FEMS Microbiology Letters 58 (1989) 287-292 287 Published by Elsevier

FEM 03518

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Svet lana V. Dob r i t s a and O.S. S tupar

Institute of Biochemistry and Physiology of Microorganisms, USSR A cztdemy of Sciences, Pushchino, U.S.S.R.

Received 30 November 1988 Accepted 1 December 1988

Key words: Frankia; Actinorhiza; DNA restriction banding pattern; Plasmid profile; Elaeagnus angustifolia; Shepherdia argentea; HippophaO" rharnnoides

1. SUMMARY

Genetic variations among selected Frankia iso- lates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant (Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indis- tinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hip- popha~ rhamnoides L., did not modify the struct- ural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some

Correspondence to: S.V. Dobritsa, Institute of Biochemistry and Physiology of Microorganisms, U.S.S.R. Academy of Sciences, Pushchino, Moscow region, 142292, U.S.S.R.

aspects of variability in natural populations of Frankia are discussed.

2. I N T R O D U C T I O N

Actinomycetes of the genus Frankia are unique in their ability to produce nitrogen-fixing root nodules (actinorhizae) on non-leguminous plants. The economic and ecological value of actinorhizal plants for stabilization and nitrogen fertilization of soils, for land reclamation and reforestation as well as for biomass production is now well recog- nized. To develop optimum symbiotic associa- tions, selection of the most infective, effective and competitive Frankia strains is needed, as well as an understanding of the ecology and dynamics of soil endophyte populations. Furthermore, because of increasing numbers of Frankia isolations from diverse habitats and host plants, the uniqueness of each isolate must be confirmed. Thus, necessity for reliable Frankia strain identification is obvi- ous. However, few phenotypic criteria are availa- ble upon which it can be based_

In a previous paper [1], we described a study of Frankia isolates from various host plants of differ- ent geographical origins and showed that restric-

0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

288

tion enzyme analysis of total genomic DNA can be useful for estimating genetic diversity among Frankia spp., for identifying strains and for dif- ferentiating between phenotypically indistinguish- able isolates. In the present work, we used the same approach to compare Frankia isolates and reisolates from particular habitats. As a result, multiple infection of individual plants with differ- ent Frankia strains and the presence of more than one Frankia strain in a nodule were shown.

3. MATERIALS AND METHODS

3.1. Strains The strains of Frankia used in this study are

listed in Table 1. All of the strains were isolated from actinorhizal root nodules by the method of Lalonde et al. [2]. Those prefixed by E were iso- lated from root nodules harvested from the same 3-year-old Elaeagnus angustifolia plant which has been grown in a greenhouse in a local unsterilized soil and received no specific treatments, which

might be considered to be representative of the natural environment. In order to isolate the endo- phytes, single nodules not producing clusters were taken. In the isolate designations, the first numeral denotes a nodule number, thus isolates E23, E25 and E28, for example, were obtained from differ- ent fragments of the same nodule numbered 2.

Strains S13, S14 and S15 were isolated from root nodules collected from a single Shepherdia argentea plant grown in Kuibyshev Botanical Garden. A number of reisolates designated H14 followed by a reisolate number were obtained from 3-month-old root nodules induced by inoc- ulation of sterile 1-month-old Hippopha~" rham- noides seedlings with strain $14.

Escherichia coli V517 was provided by F.L. Macrina, Medical College of Virginia, Richmond, U.S.A.

3.2. Growth conditions All Frankia strains were grown in OS-1 medium

consisting of the following (per liter of distilled water): K2HPO4, 0.15 g; NaHzPO 4. 2H20, 0.13

T a b l e 1

C u l t u r a l a n d m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f t h e Frankia i s o l a t e s s t u d i e d

I s o l a t e M e d i u m O S - 1 M e d i u m O S - 2

G r o w t h a V e s i c l e s S p o - S o l u b l e P i g m e n t e d G r o w t h a

r a n g i a p i g m e n t c o l o n i e s

P r e c i p i - V e s i c - S p o r a n - S o l u b l e P i g m e n t e d

t a r e b les g i a p i g m e n t c o l o n i e s

E 2 3 + + ( D ) - - + + + + + + ( C ) B - +

E 2 5 + ( C ) - + - + + ( C ) C + +

E 2 8 + + ( C ) - + - + + + + ( D ) A - +

E 4 3 + ( D ) - - - + + + ( D ) B - +

E 4 5 + ( D ) - + + + + + + ( D ) B - +

E 4 8 + ( D ) - - + + + + + + ( D ) B - +

E71 + ( D ) - + - - + + + + B - +

E 7 3 + ( D ) - + + + + + + + B - +

E 7 4 + + + + + + - + + + + C - +

S13

( A N P 1 9 0 1 0 5 ) + + + + + + + - N D C

$ 1 4

( A N P 1 9 0 1 0 6 ) + + + + + + + - + + + + B + +

H 1 4 - 7 + + + + + + + - + + + + B + +

H 1 4 - 1 3 + + + + - - + - + + + + B + +

S15

( A N P 1 9 0 1 0 7 ) + + + + + + + - N D

- +

- +

+ +

- +

- +

+

4- + +

a D , d i f f u s e c o l o n i e s ( r a r e l y b r a n c h e d h y p h a e ) ; C , c o m p a c t c o l o n i e s ( h i g h l y b r a n c h e d h y p h a e ) ; w h e n n o t i n d i c a t e d , t h e c h a r a c t e r is

n o t e x p r e s s e d .

b A , h e a v y p r e c i p i t a t e ; B, p a r t i a l l y f e r m e n t e d p r e c i p i t a t e , w i t h c r y s t a l s o v e r g r o w n w i t h h y p h a e ; C , s l i g h t o r n o p r e c i p i t a t e .

c N D , n o t d e t e r m i n e d .

g; MgSO 4, 0.05 g; KC1, 0.1 g; yeast extract (Difco), 0.25 g; Bacto- t ryptone (Difco), 2.5 g; sodium acetate, 0.5 g; ethylene diamine tetraacetic acid N a . Fe(III)-salt , 0.01 g; Tween 80 (Serva), 1 ml. In OS-2 medium, Tween 80 was replaced by Tween 60. The cultures were incubated under non-shak- ing condit ions at 2 8 ° C for 3 to 4 weeks. E. coli V517 was grown with shaking in LB bro th [3] at 37 ° C overnight.

3.3. DNA preparation, digestion and separation of fragments

Total D N A was isolated f rom Frankia cells, cleaved with restriction endonucleases and ana- lyzed by agarose gel electrophoresis as described previously [1], The restriction endonucleases EcoRI, BgllI and SalI were produced in this Institute, SmaI was purchased f rom Boehringer Mannheim, F.R.G. Plasmids f rom E. coli V517 were isolated by the alkaline lysis procedure as described by Maniatis et al. [3].

4. R E S U L T S

4.1. Characterization of the Frankia isolates Strains S13, S14 and S15 isolated f rom root

nodules of the same S. argentea plant have been

1 2 3 4 5

5 4 , 2

C P

7.3

3.9

2.1

Fig. 1. Restriction banding patterns on 0.85% agarose gels of BgllI, (A) and Sinai-digested, (B) genomic DNA from Frankia isolates E23 (lane 2), E25 (lane 3), E28 (lane 4), E43 (lane 5), E45 (lane 6), E48 (lane 7), E71 (lane 8), E73 (lane 9), and E74 (lane 10). Lane 1 contains HindlII-digested lambda DNA as an M r marker (fragment sizes in kb are indicated on the left).

1 2 3 4 5 6

289

2 3 . 1 - -

0 . 4 - -

6 . 6 - -

4 . 4 - -

2 . 3 - -

2 . 0 - -

Fig. 2. Restriction banding patterns on an 0.85% agarose gel of BgllI-digested genomic DNA from Frankia strains S13 (lane 2), S14 (lane 3), and S15 (lane 6) and reisolates H14-7 (lane 4), and H14-13 (lane 5). Lane 1 contains HindllI-digested lambda DNA as an M~ marker (fragment sizes in kb are indicated on the left).

subcultured in our labora tory for more than 3 years. These strains effectively nodula te H. rhamnoides and strain S14 was used to inoculate 50 sterile H. rhamnoides seedlings. All of the seedlings were found to be nodulated, however, reisolates were obta ined only f rom 15 seedlings. All reisolates proved to be similar in their cultural and morphological properties, therefore two reiso- lates were chosen for D N A restriction analysis. A m o n g them, H14-13 differed f rom the original strain used as inoculum by the inability to form sporangia and vesicles in the medium OS-1, while H14-7 was indistinguishable f rom strain S14 (Ta- ble 1).

In another experiment, Frankia isolates were obtained f rom separate f ragments of root nodules formed on a single E. angustifolia plant, namely f rom three nodules which were numbered 2, 4 and 7. Out of 17 phenotypical ly similar isolates, 9 were selected to analyze their D N A . Those differed f rom each other in certain cultural and morpho-

290

2 3 . 1 - -

1 2 3 4 5 6 7 8 9 l O 1 2 3 4 5 6 7 8 9 1 0

c) . 4 - -

6 . 6 - -

4 . 4 - -

2 ~ 3 - -

2 . 0 - -

A B Fig. 3.0.7% agarose gel electrophoresis of intact preparations of total cellular DNA from Frankia isolates $14 (lane 2), HI4-7 (lane 3), H14-13 (lane 4), and S15 (lane 5). Lane 1 contains plasmids from E. coli V517 as M r markers (sizes in kb are indicated on the left). C, chromosomal DNA; P, plasmid DNA.

logical characteristics Table l) to a maximum degree.

4.2 DNA restriction banding patterns of the isolates from E. angustifolia

In the previous paper [1], we showed that restriction endonucleases recognizing AT-rich hexanucleotide sequences (for example, BglIl and EcoRI) cleaved total D N A from Frankia spp. into rather large fragments which are mainly between 10 and 30 kb. In contrast, upon digestion of Frankia D N A with restriction enzymes recogniz- ing GC-rich hexanucleotide sites (for example, SalI and SmaI), smaller fragments were pro- duced, which are mainly less than 7 kb. In this study, all 9 isolates from E. angusttlfolia were indistinguishable by the DNA restriction banding patterns produced by cleavage with BglII (Fig. 1A) or EcoRI (data not shown). However, when digested with Sinai (Fig. 1B) or SalI (data not presented), the Same DNAs gave banding patterns which were identical only for the 6 isolates from nodules 2 and 4 (respectively, E23, E25, and E28 and E43, E45, and E48). The patterns for isolates

E71, E73, and E74 from nodule 7 were dis- tinguishable both from each other and from the common patterns of the other 6 isolates.

4.3. DNA restriction banding patterns and plasmid profiles of the strains from S. argentea and the reisolates from H. rhamnoides

No differences were detected between the ge- nomic DNAs from strains S14 and SI5 with all of the 4 restriction enzymes tested, irrespective of their recognition sites. The results of the DNA cleavage with BgllI are shown in Fig. 2. In ad- dition, an 18.6 kb plasmid found in both strains (Fig. 3) showed homologous restriction enzyme patterns (data not presented), which further con- firmed genetic relatedness between these strains. Some of the BglII-fragments derived from the plasmid D N A are visible as more intense bands over the chromosomal background in the electro- phoregram showing fragmentation patterns of the total DNA (Fig. 2).

Restriction analysis of total genomic D N A re- vealed no visible differences between the reisolates from the tt. rhamnoides root nodules induced by

strain S14 and the original strain (Fig. 2). Plasmid profiles of the reisolates were indistinguishable from that of the initial strain as well (Fig. 3).

By contrast, strain S13 could be distinguished from strains $14 and S15 by both restriction band- ing patterns of the total DNA, obtained with all of the restriction enzymes used (Fig. 2 shows the data for Bg/lI), and by the lack of any detectable plasmid.

5. DISCUSSION

By comparing DNA restriction patterns of the Frankia co-isolates from root nodules formed on individual actinorhizal plants we confirmed the previous conclusions [1,4] that this approach can be useful for reliable Frankia strain identification. One- and two-dimensional polyacr3'lamide gel electrophoresis of soluble cellular proteins, which have been used for the same purpose [5 8], seem to be more suitable for grouping Frankia strains but may have insufficient resolution to dis- criminate between closely related strains or variants. In contrast to protein profiles, DNA restriction banding patterns do not depend on the growth conditions, culture age or morphogenetic state. Moreover, analysis of total proteins allows only a limited part of the genome to be evaluated, while structural differences in regulatory and pro- tein non-coding sequences are not considered. We believe that for identifying Frankia strains or discriminating between closely related strains, re- striction endonucleases cutting D N A mainly into large fragments (such as BglII in this study) pro- vide resolution which is comparable with that of protein patterns. At the same time, as shown for the co-isolates from E. angustifolia, more resolu- tion can be achieved by a special choice of restric- tion endonucleases which would yield, for a given group of microorganisms, comparatively small D N A fragments, however, still producing distinct bands upon electrophoresis. For Frankia spp., Sinai and Sall are examples of such enzymes.

Our data support the view [1] that restriction analysis of total genomic D N A can be a basis to observe structural changes occurring in Frankia genomes. The identical restriction patterns ob- tained with BgllI indicate that the 9 co-isolates

291

from E. angustifolia may have had a common origin. However, the differences in the patterns of SmaI-fragments may serve as a measure of vari- ation within the given closed population (or in the natural population from which it derived), show- ing at least 4 different types of divergence from an original genome sequence. The first type is rep- resented by the 6 isolates from nodules 2 and 4 and the 3 others each by a single isolate from nodule 7. Evidently, the differences are not due to single base pair substitutions which would affect single recognition sites for restriction endo- nucleases. Although unlikely, the possibility is not excluded that DNA methylation may be involved, since analogous differences between the DNAs were revealed by their digestion with Sinai and SaII.which recognize distinct sites ( C C ~ G G G and GTCGAC, respectively [9]) but both do not cleave D N A when cytosine residues are methylated in the internal CG-dinucleotides (asterisked). Most likely, however, the changes in D N A restriction banding patterns are due to more extensive re- arrangements, a nature of which remains to be elucidated.

In previous studies, certain Frankia co-isolates from separate root nodules on the same plant were shown to have identical polypeptide profiles [5,6,8] or D N A restriction banding patterns [1]. In this work, similar results were obtained with the 6 isolates from E. angustifolia nodules 2 and 4 as well as with strains S14 and S15 from S. argentea root nodules. At this level of resolution, such indistinguishable cultures, evidently, should be re- garded as clones or reisolates of the same strain, which is supported by the stability of DNA re- striction banding patterns observed after an endo- phytic passage. However, one cannot exclude that more precise analysis, for example, DNA sequenc- ing, may reveal some differences between such co-isolates. This is all the more likely since those isolates from E. angustifolia that are indis- tinguishable by DNA restriction analysis ex- hibited some, though not very stable and repro- ducible, differences in their cultural and morpho- logical properties (see Table 1). Clear differences in pigmentation, acetylene reduction, etc. were also observed between strains S14 and $15 (un- published data).

292

A m o n g the co- isolates d i s t inguishable by D N A res t r ic t ion pa t t e rn analysis and thus referred to as dis t inct strains, those f rom the E. angustifolia roo t nodules are closely re la ted, while S14 and S15 on the one h a n d and S13 on the o ther h a n d are ra the r d i s tan t ly related. These results, together wi th d a t a r epor ted by others [5,10], ind ica te tha t mul t ip le infect ion with d i f ferent Frankia s t ra ins on a single host root sys tem is widespread in na tu ra l environ- ments. F o r this reason, results of f ield in t roduc- t ion of highly effective pai rs of Frankia s t ra ins and ac t inorhiza l p lants , selected in a greenhouse, m a y be, to a great extent, de t e rmined b y the res ident Frankia popu l a t i on a l r eady presen t in the soil. Of the s trains examined in this s tudy, those isola ted f rom S. argentea showed a growth- s t imula t ing effect u p o n H. rhamnoides in bo th l abo r a to ry and field tests [11]. However , ne i ther their compet i t iveness as c o m p a r e d with indige- nous Frankia strains nor their fate in the ecosys- tem have been s tudied yet. Our results d e m o n - s t ra te the usefulness of res t r ic t ion b a n d i n g pa t - terns of to ta l D N A as highly specific Frankia strain markers in such ecological studies. Ad- d i t iona l i n fo rma t ion can be ob t a ined f rom plas- mid profi les, which is in agreement wi th previous

d a t a [10]. There have been specula t ions [12-14] that indi-

v idual root nodules of ac t inorhiza l p lan t s m a y con ta in more than one Frankia strain, ba sed on var iab le hos t ranges of isolates f rom the same nodule . However , only Benson and H a n n a [5] ev idenced dual occupancy of one Alnus incana spp. rugosa nodule by two Frankia s t ra ins be long- ing to dis t inct p ro te in gel -e lec t rophores is groups. Our da ta showed that the E. angustifolia nodule n u m b e r e d 7 was inhab i t ed by at least 3 di f ferent Frankia strains. I t is unknown whether such mixed cul tures arise in p l an t a f rom a single ini t ia l s t ra in as a result of some muta t iona l events or resul t f rom coinfect ion with d i f ferent strains. The la t te r seems to be less p r o b a b l e because all of the 9 isolates f rom E. angustifolia, inc luded here, fai led to nodu la t e their host p lant , whereas some of the remain ing 8 co- isolates did infect Elaeagnus species (unpub l i shed data). A t any rate, a prac t ica l consequence is that the avai lable Frankia i so la t ion p rocedures do not exclude the poss ib i l i ty of ob-

ta in ing an assoc ia t ion of several strains. In par - t icular , the he te rogene i ty among di f ferent clones of the same s t ra in [15] and the emergence of spon taneous var ian ts [4,16-18], which have been a t t r i bu ted to a high level of genet ic ins tabi l i ty in cul tures of Frankia spp., may have resul ted f rom select ion of pre-ex is t ing m i n o r popu la t ions in mixed cultures.

A C K N O W L E D G E M E N T

We would l ike to thank S.N. N o v i k for assis- tance with growing plants .

R E F E R E N C E S

[1] Dobritsa, S.V. (1985) FEMS Microbiol. Lett. 29, 123-128. [2] Lalonde, M., Calvert, H.E. and Pine, S. (1981) in Current

Perspectives in Nitrogen Fixation (Gibson, A.H. and Newton, W.E., eds.), pp. 296-299, Austrahan Academy of Science, Canberra.

[3] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[4] An, C.S., Riggsby, W.S. and Mullin, B.C. (1985) Plant Soil 87, 43-48.

[5] Benson, D.R. and Hanna, D. (1983) Can. J. Bot. 61, 2919-2923.

[6] Gardes, M. and Lalonde, M. (1987) Physiol. Plant. 70, 237-244.

[7] Simon,L, Bousquet, J., Gardes, M., St-Laurent, L. and Lalonde, M. (1988) FEMS Microbiol. Lett. 51, 13-18.

[8] Benson, D.R., Buchholz, S.E. and Hanna, D.G. (1984) Appl. Env. Microbiol. 47, 489-494.

[9] Roberts, R.J. (1983) Nucl. Acids Res. 11, 135-167. [10] Simonet, P., Normand, P., Moiroud, A. and Lalonde, M.

(1985) Plant Soil 87, 49-60. [11] Stupar, O.S., Novik, S.N., Aseyev, A.N., Tomashevskii,

A.Yu., Dobritsa, S.V. and Kalakoutskii, L.V. lzvestiya AN SSSR, ser. biol., in press (in Russian).

[12] Zhang, Z., Lopez, M.F. and Torrey, J.G. (1984) Plant Soil 78, 79-90.

[13] Reddell, P. and Bowen, G.D. (1985) Plant Soil 88, 275-279. [14] Baker, D.D. (1987) Physiol. Plant. 70, 245-248. [15] Burggraaf, A.J.P. and Valstar, J. (1984) Plant Soil 78,

29-43. [16] Tisa, L., McBride, M. and Ensign, J.C. (1983) Can. J. Bot.

61, 2768-2773. [17] Parson, W.L., Robertson, L.R. and Carpenter, C.V. (1985)

Plant Soil 87, 31-42. [18] Lechevalier, M.P., Labeda, D.P. and Ruan, J.-S. (1987)

Physiol. Plant. 70, 249-254.