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 ectomy- corrhizas. 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