Louise M. Nelson

PLANT GROWTH-PROMOTING RHIZOBACTERIA: EFFECTS ON GROWTH AND NITROGEN FIXATION OF LENTIL (LENS ESCULENTA MOENCH) AND PEA (PISUM SATIVUM L.)

Abstract The potential of a number of putative plant growth-promoting rhizobacterial strains to enhance the growth and nitrogen fixation of western Canadian cultivars of lentil ( Lens esculenta Moench) and pea ( Pisum sativum L.) was assessed in field and laboratory studies. Nine strains were tested on a single cultivar of lentil (Eston) and pea (Trapper) in the field. None of the strains had any effect on growth of pea in the field, but in lentil inoculated with one or more of the rhizobacterial strains, there were significant increases in emergence, vigor, nodulation, C 2 H 2 reduction activity and root weight. Further laboratory studies conducted with the two best strains, G2-8 and G11-32 and lentil cv. Eston used four in vitro cultivation systems to determine the optimal conditions for plant growth enhancement. There was variation among experiments and cultivation systems but growth stimulation similar to that in the field was observed in trials with a sand column system. The best plant growth-promoting strain for Eston under in vitro conditions, G2-8, was then tested with lentil cv. Laird and plants inoculated with G2-8 had higher root and shoot dry weights and greater acetylene reduction than control plants in pot and sand column systems. Leonard jar and growth pouch systems were not satisfactory methods for observing growth enhancement of lentil by these bacterial strains, probably due to the slow growth rate of plants and watering method in the former system and the large seed size and short assay time in the latter. Results from these experiments suggest that strains such as G2-8 and G11-32 may be of value as adjunct inoculants for lentil, but effects are dependent on the cultivar and cultivation system used.

Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions

Most of the production of lentil (Lens culinaris) on the Great Plains occurs on soils that are free of indigenous Rhizobium leguminosarum. Inoculation is required to increase yields through N2 fixation. A screening program to evaluate the effectiveness of R. leguminosarum strains for lentil was initially carried out under controlled environments followed by an evaluation under field conditions. In two separate growth room experiments, the effectiveness of 185 and 24 different strains of R. leguminosarum were tested for Laird and Eston lentil. Significant differences between strains in number of nodules, shoot weight and nitrogenase activity (acetylene reduction activity, ARA) were found for lentil grown for 5 weeks. When lentil were grown for 7 weeks, significant differences between strains in number of nodules, total plant weight, total N, and % N were observed.Fourteen strains plus Nitragin ‘C’ inoculant were selected for further field testing on Eston and Laird lentil at two locations in 1986 and one site in 1987. Inoculation increased yield up to 135%. Percent Ndfa and total N2 fixed ranged from 0 to 76 and 0 to 105 kg ha-1, respectively. N2-fixing activity was site specific and higher spring soil NO3-levels resulted in lower N2-fixing activity. Depending on site and growing conditions, strains 99A1 and I-ICAR-SYR-Le20 appeared to be superior to the other strains tested. A good agreement was found between the estimates for N2 fixation based upon the 15N-isotope dilution and the classical N difference methods. Number of nodules, dry weight of nodules and ARA of Eston and Laird lentil grown under growth room conditions failed to show positive correlations with total dry matter production, total N or total N2 fixed of field grown lentil. However, total plant weight and total N of lentil grown under growth room conditions were highly correlated with field parameters, and were the most reliable screening parameters for the selection of superior rhizobial strains.

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring β-glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.

Field and laboratory studies of Triticum aestivum L. inoculated with co-existent growth-promoting Bacillus strains.

Abstract We have shown previously that six strains belonging to the genus Bacillus isolated from the rhizosphere of spring wheat cv. Katepwa were able to promote root growth of Katepwa but not of the parental cv. Neepawa under sterile conditions in test tubes. Further studies with strains 5A1 or 3A have shown that the growth promotion can be demonstrated under a variety of growth conditions, in sterile Leonard jars, non-sterile pots or in the field. A heat-labile factor may be involved in the mode of action because autoclaving 5A1 cells inhibited growth promotion but addition of supernatant was as effective as whole cells. IAA was detected in the supernatant using GC-MS but exogenous addition of up to 200 μg IAA per pot did not affect the growth of cv. Katepwa or Neepawa. The growth response of cv. Katepwa to inoculation with strain 5A1 was similar over a range of inoculum densities from 107 to 1010 cells per pot, but the lowest density resulted in the largest growth response. A time-course experiment with strain 5A1 and cv. Katepwa indicated that growth promotion was initiated within 2 weeks of sowing, but that the growth response was not significant until 3 weeks after sowing. In the field, strain 3A increased the rate at which Katepwa seedlings emerged. Inoculation of the same cultivar with strain 5A1 increased shoot weight and tiller number by 38% (P

Role of uptake hydrogenase in providing reductant for nitrogenase in rhizobium leguminosarum bacteroids

Abstract The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H 2 increased the rate of C 2 H 2 reduction in the absence of added substrates. Malate also increased nitrogenase (C 2 H 2 ) activity while decreasing the effect of H 2 . At exogenous malate concentrations above 0.05 mM no effect of H 2 was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H 2 uptake was not coupled to ATP formation, H 2 increased the rate of C 2 H 2 reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C 2 H 2 reduction, but when H 2 was also included, the rate of C 2 H 2 reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H 2 uptake is coupled to ATP formation, to the nitrogenase complex.

Response of Rhizobium leguminosarum isolates to different forms of inorganic nitrogen during nodule development in pea (Pisum sativum L.)

Abstract Combined nitrogen inhibits nodule development and nitrogen fixation in Rhizobium -legume symbioses. Isolates of R. leguminosarum which had been shown to vary in symbiotic effectiveness in the presence of NN 4 NO 3 were used as inoculum for peas grown with NH 4 NO 3 KNO 3 or NH 4 Cl and harvested between 14 and 24 days after planting. Plants grown in 2m m NH 4 NO 3 showed inhibition of nodule development and function 17 days after seeding but there were no significant differences (SDs) between isolates of high and low effectiveness. In the presence of 5 m M NH 4 NO 3 nodule development and C 2 H 2 reduction were severely inhibited from 14 days onwards. Plants inoculated with isolates of high effectiveness had more tap-root nodules, higher C 2 H 2 reduction rates and higher leghemoglobin content than those inoculated with isolates of low effectiveness. In plants grown with 5 m m KNO 3 or 5 m m NN 4 Cl, NO 3 − had the major inhibitory effect on nodulation and nodule development, but NH 4 + began to show inhibitory effects on nodule development after 17 days. There were no SDs between isolates of low and high effectiveness at these concentrations of NO 3 − and NH 4 + . Differences in symbiotic response of R. leguminosarum to combined nitrogen are small and appear during nodule development rather than at nodule initiation.

Variation inRhizobium leguminosarum response to short term application of NH4NO3 to nodulatedPisum sativum L.

SummaryThis study was conducted to determine the effect of short term application of NH4NO3 on nodule function and to determine whether the rhizobial isolate used was a significant factor in this effect. Pea plants were inoculated with 10 differentRhizobium leguminosarum isolates and grown for 3 weeks in N-free medium before addition of 0, 1, 2 or 5 mM NH4NO3 for 2 to 7 days. Acetylene reduction and leghemoglobin content decreased with increasing exposure time to NH4NO3 and with increasing concentration of NH4NO3. NH4+ and NO3− depletion from the nutrient medium were assayed in plants exposed to 5 mM NH4NO3 and mean uptake rates were similar for each ion. There were significant differences among isolates in the rate of decrease of C2H2 reduction with increasing NH4NO3 concentration (C2H2 reduction responsiveness to NH4NO3) 4 and 7 days after addition of NH4NO3 but no differences after 2 days of exposure to NH4NO3. There were significant differences among isolates in NH4+ depletion from the nutrient medium but these differences were not correlated with the differences observed in C2H2 reduction. Ranking of the isolates for C2H2 reduction responsiveness to NH4NO3 applied to plants with nodules was different from that obtained when NH4NO3 was applied at seeding. Isolates with varying sensitivity to NH4NO3 may be useful tools for determining the mechanisms responsible for inhibition of symbiotic N2 fixation by combined nitrogen.

TISSUE CULTURE BIOASSAY FOR PLANT GROWTH PROMOTING RHIZOBACTERIA

Abstract Plant growth promoting rhizobacteria (PGPR) Pseudomonas putida strains G2-8 and Gll-32 were tested for growth promotion of soybean callus in a tissue culture bioassay. When grown on a nutrient medium without naphthaleneacetic acid or kinetin. PGPR did not affect callus biomass. However, when growth regulators were included in the growth medium, but vitamins (thiamine HCl, nicotinic acid, pyndoxine HCl. and myoinositol) omitted, inoculation with strain Gl1 -32 significantly increased the callus biomass. Strain G2-8 decreased the callus biomass without vitamins, but the effect was not significant in any single experiment. Results are discussed in relation to the usefulness of tissue-culture based bioassays for PGPR and the mechanism by which PGPR stimulate plant growth.

Interactions among nitrogen-transforming bacteria and nitrogen-fixingPisum sativum L. in laboratory sand columns

An open laboratory sand column system was developed to study interactions between N2-fixing pea,Pisum sativum L. and the nitrifying organisms,Nitrosomonas europaea andNitrobacter winogradskyi or the denitrifier,Pseudomonas fluorescens in the presence of NH4-ion or NO3-ion, respectively. In columns watered continuously with 1 mM (NH4)2SO4, nitrification began immediately after inoculation and NO3-ion production was not inhibited by the plants. Above ambient levels of nitrous oxide were detected in all columns containing nitrifiers. In the presence of plants, nitrifiers were evenly distributed throughout the column; in their absence, they were found predominantly within the top 8 cm of the column. When15N-enriched (NH4)2SO4 was added to columns containing nitrifiers and plants,15N-enriched NO3-ion was detected in the xylem sap. Nitrogenase activity (as indicated by acetylene reduction rates and atom %15N of shoot N) was inhibited more in the presence of nitrifiers and NH4-ion than with NH4-ion alone. In columns continuously watered with 2 mM KNO3 and containing a denitrifier, low levels of denitrification were detected in columns maintained at 21% O2 or at 5% O2. Nitrate uptake was inhibited from days 26 to 31 in peas grown at 5% O2. Denitrification may have been limited by available carbon in the root exudates or by competition with roots for NO3-ion and rates were insufficient to counteract the inhibitory effects of NO3-ion on symbiotic nitrogen fixation.

Localization of NIF - and HUP -Specific Sequences on the Plasmids of Newly Isolated Strains of Rhizobium Leguminosarum

Thirtyfive strains of R. leguminosarum were isolated from pea plants grown at two different locations. Location “B” was a garden bed in which pea plants had been grown annually for at least 4 years. Location “W” was a meadow in which pea plants had not been grown for a number of years. The plasmid content of the isolated strains has been analyzed previously (Tichy, Lotz 1981).