Pergamon

Soil Biol. Biochem. Vol. 26, No. 5, pp. 547-552, 1994 Copyright 0 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 003%0717/94 $7.00 + 0.00

DIVERSITY OF FRANKIA NODULE ENDOPHYTES OF THE ACTINORHIZAL SHRUB CEANOTHUS AS ASSESSED

BY RFLP PATTERNS FROM SINGLE NODULE LOBES

DWIGHT D. BAKER’* and BETH C. MULLIN’

‘Panlabs Incorporated, 11804 North Creek Parkway South, Bothell, WA 98011-8805, U.S.A. and *University of Tennessee, Department of Botany, Center for Legume Research, Knoxville,

TN 37996-1100, U.S.A.

(Accepted 2 October 1993)

Summary-Root nodules of the actinorhizal shrub Ceanothus were collected from seven sites from its native range. DNA was extracted from individual nodule lobes using a cetyl trimethyl ammonium bromide (CTAB) extraction procedure. Three DNA probes were used in combination with two restriction endonucleases to evaluate the extent of restriction fragment length polymorphism (RFLP) diversity within and among populations of Ceanothw endophytes. We observed some diversity using a nif DH gene probe; however there was no correlation of RFLP pattern and geographic site. Using two random Frankia probes, we observed more diversity among the Ceanothus endophytes than with the nifprobe. Differences in RFLP patterns were observed among plants at a single geographic site and between geographical sites. The results demonstrated that considerable diversity exists among Frankia strains symbiotic with Ceanothus, as has been shown for pure-cultured Frankia strains isolated from other actinorhizal genera. We have also demonstrated the usefulness of this method for the study of Frankia ecology in planta.

INTRODUCTION

The symbiotic nitrogen-fixing actinomycetes of the genus Frankia have been problematic to study be- cause they are difficult to isolate and slow-growing in pure culture (Baker, 1989; Baker and Mullin, 1992). Because infective Frankia strains in pure culture exist for only less than half of the known actinorhizal plants, an understanding of the diversity which exists among Frankia strains is severely limited. Consider- able genetic diversity exists among pure-cultured Frankia strains based on their host specificity (sensu Baker, 1987) groups 1 (Simonet et al., 1989; Faure- Raynaud et al., 1990) and 3 (Jamann et al., 1992). To date, little diversity has been found among pure-cul- tured Frankia strains of host specificity group 2 (Nazaret et al., 1989; Maggia et al., 1990, 1992).

The diversity observed among pure-cultured Frankia strains may not accurately reflect diversity of Frankia populations in soil or even of endophyte populations in actinorhizal root studies. Therefore, we studied Frankia endophytes directly from field- collected root nodules to assess the extent of diversity as measured by molecular DNA methods. We chose to study endophytes of the actinorhizal host Cean- othus because no infective pure-cultured strains have been identified for this species and therefore diversity has not been assessed by studies of pure-cultured strains.

*Author for correspondence.

Ceanothus is an exclusively American genus of the family Rhamnaceae with ca 55 spp distributed south-north from Guatemala to southern Canada and west-east bounded by the Pacific and the At- lantic Oceans. C. americanus, the subject of our study is a low actinorhizal shrub found predominantly in the north-east quadrant of the range of the genus, Tennessee being well within this range.

MATERIALS AND METHODS

Nodule collections

Root nodules of Ceanothus americanus were col- lected at seven locations in Eastern Tennessee ident- ified in Table 1. Nodules were placed on ice immediately after excavation and were transferred to - 20°C upon arrival back at the laboratory. Nodules were stored at - 20°C for several weeks before DNA extraction.

DNA isolation

Total DNA was isolated from root nodules using a procedure modified from Rogers et al. (1989). Nodule pieces were washed in distilled water prior to DNA isolation to remove soil particles. The amount of nodule tissue used for isolation ranged from 3 to 25 mg. The nodule pieces were frozen in liquid N, and powdered by crushing in a micro-centrifuge tube with a wooden applicator stick. Then 0.5 ml of 2% cetyl trimethyl ammonium bromide (CT’AB) extraction buffer (2% CTAB; Tris-HCl 100 mM; EDTA 20 mhr;

547

548 DWIGHTD.BAKER and BETHC. MULLIN

NaCl 1.4 M; pH 8.0) was added to the centrifuge tube and this was kept at 65°C for 1 h. Following this protein was extracted using an equal volume of chlorofo~:isoamyl alcohol (24: 1). Then 0.1 volume of 10% CTAB extraction buffer (10% CTAB; NaCl 0.7 M) was added to the aqueous phase and additional protein was extracted using chloroform : isoamyl al- cohol until no protein was observed at the phase interface (usually a single repetition was sufficient). DNA was precipitated from the final aqueous phase by the addition of 2 vol of 100% cold ethanol, storage at -20°C for more than 2 h, and centrifugation at 11,OOOg. The precipitated DNA was resuspended in TE buffer (Tris-HCI 10 mM; EDTA 1 mM; pH 8.0). A final DNA precipitation was made by adding suffi- cient 5 M NaCl to make a final concentration of 0.1 M, and 2 vol of 100% cold ethanol. The DNA pellet was resuspended in 50 1.11 of TE buffer and stored at - 20°C until use. Approximately 25-30 nodules were used from each site for this study. DNA isolated from Frankiu strain AvcIl (strain DDB 01020110) by the CTAB procedure was used as a reference DNA on each gel.

RFLP analysis

Approximately 100 ng of total nodule DNA or 200 ng of AvcIl DNA (see above) was digested with the restriction endonucleases, Sal1 or BamHI (Gibco BRL), according to the instructions provided by the manufacturer. Digested DNAs were separated on 0.8% agarose gels and blotted to nylon membranes (Hybond N, Amersham). Blots were hybridized with each of three probes. The first probe was a gel purified 3.2 kb HindIII-EcoRI fragment (pAS1) from pSA30 (Cannon eb aZ., 1979) which contains nifDH and portions of nifK from ~l~~s~ei~~. The second and third probes were randomly-selected pieces of DNA from a Frankia cosmid clone library of strain FaCl (Ligon and Nakas, 1987). Probe No. 2 was a gel purified 1.7 kb BamHI fragment isolated from cos- mid clone 10E and probe number 3 was a gel purified 2kb BamHI fragment from cosmid clone 3C. Probes were labeled using a random primers kit (Gibco BRL) and 32P-labeled deoxy-cytidine-S-triphosphate (dCTP). Hybridizations were performed at 65°C ac- cording to procedures provided by Amersham except that prehybridization solutions contained 10 ItIM EDTA and prehybridization was carried out for at least 16 h followed by a 24 h hybridization. Hy- bridized membranes were washed once for 15 min in

Table 1. SourCe locations of Ceanorhus root nodules

Site number Location Latitude Longitude

1 Tailassee, site 1 33-33 83% 2 T&wee, site 2 3.5-33 83-58 3 Knoxville, Sharp’s

Ridge 36-00 8374 4 Walland 35”44’ 83”49’ 5 Clinton 3579 83 ‘58’ 6 Cumberland Plateau 35-56 84”55 7 Crossvilla 36”oo 85’02

2 x SSC, once for 30 min in 2 x SSC + 0.1% sodium dodecyl sulfate (SDS), and 3 times for 30 min each in 1 x SSC (150 mM NaCl, 15 mM Na citrate, pH 7.0) + 0.1% SDS. After exposure to X-ray film at -70°C in the presence of two intensifying screens, blots were stripped by boiling in 0.1% SDS before successive hybridizations.

RESULTS

The CTAB extraction procedure we used yields cu 50 ng of total DNA mg-’ nodule tissue. Of this ca 25 ng is Frankiu DNA (Mullin et al., 1983). CTAB-extracted host plant genomic DNA from nod- ules is not susceptible to restriction digestion and upon electrophoresis migrates to a position identical to total undigested DNA. CTAB-extracted Frunk~a DNA from nodules is susceptible to digestion by several restriction enzymes, yielding clean RFLP patterns following hybridization. In earlier unpub- lished trials we had found using pure-culture Frankia strains and nodules formed from these strains that the restriction fragment patterns of Frankiu DNA ex- tracted from nodules are identical to patterns gener- ated from the respective pure cultured strains. The presence of host plant DNA does not interfere with the proper migration of Frankia restriction fragments nor do any of the probes used hybridize to host plant DNA under the hyb~dization conditions used in our study.

The nodules of C. americanus collected in the Southern Appalachians contained endophytes ex- hibiting diverse RFLP patterns. Figure 1 shows an example of the diversity in RFLP patterns observed using the nif DH probe in combination with the restriction endonucleases Sal1 or BamHI. In general AvcIl reference DNA exhibited a stronger hybridiz- ation signal than the Ceanothus nodule DNA when both were hybridized to the nif DH probe. Diversity in patterns was observed between sites, but this was not consistent by site. In addition to the diversity observed among Ce~othu~ endophytes in this study, the patterns we observed were different from those for Alnus endophytes (Baker and Mullin, unpub- lished results).

The diversity in RFLP patterns was similar when a random Frankia probe was used in combination with either SalI- or BamHI-digested DNAs. This second probe, like pAS1, generally gave a stronger hybridization signal with the AvcIl reference DNA than with Ceanothus endophyte DNA. Using this probe it was possible to observe differences in RFLP patterns between plants at a single site (Fig. 2).

A greater amount of diversity in RFLP patterns was observed with the third probe tested, also a random Frankia probe {Fig. 3). Unlike the other two probes tested, this probe gave a stronger hybridiz- ation signal with Ceanothus endophyte DNA than with the AvcIl reference DNA. In all of the figures, it can be observed that there was diversity in RFLP

Fig.

1.

Cea

noth

us

nodu

le

DN

As

dige

sted

w

ith

Sal1

(a

) or

B

amH

I (b

) an

d hy

brid

ized

w

ith

pAS1

(n

if D

H).

Lan

es

1-7,

D

NA

s fr

om

plan

ts

at

site

s l-

7,

resp

ectiv

ely.

L

ane

8: D

NA

of

pu

re-c

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red

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in

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As

in

the

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s on

th

e tw

o ge

ls

are

not

from

th

e sa

me

indi

vidu

al

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ts.

Fig.

2.

Ceu

noth

us n

odul

e D

NA

s di

gest

ed

with

Sal

1 an

d hy

brid

ized

w

ith p

robe

2

(a r

ando

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Frun

kia

prob

e).

Lan

es

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D

NA

s fr

om p

lant

s at

site

s l-

7,

resp

ectiv

ely.

L

ane

8: D

NA

of

pur

e-cu

lture

d F

run

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str

ain

Avc

Il.

DN

As

in t

he l

anes

on

the

two

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(a)

and

(b)

are

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fr

om

the

sam

e in

divi

dual

pl

ants

, th

us

one

can

obse

rve

diff

eren

ces

in R

FLP

patte

rns

at a

sin

gle

site

(L

anes

3

and

4).

Fig.

3.

Cea

noth

us

nodu

le

DN

As

dige

sted

w

ith

Sal1

(a

) or

B

amH

I (b

) an

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w

ith

prob

e 3

(a s

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ndom

Fr

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. L

anes

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As

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tes

l-7,

re

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Lan

e 8:

DN

A

of

pure

-cul

ture

d F

run

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st

rain

A

vcIl

. D

NA

s in

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e la

nes

on

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ar

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and

diff

er

only

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the

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clea

se

used

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N

ote

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the

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can

be

seen

to

be

un

ique

.

DWIGHTD.BAKER and BETHC.MULLIN 5.52

patterns among plants at a single site as well as diversity between sites.

DISCUSSION

Ceanothus americanus is an actinorhizal shrub found growing in isolated patches at the edge of woodlands and along road cuts throughout Eastern Tennessee as well as similar locations within the Appalachian Mountain region. This region, which has a complex geological history, is characterized by acidic, well-drained, highly leached and rather infer- tile soils of sandstone, shale and limestone origin. The Ceanothus populations identified for study, with the exception of the Cumberland and Crossville popu- lations, were found on the sides of very steep slopes amidst herbaceous roadside weeds. Most of the plants sampled appeared to be quite old with well developed crowns and extensive root systems. The success of this species in these environments is likely to be related to its nitrogen-fixing symbiotic associ- ation with Frankia.

Little is known about the frankiae which inhabit Ceanothus nodules, in part because no pure-cultured infective Frankia strains currently exist for Cean- othus. Ours was the first attempt to assess the diver- sity of endophytes within the Ceanothus nodules and the first report of a rapid sensitive method for genomic RFLP analysis of frankiae in planta.

Our study has shown that diversity among endo- phytes in Ceanothus populations can be observed between geographically-distinct populations as well as among plants at a single geographical location. Similar diversity has been demonstrated for other Frankia populations based on a variety of techniques, including RFLP analysis of cultured Frankia strains (Benson and Hanna, 1983; Jamann et al., 1992; Maggia et al., 1992). Comparison of RFLP patterns between the Ceanothus nodule endophytes and pure- cultured strains indicates that the patterns seen in Ceanothus nodules are distinct from those seen in isolates from other actinorhizal host plants. How- ever, because our study was limited to a very small portion of the native range of Ceanothus it is probable that additional RFLP patterns will *be found in endophytes surveyed from a larger area.

We have demonstrated that the CTAB extraction procedure is a rapid efficient method for the extrac- tion of endophyte DNA from actinorhizal nodule lobes in a form that allows it to be used for RFLP analysis. This technique provides the most sensitive method to date for the detection of genomic Frankia RFLP patterns and is likely to be useful for a broad range of diversity and competition studies.

Acknowlednements-Funding for this project was provided in part by-a grant from the Lexemual and Esther Hesler Visitine Professor Endowment Fund to DDB. We thank Aphak&n Nittayajam for assistance with laboratory pro- cedures and Jim Minesky and Steven Kohls for assistance with the field collections.

REFERENCES

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Baker D. D. (1989) Methods for the isolation, culture and characterization of the Frankiaceae, soil actinomycetes and symbionts of actinorhizal plants. In Zsolution of Biotechnological Organisms from Nature (D. Labeda, Ed.), pp. 213-236. McGraw Hill, New York.

Baker D. D. and Mullin B. C. (1992) Actinorhizal sym- bioses. In Bio~ogjcu~ Nitrogen Fixation (G. Stacey, R. H. Burris and H. J. Evans, Eds), pp. 259-292. Chapman and Hall, New York.

Benson D. R. and Hanna D. (1983) Frankiu diversity in an alder stand as estimated by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis of whole-cell pro- teins. Canadian Journal of Botany 61. 29192923.

Cannon F. C., Riedel G. E. and~Ausube1 F. M. (1979) ~erlapping sequences of Klebs~e~~a pneumoniae nifDNA cloned and characterised. Molecular and General Genetics 174, 59-66.

Faure-Raynaud M., Bonnefoy Poirier M. A. and Moiroud A. (1990) Diversitv of Frankia strains isolated from . , actinorhizae of a single Atnus rubra cultivated in nursery. Symbiosis 8, 147.-160.

Jamann S., Femandez M. P. and Moiroud A. (1992) Genetic diversity of Ela~gnac~e-inf~tive Frankia strains isolated from various soils. Acta Oecologia 13, 3955405.

Ligon J. M. and Nakas J. P. (1987) Isolation and character- ization of Frunkia sp. strain FaCl genes involved in nitrogen fixation. Applied and Environmental Micro- biology 53, 2310-2327.

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Maggia L., Prin Y., Picard B. and Goullet P. (1990) Esterase patterns of 60 isolates of Frankia from Casuarina equiseti- foha grown in Senegambia. In Advances in Casuarina Besearch and Utilization (M. H. El-Lakany, J., W. Tum- bull and J,. L. Brewbaker, Eds), pp. 141-148. Desert Development Center, American University, Cairo.

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Simonet P., Thi Le N., Moiroud A. and Bardin R. (1989) Diversity of Fran&a strains isolated from a single alder stand. Plant and Soil 118, 13-22.