Title: <i>Herbaspirillum</i> colonization increases growth and nitrogen accumulation in aluminium‐tolerant rice varieties
Abstract: New PhytologistVolume 154, Issue 1 p. 131-145 Free Access Herbaspirillum colonization increases growth and nitrogen accumulation in aluminium-tolerant rice varieties Prasad Gyaneshwar, Prasad Gyaneshwar Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines; Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorEuan K. James, Corresponding Author Euan K. James Centre for High Resolution Imaging and Processing, MSI/WTB Complex, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK;Author for correspondence: Euan K. James Tel: +44 (0)138 234 4741 Fax: +44 (0)138 234 5893 Email: [email protected] for more papers by this authorPallavolu M. Reddy, Pallavolu M. Reddy Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines;Search for more papers by this authorJagdish K. Ladha, Jagdish K. Ladha Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines;Search for more papers by this author Prasad Gyaneshwar, Prasad Gyaneshwar Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines; Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorEuan K. James, Corresponding Author Euan K. James Centre for High Resolution Imaging and Processing, MSI/WTB Complex, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK;Author for correspondence: Euan K. James Tel: +44 (0)138 234 4741 Fax: +44 (0)138 234 5893 Email: [email protected] for more papers by this authorPallavolu M. Reddy, Pallavolu M. Reddy Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines;Search for more papers by this authorJagdish K. Ladha, Jagdish K. Ladha Crop, Soil and Water Sciences Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines;Search for more papers by this author First published: 04 April 2002 https://doi.org/10.1046/j.1469-8137.2002.00371.xCitations: 118AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • Varieties of rice (Oryza sativa) differing in tolerance to aluminium (Al) were evaluated for their N-fixation ability after inoculation with a gusA-marked strain of Herbaspirillum seropedicae Z67. • Under axenic conditions, by 30 d, inoculation resulted in enhanced nitrogenase activity, d. wt, total N and total C content only in the Al-tolerant varieties, and one (cv. Moroberekan) showed significantly more 15N2 incorporation than an Al-sensitive variety ('IR45'). There were no differences in the number of the bacteria colonizing the different varieties, but the Al-tolerant ones secreted larger amounts of C in their root exudates, and bacteria colonizing the roots of cv. Moroberekan strongly expressed gusA and NifH proteins. • Under glasshouse conditions, by 30 d, inoculation resulted in increased growth of both cvs IR45 and Moroberekan, but the latter showed significantly greater nitrogenase activity and 15N dilution. In a long-term experiment, by 120 d, cv. Moroberekan showed a significant increase in N content after inoculation. • Herbaspirilla were localized on and within roots and aerial parts of cvs Moroberekan and IR45 under both growth conditions. The role of N fixation in growth promotion of rice by H. seropedicae is discussed in terms of availability of C. Introduction Associative biological nitrogen fixation is a significant feature of the lowland rice systems that provide more than 80% of the world's total rice production. However, rice yields in these systems are very low and must be increased by about 50% in order to meet the projected demands in 2020 (Ladha & Reddy, 2000). This could necessitate a doubling in the use of N fertilizers, which is neither desirable nor sustainable (Ladha & Reddy, 2000). A potential alternative is to increase the contribution made by N fixation. Diverse diazotrophic bacteria abound in the rice rhizosphere (Ueda et al., 1995a,b), and many endophytic N2-fixing bacteria have also been isolated from surface-sterilized tissues of rice (Barraquio et al., 1997; Engelhard et al., 2000; Stoltzfus et al., 1997; Gyaneshwar et al., 2001). The possibility that all or some of these bacteria could be contributing to the N-balance of wetland rice has been given credence by recent 15N dilution studies, both with uninoculated (Boddey et al., 1995; Wu et al., 1995; Shrestha & Ladha, 1996; Malarvizhi & Ladha, 1999) and inoculated (Baldani et al., 2000; Mehnaz et al., 2001; van Nieuwenhove et al., 2001) plants. This associative N fixation is, however, highly variable, ranging from 0 to 36% of total N derived from air, depending on the rice variety (Shrestha & Ladha, 1996; Malarvizhi & Ladha, 1999). The reasons for the high variability in biological N fixation (BNF) associated with grasses are not well understood. It has been suggested that associative N2 fixation, unlike symbiotic N fixation in legumes, is more likely to be affected by genotype and environment (G × E) interactions, since the diazotrophs are only loosely associated with the plant and thus are more vulnerable to changes in the environment (Roger & Ladha, 1992; Malarvizhi & Ladha, 1999). Another possibility is that the low levels of N fixed by both associative and endophytic diazotrophic bacteria result from a limitation of C and energy in the rhizosphere and/or inside the plant. This proposition is supported by several observations. Egener et al. (1999) showed, in rice, that nifH expression by Azoarcus was enhanced in the presence of 5 mg l–1 malate in the assay medium, and rice seedlings inoculated with Azorhizobium caulinodans showed acetylene reduction activity only in the presence of externally supplemented C (van Nieuwenhove et al., 2000). Similarly, studies with wheat and maize inoculated with Azospirillum brasilense (Vande Broek et al., 1993) and Klebsiella pneumoniae (Chelius & Triplett, 2000), respectively, have shown that expression of nifH and/or nitrogenase Fe protein is dependent on additional C being added to the rooting medium. Using acetylene reduction and 15N dilution assays, Christiansen-Weniger et al. (1992) demonstrated that aluminium (Al) tolerant wheat varieties had significantly higher nitrogenase activity as compared with Al-sensitive varieties under axenic conditions and when inoculated with A. brasilense. As tolerance to Al-toxicity in many plants is linked with the enhanced biosynthesis and secretion of organic acids by roots (Pellet et al., 1995; Ma et al., 2000), the study of Christiansen-Weniger et al. (1992) supports the suggestion that grass-associated N fixation could be increased by enhancing the availability of C to the associated and/or endophytic bacteria. In consideration of this, the present study was aimed at determining if availability of C limits N fixation in rice under both axenic and glasshouse conditions, and how this may be overcome by using varieties that are Al-tolerant. Each variety was inoculated with a gusA-marked strain of Herbaspirillum seropedicae Z67 (Barraquio et al., 1997), a diazotroph that was originally isolated from the rhizosphere of rice by Baldani et al. (1986), and which has since been shown to be both an aggressive colonizer of its interior (James et al., 2000; James, 2000) and to promote its growth (Baldani et al., 2000). Materials and Methods Inoculation of rice seedlings with Herbaspirillum seropedicae Z67-gusA Herbaspirillum seropedicae Z67 marked with a transposon based gusA (Barraquio et al., 1997) was maintained on JNFb medium (Olivares et al., 1996) containing spectinomycin (100 µg ml–1) and naladixic acid (10 µg ml–1). Resistance to spectinomycin is encoded by transposon mTn5ssgusA21 (Wilson et al., 1995) and resistance to naladixic acid is a natural property of this bacterium. The herbaspirilla were grown in Luria broth (LB) (10 g−1 l tryptone, 5 g−1 l yeast extract, 10 g−1 l NaCl supplemented with spectinomycin (100 µg ml–1) until they reached an optical density of 0.6. Cells were then harvested by centrifugation (5000 g, 5 min), washed twice with normal saline (0.9% w : v), and resuspended in N-free Fahraeus medium (FM) (Fahraeus, 1957) for use as an inoculum. Five rice varieties ('Moroberekan', 'IRAT104', 'Azucena', 'IR43' and 'IR45') differing in Al-tolerance were selected for axenic experiments: 'Moroberekan', 'IRAT104' and 'Azucena' are considered to be Al-tolerant; 'IR43' and 'IR45' are Al-intolerant (Khatiwada et al., 1996). Dehulled seeds were surface-sterilized in 70% ethanol for 5 min followed by 0.2% mercuric chloride for 30 s, and were washed three times with sterile water. Seeds were germinated on 0.1% (w : v) tryptic soy agar plates, and uncontaminated seedlings transferred to 80 ml glass tubes with 20 ml N-free Fahreus medium and inoculated with 1 ml of suspension containing c. 107 herbaspirilla. Seedlings were placed in a growth chamber for 30 d after inoculation (DAI), but at 7 DAI the plastic top was removed from the tubes and replaced with laboratory film containing a small hole allowing shoot emergence. The plants were maintained in a growth chamber (14 h light/10 h dark cycle, irradiance level 50 µmol m–2 s–1, day/night temperatures 27°C/25°C). Uninoculated plants served as controls. For the glasshouse experiments, 5-d-old seedlings of cvs. Moroberekan and IR45 were treated with a suspension of herbaspirilla cells containing 108 cfu ml–1. After coating for 15 min the seeds were allowed to germinate for 5 d before transplanting into soil amended with or without 15N-labelled urea. At this stage the seedlings had approximately 106 cfu g–1 d. wt of herbaspirilla. The glasshouse experiments were conducted during the dry season, between February and May. Plant growth-promoting activity of Herbaspirillum seropedicae Z67-gusA Plant growth promotion by H. seropedicae Z67 under both axenic and glasshouse conditions was determined by comparing the dry weights, total N and total C of the inoculated plants with uninoculated control plants. All plants were harvested at 30 d after inoculation (DAI) or transplantation (DAT), except for the final glasshouse experiment which was harvested at maturity (120 DAT). The roots and aerial parts (and grain in the long-term experiment) were dried to a constant weight in an oven and ground to a fine powder for estimation of N and C content using a Perkin-Elmer 2400 CHN analyser (Perkin-Elmer, Norwalk, CT, USA) (Jimenez & Ladha, 1993). Collection and analysis of root exudates In a separate experiment, seedlings germinated from surface-sterilized seeds were transferred to glass tubes containing N-free liquid Fahreus medium and were allowed to grow for 10 d in a growth chamber with a 14 h light/10 h dark cycle and day/night temperatures of 27°C/25°C, with an irradiance of 50 µmol m–2 s–1. The C content of the root exudates was measured by collecting the exudates in the rooting medium, which was then freeze-dried and analysed using a Perkin-Elmer 2400 CHN analyser (Jimenez & Ladha, 1993). Nitrogenase (acetylene reduction) activity The acetylene reduction activity of inoculated plants was determined according to Ladha et al. (1986). Ten seedlings from each axenic treatment were taken at 10 DAI and washed twice with sterile distilled water to remove loosely associated bacteria. The seedlings were then transferred to fresh, N-free, liquid Fahreus medium. With the Al-sensitive varieties the Fahreus medium of an additional set of seedlings was supplemented with 10 mm sodium malate as a C source. The tubes containing the plants were sealed with a rubber seal and 10% of the headspace volume was replaced with acetylene. They were then returned to the growth chamber and incubated in the dark for 12 h at 30°C, after which any ethylene produced was determined using a Hitachi 164F gas chromatograph (Hitachi Instruments Service Co, Tokyo, Japan). Uninoculated plants and tubes not injected with acetylene served as controls. Glasshouse-grown plants were removed from the soil, washed with tap water to remove the soil, and transferred to glass tubes within which their acetylene reduction activity was determined as above. Incorporation of 15N2 into inoculated rice seedlings In a separate experiment, seedlings of the varieties 'Moroberekan' and 'IR45' derived from surface-sterilized seeds were inoculated with H. seropedicae Z67-gusA in 80 ml glass tubes and allowed to grow for 7 d. The tubes were then sealed with Suba seals and 5% of the headspace volume of half the number of tubes was replaced with 15N2 (99.5%, Monsanto Research Corp, Miamisburg, OH, USA), while the other set of tubes contained normal air. Tubes were returned to the growth chamber and after 3 d of incubation the plants from both sets of tubes were harvested and dried at 70°C to a constant weight in an oven before being ground to a fine powder that was analysed for its 15N content with a mass spectrometer (VG-Model 903) equipped with a Dumas elemental analyser (Roboprep-CN 7001, Europa Scientific Ltd, Crewe, UK). Uninoculated seedlings served as controls. The total amount of 15N fixed was calculated according to Nayak et al. (1986) as (15N atom percentage excess of sample)/(15N atom per cent excess of gas) × total N, and the percentage N derived from air (%Ndfa) was calculated as (15N atom per cent excess (tissue) – 15N atom percentage excess (background))/(15N atom per cent excess (atmosphere) –15N atom per cent excess (background)) × 100, where 15N atom per cent (background) = 0.010 ± 0.006 and 15N atom per cent excess of atmosphere = 4.9. Measurement of N fixation under glasshouse conditions using soil amended with 15N-labelled urea The 15N-labelled soil in the glasshouse experiments was the Bulacan soil used previously in the studies of Shrestha & Ladha (1996) and Malarvizhi & Ladha (1999). It had originally been amended with 15N-labelled urea that had been stabilized under flooded conditions for 7 wk before planting (Malarvizhi & Ladha, 1999). The soil had the following characteristics: pH 6.3, organic C = 1.3 g kg–1, Kjeldhal N = 0.11 g kg–1, available Olsen P = 33 mg kg–1, 15N atom per cent excess = 0.158. Twelve kilograms of the wet soil was placed into each plastic tray and supplemented with 250 mg P and 230 mg K as KH2PO4 before transplanting four 7-d-old rice seedlings, with or without inoculation with H. seropedicae Z67-gusA. The plants were sampled at 30 DAT (10 plants per treatment), washed with tap water and dried in an oven to a constant weight. The roots and shoots were separated and ground to a fine powder, which was then used for analysis of total N and 15N content as described earlier. The percentage Ndfa was calculated according to Shrestha & Ladha (1996) as (15N atom percentage excess test genotype)/(15N atom percentage excess of genotype with highest 15N enrichment) × 100. Long-term glasshouse experiment Seedlings of cvs Moroberekan and IR45 were inoculated with either live or heat-killed H. seropedicae Z67 and grown in a glasshouse as described previously, except that the Maahas soil used in this experiment was not amended with 15N-labelled urea. Enumeration of bacteria Axenically grown plants were sampled at 10 DAI. Loosely attached bacteria were removed by washing the roots in sterile water. The roots were then immersed in 5 ml sterile distilled water and vortexed for 30 s. The resulting solution was serially diluted and plated on JNFb (without bromothymol blue) agar plates containing spectinomycin (100 µg ml–1), naladixic acid (10 µg ml–1) and 5-bromo-4-chloro-3-indolyl-β-glucoronide (X-gluc) (40 µg ml–1). Counts of bacterial colonies were then determined, and these counts were assumed to be those bacteria that were closely associated with the root surface. In another set, the roots were surface-sterilized by immersion in 95% ethanol for 5 min, followed by treatment with 3% calcium hypochlorite containing 0.1% sodium dodecyl sulphate (SDS) for 1 min. After three washes with sterile distilled water, followed by maceration in saline, the homogenate was serially diluted and plated on JNFb agar as described above. In the case of the glasshouse experiment, plants were sampled at 0, 10 and 30 DAT and examined for the presence of bacteria on and within the roots and aerial parts as for the axenically grown plants. To enumerate the total population of heterotrophic bacteria in washed, but not surface-sterilized plants, the solution was plated onto Luria agar plates. Identification of bacteria isolated from glasshouse-grown plants Bacteria isolated from roots and aerial parts at 10 and 30 DAT were analysed by fingerprinting using BOX-polymerase chain reaction (PCR) amplification fragment length polymorphism as described by Verslovic et al. (1994). The BOX A1R primer (5′-CTACGGCAAGGCGACGCTGACG-3′) was used at 50 pmol with 100 ng template DNA in a 25-µl PCR reaction mixture containing 1.25 mm of each dNTP, 2 U AmpliTaq DNA polymerase (Perkin Elmer) in a reaction buffer with 10% dimethyl sulphoxide (DMSO) (v/v). The reaction buffer stock (5×) contained 83 mm ammonium acetate, 335 mm Tris-HCl, 33.5 mm MgCl2, 33.5 µm EDTA, 150 mmβ-mercaptoethanol, 850 µg ml–1 bovine serum albumin (BSA), pH 8.8. Amplification for PCR was performed in a BIOMETRA Uno-Thermocycler (Biometra, Göttingen, Germany) with an initial denaturation (95°C, 7 min) followed by 30 cycles of denaturation (95°C, 30 s); annealing (52°C, 1 min) and extension (65°C, 8 min), with a single final extension step (65°C, 16 min). After the reaction, the samples were separated on 1.5% agarose gels, stained with ethidium bromide and visualized on a UV transilluminator. Microscopy At least three seedlings from three independent inoculations were collected at 10 DAI, washed with sterile distilled water and stained for GUS activity in 50 mm potassium phosphate buffer (pH 7.0) containing 400 µg ml–1 X-gluc for 4–6 h (Jefferson et al., 1987). After staining, the leaves were cleared by immersion in 90% ethanol for 15 min. For glasshouse grown plants, the material was sampled at both 10 and 30 DAT, and then washed and incubated in the same assay system as for axenic plants, but in this case it was supplemented with chloramphenicol (200 µg ml–1) to inhibit the induction of β-glucuronidase by native plant-associated bacteria (Wilson et al., 1995). With both laboratory and glasshouse materials, roots and shoots showing blue colour were cut into small pieces (1–2 mm) and fixed in 4% glutaraldehyde (in 50 mm phosphate buffer, pH 7.0, containing 0.1% (v : v) Triton-X-100 (Sigma Chemical Co., St. Louis, MO, USA). The samples in fixative were then immediately placed under vacuum for 30 min, followed by overnight storage at atmospheric pressure. Inoculated and uninoculated roots were then prepared for examination on the cold stage of a Hitachi 4700 field emission-scanning electron microscope (FE-SEM). This involved plunging them into liquid N2 slush, etching at –95°C for 15 min to remove surface water, and then sputter-coating with 5 nm of gold-palladium in an Oxford Alto cryo-preparation chamber (Gatan, Oxford, UK). The samples were then examined at –130°C at an accelerating voltage of 5 kV. Further samples were incubated for 2 h in a polyclonal antibody raised against H. seropedicae Z67 (James et al., 1997) diluted 1 : 400 in IGL buffer (James et al., 1994), followed by 1 h in a 1 : 50 dilution (in immunogold labelling (IGL) buffer) of 15 nm gold particles conjugated to goat antirabbit antibodies (Amersham, Aylesbury, UK). The gold labelled roots were then dehydrated in an ethanol–acetone series, and critical point dried in a Bal-Tec CPD 030 critical point dryer (Balzers, Fürstentum, Liechtenstein). Before being viewed under the FE-SEM, the samples were coated with 2 nm of chromium in a Cressington 208 sputter coater (Cressington Scientific Instruments Ltd, Watford, UK), followed by 5 nm of carbon in an Agar Turbo carbon coater. The samples were viewed in the FE-SEM at an accelerating voltage of 30 kV, with or without a yttrium aluminium garnet (YAG) back scattered electron (BSE) detector inserted. Other X-gluc-stained samples were prepared for light and transmission electron microscopy (TEM) according to James et al. (1997). The identity of the bacteria in the sections was confirmed by immunogold labelling (IGL) with a polyclonal antibody raised against H. seropedicae Z67. The cross-reaction of the antibody against various bacteria commonly isolated from the rhizosphere/interior of rice was determined by enzyme-linked immunosorbent assay (ELISA), and no cross-reactions with bacteria other than H. seropedicae Z67 were observed at any dilution. To detect in situ expression of nitrogenase, an antibody raised against the Fe (nifH)-protein of the nitrogenase enzyme complex of Rhodospirillum rubrum (a gift from Dr P. Ludden, Madison, WI, USA) was also used for IGL (diluted 1 : 100 in IGL buffer). Controls for IGL with the anti-H. seropedicae or anti-nitrogenase Fe-protein antibodies were: omission of the primary antibody; and substitution of the primary antibody with non-immune serum, diluted appropriately in IGL buffer (1 : 400 or 1 : 100). Data analysis The data were compared by analysis of variance or t-tests. Results Effect of inoculation on growth and N fixation of rice seedlings under axenic conditions Inoculation with H. seropedicae Z67 resulted in enhancement of the d. wt of all the Al-tolerant varieties, ranging from 38 to 54% for roots and from 22 to 50% for shoots (Table 1). These d. wt increases were accompanied by significant enhancements in total C (35–50% for roots and 13–35% for shoots) and total N (29–61% for roots, 37–85% for shoots). By contrast, inoculation had no significant effect on the growth, total C and total N contents of the Al-sensitive varieties, IR43 and IR45 (Table 1). Table 1. Effect of inoculation with Herbaspirillum seropedicae Z67-gusA on growth, C and N contents of five rice varieties grown under axenic conditions for 30 d Variety Inoculation D. wt (mg per plant) Total C (mg per plant) Total N (mg per plant) Root Shoot Root Shoot Root Shoot 'Moroberekan' – 2.76 6.91 1.12 3.51 0.10 0.16 (0.37) (1.24) (0.16) (0.28) (0.03) (0.08) + 4.18** 10.37** 1.69* 4.37* 0.14** 0.29** (0.62) (1.45) (0.14) (0.32) (0.03) (0.10) Increase (%) 51.4 50.0 50.4 24.5 45.9 85.4 'IRAT104' – 5.88 12.65 2.17 4.61 0.07 0.36 (0.95) (1.05) (0.28) (0.20) (0.02) (0.03) + 9.04** 15.47** 3.16* 5.20* 0.13** 0.49* (1.26) (1.22) (0.25) (0.24) (0.04) (0.06) Increase (%) 53.7 22.3 45.2 12.7 61.2 37.0 'Azucena' – 4.04 9.17 1.60 3.62 0.05 0.24 (0.52) (1.22) (0.16) (0.35) (0.01) (0.03) + 5.56* 12.94* 2.16* 4.87* 0.07* 0.36** (0.46) (1.45) (0.28) (0.41) (0.01) (0.04) Increase (%) 37.6 41.1 34.7 35.0 28.8 48.3 'IR43' – 3.00 11.23 0.88 2.24 0.04 0.27 (0.68) (1.14) (0.18) (0.28) (0.01) (0.05) + 4.04 11.89 1.04 2.57 0.05 0.28 (0.52) (1.06) (0.26) (0.24) (0.01) (0.02) 'IR45' – 3.80 11.85 0.65 1.92 0.03 0.26 (0.55) (1.12) (0.15) (0.32) (0.01) (0.05) + 4.05 12.23 0.92 2.11 0.05 0.30 (0.61) (1.28) (0.22) (0.47) (0.01) (0.06) *, **For each variety, values for inoculated plants are significantly different from controls at P = 0.05 and P = 0.01, respectively. Values are means of two experiments with 10 replicates. Standard deviations are in parentheses. The Al-tolerant varieties 'Moroberekan', 'IRAT104' and 'Azucena' showed substantial acetylene reduction activity when inoculated with H. seropedicae Z67, ranging from 0.8 to 1.3 µmol C2H4 g–1 d. wt h–1 (Table 2). By contrast, no activity was detected with the Al-sensitive varieties, IR43 and IR45, except when carbon (10 mm sodium malate) was supplemented in the growth medium (Table 2). Addition of carbon to the Al-tolerant varieties did not significantly affect their nitrogenase activity (Table 2). With all the varieties, both Al-tolerant and sensitive, activity was abolished after surface-sterilization. Table 2. Nitrogenase (acetylene reduction) activity of five rice varieties after inoculation with Herbaspirillum seropedicae Z67-gusA under axenic conditions Variety Tolerance to Al Nitrogenase activity (µmol C2H4 g–1 d. wt h–1)1 Uninoculated Inoculated Inoculated + 10 mm sodium malate Inoculated and surface sterilized 'Moroberekan' Tolerant Nil 1.28 (0.45) 1.35 (0.65) Nil 'IRAT104' Tolerant Nil 0.83 (0.50) 1.23 (0.55) Nil 'Azucena' Tolerant Nil 1.04 (0.62) 1.24 (0.48) Nil 'IR43' Sensitive Nil Nil 0.91 (0.35) Nil 'IR45' Sensitive Nil Nil 0.79 (0.42) Nil Values are means of two separate experiments with 10 replicates. Standard deviations are in parentheses. 1Assays were performed at 10 d after inoculation with Herbaspirillum seropedicae Z67-gusA. The Al-tolerant variety 'Moroberekan' showed significantly higher incorporation of 15N2 compared with the Al-sensitive variety 'IR45' when inoculated with H. seropedicae Z67 (Table 3). The total N fixed was also significantly higher in 'Moroberekan' than in 'IR45'. Moreover, upon inoculation, the percentage N derived from air was only 0.42 for 'IR45', whereas 'Moroberekan' could derive 4.66% of its N from air (Table 3). Table 3. Effect of inoculation with Herbaspirillum seropedicae Z67-gusA on incorporation of 15N2 into two rice varieties grown under axenic conditions Variety Inoculation 15N atom % excess1 Total 15N fixed (µg per plant) % N derived from air 'Moroberekan' − 0.028 1.406 0.038 'Moroberekan' + 0.129* 15.993* 4.659* 'IR45' − 0.006 1.033 0.041 'IR45' + 0.059* 3.585* 0.418* LSD (5%) 0.009 0.935 0.360 * For each variety, values for inoculated plants are significantly different from controls at P = 0.05. 1 Assays were performed at 10 d after inoculation with Herbaspirillum seropedicae Z67-gusA and 3 d after injection with 15N2. Values are means of two separate experiments with three replicates each. Enumeration and localization of Herbaspirillum seropedicae Z67-gusA under axenic conditions There were no significant differences between Al-tolerant and Al-sensitive varieties in the number of herbaspirilla that could be isolated from either the surface or from the internal tissues of inoculated plants (Table 4). In all cases, the bacterial numbers on the root surface were approximately log 5 cfu g–1 d. wt whereas only log 2–3 cfu g–1 d. wt could be isolated from the surface of the shoots. In addition to the surface bacteria, log 3 cfu g–1 and log 1 cfu g–1 d. wt of herbaspirilla could be re-isolated from surface-sterilized roots and shoots, respectively. No bacteria could be isolated from the uninoculated control plants under the test conditions. Table 4. Enumeration of Herbaspirillum seropedicae Z67-gusA colonizing five rice varieties grown under axenic conditions Rice variety Total number of bacteria (log cfu g–1 d. wt) Root surface Root internal Shoot surface Shoot internal 'Moroberekan' 5.4 3.2 3.0 1.5 'IRAT104' 5.2 3.1 2.8 1.8 'Azucena' 5.0 3.4 2.5 1.9 'IR43' 5.6 3.0 2.8 1.2 'IR45' 5.0 3.1 2.5 1.3 Standard deviation 1.2 0.6 0.9 1.0 Values are means of two separate experiments with three replicates. Counts were performed at 10 d after inoculation with Herbaspirillum seropedicae Z67-gusA. The physiological status of the inoculated bacteria associated with the seedlings was determined at 10 DAI by staining for glucuronidase (GUS) activity. Roots of Al-tolerant varieties inoculated with H. seropedicae Z67-gusA showed more a intense blue colour of GUS than the roots of corresponding Al-sensitive varieties (Fig. 1a,b). This was also true under nonsterile conditions (Fig. 1c,d; data not shown). Uninoculated control plants showed no blue coloration (not shown). Ultrastructural studies with material frozen for cryo-scanning electron microscopy showed that the deeply stained roots of cv. Moroberekan had a much denser surface population than the lighter-stained roots of cv. IR45 (Fig. 2a,b); the identity of the bacteria viewed under the SEM being confirmed by immunogold labelling with an antibody raised against H. seropedicae Z67 (Fig. 2c,d). The difference between the varieties in terms of surface bacterial populations was further confirmed when GUS-stained roots were sectioned for light microscopy and transmission electron microscopy (Fig. 3a–d), with the roots of cv. Moroberekan having a dense coating of bacterial colonies and those of cv. IR45 having relatively few (Fig. 3b). Interestingly, only a few bacteria were observed within the root interior of either variety (Fig. 3a–c). In cv. Moroberekan, single bacteria were occasionally seen within exodermal cells (Fig. 3a,c), whereas cv
Publication Year: 2002
Publication Date: 2002-04-01
Language: en
Type: article
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