11
This article was downloaded by: [195.113.207.86] On: 31 March 2014, At: 03:41 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Soil Science and Plant Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tssp20 Isolation of frankia strains from root nodules of myrica rubra Tora Hiyoshi a b , Hideo Sasakawa a c & Michihiko Yatazawa a d a Department of Agricultural Chemistry, School of Agriculture , Nagoya University , Chikusa- ku, Nagoya , Japan b Iwata Experimental Station, Japan Tobacco Inc. , Iwata-gun, Shizuoka , Japan c Department of Agricultural Chemistry, School of Agriculture , Nagoya University , Chikusa- ku, Nagoya , Japan d Aichigakuin University , Nisshin-cho, Aichi , Japan Published online: 04 Jan 2012. To cite this article: Tora Hiyoshi , Hideo Sasakawa & Michihiko Yatazawa (1988) Isolation of frankia strains from root nodules of myrica rubra , Soil Science and Plant Nutrition, 34:1, 107-116, DOI: 10.1080/00380768.1988.10415584 To link to this article: http://dx.doi.org/10.1080/00380768.1988.10415584 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

Isolation of frankia strains from root nodules of myrica rubra

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Page 1: Isolation of               frankia               strains from root nodules of               myrica rubra

This article was downloaded by: [195.113.207.86]On: 31 March 2014, At: 03:41Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Soil Science and Plant NutritionPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tssp20

Isolation of frankia strains from root nodules of myricarubraTora Hiyoshi a b , Hideo Sasakawa a c & Michihiko Yatazawa a da Department of Agricultural Chemistry, School of Agriculture , Nagoya University , Chikusa-ku, Nagoya , Japanb Iwata Experimental Station, Japan Tobacco Inc. , Iwata-gun, Shizuoka , Japanc Department of Agricultural Chemistry, School of Agriculture , Nagoya University , Chikusa-ku, Nagoya , Japand Aichigakuin University , Nisshin-cho, Aichi , JapanPublished online: 04 Jan 2012.

To cite this article: Tora Hiyoshi , Hideo Sasakawa & Michihiko Yatazawa (1988) Isolation of frankia strains from root nodulesof myrica rubra , Soil Science and Plant Nutrition, 34:1, 107-116, DOI: 10.1080/00380768.1988.10415584

To link to this article: http://dx.doi.org/10.1080/00380768.1988.10415584

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Page 2: Isolation of               frankia               strains from root nodules of               myrica rubra

Soil SCL Plant Nutr., 34 (1), 107-116, 1988

ISOLATION OF Frankia STRAINS F R O M ROOT NODULES OF Myrica rubra

Toru HIYOSHI,* Hideo SASAKAWA,** and Michih iko YATAZAWA***

Department of Agricultural Chenlistry, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464 Japan

Received May 11, 1987

Frankia strains were successfully isolated from Myrica rubra root nodules using a double- layer agar system consisting of an upper layer of 1~ agar which included the endophytes and a lower layer of 1.5~ agar which contained the nutrients. Colonies appeared within 1 month and their diameter increased from 0.5 to 1.0rnm 2 months after the inoculation. Hyphae, sporangia, and vesicles were observed in the coIonies. The Frankia isolates grew well in liquid media supplemented with vitamins including biotin and p-aminobenzoic acid (modified Qmod medium; see LALONDE and CALVERT 1979). The Frankia isolates grown in N-deficient modified Qmod medium exhibited an acetylene reducing activity.

Nodules were formed on the roots of 5lyrica rubra by inoculation of the Frankia isolates and the acetylene reducing activity of the nodules amounted to 16 pmol C-.H4.(g fr wt)-t.h-L No nodules ~ere formed on Coriaria japonica after the inoculation of the isolates. These results suggest that the Frankia strains isolated exhibit a host specificity to Myrica rubra.

Key Words: Myrica rttbra, isolation of Frankia, cultural characteristics, nodulation.

Act inorhiza l plants include a wide range of woody dicotyledonous species whose

roots are susceptible to infection by the f i lamentous soil bacteria belonging to the

genus Frankia of the Act inomycetales . At present, the host plants consist of more

than 200 species distr ibuted among 23 genera in eight families (TORREY 1985). In

Japan, 5 genera in five families, that is, Casuarina, Coriaria, AhTus, Myrica, and Elaeag- nus grow naturally.

In 1950's UEMURA (1952, 1964) isolated act inomycetal endophytes f rom the nodules

of nonleguminous plants including Myrica rubra, but they did not succeed in inducing

nodula t ion in the host plants by inoculat ion of the isolates. The Frankia endophytes

were successfully isolated f rom roo t nodules of Comptonia peregrina by CALLAHAM

et al. (1978). Thereaf ter numerous strains of Frankia were isolated f rom various

plants (BURGGRAAF et al. 1981 ; BENSON 1982; DIEM et al. 1982; BURGGRAAF and SHIPTON

1983; ZANG et al. 1984). Recently JtABIN et aL (1985) reported that nodules were not

* Present address: Iwata Experimental Station, Japan Tobacco Inc., Iwata-gun, Shizuoka, 438 Japan.

** To whom correspondence should be addressed. *** Present address: Aichigakuin University, Nisshin-cho, Aichi, 470-01 Japan.

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108 T. HIYOSHI, H. SASAKAWA, and M. YATAZAWA

formed in Myrica rubra after the inoculation of Frankia isolated from nodules of the same species.

In this paper, we describe the isolation of Frankia strains f rom Myrica rubra nodules, as well as their cultural characteristics and nodulation induced by the Frankia isolates.

MATERIALS AND METHODS

Plant for endophyte isolation. Two-year-old Myrica rubra seedlings were com- mercially acquired. After loose soil fragments were removed in a stream of tap water and after the presence of nodules on the roots was confirmed, the plants were grown in N-deficient Arnon-Hoagland nutrient solution (pH 6.0) in a phytotron under a day/ night temperature regime of 30~176 and natural light conditions for 5 months. Nutrient solution was renewed every 2 weeks.

Media. The composition of the Frankia basal medium (FM) was identical with that described by BENSON (1982). In our experiments, 0.3~, (w/v) sodium pyruvate or 0 . 7 ~ (w/v) sodium succinate and 0.3% (w/v) casamino acid were added to FM as carbon and nitrogen sources, respectively (FMS). Vitamins and sodium pyruvate were filter-sterilized before use. The composition of the Qmod medium was identical with that described by LALONDE and CALVERT (1979). The Qmod medium was slightly modified by the addition of the following vitamins in 1 liter of the medium (modified Qmod): 0.1 mg thiamine-HCl, 0.5 mg nicotinic acid, 0.5 mg pyridoxine-HCl, 0.5 mg biotin, and 0.1 mg p-aminobenzoic acid. In the N-deficient modified Qmod medium the amounts of yeast extract and L-a,-lecithin (P-5638, Sigma) were reduced to 1/10 and 1/5, respectively and no Bacto-peptone (Difco) was added. Sodium propionate (1.0 g/liter) was added instead of glucose as needed.

Isolation and cultivation of endophytes. Newly developed nodules during growth in water culture were detached from the roots and washed thoroughly with cold distilled water by vigorous shaking. Nodule lobes were cut on ice at a distance of approxi- mately 2 m m from the tip with a single-edged razor blade. Soil particles and dead plant tissue residues on the lobes were carefully removed with tweezers and a needle to reduce the contamination. The lobes were washed with cold distilled water and 50 mM sodium phosphate buffer (pH 7.0), and the 1-mm tips of the lobes were used for endophyte isolation. Five to 6 lobe tips were surface-sterilized with a 1.0~o sodium hypochlorite solution for 5 rain and rinsed with sterile distilled water and the above phosphate buffer several times each at 4~ All further manipulations were carried out at 4~ aseptically.

The epidermis of the lobes was peeled in a drop of the phosphate buffer containing 1 0 ~ polyvinylpyrrolidone (PVP, av. tool. wt 40,000, Sigma) with a single-edged razor blade under a stereoscopic microscope. After being rinsed with the phosphate buffer containing 10~ PVP, the peeled lobes were homogenized with 10 ml of the above buffer in a mortar to release the endophytes. The homogenate was passed through two

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Isolation of Myrica rubra Frankia 109

Fig. 1. Apparatus used to isolate endophytes from root nodule hornogenates. A: syringe; B: filter with 45/~m nylon mesh screen; C: filter with 25 t~m stainless-steel mesh screen.

screens with nominal openings of 45 and 25 ttm, using the apparatus illustrated in Fig. 1. Large particles retained on the 45 t~m screen were washed once with 50 ml of FMS. Endophytes retained on the 25 ym screen were released in the FMS by vigorous shaking.

For the isolation of Frankia strains in the agar medium, the double layer agar technique described by DIEM et al. (1982) was used with slight modifications. The suspension of endophytes was mixed in 15.5 ml of 1.0~/o agar-FMS, previously auto- claved and cooled. Before the agar solidified, 2 rnl each of the mixture was imme- diately poured on a 1.5% agar-FMS plate in petri dishes and spread to achieve a uni- form distribution of the endophytes by swirling the dishes. To prevent drying, the dish edges were wrapped with Parafilm and the dishes were incubated at 30~ in the dark.

When actinomycetal colonies were visible, some colonies in the upper agar layer were carefully separated with a single-edged razor blade and transferred into sterile test tubes (2.5 cm in diameter, 20 cm long) containing 10 ml of the modified Qmod medium. The agar block was crushed with a spatula to facilitate the proliferation of the Frankia isolates and cultured for 2 months at 30~ in the dark. The 2-month-old cultures were transferred into newly prepared modified Qmod medium and cultured for another 2 months under the same conditions. The cultures were harvested by centrifugation at 6,000 x g for 15 rain, washed with sterile distilled water 4 times and homogenized with a pipet to prepare the inoculum suspension.

Nodulation procedures. Seeds of Myrica rubra and Coriaria japonica were surface- sterilized with a 5% sodium hypochlorite solution for 20 rain. The seeds were asepti- cally incubated in a 25 ppm gibberellin (GAs) solution at 25~ for 2 weeks in the dark to stimulate germination. Three or 5 germinated seeds were transplanted onto a sterile Seed-Pak growth pouch (Div. of American Hospital Supply Corp., Evanston, Ill.) containing 30 ml of N-free Arnon-Hoagland nutrient solution. When the roots reached a length of 5 cm after 1 month of cultivation, an appropriate amount of the Frankia isolates was inoculated. Plants were cultivated in a growth chamber under a day/night length of 14 h/10 h and artificial light of 4,000 lx at 25~ The Seed-Pak

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110 T. FIIYOSHI, H. SASAKAWA, and M. YATAZAWA

growth pouch was covered with aluminum foil to prevent light irradiation to the roots. Measurement of nitrogenase activity. Nitrogenase activity was measured by the

acetylene reduction method. The Frankia isolates cultured in the N-deficient modified Qmod medium in a test tube (1.8 cm in diameter, 1.8 cm long) for 2 months were sealed with a rubber stopper and incubated in air containing 0.I atm of acetylene at 30~ in the dark. Nodulated roots were incubated in a 50-ml Erlenmeyer flask with the same gas phase as for the Frankia isolates at 25~ in the dark. The amount of ethylene evolved was determined by gas chromatography under the same conditions as those described by HOSODA et al. (1978).

Scanning electron microscopy. Frankia isolates grown in agar-FMS were fixed with 2 .5~ paraformaldehyde and 2.0~o glutaraldehyde in 50 mm cacodylate buffer (pH 7.4) at 4~ for 12 h. After washing with the same buffer containing 3 ~ sucrose, post fixation with 2~o osmium tetroxide for 12 h at 4~ and dehydration in an alcohol series, the samples were freeze-dried with liquid carbon dioxide. Then the samples were cut with a single-edged razor blade and the surfaces were sputter-coated with gold particles.

RESULTS

Colony development and moJTholog)' of Frankia isolates Small colonies were visible in the agar plates after 1 month and their diameter

increased from 0.5 to 1.0 mm 2 months after the inoculation of the endophytes isolated from the nodules. The distribution of the colonies which appeared on the plates is shown in Table 1. Almost all the colonies consisted of actinomycetales with filamen- tous hyphae, and very few bacterial and fungal colonies were observed in the plates. The actinomyceta! colonies in the plates developed much faster in agar-FMS when pyruvate was added compared with succinate as a carbon source (data not shown). To confirm that the colonies appearing on the plates were Frankia strains, hyphal outgrowth from the central clusters was examined under the microscope. Hyphae generally grew fi'om several points on the central cluster. The actinomycetal colonies appeared to contain hyphae, sporangia, and vesicles (Fig. 2). Sporangia were espe- cially observed on aged hyphae in the center of the colonies and numerous spores were detected in mature sporangia (Fig. 2C). In contrat, vesicles were observed on young hyphae growing out of the cluster in the center of the colonies (Fig. 2D).

Table 1. Number of microbial colonies isolated from nodules of Myrica rubra.

Carbon s o u r c e Actinomycetales Bacteria Fungi

Pyruvate 183 0 1

Succinate 162 3 2

Microorganisms were isolated in agar plates containing Frankia basal medium supplemented with pyruvate or succinate.

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I s o l a t i o n o f Myrica rubra Frankia III

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Page 7: Isolation of               frankia               strains from root nodules of               myrica rubra

112 T. HIYOSHI, H. SASAKAWA, and M. YATAZAWA

Growth of Frankia isolates Frankia isolates did not grow at all in liquid F M S conta in ing pyruvate dur ing

several months of culture. In F M S supplemented with p rop iona te instead of pyruvate

as a ca rbon source, a lmos t all the isolates grew slowly, but some did not. The growth of the isolates was compared between the modified Q m o d med ium conta in ing pro-

p iona te or glucose as a carbon source. Al though the isolates grew well in both media,

their growth was slightly better on glucose than on propiona te . The colonies developed

in the med ium conta in ing glucose easily dis integrated by gentle shaking, unlike those

grown in the med ium conta in ing prop iona te .

Nodulation by inoculation of Frankia isolates and nitrogenase activity A few nodules with a br ight red color were formed on the roots of Myriea rubra

2 months after the inocula t ion of the Frankia isolates (Fig. 3). Nodu la t i on rat io was

8 5 ~ and no nodules were formed on the non- inocu la ted plants (Table 2). No nodule

fo rmat ion was observed in Coriaria japonica after the inocula t ion of the Frankia iso- lates f rom Myrica rubra nodules (Table 2).

The acetylene reducing activi ty of the Myrica rubra roo t nodules increased l inearly

Fig. 3. Nodules formed in Myrica rubra seedlings by inoculation of Frankia isolates. A: Myriea rubra seedlings growing in a plastic pouch containing a N-deficient culture medium for 2 months after inoculation of Frankia isolates. B: nodules formed on Myrica rttbra roots. Arrows show nodules. Scale bars indicate 1 era.

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Isolation of Myrica rubra Frankia 113

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Fig. 4. Acetylene reducing activity of MyHca rubra root nodules formed by inoculation of Frankia isolates. The nodulated roots of 3-month-old seedlings were used for assay. Vertical lines indicate standard deviation (n=4).

with the incubation time and reached a value of 60 nmol C2H4.plant-l .h -1 and 16 /~mol C~H4"(g fr wt nod) 1.h -1 (Fig. 4). Frankia isolates cultured in the N-deficient modified Qmod medium showed an acetylene reducing activity under free-living con- ditions and the activity increased linearly until 24 h after a 3-h lag period (data not shown).

DISCUSSION

The results obtained demonstrate that Frankia isolates from Myrica rubra were able to grow rapidly in the modified Qmod medium supplemented with vitamins in- cluding biotin and p-aminobenzoic acid, as well as to induce nodulation on the host plant (Fig. 3 and Table 2), and that the nodules displayed a high acetylene reducing activity (Fig. 4). These results indicate that the Frankia isolate is an effective strain and that the acetylene reducing activity in the newly formed young nodules is com- parable to that in the nodules of leguminous plants.

UEMURA (1964) and JIABIN et al. (1985) attempted to isolate effective Frankia strains from Myrica rubra nodules, but they did not succeed. The age of the nodules they used for endophyte isolation was not clearly indicated. In the current study, lobe tips of newly developed nodules in water culture were used for the isolation of the endophytes and the outer cortical layers of the nodule lobes were peeled off to eliminate most of the contaminants (DIEM and DOMMERGUES 1983). Moreover, the double layer agar technique described by DIEM et al. (1982) was used with slight modifications. The double layer agar technique is advantageous in that (i) contaminants do not nec- essarily overgrow the Frankia colonies, and (ii) microaerophilic Frankia colonies are not directly exposed to the atmosphere. In the current experiments, Frankia colonies were

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114

Table 2.

T. HIYOSHI, H. SASAKAWA, and M. YATAZAWA

Nodulation on roots of Myrica rubra and Coriariajaponica seedlings by inoculation of Frankia isolates from Myrica rubra.

Plant Inoculated Non-inoculated

M. rubra 18/21 0/10 C. japonica 0/10 0/10

Figures indicate the number of nodulated plants/total number of plants used.

formed inside the agar medium and hyphae never grew out of the agar. The Frankia isolates also grew in a liquid medium and appeared to require biotin and/or p-amino- benzoic acid for adequate growth in liquid culture.

The color of the nodules formed by the inoculation in Myrica rubra was bright red. APPLEBY et al. (1983) recently reported that a dimeric hemoglobin was purified f rom nitrogen-fixing nodules formed by the association of Rhizobium with a nonlegu- minous plant, Parasponia. It remains however to be determined whether hemoglobin is present in nitrogen-fixing nodules formed by the association of Frankia. Accord- ingly, the red compound(s) in the Myrica rubra nodules may not be a respiratory pig- ment such as leghemoglobin.

The specificity of actinomycetal Frankia for the host plants had been studied (BAKER and TORREY 1979, 1980; LALONDE and CALVERT, 1979; RODRIGUEZ-BARRUECO and MIGUEL 1979; VANDENBOSCH and TORREY 1983). Recently, JIABIN et al. (1985) suggested that Frankia endophytes can be divided into two groups: Alunus group and Elaeagnus group. It is unclear to which group the Frankia strain from Myrica rubra

belonged, because the Frankia strain did not nodulate any of the plants tested. In the current experiment, the Frankia strains isolated from Myrica rubra induced nodu- lation on the host plants but not on the Coriaria japonica plants (Table 2). These results suggest that the Frankia strains from Myrica rubra exhibit a rather high host specificity.

Free living Frankia strains grown with appropriate nutrients displayed an acety- lene reducing activity and the activity which was closely related to the development of vesicles (TJEPKEMA et al. 1980; BURGGRAAF and SIre'TON 1983; MURRY et al. 1984). In this experiment, the density of the vesicles was obviously higher in the Frankia iso- lates grown in N-deficient medium than in those grown in a medium with adequate supply of N (data not shown), indicating the presence of a relationship between the acetylene reducing activity and the development of vesicles. The fact that free-living Frankia strains showed an acetylene reducing activity suggests that the Frankia sp. are albe to fix dinitrogen in soils.

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Isolation of Myrica rubra Frankia 115

REFERENCES

APPLEBY, C.A., TJEPKEMA, J.D., and TRINICK, M.J. 1983: Hemoglobin in a nonleguminous plant, Parasponio: Possible genetic origin and function in nitrogen fixation. Science, 220, 951-953

BAKER, D. and TORREY, J.G. 1979: The isolation and cultivation of actinomycetous root nodule endo- phytes. In Symbiotic Nitrogen Fixation in the Management of Temperate Forests, p. 38-56, Ed. J.C. Gordon, C.T. Wheeler, and D.A. Perry, Oregon State University, Corvallis, Oreg.

BAKEa, D. and TORREY, J.G. 1980: Characterization of an effective actinorhizal microsymbiont, Frankia sp. AvcI1 (Aetonomycetales). Can. J. Microbiol., 26, 1066-1071

BENSON, D.R. 1982: Isolation of Frankia strains from alder actinorhizal root nodules. Appl. Environ. Microbiol., 44, 461~.65

BtmGGRAAF, A.J.P., QtnsvEL, A., TAK, T., and VALSTAR, I. 1981 : Methods of isolation and cultivation of Frankia species from actinorhizas. Plant Soil, 61, 157-168

BURGGRAAF, A.J.P. and SmPTON, W.A. 1983: Studies on the growth of Frankia isolates in relation to infectivity and nitrogen fixation (acetylene reduction). Can. J. Bot., 61, 2774-2782

CALLArI~, D., TREDICI, P.D., and TORREY, J.G. 1978: Isolation and cultivation in vitro of the actino- myccte causing root nodulation in Comptoaia. Science, 199, 899-902

DIEM, H.G. and DO/VlMERGUES, Y. 1983: The isolation of Frankia from nodules of Casuarhm. Can. J. Bot., 61, 2822-2825

DIEM, H.G., GAtrrmER, D., and DOMMERGUES, Y.R. 1982: Isolation of Frankia from nodules of Casuarina equisetifolia. Can. J. Microbiol., 28, 526-530

DILLON, J.T. and BAKER, D. I982: Variations in nitrogenase activity among pure-cultured Frankia strains tested on actinorhizal plants as induction of symbiotic compatibility. New Phytol., 92, 215-219

HOSODA, N., LEE, K.K., and YATAZAWA, M. 1978: Carbon dioxide, oxygen, and light on nitrogen-fixing activities in Japan clover Kmnmerowia striata. Soil Sci. Plant Nutr., 24, 113 -119

JIABIN, H., ZHEYING, Z., GUANXONG, C., and HUICHANG, L. 1985: Host range of Frankia cndophytes. Plant Soil, 87, 61-65

LALONDE, M. and CALVERT, H.E. 1979: Production of Frankia hyphae and spores as an infective in- oculant for Ahms species. In Symbiotic Nitrogen Fixation in the Management cf Temperate Forests, p. 95-110, Ed. J.C. Gordon, C.T. Wheeler, and D.A. Perry, Oregon State University, Corvallis, Oreg.

MURKY, M.C., FONIAtNE, M.S., and TORREY, J.G. 1984: Growth kinetics and nitrogenase induction in Frankia sp. HFPArI3 grown in batch culture. Plant Soil, 78, 61-78

RODRIGUEZ-BARRUECO, C. and MIGUEL, C. 1979: Host plant-endophyte specificity in actinomycete- nodulated plants. In Symbiotic Nitrogen Fixation in the Management of Temperate Forests, p. 143-159, Ed. J.C. Gordon, C.T. Wheeler, and D.A. Perry, Oregon State University, Corvallis, Oreg.

TJEVKEMA, J.D., OP, ME~OD, W., and TORREY, G. 1980: Vesicle formation and acetylene reduction activity in Frankia sp. CPI1 cultured in defined nutrient media. Nature, 287, 633 635

TORREY, J.G. 1985: The site of nitrogenase in Frankia in free-living culture and in symbiosis, bt Cur- rent Plant Science and Biotechnology in Agriculture--Nitrogen Fixation Research Progress, p. 293 299, Ed. H.J. Evans, P.J. Bottomley, and W.E. Newton, Martinus Nijhoff Publishers, Dord- recht

UEMURA, S. 1952: Studies on the root nodules of alders (Abuts spp.). IV. Experiment on isolation of actinomycetes from alder nodules. Bull. Forest Exp. Stn., Jpn., 52, 1-22 (in Japanese with English summary)

UEMURA, S. 1964: Isolation and properties of microorganisms from root nodules of non-leguminous plants. A re~,iew with extensive bibliography. Bull. Forest E.~T. Stn., Jpn., 167, 59-98

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116 T. HIYOSHI, H. SASAKAWA, and M. YATAZAWA

VANDENBOSCH, K.A. and TORREY, J.G. 1983: Host-endophyte interactions in effective and ineffective nodules induced by the endophyte of Myrica gale. Can. J. Bot., 61, 2898-2909

ZANG, Z., LOPEZ, M.F., and TORREY, J.G. 1984: A comparison of cultural characteristics and infectivity of Frankia isolates from root nodules of Casuarflla species. Plant Soil, 78, 79-90

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