Effects of boron on nodule development and symbiotic nitrogen fixation in soybean plants

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1 Soil Science and Plant Nutrition ISSN: (Print) (Online) Journal homepage: Effects of boron on nodule development and symbiotic nitrogen fixation in soybean plants Masumi Yamagishi & Yukio Yamamoto To cite this article: Masumi Yamagishi & Yukio Yamamoto (1994) Effects of boron on nodule development and symbiotic nitrogen fixation in soybean plants, Soil Science and Plant Nutrition, 40:2, , DOI: / To link to this article: Published online: 04 Jan Submit your article to this journal Article views: 372 View related articles Citing articles: 33 View citing articles Full Terms & Conditions of access and use can be found at

2 Soil Sci. Plant Nutr., 40 (2), , Effects of Boron on Nodule Development and Symbiotic Nitrogen Fixation in Soybean Plants Masumi Yamagishi l and Yuki a Yamamoto 2 Department of Agronomy, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464 Japan Received May 19, 1993; accepted in revised form October 4, 1993 Effects of boron (B) on soybean growth, nodule development, and nitrogen fixation were studied. When soybean plants were grown in B-free medium for 35,40, or 72 d, B deficiency symptoms appeared. Nodules of B-deficient soybeans were damaged and showed low acetylene reduction activities. Soybean seed production was seriously depressed. Production of whole plant, nodules and pods was maximum at concentrations of p.g B L-I ( P.M). At concentrations of p.g B L -\ nitrogen fixation on a plant and nodule weight basis was markedly enhanced. Element absorption into soybean plants grown without boron and at 22, 44, 440, and 1,300 p.g B L -I was examined. At p.g B L -I, the total amount of K, Ca, and Mg absorbed into soybean plants was relatively high, although the concentrations of these elements on a dry weight basis were almost the same. Boron concentration in the whole soybean plant increased when the amount of B in the culture media increased. The B exogenously supplied accumulated in the leaves of soybean plants. At a B concentration of 1,300 p.g L -I, soybean plants were damaged, followed by a decrease in nitrogen fixation. To study the primary action of B, B was removed from the culture medium during the last 2 or 3 weeks of the 6 week culture. Symptoms associated with B removal appeared at the root tips. The dry weight of the whole plant and the total nitrogen fixation activity per plant were also reduced as compared with those of control plants. However, since the large-sized nodules detached from the basal portions of primary roots already contained a sufficient level of B for nitrogen fixation, this activity was not reduced by B removal for the last 2 weeks of culture. On the contrary, the young nodules which were still small, showed a decrease in nitrogen fixation activity after B removal. These results clearly indicate that B removal influenced more severely the developing nodules than the developed mature nodules and that B is important for the development and nitrogen fixation of nodules. Key Words: soybean. boron deficiency, boron nutrition, nitrogen fixation, nodule, Boron (B) is an essential element for all vascular plants (Tracheophyta), and some Present addresses: 1 Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichimachi, Ishikawa, 921 Japan; 2 Biology Laboratory, Nagoya Gakuin University, Seto, Japan.

3 266 M. Y AMAGISHI and Y. YAMAMOTO diatom species also require B (Lovatt 1985). A cyanobacterium, Anabaena sp. PCC 7119, does not always require B except under N 2 -fixing conditions (Mateo et al. 1986). There are many observations on the B requirements of leguminous plants which grow by symbiotic nitrogen fixation (soybean: Hodgkiss et al. 1942; soybean and other crops: Oertli and Roth 1969; fenugreek: Molgaard and Hardman 1980; peanuts, alfalfa, and soybean: Lombin and Bates 1982; alfalfa and white and red clovers: Sherrell 1983; alfalfa, red clover, and timothy: Gupta 1984). According to these reports, the B requirement varies from species to species; that is, alfalfa, fenugreek, and peanuts require high levels of B in nutrient solutions for suitable growth, whereas soybean grows with relatively low B levels. Molgaard and Hardman (I980) reported that the inoculated bacteria formed healthy and pink nodules on roots of fenugreek irrespective of B concentrations (either 30 or 330 j.lg B L -I) in the culture solution, and no damage was observed on the nodules, whereas symptoms of B deficiency appeared on the roots in a solution containing 30 j.lg B L -I. However, the other reports on B requirement of leguminous plants did not deal with the relationship between B nutrition and symbiotic nitrogen fixation. Information on B requirement for nitrogen fixation in legume nodules is still limited. In this paper we report the effects of B on growth, nutrient absorption, nodule development, and nitrogen fixation of soybean, and show that B deficiency reduces the development and nitrogen fixation of soybean nodules. MATERIALS AND METHODS Plant materials. Surface-sterilized seeds of soybean (Glycine max L. Merr., cv. T -202) were incubated at 30T overnight in a suspension of Bradyrhizobium japonicum strain 009, which had been isolated at the experimental farm at Nagoya University, and sown in vermiculite. The B. japonicum 009 was previously cultured during a week on slants of yeast-mannitol-agar medium, which contained 0.7 K 2 HP0 4, 0.3 KH 2 P0 4, 0.2 NaCl, 0.1 MgS0 4 7H 2 0, 0.1 CaS0 4 ' 2H 2 0, 10 mannitol, 10 agar, 1.0 yeast extract (ph 7.0) in g L -I, and was used as inoculum. At 7 to 10 d after sowing, seedlings of uniform size were selected and subjected to water culture. Four seedlings were grown in a 1/5,000-a wagner pot filled with 2.5 L of nitrogen-free culture media which contained KH 2 P0 4, KCl, MgS0 4 7H 2 0, CaCl 2 2H 2 0, 1.0 MnS0 4 H 2 0, 0.25 ZnS0 4 7H 2 0, 0.25 CuS0 4 ' 5H 2 0, 0.05 Na 2 Mo0 4 '2H 2 0, 72 Fe(III)-EDTA in mg L -I, supplemented with B as H 3 B0 3 at various concentrations (expression 44 j.lg B L -I means that 0.25 mg H 3 B0 3 L -I was added to the media). The ph of the media was adjusted to ph 6.5 with 0.1 M NaOH and the media were renewed every week. The solutions were prepared with distilled water and plastic culture pots and containers of stock solutions were used all the time to prevent B contamination. The cultivation was performed in a greenhouse under natural daylight conditions. Growing period and season in each experiment are described in the footnotes of the tables and figures. Nitrogen fixation activity. Nitrogen fixation activity was determined by the acetylene reduction assay, as described previously (Kawai and Yamamoto 1986). The whole root with nodules and the nodules detached from root were, respectively, placed in given flasks. The air in the flasks was replaced with 71 kpa Ar, 19 kpa O 2, and 10 kpa C 2 H 2 and the flasks were shaken at 30'C. One milliliter of the gas sample was withdrawn with a syringe and analyzed for ethylene content with a gas chromatograph equipped with Unibeads A column. After the assay, the fresh weight of the nodules was measured.

4 Effects of Boron on Soybean Nodules 267 Assay of B content. The soybean plants after sampling were washed with distilled water and air-dried at 70"C for 3 d. Dry weights of the plants were measured. The dried plant materials were crushed into a powder. Boron was extracted with 0.5 M HCl from the dried plant powder and the concentration was determined by a colorimetric method using curcumin-oxalic acid reagent (Yoshida and Yoshida 1965). Assays of K+, Ca 2 +, and Mg2+. The plant powder (200 mg) was SUbjected to wetdigestion as described by Kushizaki and Kiuchi (1976). Contents of Ca and Mg were determined by atomic absorption analyses, and K content was measured by flame photometry, usmg a Hitachi Atomic Absorption Photometer (Z-8000 type) (Kawai and Yamamoto 1986). RESULTS Effects of B concentration on growth and nitrogen fixation Complete absence ofb (0 j.lg B L -1) in the nutrient solution resulted in significantly low dry weights of the whole plant and nodules as compared with the cultures containing B (Fig. la, B, C). Symptoms of B deficiency appeared in the plants cultivated in the B-free culture media. Primary symptoms of B deficiency occurred at the root tips (Fig. 2) as generally recognized in B deficiency. Root elongation ceased and lateral roots appeared at the tip to 0l1.5fi A' 2.5 Ol f\ :; 1 d W h o J i e P J.! 'f 1.5 Whole Plant ] 2. 0 'tf ''01! 15r. Q..c: 1 0 r:d... e I o C'1.O 0.1.IV\. 0..:c I>t. 0.3 Nodules f 51 'OJ 0.0: Nodules C' 6 J r Boron Concentration Boron Concentration (p.g L') (p.g L') D o ,300 Boron concentration (p.g V)!;... '".g.s 15: [ 2 i 0 r E : 10 : '... - Jb] - 0!; Ei 5 "=u -< Boron concentration (llg V) Boron Concentration (p.g L") Fig. 1. Effects of B concentrations in culture media on dry weights of whole plant and nodules (A, B, and C) and on acetylene reduction by soybean nodules (ethylene production per plant) (0, E, and F). A and D: Soybean plants were sowed on Nov. 16, 1986, subjected to water culture from Nov. 24, and sampled on Dec. 12 (40-d-old plants). Band E: Sowing on Mar. 10, 1987, water culture on Mar. 18, sampling on Apr. 14 (35-d-old). C and F: Sowing on May I, 1987, water culture on May 9, sampling on Jun. 11 (72-d-old). Each point and vertical bar indicate mean and standard error (SE) of eight plants, respectively. :..!:! '" :s

5 268 M. Y AMAGISHI and Y. YAMAMOTO portion. Several days later, shoot growth stopped, the growing points became brown and then died (Fig. 3). The young leaves were yellow (Fig. 3). The nodules of the B-starved plants were small and their surface was brown (Fig. 2). Low acetylene reduction activities were detected in the plants grown without B (Fig. I D, E, F). Dry weights of the whole plant, nodules (Fig. la, B, C) and pods (Fig. 4) were relatively high at concentrations of J1g B L -1 ( J1 M). At concentrations of J1g B L -1, nitrogen fixation activity on a plant and nodule weight basis increased and reached maximum values (Figs. ID, E, F and 5). Figure 6A shows the B contents (J1g per plant or organ), and Fig. 6B indicates the B concentrations based on dry weight. Boron concentrations in the nodules of the plants grown without boron and with 44 J1g B L -1 were 12.8± 1.9 and 18.0± 1.3 j1.g g-l (mean±standard error), respectively. When the B concentra- Fig. 2. A: Boron-deficient soybean roots in late June (35-d-old). Primary symptoms of B deficiency appeared at root tips. Root elongation ceased and lateral roots appeared at the tip portion. Nodules of B-deficient soybean plants were small and their surfaces were brown in color. B: Control soybean roots. Fig. 3. Boron-deficient soybean shoots in late June (35-d-old). Shoot apices were brown in color and younger leaves displayed yellowmg.

6 Effects of Boron on Soybean Nodules 269 ril 'C CI 3 = CI....c CII.Q:j..... (:l o Boron concentration (Ilg L-') Fig. 4. Effects of B concentrations in the culture media on dry weight of pods. Each point and vertical bar indicate mean and SE of eight plants, respectively. The soybean plants were grown as indicated in Fig. I C and F ' ril :e CIl... -; 20 Il/I 'C 0 I: I: CJ "C = 10 CII CIl I: CIl :c... Q e 0 CIl o E; Boron concentration (Ilg L-') Fig. 5. Effects of B concentrations in the culture media on acetylene reduction by soybean nodules (ethylene production per fresh weight of nodules). Each point and vertical bar indicate mean and SE of eight plants, respectively. The soybean plants were grown as indicated in Fig. I Band E.... 'I: 150r-r-r-rl , Il/I CII.. A o.. o "i"' 100 -= Il/I 'a. CII C 50 o ,300 Boron concentration (fig L-') Boron concentration (fig L-') Fig. 6. Effects of B concentrations in the culture media on B contents in soybean plant. A: Boron contents of each organ and whole plant. B: Boron contents per dry weight of each organ and whole plant. Each point and vertical bar indicate mean and SE of eight plants, respectively. The soybean plants were grown as indicated in Fig. IA and D. tions in the culture media were varied, the B concentrations in leaves markedly changed (Fig. 6B). Boron exogenously supplied accumulated in large amounts in the leaves of the soybean plants as reported previously (Oertli and Roth 1969). At B concentrations of jj.g L-I, the total amount of K, Ca, and Mg accumulated in soybean plants was relatively high (Fig. 7 A), although the concentrations of these elements on a dry weight basis were almost the same (Fig. 7B). The suppression of growth and acetylene reduction appeared at B concentrations higher than 440 jj.g L -I in the culture media (Fig. la, D). At a B concentration of 1,300 jj.g L -I, marginal areas of soybean leaves became yellow with brown spots (Fig. 8), followed by a decrease in seed production (data not shown). In further experiments, 44 jj.g B L -I in the culture medium was used as the control. Effects of B removal for the last 2 or 3 weeks of the culture period When B was removed from the culture medium for the last 2 weeks of the growth period,

7 270 M. Y AMAGISHI and Y. YAMAMOTO 'a til -= , Boron concentration (pg L-I) 5 Q... -=!oj = Q U Fig. 7. Effects of B concentrations in the culture media on K, Ca, and Mg contents in soybean plant. A: K, Ca, and Mg contents per plant. B: K, Ca, and Mg concentrations based on dry weight of plant. Each point and vertical bar indicate mean and SE of eight plants, respectively. The soybean plants were grown as indicated in Fig. I A and D. o ,300 Boron concentration (pg L- 1 ) Fig. 8. Upper surface of leaves of soybean grown in the medium containing 1,300 Ilg B L -I, in late June (35-d-old). Marginal areas of leaves were yellow and a part of these areas turned brown. plant dry weight and nodule fresh weight of the plants grown in the medium where B had been removed (hereafter designated as - Boron medium), decreased by about 20 and 30%, respectively compared with those of the control (B cont.) plants (Fig. 9A, B), while the numbers of nodules did not change significantly (Fig. 9B). The acetylene reduction activity was also about 50% lower after B removal for 2 weeks (Fig. 9C). Boron deficiency symptoms on roots were not recognized at 1 week after B removal but appeared within 2 weeks. When relatively large nodules in the basal portion of the primary root were detached and used for the measurements of acetylene reduction activity, no significant differences were observed between the control plants and the plants grown in the - Boron medium for the last 2 weeks of the period of culture (Table 1), presumably because a sufficient level of B had already been incorporated in the relatively large-sized nodules in the - Boron medium (Table 1). All the nodules detached from the plants cultured in the complete (control) medium and the - Boron medium for the last 3 weeks of the culture period were, respectively, classified into three groups according to their sizes, and thereafter the acetylene reduction activity of each class of nodules was assayed (Table 2). Symptoms of B deficiency were

8 Effects of Boron on Soybean Nodules 271 : :c OJ: 'u : 1 a. a..!! '" "'" = o c... 2 o 1.5.c '" 0.5 B :E: '" Cj :; :; m] o n t oro : Boron ] Dll E ai:': :& Q '" E Q a a a 7 Duration (d) Duration (d) Duration (d) Fig. 9. Effect of B removal for the last 7 or 14 d of culture on dry weight of whole plant (A), fresh weight and number of soybean nodules (B), and acetylene reduction of nodules (C). Each point indicates mean of eight plants and bar is SE. Soybean plants were sowed on lui. 10, 1987 and transferred to water culture on lui. 20. Boron was removed from the solution (- Boron) or not (Cont.) on Aug. 7 (0 d) and soybeans were sampled on Aug. 7 (Od), Aug. 14 (7d), and Aug. 21 (14d). c.14 Table 1. Boron content and acetylene reduction of relatively large-sized nodules detached from soybean plants cultured in the complete medium or the medium where boron was removed for the last 2 weeks of culture. Culture condition Dry weight of whole plant (g) Fresh Boron Acetylene No. of weight of content reduction nodules the nodules in the activity used per plant nodules (Jlmol g-1 fresh (g) (Jlg g-i) nodules h- 1 ) Medium where boron was removed 1.97±0.12" 7.4±0.6' 0.28±0.05" 22.2±0.3 B 5.8±0.6" Complete medium 2.30± ± ± ± ± 1.0 Soybean plants were sowed on Aug. 28,1987. Water culture was started on Sep. 4. The plants were transferred to the complete medium or the medium where boron was removed for the last 2 weeks and sampled on Oct. 9 (42-d-old). Mean±standard error of eight plants. Table 2. Acetylene reduction in relation to the size of nodules detached from soybean plants cultured in the complete medium or the medium where boron was removed for the last 3 weeks. Dry weight Nodule Fresh weight Acetylene reduction Culture condition of whole diameter of nodules activity plant (g) (mm) (g) (Jlmol g-1 fresh nodules h- 1 ) >3 0.35±0.06" 2.48±0.3I a (64%b) Medium where boron was ± ± 1.23 (76%) removed 1.25±0.IO a < ±O.OO 1.40±0.70 (47%) Complete medium (control) 1.16±0.08 Total 0.50± ±0.38 (74%) >3 2-3 <2 Total 0.30± ± ± ± ± ± ± S±0.35 Soybean plants were sowed on Oct. 20, 1987, subjected to water culture from Oct. 30, transferred to the complete medium or the medium where boron was removed for the last 3 weeks and sampled on Dec. I (42-d-old). a Mean±standard error of eight plants. b Percent against control (complete medium).

9 272 M. Y AMAGISHI and Y. YAMAMOTO observed on roots of the soybean plants grown in the - Boron solution, while the dry weights of the whole plants were not different between the control plants and the - Boron plants. Boron removal decreased the acetylene reduction activity per g fresh weight of total nodules by 26%. In the class of nodules less than 2 mm in diameter, B removal decreased the acetylene reduction activity by 53%, and this proportion of the decrease was larger than that in the other two classes (36 and 24%). Acetylene reduction activities of large-sized nodules detached from the basal portion of the primary root were almost the same in the - Boron plants and the control plants (Table I). On the other hand, the activity of the large-sized nodules detached from all the portions of roots decreased by 36% by B removal (Table 2). These discrepancies were presumably due to the difference in the sampling portion of the nodules. In the latter case, some of the large-sized nodules formed between the middle and tip portions of roots had presumably matured in the late period of culture and these nodules supposedly had relatively low B contents, hence, showed a relatively low acetylene reduction activity. DISCUSSION Soybean requirement for B is lower than that of other leguminous plants, and soybean can grow at low levels of B in nutrient solutions (see introduction). Oertli and Roth (1969) reported that B toxicity symptoms appeared in soybean plants at B concentrations higher than 2 J,lg g-l, while deficiency symptoms of soybeans grown in 0 J,lg B g-l could not be detected in water culture. In the present experiments, however, B deficiency symptoms were apparently observed in soybean plants grown in the B-free culture medium kept in plastic pots as evidenced by the interruption of root tip growth and appearance oflateral roots (Fig. 2). In tomato, lateral root appearance is known to be the most typical symptom of B deficiency (Kouchi and Kumazawa 1975a). Boron deficiency symptoms further appeared on root nodules. The development (Fig. 2) and nitrogenase activity (Figs. 1 and 5) of the nodules were reduced. However, it remains to be determined whether B starvation directly affected the nodules, since the long-term culture in the B-free medium severely reduced the growth of the whole plant. Therefore, short-term B removal was carried out to identify the primary effect of B. The development of the nodules and nitrogenase activity were reduced by B removal for the last 2 weeks of the period of culture, followed by the decrease of the whole plant weight compared with the control (Boron cant.) plants (Fig. 9). On the other hand, the nitrogenase activity of the relatively large-sized nodules detached from the basal portion of the primary root was not different between the short-term B removal and the control plants (Table 1). The nodules in the basal portion already contained sufficient amounts of B before B removal, and this level did not decrease by B removal for 2 weeks (Table 1), because transport of B from the older to the younger plant parts did not occur (Dugger 1983). This fact suggests that the reduction of shoot growth by short-term B removal did not influence the nitrogen fixation activity in the large-sized nodules. The nitrogenase activity of the small-sized nodules was reduced by short-term B removal, whereas the dry weight of the whole plant did not change appreciably (Table 2). The small-sized nodules which were formed in the late period of the culture, appeared to contain low levels of B. These results indicate that B removal affected more severely the developing nodules than the developed mature nodules and that B is important for the development and nitrogen fixation of nodules.

10 Effects of Boron on Soybean Nodules 273 Kouchi and Kumazawa (l975b) and Kouchi (1977) reported that B nutntlon was associated with cell division and enlargement of the root tip cells. In B deficiency, it is likely that the reduction of cell division and enlargement of nodule cells had occurred and that thereafter nodule development had been suppressed. Most of B is present in membranes (Tanada 1983) and B is considered to be related to membrane functions in higher plants. Schon et al. (1990) reported that the addition ofb induced a hyperpolarization of the plasma membrane and increase of membrane permeability to potassium. Boron deficiency in a heterocystous cyanobacteri urn Anabaena sp. pee 7119 disturbed the function of the heterocyst which plays a role in the protection for O 2 diffusion and then depressed the nitrogenase activity (Mateo et al. 1986; Garcia-Gonzalez et al. 1988). If B deficiency in nodules affects the membrane of the nodule cells, it is possible to consider that the O 2 diffusion barrier which regulates free O 2 diffusion from outside into the infected zone of the nodules may be disrupted. As a result, subsequently, the nitrogenase activity of bacteroids may decrease. Acknowledgments. This study was partly supported by Grants-in-Aid for Scientific Research to Y. Yamamoto from the Japanese Ministry of Education, Science and Culture. REFERENCES Dugger, W.M. 1983: Boron in plant metabolism. In Encyclopedia of Plant Physiology, New Series Vol. 15B, Inorganic Mineral Nutrition, Ed. A. Pirson and M.H. Zimmermann, p , Springer-Verlag, Berlin, Heidelberg, New York, Tokyo Garcia-Gonzalez, M., Mateo, P., and Bonilla,!' 1988: Boron protection for 0, diffusion in heterocysts of Anabaena sp. PCC Plant Physiol., 87, Gupta, U.c. 1984: Boron nutrition of alfalfa, red clover, and timothy grown on podzol soils of eastern Canada. Soil Sci., 137, Hodgkiss, W.S., Hageman, R.H., and McHargue, J.S. 1942: The amount of boron absorbed by soybean plants and its effect on their growth. Plant Physiol., 17, Kawai, Y. and Yamamoto, Y. 1986: Increase in the formation and nitrogen fixation of soybean nodules by vesicular-arbuscular mycorrhiza. Plant Cell Physiol., 27, Kouchi, H. 1977: Rapid cessation of mitosis and elongation of root tip cells of Vicia jaba as affected by boron deficiency. Soil Sci. Plant Nutr., 23, Kouchi, H. and Kumazawa, K. 1975a: Anatomical responses of root tips to boron deficiency. I. Effects of boron deficiency on elongation of root tips and their morphological characteristics. Soil Sci. Plant Nutr., 21, Kouchi, H. and Kumazawa, K. 1975b: Anatomical responses of root tips to boron deficiency. II. Effect of boron deficiency on the cellular growth and development in root tips. Soil Sci. Plant Nutr., 21, Kushizaki, M. and Kiuchi, T. 1976: Wet combustion. In Assay Methods of Plant Constituents (Saibai Shokubutsu Bunseki Sokutei-ho), p. 61, Yokendo, Tokyo (in Japanese) Lombin, G.L. and Bates, T.E. 1982: Comparative responses of peanuts, alfalfa, and soybeans to varying rates of boron and manganese on two calcareous Ontario soils. Can. 1. Soil Sci., 62, 1-9 Lovatt, c.j. 1985: Evolution of xylem resulted in a requirement for boron in the apical meristems of vascular plants. New Phytol., 99, Mateo, P., Bonilla,!., Fernandez-Valiente, E., and Sanchez-Maeso, E. 1986: Essentiality of boron for dinitrogen fixation in Anabaena sp. PCC Plant Physiol., 81, Molgaard, P. and Hardman, R. 1980: Boron requirement and deficiency symptoms of fenugreek (Trigonella joenum-graecum) as shown in a water culture experiment with inoculation of Rhizobium. J. Agric. Sci., Camb., 94, Oertli, J.J. and Roth, J.A. 1969: Boron nutrition of sugar beet, cotton, and soybean. Agron. J., 61, Schon, M.K., Novacky, A., and Blevins, D.G. 1990: Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to K +. Plant Physiol., 93,

11 274 M. Y AMAGISHI and Y. YAMAMOTO Sherrell, e.g. 1983: Boron deficiency and response in white and red clovers and lucerne. NZ. 1. Agl'ic. Res., 26, Tanada, T. 1983: Localization of boron in membranes. l. Plant NutI'., 6, Yoshida, Y. and Yoshida, S. 1965: Rapid assay of boron in plants by extraction method. lpn. l. Soil Sci. Plant NutI'., 36, (in Japanese)