Plant and Soil 144: 113-116, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherhmds. PLSO 9485

Actinorhizal host-specificity of Chinese Frankia strains

D A Z H I D U and D W I G H T D. BAKER Shanxi Institute of Biology, Taiyuan, Shanxi, Peoples' Republic of China and Yale University, School of Forestry and Environmental Studies, New Haven, CT 06511, USA

Received 23 January 1992. Revised April 1992

Key words: actinorhizal plants, cross-inoculation, Frankia, host specificity

Abstract

Cross-inoculation studies of numerous Frankia strains of Asian origin, primarily Chinese, were undertaken to determine whether previously described host specificity groups (HSG) were valid. Each strain was inoculated onto seedlings of Alnus glutinosa, Casuarina cunninghamiana, Elaeagnus angus- tifolia and Myrica cerifera in solution culture, and the presence of nodules scored after eight weeks. All strains tested fell into three of the four HSG descriptions of Baker (1987). Our results indicate that among pure cultured Frankia strains derived from Asia there exists less diversity than those of North America and Europe, in regard to host specificity.

Introduction

Baker (1987) inoculated 6 actinorhizal plants with 50 pure cultured Frankia strains. From his results, he identified four host-specificity groups (HSG). HSG 1 included those strains which were capable of nodulating Alnus, and Comp- tonia and the promiscuous host genus Myrica. HSG 2 included those strains which nodulated Casuarina and Myrica. HSG 3 and 4 included strains which nodulated the genera of the Elaeagnaceae, but could be distinguished by their abilities to nodulate the genus Myrica. Strains of the latter group were not capable of nodulating Myrica whereas those of the former were. Torrey (1990) found general agreement with these groups, including the results of addi- tional strains. However, the strains cited by Baker (1987) and Torrey (1990) were isolated almost exclusively from the United Stated. To verify the universality of the defined HSG for a wider selection of Frankia strains, we undertook a study of numerous Frankia strains from the geographically distinct areas of China and South- east Asia.

Materials and methods

Seedlings

In our experiment we utilized four typical ac- tinorhizal plants that have been studied widely. Seeds of Alnus glutinosa were germinated for approximately 6 weeks on a mixed medium of sand and vermiculite. Seeds of Elaeagnus angus- tifolia were germinated for approximately 3 weeks in the same manner. When seedlings of either developed four or more leaves, the seed- lings were transferred to hydroponic culture jars. Myrica cerifera and Casuarina cunninghamiana cuttings were rooted in one-quarter strength Hoagland's nutrient solution (Hoagland and Arnon, 1950). After about four to six weeks the young plants were transferred to hydroponic cul- ture jars. Modified Crone's solution (Bond 1951) was used as the nutrient medium for all inocula- tion tests. Three plants were placed in glass jars each containing 400 mL of the nutrient solution. A small amount of combined nitrogen as nitrate (50 mg L ]) was added to the nutrient solution to maintain the health of the plants. All plants

114 Du and Baker

were permitted to grow in the nitrogen-deficient nutrient solution for two weeks before inocu- lation.

Frankia strains

The Frankia strains studied in this experiment are listed in Table 1. The majority of the strains were isolated from sites in the Peoples' Republic of China, although several were from other Southeast Asian countries. In addition, ten well- characterized strains primarily from the United States were included to serve as controls. Each of the strains was assigned an arbitrary 'blind' number so that no prejudice would influence the results. All of the strains were cultivated on

DPM medium (Baker and O'Keefe 1984), ex- cept for AvcI1 and AvsI3 which were cultivated on Frankia broth (FB) medium (Baker and Tor- rey 1979). Actively growing cells between two and six weeks of age were used for inoculation.

Inoculation tests

Cells of Frankia were collected by centrifuga- tion. After homogenization in a Potter- Elvehjem tissue grinder the cells were diluted to a final concentration of 0.01 mL (packed cell volume)/mL. For each Frankia strain, six seed- lings of each host species were inoculated. Con- trol plants were inoculated with sterile distilled water. The seedlings were grown in a glasshouse

Table 1. Frankia strains used in this study

Strain Trivial Source host Geographical registry No. designation origin

IAE 01438207 Acc8207 IMB 01040002 Af2 IAE 01078204 Agc8204 IAE 01098201 Ahc8201 IAE 02020001 Cc01 IAE 020600234 Ce24 SIB 02060513 Ce1513 DAB 020603 1995 ORS 021001 Cjl-82

JCT 287 DAB 021004 1960 SIB 13010118 Ea118 IAE 13310107 Egcl07 IMB 13312061 K2061 IMB 13271510 K1510 SIB 13320273 Em273 SIB 13320373 Em373 IAE 13131211 Emocl211 IAE 13360085 Eoc85 SIB 13250131 Eul31 IAE 14010016 Hrl6 IAE 14010021 Hr21 IAE 14010034 Hr34 IAE 14010037 Hr37 SIB 14010104 Hr104 1MB 16092115 K2115 IAE 16248302 Mrc8302 HFP 022801 AIlI1 DDB 01301610 ArhI2 DDB 01020110 AvcI1 DDB 01360610 AvsI3 DDB 02060110 Cella HFP 020604 CgI4

WgCcl.17 HFP 161105 M + gI5

Alnus cremastogyne Alnus firma Alnus glutinosa Alnus hirsuta Casuarina Casuarina Casuarina Casuarina Casuarina Casuarina Casuarina Elaeagnus Elaeagnus Elaeagnus Elaeagnus Elaeagnus Elaeagnus Elaeagnus Elaeagnus

cunninghamiana equisetifolia equisetifolia equisetifolia junghuhniana spp. junghuhniana angustifolia gonyanthes gonyanthes griffithii moUis mollis multiflora oxycarpa

Elaeagnus umbellata Hippopha~ rhamnoides Hippopha~ rhamnoides Hippopha~ rhamnoides Hippopha~ rhamnoides Hippopha~ rhamnoides Myrica esculenta Myrica rubra Allocasuarina lehmanniana Alnus rhornbifolia Alnus viridis ssp. crispa Alnus viridis ssp. sinuata Casuarina equisetifolia Casuarina glauca Colletia cruciata Myrica gale

Sichuan, China China Sichuan, China China Guangdong, China Guangdong, China Guangdong, China Thailand Thailand Australia Thailand Shanxi, China China Yunnan, China Yunnan, China Shanxi, China Shanxi, China China China Shanxi, China Liaoning, China Liaoning, China Liaoning, China Liaoning, China Shanxi, China Yunnan, China Jiangsu, China Florida, USA California, USA Ontario, Canada Washington, USA Hawaii, USA Egypt The Netherlands Massachusetts, USA

Actinorhizal host-specificity of Chinese Frankia strains 115

with supplemental lighting. The nutrient solution was totally replaced every two weeks throughout the duration of the experiment. The growth con- ditions for the seedlings were as follows: photo- period 16 hours light, 8 hours dark; temperature 24-28°C day temperature, 18-22°C night tem- perature. Nodulation results were recorded 4 to 8 weeks after inoculation. Experiments were ter- minated after 8 weeks. Tests in which differences were observed between the 6 seedlings were repeated until all seedlings showed the same response.

Results and discussion

Nodulation was observed to occur for most plants within the first two weeks after inocula- tion. Nodulation of Casuarina was observed to take longer, but nodules always could be ob- served at 4 weeks. Control plants were not ob- served to nodulate. Results at 8 weeks for the cross-inoculations are listed in Table 2.

Although large geographical and ecological differences exist between the sites in China and in the USA from which the strains were isolated, the results of our study demonstrated that all of the strains belonged to the first 3 of the 4 host-specificity groups established by Baker (1987). These results verify the universality of these host-specificity groups of Frankia. None of the strains tested belonged to HSG 4, as defined by Baker (1987).

Host specificity among the currently available pure cultured Frankia strains appears to be quite conservative. The diversity of strains in regard to host specificity in Asian strains appears to be no different than for North American and European strains. It is possible that when additional strains are isolated that adjustments to HSG assign- ments may be required. However because of the large number of strains studied and wide geo- graphical distribution of the tested strains, it is unlikely that major changes will be necessary. Because no infective Frankia strains have been isolated from the actinorhizal Rosaceae, Rham- naceae, Coriariaceae or Datiscaceae, our under- standing of host specificity of all actinorhizal plants must be considered incomplete.

Table 2. R e s u l t s o f c r o s s - i n o c u l a t i o n s t u d i e s

H o s t s p e c i e s : Alg l a Cacu Elan M y c e H S G h

S t r a i n

A c c 8 2 0 7 + • - - + 1

A f 2 + - - + 1

A g c 8 2 0 4 + - - + 1

A h c 8 2 0 1 + - - t 1

CcO I - + - + 2

C e 2 4 - + - + 2

C e I 5 1 3 - - + + 3

1 9 9 5 - + - -4- 2

C j l - 8 2 - + - + 2

J C T 2 8 7 - + - + 2

1 9 6 0 - + - ~- 2

E a 1 1 8 - - + -~- 3

E g c 1 0 7 - - + + 3

K 2 0 6 1 - - + + 3

K 1 5 1 0 - - + +- 3

E m 2 7 3 - - + ~ 3

E m 3 7 3 - - + + 3

E m o c 1 2 1 1 - - + + 3

E o c 8 5 - - + + 3

E u 131 - - + + 3

H r 1 6 - - + + 3

H r 21 - - + + 3

H r 3 4 - - + + 3

H r 3 7 - - + + 3

H r 1 0 4 - - + + 3

K 2 1 1 5 - - + + 3

M r c 8 3 0 2 + - - + 1

A I I I 1 - + - + 2

A r h I 2 - - + + 3

A v c I 1 + - - + 1

A v s I 3 + - - + 1

C e l l a - - + + 3

C g l 4 - + - + 2

W g C c 1 . 1 7 - - + + 3

M + g l 5 + - - + 1

H o s t s p e c i e s : A l g l = A l n u s glut inosa, Cacu = Casuarina

c u n n i n g h a m i a n a , Elan = Elaeagnus angust i fol ia, M y c e =

Myr ica ceri fera.

b H S G = H o s t - s p e c i f i c i t y g r o u p s .

" + = A l l 6 s e e d l i n g s n o d u l a t e d ; - = A l l 6 s e e d l i n g s u n n o d u -

l a t c d .

Acknowledgements

The authors thank Drs Ruan Ji-Sheng and Zhang Zhong-ze for providing some of the strains used in this study. This research was funded in part by Project No. INT-8901155 from the U.S. National Science Foundation to DDB.

116 Actinorhizal host-specificity o f Chinese F r a n k i a strains

References

Baker D D 1987 Relationships among pure cultured strains of Frankia based on host-specificity. Physiol. Plant. 70, 245 -248.

Baker D and O'Keefe D 1984 A modified sucrose fractiona- tion procedure for the isolation of frankiae from actinor- hizal root nodules and soil samples: Plant and Soil 78, 23-28.

Baker D and Torrey J G 1979 The isolation and cultivation of actinomycetous root nodule endophytes. In Symbiotic Ni- trogen Fixation in the Management of Temperate Forests. Eds. J C Gordon, C T Wheeler and D A Perry. pp 38-56.

Oregon State University Forest Research Lab., Corvallis, OR.

Bond G 1951 The fixation of nitrogen associated with the root nodules of Myrica gale L., with special reference to its pH relation and ecological significance. Ann. Bot. 15, 447-459.

Hoagland D R and Arnon D I 1950 The water culture method for growing plants without soil. Circ. Agric. Exp. Station no. 347.

Torrey J G 1990 Cross-inoculation groups within Frankia and host-endosymbiont associations. In The Biology of Frankia and Actinorhizal Plants. Eds. C R Schwintzer and J D Tjepkema. pp 83-106. Academic Press, New York.