Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Yields and nitrogen nutrition of intercropped maize and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi)

Maize (Zea mays L.) and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi) were grown in intercrop and monoculture on Tropaqualf soils under rainfed conditions in Northern Thailand yearly from 1983 to 1986. De Wit's replacement design was used to compare intercrops and monocultures with a constant plant density equivalent to 80 000 maize or 160 000 ricebean plants ha⁻¹. Combined nitrogen was applied at varying levels to 200 kg N ha⁻¹. In the final two seasons the intercrop ratio of maize: ricebean was also varied. At the time of maize maturity intercrops yielded upt 49 kg ha⁻¹ more N in the above ground plant parts than the best monoculture. Dry matter, grain and nitrogen yield of maize and ricebean in intercrop relative to their monoculture yields (RY, relative yield) were significantly greater than their respective share of the plant population. Relative yield totals (RYT) for grain, dry matter and nitrogen were always greater than 1. Nitrogen uptake per maize plant increased with progressive replacement of maize by ricebean plants. This increase was similar to that obtained by applying combined N. Available soil nitrogen tended to decrease with increasing maize:ricebean ratio. Increasing the maize:ricebean ratio increased the % of nitrogen derived from fixation in ricebean, the increase being equivalent to that obtained by decreasing combined nitrogen application. Approximately the same amount of fertilizer and soil nitrogen was taken up by maize plus ricebean in intercrop as the maize monoculture. The results suggest that the improved nitrogen economy of the intercrop resulted from the strong competitiveness of maize in the use of mineral nitrogen and the enhancement of nitrogen fixation in intercropped ricebean which made it less dependent on the depleted pool of soil nitrogen.     

The effectiveness of some Indonesian strains of Rhizobium on four tropical legumes

Nodules were collected from 14 legume species from the Indonesian Islands of South Sulawesi, Java and Sumatra. Their rhizobia were isolated and growth characteristics, nodulation ability and nitrogen fixing effectiveness were assessed against recommended commercially available Australian strains. The test legumes wereMacroptilium atropurpureum Urb. cv. Siratro,Vigna unguiculata (L.) Walp. cv Eureka,Centrosema pubescens Benth cv. Belalto andDesmodium heterocarpon (L) DC. A significant portion of the native rhizobial isolates were of the fast growing type. Dry matter and total nitrogen production forM. atropurpureum andV. unguiculata was highest when inoculated with native strains while the commerical strains produced superior dry matter production forC. pubescens andD. heterocarpon. However the total nitrogen production of native and commercial strains was not significantly different for the latter two legumes. The study indicated that a potential exists for developing inocula from local Rhizobium strains.     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Effective nodulation of rhamnaceous actinorhizal plants induced by air dry soils

Air dry soil samples stored at room temperature for more than one and a half years, were used as inocula for actinorhizal plants. Seedlings of Colletia paradoxa, Discaria americana, D. articulata and D. trinervis (Rhamnaceae) cultivated in nitrogen-free nutrient solution, were inoculated by adding dry soil to the solution. All the soil samples tested were able to induce nodulation, showing the presence of infective propagules of Frankia. Healthy growth of nodulated plants suggested the occurrence of nitrogen fixation.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.