APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1996, p. 3026–3029 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 62, No. 8
PCR-Restriction Fragment Length Polymorphism Identification and Host Range of Single-Spore Isolates of the Flexible Frankia sp. Strain UFI 132715† ERICA LUMINI
AND
MARCO BOSCO*
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Applicata, Universita ` degli Studi di Firenze, 50144 Florence, Italy Received 11 October 1995/Accepted 28 May 1996
Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch’s postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes. (6) demonstrated the absence of Alnus-frankiae in four Alnusinfective Elaeagnus-Frankia cultures, but they could not rule out the coexistence of more than one Elaeagnus-Frankia strain in the same culture, engendering the need to obtain Frankia clonal cultures. For filamentous organisms like Frankia species, purification can only be accomplished by isolating single-spore-derived colonies (27), as protoplasts may be subject to fusion before development into true colonies (24). Knowledge about Frankia sporulation (8) is poor, despite its being quite important for performing culture purification and obtaining truly clonal cultures. This is probably because of the slow growth and nutritional fastidiousness of Frankia isolates and the low germination percentage of Frankia spores, which makes isolation of any individual spore very time-consuming. To our knowledge, the only single-spore-derived Frankia isolates available in laboratories throughout the world are those obtained by Prin et al. (27) from Frankia strain CH (ORS 140102), and those reported by Beyazova and Lechevalier (5) as being derived from Frankia strains CeI5 (UFG 02060605) and CcI3 (HFP 020203). Once a Frankia isolate has been purified, and the requirements inherent in the third and the fourth of Koch’s postulates are experimentally met, its identity should be determined. Frankia phenotypic identification is best accomplished by multiple host infectivity tests (6), while Frankia genotype can be rapidly fingerprinted by PCR-restriction fragment length polymorphism (RFLP) (13, 17), even within host plant tissues. The aim of the present work was twofold: first, to define and set up a simple method to purify Elaeagnus-Frankia isolates by single-spore colony isolation; second, to validate Koch’s postulates and to identify single-spore Frankia strain UFI 132715 clones by both phenotypic (host infectivity) and genotypic (PCR-RFLP) means. Wild-type Frankia sp. strain UFI 132715 (E15), which was isolated in our laboratory in 1982 (19), was subcultured in 250-ml flasks containing modified BAP medium without nitrogen (33) at 288C in the dark. Seeds of Alnus glutinosa, Elaeagnus angustifolia, and Hippophae¨ rhamnoides which had been surface disinfected with hydrogen peroxide were germinated on water agar in petri dishes. Axenic plant incubation, inoculation tests, and strain reisolation and designation were performed according to Bosco et al. (6). Frankia strain E15 was
Members of the soil bacterium genus Frankia (Actinomycetales) can induce N2-fixing symbiotic nodules on several nonleguminous shrubs and forest trees. Frankia isolates have routinely been obtained from nodules, and host-specific responses have been used to group them into three major host infectivity groups (HSGs) (2, 12). The Alnus group includes Frankia isolates infective on Alnus, Comptonia, and Myrica spp.; the Casuarina group includes isolates infective on Allocasuarina and Casuarina spp.; and the Elaeagnus group includes isolates infective on Colletia, Elaeagnus, Hippophae¨, and Shepherdia spp. However, host specificity of most of the isolates is still unknown. Although Benson and Hanna (3) showed that individual nodule lobes may host more than one Frankia strain, and although culture purity and identity are essential prerequisites to be ensured when characterizing bacteria or plant-bacteria interactions, it is worth noting that Frankia isolates tested in the past for infectivity were seldom purified by bacteriological methods. Only recently, Torrey (32) and Benson and Silvester (4) suggested that once a Frankia strain has been isolated, it should be purified and identified to be sure that a coculture of different isolates has not been obtained. Frankia isolates may also be divided into three groups according to their adaptation to host-plant specific responses to infection (9). One group is adapted for a root hair infection process (Alnus and Casuarina HSGs), the second group is adapted for a direct intercellular penetration process (Elaeagnus HSG), but some frankiae are capable of employing both root hair infection and intercellular penetration colonization mechanisms. The latter were defined as “flexible” by Miller and Baker (20). Few authors have reported flexible or broadhost-range frankiae. Actually, only Bosco et al. (6), Dobritsa et al. (10), Lumini et al. (18), and Margheri et al. (19) have provided evidence for a broad-host-range phenotype in Elaeagnus-compatible Frankia isolates. In particular, Bosco et al.
* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Applicata, Universita` degli Studi di Firenze, Piazzale delle Cascine 27, 50144 Florence, Italy. Phone: 39 55 3288305. Fax: 39 55 330431. Electronic mail address:
[email protected]. † This work is dedicated to the memory of Angiola B. Bersano. 3026
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found to effectively nodulate 100% of E. angustifolia and H. rhamnoides plantlets and to nodulate A. glutinosa at a rate between 20 and 60% of inoculated plants. No nodulation was observed on uninoculated controls. Widespread curling of root hairs and penetration of Frankia hyphae into root hairs were observed on inoculated Alnus species (not shown). Reisolates were obtained from H. rhamnoides and A. glutinosa inoculated with Frankia strain E15 and were named E15H and E15A, respectively. They showed in vitro morphology and physiology similar to that of the parent strain E15, as well as a flexible host range. Both Frankia strains E15 and E15A were used for single-spore isolation. We noticed that gently stirring 4-week-old cultures with a magnetic bar for 10 min at 100 rpm allowed the release of mature spores from sporangia, without fragmentating the mycelium. The suspensions were then aseptically filtered through sterile Whatman no. 1 filter paper and examined microscopically to check for the absence of hyphal fragments. Spore suspensions were diluted with sterile distilled water to approximately 2 3 103 spores ml21 after evaluating spore concentration by direct count with a hemocytometer. Spore germination assays were as described by Prin et al. (27). One hundred microliters of the above spore suspensions were spread onto the surface of a thin layer of Qmod medium (14) by using sterile glass beads. An overlay of the same medium was then added to cover the spores, and plates were incubated in the dark at 288C. Germination of spores was microscopically checked every 12 h by a 203 Long Distance Working objective (Zeiss GmbH, Jena, Germany). The percentage of germinated spores was calculated after 72 h and again after 2 weeks. Spores with germ tubes exceeding half of their length were rated as germinated. For each plate, germinated and nongerminated spores within 300 microscopic fields were counted and recorded. Each value was based on mean values from three plates. Spore germination rates for Frankia strains E15 and E15A were approximately 20% on Qmod medium, confirming the results obtained by Prin et al. (27) for Frankia strain CH and contrasting those reported by Tzean and Torrey (33) for Casuarina isolates (70 to 80% germination). It is worth mentioning that both Frankia strains CH and E15 were isolated from host plants belonging to the family Elaeagnaceae. The different spore germination rates shown by Frankia isolates belonging to the Casuarina or Elaeagnus HSGs could be explained by the phylogenetic distance between Casuarinafrankiae and Elaeagnus-frankiae (9, 22) and by the overall physiological diversity of isolates belonging to these two HSGs. At 72 h, germ tubes were evident (Fig. 1A). After 2 weeks, the germination rate did not change, and microcolonies had developed (Fig. 1). Microcolonies derived from germ tubes of individual spores were axenically picked off the solid medium and deposited in sterile test tubes containing L2 medium (0.35% agar) (15). About 4 weeks later, each colony was homogenized in a sterile Potter-Elvehjem homogenizer, transferred in 5 ml of liquid medium, and incubated at 288C. Twelve of several single-spore colonies isolated from Frankia strains E15 and E15A were subcultured, and respectively named E15a, E15b, E15c, E15d, E15e, E15f, E15Aa, E15Ab, E15Ac, E15Ad, E15Ae, and E15Af. They showed no differences in growth performance, except for macroscopic variations in colony morphology and pigmentation. Colonies appeared either very compact and well delimited, or more or less diffuse. Colony pigmentation either ranged from red to yellow or was white, as also noted by Prin et al. (27) for 22 Frankia strain CH single-spore derivatives. It is important to note that the above phenotypic characteristics are not sensitive enough for Frankia strain identification. So, the host range of single-spore cultures was checked as above (6). Single-spore isolates of Frankia
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FIG. 1. Phase-contrast micrograph of the development of Frankia E15 single-spore colonies on thin-layer agar medium. (A) Germ tube from a single spore at day 3. (B) Single germinated spore at day 5. (C) Branching hyphae from germinated spore at day 7. (D) Hyphal colony and vesicles (v) formed on a short lateral stalk at day 14. Bar, 10 mm.
strains E15 and E15A fulfilled the four Koch’s postulates by infecting both Alnus spp. and Elaeagnus spp., clearly demonstrating the flexible phenotype of the mother strain. A light microscopic examination of monoxenic nodules showed that all E15-derived Frankia strains differentiate diazo-vesicles inside the nodules of both Alnus spp. and Elaeagnus spp. However, in our experiments, nodulated Alnus plantlets often remained small and showed very low levels of acetylene reduction activity. No sporangia were detected inside nodules induced on Alnus spp. or Elaeagnus spp. DNA extraction and purification from Frankia cultures were performed according to Simonet et al. (30). Total nucleic acids were also extracted from some of the nodules obtained during plant infectivity tests. In order to prevent the inhibition of the PCR by oxidized nodule-born polyphenolic compounds, each single nodule lobe was peeled under a layer of sterile TENP (50 mM Tris, 20 mM EDTA disodium salt [pH 8.0], 100 mM NaCl, 1% [wt/vol] polyvinylpolypyrrolidone [PVPP]) buffer (6) by using a stereomicroscope. Nodule cortical tissues were ground in 500 ml of TEN-CPP (100 mM Tris, 20 mM EDTA disodium salt [pH 9.5], 1.4 M NaCl, 2% [wt/vol] cetyltrimethyl ammonium bromide, 0.5% [wt/vol] polyvinyl pyrrolidone, 0.5% [wt/vol] PVPP) buffer by using Eppendorf minigrinders and were then incubated for 60 min at 658C in the same buffer. Debris were sedimented (10 min, 7000 3 g), and the supernatant was chloroform extracted twice, DNA precipitated with one volume of chilled propanol in the presence of 0.3 M NH4 acetate, and resuspended in 10 ml of pure water. PCR (21) DNA amplifications were carried out in a final
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FIG. 2. Position of primers and diagram of nifD-nifK seminested PCR protocol. Abbreviations: 685, primer FGPD685-85; 700, primer FGPK7009-92; 597, primer FGPK5979-146; IGS, nifD-nifK intergenic spacer.
volume of 25 ml by the Perkin-Elmer GeneAmp PCR System 9600 (The Perkin-Elmer Co.) with GeneAmp PCR core reagents (The Perkin-Elmer Co.) at the following optimized conditions: reaction buffer II (50 mM KCl, 10 mM Tris-HCl [pH 8.3]), 1.5 mM MgCl2, 0.65 M glycerol, 20 mM (each) deoxynucleoside triphosphate, 0.1 mM (each) primer, and 1 U of AmpliTaq. Templates consisted of 25 ng of purified DNA from Frankia cultures or 1/10 volume of single-lobe-extracted total nucleic acids. Thermal profile was as described by Jamann et al. (13). Two different regions of the Frankia genome were analyzed. In the first experiment the V7 hypervariable region of the 16S ribosomal genes was analyzed by using primer FGPS989ac-79 (59 GGGGTCCGTAAGGGTC 39) or FGPS 989e-80 (59 GGGGTCCTTAGGGGCT 39) (6) in association with reverse primer FGPL19739-73 (59 ATCGGCTCGAGGT GCCAAGGGATCCA 39), described by Rouvier et al. (28) and now renamed FGPL509-73 (23), in order to check whether the mother strain E15 and its derivatives do harbor the Elaeagnus rrs signature (6, 22). We demonstrated that single-spore strains, as well as wild-type strain E15, nodule microsymbionts, and plant reisolates E15A and E15H were typical Elaeagnus group Frankia strains, harboring the Elaeagnus rrs signature. In fact, they were positively recognized by the primer pair FGPS989e-80–FGPL509-73 but not by the pair FGPS989ac79–FGPL509-73. In contrast, Frankia alni AcI4 (UFI 010104) yielded an amplification band (985 bp) only when the primer pair FGPS989ac-79–FGPL509-73 was used (not shown). In a separate experiment, seminested amplified nif DNA restriction analysis (SANDRA) was performed. In the first step, primers FGPD685-85 (59 CACTGCTACCGGTCGAT GAA 39) and FGPK7009-92 (59 CGAGGTAGGTCTCGAAA CCGG 39) (13) were used to amplify the Frankia nifD-nifK intergenic region. In the second step, 1 ml of a 1023 dilution of the above reaction products was reamplified by using FGPK5979-146 (59 GTGCGAGCCCACGAAGCTCGGNGT GTG 39) (23) as a reverse primer, instead of FGPK7009-92, in order to confirm the amplification of the correct target sequence. The relative position of these primers is shown in Fig. 2. To analyze the seminested nifD-nifK intergenic region PCR products, 20 ml samples from independent reactions were separately cleaved (twice) with RsaI and HaeIII restriction endonucleases according to the producer’s directions (Boehringer GmbH, Mannheim, Germany). PCR products and restriction patterns were revealed by horizontal gel electrophoresis as previously described (6). The nifD-nifK intergenic regions of the 12 single-spore strains, the wild-type strain E15, its plant reisolates, and some nodule microsymbionts were successfully amplified, yielding expected fragments of about 1,430 bp with
FIG. 3. (A) Agarose gel electrophoresis of seminested nifD-nifK PCR products obtained with primers FGPD685-85 and FGPK5979-146. Abbreviations: Dfs, DNA free sample (negative control); Nod E15e, DNA extracted from a lobe of a nodule induced on A. glutinosa by single-spore strain E15e; M, DNA Molecular Weight Marker VI (Boehringer GmbH). (B) Molecular identity of wild-type Frankia strain E15 and eight of its derivatives, as shown by SANDRA with RsaI endonuclease.
the primer pair FGPD685-85–FGPK7009-92 and of 1,330 bp with the seminested pair FGPD685-85–FGPK5979-146 (Fig. 3A). Restriction patterns obtained by SANDRA with RsaI are shown in Fig. 3B. The faint high-molecular-weight band showed by some of the lanes was due to occasionally incomplete DNA restriction, as the sum of restriction fragment sizes was larger than the size of unrestricted PCR product, and does not change the overall conclusion of the work. The 12 clonal Frankia cultures obtained from Frankia strains E15 and E15A were identified by SANDRA, the improved PCR-RFLP protocol. On the one hand, all 12 single-spore strains, as well as endophytic E15e, could be clearly assigned to one single PCRRFLP group, i.e., the same group as the mother strain E15 and its derivatives E15A and E15H (Fig. 3). On the other hand, their PCR-RFLP pattern was different from the pattern obtained from each of the eight Frankia reference strains (Fig. 4). These results are consistent with the results obtained by Lumini et al. (17), who compared PCR-RFLPs and DNA-DNA hybridizations against the same reference strains used here. Previous comparison of Frankia wild-type strains with respective reisolates from plants or cultures showed that Frankia strains can be somewhat mutable (5, 7, 16, 25, 29, 31) or quite stable (5, 6, 27). However, a few authors dealt with experimentally achieved “monofrankial” cultures. Actually, it may be possible for a slowly growing nodule isolate to be masked by a fast-growing contaminant one. Physiologic and genotypic characters may be attributed to such a fast-growing subculturedominant coisolate, while nodulation may actually be caused by the slowly growing less obvious one, which is then reisolated. In our opinion, unless clonal purity has been achieved by bacteriological methods, no evidence is obtainable for the mu-
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NOTES
6.
7. 8. 9.
10. 11.
12. 13.
FIG. 4. RsaI-SANDRA patterns of the single-spore Frankia strain E15e and eight Frankia reference strains. Abbreviations: HrI1, UFI 140101; 2.1.7, UGL 140101; SCN10a, ULQ 190201001; Ea112, ULF 130100112; HRX401a, ULF 140104001; EUN1f, ULQ 132500106; HRN18a, ULF 140101801; L, 10-bp DNA ladder (Gibco-BRL); M, DNA Molecular Weight Marker VI (Boehringer GmbH).
14.
15. 16.
tability of Frankia strains, as the liquid Frankia isolation procedures (14) do not exclude the possibility of obtaining a coculture of different isolates (11). Thus, we believe that only single-spore-derived isolates should be considered as pure cultures of Frankia spp. We have characterized and monitored 12 Frankia single-spore isolates over several years by means of both genotypic (PCR-RFLP) and phenotypic (host range) methods while observing neither phenotypic nor genotypic variability. In the light of our experience, single-spore Frankia purification methods are sufficiently easy to work with to be applied to other Frankia reference strains, including those of the Alnus group (1). Single-spore purification should be considered a fundamental prerequisite for the molecular characterization of Frankia spp. In particular, clonal Frankia cultures are essential for the development of Frankia mutagenesis and transformation systems. The availability of clonal flexible Frankia cultures should make molecular interactions between Frankia and actinorhizal plants open to direct and safe experimentation. In fact, the selecting role played by Frankia nod genes toward host plants of different genera may be more easily understood if purity and the identity of cultures are certain. We thank M.C. Margheri for Frankia strain UFI 132715 and for helpful discussions, H.G. Diem, A. Moiroud, and C.T. Wheeler for reference Frankia strains, and L. Calamo Porcino for nodule digging. We also thank R. Materassi and F. Favilli for critical review of the manuscript and Susan Hitchin for proofreading. This work was supported by grant 95.03224.CT06 from the Italian National Research Council, Rome.
17. 18.
19.
20.
21. 22.
23. 24. 25.
26.
27.
28.
29.
30.
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