Host specificity of nodulation inRhizobium sp. (Cicer) as revealed by Tsr bioassay

Bioassay based on thick and short root (Tsr) and hair deformation (Had) phenotypes were used to test the activity of Nod factors produced byRhizobium sp. (Cicer) strains HS-1, Rcd-301, IC-59, IC-76 and Ca-181 on chickpea (Cicer arietinum) cv. ‘C-235’. Nod⁻ mutants ofRhizobium sp. (Cicer) did not produce Tsr⁺ and Had⁺ phenotypes on chickpea, indicating the requirement of nodulation genes for their appearance. The strain HS-1 treated with root exudates of pea (Pisum sativum), berseem (Trifolium alexandrinum) and lucerne (Medicago sativa) failed to produce the Tsr⁺ and Had⁺ phenotypes on chickpea. ConverselyR. leguminosarum bvs.viciae andtrifolii, R. meliloti, Rhizobium sp. (Sesbania), andRhizobium sp. (Cajanus) induced with chickpea root exudates did not show Tsr⁺ and Had⁺ phenotypes on chickpea. It appears that host specificity inRhizobium sp. (Cicer)-chickpea symbiosis is regulated by the production of host-specific factors which are not active on heterologous hosts.     

Comparative response ofPisum sativum nodulated with indigenous soilRhizobium populations and/or co-inoculated with aRhizobium leguminosarum strain

No significant differences in the acetylene-reducing activity and evolution of H₂ and CO₂ nodulated roots ofPisum sativum inoculated with soilRhizobium populations from two soils with different acidities (Ruzyně soil 7.6; Lukavec soil 4.9) were observed.Rhizobium population from Lukavec soil formed nodules, exhibiting a higher H₂ evolution. Co-inoculation with the Hup⁺ strain 128C30 (7×10⁷ cells per seedling) eliminated, to some extent, the effect of soil populations on physiological activity. Translated by Č. Novotny     

Hydrogenase in legume root nodule bacteroids: Occurrence and properties

Summary The hydrogenase found in Rhizobium bacteroids is compared with that found in Azotobacter and found, in all respects examined, to be similar. When three host species were inoculated with Rhizobium, strain 311, different amounts of hydrogenase activity were found in Pisum sativum and Vicia bengalensis while the enzyme was absent from nodules of Vicia faba. Of four different strains of Rhizobium examined only two strains possessed the hydrogenase when present in pea root nodules. The role of the hydrogenase in nitrogen fixation is discussed and it is tentatively concluded that the overall efficiency of the nitrogen fixation process is increased by its presence.     

Fungal microflora of seeds of Pisum sativum L. and its control

Five varieties ofPisum sativum L. were tested for seedborne fungi. Tests were conducted by standard International Seed Testing methods. It was observed that fungi likeAlternaria, Aspergillus, Rhizopus, Mucor andFusarium were associated with all the five varieties tested.Fusarium andRhizopus were dominant in all the varieties. They were also responsible for reduction in germination percentage of seeds. Early December was selected for testing the efficacy of certain fungicides (namely, Agrosan G.N., Ceresan, Copper carbonate, Tafasan, Tillex, Stardex and Sulphur) against seedborne fungi ofPisum sativum L., Agrosan G.N. and Ceresan gave the better control of seedborne pathogens without any adverse effect on germination.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Root-hair infection and nodulation of four grain legumes as affected by the form and the application time of nitrogen fertilizer

The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in bothVicia faba andPisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation ofV. faba, Phaseolus vulgaris andVigna sinensis. The inhibitory effect of urea on nodulation ofP. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events ofRhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.     

The Involvement of Endogenous Phenolic Compounds in the Response of Pea Seedling Roots to Inoculation with Rhizobium leguminosarum at Various Temperatures

Endogenous phenolic compounds (PC) affecting Rhizobium leguminosarum bv. viceae propagation were isolated from the roots of etiolated pea (Pisum sativum L.) seedlings before and within one or two day after inoculation. It was established that, during the first day after inoculation, PC-induced stimulation of bacterial growth in roots was replaced by its inhibition, which was somewhat more pronounced at 8°C. The ratio between PC fractions was also changed during the first day after inoculation, especially strongly at low temperature; and this was evidently the cause for Rhizobium growth inhibition in root cells.     

Growth of three legume trees inoculated with VA mycorrhizal fungi and Rhizobium

Effectiveness among four VA mycorrhizal fungi and Rhizobium (R) in promoting growth of three legume trees in a P-deficient soil was studied.Glomus fasciculatus + R andGigaspora margarita + R were most effective forAcacia mangium andAlbizia falcataria (syn.:Paraserianthes falcataria). Scutellospora persica + R,Gigaspora margarita + R andGlomus fasciculatus + R were most effective forAcacia auriculifornis. Consistently poor growth was attained by seedlings inoculated withSclerocystis clavispora + R,Rhizobium alone, or by uninoculated seedlings.     

Attachment of Rhizobium to Legume Roots as the Basis for Specific Interactions

Experiments were conducted to elucidate the basis of the observation that different strains of Rhizobium infect particular legumes. Rhizobia specific for a variety of legumes were grown with ¹³PO^2?₄ and exposed to pea roots (Pisum sativum L.), R. leguminosarum 128C53, which nodulates pea, did not attach to the roots in greater numbers than those strains of rhizobia incapable of infecting pea roots. A complex of R. leguminosarum 128C53 conjugated to a fluorochrome-labeled antibody exhibited a striking attachment to the tips of pea root hairs, where infection normally occurs, but this fluorescent complex also bound to the root hairs of Canavalia en siformis DC., Lupinus polyphyllus Lindl., Trifolium pratense L., and Medicago sativa L., which are not infected by this bacterium. A reproducible, quantitative technique developed for studying interactions between fluorochrome-labeled lectins and rhizobia revealed no relationship between lectin-Rhizobium interactions and the capacity to infect a plant. The data are interpreted as suggesting that simple attachment of Rhizobium to a legume root is not the basis of host-symbiont specificity in this system.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.     

Crop husbandry at Miri Qalat Makran, SW Pakistan (4000–2000 B.C.)

Large scale sampling for plant remains at Miri Qalat indicates that agriculture based on naked wheat and naked and hulled barley was practised between the 4th and the 2nd millennia B.C. Other cultivated plants identified areLens culinaris (lentil),Pisum sativum (pea),Linum usitatissimum (flax),Vilis vinifera (grape) andCoriandrum salivum (coriander). The only summer crop,Sesamum indicum (sesame), appears during the second half of the 3rd millennium. Gathered edible fruits includeCordia, Grewia andNannorrhops ritchieana.Phoenix dactylifera (dates) may also have been gathered rather than cultivated.     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens

Acidic exopolysaccharides and O-antigen containing lipopolysaccharides were isolated from Rhizobium japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, cowpea Rhizobium sp. and a non-nodulating soil bacterium. Lectins from seeds of soybean (Glycine max), garden pea (Pisum sativum), lentil (Lens culinaris), alfalfa (Medicago sativa), field bean (Phaseolus vulgaris), jackbean (Canavalia ensiformis) and from wheat germ were tested for their capacity to precipitate rhizobial exopolysaccharides and lipopolysaccharides in the Ouchterlony double diffusion test. Soybean lectin precipitated exclusively with the exopolysaccharide of R. japonicum, whereas the lectins from pea and lentil precipitated exopolysaccharides from all the fast growing strains of Rhizobium. Host range specific interactions between lipopolysaccharides and lectins were observed in the pea/lentil-R. leguminosarum and in the alfalfa-R. meliloti systems. Concanavalin A precipitated the exopolysaccharides of all fast growing strains of Rhizobium, the exopolysaccharide of the cowpea strain and several lipopolysaccharides of different Rhizobium species and thus did not show any correlation between polysaccharide binding and symbiotic specificity. Non-leguminous wheat germ agglutinin did not precipitate any of the rhizobial polysaccharides tested and the lipopolysaccharide of the soil bacterium did not precipitate with any of the lectins examined.Abbreviations Con A Concanavalin A - CPC cetylpyridinium chioride - EPS exopolysaccharide - FITC fluorescein isothiocyanate - KDO 2-keto-3-deoxyoctonic acid - LPS lipopolysaccharide - PBS phosphate-buffered saline - PS polysaccharide     

Biocontrol of wilt disease complex of pea using Pseudomonas fluorescens and Rhizobium sp.

Biocontrol of wilt disease complex of pea caused by the root-knot nematode Meloidogyne incognita and Fusarium oxysporum f. sp. pisi was studied on pea (Pisum sativum L.) using plant growth-promoting rhizobacterium Pseudomonas fluorescens and root nodule bacterium Rhizobium sp. Inoculation of M. incognita and F.oxysporum alone caused significant reductions in plant growth over un-inoculated control. Reduction in plant growth caused by M. incognita was statistically equal to that caused by F. oxysporum. Inoculation of M. incognita plus F. oxysporum together caused a greater reduction in plant growth than the sum of damage caused by these pathogens singly. Inoculation of P. fluorescens and Rhizobium sp. individually or both together increased plant growth in pathogen inoculated and un-inoculated plants. Inoculation of P. fluorescens to pathogen-inoculated plants caused a greater increase in plant growth than caused by Rhizobium sp. Application of Rhizobium plus P. fluorescens caused a greater increase in plant growth than caused by each of them singly. Inoculation of P.fluorescens caused higher reduction in galling and nematode multiplication than caused by Rhizobium sp. Use of Rhizobium plus P. fluorescens caused higher reduction in galling and nematode multiplication than their individual inoculation. Plants inoculated with both pathogens plus Rhizobium showed less nodulation than plants inoculated with single pathogen plus Rhizobium. Inoculation of Rhizobium plus P. fluorescens resulted in higher root-nodulation than inoculated only with Rhizobium. Wilting indices were 4 and 5, respectively, when plants were inoculated with F. oxysporum and F. oxysporum plus M. incognita. Wilting indices were reduced maximum to 1 and 2, respectively, when plants inoculated with F.oxysporum and plants with both pathogens were treated with P. fluorescens plus Rhizobium.     

Potential for improving pea production by co-inoculation with fluorescent Pseudomonas and Rhizobium

Seed bacterization with five plant growth promoting fluorescent Pseudomonas strains isolated from Indian and Swedish soils and three Rhizobium leguminosarumbiovar viceae strains isolated from Swedish soils were shown to promote plant growth in Pisum sativum L. cv. Capella. Co-inoculation of the fluorescent pseudomonads and Rhizobium improved plant growth in terms of shoot height, root length and dry weight. Both the fluorescent pseudomonads and Rhizobium were shown to exhibit a wide range of antifungal activity against pathogens specific to pea. Seed bacterization with plant growth promoting strains alone and together with a rhizobial isolate, R 361-27 reduced the number of infected peas grown in Fusarium oxysporum infested soils. We found that the introduced organisms were able to colonize the roots, which was confirmed using immunofluorescence staining and drug resistant mutant strains. In a synthetic culture medium, all the plant growth promoting fluorescent pseudomonads strains produced siderophores, which shown to express antifungal and antibacterial activity. Our results suggest the potential use of these bacteria to induce plant growth and disease suppression in sustainable agriculture production systems.     

Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A

We report here that the pea early nodulin genes PsENOD5 and PsENOD12A are induced during the interaction of pea roots and the endomycorrhizal fungus Gigaspora margarita. Using the pea nodulation mutant Sparkle-R25, which is mutated in SYM8, it is shown that SYM8 is essential for the induction of PsENOD5 and PsENOD12Ain pea roots interacting either with Rhizobium or the endomycorrhizal fungus Gigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor-activated signal transduction cascade.     

Sucrose synthase levels do not limit or regulate carbon transfer in the arbuscular mycorrhizal symbiosis

Abstract

Sucrose synthase (SuSy) is the main sucrose breakdown enzyme in plant sink tissues, including nodules, and is a possible candidate for the diversion of plant carbon to arbuscular mycorrhizal (AM) fungi in roots. We tested the involvement of SuSy in AM symbiosis of Glomus intraradices and Pisum sativum (pea). We observed that peas deficient in the predominant root isoform of SuSy were colonized successfully by AM fungi similar to wild-type roots. SuSy protein levels did not increase in roots as AM symbiosis developed, although SuSy protein levels did increase in nodules as the rhizobium symbiosis developed. Our results lead us to conclude that, unlike nodule symbiosis, SuSy protein does not limit or regulate carbon transfer in the AM symbiosis.