J.E. Cooper

Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection

Abstract In the formation of legume-rhizobia symbioses flavonoids released from roots and seeds are best known as inducers of the bacterial nodulation genes that control the synthesis of reciprocal chitolipooligosaccharide signals (Nod factors) to the prospective host plant. However, successful symbiotic development requires the transmission of a variety of other signals from rhizobia to plant roots and flavonoids initiate or mediate the production of most of them. This review considers the wide range of rhizobial responses to these secondary plant metabolites in the early phases of symbiotic interaction, including chemotaxis, growth stimulation, degradation, Nod factor synthesis, protein secretion by type I and III systems, surface polysaccharide production and expression of many new genes and proteins whose functions are only beginning to be analysed. Attention is also drawn to aspects of flavonoid-rhizobia interaction, such as release of compounds from inoculated roots and the reception of nodulation gene inducers by regulatory NodD proteins, that will need to be revisited by researchers if a complete understanding of the molecular dialogue between the partners is to be achieved.

The occurrence and possible sources of nitrite in a grazed, fertilized, grassland soil

Concentrations of NO2−N in land drainage and river waters in Northern Ireland in recent years have frequently exceeded EEC guide values. Very little information exists to indicate if and when NO2− accumulates in soil solution, and whether NO2− from the soil profile is the source of NO2− in drainage and river waters. The occurrence of NO2− in the field was studied and laboratory incubation experiments carried out to determine the possible sources of NO2− in grassland soil. Field studies were carried out to determine the occurrence and spatial variability of NO2− in a grazed, grassland soil. Plots receiving either 100 or 500 kg N ha−1 yr−1 were systematically sampled in May and October 1992. Concentrations of NO2− in soil were highly variable and ranged from 0 to 2.747 μg N g−1, the data being significantly skewed to the right. Correlation matrices and stepwise multiple regression analyses showed relationships between NO2− and a number of soil variables. Nitrite appeared to be related to variables which indicated its occurrence as a result of nitrification of either fertilizer- or urine-derived NH4+. Nitrate was repeatedly correlated to NO2− concentrations, suggesting that both nitrification and nitrate reduction may be responsible for NO2− formation. Spatially, nitrite occurred at random, basic geostatics producing only one variogram, showing an increase in NO2− concentrations with an increase in distance between sampling points. There was no pattern to the distribution of NO2− with depth, indicating differences in the ratios of the rates of NO2− production and consumption. Numbers of NH3-oxidizers were consistently higher than numbers of NO2−-oxidizers, with some degree of variation between samples. The microbial aspects of NO2− formation are discussed, including partial recycling of NO2− via the NO3− pool, and possible causes of NO2− accumulation due to the inhibition of NO2−-oxidizing bacteria. Laboratory incubation studies were carried out in which measurable NO2− flushes were induced. Increasing soil pH and NH4+ concentrations produced large NO2− flushes, which peaked after about 17 days of incubation, then rapidly declined. Soil incubated with urea produced NO2−N concentrations equivalent to those encountered in the field, suggesting that NH4+ oxidation accounts for a significant proportion of NO2− formed in this soil.

Denitrification in arable soils in relation to their physico-chemical properties and fertilization practice

Abstract Increased use of N-fertilizers in the Czech Republic in recent decades has been accompanied by increased amounts of N compounds in the general environment, and also by shifts in the rates of biological N-processes in agricultural soils. Knowledge of these changes, including rates, distribution and variability of denitrification in soils in the Czech Republic is, however, very limited. In the period 1993–1995, potential denitrification (i.e. denitrifying enzyme activity (DEA) and denitrification potential (DP)), was determined at 13 different sites, in soils with differing physico-chemical and biological properties, but which were typical of large areas in the Czech Republic. In 1994–1995, more detailed research on four differently fertilized plots of a sandy-loam cambisol was conducted on nine occasions in order to analyze seasonal effects and to establish if more than 20 yr of fertilization and selected environmental factors had influenced denitrification. This research was completed in 1996, when 12 differently fertilized plots of the same soil were examined. When a range of soils was investigated, DEA reached values between 15 and 680 ng N 2 O-N g −1 h −1 , corresponding roughly to 1.3–16 mg N kg −1 d −1 , and DP reached values between 6.6–52 mg N kg −1 d −1 . No significant relationship was found between DEA and DP, indicating the independence of the existing metabolic activity of the denitrifier community (DEA) and its potential for rapid development if the environmental conditions change in its favor (DP). While DEA did not correlate with any of the measured soil properties, DP correlated highly significantly with soil pH (H 2 O) and organic carbon content. Detailed research on a sandy loam cambisol showed (i) a large temporal variability of DEA and DP, (ii) an influence of long-term fertilization on denitrification characteristics; DEA and DP responded differently: while DEA increased with all fertilization rates, DP increased in moderately fertilized soils, but decreased significantly in a highly fertilized soil and (iii) a positive correlation of DP with many soil properties, including pH, microbial biomass and dehydrogenase activity.

Screening clover and Lotus rhizobia for tolerance of acidity and aluminium

Abstract Clover rhizobia (55 strains) were screened for tolerance of acidity and Al, using the technique of Keyser and Munns (1979). Assessment of visible turbidity after 14 days indicated three strains tolerant of pH 4.5 (although growth rate was reduced), 25 strains tolerant of 5μ m Al and no strains tolerant of 50 μ m Al at pH 5.5.50 μ m Al caused a decrease in the numbers of acid-tolerant strains at pH 4.5. Tolerance of acidity or Al was not associated with the pH or Al status of the soil from which a strain was isolated. Screening of eight strains of clover rhizobia and nine strains of Lotus rhizobia using turbidity assessment and viable counts indicated seven strains of clover rhizobia with different degrees of tolerance of 20 μ m Al but none tolerant of 50 μ m Al at pH 5.5. All Lotus rhizobia (both slow- and fast-growers) were tolerant of 20 and 50 μ m Al at pH 5.5, with 50 μ m Al causing a reduction in growth rate. Subculturing of strains in non-stressed and stressed media had no effect on the response to 50 μ m Al at pH 5.5.

Aluminium toxicity and multiplication of Rhizobium trifolii in a defined growth medium

Abstract The responses of six strains of R. trifolii to pH, Ca, Al and phosphate were studied in the defined medium of Munns and Keyser (1981) supplemented with vitamins. Wild-type strains responded in the same way as rifampicin-resistant mutant strains. Multiplication was inhibited by acidity alone at pH4.3, and by 50 μM Al at pH 5.5 (despite the precipitation of Al). This inhibitory effect of Al was overcome by increasing the pH above 6.0, or by increasing the phosphate concentration from 10 to 100 μm. Ca concentration had no effect, nor did holding the medium at 22°C for 36 weeks before inoculation. There was variation between strains in their responses to concentrations of A1 Results from these studies and from chemical analyses indicate that the inhibitory effect of Al under partially-neutralized conditions is not due to a direct effect of monomeric A1, nor to an indirect effect of polymeric A1 in reducing the concentration of phosphate, but is due to a direct effect of polymeric A1.

Nitrification in soils incubated with pig slurry

Abstract Incubation studies (5 weeks at 30°C) of nitrification were made in an acid (pH 5.8) and a neutral (pH 7.1) soil receiving varying concentrations of pig slurry and (NH 4 ) 2 SO 4 solution. Mineral-N and pH changes were observed at weekly intervals and inorganic salts media were used to obtain separate estimates of the numbers of NH 4 -N- and NO 2 -N-oxidizing bacteria. In the acid soil, pig slurry NH 4 -N was nitrified to a greater extent than (NH 4 ) 2 SO 4 . In the neutral soil, slurry additions resulted in the accumulation of NO 2 − -N and, in one case, the complete inhibition of nitrification for 4 weeks. Slurry raised the pH of both soils more than (NH 4 ) 2 SO 4 and nitrification in the acid soil was most rapid in a 2 week period of elevated pH following slurry applications. Numbers of Nitroxomonas isolated from the acid soil were considered high enough to account for NH 4 -N oxidation in slurry-treated samples. Numbers of nitrifiers recovered from the incubated neutral soil samples were variable but frequently high enough (>10 4 /g dry soil) to account for observed rates of nitrification. Results are discussed in relation to heterotrophic nitrification in soils, and the practical implications of spreading slurry on agricultural land.

Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia

Abstract The nodulation of Lotus pedunculatus and the multiplication of three Rhizobium loti (fast-growing, acid-producing) and two Bradyrhizobium (slow-growing, alkali-producing) strains was investigated in acidified rooting solution. R. loti strains multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply. No difference in growth rate between R. loti and Bradyrhizobium strains was apparent in rooting solution at pH 6.7. Similar responses to pH were observed in yeast extract-mannitol broth except that Bradyrhizobium strains multiplied more slowly than R. loti at pH 6.7. All strains nodulated L. pedunculatus growing in acid (pH 4.5) rooting solution when presented as single cultures. Following inoculation with 1:1 mixtures of R. loti and Bradyrhizobium strains, R. loti formed 93% of nodules at pH 4.5 and significantly fewer nodules (66%) at pH 6.7. These results demonstrate a competitive advantage for acid-tolerant strains over acid-sensitive strains in nodulation of their lost legume at pH 4.5.

Acidity, aluminium and multiplication of Rhizobium trifolii: Effects of initial inoculum density and growth phase

Abstract Factors influencing the response of Rhizobium strains to acidity and aluminium were studied in an arabinose-galactose-glutamate liquid medium. Rhizobium trifolii strains HP3 and BEL 1192 multiplied at the same rate at pH S.S. BEL1192 multiplied at a slower rate at pH 4.5, and all produced visible turbidity from a low initial cell density. HP3 multiplied at pH 4.5 but produced no turbidity. The pH value of the medium increased when cell densities reached 106-107 cfu ml−1. Diauxic growth responses occurred, only at pH 4.5, and HP3 demonstrated a population effect at pH 4.5 with visible turbidity produced from high initial cell densities. 50 μm Al caused a decrease in numbers of BEL 1192 at pH4.5 and HP3 at pH 5.5 irrespective of initial cell densities. 50 μ m Al was more toxic to HP3 cells in log phase than in stationary phase.

Acidity, aluminium and multiplication of Rhizobium tripolii: Possible mechanisms of aluminium toxicity

Abstract In the absence of Al the lower limit for multiplication by Rhizobium trifolii strain HP3 was pH 4.8–5.0 and for BELI192 was pH 4.4–4.6. 50 μm Al caused inhibition of multiplication of both strains in the range pH 4.6–5.6 despite precipitation of Al at pH values greater than 4.6. An increase in phosphate concentration from 10 to 100 μ m for cells which had been exposed to Al for 14 days at pH 4.5 and 5.5 did not remove the inhibitory effect of Al. Transfer of these cells to fresh Al medium caused a further decrease in numbers although transfer to non-stress medium allowed multiplication to occur. Equilibration of Al medium for 18 months or centrifugation of the medium before inoculation did not reduce the inhibitory effect of Al. Data from these experiments indicate that Rhizobium cells exposed to Al suffer an initial irreversible toxic effect and cells which survive this stage, perhaps due to environmental variation, then suffer a bacteriostatic effect. Calculation of ion activity products p AL + 3 p OH and p Al + p PO 4 and comparison with solubility products for AI(OH) 3 and AIPO 4 successfully predicted the precipitation of Al in the medium, but not the conditions under which Al was toxic to Rhizobium . Therefore, both the toxic form of Al and the mechanism(s) of toxicity remain unclear.

Variation in speed of infection of “no root hair zone” of white clover and nodulating competitiveness among strains of Rhizobium trifolii

Abstract Using a marking technique to determine the location of infectible regions on the roots of white clover, nodulation frequency profiles for three strains of Rhizobium trifolii were obtained every 2 days for 10 days at pH 6.7 and 5.0 in N-free rooting solution. For all strains, the first nodules were formed in the region of the primary root which bore no root hairs (NRH zone) at the time of inoculation. Strains differed significantly in the speed with which they nodulated this zone. There were also similar significant differences in the percentage of plants nodulated by each strain in the NRH zone 10 days after inoculation. At both solution pH values, there was a positive correlation between the speed with which a strain nodulated the NRH zone in single culture and its nodulating competitiveness in mixed culture.