J. Brockwell

Manipulation of rhizobia microflora for improving legume productivity and soil fertility: A critical assessment

Inputs of biologically fixed nitrogen derived from the symbiotic relationship between legumes and their root-nodule bacteria into terrestrial ecosystems amount to at least 70 million metric tons per year. It is obvious that this enormous quantity will need to be augmented as the worlds population increases and as the natural resources that supply fertilizer nitrogen diminish. This objective will be achieved through the development of superior legume varieties, improvement in agronomic practice, and increased efficiency of the nitrogen fixation process itself by better management of the symbiotic relationship between plant and bacteria. This paper considers ways and means by which populations of root-nodule bacteria, established and introduced, can be manipulated ecologically, agronomically, edaphically and genetically to improve legume productivity and, as a consequence, soil fertility.

Evaluation of the symbiotic nitrogen-fixing potential of soils by direct microbiological means

A method for estimating the nitrogen-fixing capacity of a population of rhizobia resident in soil is presented. legume test plants, growing under microbiologically-controlled conditions in test tubes packed with a vermiculite substrate moistened with a nitrogen-free plant nutrient solution, are inoculated directly with a suspension of the soil under examination. Rhizobia in the soil nodulate the test plants, and the amount of foliage dry matter produced in the 28 days after inoculation is regarded as an index of their effectiveness. An inoculum of at least 30, and preferably 100, rhizobia is needed to ensure that nitrogen fixation is not masked by delayed nodulation. The new method is tentatively described as the ‘whole-soil inoculation’ technique.Appraisals were made withTrifolium subterraneum L. andRhizobium trifolii and withMedicago sativa L. andR. meliloti. Soil-borne pathogens did not interfere with plant growth. The whole-soil inoculation technique was less tedious and time-consuming than an alternative method which involved extracting representative isolates from the soil and testing their effectiveness individually, and appeared to give more realistic values for the nitrogen-fixing capacity of the soil as a whole. Used in association with a field experiment, the whole-soil inoculation technique confirmed microbiologically that there had been an agronomic response to surface application of inoculant to poorly-nodulatedT. subterraneum pasture.It is submitted that this technique for determining the effectiveness of rhizobia in soil, combined with a plant-infection method for counting rhizobia, can be a reliable guide to the need for inoculation in the field.

Studies of field populations of rhizobium: Effectiveness of strains of rhizobium trifolii associated with trifolium subterraneum L. pastures in South-Eastern Australia

Abstract Field populations of Rhizobiuin trifollii from eight regions in south-eastern Australia were sampled over a period of 5 years from 1966 to 1970. The R. trifolii isolates were tested under bacteriologically-and environmentally-controlled conditions for effectiveness of nitrogen fixation in combination with Trifalium subterraneum L. cv. Bacchus Marsh; the effective strain TA1 was used as a standard of comparison. Mean effectiveness of the R. trifolii populations for any region at any sampling varied between 62 and 93 percent of the effectiveness of the standard strain. The principal feature was the large variability within sampling sites, between sites within paddocks, and between paddocks within regions. In addition there was some variability with time in the range of effectiveness values of isolates within a site and in the absolute values for both sites and paddocks. Effectiveness values were not related to soil pH, size of population of R. trifolii, inoculation procedure at sowing, age of pasture, annual rate of fertilizer application, or mean annual rainfall.

The number of Bradyrhizobium SP. (Lupinus) applied to seed and its effect on rhizosphere colonization, nodulation and yield of lupin

Abstract In field experiments we investigated the roles of inoculum potential of Bradyrhizobium sp. ( Lupinus ) and quantity of peat used to inoculate seed on the nodulation and yield of lupins. Within the range 0.125–3 times the Australian recommended rate of peat application (2.5 g peat kg −1 lupin seed), the amount of peat had no effect on nodulation or grain yield. In the first experiment, seven inoculum potentials were applied within the range log 10 0.32–6.28 bradyrhizobia per seed in 7, 10-fold increments which spanned the recommended rate of login 5.55 per seed. Inoculum potentials of log 10 6.27 and 5.27 improved the colonization of lupin rhizospheres and increased early nodulation, nodule number and nodule mass. Nodule mass was increased from 65 to 393 mg plant −1 at 43 days by increasing the inoculum from log 10 4.27 to 6.27 bradyrhizobia seed −1 . Grain yield and % N in the grain were not significantly different ( P > 0.05) between potentials of logio 4.27 and 6.27. In the second experiment, higher potentials of 6.80 and 7.28 further improved rhizosphere colonization and increased nodule mass. Studies of the survival of the inoculum, during inoculation, sowing and in the soil, identified a large mortality factor; 95% of bacteria died between inoculation and sowing and of those surviving, 83% died after 22.5 h in the soil. These observations have important implications for setting new standards for commercial inoculants and for emphasizing the care needed in handling inoculated seed to reduce the death of bradyrhizobia in the period between inoculation and sowing.

Ecological studies of root-nodule bacteria introduced into field environments—V.A critical examination of the stability of antigenic and streptomycin-resistance markers for identification of strains of Rhizobium tripolii

Abstract Marked strains of Rhizobium trifolii, distinguishable from other strains antigenically and by streptomycin resistance, were introduced by seed inoculation of subterranean clover (Trifolium subterraneum L.) into a field environment having a natural population of R. trifolii. Isolates from nodules obtained periodically during the following 41 months were classified using both methods of identification in parallel. This procedure made it possible to determine the reliability of each method independently. There was a gradual disappearance of the inoculum strains which occurred more rapidly in plots of cv. Woogenellup than in plots seeded with cv. Mount Barker. At five harvests, there was 95% (or greater) correspondence between inoculum survival using either method of identification. There was evidence that a small proportion of the progeny of the inocula sustained independent loss of antigenic character and/or streptomycin resistance in the field or, alternatively, that strains occurring naturally acquired these characteristics. A few nodules contained more than one strain of rhizobia. These exceptions occurred at low frequency and did not interfere substantially with identification results. It is concluded that gel immune diffusion serology and the use of streptomycin-resistant mutants are both reliable methods for identifying strains of rhizobia reisolated from field environments.

Contributions of fixed nitrogen and soil nitrate to the nitrogen economy of irrigated soybean

Abstract Effects of soil nitrate and numbers of Bradyrhizobium japonicum on the development and functioning of a soybean symbiosis and on crop production were studied in a field experiment at Breeza, New South Wales, Bragg soybean was grown with irrigation on soil, initially free of B. japonicum , with four rates of fertilizer-N (0, 100, 200, 300 kg N ha −1 as ammonium nitrate applied 6 weeks before sowing to provide four concentrations of soil nitrate) and four rates of inoculation [nil, normal (n). 100n, 1000n]. The inoculant strain was B. japonicum CB1809. Observations were made on nodulation, the relative abundance of ureidcs in xylem exudates as an index of N 2 fixation, dry matter and seed yield, and total nitrogen in shoots and seed. Results showed clearly that soil nitrate repressed nodulation, that the effect was magnified as soil nitrate concentrations increased, but that inhibition was substantially ameliorated by increased numbers of rhizobia. The relative abundance of ureides in xylem exudates responded similiarly. The highest yields of dry matter and of N in shoots and in seed occurred at the highest rates of inoculation (100n, 1000n) at intermediate and high soil nitrate (N100, N200, N300); at low soil nitrate (N0), yields were increased by inoculation per se but not by the rate used. Uninoculated plants did not nodulate and yields in these plots reflected concentrations of soil nitrate. Data suggested that soil nitrate and N 2 fixation were not always complementary in meeting the N requirements of the growing crop. Absence of rhizobia. except at the highest rate of nitrate, and repression of nodulation at the normal rate of inoculation by intermediate concentrations of nitrate resulted in reduced N yields because of insufficient N supply to the crop during the final stages of growth.

Use of wild soybean (Glycine ussuriensis Regel and Maack) as a test plant in dilution—nodulation frequency tests for counting Rhizobium japonicum

Abstract In cross-inoculation tests with Glycine ussuriensis Regel and Maack and soybean, Glycine max (L.) Merrill, it was found that the species were very similar symbiotically. This finding is consistent with cytogenetic evidence that taxonomically G. ussuriensis and G. max are one species. A dilution-nodulation frequency technique (plant-infection test) is described whereby G. ussuriensis , a small-seeded species, is grown in cotton-wool-plugged specimen tubes as a test plant for identification and enumeration of Rhizobium japonicum . In a pure culture comparison with direct counting (agar plate counts), the plant-infection test gave an accurate estimate of numbers provided that a plant nutrient agar substrate was used for growing the test plants. This indicated that the probability of a single rhizobial cell initiating nodulation of the test plant was close to P = 1 . With a vermiculite substrate, the plant-infection test was less reliable. Tables are presented which permit calculation of most probable numbers of nodule bacteria from the distribution of positive (nodulated) test plants in plant-infection tests based on 5- and 10-fold dilution series.

Nitrogen fixation by soybean in commercial irrigated crops of central and southern New South Wales

Amounts of symbiotic N2 fixation were monitored using the ureide-sap technique in 36 irrigated soybean crops [Glycine max (L.) Merrill], from 26 commercial operations in the Coleambally irrigation area and in the Macquarie Valley of N.S.W. during the summer of 1990–1991. Crop N yields, N2 fixation, and nodulation were measured twice during crop growth and estimates of the seasonal amounts of N2 fixed were compared with amounts of seed N removed. For each crop, a net N-balance was calculated at harvest to determine whether there was a net gain or loss of N in soil as a result of soybean cropping. All crops under study were well nodulated; however, relative sap ureides collected indicated that the proportion of soybean N derived from N2 fixation (Pfix) ranged between 21 and 94% at flowering, and 4 and 96% at seed-filling. Seasonal estimates of Pfix and amounts of N2 fixed ranged from 52 to 73% and 103 to 313 kg N ha−1 at Coleambally, and 13 to 64% and 44 to 238 kg N ha−1 in the Macquarie Valley, respectively. Following seed harvest these amounts of N2 fixation resulted in N-balance determinations of between −33 to +69 kg N ha−1 at Coleambally, and −134 to +52 kg N ha−1 in the Macquarie Valley. The vegetative residues of 11 of the 33 soybean crops for which measurements continued until final harvest were estimated to have contributed more than 15 kg of fixed N ha−1 to soil, while N2 fixation was insufficient to satisfy seed N requirements in 12 of the remaining crops. The average seasonal Pfix determinations were not greatly different at Coleambally between crops considered to be in net positive N-balance (65%) and in net negative balance (59%), and the final result was more dependent upon soil type, and the average amounts of crop N accumulated and seed N removed than reliance upon N2 fixation for growth per se. However, in the Macquarie Valley, average crop N yields and seed N removal were similar across all soil types, and there was a distinct relationship between average Pfix values and net N-balance (59 cf. 32% for crops in positive and negative balance, respectively). Crops with the highest rates of N2 fixation and greatest contributions of fixed N to soil followed several years of cereal cropping, or occurred where soybean had been double-cropped with wheat. In these situations nitrate concentrations in surface soils were low at sowing. The lowest amounts of N2 fixation and poorest N-balance arose when the 1990–1991 crop followed 2 or 3 consecutive years of soybean, at sites with a recent history of legume-based pastures, or where N-fertilizer had been applied. Only 4 of the 33 crops followed through to final harvest exhibited evidence of prolognged N2 fixation during seed maturation.

Factors affecting the potential contributions of N2 fixation by legumes in Australian pasture systems

Abstract. The amounts of foliage nitrogen (N) fixed by various annual and perennial legumes growing in Australian pastures range from <10 to >250 kg N/ha.year. Differences in N2 fixation result from variations in the proportion of the legume-N derived from atmospheric N2 (%Ndfa) and/or the amount of legume-N accumulated during growth. On-farm surveys of %Ndfa achieved by legumes growing in farmers’ paddocks in Australia indicated that N2 fixation contributed >65% of the legume’s N requirements in three-quarters of the annual legumes examined, but this decreased to two-thirds of lucerne (Medicago sativa; also known as alfalfa), and half of white clover (Trifolium repens) samples. Factors such as low numbers or the poor effectiveness of rhizobial strains in the soil, water stress, high soil concentrations of N, and nutrient disorders contribute to poor nodulation and %Ndfa values <65%, but there is also evidence that the observed %Ndfa can be dependent on the legume species present, and whether the legume is grown in a pure stand or in a mixed sward. The accumulation of legume-N relates primarily to the legume content and net productivity of the pasture. For many legume species, ∼20 kg of shoot-N is fixed on average for every tonne of herbage dry matter produced. Legume productivity can be influenced by (i) sowing and establishment techniques and other strategies that enhance the legume content in pasture swards; (ii) the amelioration of soil constraints; (iii) the use of new legume species (and host–rhizobial strain combinations) that are more tolerant of hostile soil environments than subterranean clover (T. subterraneum) or annual medics (Medicago spp); and (iv) the inclusion of perennials such as lucerne to offset the year-to-year variability in productivity and N2 fixation that is a common occurrence with annual legumes.

Competitive advantage of bacteriocin and phage-producing strains of Rhizobium trifolii in mixed culture

Abstract Bacteriocinogenic and lysogenic strains of Rhizobium trifolii were grown with sensitive strains in sterile broth or peat culture. In broth culture the producing strains strongly suppressed growth of sensitive strains. In peat culture a considerable degree of suppression was also observed, the effect being stronger when the peat was “wet” than when it was “damp”. Apparently, conditions which favour growth in peat also favour dominance by bacteriocinogenic or lysogenic bacteria in cultures of mixed strains. The results are discussed in relation to preparation of mixed-strain legume inoculants and to studies of competition between strains in the field environment.