World Journal of Microbiology & Biotechnology 15: 363±367, 1999. Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Response of four rice varieties to Rhodobacter capsulatus at seedling stage M. Elbadry* and Kh. Elbanna Department of Agricultural Microbiology, Faculty of Agriculture, Fayoum Branch, Cairo University, 63514 Fayoum, Egypt *Author for correspondence: Fax: 002084334964, E-mail:
[email protected] Received in revised form 20 February 1999; accepted 1 March 1999
Keywords: Hydroponic culture, Rhodobacter capsulatus, rice (Oryza sativa L.)
Abstract The phototrophic purple nonsulphur bacterium (PPNSB) Rhodobacter capsulatus was used to inoculate seedlings of four rice varieties Giza 159, Giza 171, Giza 176 and Giza 181, grown in hydroponic culture with or without nitrogen. After three weeks the seedling growth parameters were measured. Inoculation with R. capsulatus enhanced seedling growth of all rice varieties tested. The response to inoculation as compared to control plants (no nitrogen, no R. capsulatus) were 52% to 75% for shoot height, 47% to 100% for aerial part dry weight, 45% to 78% for aerial part N content, ÿ37% to ÿ 9% for maximum root length, ÿ4% to 8% for root system dry weight and 50% to 62% for root N content. Introduction
Microorganism
It is well established that many soil microorganisms promote plant growth due to nitrogen feeding, improving plant nutrition, changing nutrients in an available form or to production of plant growth-promoting substance (Fages 1994). Kobayashi et al. (1967) suggested a possible growth-promoting role of phototrophic purple nonsulphur bacteria (PPNSB) in rice ®elds. Maudinas et al. (1981) showed that in the absence of combined nitrogen, but in the presence of the diazotrophs Azotobacter vinelandii and Rhodopseudomonas capsulata (now Rhodobacter capsulatus) in a liquid medium, rice plants can bene®t nitrogen for germination up to ear stage. However, so far, other investigators have not con®rmed this result. In the present work, we aimed to study the response of sterile pregerminated seeds of four rice varieties to inoculation with a PPNSB, Rhodobacter capsulatus during growth for 20 days in hydroponic cultures involving complete as well as nitrogen-de®cient sterile nutrient solution.
A strain of the anoxygenic phototrophic purple nonsulfur bacterium Rhodobacter capsulatus DSM 155 was used in the inoculation experiments. Rice nutrient solution (Heulin et al. 1987) Solution A. (g lÿ1 ): ZnSO4 7H2 O, 0.42; MnSO4 H2 O, 1.30; Na2 MoO4 2H2 O, 0.75; H3 BO3 , 2.8; CuSO4 7H2 O, 0.026; CoSO4 7H2 O, 0.07. Solution B. (g lÿ1 ): MgSO4 7H2 O, 2.00; CaCl2 2H2 O, 2.00; FeSO4 7H2 O, 0.44; EDTA, 0.40; Solution A, 20 ml. Solution C. (g lÿ1 ): K2 HPO4 , 90; KH2 PO4 , 60. Final nutrient solution: Solution B, 50 ml; Solution C, 15 ml; distilled water, 1000 ml. For N-treatments, the ®nal nutrient solution was supplemented with 40 ppm nitrogen as NH4 NO3 . Seedling growth unit
Materials and Methods Rice seeds Rice (Oryza sativa L.) seeds of dierent four varieties, Giza 159 (Japonica hybrid); Giza 171 (Japonica hybrid); Giza 176 (Indica/Japonica hybrid) and Giza 181 (Indica hybrid) were kindly supplied by the Institute of Rice Research, Sakha, Egypt.
The seedling growth unit used in the laboratory inoculation experiment is made of two parts (Figure 1). The upper part (A) is a plastic cup of 7 cm diameter and 9.2 cm height with a pored bottom. This part supports the germinating seeds and the aerial parts of the rice seedling. The lower part (B) is a 650 ml glass vessel, with a mouth larger than the base of the plastic cup (part A). At the start of the experiment, the two parts of the unit were sterilized separately. The glass vessels containing
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Figure 1. Seeding growth unit.
the rice nutrient solution were autoclaved at 121 C for 20 min, and the plastic cups were sterilized by ethanol. Thereafter, the plastic cup was tightly placed over the mouth of the glass vessel, so that the solution level reached the rice plants continuously. The germinated rice seeds were transferred aseptically to the upper part (A), one seed for each hole. Preparation of inoculum Bacterial inocula were prepared immediately before inoculation. R. capsulatus DSM 155 was grown photosynthetically on Malik's medium (1983). The cells were collected at the exponential growth phase by centrifugation at 10,000 rev/min for 10 min and the cell pellets were washed twice with sterilized distilled water. The cells were resuspended in a medium having the same composition as the growth medium except that NH4 Cl was omitted. The cultures were grown in completely ®lled glass bottles of 1000-ml capacity and incubated as above by degassing and ¯ushing with nitrogen. The cells were centrifuged at the logarithmic growth phase and the cell pellets were washed twice with sterilized distilled water and then resuspended in phosphate buer 0.05 M, pH = 7. Hereafter these starved bacterial cells will be referred to as ``inoculum''. The inoculum contained about 2:2 108 cells mlÿ1 . A sterile buer solution of the same volume was applied to the uninoculated treatments. Plant experiment In this experiment, four dierent rice varieties were used: G 159, G 171, G 176 and G 181. Rice seeds of approximately similar size were surface sterilized with a 0.1% mercuric chloride solution for 1 min; followed by washing thoroughly with several changes of sterile distilled water (Yoshida et al. 1976) and allowed to ger-
M. Elbadry and Kh. Elbanna minate on nutrient agar in Petri dishes for 3 days at 30 C in the dark. Ten contaminant-free uniformly germinated seeds were aseptically transferred to sterilized growth assemblies (Figure 1) containing 600 ml of the sterile nutrient solution with or without nitrogen. Nutrient solutions were either inoculated immediately with 60 ml R. capsulatus inoculum to yield approximately 2:2 107 cells mlÿ1 of nutrient solution or received the same volume from the sterile buer solution. The ®nal volume of the solutions in the growth assembly was sucient to cover the rice seeds. The experiment comprised four treatments for each rice variety: (1) nutrient solution, (2) nitrogen-free nutrient solution, (3) inoculated nutrient solution, and (4) inoculated nitrogen-free nutrient solution. Three replicate assemblies were used for each treatment. The plants were grown in a cabinet under natural light conditions at 29
2 C at day and 20
2 C at night. The water lost through evaporation and transpiration was replaced by sterilized distilled water. Measurements of the growth parameters of rice seedlings were made on days 3, 6, 9, 13, and at day 20 after emergence for shoot height, and maximum root length. At the end of the 20-day experimental period, the plants were removed gently from the assemblies and washed in distilled water to remove all the visible bacterial cells. The shoot and root portions were separated, and the growth measurements were taken. All the ten plants in each assembly were taken together for measurements of fresh and dry weight (DW), and all the plants in each treatment were pooled for nitrogen determination by the micro-Kjeldahl technique. Statistical analysis Data obtained from dierent varieties and fertilization treatments were subjected to analysis of variance. Multiple mean comparison was made by Duncan multiple range test. All analyses were conducted using the general linear model procedure of SAS (SAS Institute 1988). Results and Discussion The hydroponic culture technique was used in this study to avoid the extreme complexity of plant-bacteria interaction in soil. Among the PPNSB, R. capsulatus was used in the present work to inoculate rice plants. Several bacterial properties were considered in choosing R. capsulatus as the inoculum. For example, the N2 -®xing activity of all the known species of the family Rhodospirillaceae (PPNSB), has been investigated by Madigan et al. (1984). They found that the tested R. capsulatus strains were more tolerant to O2 while ®xing N2 chemotrophically and showed the highest nitrogenase activities as compared to other members of this family. Also, rates of 15 N2 -®xation in the dark have been reported to range from 5% of rates under illumination in Rhodospirillum rubrum (Kamen & Gest 1949) to a
R. capsulatus and rice seedlings
Figure 2. Figure 3. Figure 4. Figure 5.
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Eect of R. capsulatus on shoot height and root length of rice seedlings (cultivar Giza 159). * The dierence is signi®cant at 5% level. Eect of R. capsulatus on shoot height and root length of rice seedlings (cultivar Giza 171). * The dierence is signi®cant at 5% level. Eect of R. capsulatus on shoot height and root length of rice seedlings (cultivar Giza 176). * The dierence is signi®cant at 5% level. Eect of R. capsulatus on shoot height and root length of rice seedlings (cultivar Giza 181). * The dierence is signi®cant at 5% level.
high value of 40% in R. capsulatus (Kobayashi & Haque 1971). The results of the eects of R. capsulatus inoculation on rice seedling development of four rice varieties are presented graphically in Figures 2±5. Regardless of rice variety, bacterial inoculation caused a signi®cant increase, over the corresponding control (U±N), in shoot height of rice seedlings at all the growth stages studied. However, it was found that the stimulation eect increased steadily during the time course of growth. The explanation may be that during the ®rst few days of
seedling growth, it was still dependent on seed reserves for nutrient source, or/and that the amount of the nitrogen ®xed by R. capsulatus was still low due to the small amount of exudates released from the very young rice seedling roots (Table 1). In contrast to shoots (Table 1), roots of the inoculated rice seedlings (I±N) were shorter as compared to those of the corresponding control seedlings (U±N), but the dierences in maximum root lengths were not always signi®cant, particularly at the end of the 20 day experimental period. Murty & Ladha (1988) showed that
0.43c 0.71b 0.75a 0.76a 10.4ab 11.2a 9.2b 10.1ab 11.2a 7.1b 5.7c 4.7c 0.42d 0.68c 0.71b 0.75a 11.5a 11.3a 8.7b 9.1b
* Nutrient solution was inoculated with 10% R. capsulatus culture (2.2 ´ 108 ml)1); U= uninoculated, I = inoculated. ** N-fertilization with 40 mg NL)1 as (NH4)2 SO4; )N = No nitrogen, +N = Nitrogen supplemented plants. (1) Means in a column not followed by a common letter are signi®cantly dierent at 5% level.
10.7a 9.4a 8.9a 6.2b 0.46d 0.69c 0.72b 0.82a 9.6a 9.8a 7.9b 7.6b 11.6a 7.60b 6.0c 4.5c 0.44b 0.71a 0.71a 0.73a 9.9a 9.5a 9.3a 8.8a 10.6a 9.6ab 7.9bc 6.4c )N )N +N +N 2. Root length U I U I
5.6c 9.9b 13.6a 13.6a 8.6c 13.3b 15.6a 15.9a 0.36c 0.64b 0.63b 0.76a 6.4c 10.2b 15.6a 14.4a 11.4c 19.9b 22.9a 24.8a 0.42d 0.61c 0.58b 0.72a 5.4c 10.8b 13.1a 13.2a 14.9b 23.9a 22.9a 24.6a 0.35d 0.58c 0.61b 0.73a 7.3c 10.7b 13.2a 13.4a 15.10c(1) 23.00b 23.60ab 25.30a )N )N +N +N U I U I
1. Shoot height
DW (mg plant)1) Height (cm) Height (cm)
DW (mg plant)1)
N(%)
Height (cm)
DW (mg plant)1)
N(%)
Height (cm)
DW (mg plant)1)
N(%)
G181 G176 G171 G159 Inoculation*
Nitrogen Fertilization**
Cultivars Treatment
Table 1. Eect of Rhodobacter capsulatus inoculation and/or nitrogen fertilization on shoot height, root length, dry weight and N% of rice cultivars grown in hydroponic culture.
0.37c 0.62b 0.61b 0.74a
M. Elbadry and Kh. Elbanna N(%)
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Azospirillum inoculation of rice under hydroponic conditions signi®cantly reduced the root length of rice in agreement with our results. In the present work, it was observed that when root elongation was decreased, an increased number of new roots were initiated from the crown. Barbieri et al. (1986) found that inoculation of wheat with Azospirillum brasilense caused increased production of lateral roots and decreased lengths of main roots, while Tien et al. (1979) found similar eects of A. brasilense and plant hormones on lateral root production and main root elongation by pearl millet. As to the number of leaves per rice seedling, it was found to increase in all stages except at 3 days. In the case of the nitrogen-supplemented treatments, inoculation also generally increased both shoot height and leaf number, and decreased maximum root length of rice seedlings. However, the eects of inoculation in the presence of nitrogen were considerably less than those found in the nitrogen-free treatments. Results clearly indicate that inoculation with R. capsulatus invariably improved seedling growth, as indicated by increase in dry weight (DW) of the aerial part and shoot height compared to the control. Inoculation with R. capsulatus caused a marked increase in the nitrogen percentage in roots of seedlings in all the rice varieties tested. Similar eects resulted from N addition (Table 1). The pronounced increase in seedling, dry weight, nitrogen percentage and consequently nitrogen content suggest that R. capsulatus could ®x atmospheric nitrogen and the rice plants could bene®t from the ®xed nitrogen. These results clearly indicate that inoculation with R. capsulatus improved growth of seedlings of all the rice varieties included in this study, although there were variations between rice cultivars. This result is in harmony with the ®ndings of others (Lee et al. 1977; Ladha et al. 1986). Rice seedlings inoculated with R. capsulatus or/and supplemented with combined nitrogen were healthy, vigorous and taller with a well developed dense root system and green leaves as compared to the slow and poor growth of the untreated seedlings which suered from yellowing, a typical symptom of nitrogen de®ciency. The R. capsulatus cells multiplied and formed a thin red coloured layer on the root surfaces of the inoculated rice seedlings. This indicates that the R. capsulatus cells may be chemotactic to some rice root exudates (e.g. organic acids) which can be used as a carbon source for their growth. Root exudates of a cultivated rice variety have been investigated by Waschuetza et al. (1992). The main root exudates were the sugars ± glucose and ribose; the organic acids malate, acetate and pyruvate; and the amino acids serine, glycine, alanine and aspartic acid. Inoculation with R. capsulatus also caused signi®cant increases in nitrogen percentage in the aerial parts of rice seedlings. The increases were approximately similar to that caused by nitrogen addition (Table 1).
R. capsulatus and rice seedlings Inoculation (I ± N) and N addition (U+N) resulted in a decrease in the maximum root length of seedlings of all the rice varieties tested as compared to untreated plants (U ± N). Inoculation generally gave seedlings with signi®cantly longer roots relative to those of the N-supplemented seedlings. Concerning the dry weight of root system, the eects of inoculation were not so pronounced either in the absence or the presence of treatments (U+N). Inoculation treatments (I ± N) generally showed a signi®cant increase in the dry weight of root systems. However, no signi®cant dierences were found between (U+N) and (I+N) treatments. It becomes evident that the observed N gains and vegetative growth enhancement of the inoculated rice seedlings are primarily due to N2 -®xation and that inoculation gives bene®ts to rice seedling growth almost commensurate with the addition of 40 ppm of nitrogen. The promotion of satisfactory seedling development is an important stage in crop development, essential for achieving optimal populations, and in turn, maximum yield. Therefore, the results obtained have been highly encouraging and provide good grounds for conducting further trials. References Barbieri, P., Zanelli, T., Galli, E. & Zanetti, G. 1986 Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen ®xation and indole-3-acetic acid production. FEMS Microbiology Letters 36, 87±90. Fages, J. 1994 Azospirillum inoculants and ®eld experiments. In Azospirillum/Plant Association, ed. Okon, Y., pp. 87±109. CRC Press. ISBN 0-8493-4925-7. Heulin, T., Guckert, A. & Balandreau, J. 1987 Stimulation of root exudation of rice seedlings by Azospirillum strains: Carbon budgets under gnotobiotic conditions. Biological Fertilizers and Soil 4, 9±14.
367 Kamen, M.D. & Gest, H. 1949 Evidence for a nitrogenase system in the photosynthetic bacterium Rhodospirillum rubrum. Science 109, 560. Kobayashi, M. & Haque, M.Z. 1971 Contribution to nitrogen ®xation and soil fertility by photosynthetic bacteria. Plant and Soil (Special volume) 443±456. Kobayashi, M., Takahashi, E. & Kawaguchi, K. 1967 Distribution of nitrogen-®xing microorganisms in paddy soils of Southeast Asia. Soil Science 104, 113±118. Ladha, J.K., Tirol, A.C., Daroy, M.L.G., Caldo, G., Ventura, W. & Watanabe, I. 1986 Plant-associated N2 ®xation (C2 H2 -reduction) by ®ve rice varieties, and relationship with plant growth characters as aected by straw incorporation. Soil Science and Plant Nutrition 32, 91±106. Lee, K.K., Castro, T. & Yoshida, T. 1977 Nitrogen ®xation throughout growth and varietal dierences in nitrogen ®xation by the rhizosphere of rice planted in pots. Plant and Soil 48, 613±619. Madigan, M., Cox, S.S. & Stegeman, R.A. 1984 Nitrogen ®xation and nitrogenase activities in members of the family Rhodospirillaceae. Journal of Bacteriology 157, 73±78. Malik, K.A. 1983 A modi®ed method for the cultivation of phototrophic bacteria. Journal of Microbological method 1, 343±352. Moudinas, B., Chemardin, M., Yovanovitch, E. & Gadal, P. 1981 Genotobiotic culture of rice plants up to ear stage in the absence of combined nitrogen source but in the presence of free living nitrogen ®xing bacteria Azotobacter vinelandii and Rhodopseudomonas capsulata. Plant and soil 60, 85±97. Murty, M.G. & Ladha, J.K. 1988 In¯uence of Azospirillum inoculation on the mineral uptake and growth of rice under hydroponic conditions. Plant and Soil 108, 281±285. SAS Institute 1988 SAS/Stat user's guide. Release 6, 3rd edn, Cary, NC: SAS Institute Inc., USA. Tien, T.M., Gaskins, M.H. & Hubbell, D.H. 1979 Plant growth substances produced by Azospirillum brasilense and their eect on the growth of pearl millet (Pennisetum americanum L.). Applied and Environmental Microbiology 37, 1016±1024. Waschuetza, S., Hofmann, N., Niemann, E.G. & Ferdrik, I. 1992 Investigation on root exudates of Korean rice. Symbiosis 13, 181± 189. Yoshida, S., Forno, D.A., Cock, J.H. & Gomez, K.A. 1976 Laboratory Manual for Physiological Studies of Rice. IRRI 3rd edn, Philippines.
