Plant and Soil 218: 273284, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

273

Repeated benecial effects of rice inoculation with a strain of Burkholderia vietnamiensis on early and late yield components in low fertility sulphate acid soils of Vietnam
V. Trn Van1 , O. Berge2 , S. Ng K3 , J. Balandreau1 and T. Heulin2
Microbienne du Sol, UMR 5557 CNRS-Universit e Lyon I, 43 Bd du 11 Novembre, 69622 Villeurbanne ere (LEMIR), cedex, France; 2 CEA/Cadarache-DSV-DEVM, Laboratoire dEcologie Microbienne de la Rhizosph` edologie BioloUMR 163 CEA-CNRS 13108 St Paul lez Durance cedex, France; 1,2 Previous address: Centre de P gique, UPR 6831 du CNRS associ ee a ` lUniversit e H. Poincar e Nancy I, BP 5 54501 Vanduvre l` es Nancy, Cedex, France and 3 Institute of Tropical Biology, Vietnam National Center for Natural Science and Technology, 1 Mac Dinh Chi Street, H Chi Minh City, Vietnam
Received 5 July 1999. Accepted in revised form 16 November 1999
1 Ecologie

Key words: proteo-bacteria, Burkholderia vietnamiensis, inoculation, lowland rice, nitrogen nutrition, PGPR effect, acid sulphate soil, yield

Abstract TVV75, a strain of Burkholderia vietnamiensis, was isolated from an acid sulphate soil of south Vietnam, and selected for its high in vitro nitrogen xation potential. This plant growth-promoting rhizobacterium (PGPR) had been used in a previously reported pot experiment. It was used in two new pot experiments and four eld experiments to inoculate lowland rice at sowing and at transplanting, in three different South Vietnam acid sulphate soils. We rst studied the effect of inoculation during early plant growth in nurseries. Seedlings were then transplanted both to eld and pots. Treatments included two levels of inoculation (inoculated vs uninoculated) and three levels of N fertilizer (0, recommended rate and half this rate), in a randomized block design with six replicates. In all four experiments nitrogen appeared to be the limiting factor for yield. Inoculation had already had a strong benecial effect at the transplanting stage (day 24), as measured by shoot weight (+33%) root weight (+57%), and leaf surface (+30% at day 14). Final results indicated that inoculation of rice with B. vietnamiensis TVV75 signicantly increased several yield components, resulting in a nal 13 to 22% increase in grain yield. A late yield component, 1,000 grain weight, was signicantly increased by inoculation, but not by nitrogen fertilizers, in all pot and eld experiments, indicating a long-lasting effect of the inoculated bacteria. It was possible to evaluate the nitrogen fertilizer equivalent of inoculation (NFEI): at the medium rate of N fertilizer, inoculation ensured a yield equivalent to that obtained in the uninoculated control with 25 to 30 kg more nitrogen fertilizer. Comparison of the local cost of NFEI kg N-fertilizer and the cost of inoculation would help in making the decision to inoculate.

Introduction In Vietnam, most aspects of rural social and economic life are centered around rice production. It is grown on 6.3 million ha (statistical publishing house, Hni, 1986 and 1992). In 1992, the 24.5 million T rice yield accounted for 43% of the gross value of agricultural products. This production needs to increase to match the demand of the increasing population. In Vietnam,

2.6 million ha are acidic soils, of which 1.7 million ha are located in the Mekong rice basin. Chemical constraints for crop production on acidic soils can be overcome to some extent by the addition of lime and fertilizers. Nevertheless, when rice is grown in these areas, plant growth and development are impaired and nal grain yield is lower. For many farmers, the use of pesticides and chemical amendments is too costly, especially nitrogen which is the most frequent lim-

274 iting factor of rice production. This is all the more so as up to 5070% applied N can be lost through nitrication-denitrication. To overcome these limitations it is possible to use selected cultivars and to organize the timing of N application better to match the plant demand, or the efciency of the plant use of applied N. Another potentially attractive practice is seed inoculation with bacteria, which selectively decrease N needs through nitrogen xation and other plant growth promotion mechanisms. In a previously published study (Trn Van et al., 1996) of dominant nitrogen-xing bacteria associated with rice grown in a phytotron on a Vietnamese acid sulphate soil, N2 xing bacteria were counted and isolated using the "spermosphere model" technique (Thomas-Bauzon et al., 1982). Under these conditions N2 xers represented 40% of total cultivable bacteria in the rhizosphere soil sample studied, and in addition to enterobacteriaceae, predominant taxa were Azospirillum lipoferum, A. brasilense and Burkholderia, the latter being a new species, subsequently described as B. vietnamiensis (Gillis et al., 1995). Among the isolates, strain TVV75 of B. vietnamiensis was the most efcient in reducing acetylene, could inhibit phytopathogenic fungi (Trn Van, 1994a), and produced a new and efcient siderophore (Meyer et al., 1995). This strain was subsequently used in a pot assay, as a seed inoculant on an acid sulphate soil receiving different rates of N fertilizer. The results of this pot experiment have already been published: inoculation resulted in a nal 20% yield increase through positive effects on most yield components (Trn Van et al., 1994b). We report here results obtained in four eld assays, two of them being paralleled by new pot assays.
Table 1. Physical and chemical characteristics of the three soils (upper 0-16 cm horizon) Soils characteristics Hoc Mn Nh` a B` e B` inh Chanh (summer) (autumn) (autumn) pH (H2 O) 4.8 pH (KCl) 3.9 Soil texture (% soil) Fine sand (50-200 m) 1.2 Coarse silt (2050 m) 4.6 Fine silt (220 m) 32.2 Clay (<2 m) 60.7 Organic C (% soil) 1.8 Total N (% soil) 0.11 Organic matter (% soil) 2.8 C/N 16.5 Exchangeable cations and CEC (me/100g) Na+ 1.4 K+ 0.2 Ca++ 2.3 Mg++ 4.0 CEC 14.2 Extractable elements () Tamm reagent a Fe 5.3 Al 1.2 Si 0.2 Mehra-Jackson reagent b Fe 13.5 Al 1.2 Si 0.6 Fe crystalline 7.8 3.4 3.0 0.2 1.7 30.9 66.0 0.8 0.04 1.7 20.5 3.5 0.4 1.5 4.4 14.8 4.8 3.8 1.3 5.3 31.5 58.3 1.8 0.12 2.5 14.8 1.2 0.2 2.3 3.8 14.9

1.8 1.2 0.1 19.8 1.2 0.7 18.0

6.0 1.3 0.2 12.4 1.3 0.5 6.4

Materials and methods Site locations Trials were set up during the 1991-1992 cropping season in three locations in the vicinity of H Chi Minh City: two in Hoc Mn, one in Nh B, and one in Bnh Chanh, situated respectively 13 km north, 8 km south-east and 23 km south-west of H Chi Minh City. These sites are representative of common lowland rice cropping systems: in Hoc Mn, an irrigated area, three modern cultivar rice crops are grown every year. In Bnh Chanh two crops a year are grown: modern short cycled cultivars in summer, traditional rice in the autumn. In Nh B only one crop (local rice) is grown.

a Extraction with Tamm reagent (oxalic acid + ammonium oxalate, pH 3.3) for the amorphous and poorly crystallised constituents (hydroxides, hydrous oxides and oxides of iron, aluminium and silica). b Extraction with Mehra-Jackson reagent (sodium citrate-sodium bicarbonate-dithionite) for the ferric crystalline, cryptocrystalline oxihydroxide and aluminium remaining in these oxides.

In the latter two locations rice can be grown only after soil toxic compounds have been leached away by rain. Soils Nh B soil is a saline acid sulphate soil (USDA: Umbric Suldic Sulc Tropaquepts-salic), and Hoc Mn soil is of the acid sulphate type (USDA: Humic Tropaquults). Bnh Chanh soil is a slightly acid sulphate alluvial soil without annual deposits (USDA: Humic Rhodic Tropaquepts) under the inuence of the nearby acid sulphate soil area. These soils have developed

275 on sediments of the Saigon and Dong Nai river. Soil analyses were carried out in the Centre de Pdologie Biologique, Vanduvre ls Nancy, France. Physical and chemical properties of the 0- to 16-cm horizon are summarized in Table 1. Nh B soil is less fertile, having a lower pH and organic matter content, a higher C/N ratio and a slightly higher salt content than the other two soils. These soils were also used in pot experiments. Bacterial strain Strain TVV75 has been isolated from the rhizosphere of rice cv. Huyt Rng growing in a phytotron on Bnh Thanh soil, a saline acid sulphate soil comparable to Nh B soil (Trn Van et al., 1996). Under these conditions actively nitrogen-xing 8 days old rice plants were obtained: from them, macerated rhizosphere samples were prepared, serially diluted and inoculated to spermosphere models (Thomas-Bauzon et al., 1982), incubated under 1% acetylene in order to evaluate most probable diazotroph numbers. The nitrogen-xing (ARA) community amounted to 40% of the total microora. Isolates were obtained from tubes of the highest ethylene positive dilution on a nitrogen-free medium, most of them belonging to a new species of Burkholderia, ultimately described as B. vietnamiensis (Gillis et al., 1995). Of these, strain TVV75 (type strain of the new species) proved to be the most active in reducing acetylene and was retained for further inoculation experiments. Production of the bacterial inoculum Strain TVV75 of B. vietnamiensis was stored at 20 C in 50% (vol/vol) glycerol. When required for eld experiments, cells were removed from the freezer and plated on Luria-Bertani (LB) and PCAT agar medium, a medium elective for Burkholderia strains (Burbage and Sasser, 1982). After incubation for 24 h at 30 C, purity was checked and cells were used to inoculate 10-liter asks containing 5 litres of sterile Luria-Bertani broth. To obtain bacterial cultures in late log phase, asks were incubated for 24 h up to a density of 8 109 CFU mL1 on a rotary shaker at 30 C. Bacterial cells were harvested by centrifugation (7,000 g for 20 min). After removal of the culture medium, the bacterial pellet was washed in sterile water and centrifuged again (7,000 g for 20 min). Bacterial cells were then resuspended in sterile saline solution (8.5 g KCl L1 ) to one tenth of the original volume (500 mL); the inoculation mixture thus contained approximately 8 1010 CFU mL1 . Seed pregermination, inoculation, nurseries It is the local custom to pregerminate rice seeds for two days, in 34 kg bags periodically dipped in water tanks. After two days, the rst inoculation was performed by adding the inoculum in quantities theoretically sufcient to ensure 8 108 cells per seed. Water was then added to keep the seeds completely immersed for 18 h at room temperature to allow bacterial cells to adhere to seeds and roots. After 18 h, inoculated pregerminated seeds were sown in nursery beds, where they grew for 25 to 50 days, depending on the experiment. Pregerminated inoculated seed aliquots (50 seeds) were sampled, macerated with sterile pestle and mortar, serially diluted and inoculated to plates of PCAT medium to evaluate the numbers of Burkholderia cells. In a previous study (Viallard et al., 1997), on a total number of 57 bacterial strains growing on PCAT, 56 proved to be actual Burkholderia. In that study, all Burkholderia reference strains could grow on PCAT with the exception of 4 collection strains indicating an acceptable selective power of this medium. At transplanting, young plantlets were inoculated again by dipping them in large tanks for 18 h in a similar bacterial suspension in quantities sufcient to ensure 108 cells per plantlet. Reinoculated plants were then transferred to pots or eld plots. Control seeds and plants without bacterial inoculum were given a KCl solution (0.85%), in a similar manner. In nurseries, control and inoculated plots were separated by mud levees. Small amounts of nitrogen were added, once or twice, when necessary to avoid yellowing of plantlets: they represented from 1 to 11 g N per m2 depending on the experiment and they were identical for all treatments, thus avoiding bias. Rice In Nh B and Bnh Chanh, seeds of local rice cultivars were obtained from the farmers: cv. Nng Huong in Nh B and cv. Nng Thom in Bnh Chanh. These cultivars have a long growth cycle (5 to 6 months) allowing one crop per year, rarely two, with a yield of 3- to 5-T ha1 . They have a long straw (1.3to 1.5-m) and are photoperiod-sensitive. They are not heavily fertilized (50- to 80-kg urea-N ha1 ). In Hoc Mn, improved cultivars have been used: OM5971 for the summer experiments and TL008 for

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Table 2. Lay-out of the eld experiments. Pot experiments have been run in parallele with B` inh Chanh and Hoc Mn (winter) eld experiments Experiment Nh` a B` e (autumn) N` ang Huong 160 6 58 30 11 July 22, 1991 Sept. 9 Dec. 29 B` inh Chanh (autumn) N` ang Thom 159 6 24 18 31 July 20, 1991 Sept. 7 Dec. 27 Hoc Mn (summer) OM 5971 106 6 20 22 21 June 5, 1991 July 2 Sep. 20 Hoc Mn (winter) TL008 108 6 20 20 25 Nov. 24, 1991 Dec. 22 March 9, 1992

Rice cultivar Growth period (days) Replicates Plot size (m2 ) Distance (cm) between rows Hills/row Sowing time Transplanting time Harvest time

Table 3. Numbers of bacteria attached to rice seedlings (after 18 h) and plantlet roots (at transplanting) of various cultivars. Inoculum contained enough bacteria to ensure 8 108 colony forming units (CFU) B. vietnamiensis TVV75 per seed. Counts were done on PCAT medium Experiments Cultivar Number of bacteria 106 CFU seedling1 106 CFU g plantlet (3 replicates) fresh root1 (5 replicates) 3 1a (0.4)b 0.6 0.2 (0.1) 5 2 (0.6) 24 7 (3.0) 140 2 20 4 12 2 94 23

Nh` a B` e (autumn) B` inh Chanh (autumn) Hoc Mn (summer) Hoc Mn (winter)

N` ang Huong N` ang Thom OM 5971 TL 008

a Each value represents mean SD b Values between brackets indicate the percentage of bacteria attached as compared to the

number of bacteria inoculated

winter experiments. These cultivars were obtained respectively from the Agronomy Station of IBE (Institut de Biologie Exprimentale) in H Chi Minh City and from the Genetic Department of H Chi Minh City University. They are short cycled (105- to 110-days) high yielding (6- to 7-T ha1 ) varieties, with a short stem (<90 cm); they are not photoperiod-sensitive and thus can be grown all the year round. They require irrigation and high fertilization rates (up to 150-kg urea-N ha1 ). These cultivars were chosen as they perform well on acid sulphate soils in this region. Experimental set up Nursery The nursery beds (one per site) had a surface representing 10% of the experimental area. The nurseries were ooded to 2 cm water above the soil surface after

seedling emergence. A detailed study of nursery seedlings was performed in Hoc Mn in winter at 14 and 24 days after sowing. At each sampling time, 144 seedlings per treatment were sampled to determine shoot height, shoot and root dry weights. Leaf area and tiller number per plant were measured on another set of 100 seedlings from each treatment. Transplanting Rice seedlings were transplanted to the eld plots at the three-leaf stage, i.e. 25 to 50 days after sowing, depending on the cultivar. The plots were square, with areas depending upon the site (Table 2). Three seedlings were transplanted per hill, the distance between hills depending upon the cultivars (Table 2). Plots were surrounded by mud levees and separated by 50 cm large ditches. Experimental plots were separated from farmers elds by a buffer zone representing

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Table 4. Effect of seed inoculation of rice cultivar TL008 with B. vietnamiensis TVV75 on different parameters: shoot height, dry weights of shoots and roots, leaf surface, tiller number per plant. Hoc Mn nursery beds in Winter, at two times after sowing Number of replicates Day 14: Shoot height (cm) Shoot weight, (mg plant1 ) Root weight (mg plant1 ) Leaf surface (cm2 plant1 ) Day 24: Shoot height (cm) Shoot weight (mg plant1 ) Root weight (mg plant1 ) Leaf surface (cm2 plant1 ) Tiller number per plant Treatments Control Inoculated 18.4 0.4 a 23.9 2 a 5.1 0.5 a 7.0 0.5 a 29.9 0.5 a 67.5 6.2 a 8.8 0.6 a 17.5 1.1 a 1.8 a 21.6 0.3 b 29.3 2.2 b 8.1 0.6 b 9.1 0.6 b 33.7 0.5 b 90.1 5.6 b 13.8 0.8 b 18.8 0.8 b 2.0 b Inoculation effect

144 72 72 72 144 72 72 72 100

+17% +23% +59% +30% +13% +33% +57% +8% +13%

Means condence intervals (95%). Figures followed by the same letter on the same line, are not signicantly different at a 5% threshold (LSD). For the tiller number per hill a 2 test showed the difference to be signicant (P=5%).

half the area of the experimental area and cultivated with rice. All experiments comprised two levels of inoculation (inoculated vs control) and three levels of nitrogen fertilization (control, recommended rate, half this rate). These six treatments were applied in six replicates. Inside blocks, the six treatment plots were randomized. Plots were weeded by hand after water removal at each fertilizer application. Pot experiments were run simultaneously in Hoc Mn (Winter) and Bnh Chanh (Autumn). One or three seedlings were transplanted into each pot. Pots were 32.5 cm in diameter and 37 cm high. In Bnh Chanh six replicate pots were set per treatment, each containing 12 kg wet soil. In this pot experiment, pots were set out following exactly the same design as the corresponding eld plots. In Hoc Mn there were twelve replicate pots per treatment, containing 5 kg dry soil, and pots were completely randomized.

- In the eld, grain yield was determined on the whole plot, leaving out the two border rows to avoid border effects. Plants were harvested by hand, sun-dried and weighed. Yield was expressed as weight of paddy at a 14% water content. Yield was determined for the six replicates of each treatment. For the estimation of yield components, three sub-plots per plot were randomly selected, and 4 hills per sub-plot were sampled. Each sample contained plants and the adhering clod. All samples were kept in plastic bags and processed in the laboratory to determine the plant height (from soil surface to the tip of the upper leaf), the number of fertile tillers, the number of lled grains by panicle, and the dry weight of 1,000 grains (oven-dried for a week at 60 C ). Statistical analysis A rst variance analysis was performed on data collected during the nursery stage. Data were the means of 72, 100 or 144 replicates, and one-way analysis of variance was used to determine whether inoculation had a signicant effect on plant growth. After transplanting, the data for each treatment (six replicates) were subjected to a variance analysis with two factors (inoculation and nitrogen) for the pot experiment in Hoc Mn and with three factors (nitrogen, inoculation and block) for the other experiments, using the Statgraphics software (Release 5.0, Uniware STSC, Inc.). When analysis of variance showed signicant treatment effects, the Least Signi-

Measurements Data collected comprised: - Most probable number (MPN) B. vietnamiensis present on inoculated seeds, using the PCAT medium. - In nursery, on the fourteenth and twenty fourth days after sowing, shoot and root dry weights of 72 plantlets were measured along with leaf areas. Shoot height was also measured on 144 plantlets. On day 24, tiller numbers per plant were measured on 100 plantlets.

278 cant Difference (LSD, p<0.05) test was applied to make comparisons between treatments. no signicant effect of N but a signicant effect of inoculation (Table 5). In the two experiments, there was no interaction between inoculation and N fertilizer rates, and inoculation caused a signicant (p0.05) positive effect on yield and all its measured components (1,000 grain weight, number of tillers per pot, and number of lled grains per pot) (Table 5). In Hoc Mn (winter) inoculation signicantly increased yield by 19%, the weight of 1,000 grains by 3%, the number of panicles by 11%, and the number of lled grains per pot by 17% (Table 5). Table 5 also shows a substantial effect of B. vietnamiensis TVV75 on the number of fertile tillers at Binh Chanh (22% above the uninoculated control). When a detailed LSD analysis was performed to evaluate separately the effects of inoculation at different nitrogen levels (Tables 6a and 6b), most inoculation effects were below the signicance level for the different yield components. In Hoc Mn (winter), inoculation with B. vietnamiensis was not accompanied by a signicant increase in the three yield components (Table 6a), whereas in Bnh Chanh at 44 kg N ha1 , there was a signicant effect of inoculation on all yield components (Table 6b). Inoculation effect, eld experiments Results of the variance analyses of the four eld experiments are shown on Table 7. The experimental layout and proper maintenance were good enough to show very rare block effects. N fertilizer always had an effect on yield, tillering and lled grains, but no effect on the weight of 1,000 grains. In all experiments, the variance analysis showed that inoculation had a signicant effect on all yield components. It is interesting to note that inoculation positively affected grain lling whereas nitrogen fertilization had no effect on this late yield component. In Hoc Mn, where more nitrogen fertilizer was applied, this effect of inoculation on the weight of 1,000 grains tended to depend on the rate of nitrogen applied, being signicant mostly in the controls without fertilizer (Tables 8c and 8d). Inoculation signicantly increased the grain yield in Nh B (20%) and in Hoc Mn in summer (22%) (Table 7). Yield increases were lower but still signicant in Bnh Chanh (13%), and in winter at Hoc Mn (15%). In all four experiments, signicant increases were seen in all measured yield components: 1,000 grain weight, number of panicles per m2 , number of lled grains per panicle (Table 7). When results obtained for the different N rates are considered sep-

Results Inoculation Before inoculation, unsterilized rice seeds were shown to be naturally devoid of bacteria able to grow on PCAT. The B. vietnamiensis TVV75 bacterial inoculum was initially added at approx. 800 106 CFU per seedling. The levels of Burkholderia cells actually found on pregerminated seeds after 18 h incubation with this inoculum ranged from 0.60.2 to 247.0 106 CFU per seedling depending on the experiment (Table 3). Bacteria found on seeds represented 0.1 to 3% of added bacteria. It was not possible to prove that PCAT growing bacteria actually were the inoculated strain. In cultivars OM 5971 and Nng Huong, the colonisation was similar after the rst inoculation. Nevertheless, at transplanting cv. Nng Huong had 12 times more bacteria (1402 vs. 122 106 CFU) per g of root, this difference being reinforced by the fact that its root system was obviously more developed than that of cv. 5971. It thus seems that different cultivars have different and unpredictable behaviours when inoculated with the same bacterium. Inoculation effect in nursery In all nurseries, inoculation had a visible effect on plantlet growth. Differences due to inoculation were observed at the three sites with both the local and the modern cultivar. This effect was quantied in Hoc Mn in the winter experiment (Table 4). Plants inoculated with B. vietnamiensis TVV75 exhibited 23% and 59% signicant (p0.05) increases for shoot and root weights respectively, over uninoculated plants. Shoot heights were 17% larger. These effects were still visible at day 24 after sowing. Inoculation had a signicant effect on the leaf surface at day 14 after sowing. Ten days later, this effect had diminished, probably due to inter-plant competition (Table 4). Inoculation effect in pots Variance analyses were peformed on the two pot experiments (Table 5). N fertilizer had an overall effect on yield, tillering and lled grain numbers in the two experiments. For the weight of 1,000 grains, there was

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Table 5. Effect of rice inoculation by Burkholderia vietnamiensis TVV75, on yield and yield components in the Hoc Mn and B` inh Chanh pot experiments: results of the variance analysis with three factors: inoculation (inoc), N fertilizer rate (fert), block (bl), global means for yield and yield components Experiments Variance analysis Effects of the factors inoc fert bl Global means Inoculation effect Means per treatment control inoc 60 NS NR NR NR NR 15.11.5 22.40.2 9.81 67368 36 NS NS NS NS 19.81.3 21.00.3 9.81.1 94330 30 13.8 22.1 9 621 30 16.5 22.7 10 725 +19 +3 +11 +17 increase /control (%)

Hoc Mn, winter 3 pl pot1: Grain yield pot1 (g) 1000 gr weight (g) Panicles pot1 Filled grains pot1 B` inh Chanh, autumn 1 pl pot1: Grain yield pot1 (g) 1000 gr weight (g) Panicles pot1 Filled grains pot1
number of replicates

18 18.3 20.5 9 896

18 21.3 21.6 11 990 +16 +5 +22 +11

= signicant (P0.05); NS = not signicant. NR: not relevant (pots were completely randomised in Hoc Mn) Table 6. Effects of inoculation with B. vietnamiensis TVV75 on the yield components at the three different nitrogen levels 6a. Winter pot experiment, Hoc Mn (10 replicates). N fertilizer Kg N ha1 0 45 90 1,000 grain weight (g) Control Inoculated 21.6a 0.4 22.4ab 0.7 22.2ab 0.6 22.6b 0.8 22.8b 0.4 22.7b 0.4 Panicles pot1 Control Inoculated 6a 1 10b 1 11bc 1 7a 1 11bc 1 12c 1 Filled grains pot1 Control Inoculated 341a 45 577bc 51 944d 77 466ab 62 674c 79 1036d 202

I/C +5 +1 +3

I/C +16 +11 +7

I/C + 36 + 17 +10

6b. Autumn pot experiment, B` inh Chanh (6 replicates). N fertilizer Kg N ha1 0 44 88 1,000 grain weight (g) Control Inoculated 20.3a 0.5 20.7a 0.5 20.6a 0.5 21.5ab 1 21.3b 0.2 21.9b 0.7 Panicles pot1 Control Inoculated 6a 2 9ab 1 13c 1 7a 1 12c 2 14c 2 Filled grains pot1 Control Inoculated 742a 80 888a 37 1059bc 57 794a 114 988b 161 1187c 132

I/C +6 +3 +6

I/C +11 +35 +8

I/C +1 + 17 +12

Mean condence intervals (95%). Means followed by the same letter on the same line within each nitrogen level, are not

signicantly different at a 5% threshold (LSD test). I/C = increase due to inoculation over the control (%).

arately, the yield components of inoculated plots were often higher than in the uninoculated plots whatever the location: increases were rarely signicant in Hoc Mn with modern cultivars (Tables 8c and 8d), but they were signicant at the three different nitrogen

levels in Nh B and in Bnh Chanh with the local cultivars (Tables 8a and 8b). Yields in Hoc Mn are lower than in the other locations (Table 7), but one must keep in mind that in this

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Table 7. Global effect of rice inoculation by Burkholderia vietnamiensis TVV75 on the grain yield and its components, in the four eld experiments. Results of the variance analysis with three factors: inoculation (inoc), N fertilizer rate (fert), block (bl). Global means represent pooled data obtained from the 36 individual plots. The 6 treatments include inoculation vs control (Contr), in combination with three N rates. Each treatment was applied in 6 replicate blocks. Inside blocks individual treatments were completely randomised Experiments Variance analysis Effect of the factors Inter action inoc fert bl fert/ inoc 4.50.3 23.80.1 2084 1072 4.20.3 21.50.1 2345 923 2.30.2 23.40.1 2046 643 3.640.2 22.90.1 2478 732 Global means Inoculation effect Means per increase treatment Inoc/ Control Contr Inoc (%)

Nh` a B` e, autumn Grain yield T ha1 1000 grains weight (g) Panicles m2 Filled grains panicle1 B` inh Chanh, autumn Grain yield T ha1 1000 grains weight (g) Panicles m2 Filled grains panicle1 Hoc Mn, summer Grain yield T ha1 1000 grains weight (g) Panicles m2 Filled grains panicle1 Hoc Mn, winter Grain yield T ha1 1000 grains weight (g) Panicles m2 Filled grains panicle1

NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS P=7.4% NS NS

4.1 23.5 201 103 3.9 21.3 224 90 2.3 23.1 198 60 3.4 22.6 237 70

4.9 24.1 214 111 4.4 21.6 244 95 2.8 23.7 209 68 3.9 23.1 257 76

+20 +3 +7 +8 +13 +1 +9 +6 +22 +3 +6 +13 +15 +2 +8 +9

=signicant (P0.05), NS = not signicant.

Numbers are means of 18 measurements in the four site experiments.

location modern cultivars have been used which allow several crops a year.

Discussion and conclusion B. vietnamiensis TVV75 had been previously identied as a promising bacterial candidate for inoculation assays with its high rhizospheric population, its high nitrogenase activity, and its ability to inhibit fungal plant pathogens and to produce efcient new siderophores and IAA (Trn Van et al., 1994b, 1996, Meyer et al., 1995, Tabacchioni et al., 1993). It was decided to use it to inoculate rice in Vietnam on an acid sulphate soil similar to its soil of isolation. In a rst pot experiment, inoculation resulted in a nal 20% yield increase through positive effects on most yield

components (Trn Van et al., 1994b). We report here results obtained in four subsequent eld assays, two of them being paralleled by new pot assays. Here also, TVV75 signicantly increased several yield components, resulting in a nal 1322% increase in grain yield. During the initial inoculation step, only a small proportion (ca 1%) of the initial bacteria were retained by the seeds (Table 3). Since strain TVV75 had been isolated from a local cultivar (Huyt Rng), it was expected that it would colonize a local cultivar better than a modern one. In fact, its colonizing ability was maximum with the modern cultivar TL 008 but low with the other modern cultivar, OM 5971, and the two local cultivars, including Nng Huong (Table 3). Twenty four days later, root colonization in inoculated treatments represented 107 to 108 bacteria per g of

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Table 8. Effects of inoculation with B. vietnamiensis TVV75 on the yield components at maturity and at the three different nitrogen levels. Means of 72 individual measurements 8a. Autumn eld experiment, Nh` a B` e. N fertilizer Kg N ha1 0 26 52 1,000 grain weight (g) LSD=0.3 Control Inoculated 23.5a 0.3 23.4a 0.2 23.6a 0.1 24.0b 0.2 24.2b 0.3 24.2b 0.1 Panicles m2 LSD=10.5 Control Inoculated 179a 10 202b 9 223c 9 202b 5.2 206b 6.5 235d 6.4 Filled grains panicle1 LSD=7.2 Control Inoculated 97a 3 102ab 6 109c 5 101a 6 112c 5 121d 5

I/C +2 +3 +3

I/C + 13 +2 +5

I/C +4 + 10 + 11

8b. Autumn eld experiment, B` inh Chanh. N fertilizer Kg N ha1 0 44 88 1,000 grain weight (g) LSD=0.25 Control Inoculated 21.3a 0.1 21.2a 0.2 21.4ab 0.2 21.5bc 0.2 21.7c 0.2 21.7c 0.2 Panicles m2 LSD=10.9 Control Inoculated 198a 4.0 229b 8.7 244c 8.1 219b 7.8 251cd 6.5 261d 8.7 Filled grains panicle1 LSD=8.5 Control Inoculated 83a 4.1 93bc 10.8 94c 5.9 85a 6.1 95c 8.8 106d 7.8

I/C +1 +2 +1

I/C + 11 + 10 +7

I/C +2 +2 + 13

8c. Summer eld experiment, Hoc Mn. N fertilizer Kg N ha1 0 39 77 1,000 grain weight (g) LSD=0.27 Control Inoculated 23.2a 0.1 23.2a 0.2 23.0a 0.2 23.8c 0.3 23.4ab 0.2 23.9c 0.2 Panicles m2 LSD=19.3 Control Inoculated 182a 13 195ab 14 218c 17 187a 10 208bc 17 233cd 18 Filled grains panicle1 LSD=8.6 Control Inoculated 57a 4 59ab 5 63ab 6 64ab 8 67bc 8 73c 8

I/C +3 +1 +4

I/C +3 +7 +7

I/C + 12 + 14 + 16

8d. Winter eld experiment, Hoc Mn. N fertilizer Kg N ha1 0 45 90 1,000 grain weight (g) LSD=0.29 Control Inoculated 22.6a 0.2 22.5a 0.2 22.8ab 0.2 23.20c 0.1 23.0bc 0.3 23.90b 0.2 Panicles m2 LSD=21.3 Control Inoculated 207a 12 237bc 18 266d 21 227ab 10 254cd 9 290e 20 Filled grains panicle1 LSD=5.5 Control Inoculated 65a 4 67ab 5 79bc 5 69ab 4 74b 4 84c 4

I/C +3 +2 +1

I/C + 10 +7 +9

I/C +6 + 10 +6

Means condence intervals (95%). Means followed by the same letter on the same line within each nitrogen level, are not signicantly different at a 5% threshold (LSD test). I/C = increase due to inoculation over the control (%).

fresh roots, a density higher than that reported by Di Cello et al. (1997) on roots of uninoculated maize, comparable to that found by Nacamulli et al. (1997) on maize inoculated by B. cepacia MCI 7, by Chiarini et al. (1998) on Sorghum inoculated by B. cepacia PHP7, but lower than that found by Hebbar et al. (1992) on roots of maize inoculated by B. cepacia strain 526. It must be emphasized that strain TVV75 is a local isolate, which ensures a good adaptation to local conditions and explains its good colonization ability. It was assumed that its natural abundance in soil (Tran Van et al., 1996) is also a guarantee of its lack of pathogenicity for man.

It has been observed (no quantitative data) that inoculation accelerated the germination process. Other PGPR have been shown to promote emergence of host plants (Kloepper et al., 1986, Freitas and Germida 1990, Xu and Gross, 1986, Turner and Mackman, 1991), and have even been named emergence promoting rhizobacteria: TVV75 must be considered as one of them. Inoculation had already increased the plant growth at the nursery stage: differences between inoculated treatments and the controls were visible in all experiments. This effect has been quantied only in Hoc Mn: in the nursery, signicant increases of shoot and

282 ponent is left unchanged by inoculation. The increase observed here is indicative of an effect of inoculation on very late yield components also. It is interesting to note that inoculation positively affected grain lling whereas nitrogen fertilization had no effect on this late yield component: one can speculate that inoculated bacteria are still able to positively affect the nutrition of plants at a time when fertilizers are depleted. This has been already observed in rice eld inoculation studies by Omar et al. (1989, 1992). At harvest, the overall result of the four eld and three pot (including the previously reported pot assay) inoculation experiments was a nal increase in yield ranging from 13% (Bnh Chanh, autumn) to 22% (Hoc Mn, summer) due to the accumulation of the above mentioned positive effects on different yield components. In conclusion, the action of inoculated bacteria begins very early and lasts for a very long time, as both early and late yield components are affected. Three inoculation experiments using Burkholderia have already been reported: (1) Bevivino et al. (1994) compared clinical and environmental Burkholderia strains for their ability to colonize Cucumis sativus roots and observed a dry weight increase in root biomass (at day 40) following inoculation of strain TVV75. (2) Chiarini et al. (1998) did inoculation trials of Sorghum bicolor with strain PHP7, a Burkholderia strain related to Burkholderia cepacia. At day 60 they could demonstrate an increase in root weight following inoculation. (3) Germida and Walley (1996) used two Burkholderia strains to inoculate spring wheat in Saskatchewan: results obtained were rather inconsistent and inoculation produced mostly negative effects. In the experiments of Omar et al. (1989, 1992) in Egyptian soils, benecial effects of inoculation of N2 -xing bacteria were seen only where N was the limiting factor of yield. This is the case here, as yield depends upon N fertilizer rates in all treatments (Table 5 and 7). This conrms the advantage of N2 xing bacteria as a seed inoculant, under conditions where crops respond to N, i.e. under most normal conditions of modern agriculture. Along the same line, if N is the limiting factor of growth, it is also the limiting factor of photosynthesis and thus of exudation rates: ultimately, it is conceivable that N xation could be positively correlated to available N, especially when photosynthesis is high, i.e. near the end of the vegetative part of the growth cycle. This could explain why a signicant interaction between inoculation and

Figure 1. B` inh Chanh eld experiment, autumn: Rice cv. N` ang Thom inoculated by B. vietnamiensis TVV75. N is limiting the yield, and there is a signicant effect of inoculation. The same yield (4.4 T ha1 ) can be obtained either under regular practice, using 68 Kg N ha1 or using inoculation and only 43 kg N ha1 . The NFEI (nitrogen fertilizer equivalent of inoculation) is thus 25 Kg N ha1 . Given the inoculation cost and the cost of N fertilizers, the NFEI value is a help to reach a decision about the feasibility of inoculation.

root mass due to inoculation reached 23% and 59% respectively, before the second inoculation (day 24 after sowing). This is indicative of a very early effect of bacteria on their host plant. It is reminiscent of results obtained by Jacoud et al. (1999) showing that a very short and early contact of maize with PGPR bacteria was enough to ensure a signicant effect on subsequent growth. Tillering is the next important step in rice development, and the tiller number per plant was signicantly increased (+13%) in Hoc Mon nursery 24 days after sowing (Table 4). This effect has been observed by several authors in comparable inoculation trials, e.g. in wheat with Azospirillum, Bacillus (Reynders and Vlassak, 1982, Chanway and Nelson, 1990), Burkholderia (De Freitas and Germida, 1990) and Paenibacillus polymyxa (Gouzou, 1992). During eld growth inoculated plants looked healthier. This effect was not quantied but could correlate with the in vitro observation that strain TVV75 could inhibit growth of several fungal plant pathogens (data not presented). Moreover, inoculated plots set owers earlier, an observation already made in similar inoculation studies performed on maize (Berge et al., 1990), rice (Omar et al., 1989) sorghum and millet (Smith et al., 1984). More unusual is the increase in average grain weight that we observed (Tables 5 and 7). In most reported eld inoculation experiments this yield com-

283 N fertilization is sometimes seen (Table 7) for the late yield components (weight of 1,000 grains). This study did not address the debated question of the mechanism involved. Strain TVV75 has been selected for its nitrogenase activity in association with rice under gnotobiotic conditions (Trn Van et al., 1996). If its nitrogenase activity plays a role in the growth stimulation process observed, it is not surprising to nd it limited to situations where N is limiting. Hormone production has also very often been evoked to explain inoculation effects of PGPR. Hormone-like compounds have indeed, been found when looked for, including in TVV75 (Tabacchioni et al., 1993). Results presented here do not contradict any of these two possible mechanisms for the effects. Furthermore, it could be an adaptive trait for good rhizosphere inhabitants to have both the ability to increase root growth and to x nitrogen. The two characters are linked, as nitrogen xation is energy limited and thus depends on the photosynthate derivation towards roots, causing more root growth as well as more root exudation (Heulin et al., 1987). In the context of a foreseeable less polluting agriculture, seed inoculation could represent an interesting alternative to the use of high rates of N fertilizers. As represented on Figure 1, a similar yield can be obtained with less added N if seeds are inoculated: this denes the nitrogen fertilizer equivalent of inoculation (NFEI). Comparison of the cost of NFEI kg N-fertilizer (including eventual penalties) to the cost of inoculation would help in making the decision to inoculate. their contribution to the administrative work required by this project. J L Hubert also contributed to the statistical analyses.

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Acknowledgements This work was funded by the European Union project STD 2A-0119F of DG XII. The rst author wishes to thank his students L. Nguyn Hoi, L. Nguyn Ngoc Huyn, P. Nguyn Van and L. Mai Ngoc as well as the agronomists who assisted him: N. Nguyn Van, National Consultant on Soils in Vietnam, N. Nguyn Xun, H. Vu Ngoc for expert technical assistance in soil identication experiments. P. Phan Van, D. Nguyn Trong, T. Dinh Van, H. Mai Van, U. Nguyn Chi, C. Nguyn Van for eld assistance throughout the course of this work. We thank the farmers for their participation in the different eld experiments. We also gratefully acknowledge Mrs C. Ginsburger (Centre de Pdologie Biologique du CNRS, Nancy, France) and Mr Nol, regional delegate of CNRS in Nancy, for

284
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