natural diversity of nodular microsymbionts of myrica rubra

Plant and Soil 262: 229–239, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Natural diversity of nodular microsymbionts of Myrica rubra

X.H. He1,2,3, L.G. Chen1, X.Q. Hu1 & S. Asghar1

1Department of Horticulture, Zhejiang University, Hangzhou, Zhejiang 310029, P.R. China. 2Department ofHorticulture, Guangxi University, Nanning, Guangxi 530004, P.R. China. 3Corresponding author∗

Received 19 March 2003. Accepted in revised form 4 November 2003

Key words: actinorhizal, diversity, Frankia, Myrica rubra, nifD-nifK IGS

Abstract

Myrica is often considered a promiscuous actinorhizal genus. However, there are large differences in diversityamong Myrica spp., and M. gale does not exhibit such promiscuity in its natural environment. In order to understandthe diversity of nodular microsymbionts of M. rubra in natural environments and whether or not the M. rubra is a‘promiscuous’ host, we studied the natural diversity of nodular microsymbionts of different cultivars of M. rubra.15 nodules from nine horticultural cultivars of M. rubra were collected in 7 sites of eastern, southeastern, centraland northern part of Zhejiang province, China. Unisolated strains were compared by sequence analyses of theirnifD-nifK intergenic spacers and PCR amplification protocol on nodules. Phylogenetic relationships among nodularFrankia strains were analyzed by comparing sequences of their nifD-nifK intergenic spacers and reference strains.There is a high degree of diversity among nodular Frankia symbionts of M. rubra. Frankia strains from cluster I andcluster III were found in nodules from many different cultivars of M. rubra. Furthermore, there were sometimes twostrains which belong to different infective clusters of Frankia in the same nodule, and Frankia strains of cluster Iwere often dominant strains when there were two strains. M. rubra can thus be considered to be promiscuousin nature. Identical sequences in nodules from different plants at widely separated sites were commonly found,indicating that some strains are cosmopolitan. Geographic separation, host selectivity for Frankia symbionts andsoil environment may account for the diversity of Frankia strains and differences in Frankia populations found inM. rubra nodules. Several very closely related local Frankia populations in M. rubra nodules could be distinguishedfrom one another by our approach.

Introduction

Slowly growing actinomycetes of genus Frankia canestablish a nitrogen-fixing symbiosis with a widerange of woody dicotyledonous plants. This nitrogen-fixing symbiosis is known to occur in more than200 species of plants belonging to 24 genera andeight families that are called actinorhizal plants (Ben-son and Silvester, 1993). One family, the Myricaceaein the subclass Hamamelidae, is considered the firstactinorhizal family to have emerged during the lateCretaceous (Maggia and Bousquet, 1994). Myrica,subsectioned to the Myricaceae family, is divided into35 species, with 28 reported to be actinorhizal (Bond,1983).

∗FAX No: +86-571-86971390. E-mail: [email protected]

The strains that infect Myrica spp. often groupedwith Alnus-infective strains (cluster I). However, someresults showed strains isolated from or identifiedin Myrica spp. (M. pensylvanica and M. cerifera)nodules had close relationships with Elaeagnaceae-infective strains (cluster III) (Clawson et al., 1999;Zimpfer et al., 1999), so Myrica spp. are often con-sidered to be ‘promiscuous’ host (Baker, 1987; Torrey,1990; Clawson et al., 1999), while M. gale has lowdiversity of strains and does not exhibit promiscuity inits natural environment (Huguet et al., 2001).

Six species of Myrica has been reported in Chinawhich include M. esculenta Buch-Ham., M. edeno-phora Hance, M. nana Cheval, M. rubra Bieb. etZucc, M. integrifolia Roxb and M. arboresceus S.R.Li and X.L. Hu. Sp.nor (Zhang et al., 2001). Among

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them, M. esculenta Buch-Ham., M. nana Cheval &M. rubra Bieb. et Zucc. can fix nitrogen in symbi-osis with actinomycete Frankia, but the relationshipof Frankia strains from the three species has not beenstudied (Ding et al., 1999). Up to date, only Myricarubra has been artificially cultivated on large scale.M. rubra (Chinese tree berry, red bayberry) which isan important native fruit-producing endemic speciesof China and is seldom planted in other countries, isa tropical and subtropical fruit and has habituated tothe warm and humid environments. It is mainly dis-tributed in the south of the Yangtze River includingTaiwan and Hainan, with a similar extent of citrusfruit, loquat, tea and bamboo. Provinces of Guang-dong, Fujian and Jiangsu also produce an appreciableamount of red bayberry, but the majority is heav-ily concentrated in Zhejiang Province, where the redbayberry is the second large fruit crop, next to thecitrus (Li et al., 1992). According to statistics (2001),the total acreage of this fruit in Zhejiang Province is46667 ha. which is 21.88% of the total area of chinaunder this fruit (total 213,333 ha.). There are 305 cul-tivars and 120 clones of M. rubra in China and mostof them originate from and were selected in Zhejiang(Liang, 2002). He et al. (2002) found genetic diversityin Frankia isolates from M. rubra by PCR amplifiedfrom 16S rDNA and RFLP, but the diversity of nodularmicrosymbionts of M. rubra in natural environments isunknown.

Molecular characterization tools based on nif se-quences have been used for Frankia detection in rootnodules or in soil (Jamann et al., 1993; Nalin et al.,1995, 1997, 1999; Lumini et al., 1996). The nifgenes, which code for the nitrogenase responsiblefor nitrogen fixation, are a well-characterized regionin the Frankia genome. The nif region is a poten-tially good genomic marker to detect nonculturedmembers of Frankia provided they have nif genesof course and can be used to approximate the taxo-nomic position of strains and confirm their placementin the genus Frankia. In the nif region, the nifD-nifKIGS seems more appropriate to discriminate amongFrankia strains than the nifH-nifD IGS because it isa long and more variable fragment (Normand et al.,1988; Navarro et al., 1997; Lumini et al., 1999; Nalinet al., 1999). Rouvier et al. (1996) examined the IGSregions of the 16S-23S rDNA and nifD-nifK to assessthe diversity of Casuarina and Allocasuarina usingPCR-RFLP analysis and found host specificity andhigh levels of diversity, and the groupings based onthe nif and rrn DNA regions were composed of the

same strains while Lumini et al. (1999) compared 67Frankia strains of the three host specificity groups byPCR-RFLP analysis of both the 16S-23S rDNA IGSand nifD-nifK IGS and the results indicated that ingeneral the nifD-nifK IGS proved to be more poly-morphic than rrn target for both Alnus and Elaeagnus.Therefore the nifD-nifK IGS region was frequentlyused to characterize Frankia strains by PCR-RFLP.

Zhejiang province is the largest producing areaof M. rubra in China and the world. Zhejiang ownsthe rich germplasm of M. rubra, among which twopromising commercial cultivars i.e biji and dongkuiwere selected in the Zhejiang province. In order tounderstand the diversity of nodular microsymbionts ofM. rubra in the natural environment and whether ornot the M. rubra is a ‘promiscuous’ host, we studiedthe natural diversity of nodular microsymbionts of dif-ferent cultivars of M. rubra from different regions ofZhejiang province, China, and related the strain’s sym-biosis with M. rubra to other Frankia strains by usingsequences of the nifD-nifK IGS as well as specificamplification.

Materials and methods

Nodules and Frankia strains

Nodules from M. rubra plants were harvested fromdifferent orchards where other actinorhizal specieswere not found in the main producing areas of Zheji-ang Province (Table 1 and Figure 1). Zhoushan city,Xiangshan county and Cixi town are located in theeastern part of Zhejiang. The three regions are morethan 100 km apart, with Zhoushan city being situatedon an island in the Eastern Sea. Linhai Town and Li-andu District are in the southeastern and southwesternpart of Zhejiang respectively and they are more than200 km apart. Lanxi Town and Anji County are in thecentral and northern part of Zhejiang respectively andthey are more than 200 km apart. Their climate andsoil environment are different. The reference strainsused are described in Table 1.

DNA extraction from nodules

DNA from root nodules was directly extracted ac-cording to Huguet et al. (2001). After washing thenodules with water, a single nodule was selected,and the outer layers were removed. Each nodulewas crushed in 300 µL of TCP extraction buffer(100 mM Tris-HCl [pH 7], 0.5 M NaCl, 50 mM EDTA

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Table 1. Origin of nodules and isolated Frankia strains in this study

Strain or nodule GenBank Host plant Geographical origin source or

accession No. and soil type reference

M. rubra nodules/strains

MBJ1 AY312244 M. rubra var. biji Anji County/laterite This study

MBJ2 AY312245 M. rubra var. biji Cixi Town/loess This study

MBJ3 AY312246 M. rubra var. biji Linhai Town/loess-lik loam This study

MBJ4-L� AY312247 M. rubra var. biji Lanxi Town/loess-like loam This study

MBJ4-S� AY312248 M. rubra var. biji Lanxi Town/loess-like loam This study

MBM AY312249 M. rubra var. baimei Cixi Town/loess This study

MDK1 AY312250 M. rubra var. dongkui Xiangshan County/loess This study

MDK2-L AY312251 M. rubra var. dongkui Linhai Town/loess-like a loam This study

MDK2-S AY312252 M. rubra var. dongkui Linhai Town/loess-like a loam This study

MDK3-L AY312253 M. rubra var. dongkui Lanxi Town/loess-like a loam This study

MDK3-S AY312254 M. rubra var. dongkui Lanxi Town/loess-like a loam This study

MMY1 AY312255 M. rubra var. muye Lanxi Town/loess-like loam This study

MMY2 AY312256 M. rubra var. muye Lanxi Town/loess-like loam This study

MSM-L AY312257 M. rubra var. shuimei Liandu District/loess This study

MSM-S AY312258 M. rubra var. shuimei Liandu District/loess This study

MWD AY312259 M. rubra var. wandao Zhoushan Town/sandy loam This study

MWY-L AY312260 M. rubra var. wumei Xiangshan County/loess This study

MWY-S AY312261 M. rubra var. wumei Xiangshan County/loess This study

MYL-L AY312262 M. rubra var. yangliu Lanxi Town/loess-like loam This study

MYL-S AY312263 M. rubra var. yangliu Lanxi Town/loess-like loam This study

MDZ-L AY312264 M. rubra var. zaoda Linhai Town/loess-lik loam This study

MDZ-S AY312265 M. rubra var. zaoda Linhai Town/loess-lik loam This study

Mrp182 AY115490 M. rubra var. biji Cixi Town/loess This study

Gymnostoma nodules

RPL161(U) U63334 G. leucodon Riviere des Pirogues (New Caledonia) Nalin et al(1999)

TC24 (U) U63692 G. chamaecyparis Tontouta (New Caledonia Nalin et al(1999)

CN61(U) U63693 G. nodiflorum Canala (New Caledonia Nalin et al(1999)

MG59(U) U63691 G. glaucescens Me Aiu (New Caledonia Nalin et al(1999)

KP54 (U) U63694 G. poissonianum Kouaoua (New Caledonia) Nalin et al(1999)

Elaeagnaceae strains�EUN1f (6) L37664 Elaeaguns umbellata Illinois, US Nalin et al(1999)

HRN18a(7) U63696 Hippophae rhamnoides Alps (France) Nalin et al(1999)

EaN1-pec(5) U63698 E. angustifolia Ohio, US Nalin et al(1999)

EaI-12 (4) U63697 E. angustifolia Ecully (France) Nalin et al(1999)

SCN10a(U) U63695 Shepherdia canadensis Quebec (Canada) Nalin et al(1999)

EaI1 AJ251388 E. angustifolia France Nalin et al(1999)







Casuarina strains

CcI3 (9) U63699 C. cunninghamiana Florida Nalin et al(1999)

D11(atypical)(U) U63700 C. equisetifolia Dakar (Senegal) Nalin et al(1999)

Alnus strains

ArI (1) L35557 A. rubra Oregon Nalin et al(1999)

FaC1(U) U53363 A. viridis USA Nalin et al(1999)

nodule-L(S) �, ‘L’ and ‘S’ stand for clone of long fragment and short fragment in eletrophoresis pattern, respectively�Genomic species described by Fernandez et al(1989) are in parentheses. U, undetermined species.

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Figure 1. Map of M. rubra distribution in China comprising collection of sites of root nodules of M. rubra. Left: Map of P.R.China; Arabicnumbers from 1 to 15 represent Yunnan, Sichuan, Chongqing, Guizhou, Guangxi, Guangdong, Hunan, Jiangxi, Fujian, Zhejiang, Hubei, Anhui,Jiangsu, Hainan and Taiwan provinces respectively where M. rubra is distributed. Right: Sites map of collection of root nodules of M. rubra.in Zhejiang province, Hangzhou is the capital of Zhejiang Province.

[pH 8], 2% [wt/vol] cethyltrimethylammonium brom-ide [Amresco], 1% [wt/vol] polyvinylpolypyrrolidone[Sigma]). The homogenate was incubated at 65 ◦C for1 h and centrifuged twice at 6, 000 × g for 5 min. Thesupernatant was extracted with an equal volume ofchloroform-isoamyl alcohol (24:1, vol/vol) and cent-rifuged at 13, 000 × g for 20 min. DNA from theaqueous phase was precipitated in ethanol for at least2 h at −20 ◦C. The sample was then centrifuged at13, 000 × g for 30 min, and the resulting DNA pelletwas washed with 70% (vol/vol) ethanol, air dried anddissolved in 10 µL of Tris-EDTA [TE] buffer [pH 7.5].

PCR amplification

Two, 20-base oligonucleotides were used as primers.These oligonucleotides have the following sequences:forward primer FGPD1387, 5′-ATGGACATCGCCATCAACAG-3′ and reverse primer FGPK96, 5′-CTCGAACTGCTTCTGGTAGA-3′. The sequencesof the primers were compared with the correspond-ing regions of nifD and nifK of other Frankia strainsand nifHDK sequence of Mrp182 (an isolate froma Myrica rubra root nodule) (GenBank acessionNo. AY115490) to ensure specificity. PCR amplic-

ation was performed in 0.2 mL Eppendorf tubes ina total volume of 50 µL containing template DNA(approximately 0.1 µg), 0.1 mmol/L deoxynucleos-ide triphosphate, 5 µL 10× PCR Buffer for Taq Plus,50 µmol/L each of primer, 2.5 U of Taq plus DNApolymerase [Sangon]. DNA amplication was carriedout in a PCR Express (Hybaid, Thermo ElectronCorp.) with the following procedure: initial denatur-ation for 5 min at 95 ◦C; 35 cycles of denaturation(1 min at 94 ◦C), annealing (40 sec at 52 ◦C), and ex-tension (90 sec at 72 ◦C); and a final extension (10 minat 72 ◦C). PCR amplification of DNA was checked byagarose gel electrophoresis (2%,wt/vol) with ethidiumbromide in TAE buffer with 5 µL of PCR product.

Cloning and DNA suquencing

Recombinant plasmid was obtained by standard meth-ods (Sambrook et al., 1989). After agarose gel elec-trophoresis, the band was cut off and purified withQIAquickTM Gel Extraction Kit (Germany). The puri-fied amplicons were cloned in pUCm-T using Escheri-chia coli TG1 as the host and sequenced by ShanghaiShenyou Biotechnology Co., Ltd.

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Figure 2. Specific amplication with primers FGPD1387 andFGPK96. A: lane 1–12: PCR amplified product of nodule DNAof MWD, MWY, MDK1, MBJ1, MBJ2, MBM, MSM, MMY1,MBJ4, MDK3, MYL and MMY2 respectively, lane 13: DL2000DNA marker. B: lane 1–6, PCR amplified product of nodule DNAof MDK2, MDK2, MDZ, MDZ, MBJ3, MBJ3 respectively, lane 7:DL2000 DNA marker.

Alignment of sequences and cluster analysis

The sequences were aligned and clustered byCLUSTAL W (1.82) and DNAMAN Version 4.0.

Results

PCR amplification

Using a pair of primers FGPD1387 and FGPK96,one or two bands were obtained from root nod-ules of different cultivars of M. rubra by PCR. Theshort fragment band was almost the same, but thelong fragment band was different among cultivarsor regions(Figure 2A,B). The electrophoresis res-ults showed the long fragment band is stronger andbrighter than the short fragment band if there are twobands by PCR amplified from a nodule (Figure 2A,B).There are differences in PCR amplified products ofthe nodules of the same cultivar collected from differ-ent regions, such as M. rubra var. biji and M. rubravar. dongkui, or of the different cultivars from thesame orchard, such as nodules of MMY and MDK3,or MYL and MBJ4, where the distance apart of twotrees was less than 30 m. The nodular microsymbiontsof different cultivars from southeastern part and cent-ral part of Zhejiang province usually had a commonshort fragment band that was not a dominant strainif there were two bands. Three conclusions may beinferred from the Figure 2. Firstly, there is high di-versity among nodular microsymbionts of M. rubra.

Secondly, two strains can infect the same root nodule.Thirdly, we can assess the population, richness anddominant strain of Frankia that inhabit root nodulesusing PCR based on nif DK IGS.

Sequence analysis

After agarose gel electrophoresis, the ‘desired’ bandswere cloned and sequenced. There were differencesamong the size and base composition of the nifD-nifK intergenic spacer in microsymbionts of differenthorticultural cultivars of M. rubra from different sites(Figure 3). The size of nifD-nifK IGS is from 123 bpto 269 bp (Figure 3). The sizes of nifD-nifK IGSamong the short fragment bands of agarose gel elec-trophoresis (Figure 2) are all 123 bp, the differencesbeing due to the size of nifD-nifK IGS among thelong fragment bands of agarose gel electrophoresis(Figure 2). There are differences in the size and basecomposition of the nifD-nifK IGS of the nodules of thesame cultivar collected from different regions, suchas M. rubra var. biji and M. rubra var. dongkui, orof the different cultivars from the same orchard, suchas nodules of MMY and MDK3, or MYL and MBJ4from the same orchard. The nucleotide acid alignmentamong the nifD-nifK IGS of long fragment bands ismore than 90% and that among the nifD-nifK IGS ofshort fragment bands is more than 97%, but the nuc-leotide aligment based on the nifD-nifK IGS betweenlong fragment bands and short fragment bands is lessthan 60%.

Comparison of the nifD-nifK IGS from nodularsymbionts of M. rubra with those obtained from otherFrankia strains shows more variations in size and com-position in the nifD-nifK IGS (data is not shown). Thusthe nifD-nifK IGS regions is a highly variable genomicmarker for the detection and characterization of mem-bers of the Frankia genus based on its specificity andits narrow distribution among phylogenetic neighborsof Frankia (Nalin et al., 1999).

The phylogenetic tree analysis based on nifDKIGS sequence suggests that M. rubra-infective Frankiasymbionts be grouped into two clusters. M. rubra-infective Frankia strains with long fragments (sizeof nif DK IGS≥ 205 bp ) are phyletically close toArI3 and FaC1, two Frankia strains isolated from Al-nus, so they can be grouped with the Alnus-infectivestrains, viz. cluster I (Figure 4). The result is con-sistent with previous result obtained by other meth-ods where the strains that infect Myrica. spp. oftengrouped with Alnus-infective strains (Huguet et al.,

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Figure 3. Nucleotide alignment of the nifDK intergenic spacer of the Frankia symbionts of different horticultural cultivars of M. rubra. Thestop codon of nifD and start codon of nifK are bold and underlined. Note: nodule-L(S), ‘L’ and ‘S’ stand for clone of long fragment and shortfragment in eletrophoresis pattern, respectively.

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Figure 3. Continued.

2001). Whereas, M. rubra-infective Frankia strainswith short fragment (123 bp of nif DK IGS) are phylet-ically close to Elaeagnus strains, so they are classifiedinto Elaeagnus-infective Frankia strains, viz. clusterIII (Figure 4).

Discussion

Normand et al. (1996) classified Frankia strains intofour clusters by comparative sequence analysis ofPCR-amplified 16S rDNA . Cluster I was a very largegroup comprising Frankia alni and related organisms(including Alnus rugosa Sp7 microsymbionts that are

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Figure 3. Continued.

seldom isolated in pure culture), to which Casuarina-infective strains, a Myrica nagi microsymbiont, andother effective Alnus-infective strains were related.Cluster II contained unisolated microsymbionts ofDryas, Coriaria, and Datisca species. Cluster III in-cluded Elaeagnus-infective strains and cluster IV wascomprised of ‘a typical’ strains (a group which in-cluded an Alnus-infective, non-nitrogen-fixing strain).Later on, Clawson (1998, 1999) studied natural standsof Myrica and came up with a system of groups (in-stead of clusters) similar to that of Normand et al.except that in it were interverted # II and #III. Be-cause the system of clusters was used extensively, wefollowed Normand’s (1996) system of clusters in ourpaper.

Clawson et al. (1999b) reported that Frankia fromclusters I, III, and IV were found in M. pensylvanicaby cluster analysis of partial 16S rDNA sequence. TheFrankia strains of nodules from M. gale and C. per-egrina are classified into cluster I. M. gale noduleshave low diversity of strains and C. peregrina nod-ules have an intermediate level of diversity of strains.Huguet et al. (2001) found that M. gale strains fromall three sites belonged to cluster I and were low indiversity for a host genus. Bloom et al. (1989) reportedhighly divergent strains were isolated from the sameM. pensylvanica and found to inhabit the same noduleby DNA restriction patterns and DNA-DNA solutionhybridization, and the Frankia strains which infect thesame host plant are not only phenotypically differentbut also genetically diverse, while the relationship of

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Figure 4. Phylogenetic Neighbor-Joining tree based on nifDK intergenic spacer sequences. The bar represents 0.05 substitution per site. Thenumbers are the percentages of bootstap replicates in which the clusters were found.

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one strain with another could not be determined at thetime. We also found there are differences in phen-otypes and gene types among Frankia isolates fromthe same nodule cluster of M. rubra var biji at dif-ferent times and these isolates may be in differentgene groups by PCR-RFLP (He et al., 2002). Theexperimental results showed that Frankia strains fromcluster I and cluster III were found in the same nodulefrom many different cultivars of M. rubra. The speciescan thus be considered to be promiscuous in nature(Torrey, 1990) and possibly a reservoir for differentmajor lineages of Frankia.

M. rubra originated in China and is mainly dis-tributed in China; very few plants of M. rubra areplanted in other countries. It has successfully adap-ted to tropical and subtropical dry soils, sand dunes,water-saturated fields and karst soils as a fruit or foresttree for land stabilization and soil improvement in theregions of southern part of the Yangtze River in China.M. rubra is classified into six horticultural species andmany cultivars (Miao and Wang, 1987). Our study in-dicates that there is high diversity in nodular Frankiasymbionts of M. rubra. The Frankia strains of differ-ent horticultural cultivars of M. rubra from the samesite are very different, as are the Frankia strains of thesame cultivar from different sites.

Two strains which belong to different infectiveclusters of Frankia are often found in a nodule ofM. rubra in the southeastern part and central part ofZhejiang province, and M. rubra-infective Frankiastrains with long fragment (size of nif DK IGS≥205 bp ) are often dominant strains or dominant popu-lation if there are two strains in a nodule. The nodularFrankia symbionts of different cultivars in these re-gions usually have a common Frankia strain with123 bp of nif DK IGS, but the strain is not a domin-ant strain if there are two strains in a nodule. Frankiastrain of 205 bp nif DK IGS is often found in thenodules of M. rubra in the eastern part and northernpart of Zhejiang province and is a dominant strain. Thecommon occurrence of identical sequences in nodulesfrom different plants at widely separated sites indic-ates some strains are cosmopolitan (Figures 2 and 3).Their distribution may best be described in terms of ageographical mosaic, and their interactions with theirhosts take place within the constraints of each part ofthe mosaic (Clawson et al., 1999b).

The above results indicate that the diversity ofFrankia strains in the nodules of M. rubra may resultfrom the interactions among host plant, soil environ-ment, geographical source and Frankia strains.

Acknowledgements

Thanks are expressed to Dr D. Myrold (Department ofCrop and Soil Science, Oregon State University) forreviewing the manuscript. This work was supportedby grant #302362 from the Natural Science Fundationof Zhejiang Province.

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Section editor: F.R. Minchin

natural diversity of nodular microsymbionts of myrica rubra

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