Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.     

Danish Rhizobium leguminosarum strains nodulating 'Afghanistan' pea (Pisum sativum)

A wild pea ( Pisum sativum L.) native to Afghanistan normally known to be resistant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection under aseptic conditions. Five types differing in isoenzyme composition pattern were found among 15 isolates from 'Afghanistan' nodules. None were identical with the 'Tom' strain from Turkey, which also forms effective nodules with 'Afghanistan'. The five types were also different with respect to isoenzyme pattern from Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field.     

Nodulation and nitrogen fixation mutants of pea,Pisum sativum

Summary The 36 mutants which did not nodulate and 24 mutants which formed inefficient nodules with no or very low acetylene reduction activity were isolated among 86,000 M₂-seedlings of Finale pea, Pisum sativum L., after treatment with chemical mutagens. One mutant was found for approximately every 50 chlorophyll mutants. Most mutations were induced by ethyl methanesulfonate; some by diethyl sulfate, ethyl nitrosourea and acidified sodium azide. Putative mutants were selected as nitrogen deficient plants, yellowing from the bottom and up, when M₂ seedlings were grown in sand with a Rhizobium mixture and PK fertilizer. The mutants were verified in the M₃ generation by acetylene reduction assay on intact plants.     

Hollow heart of pea (Pisum sativum)

Book reviewed in this article: The Way Ahead in Plant Breeding. Proceedings of the Sixth Congress of Eucarpia. Edited by F. G. H. Lupton , G. Jenkins and R. Johnson . Basic Electron Microscope Techniques. By M. A. Hayat . The Logit Transformation (with special reference to its uses in bioassay). By W. D. Ashton . Families of Frequency Distributions. By J. K. Ord .     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Embryogenesis of the pea (Pisum sativum)

Summary The most striking internal feature of the suspensor cells inPisum is the abundant occurrence of a plastid containing spherical bodies consisting of intertwined bundles of tubules. These tubular complexes are not typical prolamellar bodies and they are not converted into grana. Cytochemical reactions indicate that they are proteinaneous. The participation of this plastid in the possible nutritional function of the suspensor is discussed but it is pointed out that critical experimental evidence is needed before the role of the suspensor and its contents in embryogenesis can be understood.Supported by a grant from the Australian Research Grants Committee.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.     

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Characterization of a lectin-binding storage protein from pea (Pisum sativum)

From the storage proteins of the pea (Pisum sativum), the fraction which interacts with the pea lectin by the sugar-binding site was studied. By electrophoretical subunit patterns and other criteria, this fraction resembles the group of the 7S storage proteins (vicilins). The fraction was resolved into subunits by micropreparative SDS PAGE. The N-terminal sequences of the individual subunits were determined. Most of these are identical with published vivilin subunit sequences; therefore this lectin-binding fraction belongs to the vicilins. Selected subunits and tryptic fragments were analysed for amino-acid compositions. Though unequivocal assignments to vicilin segments were possible, significant differences could be recognized, in particular in the tryptic fragments.     

Boron alleviates aluminum toxicity in pea (Pisum sativum)

One important target of boron (B) deficiency and aluminum (Al) toxicity is cell wall. Thus we studied the hypothesis that B is capable of alleviating Al toxicity in pea (Pisum sativum). Short-term and prolonged Al exposure to pea roots at different B levels was carried out on uniform seedlings pre-cultured at a low B level. When seedlings with a low B level were supplied with or without B for 1 and 2 days before 24 h Al exposure, roots were longer while root diameter was thinner after B addition especially for 2 days even with exposure to Al; root elongation was inhibited while root diameter was enlarged by Al exposure. Callose induction by Al toxicity was higher with B added, but this was reversed after the removal of the cotyledons. Hematoxylin staining was lighter in the root tips given B, and Al content in the root tips and cell walls dropped after exposure to B. This indicates that B alleviated Al toxicity in the root tips during short-term Al exposure by decreasing Al binding in root cell walls. An increase in chlorophyll and biomass and reduced chlorosis were found at the higher level of B during prolonged Al treatment, which was coincided with the decreased Al contents, indicating that B alleviated Al toxicity to shoots. B supplementation alleviates some of the consequences of Al toxicity by limiting some Al binding in cell walls, resulting in less injury to the roots as well as less injury to the shoots.     

Characterization of two novel defense peptides from pea (Pisum sativum) seeds

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC(50) values ranging from 0.04 to 22 microg/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca(2+), Mg(2+), Na(+), and K(+). Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group.     

Protochlorophyllide forms in non-greening epicotyls of dark-grown pea (Pisum sativum)

Low-temperature fluorescence emission spectra of 6.5-day-old dark-grown epicotyls of pea ( Pisum sativum ) revealed the presence of protochlorophyll(ide). The upper part of the epicotyl contained 30% of the protochlorophyll(ide) content per fresh weight found in pea leaves, whereas the lower part contained 3%. Three discrete spectral forms of protochlorophyll(ide) were clearly distinguished after Gaussian deconvolution of fluorescence excitation and emission spectra. Adding the satellite bands of the Qy_(0-0) transitions (the emission vibrational (Emv) bands with correlated amplitudes, gave the following delineation: Ex439–Em629–Emv684, Ex447–Em636–Emv700 and Ex456–Em650–Emv728. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunodetection of whole tissue extracts of the epicotyl indicated the presence of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). Electron micrographs showed prolamellar bodies in at most 11 % of the plastid profiles of the epicotyl cells. These prolamellar bodies were smaller, and many of them showed less regular structure than those of the leaves. Taken together, the results indicate that the protochlorophyll(ide) in epicotyls is arranged in a different way than in leaves.     

Physiology of acclimation to salinity stress in pea (Pisum sativum)

Pea (Pisum sativum L.) seedlings were grown in half strength Hoagland solution and exposed to 0, 10, 25 mM NaCl and 2.5% PEG 6000 for 1 week (pre-treatment). Thereafter plants were exposed to 0 and 80 mM NaCl for 2 weeks (main treatment). The control plants were maintained in half strength Hoagland solution without NaCl. Various physiological parameters were recorded from control, pretreated and non-pretreated plants. There was no negative effect of the pre-treatments on growth (total fresh and dry matter production), and plants pre-treated with 10 mM NaCl had biomass accumulation equal to control plants. The beneficial effect of salt acclimation was also evident in the prevention of K⁺ leakage and Na⁺ accumulation, primary in roots, suggesting that here the physiological processes play the major role. 2.5% PEG 6000 was not as efficient as salt in enhancing salt tolerance and acclimation appears to be more related to ion-specific rather than osmotic component of stress. We also recorded an increase of the xylem K/Na in the salt acclimated plants. Therefore, the present study reveals that short-term exposure of the glycophyte P. sativum species activates a set of physiological adjustments enabling the plants to withstand severe saline conditions, and while acclimation takes place primary in the root tissues, control of xylem ion loading and efficient Na⁺ sequestration in mesophyll cells are also important components of this process.