Siderophore production and utilization byRhizobium trifolii

Summary Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.     

Strain identification inRhizobium meliloti using the antibiotic disk susceptibility test

Summary The antibiotic disk susceptibility test was used to measure the variation in the intrinsic resistance of 49 strains ofRhizobium meliloti to 9 antibiotics. Several strains had unique patterns of resistance. However, during cluster analysis, when a minimum Euclidean distance equal to 4 was used as a discriminating tool, the strains were grouped in 12 groups. The largest group contained 74% of the strains but 9 strains (2 very effective, 4 effective and 3 ineffective) showed very unique patterns of resistance and formed 9 distinct groups.R. meliloti strains in general showed high intrinsic resistance to the 9 antibiotics tested.Contribution no. 189 Station de Recherches, Agriculture Canada.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Studies of natural populations and mutants of rhizobium in the improvement of legume inoculants

Summary A study has been made of the symbiotic effectiveness ofRhizobium trifolii in fields ofTrifolium subterraneum in south-eastern Australia, with the purpose of providing background information for a programme of inoculant strain improvement. The strains found varied widely in symbiotic effectiveness. The distribution patterns of effectiveness varied from year to year and from locality to locality. From 24 to 65% of strains ofR. trifolii from different localities exhibited antagonism to selectedR. trifolii indicator strains. These reactions were mainly due to mild antibiotic effects but bacteriocinogenic strains and strains producing specific, virulent or wide-range, temperate phages, were also common. Factors to be considered in the selection and evaluation of inoculant strains from among natural populations are discussed and the possible role of genetic manipulation is examined. It is concluded that the general application of DNA transformation and transduction is restricted because of the limited degree of DNA homology among strains, the limited host-range of transducing phages and the lack of suitablein vitro screening procedures for symbiotic characters. A mutational model is presented in which characters known to be associated with symbiotic effectiveness would be manipulated by mutation and back-mutation to effect quantitative increases in effectiveness.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Nodulation ofTrifolium subterraneum byRhizobium leguminosarum

Summary Four cultivars ofTrifolium subterraneum were nodulated by five strains ofRhizobium leguminosarum; all combinations except one gave 100% nodulation. Rates of nodule formation and total nodule numbers were similar to those with an effectiveR. trifolii strain. The nodules were more commonly associated with lateral roots and were ineffective in nitrogen fixation.     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Competition studies withRhizobium trifolii in laboratory experiments

Summary Competition studies were carried out in soil cores by comparing the nodule forming ability of antibiotic resistance marked strains, inoculated at three inoculum levels onTrifolium repens versus an effective indigenous Rhizobium population of 1.5×10⁵/gm. It was seen that the indigenous marked isolate G1067 formed a high proportion of nodules sampled (>90%) at all three inoculum levels (10⁵, 10⁷ and 10⁹ cells/seed) wherein the introduced foreign strain G1032 formed >50% of the nodules at the highest inoculum level.In a test tube experiment, competition for nodule sites was examined by inoculating mixtures of twoR. trifolii strains at different input levels on two cultivars ofTrifolium repens var. Huia and Titan. It was seen that G1032 was less competitive than G1006 and G1067 on cv. Huia but was more competitive on cv. Titan than the other two strains.     

The response of white clover to different strains ofRhizobium trifolii in hill land reseeding: A second trial

Summary S. 184 white clover was surface seeded into natural molinia pasture on wet stagnogley soil containing no indigenousRhizobium trifolii. Seedlings were ‘spray inoculated’ after emergence with each of three strains ofR. trifolii. The best of these treatments produced an eight fold improvement in dry matter in the seeding year, followed by a 28% improvement in the following year. The results confirm the potential benefits which may be achieved by inoculating clover with suitable strains of rhizobia. The data are compared with a previously reported trial on an adjacent site where benefits were much greater in the first year. The difference is attributed to the overall advantages conferred in the present trial by much higher seedling populations and less severe competition from native species in the establishment phase.     

Nodulation of Trifolium repens with modified Bradyrhizobium and the nodulation of Parasponia with Rhizobium leguminosarum biovar trifolii

Thirty one strains of Rhizobium leguminosarum biovar trifolii isolated from the North and South American continents, New Guinea, USSR, Turkey and Australia, nodulated P. andersonii ineffectively when grown in plant growth tubes and in Leonard jars. Nodules were slow to form, sometimes taking over 100 days. Reisolates of R. leguminosarum biovar trifolii from P. andersonii nodulated Trifolium repens and their identity was confirmed using serological techniques. Dual occupation of nodules by Rhizobium and Bradyrhizobium in P. andersonii is reported. The reduced effectiveness of the Bradyrhizobium symbiosis depended on the relative numbers of Rhizobium occupants in this dual system. R. leguminosarum biovar trifolii and Bradyrhizobium strains from Parasponia were able to co-exist in nodules on P. andersonii and maintain similar populations in the rhizosphere and on culture media. Bradyrhizobium strains, separated from R. leguminosarum biovar trifolii, were able to initiate and form nodule-like structures on T. repens. Bradyrhizobium bacteria were identified as the sole occupants of the cells of the nodule-like structures on Trifolium repens using an immunogold labelling technique applied to ultrathin sectins. The re-isolates of Bradyrhizobium obtained from these nodule-like structures on T. repens were able to effectively nodulate P. andersonii.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Some properties of arctic rhizobia

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.     

Characterization ofBradyrhizobium sp. (Astragalus sinicus L.) using serological agglutination,intrinsic antibiotic resistance,plasmid visualization,and field performance

Eleven isolates ofBradyrhizobium sp. (Astragalus sinicus L.), originating from plants ofAstragalus sinicus (Chinese milkvetch) in China and Japan, were characterized using serological agglutination, intrinsic antibiotic resistance, and plasmid visualization. Serological agglutination, using antisera against three isolates effective onA. sinicus and two isolates effective on otherAstragalus spp., showed that there may be two serogroups among the eleven isolates effective onA. sinicus, and that the antigenic components are heterogeneous. Results of both intrinsic antibiotic resistance and plasmid visualization demonstrated similarities between four of the isolates.Ten isolates were further evaluated for effectiveness on the host,A. sinicus, in a field study in Puyallup, Washington (47°N, 122°W). This study showed that the Chinese commercial inoculum and one field isolate were about ten times more effective than the control based on plant dry weight and seed yield. Eight other isolates were less effective.     

Transfer of nodulation ability in Rhizobium using R68.45 derived plasmids

Summary Nodulation ability was transferred from Rhizobium meliloti L5.30 to the non-nodulating mutant Rhizobium trifolii 24K using plasmid R68.45. Transconjugants selected for the carbenicillin resistance (cb ^r) marker became simultaneously capable of nodulating clover and showed changes in phage sensitivity. Besides the indigenous plasmid of 90 MD (pUCS201), the nodulating transconjugants harbored the newly introduced plasmid pUCS202 (ca. 40 MD). After treatment of the transconjugants with curing agents the simultaneous loss of antibiotic resistance and ability to form nodules were associated with the disappearance of pUCS202. nod and cb ^r genes were cotransferred into R. trifolii strains by conjugation and transformation. There is genetic evidence that the nod gene(s) was integrated into R68.45.     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

Intrinsic multiple antibiotic resistance markers for competitive and effectiveness studies with various strains of mung bean rhizobia

Ten strains ofRhizobium sp. with multiple antibiotic resistance markers were used for competitive and ef ficiency studies with mung bean var. ML 5. All the strains showed significant increase in grain yield and so also for nitrogenase activity except MO 5. Nitrogenase activity correlated very well with grain yeild. The compatibility of strains varied from 17 to 50%. The intrinsic multiple antibiotic markers for strain identification were found to be stable after passing through soil and host conditions and could be used for ecological studies. It was further revealed that the overall efficiency of a strain is the combined effect of characters like compatability, competitiveness and inherent capacity to fix nitrogen.     

Intra and inter generic homology in polysaccharide structure ofRhizobium andAlcaligenes

A study was conducted on the structure of extracellular, water-soluble polysaccharides from 5 different strains ofRhizobium viz. R. trifolii J60 andR. meliloti strains J7017, 202, 204 and 207. All these polysaccharides were found to contain glucose and galactose in the approximate molar ratio of 7:1. Methylation analysis revealed these polysaccharides to contain (1 → 3), (1 → 6), (1 → 4), (1 → 4, 1 → 6)-linked D-glucose residues, (1 → 3)-linked D-galactose and nonreducing terminal D-glucose attached to pyruvate. These polysaccharides were also found to be acylated by both acetyl and succinyl residue. This structure was found to be similar to that of succinoglycan, a succinic acid-containing water-soluble, extra-cellular polysaccharide elaborated byAlcaligenes faecalis var.myxogenes 10C3. This similarity in structure of polysaccharides from two different species ofRhizobium and also the polysaccharide produced byAlcaligenes has been discussed.