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When seedlings grown at low (6 or 12 °C) or high (36 °C) root temperatures were transferred to 24 °C the rates of root hair infection increased rapidly, generally to above that of plants kept at 24 °C. This 'switching-on' of the infection process occurred irrespective of the time of transfer between 6 and 14 d, but transfers from 36 to 24 °C, at times later than 6 d showed a small lag period before the numbers of infections increased. Responses to late transfer at 22 d from either 6 or 36 °C to 24 °C were less striking. A reverse transfer from 24 °C to 6 or 36 °C at any time later than 4 d caused no change in the infection rate. When plants at 6 °C were moved at different times to temperatures in the range of 12-36 °C for 8 h and then returned to 6 °C (double transfer) rates of infection were most increased at 30 °C; 36 °C was inhibitory. The greatest and the quickest response was with plants transferred at 2 d; transfer at 10 d had no effect. When plants grown at 6 °C for 6 d were exposed to 24 °C for 0-5, 1 ... 24 h and then returned to 6 °C rates of infection were enhanced compared with plants held at 6 °C; responses were directly proportional to time (>1 h) spent at 24 °C. Rates of infection were greatest immediately after transfer and thereafter declined.

A plasmid-cured derivative of Rhizobium loti strain NZP2037, strain PN4010, was significantly more effective in N2 fixation and showed an improved capacity to compete with other root nodule bacteria for nodulation of Lotus pedunculatus, relative to that of strain NZP2037. Reintroduction of the plasmid pRlo2037 into PN4010 resulted in a return to the NZP2037 level of symbiotic effectiveness and competitiveness. The enhanced effectiveness and competitiveness of PN4010 for L. pedunculatus was associated with its increased capacity to form nodules. Strain PN4010 did not differ from NZP2037 in its growth characteristics in pure culture, in its resistance to L. pedunculatus root flavolan or in its ability to multiply in the L. pedunculatus rhizosphere in the presence of a competing strain of R. loti. An examination of 35 R. loti strains (including 25 field isolates) revealed two strains (NZP2014 and NZP2042) that did not contain a single large indigenous plasmid; both were significantly more effective in N2 fixation with L. pedunculatus than was NZP2037 or two other Nod+ Fix+ plasmid-containing strains of R. loti. Transfer of pRlo2037 into NZP2014 and NZP2042 reduced their effectiveness for L. pedunculatus.

Summary Cross-inoculation group specificity in Cicer arietinum L. (chickpea) was studied. Seventy-one strains of root nodule bacteria of C. arietinum were examined for nodulation on 87 species of legumes. These species represent all the known cross-inoculation groups and were selected from various tribes and genera of family Fabaceae and Mimosaceae. In a reciprocal cross-inoculation study, 287 strains of root nodule bacteria from 52 of the 87 species, were examined on various genotypes of C. arietinum. C. arietinum and its root nodule bacteria did not show cross inoculation affinity with any of the members of the known cross-inoculation groups: alfalfa, clover, pea, bean, soybean, lupin-lotus and cowpea miscellany; except for some loose non-reciprocal kinship with Sesbania, which in its turn has strong affinity with the cowpea miscellany. However, under natural conditions in soil, even the loose non-reciprocal kinship between Cicer and Sesbania was rare. For practical reasons, it is suggested that C. arietinum and its root nodule bacteria should be considered in a separate cross-inoculation group. ‘Cross-inoculation host specificity’ appears to be of some help in the revision of the taxonomy of order Leguminosales and supports the placement of Cicer in a monogeneric tribe Cicereae.

Leakage of materials reacting with the fluorescent probe 1-anilino-8-naphthalcne sulphonate (ANS) from root-nodule bacteria subjected to desiccation and rehydration suggests that death of these bacteria is associated with changes in membrane permeability. The slow-growing rhizobia and the fast-growing Rhizobium meliloti differ from other fast-growing bacteria of the R. leguminosarum group in that they exhibit little fluorescence in the presence of ANS. This probably reflects differences between these groups of bacteria in the polar/non-polar nature of the outer cell envelopes. Following desiccation and rehydration, all groups of root-nodule bacteria fluoresced strongly in the presence of ANS. Repair of desiccation-induced damage to the cell envelopes of R. japonicum QA372. R. trifolii SU297/32B and R. meliloti CC131 was not observed using a range of screening agents. However, repair of significant numbers of lysozyme-sensitive bacteria was observed with R. leguminosarum TA101 within an hour of rehydration.

A comparison was made between 65 strains of root nodule bacteria from indigenous New Zealand legumes and 45 reference strains including: Rhizobium trifolii, R. phaseoli, R. leguminosarum, R. meliloti, and both “acid-producing” and “non-acid-producing” strains from the Lotus-Lupinus-Ornithopus cross-inoculation group. The strains were classified into 10 clusters on the basis of 37 morphological, cultural, and physiological tests. Relationships disclosed among the reference strains were in accordance with current ideas on Rhizobium taxonomy. The indigenous strains were well separated from both the trifolii-leguminosarumphaseoli complex and R. meliloti but acid-producing strains from the Lotus-Lupinus-Ornithopus cross-inoculation group segregated with the indigenous strains. The principal characters differentiating the clusters are discussed. The base composition of DNA from representative indigenous strains was determined and base sequence homology studied. The relative reassociation at 65°c in 0.2...

In root nodule cells of Sophora flavescens and Cytisus scoparius, normal mitoses were observed even in such cells that had been invaded by bacteria. Invading bacteria were found outside the spindle region in each mitotic stage. These seem to imply some type of boundary system in the spindle region which prevents the passage of the bacteria, and this, in turn, seems to prevent these bacteria from interfering with the normal mitotic chromosome movement.

Rhizobium trifolii isolates were obtained from nodules of white clover (Trifolium repens L.) growing within a grazing trial in steep, North Island hill country. The white clover was from each of 6 experimental treatments involving 2 fertiliser application regimes and 3 grazing managements. ‘Grasslands Huia’ and the resident Kentish-type white clover were present in pastures in variable proportions. Effectiveness of isolates, measured using Huia as the host plant and compared with a standard strain, ranged from 3 to 170%. Of 285 isolates tested, 9% were at least 20% more effective than the standard strain. Influence of experimental treatments on effectiveness of isolates appeared to be indirect, acting through effects on proportion of Huia and the resident white clover present at sample sites.

Significant correlations exist between the ability of root-nodule bacteria to fix molecular nitrogen and some of their properties (accumulation of riboflavin, cobalamine, ATP, cell pyrolysis, effect on plant vegetation characteristics) which can be used for quickly assessing the activity of the process in individual strains of rhizobia.

Summary The study on cultural and biochemical characteristics of 115 strains of root nodule bacteria of chickpea (henceforth named Cicer-Rhizobium ), suggest that only few characteristics, like the absence of growth on yeast extract mannitol agar (YEMA) in 24 h, no or little growth on glucose peptone agar without change of p H of the medium in 48 h, inability to produce 3-ketolactose, to precipitate calcium glycero-phosphate, to form serum zone coupled with acid production in litmus milk, to tolerate 20 meq Mn ++ /1 lactose medium and p H 10.0 in Hofer's alkaline broth, could be of diagnostic value in the identification of Rhizobium . In other characteristics, the strains were found to vary. The Congo-red colour differentiation was better obtained by pour plate method than by streaking the culture on YEMA plates. The production of acid or alkali on mannitol was found to be independent of the properties of soil from where the strains were isolated; and this did not substantiate Norris's hypothesis that Rhizobium , associated with tropical legumes, produces alkali. The position of Cicer-Rhizobium as a link between the group of fast growing Rhizobium from clover, pea, and beans and the slow growing group of Rhizobium from soybean, lupin, and cowpea miscellany has been indicated.

Rhizobium trifolii organisms associated with eleven species ofTrifolium were isolated and studied by means of antigen-antibody agglutination and immune diffusion techniques. Ten serologic types were differentiated from the eleven species of nativeTrifolium.

Eighteen strains of Rhizobium lupini were shown to form effectively nitrogen-fixing root nodules in Lotus uliginosus whose rhizobia appeared culturally similar to R. lupini. Six strains formed effective or (in one case) semi-effective nodules in Ornithopus sativus.

The effect of the air content of the medium on the development and function of the nodule was studied with sterile cultures of peas inoculated with their appropriate strain of nodule bacteria.In water cultures the plant grows well without added nitrogen, even though all nodules are submerged, if air is bubbled through the medium during the period of growth. Abundant nodulation occurs also in nonaerated liquid cultures; the nodules, however, are much smaller than in aerated cultures, and the plant grows poorly. Subsequent aeration of such cultures results in a distinct increase in the size of the nodules and improvement in the growth of the plant.Nodulation is completely prevented by passing a stream of gaseous N through the medium. That oxygen is indispensable for the function of the nodules is shown also by the fact that the peas grow excellently on added nitrogen in non-aerated cultures.

The failure of pea (Pisum sativum) to grow in ‘goradu’ soil of Central Gujarat without artificial inoculation of the seed with the specific root nodule bacteria has been investigated. The study of a large number of soil fungi isolated from ‘goradu’ soils failed to show that they had any selective antibiotic activity against the pea rhizobia. It has been explained that the high sand content of this soil and the lower thermal death point of the pea rhizobia are the probable causes for the failure. The high sand content does not give any protection to the pea rhizobia against the high temperatures of the summer months with the result that their number gets so reduced and are not able to nodulate plants sufficiently.

Four distinct types of colonial mutation were obtained by X-ray irradiation of a parasitic strain of alfalfa Rhizobium. Two types were procured from an effective strain. Colonial morphology remained stable throughout serial transfer on artificial medium, but changed considerably after one plant passage. Three variants derived from the parasitic parent were found to be efficient in nitrogen fixation when tested by sterile plant growth procedures and the Virtanen technique. This efficiency increased during the course of two plant passages. Colonial variation and effectivity changes occurred independently of one another.

SUMMARY: The application of computer techniques to study a group of organisms comprising 83 strains of the genus Rhizobium and 38 strains belonging to the genera Agrobacterium, Chromobacterium, Beijerinckia and Bacillus is described. One hundred different features including vitamin, carbohydrate and nitrogen nutrition, antibiotic sensitivities, morphological and colonial characteristics, and infective attributes were used to determine the relationships between these organisms. Members of the genera Chromobacterium, Bacillus and Beijerinckia showed little affinity with the other species studied, or with each other. The results obtained for the genus Rhizobium indicate the need for major taxonomic changes. These changes would include: (1) the consolidation of R. trifolii, R. leguminosarum and R. phaseoli into a single species, R. leguminosarum; (2) The consolidation of A. radiobacter and A. tumefaciens and their inclusion as R. radiobacter in the genus Rhizobium. The creation of a new genus Phytomyxa to contain strains of slow-growing root-nodule bacteria is also proposed.

Trichloro-, dichloro- and monochloroacetic and α-dichloro-, α-monochloro-and β-monochloropropionic acids as sodium salts in 0.10–1.0mm concentration accelerated the growth of a strain of Rhizobium leguminosarum in a liquid synthetic medium with biotin as sole accessory growth factor. The strongest effect was shown by β-monochloropropionate. Concentrations of 10mm and above were inhibitory. α-Monochlorobutyrate did not accelerate growth.

The Virtanen technique was used to detect effective and ineffective rhizobia. The rhizobia studied varied widely in their action towards different varieties of the same host species. Maximum leghemoglobin concentrations occurred just prior to blossoming in field legumes, but much earlier in greenhouse legumes cultivated under conditions of this experiment. The nodules of the ineffective strains of legume bacteria differed from those produced by effective strains in color, size, distribution, rate of destruction of the bacteroid area, and presence or absence of rodlike bacterial forms. Experimental evidence is presented to show that a supposedly parasitic strain of alfalfa organism can exist.

The survival over summer of field-grown root nodule bacteria was studied in the field and the laboratory during the course of an investigation into a nodulation problem of annual clovers. Dry field soils containing Rhizobium trifolii and R. lupini were subjected to a range of temperatures in the laboratory, the bacteria surviving 6 h exposure to temperatures as high as 80°C. Soil temperatures during summer were recorded at different depths. Populations of rhizobia were estimated at these depths from the end of the growing season (October) to early autumn (April) in plots which had carried dense swards of subterranean clover and Serradella. High populations of R. lupini were maintained in the Serradella plots throughout the summer. Populations of R. trifolii in the subterranean clover plots were initially much lower, and declined with both time and depth. The problem known as ‘second-year clover mortality’ is primarily due to low numbers of clover rhizobia in the soil at the end of the growing season. This situation is aggravated over the long hot dry summer, when there is a further decline in numbers.

SUMMARY The growth and survival in peat culture of Rhizobium spp. from 3 inoculation groups was markedly affected by the gas exchange properties of the container. Complete restriction of aeration caused rapid reduction in numbers after only 1 week. Low moisture contents restricted growth but higher levels up to 60% (on a wet weight basis) favoured survival. Within the range tested the size of the inoculum was unimportant in determining the number of rhizobia in sterilized peat provided sufficient time elapsed for multiplication. Storage temperatures between 4° and 26° affected the rate of growth but not survival of rhizobia in pure culture for periods up to 26 weeks provided moisture was not limiting.

SUMMARY: The vitamin requirements of 63 strains of Rhizobium and 18 of Agrobacterium were examined. R. trifolii, R. leguminosarum and R. phaseoli responded in most cases to thiamine, biotin, and calcium pantothenate. Strains of R. meliloti and the slow-growing rhizobia sometimes responded to biotin, but not to the other vitamins. An unknown growth factor, active for the slow-growing rhizobia, was detected. Three strains of Agrobacterium required thiamine.

SummaryAn experiment was conducted on peanut inoculation with Rhizobium inoculum, introduced below the planting level at three different soil depths (8, 12 and 15 cm.), to ensure more favourable moisture conditions for proliferation of the root nodule bacteria, and to obviate the need for frequent irrigation. Inoculation on both sides of the peanut rows, after the plants had emerged, was also tested. The number and weight of nodules per plant decreased with increase in placement depth. Side inoculation after emergence of the plants resulted in significantly less plant infection than placement of the inoculum at 8 and 12 cm. depths. Peanut yield and quality after inoculation at 15 cm. depth were significantly lower than at 8 cm. inoculation.

The associated growth of peas and different non-legumes (barley, oats, wheat, and potato) was investigated both under sterile conditions and in ordinary pot cultures. In the former case the medium consisted of quartz sand, and in the latter of clay or sandy loam soil, poor in soluble nitrogen. It is shown that nitrogen was transferred from the pea nodules to the non-legumes.In sterile cultures, where the excreted amino-acids cannot undergo decomposition, oats, wheat and barley utilized only a part of the excreted nitrogen, generally less than 50 per cent. In some instances, however, the barley took up considerably over 50 per cent of the excreted nitrogen. There was an apparent accumulation of aspartic acid in the medium of the associated cultures. In pot cultures, where the amino-acids may have been broken down by contaminating micro-organisms, the transfer of the excreted nitrogen to the potato exceeded 90 per cent.All experiments show that with increasing ratio of non-legumes to legumes the growth of the peas suffers, obviously from lack of nitrogen. Under these conditions, the excretion from the nodules increases to such an extent that the peas receive only a small fraction of the nitrogen fixed by the nodules. The major part of the carbohydrate material furnished by the pea for the amino-acid synthesis in the nodules is excreted into the medium. This naturally causes disturbances in the growth of the pea.

Growth of Lotus nodule bacteria (Strain LC 296) was temporarily suppressed in a yeast-extract mineral-salts medium containing 2,4-DB at 50 and 100 µg/ml. However, the 2,4-DB concentration needed to completely suppress the growth of the bacteria appeared to be above 500 µg/ml. When added during the logarithmic phase of growth 2,4-DB at 50 and 100 µg/ml did not inhibit the growth of the cells, so that the growth-inhibiting effect of the herbicide apparently was directed primarily against lag phase cells. Pre-incubation of rhizobia in a medium containing 2,4-DB at concentrations up to 10 µg/ml did not affect the capacity of the bacteria to effectively nodulate their host plant. Attempts to induce the rhizobia to utilize 2,4-DB as a sole carbon source failed. Nevertheless, when theLotus nodule bacteria (LC 296) were grown in a medium containing 2,4-DB-1-C14, C14O2 was released. Similar results were obtained with other strains of the Lotus rhizobia and other species of Rhizobium. C14O2 evolution was also demonstrated when radioactive 2,4-DB was added to both intact (excised) and crushed trefoil nodules. The results indicate that the nodule bacteria are capable of degrading 2,4-DB; although the mechanism is still obscure it does not appear to be via cleavage of the ether link.

Bacteriophage for clover nodule bacteria can be found on roots and nodules of all naturally grown clover plants and also in the soil surrounding the roots, but not in soil without clover plants. Alternative hosts for the phage of clover bacteria are pea bacteria, and vice versa. The bacteria and the phage are heterogeneous in the sense that only a proportion of strains of clover bacteria and of pea bacteria are susceptible to lysis by a given race of phage, and only a proportion of races of phage can lyse a given bacterial strain. There does not seem to be any association between the susceptibility of bacterial strains to lysis by phage and any other features such as antigenic structure and effectiveness in nitrogen fixation. There may be an association with avirulence, i.e. inability to infect the host plant. The behavior of phage–bacterial mixtures depends on the surrounding medium. The longevity of phage in soil or in a soil-like medium such as a vermiculite mixture is relatively short, and the effect of phage can be localized so that phage-susceptible bacteria and the phage can exist close to each other without any apparent interaction. However, as long as the phage is present, phage-resistant bacterial mutants are usually present also. The phage-resistant mutants may also be mutants in other respects such as effectiveness in nitrogen fixation. In the presence of weakened phage, bacterial mutants were found to occur that differ from the parent form in effectiveness but resemble it in susceptibility to the phage.