Isozyme Variation in Wild and Cultivated Species of the Genus Trifolium L. (Leguminosae)

Isozyme patterns for PGI (phosphoglucoisomerase), MDH (malatedehydrogenase), EST (esterase) and SOD (superoxide dismutase)were evaluated in 36 accessions of eight Trifolium species,namely T. incarnatum, T. polymorphum, T. pratense, T. repens,T. resupinatum, T. riograndense, T. subterraneum and T. vesiculosum.Similarity between species and accessions was estimated by Jaccard’ssimilarity index based on presence or absence of bands. TheUPGMA method was utilized for the groupings and dendrogram constructionwith the aid of the NTSYS-PC program. Interspecific similaritywas low (J 0.351). The dendrogram presented eight groups, eachone corresponding to a taxonomic species. Most of the accessionsof the same species grouped together at J > 0.50, exceptT. riograndense, T. repens and T. pratense. These three speciesshowed the lowest similarity between their accessions, reflectinghigher intraspecific variation. Some accession-specific bandswere identified. The species groupings are consistent with traditionaltaxonomic species delimitation. Therefore isozyme patterns,especially when several systems are employed, are useful andreliable biochemical markers for the taxonomic delimitationand characterization of Trifolium germplasm. Copyright 2000Annals of Botany Company Genetic diversity, germplasm characterization, isoenzymatic variability, biochemical markers, isozymes,Trifolium     

Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting

The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species could have been the donor of one of two genomes of the allotetraploid T. repens. In addition, the dendrograms, produced by NTSYS-pc, also indicated close affinity of T. nigrescens and T. occidentale to the accessions of T. repens. These data is congruent with karyological and phylogenetic affinities between the white clover and T. occidentale. The relationships between the examined accessions, in the T. repens gene pool, may be regarded to indicate the presence of shared alleles between T. repens, T. occidentale and T. uniflorum. Further, T. occidentale showed close phylogenetic relations to T. pallescens.     

Isoflavone synthase (IFS) gene phylogeny in Trifolium species associated with plant isoflavone contents

This study presents the results of the identification and quantification of 12 isoflavones (prunetin, irilone, pseudobaptigenin, glycitein, daidzin, genistin, daidzein, pratensein, puerarin, biochanin A, formononetin and genistein) in 23 species of Trifolium (T. arvense, T. pratense, T. ligusticum, T. striatum, T. lappaceum, T. angustifolium, T. hirtum, T. subterraneum, T. isthmocarpum, T. stellatum, T. mutabile, T. strictum, T. fragiferum, T. alexandrinum, T. tomentosum. T. nigrescens subsp. petrisavii, T. nigrescens, T. glomeratum, T. subterraneum subsp. brachycalycinum, T. cherleri, T. resupinatum, T. campestre and T. repens). Isoflavones were extracted by an MSPD method and analyzed with HPLC coupled with a diode-array detector. The evaluation of molecular phylogeny of the IFS gene and the relation with isoflavone content was also performed. Five species (T. subterraneum subsp. brachycalycinum, T. alexandrinum, T. pratense, T. subterraneum and T. lappaceum) were identified with high levels of biochanin A (431–83 mg/kg), formononetin (72–365 mg/kg) and genistein (9–509 mg/kg), which could be utilized as alternative sources for the nutraceutical industry. Genetic phylogeny for the IFS gene was found in the species studied, with 20 out of 23 species having been divided into two clades, while the remaining three were genetically distant. Based on our results, we confirm the direct correlation between IFS gene polymorphism and isoflavones content in species of Trifolium particularly noted for formononetin. Therefore, the IFS gene can be utilized for screening Trifolium genotypes for formononetin. The relation of the three isoflavones' contents and the molecular phylogeny of plants determined by the IFS sequences, as a screening marker for plants with high isoflavone contents in Trifolium species, are to the best of our knowledge described for the first time.     

Isoflavonoid and stilbene phytoalexins of the genus Trifolium

The fungus-inoculated leaflets of 55 Trifolium species have been examined for the presence of isoflavonoid and non-flavonoid phytoalexins. Isoflavonoid derivatives belonging to the pterocarpan (medicarpin, maackiain and 4-methoxymaackiain) and isoflavan (vestitol, isovestitol, sativan, isosativan and arvensan) classes were isolated from 50 species whilst T. campestre and T. dubium accumulated the trihydroxy stilbene, transresveratrol. Three other species (T. badium, T. scutatum and T. spadiceum) apparently did not produce phytoalexins in response to fungal inoculation. The distribution and biosynthetic relationship of Trifolium phytoalexins is discussed and the genus compared with others belonging to the Trifolieae and related tribes.     

Evidence of Three Subspecies in Trifolium nigrescens Viv.

Germplasm accessions of Trifolium nigrescens Viv. were foundto belong to three distinct taxa which differed in morphology,hybrid seed-set in crosses among themselves and with white clover(T. repens L.), chromosomal distribution of rDNA genes, andrDNA internally transcribed spacer (ITS) region sequences. Thesetaxonomic groups correspond with T. nigrescens ssp. nigrescens,T. nigrescens ssp. petrisavii var.petrisavii and var. meneghinianum.The existence of var. meneghinianum has been rejected or ignoredin recent taxonomic treatments. The present study indicatesthat it is very distinctive and warrants at least subspeciesstatus. Phylogenetic analysis based on ITS sequences and podmorphology placed ssp. nigrescens as progenitor to the others.ITS sequences placed white clover closer to ssp. petrisaviibut neither DNA sequences nor rDNA chromosome locations clearlyshowed whether var. petrisavii or var. meneghinianum was closerto white clover. In contrast, results of hybridization studiesshowed that ssp. nigrescens gave full seed-set when pollinatedwith T. repens whereas both ssp. petrisavii var. petrisaviiand ssp. petrisavii var. meneghinianum gave poor seed-set. Allthree taxa showed close relationships with white clover andnone can clearly be shown to be a direct ancestor of T. repens.Copyright 2001 Annals of Botany Company Fabaceae, Trifolium nigrescens, Trifolium repens, internal transcribed spacer region (ITS), nuclear ribosomal DNA, interspecific hybridization     

Effects of leguminous ground cover competition on red birch and soil nutrient status in the nursery

Legumes as ground cover are regularly planted to increase nitrogen economy of crops and to improve soil. In the present study various clover species were evaluated as vegetative ground cover in nursery field production of micropropagated red birch (Betula pubescensEhrh. f. rubraUlvinen f. nova) in two 2-year experiments. The clover species and cultivars, Trifolium pratenseL. ‘Bjursele’, T. repens L. ‘Jogeva’, T. repens L. ‘Sonja’, T. hybridum L. ‘Frida’, T. incarnatum L. ‘Opolska’, T. resupinatum L. and T. subterraneum L. were compared to grass sod Festuca rubra L. ‘Ensylva’ and to a coverless ground (control). The last one was kept weed free by hand hoeing. Birch (leaves, stems, branches and roots) and soil nutrient concentrations (N, P, K, Ca, Mg and Fe) were analysed and nutrient ratios in birch determined. The annual clovers, T. incarnatumL., T. resupinatumL. and T. subterraneumL., provided about the same nutrient status in birch as did the control. Perennial clovers and grass were strong competitors with trees. High levels of P and Mg in birch leaves relative to N concentration were typical for poorly growing seedlings. Neither annual nor perennial clovers did generally improve soil nutrient status. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro culture and somatic embryogenesis of four Trifolium species

Callus cultures were induced from hypocotyl sections of Trifolium incarnatum L., T. vesiculosum Savi., T. ambiguum Bieb., and T. repens L. Callus production was the greatest for T. incarnatum and T. ambiguum on Phillips and Collins L2 medium with 0.06 mg/l picloram and 0.1 mg/l 6-benzylaminopurine and for T. vesiculosum and T. repens on Gamborg B5 medium with 1.25 mg/l 2,4-dichlorophenoxyacetic acid (2.4-D), 0.5 mg/l naphthaleneacetic acid (NAA), and 0.5 mg/l 6-furfurylaminopurine (kinetin). Proembryos or somatic embryos were formed in all four species though germination and subsequent plantlet formation only occured in t. incarnatum and T. vesiculosum. Mature plants were obtained via somatic embryogenesis for T. incarnatum. The regenerated plants were fertile and had the normal diploid number of chromosomes (2n = 14).     

The Effects of Rootzone Salinity and Hypoxia on Shoot and Root Growth in Trifolium Species

The effect of concurrent salinity (0-60 mM NaCl) and rootzonehypoxia (flooding for up to 15 d) on shoot and root growth andshoot ion concentrations of six species of Trifolium (T. subterraneumL., T. fragiferum L., T. michelianum Savi., T. isthmocarpumBot., T. purpureum Lois., and T. repens L.), was studied intwo greenhouse experiments. There was a significant salinityx flooding effect for shoot yield but no significant salinityx flooding x species interaction although individual speciesdiffered significantly (P < 0·001) in their growthresponse to the saline or flooded conditions separately. Concentrationsof Na and Cl in the shoots of all species increased with increasingperiods of saline flooding and there was a significant salinityx flooding interaction. Sodium and Cl concentrations were significantlyhigher (P < 0·001) in T. purpureum, the species inwhich shoot growth was most depressed by saline flooding, thanother species. In T. michelianum, T. fragiferum and T. repens,fresh and dry weight of roots increased with flooding underboth saline and non-saline conditions while in T. subterraneumroot growth decreased. A significant proportion of the increasedroot growth in the first three species occurred as new adventitiousroots. These roots had higher percentages of internal gas spaceswithin the root tissue even in the presence of salinity comparedwith roots from non-flooded conditions. There were also significantlymore gas spaces in the total root tissue in T. fragiferum andT. repens under saline-flooding than in roots of T. subterraneum.Electron micrographs of the root cross sections illustratedthe presence of these gas spaces or aerenchyma. Trifolium fragiferum, T. repens and T. michelianum are morelikely to be suited to growth in soils prone to high salinityand to flooding than are T. subterraneum, T. purpureum and T.ishmocarpum.Copyright 1993, 1999 Academic Press Trifolium subterraneum, Trifolium fragiferum, Trifolium michelianum, Trifolium isthmocarpum, Trifolium purpureum, Trifolium repens, salinity, flooding, hypoxia, adventitious roots, aerenchyma, subterranean clover, white clover, strawberry clover, purple clover, balansa clover     

Ecogeographical analysis of the distribution of six Trifolium species in Sardinia

Collections of six species of Trifolium (Fabaceae) were made at 16 sites across the island of Sardinia in June/July 1998 and detailed ecogeographic information was recorded at each site. The six species collected were the inbreeding T. glomeratum, T. subterraneum and T. tomentosum, and the outcrossing T. nigrescens, T. michelianum and T. resupinatum. A principal components analysis and canonical variate analysis were conducted to group the sites, using the ecogeographical variables collected at each site. The variables that were found to be most important in grouping the sites were: mean January and July temperature, mean annual rainfall, latitude, longitude and soil pH. None of the six species were limited to only one ecogeographical group. However, it was often found to be only one or two variables that were determining their distribution. It is suggested that more detailed analysis of ecogeographic data of collection sites will become vital for improved planning of future collection missions, as the cost of collections and genetic erosion increases.     

Responses of genotypes from species of Trifolium, Ornithopus, Biserrula and Hedysarum to a highly virulent race of Phytophthora clandestina and new sources of resistance

Thirty‐six genotypes, including 15 cultivars and 10 breeding lines of Trifolium subterraneum, a single genotype of each of seven other species of Trifolium (viz. Trifolium dasyurum, Trifolium glanduliferum, Trifolium incarnatum, Trifolium michelanium, Trifolium purpureum, Trifolium spumosum and Trifolium vesiculosum), Biserrula pelecinus, Hedysarum coronarium, Ornithopus compressus and Ornithopus sativus were screened under controlled environmental conditions for resistance to root disease caused by the most pathogenic race of Phytophthora clandestina occurring in Australia, namely race 177. This is the first time any of these genera/species other than T.subterraneum has ever been screened for its response to P. clandestina. The root disease caused by P.clandestina is the first report of susceptibility to this pathogen for the seven other species of Trifolium and also for B.pelecinus, H.coronarium and O.sativus. Within T.subterraneum, a very high level of resistance was identified in cvs Denmark, Junee and Meteora [scores ≤1.5 (0–5 scale where 0 = no disease) across two separate screening tests] and in the breeding lines SL027 and SM023 (scores ≤1.3 across two separate screening tests). Six of the seven other species of Trifolium (viz. T.dasyurum, T.glanduliferum, T.incarnatum, T.michelanium, T.purpureum and T.spumosum) showed a high level of resistance (scores ≤0.8 across two separate screening tests), while T.vesiculosum showed a disease score of ≤1.2 across both screening tests. O.compressus showed no disease in either test, and O.sativus showed a disease score of ≤0.7 across both screening tests. H.coronarium was susceptible with a disease score of ≤2.8 across two separate screening tests, while B.pelecinus was highly susceptible with disease scores of 3.5 and 4.6 in these tests. The high levels of resistance identified against P.clandestina are useful sources of resistance that can be exploited commercially, either directly to minimise damage from this disease or as parents in breeding programs to develop cultivars within the genera/species tested with improved resistance to this highly pathogenic race of P.clandestina.     

Growth and root dry matter allocation by pasture legumes and a grass with contrasting external critical phosphorus requirements

Background and aims

This work aimed to quantify the critical external requirement for phosphorus (P) (i.e. extractable-P concentration required for 90 % of maximum yield) for a number of temperate legume species and understand differences in dry matter allocation, P distribution and P acquisition efficiency among these species.

Methods

Shoot and root growth of five legume and one grass species was assessed in response to six rates of P mixed into the top 45 mm of soil in a pot experiment. Dactylis glomerata and Trifolium subterraneum were used as benchmark species; they are commonly grown together in mixed temperate pastures and have low and high critical external requirements for P, respectively. Growth was compared with four alternative legume species: Ornithopus compressus, Ornithopus sativus, Biserrula pelecinus and Trifolium hirtum, that have root morphologies better suited to soil exploration and nutrient acquisition than that of Trifolium subterraneum.

Results

Dactylis glomerata, Ornithopus compressus and Ornithopus sativus had maximum yields equal to or greater than Trifolium subterraneum but achieved this at rates of P less than half that of Trifolium subterraneum. Biserrula pelecinus and Trifolium hirtum had critical P requirements between that of Trifolium subterraneum and the Ornithopus species, but also had lower yields. Root dry matter of Dactylis glomerata and the Ornithopus species in the fertilised soil layer was only marginally changed in response to low P supply. In contrast, Trifolium subterraneum, Trifolium hirtum and to a lesser extent Biserrula pelecinus markedly increased root dry matter allocation to this soil layer. Species with lower critical P requirements were able to take up more P per unit root dry mass than those with higher critical P requirements, particularly at lower levels of P addition.

Conclusions

The high P acquisition efficiencies of the Ornithopus species and Dactylis glomerata were likely to have contributed to their low critical external P requirements. It was surmised that differences in root morphology traits underpin the differences in acclimation to low P stress and P acquisition efficiency among the species.

     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

Nitrogen fixation, stomatal response and transpiration in Medicago sativa, Trifolium repens and T. subterraneum under water stress and recovery

Stomatal behaviour, transpiration and nitrogen fixation were investigated in Medicago sativa L. (cvs. Tierra de Campos and Aragon, Hidalgo-Maynar 1966), Trifolium repens L. (cv. Aberystwyth S-184) and Trifolium subterraneum L. (cv. Clare) subjected to drought by withholding water and then to three days’ recovery after rewatering. Dawn leaf water potential was measured with pressure chamber, stomatal response with a diffusion porometer and nitrogen fixation by using acetylene reduction technique. At low water potentials, the leaf resistance was higher in Medicago than in Trifolium. As water stress developed all species decreased their transpiration, T. subterraneum being the one most affected by moderate deficits. During water stress ‘Tierra de Campos’ always maintained higher acetylene reduction levels than ‘Aragon’ and the Trifolium species, except for the lowest water potentials. During recovery from water stress only ‘Tierra de Campos’ reached predeficit transpiration rates. In ‘Tierra de Campos’ acetylene reduction recovery after rewatering was more rapid and intense than in ‘Aragon’. It is concluded that, of the plants investigated, ‘Tierra de Campos’ was best adapted to water deficits.     

Cytological characterization of Vicia oroboides Wulfen in Jacq

Vicia oroboides, a rare taxon belonging to section Atossa of subgenus Vicia, was recovered and analysed by means of cytological and karyological methods with the aim of both characterising this species and integrating our knowledge on phylogeny of subgenus Vicia. Automated karyotype analysis and nuclear DNA content have been determined after Feulgen’s reaction; chromosome banding was performed by fluorochrome staining to evidence heterochromatic blocks along the chromosome complement. The chromosome number is in line with the values of the species of section Atossa; the GC- and AT-rich sites were identified by CMA and DAPI staining. Karyomorphological parameters, based on symmetry indices, provide information about the phylogenetic position of this species inside the subgenus Vicia. DNA content is reported for the first time.     

On the origin of the cyanogenic polymorphism in Trifolium repens L.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium. The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens . We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens . The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens , as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li . As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens .     

Additional Hosts for the Ring Nematode,Criconemella xenoplax

Some common legumes and weeds indigenous to peach orchards in South Carolina were tested in greenhouse experiments to determine their host suitability for Criconemella xenoplax. Legumes that were hosts for the nematode were dwarf English trefoil (Lotus corniculatus var. arvensis), big trefoil (L. uliginosis), birdsfoot trefoil (L. corniculatus), narrowleaf birdsfoot trefoil (L. tenuis), ball clover (Trifolium nigrescens), rose clover (T. hirtum), subterranean clover (T. subterraneum), striate lespedeza (Lespedeza striata), and partridge pea (Cassiafasciculata). Most nonleguminous plants tested did not support population increases, but small increases were observed on orchardgrass (Dactylis glomerata), broadleaf signalgrass (Brachiaria platyphylla), purslane (Portulaca oleracea), and Carolina geranium (Geranium carolinianum). Results indicate that leguminous plants probably should not be used as ground cover or rotation crops for plants that are injured by C. xenopax.     

Chromosome number and genetic system in several Trifolium species related to T. alexandrinum

Chromosome counts have been done in 12 species of Trifolium, closely related to T. alexandrinum L., and the chiasma frequency in meiosis, pollen fertility, and seedset upon selfing was estimated. In all species, the basic number of chromosomes was 2n=16 arranged in eight bivalents. Two species, T. salmoneum Mout, and T. berytheum Boiss., have 0–7 B-chromosomes in eight out of nine plants examined. The frequency of B-chromosomes was somewhat correlated with pollen fertility. Chiasma frequency ranged between 10.8 and 16.1 per cell with specific and ecotypic differentiation. Except for T. alexandrinum, the species analyzed were completely (or, in a few cases, highly) self-incompatible.Contribution from the Volcani Institute of Agricultural Research, Bet Dagan, Israel. 1970 Series, No. 1677 E. This research was financed by a grant from the U.S.D.A. under P.L. 480, Contract A10-CR-56.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

Selection and symbiotic properties ofRhizobium leguminosarum biovarphaseoli strains harboring pRtr5a

TheRhizobium leguminosarum biovartrifolii symbiotic plasmid pRtr5a has been transferred toR. leguminosarum biovarphaseoli RCR 3644-S1. The transconjugant selection had been done byTrifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix⁺ onT. repens andT. alexandrinum, Fix^– onT. subterraneum, and formed a few small white and Fix^– nodules onPhaseolus vulgaris. It is shown that this nodulation onP. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage throughTrifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.