Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1982, p. 124-128 0099-2240/82/010124-05$02.00/0 Vol. 43, No. 1 Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice W. L. BARRAQUIO, M. R. DE GUZMAN, M. BARRION, AND I. WATANABE* The International Rice Research Institute, Los Bafnos, Laguna, Philippines Received 4 June 1981/Accepted 21 September 1981 Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucoseyeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice. Yoshida and Ancajas (11) reported that nitrogen fixation (acetylene reduction) in rice fields was more pronounced in flooded than in dryland conditions. Acetylene reduction activity of the excised rice root was also higher in wetland rice than in dryland rice. The comparison of the nitrogen-fixing population was not made between the wetland rice soil and dryland soil. Barraquio and Watanabe (1) found that the incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria isolated from washed roots was higher in wetland plants, including Oryza sativa, 0. punctata, 0. australiensis, and Monochoria vaginalis, than in dryland plants, including Cassia tora, maize, sorghum, Panicum maximum, Digitaria smutsii, Paspalum plicatulum, and 0. sativa, grown in dryland conditions. The incidence of nitrogen-fixing bacteria from dryland rice root gave the intermediate figures between other dryland plants and wetland rice. In this experiment, the same rice varieties were not, however, used to compare dryland rice with wetland rice, and the rices were grown in dryland conditions during the wet season, when the dryland was temporarily flooded. To elucidate further the difference between the population of nitrogen-fixing bacteria associated with rice grown in wetland and dryland conditions, the same varieties were grown in flooded and dryland conditions during the dry season. This paper reports the results of this comparison. MATERIALS AND METHODS Field layout. The dryland and wetland (flooded) plots were located side by side in the International Rice Research Institute's (IRRI) field. Each plot had six subplots 5.5 by 6.0 m each. Two rice varieties, IR5 and OS4, were grown in three replicate plots. For dryland cultivation, seeds were sown 15 days before transplanting the rices to flooded plots. On the same day, the seeds were sown in a nursery bed for wetland growth. No chemical nitrogen fertilizer was applied. The spacing of wetland rice was 20 by 20 cm, and dryland rice was sown on the rows 40 cm apart. Samplings were made 2 weeks after transplanting wetland rice and thereafter at 2-week intervals. For water culture acetylene reduction activity (ARA), two hills from each subplot were dug out with adhering soil from the flooded plots. From dryland plots, a 10- by 10-cm cubic soil block including three or four plants was dug out. Two blocks were taken from each subplot. For ARA assays, two sites from each subplot were chosen. For microbial counting, the plants were taken the same day as samples for water culture ARA assays were taken. At early stages of rice growth, 10 to 15 hills of wetland rice or the same number of plants of dryland rice were dug out. At later stages, three to five plants were taken. The plants from three subplots were pooled, and 10- to 20-g (fresh weight) samples of rhizosphere soil, basal shoots, and histosphere (rhizo- 124

VOL. 43, 1982,.mol C2H4/hill N2-FIXATION BY DRYLAND AND WETLAND RICE 125 0.5 0.4 0.3 0.2 0.1 21 4 51 61 71 Samp rg 50 100 days FIG. 1. Acetylene reduction activity associated with IR5 and OS4 grown in wetland and dryland conditions. (A); Field in situ activity for 24 h (B) water culture activity for 5 h. Days are counted from the seeding of rice. The curved arrows in B indicate the heading stages. The arrows under abscissae indicate the sampling times. The vertical bars indicate the standard errors of the averages in logarithmic scale. plane) were taken. Processing these samples was as described by Watanabe et al. (7). Rhizosphere soil refers to the soil fraction attached to the root, and the histosphere sample was obtained by washing the root several times and then macerating it. ARA of plants. After washing of the roots by tap water to remove the bulk of adhering soil, rice plants from each subplot site were transferred to a 3-liter pot with nitrogen-free mineral solution and grown for 1 day before ARA assays. ARA assays were made for 5 h by the method of Watanabe and Cabrera (8). Acetylene was added to both liquid and gas phases within an assay chamber. In flooded fields, ARA assays were conducted by the method of Lee and Watanabe (4, 10). The surface soil and basal shoot about 10 cm above the ground were covered by aluminum foil to prevent photodependent ARA. The assays were started the same day as water culture ARA assays were made and continued for 24 h. From dryland fields, the cubic soil block (10 by 10 by 10 cm), including three to four dryland rice plants, was dug out. Two blocks were taken from each subplot. The surface soil and basal portion of the shoots were covered by aluminum foil, and the soil block with rice plants was enclosed in a plastic bag. Five liters of 20%o C2H2 containing ambient air was introduced into the plastic bag. Assays were continued for 24 h in the field. The gasses were analyzed for C2H4 by the method of Lee and Yoshida (5). Microbial counting. Semisolid glucose-yeast extract and malate-yeast extract media (6, 7) were used for most probable number (MPN) counting of aerobic nitrogen-fixing bacteria. Tenfold dilution (four tubes per dilution) was used. In some cases, malate-yeast extract medium was modified by the method of Dobereiner and Baldani (3). The use of the modified media is described in the figures. ARA (24 h) was used to detect positive tubes in nitrogen fixation after 3 days of growth. Tryptic soy broth agar (TSA) plates, including

126 BARRAQUIO ET AL. 0.1% tryptic soy broth (Difco) and 1.5% Noble agar (Difco), were used for counting aerobic heterotrophic bacteria (6). A total of 100 colonies from TSA plates for each site of plant every two sampling times was fished, and their ARA was tested for 24 h at 35 C in 72- h-old glucose-yeast extract semisolid culture. RESULTS AND DISCUSSION Field ARA. The results of field 24-h ARA assays of wetland and dryland rice are shown in Fig. 1. The difference between wetland rice and dryland rice was clear, particularly after the middle stage of rice growth. IR5 gave higher ARA than did OS4. Because the endogenous ethylene evolution was not measured in dryland conditions, ethylene formation from acetylene associated with dryland rice could have been much less than the values shown in Fig. 1. In wetland conditions, daily endogenous ethylene evolution seldom exceeded 1,umol of C2H4 per rice hill; hence, the observed ethylene evolution, particularly at later stages of rice, was due to nitrogen fixation. Yoshida and Ancajas (11) measured the ARA of excised root and soil from planted plots and showed higher nitrogen fixation in wetland conditions. APPL. ENVIRON. MICROBIOL. Water culture assays. Up to 70 days after seeding, the ARA was low, and no difference between the two growing conditions was found. Thereafter, the ARA of wetland rice became much higher than that of dryland rice, which is a result found by field ARA assays. IR5 gave a higher ARA than did OS4. Because the rice plants were grown in water culture for 24 h before ARA assays, and this incubation might have modified microflora, the exact comparison of water culture ARA with field ARA would be difficult. However, both methods gave an identical trend of ARA changes due to growing conditions, growth stages, and varieties. Total number of heterotrophic bacteria. No replication was made for each sample. Therefore, about a 10-fold difference in plate counts and MPN would be meaningful. The number of aerobic heterotrophic bacteria counted on TSA plates is shown in Table 1. The number of heterotrophic bacteria in the rhizosphere soil and histosphere of IR5 was not different due to growing conditions. In the case of OS4, the counting failed for the first two samplings, but at later stages, the counts of heterotrophic bacteria in the histosphere were lower in dryland than in Number of heterotrophic bacteria on TSA plates and percentage of nitrogen-fixing bacteria TABLE 1. Sampling Site IR5 OS4 Wetland Dryland Wetland Dryland 1 Rhizosphere soil 45a (11)b 80 (2) 67 (4) 60 (1.2) Histosphere c Basal shoot 420 (9) 48 (1) 2 Rhizosphere soil 142 41 95 Histosphere 290 160 11 Basal shoot 34 5 3 Rhizosphere soil 42 (30) 100 (2) 60 (5) 81 (1) Histosphere 260 (45) 140 (<1) 1,300 (47) 120 (17) Basal shoot 67 (70) 14 (<1) 80 (14) 16 (2) 4 Rhizosphere soil 63 90 63 91 Histosphere 310 460 1,000 41 Basal shoot 17 20 27 19 5 Rhizosphere soil 66 (7) 90 (2) 80 (6) 67 (<1) Histosphere 680 (44) 520 (34) 1,400 (61) 200 (1) Basal shoot 40 (43) 20 (1) 16 (36) 10 (18) 6 Rhizosphere soil 150 91 Histosphere 1,000 540 Basal shoot 24 21 7 Rhizosphere soil 59 (24) 130 (8) Histosphere 1,700 (44) 420 (18) Basal shoot 31 (39) 150 (58) a Data are given X106 per gram (dry weight). b Numbers within parentheses indicate percentages of nitrogen-fixing bacteria. c -, Contaminated.

VOL. 43, 1982 wetland conditions. On the other hand, the counts of heterotrophic bacteria associated with basal shoots (IR5) at the early growth stages were higher in wetland conditions than in dryland conditions. This finding means that the colonization of bacteria to the basal shoots took place rapidly after transplanting rice to the flooded soil. The population at the basal shoots was at the order of 107 to 108 bacteria per g fresh weight and lower than that of the histosphere (108 to 109 bacteria per g). Number of nitrogen-fixing bacteria. For both varieties, it is clear that the number of nitrogenfixing bacteria measured on both glucose and malate media was much higher in wetland than in dryland conditions. This difference was approximately 10 times (Fig. 2 and 3). The higher number of nitrogen-fixing bacteria in wetland condition was found in all three sites-rhizosphere soil, histosphere or root, and basal shoots. The difference due to growing conditions of rice was most pronounced in basal shoot samples. As we showed earlier (7), the MPN on glucose-yeast extract medium was higher than that on malate medium. The nitrogen-fixing bacteria grown on malate medium were predominantly Azospirillum, a genus characterized by white, undulating fine pellicles appearing 3 mm beneath the surface of semisolid medium and curved or spiral forms of cells. The difference between MPN in the two media was least pronounced in the basal shoot samples. A previous report (7) with a limited number of samples 8-1 log/g 7 1-2I 6 %A" 4 -/ I 1st 2sd 3.4 4ttt 5th 6th 7It FIG. 2. Number of nitrogen-fixing bacteria associated with IR5 grown wetland and dryland conditions. The number is on dry weight basis. For the second assays, the medium of Dobereiner and Baldani (3) was used. For the fifth assays, the vitamin solution was replaced by 0.05 g of yeast extract per liter. N2-FIXATION BY DRYLAND AND WETLAND RICE 127,og/g 7 6 5 4] Io/g 8 7-6 4t 01 5/ Histo4msev 2 3 4 5 6 2 3 4 5 Iog/g I JTI,/,yt Bo/,, 1 r 2 3 4 5 E FIG. 3. Number of nitrogen-fixing bacteria associated with OS4. The explanations and symbols are as in Fig. 2. showed that in basal shoots (stems) the MPN of malate-utilizing, nitrogen-fixing bacteria was almost equal to that of glucose utilizers. A similar trend was found for both varieties in both dryland and wetland conditions. Incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria. The percentage of nitrogen-fixing bacteria among aerobic heterotrophic bacteria is shown in Table 1. The incidence of nitrogen-fixing bacteria in dryland rice was much smaller than that in wetland rice. This was the case particularly at the earlier stages of rice growth. The higher incidence in wetland condition was found for all three sites, except for basal shoots of IR5 at 98 DAT. Their incidence was highest in the histosphere, followed by basal shoots and rhizosphere soil in both dryland and wetland conditions. The highest incidence of nitrogen-fixing bacteria in the histosphere was previously reported for wetland rice (6). The percentage of nitrogen-fixing bacteria in the histosphere of dryland rice was higher than that of other dryland plants, as previously reported (1). The predominant nitrogen-fixing bacteria from the histosphere grown on TSA resemble those bacteria which were described as Achromobacter type (6), but further examination revealed they are rather more close to Pseudomonas due to their flagellum type, GC content, physiological characteristics, and immunological properties (unpublished data). The presence

128 BARRAQUIO ET AL. of these bacteria among nitrogen-fixing bacteria on TSA was found exclusively in cultures from the histosphere of wetland rice. The percentage of these type of bacteria among nitrogen-fixing isolates ranged from 29 to 58%, except for the first sampling (null). The presence of these bacteria exclusively in the histosphere of wetland plants was previously reported (1). The number of nitrogen-fixing bacteria estimated by the MPN method (Fig. 2 and 3) was 10 to 100 times lower than the number estimated by direct pick-up of colonies on TSA and their ARA tests on glucose-yeast extract medium (incidence x total heterotrophic bacteria counts). The difference between the two methods was least pronounced in the rhizosphere soil samples. This discrepancy may be explained by many factors. In MPN counting, the minimum cell number to induce positive ARA after 48 h of growth was taken as a unit. Probably more than one cell would be needed to induce a positive reaction. In the direct pick-up method, 107 to 108 cells per tube were inoculated to semisolid medium. The higher inoculum may be favorable to nitrogen fixation, because the respiration by a high number of cells induces rapid oxygen depletion or low oxygen conditions in semisolid medium. The negative interaction by non-nitrogenfixing bacteria, particularly by acid formation, in MPN tubes may be another factor to make MPN a low estimate. The research reported here points out that the lower nitrogen-fixing activity of dryland rice was related to the lower number of nitrogen-fixing bacteria associated with the root and basal shoots. If the lower nitrogen-fixing activity of dryland rice were due to the difference of the root exudate, there should be a difference in the number of heterotrophic bacteria of the roots. This was not the case. There must be favorable conditions in wetland root for the selective development of nitrogen-fixing bacteria. Predominant nitrogen-fixing bacteria (6) and Azospirillum (2) fix nitrogen in microaerophilic conditions. In wetland conditions, in the root tissue or on its surface, microaerophilic conditions may develop as a balance between oxygen transfer to the root and its consumption by the root and bacteria. The microaerophilic condition developed in or on the plant tissue of the submerged parts may give favorable conditions for APPL. ENVIRON. MICROBIOL. nitrogen-fixing bacteria. On the other hand, dryland rice root would be too aerobic for nitrogenfixing bacteria. However, we need further study to explain the favorable conditions of wetland rice for nitrogen-fixing activity and population. As pointed out previously (9), the basal portion of shoots contributes greatly to nitrogen fixation associated with wetland rice. Presumably, the contribution of basal shoots is marked in the wetland rice, because the population of nitrogen-fixing bacteria at this portion is much higher in wetland rice. One experiment conducted during the ripening period in both conditions (IR5) showed that root cutting reduced field ARA 94% in dryland rice, but 60% in wetland rice, suggesting the greater contribution of basal shoots in wetland rice than in dryland rice. LITERATURE CITED 1. Barraquio, W. L., and I. Watanabe. 1981. Occurrence of aerobic nitrogen-fixing bacteria in wetland and dryland plants. Soil Sci. Plant Nutr. 27:121-125. 2. Day, J. M., and J. Dabereiner. 1976. Physiological aspects of N2-fixation by a Spirillum from Digitaria roots. Soil Biol. Biochem. 8:45-50. 3. D6bereiner, J., and V. L. D. Baldani. 1979. Selective infection of maize roots by streptomycin resistant Azospirillum lipoferum and other bacteria. Can. J. Microbiol. 25:1264-1269. 4. Lee, K. K., and I. Watanabe. 1977. Problems of the acetylene reduction technique applied to water-saturated paddy soils. Appl. Environ. Microbiol. 34:654-660. 5. Lee, K. K., and T. Yoshida. 1977. An assay technique of measurement of nitrogenase activity in root zone of rice for varietal screening by the acetylene reduction method. Plant Soil 46:127-134. 6. Watanabe, I., and W. L. Barraquio. 1979. Low levels of fixed nitrogen required for isolation offree living N2-fixing organisms from rice root. Nature (London) 277:565-566. 7. Watanabe, I., W. L. Barraquio, M. R. de Guznan, and D. Cabrera. 1979. Nitrogen-fixing (acetylene reduction) activity and population of aerobic heterotrophic nitrogenfixing bacteria associated with wetland rice. Appl. Environ. Microbiol. 37:813-819. 8. Watanabe, I., and D. A. Cabrera. 1979. Nitrogen fixation associated with the rice plant grown in water culture. Appl. Environ. Microbiol. 37:373-378. 9. Watanabe, I., D. A. Cabrera, and W. L. Barraquio. 1981. Contribution of basal portion of shoot to N2 fixation associated with wetland rice. Plant Soil 59:391-398. 10. Watanabe, I., K. K. Lee, and M. R. de Guzman. 1978. Seasonal change of N2-fixing rate in rice field assayed by in situ acetylene reduction technique. II. Estimate of nitrogen fixation associated with rice plants. Soil Sci. Plant Nutr. 24:465-471. 11. Yoshida, T., and R. R. Ancajas. 1973. Nitrogen fixation activity in upland and flooded rice fields. Soil Sci. Soc. Am. Proc. 37:42-46.