Therefore, further insights into the interaction A, Garbaye J & Frey-Klette P (2003) In situ identification of Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 between ectomycorrhizal fungi and endobacteria may be intracellular bacteria related to Paenibacillus spp. in the gained through the use of culture-independent and func- mycelium of the ectomycorrhizal fungus Laccaria bicolor tional gene-based molecular approaches. S238N. Appl Environ Microbiol 69: 4243–4248. Bucking H, Kuhn AJ, Schroder WH & Heyser W (2002) The fungal sheath of ectomycorrhizal pine roots: an apoplastic Acknowledgements barrier for the entry of calcium, magnesium, and potassium into the root cortex. J Exp Bot 53: 1659–1669. We would like to thank Drs F. Moore and D. White, and Ms Chanway CP (1998) Bacterial endophytes: ecological and P. Parkin and Ms C. Reiff for technical assistance and Dr practical implications. SYDOWIA 50: 149–170. C. Campbell for helpful suggestions on the carbon utiliza- Coombs JT & Franco CMM (2003) Isolation and identification of tion assays. Dr D. Genney is acknowledged for providing the actinobacteria from surface-sterilized wheat roots. Appl Suillus flavidus fruit body sequence. This work was finan- Environ Microbiol 69 : 5603–5608. cially supported by the Macaulay Development Trust. The Cortezzo DE & Setlow P (2005) Analysis of factors that influence Macaulay Institute receives funding from the Scottish Ex- the sensitivity of spores of Bacillus subtilis to DNA damaging ecutive Environment and Rural Affairs Department. chemicals. J Appl Microbiol 98: 606–617. Espinosa-Urgel M (2004) Plant-associated Pseudomonas References populations: molecular biology, DNA dynamics, and gene transfer. Plasmid 52: 139–150. Agerer R (1987–1993) Colour Atlas of Ectomycorrhizae. Einhorn- Frey P, Frey-Klett P, Garbaye J, Berge O & Heulin T (1997) Verlag, Gmund.¨ Metabolic and genotypic fingerprinting of fluorescent Agerer R (2001) Exploration types of ectomycorrhizae – a Pseudomonas associated with the Douglas fir-Laccaria bicolor proposal to classify ectomycorrhizal mycelial systems mycorrhizosphere. Appl Environ Microbiol 63: 1852–1860. according to their patterns of differentiation and putative Frey-Klett P, Chavatte M, Clausse ML, Courrier S, Le Roux C, ecological importance. Mycorrhiza 11: 107–114. Raaijmakers J, Martinotti MG, Pierrat JC & Garbaye J (2005) Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic Ectomycorrhizal symbiosis affects functional diversity of identification and in situ detection of individual microbial rhizosphere fluorescent pseudomonads. New Phytol 165: cells without cultivation. Microbiol4 Rev 59: 143–169. 317–328. Barbieri E, Potenza L, Rossi I, Sisti D, Giomaro G, Rossetti S, Garbaye J (1994) Helper bacteria – a new dimension of the Beimfohr C & Stocchi V (2000) Phylogenetic characterization mycorrhizal symbiosis. New Phytol 128: 197–210. and in situ detection of a Cytophaga-Flexibacter-Bacteriodes Gardes M & Bruns TD (1993) ITS primers with enhanced phylogroup bacterium in Tuberborchii Vittad, ectomycorrhizal specificity for basidiomycetes – application to the mycelium. Appl Environ Microbiol 66: 5035–5042. identification of mycorrhizae and rusts. Mol Ecol 2: 113–118. Barka EA, Gognies S, Nowak J, Audran JC & Belarbi A (2002) Gazzanelli G, Malatesta M, Pianetti A, Baffone W, Stocchi V & Inhibitory effect of endophyte bacteria on Botrytis cinerea and its influence to promote the grapevine growth. Biol Cont 24: Citterio B (1999) Bacteria associated to fruit bodies of the 135–142. ecto-mycorrhizal fungus Tuber borchii Vittad. SYMBIOSIS 26: Bent E & Chanway CP (1998) The growth-promoting effects of a 211–222. bacterial endophyte on lodgepole pine are partially inhibited Germida JJ, Siciliano SD, de Freitas JR & Seib AM (1998) by the presence of other rhizobacteria. Can J Microbiol 44: Diversity of root-associated bacteria associated with held- 980–988. grown canola (Brassica napus L.) and wheat (Triticum Bent E & Chanway CP (2002) Potential for misidentification of a aertiyum L.). FEMS Microbiol Ecol 26: 43–50. spore-forming Paenibacillus polymyxa isolate as an endophyte Griffiths RI, Whiteley AS, O’Donnell AG & Bailey MJ (2000) by using culture-based methods. Appl Environ Microbiol 68: Rapid method for coextraction of DNA and RNA from natural 4650–4652. environments for analysis of ribosomal DNA- and rRNA-

based microbial community composition. Appl Environ Paul LR (2002) Nitrogen fixation associated with tuberculate Microbiol 66: 5488–5491. ectomycorrhiza on lodgepole pine (Pinus contorta). PhD Guasp C, Moore ERB, Lalucat J & Bennasar A (2000) Utility of thesis. The University of British Columbia, Vancouver, internally transcribed 16S-23S rDNA spacer regions for the Canada. definition of Pseudomonas stutzeri genomovars and other Pearson WR & Lipman DJ (1988) Improved tools for Pseudomonas species. Int J Syst Evol Microbiol 50: 1629–1639. biological sequence comparison. Proc Natl Acad Sci USA 85: Hauben L, Vauterin L, Swings J & Moore ERB (1997) 2444–2448. Comparison of 16S ribosomal DNA sequences of all Pirttila AM, Laukkanen H, Pospiech H, Myllyla R & Hohtola A Xanthomonas species. Int J Sys Bacteriol 47: 328–335. (2000) Detection of intracellular bacteria in the buds of Scotch Heinonen-Tanski H & Holopanainen T (1991) Maintenance of fine (Pinus sylvestris L.) by in situ hybridization. Appl Environ ectomycorrhizal fungi. Techniques for Mycorrhizal Research Microbiol 66: 3073–3077.

(Norries JR, Read DJ & Varma AK, eds), pp. 413–422. Poole EJ, Bending GD, Whipps JM & Read DJ (2001) Bacteria Downloaded from https://academic.oup.com/femsec/article/56/1/34/563439 by guest on 24 September 2021 Academic Press, London. associated with Pinus sylvestris–Lactarius rufus Heinonsalo J, Jorgensen KS & Sen R (2001) Microcosm-based ectomycorrhizas and their effects on mycorrhiza formation in analyses of Scots pine seedling growth, ectomycorrhizal fungal vitro. New Phytol 151: 743–751. community structure and bacterial carbon utilization profiles Read JD & Perez-Moreno J (2003) Mycorrhizas and nutrient in boreal forest humus and underlying illuvial mineral cycling in ecosystems – a journey towards relevance. New horizons. FEMS Microbiol Ecol 36: 73–84. Phytol 157: 475–492. Johansson JF, Paul LR & Finlay RD (2004) Microbial interactions Riesenman PJ & Nicholson WL (2000) Role of the spore coat in the mycorrhizosphere and their significance for sustainable layers in Bacillus subtilis spore resistance to hydrogen peroxide, agriculture. FEMS Microbiol Ecol 48: 1–13. artificial UV-C, UV-B, and solar UV radiation. Appl Environ Khetmalas MB, Egger KN, Massicotte HB, Tackaberry LE & Microbiol 66: 620–626. Clapperton MJ (2002) Bacterial diversity associated with Shishido M, Breuil C & Chanway CP (1999) Endophytic subalpine fir (Abies lasiocarpa) ectomycorrhizae following colonization of spruce by plant growth-promoting wildfire and salvage-logging in central British Columbia. Can J rhizobacteria. FEMS Microbiol Ecol 29: 191–196. Microbiol 48: 611–625. Smith SE & Read DJ (1997) Mycorrhizal Symbiosis. Academic Koide RT, Shumway DL & Stevens CM (2000) Soluble Press, London. carbohydrates of red pine (Pinus resinosa) mycorrhizas and Sprent JI & Sprent P (1990) Nitorgen Fixing Organisms. Chapman mycorrhizal fungi. Mycol Res 104: 834–840. & Hall, London. Kulikova T, Aldebert P, Althorpe N, et al. 2004 The EMBL Sturz AV & Kimpinski J (2004) Endoroot bacteria derived from Nucleotide sequence database. Nucleic Acids Res 32: D27–D30. marigolds (Tagetes spp.) can decrease soil population densities Li CY, Massicotte HB & Moore LV (1992) Nitrogen fixing Bacillus of root-lesion nematodes in the potato root zone. Plant and sp. associated with Douglas-fir tuberculate ectomycorrhizae. Soil 262: 241–249. Plant and Soil 140: 35–40. Timonen S, Jorgensen KS, Haahtela K & Sen R (1998) Bacterial Lincoln SP, Fermor TR & Tindall BJ (1999) Janthinobacterium community structure at defined locations of Pinus sylvestris agaricidamnosum sp. nov., a soft rot pathogen of Agaricus Suillus bovinus and Pinus sylvestris Paxillus involutus bisporus. Int J Sys Bacteriol 49: 1577–1589. mycorrhizospheres in dry pine forest humus and nursery peat. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Can J Microbiol 44: 499–513. Mezgeay M & van der Lelie D (2002) Endophytic bacteria and Varese GC, Portinaro S, Trotta A, Scannerini S, LuppiMosca AM their potential applications. Crit Rev Plant Sci 21: 583–606. & Martinotti MG (1996) Bacteria associated with Suillus Miche L & Balandreau J (2001) Effects of rice seed surface grevillei sporocarps and ectomycorrhizae and their effects sterilization with hypochlorite on inoculated Burkholderia on in vitro growth of the mycobiont. SYMBIOSIS 21: vietnamiensis. Appl Environ Microbiol 67: 3046–3052. 129–147.