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Nodulation and N2 fixation effectiveness of Bradyrhizobium strains in symbiosis with Adzuki Bean, Vigna angularis

Abstract

In pot experiment, one isolate Knj from a Serbian soil, four strains of Bradyrhizobium japonicum and three strains of Bradyrhizobium spp. were examined for the effect on adzuki bean nodulation and effectiveness in symbiotic N2 fixation. All the tested strains produced root nodules in adzuki bean. Strains of B. japonicum showed high potential of N2 fixation, particularly 525 and 542. B. japonicum strains resulted 65-71% shoot dry weight and 99-138% total N content of uninoculated control with full N content (100%). No significant difference was found between the plants inoculated with Bradyrhizobium spp. strains and uninoculated control plants without N (40-42 and 42% shoot dry weight, respectively), which indicated symbiotic N2 fixation inactivity of the Bradyrhizobium spp. strains. Knj strain had the middle position (56% shoot dry weight). These data showed that B. japonicum 525 and 542 strains could be used in further investigations in order to apply them as inoculants in microbiological N fertilizers.

Bradyrhizobium; adzuki bean; nodulation; symbiotic N fixation


AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Nodulation and N2 fixation effectiveness of Bradyrhizobium strains in symbiosis with Adzuki Bean, Vigna angularis

Dušica Delić* * Author for correspondence: vukmirdusica@yahoo.com ; Olivera Stajković; Nataša Rasulić; Djordje Kuzmanović; Dragana Jošić; Bogić Miličić

Department for Microbiology; Institute of Soil Science; Teodora Drajzera 7; 11000 Belgrade, Serbia

ABSTRACT

In pot experiment, one isolate Knj from a Serbian soil, four strains of Bradyrhizobium japonicum and three strains of Bradyrhizobium spp. were examined for the effect on adzuki bean nodulation and effectiveness in symbiotic N2 fixation. All the tested strains produced root nodules in adzuki bean. Strains of B. japonicum showed high potential of N2 fixation, particularly 525 and 542. B. japonicum strains resulted 65-71% shoot dry weight and 99-138% total N content of uninoculated control with full N content (100%). No significant difference was found between the plants inoculated with Bradyrhizobium spp. strains and uninoculated control plants without N (40-42 and 42% shoot dry weight, respectively), which indicated symbiotic N2 fixation inactivity of the Bradyrhizobium spp. strains. Knj strain had the middle position (56% shoot dry weight). These data showed that B. japonicum 525 and 542 strains could be used in further investigations in order to apply them as inoculants in microbiological N fertilizers.

Key words:Bradyrhizobium, adzuki bean, nodulation, symbiotic N fixation

INTRODUCTION

Adzuki bean, Vigna angularis (Willd.) is an important legume and traditional pulse crop in East Asia, widely used as a source of protein for human nutrition, especially in developing countries. In Europe and America, it is valued for its high protein content (25%), low fat, natural sugar nutritional profile, high nutritious value and easy digestibility compared with Phaseolus vulgaris. It has following economic importance: environmental (soil improver as a green manure), animal food (forage), medicines (folklore-herb) and for human food (pulse, vegetables, beverage base) (Sato et al., 2005). The ability of adzuki bean to grow in different geographical regions brought the opportunity for introducing this legume into Serbian agricultural practice (Yee et al., 1999; Zong et al., 2003).

Adzuki bean has high physiological nitrogen (N) requirements. However, little is known about its assimilation of N from soil and atmosphere (Kimura et al., 2004). Robinson (1983) found that adzuki bean did not increase yield with inorganic N fertilization, indicating the priority of symbiotic nitrogen fixation (SNF). Adzuki bean fixed N2 in symbiosis with some bacterial strains of the genera Bradyrhizobium, Rhizobium and Sinorhizobium (Fujihara et al., 2006). The native populations of rhizobial strains are generally poor efficient in N fixation (Yadav et al., 1996; Vlassak and Vanderleyden, 1997) and inoculation with effective strains as active agent of microbiological N fertilizer is required as an important farming method. The formation of effective root nodules leads to N2 fixation, which makes N mineral fertilizers superfluous. Hence, there is a need to conduct a screening of inoculant strains for better effectiveness.

In this study, some selected rhizobial strains (525, 526. 532, 542 of B. japonicum; Knj isolate and 2001, 2801 and 5388 of Bradyrhizobium spp) were examined for their ability to promote nodulation of adzuki bean and effectiveness in SNF in order to acquire highly effective strains as the basic agent of microbiological nitrogen fertilizers.

MATERIALS AND METHODS

Bacterial strains and host plant

Four strains >f Bradyrhizobium japonicum (525, 526, 432 and 532), one isolate (Knj) of Bradyrhizobium spp, from the Collection of Institute of Soil Science, Belgrade and three Bradyrhizobium spp. strains (2001, 2801 and 5388), specific to adzuki bean from the Collection of Faculty of Agronomy, University of Buenos Aires were tested. Adzuki bean, Vigna angularis L., was used as the host-plant. The seeds (domestic population) were obtained from the "Selsem", company for breeding and seed production (Belgrade, Serbia).

Nodulation test and symbiotic properties

Strain nodulation ability was tested on adzuki bean plants in a pot experiment conducted in a semi-controlled greenhouse environment (Vincent, 1970). The clay pots were filled with fine grade (2-3 mm) sand substrate, covered with plastic wrap and sterilized by autoclaving for two hours twice. The seeds for sowing were surface sterilized by 96% ethanol (30s) and 2‰ HgCl2 (3 min), respectively and washed 6-8 times with sterile water. The seedling was performed by sterile pincers, five seeds per pot. The experiment was designed with eight inoculated and two controlled uninoculated treatments with three replications in a randomized complete block system. The seeds of each inoculated treatment were inoculated with 1 ml of suspension of single strain which were grown in yeast extract mannitol broth for 72 h at 280C. All the operations (inoculation and planting) were carried out aseptically in a laminar flow chamber. Inoculated plants were grown on sand with N-free Jensen nutrient solution. Uninoculated control plants were grown on sand with N-free (θ) and full N rate as KNO3 (0.05%, i.e. 500 mg l-1) (Nθ) Jensen nutrient solution (Vincent, 1970). The plants were watered according to need and with sterile Jensen nutrient solution (with or without N) at 15-days intervals. They were harvested in phase of the third trefoil of Nθ (six weeks). The number of nodules was recorded after uprooting the plants, followed by dipping the roots in water to remove sand without losing the nodules. Nodules were collected from the roots and counted. Their average number was expressed as number of nodules per plant. The size of nodule clusters was measured by ruler while the size of single nodules was determined by sieving successively trough screens with different openings (Fujihara et al., 2006). The cross-sections of nodules and differences in the colour of nodular tissue were used for preliminary fast screening effective and ineffective nodules. The ineffective nodules were white to light green inside, while the effective nodules were characteristically pinkish brown (Gwata et al., 2003).

The root and shoot portions of adzuki bean were separated and nodules' fresh weight, shoot and root were measured. Plants shoots, roots and nodules were dried in an oven at 700C to constant weight and the dry weight per plant was determined.

The percentage of shoot and root nitrogen was determinated by micro-Kjeldahl method and it was used to calculate total and fixed N (only for shoot) contents in mg per plant. All the data were statistically processed by the LSD and Duncan test using SPSS 10.0 computer program. Correlation analysis between parameters of nitrogen fixation was also conducted.

RESULTS

All the tested strains were able to nodulate adzuki bean. However, there were major differences in nodule number, position and the looks of nodules induced with different strains. Also, there were differences in habitus of plants. Plants inoculated with the strains of B. japonicum had dark green leaves and dark-beige, circular shape nodules (1-2 mm) in the crown of the root system, forming a cluster (0.5-1.0 cm), or being arranged in rows on the root hair. These were visual indicators of active nodules. The plants inoculated with the Knj isolate had light green leaves and dark-beige, circular shape nodules (1-2 mm) placed on the root hair, usually arranged in rows and in sporadic clusters. The interior nodules colour of B. japonicum strains and Knj was pinkish-brown, indicating effective nodules. The plants inoculated with strains of Bradyrhizobium spp. had light green leaves, single light-beige nodules (1 mm) placed on the root hair all over the initial third part of the root. White interior colour of these nodules indicated either poor effective, or ineffective nodules. The average number of nodules ranged from 21.1 to 31.4 for Knj and B. japonicum strains and from 6 to 7 per plant in Bradyrhizobium spp. strains. Similar to the average nodule number, Knj and B. japonicum strains stood out compared to Bradyrhizobium spp. strains in respect to the average nodule dry weight. The average nodule dry weight per plant ranged from 43.2 to 68.8 in strains B. japonicum and Knj and from 2.8 to 21.45 mg plant-1 in Bradyrhizobium spp. Maximum number of nodules (31.4 nodule plant-1) was induced by 526 strain, closely followed by Knj (28.8 nodule plant-1), both of them being significantly superior over all other treatments (Table 1). The highest nodule dry weight per plant was recorded with strain 532 (68.88 mg plant-1), which was significantly higher than the next highest nodule dry weight reached with 526 strain (62.17 mg plant-1). The strains of Bradyrhizobium spp. had very low nodule number as well as nodule dry weight per plant comparing to the strains of B. japonicum and Knj.

Dark green leaves and chlorotic plants with green-yellow leaves were observed in Nθ and θ controls, respectively.

The maximum shoot length of adzuki bean was recorded in the Nθ control, which was significantly superior over all other treatments. The strains 525 and 532 had the highest shoot length among inoculated plants, but without significant statistic differences. The adzuki plants inoculated with 542 strain recorded the maximum root length (31.06 cm); however, without significant differences compared to almost all other treatments. Strain 5388 showed the lowest value of both shoot and root length.

Average shoot dry weight (SDW), total and fixed N contents in plants were used for quantitative and qualitative SNF efficiency assessment (Table 2). Based on the average SDW, all the strains were classified into three significantly different declining active groups. Shoot dry weight values were in high significant positive correlation with shoot total N and fixed N content (Table 3). B. japonicum strains 525, 532, 542 and 526 of the first group were the most effective. Shoot dry weight, total and fixed N content ranged from 290-320, 4.64-6.25 and 3.81-5.71 mg plant-1, respectively.

Strain 525 showed the greatest SDW, while strain 542 had the largest amount of fixed and total N content. Symbiosis between adzuki bean plants and the Knj strain held the middle position (SDW, total and fixed N were 250, 3.17, 2.34 mg plant-1, respectively). The plants with low values of the investigated parameters were inoculated with Bradyrhizobium spp. strains; 2001, 2801 and 5388 belonging to the third group. In this group, SDW was between 180 and 210 mg plant-1, total N content 1.08-1.40 and fixed N content 0.32-0.63 mg plant-1.

No significant difference was found between the plants inoculated with the strains of the third group and θ control plants, which indicated SNF inactivity of the Bradyrhizobium spp. strains. A significant difference was found between the inoculated plants of the first and third groups. Compared to these two groups, the Knj isolate did not differ significantly for SDW, except strains 525, 2801 and 5388, but differed for the total and fixed N content.

The highest values of SDW were obtained in Nθ control with significant difference existing compared to all inoculated treatments. Symbiotic efficiency (%) is the ability of inoculated plant to fix N2 in relation to use full mineral N content (100%) of uninoculated control (Nθ). In symbiotic associations, strains of the first groups showed 65-71% SDW and 99-138% total N content of the Nθ control (Table 2). High symbiotic efficiency of adzuki bean in symbiosis with strains 525 and 542 were achieved because these strains showed 71% SDW and 138% total N content, respectively. It indicated that B. japonicum strains with high symbiotic efficiency were able to provide the plant with necessary N in process of N2 fixation.

Root dry weight and total N root content had similar trend like the same parameters of shoot, except in the case of Nθ control (Table 1). The maximum root dry weight was observed in adzuki bean inoculated with strains 542, 532 and 525 (180 mg plant-1), (I group in relation to SDW), which was significantly better than the values reached by Bradyrhizobium spp. strains (III group in relation to SDW). In contrast to shoot dry weight, where Nθ control showed significantly superior value over all other treatments, root dry weight of Nθ did not significantly differ from almost all other treatments. The highest total N content in root, as in the case of total N content in shoot was achieved by strain 542 (3.24 mg plant-1), followed by strains 525 and 532 (2.72, 2.59 mg plant-1, respectively).

DISCUSSION

There is a constant search for superior rhizobial strains, characterized by high rates of N2 fixation, high nodule efficiency and competitiveness, which would allow an increase in legume yields and N content (Hungria et al., 1996). The data reported by some authors indicated a need to measure the rhizobial population characteristics directly with the host plants of interest in order to obtain an accurate assessment of the need to inoculate (Thies et al., 1991). Bradyrhizobium spp. strains are known to nodulate specifically plants of the cross-inoculation Vigna group (cowpea group), while B. japonicum specifically nodulates Glycine max, belonging to another cross-inoculation group (Dénarié et al., 1992). In the present study, an attempt was made to test the nodulation and efficacy of B. japonicum, Bradyrhizobium spp. strains and Knj isolate specific to cowpea group on adzuki bean. All the tested strains were capable of nodulating adzuki bean, but there were different responses to the strains inoculation (Tables 1-3).

The strains of B. japonicum showed ability to establish high effective nodulation in adzuki bean according to parameters of effective symbiosis, both SDW and N content (total and fixed), which have also been applied elsewhere (Yadav et al., 1996; Mayz et al., 2003). As observed in previous studies (Deli et al., 1997), the shoot dry weight and total N content per plant among the B. japonicum strains mostly depended on the strain activity and these parameters were in high correlation (Table 3). The most active strains were 525 and 542 for these parameters. The strain 542 had significantly better quality of yield than the Nθ control, while strains 525, 532 and 526 with a quality similar to the Nθ clearly indicated the SNF efficiency of these strains. There were symbiotic associations with the potential of achieving higher shoot quality by N fixation than with using N from mineral fertilizers (Phillips and Teuber, 1985). Symbiosis between adzuki bean plants and the Knj strain held the middle position; therefore, Knj should be investigated in symbiosis with slightly more compatible adzuki bean genotypes.

Present results showed that the Bradyrhizobium spp. strains were ineffective in symbiosis with the investigated adzuki bean species, due to very low SDW values and total N content achieved by these plants, which was within the level of un-inoculated control without nitrogen indicating ineffective symbiosis. The rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group (Thies et al., 1991).

According to criteria of Gwata et al., (2003), additional confirmation that the plants inoculated with B. japonicum formed effective symbiosis in the sixth week of developement was the dark green colour of their leaves contrary to the light leaves colour of uninoculated plants without N as well as Bradyrhizobium spp. inoculated plants.

The interior pinkish brown colour and size of 1-2 mm found in the effective nodules of B. japonicum and Knj showed that their stage of development lasted for six weeks, similar to the data reported by other authors (Gwata et al., 2003; Fujihara et al., 2006). The inoculation with effective B. japonicum strains and Knj had a dramatic effect on the nodule number formed by these strains compared to ineffective Bradyrhizobium spp. B. japonicum strains induced at least three times higher and more number of nodules compared to Bradyrhizobium spp. strains. Consequently, the nodule dry weight per plant was significantly higher in B. japonicum inoculated plants. There was significant correlation between the nodule number per plant, nodule dry weight and shoot dry weight (Table 3).

However, despite such significant correlation between the nodulation and symbiotic effectiveness expressed by SDW, considering only effective strains of B. japonicum (due to the extremely poor nodulation of Bradyrhizobium spp. strains), there was no correlation between SDW and nodule number as well as SDW and nodule dry weight per plant. This was in accordance with previous results, which found that nodule number was not an appropriate trait for selection of the most effective N2 fixing rhizobium-legume association (Deli et al., 1997; Hefny et al., 2001).

As presented in Table 3, the values of root dry weight significantly correlated with total root N content, as well as with shoot dry weight of investigated strains (Hefny et al., 2001; Kuang et al., 2005). Root dry weight of plant inoculated with B. japonicum strains and Knj was similar to the Nθ control, but total N content in root of all B. japonicum strains, except strain 526, was significantly higher than those of Nθ control (Table 1), which was in agreement with previous findings (Hefny et al., 2001).

The present work on adzuki bean demonstrated a positive effect of B. japonicum strains on the nodulation and N2 fixation for this plant. The strains of B. japonicum showing high potential of N2 fixation in pot experiment could be used in further investigation for their efficiency under field condition for inoculation as microbiological N fertilizers. These results indicated that it would be possible to reduce the use of nitrogen mineral fertilization, or completely avoid it in adzuki bean production through inoculation with highly effective strains.

ACKNOWLEDGEMENTS

This study was supported by the Ministry of Science and Environmental Protection of Republic of Serbia, Project, No TR-006875B.

Received: December 06, 2006; Revised: August 24, 2007; Accepted: September 25, 2009.

  • Dénarié, J.; Debellé, F. and Rosenberg, C. (1992), Signaling and Host Range Variation in Nodulation. Annu. Rev. Microbiol, 46, 459-531.
  • Deli, D.; Lugi Z., and Jasmina, R. (1997), Determination of correlative relation ships between indicators of nitrogen fixation intensity for alfalfa, Medicago sativa L. Paper presented at 21th Meeting of the Fodder Crops and Amenity Grasses Section of Eucarpia, p. 11, 9-12 September, Kartause Ittingen, Switzerland.
  • Fujihara, S.; Terakado, J. and Nishibori, N. (2006), Accumulation of an aromatic amine, β-phenethylamine, in root nodules of ad zuki bean, Vigna angularis Plant Soil, 280, 229-237.
  • Gwata, E. T.; Wofford, S. D.; Boote, J. K. and Mushoriwa, H. (2003), Determination of effective nodulation in early juvenile soybean plants for genetic and biotechnology studies. Afr. J. Biotechnol., 2, 417-420.
  • Hefny, M.; Doliñski, R. and MaBek, W. (2001), Variation in symbiotic characters of alfalfa cultivars inoculated with Sinorhizobium meliloti strains. Biol. Fertil. Soils, 33, 435-437.
  • Hungria, M.; Nishi, C. Y. M.; Cohn, J. and Stacey, G. (1996), Comparison between parental and variant soybean Bradyrhizobium strains with regard to the production of lipo-chitin nodulation signals, early stages of root infection, nodule occupancy, and N2 fixation rates. Plant Soil, 186, 331-341.
  • Kimura, D. S.; Schmidtke, K.; Tajima, R.; Yoshida, K.; Nakashima, H. and Rauber, R. (2004), Seasonal N uptake and N fixation by common and adzuki bean at various spacing. Plant Soil, 258, 91-101.
  • Kuang, R. B.; Liao, H.; Yan, X. L. and Dong, Y. S. (2005), Phosphorus and nitrogen interactions in field-grown soybean as related to genetic attributes of root morphological and nodular traits. J. Inter. Plant. Biol., 47, 549-559.
  • Phillips, D. A. and Teuber, R. L. (1985), Genetic Improvement of Symbiotic Nitrogen Fixation in Legumes, In: Evans, H. J., Bottomley, P. J. and Newton, W. E. (Eds.) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht, pp. 11-17.
  • Robinson, R. G. (1983), Yield and composition of field bean and adzuki bean in response to irrigation, compost, and nitrogen. Agron. J., 75, 31-35.
  • Sato, S.; Hori, Y.; Yamate, J.; Saito, T.; Kurasaki, M. and Hatai, A. (2005), Protective effect of dietary adzuki bean (Vigna angularis) seed coats against renal interstitial fibrosis of rats induced by cisplatin. Nutrition, 21, 504-11.
  • Thies, J. E.; Bohlool, B. B. and Singleton, P. W. (1991), Subgroups of Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum. Appl. Environ. Microbiol., 57, 1540-1545.
  • Vincent, M. J. 1970. A Manual for the Practical Study of the Root Nodule Bacteria IBP Handbook no. 15. Blackwell Scientific Publications, Oxford.
  • Vlassak, K. M. and Vanderleyden, J. (1997), Factors influencing nodule occupancy by inoculant rhizobia. Crit. Rev. Plant Sci., 16, 163-223.
  • Yadav, A. S.; Rai, S., Upadliyay, K. K. S.; Sawhney, S. K. and Vashishat, R. K. (1996), Nitrogen fixing efficiency and rate of respiration of azide sensitive and resistant strains of Bradyrhizobium sp. (Vigna). In: Kahlon, R. S. (Ed.), Perspectives in Microbiology-1996, National Agricultural Technology Information Centre. India: Ludhiana. pp. 171-174.
  • Yee, E.; Kidwell, K. K.; Sillis, G. R. and Lumpkin, T. A. (1999), Diversity among selected Vigna angularis (Azuki) accessions on the basis of RAPD and AFLP markers. Crop Science, 39, 268-275.
  • Zong, X. X.; Kaga, A.; Tomooka, N.; Wang, X. W.; Han, O. K. and Vaughan, D. (2003): The genetic diversity of the Vigna angularis complex in Asia. Genome, 46, 647-658.
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    Author for correspondence:
  • Publication Dates

    • Publication in this collection
      12 May 2010
    • Date of issue
      Apr 2010

    History

    • Received
      06 Dec 2006
    • Reviewed
      24 Aug 2007
    • Accepted
      25 Sept 2009
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