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Effects of VA mycorrhizae and Frankia dual inoculation on growth and nitrogen fixation of Hippophae tibetana
Forest Ecology and Management 170 (2002) 307–312
Short communication
Effects of VA mycorrhizae and Frankia dual inoculation on growth and nitrogen fixation of Hippophae tibetana Chunjie Tiana,*, Xingyuan Heb, Yang Zhonga, Jiakuan Chena a
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai 200433, PR China b Shenyang Institute of Applied Ecology, Academia Sinica, Shenyang 110015, PR China Received 30 November 2000; received in revised form 27 July 2001; accepted 10 October 2001
Abstract Through biological inoculation technology, the joint symbiosis of Tibetan seabuckthorn (Hippophae tibetana) in pure culture was identified and the effects of dual inoculation with Frankia and mycorrhizal fungi on the host plants in pot cultures were investigated. The results obtained from the comparative study showed that H. tibetana could form nodules and VA mycorrhiza both in pot and pure cultures. VA mycorrhizae and Frankia can stimulate the growth and the nitrogen fixation ability of host plants, respectively, yet the stimulation of the dual inoculation on the growth and nitrogen fixation ability of the host plants was more significant ðp < 0:05Þ: stronger nitrogen-fixing ability, higher VA mycorrhizal development and better growth of seedlings in VAH and HR16 dual inoculation. # 2002 Elsevier Science B.V. All rights reserved. Keywords: VA mycorrhizae; Glomus; Nitrogen fixation; Frankia; Hippophae tibetana
1. Introduction Trees harboring Frankia can fix atmospheric N2 and increase the soil nitrogen nutrient. They have been widely used in forest restoration or reconstruction and agroforestry ecosystems as the pioneer species (Callaham et al., 1978; Sprent and Parsons, 2000). These species have the advantages of high tolerance to environmental stresses and cold plateau-climate (Seiler and Johnson, 1984; Ruskin, 1984). To date, eight families and 24 genera in the world are known as the Frankia non-leguminous nitrogen-fixing trees (Sprent and Parsons, 2000). In China, about six genera and *
Corresponding author. Tel.: þ86-21-6564-2263; fax: þ86-21-6564-2468. E-mail addresses: [email protected], [email protected] (C. Tian).
46 species were recorded, among which 20 species are endemic (Huang et al., 1984). However, it has been reported that there might be a wider natural host range for Frankia worldwide. The Tibetan seabuckthorn, Hippophae tibetana Schlecht (Elaeagnaceae), is a major pioneer species widely distributed in the desert steppes in northern China. It is also of very important medical value since the seabuckthorn oil from the fruits has been identified as a new medicine (Khaidarov et al., 1991; Cao, 1999). Although the interaction between Rhizobium and mycorrhizal fungi was previously reported (Olesniewicz and Thomas, 1999), less attention was paid to the joint symbiosis of Frankia, mycorrhizae and H. tibetana, especially in identification of the joint symbiosis. We report here the joint symbiosis of Frankia, VA mycorrhizae and H. tibetana both in artificial pure and
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 7 8 1 - 2
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C. Tian et al. / Forest Ecology and Management 170 (2002) 307–312
pot cultures, and the effects of dual inoculation with Frankia and VA mycorrhizae on growth and nitrogen fixation of H. tibetana.
2. Materials and methods 2.1. Strains and dosages Frankia (strain HR16) was isolated from the nodules of H. tibetana in sandlot, Xinmin County, Liaoning Province, China, in June 1999. After culturing for 1 month, it was blended and centrifuged in a viscolizer for 5–10 min at 3000 circles per minute. Two strains of VA mycorrhizae (VAR and VAH), mainly Glomus aggregatum, were isolated from the roots of Robinia pseudoacacia and H. tibetana, respectively, in Xinmin County, Liaoning Province, China, in June 1998. To get enough for the inoculation, VA mycorrhizae were reproduced by Trifolium repens for 1 year. All strains were determined in the Laboratory of Microbiology, Shenyang Institute of Applied Ecology (SIAE), Academia Sinica. In the pot culture, the inoculation dosage of VAR and VAH mycorrhizae was 50 g (soil containing mycorrhizal spores, 6–8/g) per pot without any root. Centrifuged mycelia of Frankia were inoculated at 1 ml/100 seedlings of H. tibetana. In the artificial pure culture, the inoculation dosage of VAR or VAH was 50 spores every conical flask (500 ml). Centrifuged mycelia of Frankia were inoculated 1 ml/100 seedlings of H. tibetana. 2.2. Soil The raw soil used in the pot culture was from the earth surface (at a depth of 40 cm) in the Arboretum of SIAE. The soil is chemically similar to the soils where afforestation takes place. The soil was mixed with river sand in a ratio of 1:2. Two and a half kilograms mixed soil was put into pots of 20 cm diameter including 5 g of calcium superphosphate per pot. P-fertilization with superphosphate is part of the afforestation practice (Yao et al., 1994). The contents of organic matter in the soil was 8.1 g/ kg; total N, 0.119%; total P (P2O5), 0.29%; total K (K2O), 2.608%; available N, 87.08 mg/kg; available P, 60.74 mg/kg; available K, 129.98 mg/kg; pH 6.66.
2.3. Seeds H. tibetana seeds were surface-sterilized for 1 min in 96% ethanol and 5 min in 5% NaCLO and washed in distilled water. They were germinated in a thermostat at 28 8C. Well-germinated seeds were selected and moved into pots in which VAR and/ or VAH mycorrhizae were already inoculated. A week after the emergence of seedlings, mycelia of Frankia were inoculated. Pots were put in the greenhouse for 2 weeks and then moved out. Seven characteristics of the seedlings were measured after 6 months. In the pure culture, well-germinated seeds were selected and inseminated in test tube with sterilized agar culture. Twenty days after, the seedlings were transplanted into the conical flask with sterilized mixed culture medium of perlite and sphagnum (the quality ratio is 7:1) inoculated with VAR or VAH mycorrhizae and Frankia. The MMN liquid was sprinkled 30 ml per flask every 3 days. After 6 months, the micromorphological characters of the roots were recorded. In pot culture, six treatments were as follows: (1) inoculation with Frankia HR16; (2) inoculation with mycorrhizae VAR; (3) inoculation with mycorrhizae VAH; (4) inoculation with both VAR and Frankia; (5) inoculation with both VAH and Frankia; (6) inoculation with the control. There were five replicates for each treatment and at least five seedlings in each pot. The number of spores of VAM in control soil was 35 spores/100 g. 2.4. Morphological and physiological characters and Duncan test Seven morphological and physiological characters of H. tibetana were used to evaluate the effects of Frankia and VA mycorrhizae dual inoculation on the growth and nitrogen fixation of host plants in pot culture. Of these, the nitrogen-fixing activity was obtained by acetylene reduction assay (Hardy et al., 1973); select young roots and then cut them into 1 cm pieces. Dye the roots according to Phillips and Hayman (1970). Calculate mycorrhizal infection rate (Mark et al., 1996). After carrying out an analysis of variance (ANOVA), the Duncan test was performed to detect
C. Tian et al. / Forest Ecology and Management 170 (2002) 307–312
significant differences among the treatments with a probability of 5%.
3. Results and discussion 3.1. Joint symbiosis of Frankia, VA mycorrhizae and H. tibetana It has been reported that mycorrhiza and Frankia can form symbiosis in nitrogen fixation trees by inoculation technology, but the results vary between different inoculated strains (Gardner and Barrueco, 1995; Arveby and Granhall, 1998). Typical characteristics of the VA mycorrhizae, i.e. vesicles and arbuscules are shown in Fig. 1A. They were observed between and within the cortical cells of roots of H. tibetana inoculated with mycorrhizae in the artificial pure culture. Estimates of arbuscules longevity range from 1 to 3 weeks, so there is a high turnover rate, but it seems that they are constantly produced as long as phosphorus exchange takes place over the mycorrhizal
309
interface (Cox and Tinker, 1976). In the pot culture experiment, the vesicles of VA mycorrhizae were found between and within the cortical cells of roots of H. tibetana inoculated with mycorrhizae (Fig. 1C). Nodules in H. tibetana inoculated with Frankia (HR16) were also found (Fig. 1D). In pure cultures, the spores were found in the incubation material around the roots, and the epibotic hyphae of the VA mycorrhizae were observed on the surface of roots as well. On the other hand, the nodules in H. tibetana inoculated with the Frankia (HR16) were observed clearly (Fig. 1B). 3.2. Effects of VA mycorrhizae and Frankia on growth and nitrogen fixation of H. tibetana A number of morphological and physiological characters of H. tibetana were used in evaluating the effects of inoculation with VA mycorrhizae and Frankia on its growth and nitrogen fixation in the pot culture as shown in Table 1. In comparison with the control, the growth and nitrogen-fixing ability of
Fig. 1. Identification of the symbiosis of H. tibetana ð10 20Þ: (A) the typical characteristics of VA mycorrhiza in pure culture (vesicles and arbuscules between and within the cortical cells of roots of H. tibetana in the pure culture); (B) the nodule in H. tibetana inoculated with Frankia in pure culture; (C) the typical characteristics of VA mycorrhiza in pot culture (vesicles between and within the cortical cells of roots of H. tibetana in the pot culture); (D) the nodule in H. tibetana inoculated with Frankia in pot culture.
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Table 1 Shoot growth, mycorrhizal development and nitrogen fixation of H. tibetana seedlings 6 months after inoculation in pot culture Treatment
VAR VAH HR16 VAR þ HR16 VAH þ HR16 Control a
Charactera Shoot height (cm)
Shoot Plant fresh Number of lateral Mycorrhizal diameter (cm) weight (g/plant) roots (piece/plant) infection (%)
Dry nodule Acetylene reduction weight (g/plant) activity (ppm)
35.80 39.65 34.32 40.95 45.78 28.50
0.421 0.443 0.417 0.512 0.585 0.345
0.4076 0.5850 0.4934 0.7325 0.9858 0.1795
b c b c d a
b b b c d a
10.52 12.24 9.48 14.56 17.73 4.58
b c b d e a
12 18 10 15 22 8
b c b bc d a
45 65 42 55 75 40
b d ab c e a
b d c e f a
24.92 26.52 25.81 28.99 32.17 20.36
b b b c d a
Values denoted by the same letter are not significantly different at p < 5% level.
the plants in all the inoculated treatments were improved significantly ð p < 0:05Þ. When the treatment with VAH and HR16 and the treatment with VAH or HR16 were compared, it was found that the host plants of dual inoculation with VA mycorrhizae and Frankia had significantly more ability for growth and nitrogen fixation than that of single inoculation with VA mycorrhizae or Frankia ðp < 0:05Þ, as did the comparison between the treatment with VAR and HR16 and the treatment with HR16 or VAR. Of all, the treatment with the best effects was the one inoculated with VAH and HR16 in which the host plants had the fastest growth rate, the strongest nitrogen-fixing ability and the highest mycorrhizal development. It was reported that mycorrhizae infected roots are much more efficient in taking up soil P than uninfected roots (Stribley et al., 1980). Most of the observed changes in the growth of the host plants as a result of mycorrhizal infection are consequences of this measured uptake of P and other nutriment (Raju et al., 1990; Kothari et al., 1991). Rhizobium and mycorrhizal fungi can play an important role in the establishment of plants in soils with low nutriment levels (Biro´ et al., 2000). This study indicates that dual inoculation with mycorrhizae and Frankia can increase the growth and nitrogen-fixing ability of H. tibetana more than single inoculation with mycorrhizae or Frankia. In fact, it is easy to understand that the nodules can fix atmospheric nitrogen, but its efficiency is mostly determined by the phosphorous nutrient condition of the host plant since appropriate phosphorous nutrient support is indispensable for the process of nitrogen fixation. Mycorrhizal infection could contribute to proper phosphorous uptake in Frankia and ensure the activity of the nitrogen fixation enzyme.
Some research has shown the response of Alnus and Casuarina to endomycorrhizal inoculation (Valdes and Sanchez-Francia, 1996). It has also been reported that nodule dry weight, nitrogenase activity and nodule nitrogen content increased with an increase in P application up to quantities typical of those in soil after which it declined (Jha et al., 1993). All these showed that dual inoculation with mycorrhizae and Frankia can support both the needs for N and P and increase the growth of host plants. In fact, Frankia and mycorrhizae can do good to each other indirectly by improving the growth of host plants, but they can also affect each other by changing the microbial environment through secretion (Chatarpaul et al., 1989). In our experiment during pure culture, the inoculation was made after sterilizing the culture medium, so there are no other spores except for the inoculated strains. In pot culture, we calculated mycorrhizal infection rate according to Phillips and Hayman (1970) and Mark et al. (1996) strictly. The possibility of other intraradical spores developed by some hyphae other than endophytes is little, and this possibility did not affect comparison of results. From the results, we could find that mycorrhizal infection rate increased after inoculation, which indicated that inoculation technology can increase the potential of inoculation and so the infection rate increases. It also showed that the increases are different according to different inoculated strains. The nitrogen-fixing ability of plants inoculated with VAH and HR16 increased more significantly than that of plants inoculated with VAR and HR16 ð p < 0:05Þ. VAH was isolated from H. tibetana, while VAR was isolated from the black locust, so it also showed that indigenous fungi is more suitable to the establishment of joint symbiosis and can have more symbiotic
C. Tian et al. / Forest Ecology and Management 170 (2002) 307–312
benefits. Indigenous microbes are connected with symbiotic plants in some environments, and they are more suitable for the host plants because of the mutual benefits developed after long co-evolution (Li and Zhao, 1994). So, it is better to select the microbe isolated from the roots as the inoculation. 3.3. Implications of the joint symbiosis In most arid and semiarid regions of northern China, the seabuckthorns (Hippophae rhamnoides and H. tibetana) are the major pioneer tree species for artificial reforestation due to their ecological characteristics and economic values. In recent years, the area of manmade seabuckthorns forest has reached 3400 km2 in Qinghai Province, China (Zhang et al., 1999). However, the afforestation was limited by the conventional means because of the slow growth and low survival rate of seabuckthorns. Our study shows that VA mycorrhizae and Frankia dual inoculation should greatly improve the growth and nitrogen fixation of H. tibetana, especially when the joint symbiosis of VA mycorrhizae, Frankia and H. tibetana is formed. The joint symbiosis could be a new approach to increase the survival and growth rate of H. tibetana. However, many practical problems remain, e.g. the selection of better strains, optimization of strain dosages, and the appropriate time for inoculation need to be further studied.
Acknowledgements We would like to thank Professor Deming Su and Miss Siqi Chen for critical reading of the manuscript and Yuzhi Zhou, Guiyun Han and Qingfeng Wu for technical assistance. This work was partially supported by grants from Shenyang Institute of Applied Ecology, Academia Sinica. References Arveby, A.S., Granhall, Ulf., 1998. Occurrence and succession of mycorrhizas in Alnus incana. Swed. J. Agric. Res. 28, 117–127. Biro´ , B., Kves-Pechy, K., Voros, I., Takacs, T., Eggenberger, P., Strasser, R.J., 2000. Interrelations between Azospirillum and Rhizobium nitrogen-fixers and arbuscular mycorrhizal fungi in rhizosphere of alfalfa in sterile, AMF-free or normal soil conditions. Appl. Soil Ecol. 15, 159–168.
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Callaham, D., Del Tridici, P., Torrey, J.G., 1978. Isolation and cultivation in vitro of the actinomycete causing root nodulation in Comptonia. Science 199, 899–902. Cao, Y., 1999. Juice from Hippophae fruit and reservation techniques. Products Agro-herding 9, 116–117 (in Chinese). Chatarpaul, L., Chakravarty, P., Subramaniam, P., 1989. Studies in tetrapartite symbiosis 1: Role of ecto- and endo-mycorrhizal fungi and Frankia on the growth performance of Alnus incana. Plant Soil 118, 145–150. Cox, G., Tinker, P.B., 1976. Translocation and transfer of nutrients in vesicular–arbuscular mycorrhizae. I. The arbuscular and phosphorus transfer: a quantitative ultrastructural study. New Phytol. 77, 374–378. Gardner, I.C., Barrueco C.R., 1995. Mycorrhizal and actinorhizal biotechnology: problems and prospects. In: Varma, A., Hock, B. (Eds.), Mycorrhiza: Structure, Function, Molecular Biology and Biotechnology, pp. 469–495. Hardy, R.W.F., Bums, R.C., Hosten, R.D., 1973. Application of the acetylene–ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5, 7–81. Huang, J., Chen, G., Yang, S., 1984. Investigation of nonleguminous nitrogen fixing resources in China. In: Proceedings of the Fifth International Congress on Biological Nitrogen Fixation, The Netherlands. Jha, D.K., Sharma, G.D., Mishra, R.R., 1993. Mineral nutrition in the tripartite interaction between Frankia, Glomus and Alnus at different soil phosphorus regimes. New Phytol. 123, 307– 311. Khaidarov, K.K., Sadykov, Y.D., Lebedeva, L.D., Ismailova, M.B., 1991. Composition and pharmacological activity of essential oil derived from bunium–persicum (Boiss) B-fedsch fruits. Khim. Farm. Zh. 25, 73–75. Kothari, S.K., Marschner, H., Romheid, V., 1991. Contribution of the VAM hyphae in acquisition phosphorus and zinc by maize grown in a calcareous soil. Plant Soil 131, 177–185. Li, S., Zhao, S., 1994. Effect of VA mycorrhizal inoculation on potato. Acta Pedologica Sinica 31, 100–105 (in Chinese with English summary). Mark, B., Neale, B., Bernie, D., 1996. Working with Mycorrhizas in Forestry and Agriculture. Australian Centre for International Agricultural Research, Canberra, Australia. Olesniewicz, K.S., Thomas, R.B., 1999. Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide. New Phytol. 142, 133–140. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing and attaining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Raju, P.S., Clark, R.B., Ellis, J.R., 1990. Effects of species of VAM fungi on growth and mineral uptake of sorghum at different temperatures. Plant Soil. 121, 165–170. Ruskin, F.R., 1984. Casuarinas: Nitrogen Fixing Trees for Adverse Sites. National Academy Press, Washington, DC. Seiler, J.R., Johnson, J.D., 1984. Growth and acetylene reduction of black alder seedlings in response to water stress. Can. J. For. Res. 14, 477–480.
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Sprent, J.I., Parsons, R., 2000. Nitrogen fixation in legume and nonlegume trees. Field Crop Res. 65, 183–196. Stribley, D.P., Tinker, P., Rayner, J., 1980. Relation of internal phosphorus concentration and plant weight in plants infected by vesicular–arbuscular mycorrhizae. New Phytol. 86, 261–266. Valdes, M., Sanchez-Francia, D., 1996. Response of Alnus and Casuarina to endomycorrhizal inoculation. Rev. Mexicana Microbiol. 12, 65–67.
Yao, T., Li, X.M.X., 1994. Effect of dual inoculation with VA mycorrhizal fungi and Rhizobium on biological characteristics of Astragalus mongholicus. Acta Pedologica Sinica 31, 90–94 (in Chinese with English summary). Zhang, P., Zhao, Y., Yang, B., Chen, W., 1999. Exploitation and utilization of H. tibetana resource and the countermeasure for continuable progress in Qinghai Province. Qinghai For. Sci. Technol. 1, 31–33 (in Chinese).
Short communication
Effects of VA mycorrhizae and Frankia dual inoculation on growth and nitrogen fixation of Hippophae tibetana Chunjie Tiana,*, Xingyuan Heb, Yang Zhonga, Jiakuan Chena a
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai 200433, PR China b Shenyang Institute of Applied Ecology, Academia Sinica, Shenyang 110015, PR China Received 30 November 2000; received in revised form 27 July 2001; accepted 10 October 2001
Abstract Through biological inoculation technology, the joint symbiosis of Tibetan seabuckthorn (Hippophae tibetana) in pure culture was identified and the effects of dual inoculation with Frankia and mycorrhizal fungi on the host plants in pot cultures were investigated. The results obtained from the comparative study showed that H. tibetana could form nodules and VA mycorrhiza both in pot and pure cultures. VA mycorrhizae and Frankia can stimulate the growth and the nitrogen fixation ability of host plants, respectively, yet the stimulation of the dual inoculation on the growth and nitrogen fixation ability of the host plants was more significant ðp < 0:05Þ: stronger nitrogen-fixing ability, higher VA mycorrhizal development and better growth of seedlings in VAH and HR16 dual inoculation. # 2002 Elsevier Science B.V. All rights reserved. Keywords: VA mycorrhizae; Glomus; Nitrogen fixation; Frankia; Hippophae tibetana
1. Introduction Trees harboring Frankia can fix atmospheric N2 and increase the soil nitrogen nutrient. They have been widely used in forest restoration or reconstruction and agroforestry ecosystems as the pioneer species (Callaham et al., 1978; Sprent and Parsons, 2000). These species have the advantages of high tolerance to environmental stresses and cold plateau-climate (Seiler and Johnson, 1984; Ruskin, 1984). To date, eight families and 24 genera in the world are known as the Frankia non-leguminous nitrogen-fixing trees (Sprent and Parsons, 2000). In China, about six genera and *
Corresponding author. Tel.: þ86-21-6564-2263; fax: þ86-21-6564-2468. E-mail addresses: [email protected], [email protected] (C. Tian).
46 species were recorded, among which 20 species are endemic (Huang et al., 1984). However, it has been reported that there might be a wider natural host range for Frankia worldwide. The Tibetan seabuckthorn, Hippophae tibetana Schlecht (Elaeagnaceae), is a major pioneer species widely distributed in the desert steppes in northern China. It is also of very important medical value since the seabuckthorn oil from the fruits has been identified as a new medicine (Khaidarov et al., 1991; Cao, 1999). Although the interaction between Rhizobium and mycorrhizal fungi was previously reported (Olesniewicz and Thomas, 1999), less attention was paid to the joint symbiosis of Frankia, mycorrhizae and H. tibetana, especially in identification of the joint symbiosis. We report here the joint symbiosis of Frankia, VA mycorrhizae and H. tibetana both in artificial pure and
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 7 8 1 - 2
308
C. Tian et al. / Forest Ecology and Management 170 (2002) 307–312
pot cultures, and the effects of dual inoculation with Frankia and VA mycorrhizae on growth and nitrogen fixation of H. tibetana.
2. Materials and methods 2.1. Strains and dosages Frankia (strain HR16) was isolated from the nodules of H. tibetana in sandlot, Xinmin County, Liaoning Province, China, in June 1999. After culturing for 1 month, it was blended and centrifuged in a viscolizer for 5–10 min at 3000 circles per minute. Two strains of VA mycorrhizae (VAR and VAH), mainly Glomus aggregatum, were isolated from the roots of Robinia pseudoacacia and H. tibetana, respectively, in Xinmin County, Liaoning Province, China, in June 1998. To get enough for the inoculation, VA mycorrhizae were reproduced by Trifolium repens for 1 year. All strains were determined in the Laboratory of Microbiology, Shenyang Institute of Applied Ecology (SIAE), Academia Sinica. In the pot culture, the inoculation dosage of VAR and VAH mycorrhizae was 50 g (soil containing mycorrhizal spores, 6–8/g) per pot without any root. Centrifuged mycelia of Frankia were inoculated at 1 ml/100 seedlings of H. tibetana. In the artificial pure culture, the inoculation dosage of VAR or VAH was 50 spores every conical flask (500 ml). Centrifuged mycelia of Frankia were inoculated 1 ml/100 seedlings of H. tibetana. 2.2. Soil The raw soil used in the pot culture was from the earth surface (at a depth of 40 cm) in the Arboretum of SIAE. The soil is chemically similar to the soils where afforestation takes place. The soil was mixed with river sand in a ratio of 1:2. Two and a half kilograms mixed soil was put into pots of 20 cm diameter including 5 g of calcium superphosphate per pot. P-fertilization with superphosphate is part of the afforestation practice (Yao et al., 1994). The contents of organic matter in the soil was 8.1 g/ kg; total N, 0.119%; total P (P2O5), 0.29%; total K (K2O), 2.608%; available N, 87.08 mg/kg; available P, 60.74 mg/kg; available K, 129.98 mg/kg; pH 6.66.
2.3. Seeds H. tibetana seeds were surface-sterilized for 1 min in 96% ethanol and 5 min in 5% NaCLO and washed in distilled water. They were germinated in a thermostat at 28 8C. Well-germinated seeds were selected and moved into pots in which VAR and/ or VAH mycorrhizae were already inoculated. A week after the emergence of seedlings, mycelia of Frankia were inoculated. Pots were put in the greenhouse for 2 weeks and then moved out. Seven characteristics of the seedlings were measured after 6 months. In the pure culture, well-germinated seeds were selected and inseminated in test tube with sterilized agar culture. Twenty days after, the seedlings were transplanted into the conical flask with sterilized mixed culture medium of perlite and sphagnum (the quality ratio is 7:1) inoculated with VAR or VAH mycorrhizae and Frankia. The MMN liquid was sprinkled 30 ml per flask every 3 days. After 6 months, the micromorphological characters of the roots were recorded. In pot culture, six treatments were as follows: (1) inoculation with Frankia HR16; (2) inoculation with mycorrhizae VAR; (3) inoculation with mycorrhizae VAH; (4) inoculation with both VAR and Frankia; (5) inoculation with both VAH and Frankia; (6) inoculation with the control. There were five replicates for each treatment and at least five seedlings in each pot. The number of spores of VAM in control soil was 35 spores/100 g. 2.4. Morphological and physiological characters and Duncan test Seven morphological and physiological characters of H. tibetana were used to evaluate the effects of Frankia and VA mycorrhizae dual inoculation on the growth and nitrogen fixation of host plants in pot culture. Of these, the nitrogen-fixing activity was obtained by acetylene reduction assay (Hardy et al., 1973); select young roots and then cut them into 1 cm pieces. Dye the roots according to Phillips and Hayman (1970). Calculate mycorrhizal infection rate (Mark et al., 1996). After carrying out an analysis of variance (ANOVA), the Duncan test was performed to detect
C. Tian et al. / Forest Ecology and Management 170 (2002) 307–312
significant differences among the treatments with a probability of 5%.
3. Results and discussion 3.1. Joint symbiosis of Frankia, VA mycorrhizae and H. tibetana It has been reported that mycorrhiza and Frankia can form symbiosis in nitrogen fixation trees by inoculation technology, but the results vary between different inoculated strains (Gardner and Barrueco, 1995; Arveby and Granhall, 1998). Typical characteristics of the VA mycorrhizae, i.e. vesicles and arbuscules are shown in Fig. 1A. They were observed between and within the cortical cells of roots of H. tibetana inoculated with mycorrhizae in the artificial pure culture. Estimates of arbuscules longevity range from 1 to 3 weeks, so there is a high turnover rate, but it seems that they are constantly produced as long as phosphorus exchange takes place over the mycorrhizal
309
interface (Cox and Tinker, 1976). In the pot culture experiment, the vesicles of VA mycorrhizae were found between and within the cortical cells of roots of H. tibetana inoculated with mycorrhizae (Fig. 1C). Nodules in H. tibetana inoculated with Frankia (HR16) were also found (Fig. 1D). In pure cultures, the spores were found in the incubation material around the roots, and the epibotic hyphae of the VA mycorrhizae were observed on the surface of roots as well. On the other hand, the nodules in H. tibetana inoculated with the Frankia (HR16) were observed clearly (Fig. 1B). 3.2. Effects of VA mycorrhizae and Frankia on growth and nitrogen fixation of H. tibetana A number of morphological and physiological characters of H. tibetana were used in evaluating the effects of inoculation with VA mycorrhizae and Frankia on its growth and nitrogen fixation in the pot culture as shown in Table 1. In comparison with the control, the growth and nitrogen-fixing ability of
Fig. 1. Identification of the symbiosis of H. tibetana ð10 20Þ: (A) the typical characteristics of VA mycorrhiza in pure culture (vesicles and arbuscules between and within the cortical cells of roots of H. tibetana in the pure culture); (B) the nodule in H. tibetana inoculated with Frankia in pure culture; (C) the typical characteristics of VA mycorrhiza in pot culture (vesicles between and within the cortical cells of roots of H. tibetana in the pot culture); (D) the nodule in H. tibetana inoculated with Frankia in pot culture.
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Table 1 Shoot growth, mycorrhizal development and nitrogen fixation of H. tibetana seedlings 6 months after inoculation in pot culture Treatment
VAR VAH HR16 VAR þ HR16 VAH þ HR16 Control a
Charactera Shoot height (cm)
Shoot Plant fresh Number of lateral Mycorrhizal diameter (cm) weight (g/plant) roots (piece/plant) infection (%)
Dry nodule Acetylene reduction weight (g/plant) activity (ppm)
35.80 39.65 34.32 40.95 45.78 28.50
0.421 0.443 0.417 0.512 0.585 0.345
0.4076 0.5850 0.4934 0.7325 0.9858 0.1795
b c b c d a
b b b c d a
10.52 12.24 9.48 14.56 17.73 4.58
b c b d e a
12 18 10 15 22 8
b c b bc d a
45 65 42 55 75 40
b d ab c e a
b d c e f a
24.92 26.52 25.81 28.99 32.17 20.36
b b b c d a
Values denoted by the same letter are not significantly different at p < 5% level.
the plants in all the inoculated treatments were improved significantly ð p < 0:05Þ. When the treatment with VAH and HR16 and the treatment with VAH or HR16 were compared, it was found that the host plants of dual inoculation with VA mycorrhizae and Frankia had significantly more ability for growth and nitrogen fixation than that of single inoculation with VA mycorrhizae or Frankia ðp < 0:05Þ, as did the comparison between the treatment with VAR and HR16 and the treatment with HR16 or VAR. Of all, the treatment with the best effects was the one inoculated with VAH and HR16 in which the host plants had the fastest growth rate, the strongest nitrogen-fixing ability and the highest mycorrhizal development. It was reported that mycorrhizae infected roots are much more efficient in taking up soil P than uninfected roots (Stribley et al., 1980). Most of the observed changes in the growth of the host plants as a result of mycorrhizal infection are consequences of this measured uptake of P and other nutriment (Raju et al., 1990; Kothari et al., 1991). Rhizobium and mycorrhizal fungi can play an important role in the establishment of plants in soils with low nutriment levels (Biro´ et al., 2000). This study indicates that dual inoculation with mycorrhizae and Frankia can increase the growth and nitrogen-fixing ability of H. tibetana more than single inoculation with mycorrhizae or Frankia. In fact, it is easy to understand that the nodules can fix atmospheric nitrogen, but its efficiency is mostly determined by the phosphorous nutrient condition of the host plant since appropriate phosphorous nutrient support is indispensable for the process of nitrogen fixation. Mycorrhizal infection could contribute to proper phosphorous uptake in Frankia and ensure the activity of the nitrogen fixation enzyme.
Some research has shown the response of Alnus and Casuarina to endomycorrhizal inoculation (Valdes and Sanchez-Francia, 1996). It has also been reported that nodule dry weight, nitrogenase activity and nodule nitrogen content increased with an increase in P application up to quantities typical of those in soil after which it declined (Jha et al., 1993). All these showed that dual inoculation with mycorrhizae and Frankia can support both the needs for N and P and increase the growth of host plants. In fact, Frankia and mycorrhizae can do good to each other indirectly by improving the growth of host plants, but they can also affect each other by changing the microbial environment through secretion (Chatarpaul et al., 1989). In our experiment during pure culture, the inoculation was made after sterilizing the culture medium, so there are no other spores except for the inoculated strains. In pot culture, we calculated mycorrhizal infection rate according to Phillips and Hayman (1970) and Mark et al. (1996) strictly. The possibility of other intraradical spores developed by some hyphae other than endophytes is little, and this possibility did not affect comparison of results. From the results, we could find that mycorrhizal infection rate increased after inoculation, which indicated that inoculation technology can increase the potential of inoculation and so the infection rate increases. It also showed that the increases are different according to different inoculated strains. The nitrogen-fixing ability of plants inoculated with VAH and HR16 increased more significantly than that of plants inoculated with VAR and HR16 ð p < 0:05Þ. VAH was isolated from H. tibetana, while VAR was isolated from the black locust, so it also showed that indigenous fungi is more suitable to the establishment of joint symbiosis and can have more symbiotic
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benefits. Indigenous microbes are connected with symbiotic plants in some environments, and they are more suitable for the host plants because of the mutual benefits developed after long co-evolution (Li and Zhao, 1994). So, it is better to select the microbe isolated from the roots as the inoculation. 3.3. Implications of the joint symbiosis In most arid and semiarid regions of northern China, the seabuckthorns (Hippophae rhamnoides and H. tibetana) are the major pioneer tree species for artificial reforestation due to their ecological characteristics and economic values. In recent years, the area of manmade seabuckthorns forest has reached 3400 km2 in Qinghai Province, China (Zhang et al., 1999). However, the afforestation was limited by the conventional means because of the slow growth and low survival rate of seabuckthorns. Our study shows that VA mycorrhizae and Frankia dual inoculation should greatly improve the growth and nitrogen fixation of H. tibetana, especially when the joint symbiosis of VA mycorrhizae, Frankia and H. tibetana is formed. The joint symbiosis could be a new approach to increase the survival and growth rate of H. tibetana. However, many practical problems remain, e.g. the selection of better strains, optimization of strain dosages, and the appropriate time for inoculation need to be further studied.
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