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Agronomy Journal 92:880-886 (2000)
© 2000 American Society of Agronomy

RICE

Rhizobial Inoculation Influences Seedling Vigor and Yield of Rice

Jatish C. Biswasa, Jagdish K. Ladhaa, Frank B. Dazzob, Youssef G. Yannic and Barry G. Rolfed

a Soil and Water Sciences Div., Int. Rice Res. Inst. (IRRI), P.O. Box 3127, Makati Central Post Office, 1271 Makati City, Philippines
b Dep. of Microbiology, Michigan State Univ., East Lansing, MI 48824 USA
c Sakha Agric. Res. Stn., Kafr El-Sheikh, 33717 A. R. Egypt
d Plant-Microbe Interaction Group, Res. School of Biological Sciences, The Australian National Univ., Canberra, ACT 2601, Australia

j.k.ladha{at}cgiar.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 
Rice (Oryza sativa L.) is one of the world's most important crops. The present investigation was designed to assess the range of growth-promoting activities of various diazotrophic bacteria on rice seedling vigor, its carryover effect on straw and grain yield, and the persistence of an inoculant strain on rice roots under greenhouse conditions. Growth responses to inoculation exhibited bacterial strain–rice variety specificity that were either stimulatory or inhibitory. Growth responses included changes in rates of seedling emergence, radical elongation, height and dry matter, plumule length, cumulative leaf and root areas, and grain and straw yields. Most notable were the inoculation responses to Rhizobium leguminosarum bv. trifolii E11 and Rhizobium sp. IRBG74, which stimulated early rice growth resulting in a carryover effect of significantly increased grain and straw yields at maturity, even though their culturable populations on roots diminished to below detectable values at 60 d after planting. The test strains were positive for indole-3-acetic acid production in vitro, but only some reduced acetylene to ethylene in association with rice under laboratory growth conditions. These studies indicate that certain strains of nonphotosynthetic diazotrophs, including rhizobia, can promote growth and vigor of rice seedlings, and this benefit of early seedling development can carryover to significantly increased grain yield at maturity.

Abbreviations: ARA, acetylene reduction assay • BNF, biological nitrogen fixation • DAP, days after planting • DM, dry matter • GPA, growth-promoting activities • IAA, indole-3-acetic acid • PGPR, plant growth promoting rhizobacteria


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results
 Discussion
 REFERENCES
 
SEEDLING VIGOR is critical when competition for light, nutrients, air, and water becomes strong. Seedlings with a vigorous growth pattern can compete successfully under stress, influencing stand establishment and ultimately grain yield. The vigor parameters of a crop variety can be influenced by genetic manipulations that are time-consuming and costly, and cultural manipulations that can provide quicker, short-term boosts in crop yield by changing the physiological status of young plants that persists throughout their life cycle (Teng, 1990).

Cultural manipulations under field conditions can be achieved by delivery of a balanced fertilization, optimum water management, seed treatment, etc. Treatment of seeds with beneficial microbes can help to control disease incidence and severity (O'Sullivan and O'Gara, 1992), improve nutrient uptake efficiency (Bashan et al., 1990), and promote growth leading to enhanced yield (De Freitas and Germida, 1990).

The growth-promoting activities (GPA) of bacterial inoculants on crop plants may be manifested in several ways. For example, their production of iron-sequestering siderophores and antimicrobial compounds may hinder colonization of hosts by phytopathogens (Neilands and Leong, 1986), thereby suppressing the diseases they cause. Other mechanisms of GPA include the induction of host systemic disease resistance (Maurhofer et al., 1994), N2 fixation (Burton, 1976), solubilization of precipitated mineral nutrients (Subba Rao, 1982), and/or production of plant growth regulators (Tien et al., 1979; Bashan et al., 1990) that induce additional root hairs and/or lateral root formation (Tien et al., 1979), thereby enhancing the plant's ability to take up nutrients from soil and increase yield.

Rhizobial inoculation of legume seed is well studied, and exploitation of this beneficial N2-fixing root-nodule symbiosis represents a hallmark of successfully applied agricultural microbiology. However, much less information is available regarding the association and GPA of rhizobia with nonlegumes. In nature, rhizobia do associate with roots of nonlegumes without forming true nodules (Ladha et al., 1989; Yanni et al., 1997), but their populations decrease in number in the absence of legume-host plants (Ladha et al., 1989; Chabot et al., 1996b). Direct growth promotion of nonlegumes by rhizobia has also been reported (Hoflich et al., 1995; Chabot et al., 1996a; Noel et al., 1996; Yanni et al., 1997).

Recently, we found that R. leguminosarum bv. trifolii develops natural, intimate associations with rice roots in fields of the Egyptian Nile Delta where this cereal had been rotated with berseem clover since antiquity (Yanni et al., 1997). Some strains isolated from this association exhibit GPA on rice under microbiologically controlled gnotobiotic conditions, and inoculation trials indicate that they could increase both yield and agronomic fertilizer N-use efficiency under experimental field conditions (Yanni et al., 1997). The degree to which this association benefits rice growth varies with rice variety, cultural conditions, and inoculant strains. The mechanisms of growth promotion by rhizobia on nonlegumes that have been considered include production of phytohormones and/or phosphate-solubilizing activity (Abd-Alla, 1994; Chabot et al., 1996b), inhibition of fungal growth (Haque and Ghaffar, 1993; Nautiyal, 1997), and antagonism of the indigenous soil microflora (Schloter et al., 1997).

Further confirmation under nonsterile soil conditions is needed to exploit the potential benefits of rhizobial association with rice. Because seedling vigor is critical to overall crop performance, cultural manipulation of seedling growth by rhizobial inoculation would be a potentially useful technology for sustainable agriculture without compromising other natural resources. In this study, we have compared the GPA of various rhizobia with other cereal-associated diazotrophs on rice under greenhouse conditions. Rhizobial test strains were selected based on prior knowledge of their colonization and/or GPA on rice and to provide a wide host range. Other nonphotosynthetic diazotrophic plant growth-promoting rhizobacteria (PGPR) were included for comparison. We thoroughly evaluated various parameters of rice growth at the seedling and reproductive stages of development to gain more insight into their possible mechanisms of GPA on rice. We also investigated the survival of a marked rhizobial strain in association with rice roots in relation to their continuity of GPA in rice plants. Finally, we screened these test strains for in vitro production of the phytohormone, indole-3-acetic acid (IAA), and for N2-fixing activity in association with rice using the acetylene reduction assay (ARA).


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results
 Discussion
 REFERENCES
 
Bacterial Strains Used
Table 1 lists the strains used in this study, their plant hosts, and where they were isolated. For survival studies, Rhizobium spp. strain IRBG74 was tagged by conjugation with plasmid pFAJgusA21 containing a gusA reporter gene (Wilson et al., 1995). The IRBG74-gusA derivative was similar to its parent in growth rate on yeast mannitol agar and yeast mannitol broth at 30°C, and in nodulation frequency on the host, Sesbania cannabina.


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  		Table 1 Strains used in this study

 
Inocula Preparation
Strains E11, E12, IRBG74, Tal441, and JCB were grown in yeast mannitol broth; FS was grown in nutrient broth; and ORS571, IRBG271, USDA94, and Pal5 were grown in half-strength tryptone glucose yeast extract broth. Exponentially growing cells in shaken broth culture were collected by centrifugation for 10 min at 6°C and washed with sterile phosphate buffered saline (pH 7.0). Cell pellets were suspended in 1 mL of saline buffer, transferred to 1.5-mL Eppendorf tubes, and recentrifuged at 10000 x g for 1 min. The pelleted cells were suspended in an aqueous solution of xanthan gum (5 g kg-1) for seed inoculation.

Evaluation of Plant Growth-Promotion Responses to Inoculation
Laboratory and greenhouse experiments were performed in 1997 and 1998 at the International Rice Research Institute, Los Baños, Philippines. A completely randomized design with four replications was utilized unless otherwise indicated. Each experiment had at least two rows of border plants that were not measured. A lowland soil from IRRI's experimental farm, commonly known as Maahas clay (Aquandic Epiaquoll), was used. Its major properties were: pH (1:1 w/v water), 6.13; organic C, 12.6 g kg-1; Olsen P, 38 mg kg-1; exchangeable K, 1.4 cmolc kg-1 soil, electrical conductivity (1:1), 0.44 dS m-1; NH4–N, 26.5 mg kg-1; total N, 1.4 g kg-1; and cation exchange capacity, 20 cmolc kg-1. `Pankaj', a rice cultivar (125 d growth duration) from India, and IR74, a modern semidwarf variety (120 d growth duration) bred in the Philippines, were used in the first and second set of experiments, respectively.

One study was conducted in a greenhouse to determine if bacterial inoculation of seed affected seedling emergence. Similar-sized seeds of Pankaj rice were sorted, coated with inocula of strains E11 and IRBG74 in the proportion of 3.5 x 105 cells seed-1, and sown singly at equal depth in 1-cm2 sections of seeding trays filled with unfertilized air-dried Maahas clay soil. Soil in each treatment was moistened with an equal volume of tap water. Cumulative emergence rate was determined by counting emerged seedlings at various sampling times up to 157 h.

A second study was performed to evaluate parameters of seedling growth and development. Inoculated seeds (100 plate-1) were spread on 15-cm diameter petri dishes containing wet filter paper and germinated in the dark at 30°C. The plumule and radical lengths were measured at 120 h.

The third study examined effects of inoculation on various parameters of seedling vigor under greenhouse conditions. Pregerminated seeds (100 replication-1) of Pankaj were sown in plastic trays (35 cm by 28 cm by 11 cm) containing 6 kg air-dried soil without fertilizer amendments. Seedlings were allowed to grow up to 22 d. Additional studies with IR74 rice (designated subexperiments A, B, and C) were conducted to examine seedling vigor responses to inoculation with five selected strains (E11, IRBG74, IRBG271, ORS571, and JCB). Tap water was used to irrigate young seedlings.

At 22 d, seedling height was measured from the base of the emerged root to the top of the tallest leaf. Shoots were cut at the base of the emerged root, oven-dried at 70°C for 72 h, and weighed. Similarly, individual seedling dry weight was recorded. The relative surface areas of washed and air-dried (30 min at room temperature) roots were determined by the gravimetric method of Carley and Watson (1966), based on measurement of the amount of calcium nitrate absorbed from aqueous solution by the plant roots. Because rice root length, density, and thickness are important to nutrient uptake (Yoshida and Hasegawa, 1982; Barber, 1985), the lengths of coarse (>300 µm), medium-coarse (150–300 µm), and fine roots (50–150 µm) were measured using a Delta T-image analysis system (DIAS, Burwell, Cambridge, UK). Cleaned roots were treated with methyl violet (10 g L-1) for 5 min, washed thoroughly, cut into ~1 cm pieces, spread in petri dishes, and evaluated using a DIAS-root length module. Thresholds were set at 180 for coarse, medium, and fine roots; 150 for coarse and medium roots; and 120 for coarse roots only.

The leaf area (cm2) and leaf greenness (SPAD reading) of the upper two fully expanded leaves were measured by a LI-3000A portable area meter (LI-COR, Lincoln, NE) and by a SPAD-502 chlorophyll meter (Minolta, K. Arano & Co. Ltd., Tokyo, Japan), respectively. Total N contents in leaves and stems of IR74 rice seedlings were determined by a CHN analyzer (Jimenez and Ladha, 1993). Seedling N content was calculated as the product of N concentration and dry weight of seedlings. Since seeds for all treatments were of similar size (20 mg seed-1, 7 g N kg-1), the amount of seed-derived N present in plants would have been approximately equal in all treatments. This represented only a small proportion (<3.5%) of total N derived from soil N. Therefore, values were not corrected for individual seed N.

The second set of experiments was designed to evaluate the carryover effects of seedling vigor on yield parameters at maturity during the wet (May–August 1997) and dry (October–January 1997–1998) seasons. Representative, 22-d-old similar sized seedlings of IR74 were selected randomly from control and inoculated treatments (excluding border areas) and transplanted into pots containing 12 kg air-dried Maahas clay soil. Potted soil received N as urea, P as disodium phosphate, and K as potassium chloride at the rate of 90–26–33 kg ha-1, respectively. One-third N and full doses of P and K fertilizers were applied 1 d before transplanting. Potted soil was watered and puddled before planting. Supplemental N fertilizer was applied in two equal doses, the first at 30 d after planting, and the second at the panicle initiation stage.

Plant height was measured from the soil surface to the tallest panicle. Panicles per hill were counted and panicle length measured to 1 mm precision. Filled grains were separated from unfilled grains. Grain yield was expressed on the basis of 140 g water kg-1. Straw yields were recorded after oven drying at 70°C for 72 h.

Survival of a Rhizobium sp. IRBG74-gusA Strain in Association with Rice Roots
Survival of seed-inoculated tagged rhizobia on rice roots was investigated during the dry season under greenhouse conditions. Representative, 3-d-old similar sized seedlings of IR74 were selected randomly from control and inoculated treatments, and one seedling was transplanted into each pot containing 1 kg air-dried soil. At 0, 10, 20, 40, and 60 d after planting (DAP), duplicate plants were uprooted carefully and washed in tap water to remove adhering soil. Roots were cut at the stem base, blotted, weighed, and finally washed with 50 mL sterile phosphate buffered saline solution. Root samples were macerated in ice-cooled saline solution using a sterile mortar and pestle. Tenfold dilutions were plated in duplicate on yeast mannitol medium containing substrates for gusA (5-bromo-4-chloro-3-indoxyl-ß-D-glucuronic acid) at 75 µg mL-1 plus nalidixic acid at 30 µg mL-1 and tetracycline at 10 µg mL-1. Plates were incubated for 3 to 4 d at 30°C, and then gus expressing blue colonies with the same size, contour and texture as those of the parent strain were counted. The root-associated culturable populations of IRBG74-gusA were transformed to log10 and expressed as log (colony forming units + 1) to avoid zero values. At 20 DAP, 25 randomly selected blue colonies were inoculated onto 25 Sesbania cannabina plants (7 d old) within enclosed tubes for nodulation testing. After completion of the greenhouse experiment, plants and soil were sterilized by autoclaving at 120°C and 103.5 KPa for 1 h, as specified by Philippine Safety Regulations.

Production of IAA and Determination of Nitrogen-Fixing Activity
Test strains were grown in Tris-YMRT medium (mannitol, 10 g; CaCl2·2H2O, 0.15 g; MgSO4·7H2O, 0.25 g; TRIS [hydroxymethyl] amino methane, 1.21 g; casamino acids, 1.0 g; yeast extract, 0.2 g; water, 1000 mL; pH 6.8) for 12 to 72 h, depending on their growth rate for determination of IAA production in vitro. Cultures were centrifuged and IAA in the supernatant fluid was detected colorimetrically (Gordon and Weber, 1951). This test was repeated twice. To measure N2-fixing activity, plants were grown in N-free Fahraeus (Fahraeus, 1957) medium supplemented with 12 g agar L-1 for 31 d in a growth chamber. Three-day-old plants were inoculated with 106 cells and acetylene-reducing activity was determined at 14 and 28 d after inoculation by gas chromatography (Ladha et al., 1989). Acetylene-dependent ethylene production was compared between uninoculated and inoculated plants.

Data Analysis
Data were analyzed by analysis of variance and the treatment means were compared relative to control following Duncan's multiple range test (DMRT) or least significant difference (LSD) test. Unless indicated otherwise, differences were only considered when significant at P < 0.05.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
Discussion
 REFERENCES
 
Inoculation Responses of Rice during the Seedling Stage
Pankaj rice seedlings emerged faster during the first 100 h when inoculated with strain E11 or IRBG74 compared with the uninoculated control, although the final percentage of emerged seedlings equalized after 150 h (Fig. 1) . Plumule length at 120 h was increased due to inoculation with strains JCB or FS compared with the control, whereas it was reduced by inoculation with strains IRBG74, Tal441, USDA94, and Pal5 (Table 2) . Likewise, inoculation elicited a mixed response for seedling radical lengths, with longer radicals developing from seeds inoculated with strains E12, ORS571, and FS, and shorter radicals with strains E11, Tal441, USDA94, and Pal5 (Table 2).



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  		Fig. 1 Influence of seed inoculation with rhizobial strain IRGB74 or E11 on seedling emergence of Pankaj rice. The vertical bars indicate the standard errors of the means

 

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  		Table 2 Influence of seed bacterization on plumule and radical lengths of Pankaj rice.{dagger}

 
The types of growth responses to inoculation varied during vegetative growth stages for both rice varieties. Seedling leaves of Pankaj at 22 d after seeding had higher SPAD readings of leaf greenness when inoculated with strain FS only (Table 3) . The leaf areas were increased in plants inoculated with strain E11, E12, or Tal441, and were decreased in plants inoculated with strain IRBG74 (Table 3). Inoculation did not change seedling height relative to the uninoculated control, but plants inoculated with strain E11 or Tal441 were taller than plants inoculated with strain ORS571, IRBG271, USDA94, or Pal5. Root surface area did not differ significantly, although a 4 to 30% increase in root surface area was observed following inoculation with strains E11, E12, ORS571, JCB, Tal441, FS, and Pal5 (Table 3). In general, plants accumulated higher shoot dry matter (DM) when inoculated with strain E11, E12, or Tal441.


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  		Table 3 Influence of seed bacterization on seedling vigor parameters of Pankaj rice.{dagger}

 
Leaf area, seedling height, and seedling DM accumulation were consistently higher for IR74 rice inoculated with strain E11 or IRBG271 as compared with the uninoculated control (Table 4) . In addition, strain IRBG74 promoted accumulation of DM in this rice variety. Patterns of frequency distribution for DM accumulation based on a larger plant population are shown in Fig. 2 . Although about 45% of the uninoculated seedlings belonged to the <120 mg seedling-1 weight category, the major proportions (40–60%) of seedlings inoculated with rhizobia belonged to the 150 to 200 mg seedling-1 weight category (Fig. 2).


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  		Table 4 Influence of rhizobial inoculation on seedling parameters of IR74 rice (mean of three experiments).{dagger}

 


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  		Fig. 2 Influence of rhizobial inoculation on dry matter accumulation of IR74 rice seedlings at 22 d

 
The SPAD readings of IR74 rice leaves (average of three studies) increased in the IRBG271 treatment only, whereas the average root surface area was higher in the E11, IRBG74, and JCB treatments than in the uninoculated control (Table 4). All strains influenced the coarse root length compared with the control, but the total root lengths were different only with the IRBG74, IRBG271, and JCB treatments (Fig. 3) . The medium-coarse root lengths were greater with the IRBG271, IRBG74, and JCB treatments, and the fine root lengths were greater with the IRBG271 and JCB treatments only. The N content of IR74 rice seedlings was 22 to 52% higher in all inoculated treatments than in the uninoculated control (Table 4).



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  		Fig. 3 Influence of rhizobial inoculation on root lengths of IR74 rice seedlings. Diameters of coarse, medium-coarse and fine roots are >300 µm, 150 to 300 µm, and 50 to 150 µm, respectively

 
Inoculation Responses of IR74 Rice during the Reproductive Phase
Average grain yields of two seasons were higher when inoculated with strain E11, IRBG74, or IRBG271, and straw yields were increased when inoculated with strain E11 or IRBG74 (Table 5) . The main component contributing to the greater grain yield was the increased panicles hill-1. The total number of spikelets panicle-1 was higher in E11 and ORS571 treatments than the control. Plant height, panicle length, and 1000-grain weight of this rice variety were unaffected by inoculation.


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  		Table 5 Carryover effects of seed inoculation with rhizobial strains on yield and yield components of IR74 rice grown in pots under greenhouse conditions (average of wet and dry seasons)

 
Survival of Rhizobium sp. IRBG74 in Association with IR74 Rice
The results of viable plate counts using the appropriate selective and differential medium for the gusA reporter strain derivative of IRBG74 are shown in Fig. 4 . The population of IRBG74 declined from its initial size of 3.5 x 107 cells plant-1 on IR74 rice roots. Nodulation tests indicated that at least 75% of the blue colonies at the 20 DAP sampling could nodulate the host legume of the IRBG74 parent strain (data not shown). This inoculant strain could still be recovered from rice roots at 40 DAP but not at 60 DAP. An important implication of these results is that certain rhizobia can significantly promote rice growth even when their culturable population size declines. More studies are necessary to distinguish whether these survival kinetics reflect a true decline in its population size or its conversion to a viable but nonculturable state.



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  		Fig. 4 Surviving populations of the IRBG74-gusA strain on roots of IR74 rice following seed inoculation. The vertical bars indicate the standard errors of the means

 
The potted soil used in this experiment had a marginal background population of GusA+ bacteria with intrinsic resistance to tetracycline and naladixic acid that did not begin to increase (and hence obscure the results) until 40 DAP. Hence, the reisolation of rhizobia from plant-associated microbial communities can be facilitated when these root-nodule bacteria are marked with the gusA reporter gene and enumerated on plates containing appropriate antibiotics and substrate. However, nodulation tests of colony isolates on the appropriate legume host are still recommended for tracking marked strains of rhizobia on soil-grown rice because the background soil microflora includes other microorganisms intrinsically resistant to antibiotics and possessing gusA activity.

Assays for IAA Production and Nitrogen-Fixing Activity
The colorimetric test for IAA was positive with the culture supernatants of all test strains grown in vitro, indicating variable amounts present (1.6–2.8 µg mL-1). Tube cultures of IR74 rice inoculated with strains Tal441, IRBG271, FS, Pal5, and ORS were positive for ARA at 14 and 28 DAP as compared with uninoculated controls (data not shown). Under identical growth conditions, plants inoculated with E11, E12, IRBG74, JCB, USDA94 and the uninoculated control plants were negative in ARA tests of N2 fixation.


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
REFERENCES
 
Certain rhizobial strains exhibited significant GPA on rice seedlings under greenhouse conditions, including increased rates of germination, DM accumulation, root surface area, and N uptake. The high-quality seedlings produced because of inoculation had a carryover effect on subsequent plant growth, thus improving grain and straw yields in pot experiments, especially with inoculant strains E11 and IRBG74. Thus, earlier reports on the ability of certain rice-adapted Rhizobium leguminosarum bv. trifolii strains (Yanni et al., 1997) to promote the vegetative growth and grain yield of Egyptian rice varieties have been confirmed and extended to include other strains and rice varieties by this study.

The consistently higher DM accumulation by rice seedlings due to inoculation with rhizobial strains E11, IRBG74, and IRBG271 was conducive to increased early tiller production, which was reflected in higher grain yield. Also, the increased greenness (as a measure of chlorophyll content) of leaves and expanded root architecture resulting from inoculation would likely improve photosynthetic capacity and higher nutrient uptake efficiency (Bashan et al., 1990), respectively, which in turn would favor higher DM accumulation.

Production of IAA and biological N2 fixation (BNF) by the diazotrophic bacteria were examined as possible contributing factors of rice growth promotion. Tests for production of the auxin IAA were positive for all test strains, suggesting a potential mechanism whereby these bacteria may regulate plant growth. The levels and/or diversity of plant growth regulators produced by the different test strains during plant coculture may possibly contribute to the observed stimulatory and inhibitory growth responses of rice. This interpretation is in line with the well-known characteristic of certain phytohormones (e.g., auxin, ethylene) to elicit stimulatory plant growth responses within a narrow window of low concentration, outside of which the plant is either unresponsive or inhibited (Esashi, 1991; Jackson, 1991). Induction of longer roots with increased number of root hairs and root laterals is a growth response attributed to IAA production by other rhizobacteria, which improves their nutrient uptake efficiency (Okon, 1985).

The larger N content of seedlings resulting from inoculation may be due to increased uptake by a larger root surface area associated with additional root hairs and lateral root development and/or to BNF, either directly by the inoculant strains or indirectly by stimulating BNF activity of the associated rhizosphere community (Ladha et al., 1998). In this study, only strains FS, IRBG271, ORS571, Pal5, and Tal441 showed acetylene-reduction activity in direct association with rice roots under gnotobiotic conditions. A greenhouse study using the 15N isotope dilution technique with rice in potted soil inoculated with rhizobial strain E11, E12, IRBG74, IRBG271, USDA94, or Tal441 indicated that BNF did not make a consistent, significant contribution to the N content of the rice plant (Biswas et al., 2000). A field study in the Nile Delta using rice inoculated with strain E11 and analyzed by the {Delta}15N natural abundance technique also found no significant contribution of BNF to the N content of the harvested rice plants at maturity (Dazzo et al., 2000). In another study, strain IRBG74 marked with a transposon containing a gus-fusion with a rhizobial nifH promoter did not show nifH-gus expression in association with rice roots (Saxena et al., 2000). Considered collectively, these results plus earlier studies showing positive growth responses of rice to certain rhizobial strains even when excess combined N is present (Yanni et al., 1997; Prayitno et al. 1999) argue against BNF as an important mechanism of GPA to account for the significant N gains in this beneficial rhizobia–rice association (Biswas et al., 2000; Dazzo et al., 2000). Nevertheless, final assessment of the role of BNF in rhizobial promotion of rice growth will require the future testing of isogenic Pgp+ Fix+/Fix- strains.

In conclusion, our results and the earlier results of Yanni et al. (1997) indicate that selected rhizobial strains do promote the growth of rice in ways that could be harnessed to practical benefit for the farmer and are consistent with sustainable agricultural practices. More rhizobial strains should be screened through greenhouse and field studies to exploit their potential as PGPR that benefit rice productivity in more ways than BNF alone. Although the culturable population of inoculated rhizobia declines on rice roots, their longevity is nevertheless adequate to trigger plant growth stimulation and vigor of young seedlings that carryover to produce more productive plants, resulting in higher yields at maturity.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the German Agency for Technical Cooperation to IRRI and the U.S.–Egypt Science and Technology Joint Fund (BIO2-001-017-98) to Michigan State University and the Sakha Agricultural Research Station.

Received for publication January 20, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 




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