Conversion of wyerone to wyerol by Botrytis cinerea and B. fabae in vitro

The phytoalexin wyerone was induced to accumulate in cotyledons of Vicia faba infected with Botrytis cinerea or B. fabae. The acetylenic keto ester, wyerone, was converted to the less antifungal corresponding hydroxy ester, wyerol, by both species of Botrytis in vitro.     

A chemical dichotomy in phytoalexin induction within the tribe vicieae of the leguminosae

Using either leaf or cotyledon tissues, the phytoalexin response of over 60 representative members of the genera Lathyrus, Lens, Pisum and Vicia was determined. Twenty-nine Vicia species produced furanoacetylenes, as did the two Lens species examined. By contrast, Pisum and Lathyrus failed to produce any of these phytoalexins, but formed pisatin instead. Thus, pisatin was formed as the major phytoalexin in 29 of 31 Lathyrus species tested, representing 10 sections within the genus. This dichotomy in phytoalexin response is discussed in relation to the taxonomy of the tribe Vicieae.     

Degradation of wyerone,the phytoalexin of faba beans byRhizobium leguminosarum

Wyerone is the predominant phytoalexin produced byVicia faba. At a concentration of 100 µM, wyerone prolonged the lag phase before the onset of exponential growth of different strains ofRhizobium leguminosarum andBradyrhizobium japonicum. The response to wyerone was dose dependent, with a shorter lag phase occurring at lower concentrations. Wyerone was only moderately inhibitory towards the phytopathogenic bacteriumErwinia carotovora cv. atroseptica. HPLC analysis of the medium during bacterial growth indicated thatRhizobium leguminosarum was able to metabolize wyerone. The identification of the product as hydroxyester wyerol was confirmed by GC/MS analysis.     

Changes in wyerone acid concentrations in leaves of Vicia faba after infection by Botrytis cinerea or B.fabae

Wyerone acid was produced by leaves of Vicia faba in response to infection by both Botrytis cinerea and B.fabae. Host cell death caused by either fungus appeared to be the trigger for rapid wyerone acid synthesis, although the phytoalexin was not confined to brown cells. At B. cinerea inoculation sites wyerone acid concentration increased rapidly, at the time of fungal invasion of the epidermis, to levels greater than that completely inhibitory to mycelial growth. Wyerone acid is therefore probably the primary cause of the inhibition of B. cinerea within infected tissue. The partial blackening of B.fabae inoculation sites and surrounding peripheral tissues was accompanied by an increase in wyerone acid. There followed a striking decrease as tissues became completely blackened and invaded by B.fabae. B.fabae appeared to metabolize wyerone acid and prevent its accumulation in invaded tissues. Mycelial growth of B. fabae was less sensitive to wyerone acid than was B. cinerea. The differing abilities of B.fabae and B. cinerea to spread from lesions after both have induced wyerone acid production probably depend on both their differing sensitivities to the phytoalexin and their abilities to metabolize it to less toxic products.     

Global Wild Annual Lens Collection: A Potential Resource for Lentil Genetic Base Broadening and Yield Enhancement

Crop wild relatives (CWRs) are invaluable gene sources for various traits of interest, yet these potential resources are themselves increasingly threatened by the impact of climate change as well as other anthropogenic and socio-economic factors. The prime goal of our research was to cover all aspects of wild Lens genetic resource management like species characterization, agro-morphological evaluation, diversity assessment, and development of representative sets for its enhanced utilization in lentil base broadening and yield improvement initiatives. We characterized and evaluated extensively, the global wild annual Lens taxa, originating from twenty seven counties under two agro-climatic conditions of India consecutively for three cropping seasons. Results on various qualitative and quantitative characters including two foliar diseases showed wide variations for almost all yield attributing traits including multiple disease resistance in the wild species, L. nigricans and L. ervoides accessions. The core set developed from the entire Lens taxa had maximum representation from Turkey and Syria, indicating rich diversity in accessions originating from these regions. Diversity analysis also indicated wide geographical variations across genepool as was reflected in the core set. Potential use of core set, as an initial starting material, for genetic base broadening of cultivated lentil was also suggested.     

Biosynthesis of the phytoalexin wyerone in Vicia faba

In contrast to earlier results [1-¹⁴C] acetate, [2-¹⁴C] malonate and [n 9,10-³H] oleate show significant incorporations into wyerone and related Vicia faba phytoalexins, following infection by Botrytis cinerea.     

Wyerone Acid Phytoalexin Synthesis and Peroxidase Activity as Markers for Resistance of Broad Beans to Chocolate Spot Disease

Infection of broad bean (Vicia faba L.) leaves with Botrytis fabae (Sard.) resulted in wyerone acid phytoalexin accumulation and increase in peroxidase activities in resistant and susceptible broad bean cultivars. Rapid wyerone acid accumulation was observed in leaves of resistant cultivars that reached levels greater than twofold of the susceptible cultivars. Following infection of broad bean cultivars, either resistant or susceptible to B. fabae, wyerone acid synthesis in resistant cultivars peaked at 5 days then declined, whereas in susceptible cultivars synthesis gradually decreased. While the ethanol‐extract of wyerone acid significantly reduced B. fabae spore germination with increased concentrations, the phytoalexin had no effect on in vitro B. fabae germ‐tube growth. Peroxidase activity in infected leaves of resistant was 10 times higher than the susceptible cultivars and eight times higher in uninfected leaves of the resistant than the susceptible cultivars. Peroxidase activity in infected leaves was greater than twofold in resistant cultivars and less than twofold in susceptible cultivars, when compared with uninfected leaves. The ratio of peroxidase activity in infected to uninfected leaves increased over time in susceptible cultivars but remained unchanged in resistant cultivars. Peroxidase activity in uninfected and infected leaves and wyerone acid biosynthesis in infected broad bean plants were successfully used to ascertain the resistance and susceptibility of four broad bean cultivars to B. fabae. Using wyerone acid synthesis and peroxidase activity as preliminary markers for resistance of broad bean to chocolate spot disease, caused by B. fabae, is suggested in this study.     

The effect of pH and composition of test solutions on the inhibitory activity of wyerone acid towards germination of fungal spores

Wyerone acid at 100μg/ml prevented germination of conidia of Botrytis cinerea Fr. in aqueous extracts of leaves of Vicia faba L., but it was inactive in water and some synthetic nutrient solutions though antifungal activity appeared when the pH of these media was adjusted to 4.0 or 4.5. In pollen diffusates (V.faba or Nicotiana glutinosd) wyerone acid had little activity even at pH 4.0. In malt extract at pH 4.5 wyerone acid was active against several other plant pathogenic fungi but not against B. cinerea. It was concluded that both pH and unknown components of natural media affected the fungitoxicity of wyerone acid.     

Inter-primer binding site retrotransposon and inter-simple sequence repeat diversity among wild Lens species

Even though lentil has been an important food legume for centuries, genetic studies in lentil are still in their infancy. Genetic diversity and relationships among wild Lens species from Turkey has seldom been investigated. Additionally, a limited number of simple sequence repeat (SSR) markers have been developed for use in breeding and genetic studies of lentil crop. In this study, molecular characterization of 50 accessions mostly from Turkey, belonging to 6 wild and 1 cultivated Lens species, was performed using newly developed inter-primer binding site (iPBS) retrotransposons and inter-SSR (ISSR) markers. The 10 iPBS primers generated a total of 151 scorable bands, of which 150 were polymorphic (99.3%) with an average of 15.0 polymorphic fragments per primer. The 10 ISSR primers detected 138 scorable bands showing 100% polymorphism, with an average of 13.5 bands per primer. The average polymorphism information content (PIC) value for ISSR markers (0.97) was higher than that for iPBS markers (0.90). Lens orientalis was found to be the most diverse species, raising the possibility of wide crosses with cultivated species Lens culinaris. Cultivated varieties also showed high level of polymorphism, at 82.92% and 51.92% with ISSR and iPBS markers, respectively. Lens lamottei and Lens tomentosus were found as the least polymorphic species using both marker systems. The grouping of accessions and species within clusters were almost similar when iPBS and ISSR graphs were compared. Our data also suggested the role of iPBS-retrotransposons as ‘a universal marker’ for molecular characterization of wild and cultivated Lens species.     

Polar Movement of Indole-3-acetic Acid-C in Roots of Lens and Phaseolus

A critical review of the few papers on IAA-¹⁴C movement in roots revealed apparent contradictions, as well as flaws in experimental design that would be apt to cause artifacts. The movement of ¹⁴C from IAA-¹⁴C was studied in sections of Lens and Phaseolus roots, using a system 20 or more times as sensitive as any previously used. To make sure that our results with roots could be compared validly with published work on petioles and stems, we used the same techniques as we had earlier used for shoot structures. The results with Lens were similar in many ways to those for shoots: net movement into receiver blocks was very strongly polar, followed a linear course for several hours, and showed a velocity of the same order of magnitude as in shoots (and, in fact, very close in absolute value to that found in Coleus stem cylinders). Also, as with shoots, all the radioactivity in receiver blocks ran to the R_F of IAA. The time-course of loss of counts from donor blocks was similar to that found in shoots. The 2 most striking differences from shoots were 1) the very low percentage of added ¹⁴C that was moved into the receivers (about one-tenth of the values for bean petioles), and 2) the fact that the polar movement was acropetal in roots, rather than basipetal as in shoots. Results with Phaseolus roots were similar to those for Lens, although an additional complication with Phaseolus roots was the indication of a transitory stage of weak basipetal polarity in the first few hours after excising the section. This stage was followed in a few hours by a stronger acropetal polarity.     

Dihydrowyerone derivatives as components of the furanoacetylenic phytoalexin response of tissues of Vicia faba

Changes in concentrations of 7 wyerone derivatives in bean tissues undergoing resistant reactions to Botrytis cinerea or B. fabae and in cotyledons in response to mercuric chloride have been examined using high performance liquid chromatography. The proportion of derivatives occurring in their saturated (dihydro) forms varied between cotyledon, leaf and pod tissues and with time after inoculation. Unsaturated derivatives were always present in greater concentrations than their dihydro analogues.     

Comparison of crossability, RAPD, SDS-PAGE and morphological markers for revealing genetic relationships within and among Lens species

The phylogenetic relationships among (sub)-species in the genus Lens have been reviewed based on recent published reports. There was both a substantial level of agreement and disagreement between reports based on different analytical procedures and different plant germ plasms. Lens culinaris ssp. orientalis appeared as the wild progenitor of the cultivated lentils. A gene flow from L. odemensis and L. ervoides during lentil crop evolution was suggested. Morphological characters (quantitative and qualitative) showed a different taxonomic pattern in the genus Lens. The use of nuclear and biochemical markers (RFLPs, RAPDs, seed-protein electrophoresis) appeared to be the most consistent and reliable methods for determining genetic relationships. It is suggested that these techniques be used in combination for taxonomic analysis of the genus Lens.     

Relationships among cultivated and wild lentils revealed by RAPD analysis

RAPD markers were used to distinguish between six different Lens taxa, representing cultivated lentil and its wild relatives. Twenty-four arbitrary sequence 10-mer primers were identified which revealed robust and easily interpretable amplification-product profiles. These generated a total of 88 polymorphic bands in 54 accessions and were used to partition variation within and among Lens taxa. The data showed that, of the taxa examined, ssp. orientalis is most similar to cultivated lentil. L. ervoides was the most divergent wild taxon followed by L. nigricans. The genetic similarity between the latter two species was of the same magnitude as between ssp. orientalis and cultivated lentil. In addition, species-diagnostic amplification products specific to L. odemensis, L. ervoides and L. nigricans were identified. These results correspond well with previous isozyme and RFLP studies. RAPDs, however, appear to provide a greater degree of resolution at a sub-species level. The level of variation detected within cultivated lentils suggests that RAPD markers may be an appropriate technology for the construction of genetic linkage maps between closely related Lens accessions.On sabbatical leave from HP Agricultural University, Palampur 176 062, India     

Bowman–Birk inhibitors in Lens: identification and characterization of two paralogous gene classes in cultivated lentil and wild relatives

In order to investigate the genetic structure of lentil Bowman–Birk inhibitors (BBIs), primers were designed on pea BBI sequences. The sequences obtained from lentil DNA, using these primers, indicate that lentil possesses at least two paralogous genes. Protein sequences translated in silico from lentil DNA sequences suggest that the two coded proteins are highly similar to Pisum trypsin inhibitor TI1 and TI6 BBIs, respectively. In fact, both are double-headed inhibitors, one class showing the presence of a trypsin- and a chymotrypsin-reactive site, the other showing two trypsin-inhibition sites, similar to pea TI1 and TI6, respectively. The same primers were used to amplify sequences from the DNA of other Lens species. The results strongly support that all species of Lens possess the same classes of BBI coding genes, orthologous to those identified in the cultivated lentil. Lens nigricans showed the most diverging sequences both at the nucleotide and the amino acid level. The similarity of the two gene classes identified in the genus Lens to those of Pisum and the observations that the patterns of expression of the Lens genes are equivalent to those of pea orthologous genes, possibly imply that BBIs in Lens are coded by gene classes with similar genome organization and function to those of pea. Finally, a phyletic analysis, based on the comparison of sequences obtained from other species belonging to the Vicieae tribe of the Fabaceae family, strongly suggests that all Vicieae could have a similar genome organization and function for BBI genes, and that this could be a general rule in all the Fabaceae family.Publication of the Institute of Plant Genetics N. 50     

AFLP analysis of the diversity and phylogeny of Lens and its comparison with RAPD analysis

AFLP and RAPD marker techniques have been used to evaluate and study the diversity and phylogeny of 54 lentil accessions representing six populations of cultivated lentil and its wild relatives. Four AFLP primer combinations revealed 23, 25, 52 and 48 AFLPs respectively, which were used to partition variation within and among Lens taxa. The results of AFLP analysis is compared to previous RAPD analysis of the same material. The two methods provide similar conclusions as far as the phylogeny of Lens is concerned. The AFLP technique detected a much higher level of polymorphyism than the RAPD analysis. The use of 148 AFLPs arising from four primer combinations was able to discriminate between genotypes which could not be distinguished using 88 RAPDs. The level of variation detected within the cultivated lentil with AFLP analysis indicates that it may be a more efficient marker technology than RAPD analysis for the construction of genetic linkage maps between carefully chosen cultivated lentil accessions.     

Allozyme divergence and evolution in the genusLens

The genusLens includes 5 taxonomic species:L. culinaris is cultivated andL. orientalis, L. odemensis, L. ervoides, andL. nigricans are wild. All the species are annual and almost exlusively selfers. The wild lentils are distributed over a large geographical area and form small disjunct populations which are composed of a small number of plants. 67Lens populations were assayed electrophoretically for 9 enzyme systems; 15 enzymic genes with 37 alleles were identified. The genetic distances (D) measured between the pairs of populations indicated a significantly greater similarity between populations belonging to the same taxonomic species. Assuming the populations represent a random sample of the variability in each of the species the genetic distances (D) between the 5 taxa were calculated. The shortest genetic distance was found betweenL. orientalis andL. culinaris. Another significant feature of the data is the apparent isolation ofL. nigricans from the other 4 species. The genetic distances between theLens species are compared to the patterns of crossability barriers between them.     

An intersubspecific genetic map of<Emphasis Type="Italic"> Lens</Emphasis>

A Lens map was developed based on the segregational analysis of five kinds of molecular and morphological genetic markers in 113 F₂ plants obtained from a single hybrid of Lens culinaris ssp. culinaris × L. c. ssp. orientalis. A total of 200 markers were used on the F₂ population, including 71 RAPDs, 39 ISSRs, 83 AFLPs, two SSRs and five morphological loci. The AFLP technique generated more polymorphic markers than any of the others, although AFLP markers also showed the highest proportion (29.1%) of distorted segregation. At a LOD score of 3.0, 161 markers were grouped into ten linkage groups covering 2,172.4 cM, with an average distance between markers of 15.87 cM. There were six large groups with 12 or more markers each, and four small groups with two or three markers each. Thirty-nine markers were unlinked. A tendency for markers to cluster in the central regions of large linkage groups was observed. Likewise, clusters of AFLP, ISSR or RAPD markers were also observed in some linkage groups, although RAPD markers were more evenly spaced along the linkage groups. In addition, two SSR, three RAPD and one ISSR markers segregated as codominant. ISSR markers are valuable tools for Lens genetic mapping and they have a high potential in the generation of saturated Lens maps.Communicated by H.C. Becker     

Die Dosis-Wirkungsrelationen bei geotroper und phototroper Reizung: Vergleich von Mono- mit Dicotyledonen

Zusammenfassung der Ergebnisse Die geotropen Krümmungen von Avena-Coleoptilen und Lens-Epicotylen nehmen linear proportional der Reizdauer zu. Bei geringen Reizzeiten sind beide Objekte gleich hoch empfindlich; bei längerer Reizdauer krümmen sich die Avena-Coleoptilen etwas stärker. Lens-Epicotyle reagieren auf zunehmende phototrope Reizung mit einer ersten und einer zweiten positiven Krümmung. Zwischen beiden Krümmungsbereichen (etwa von 10 000–1 000 000 lx · sec) sind sie phototropisch unempfindlich. Negative Krümmungen treten, nie auf. Ebenso verhalten sich 12 weitere dicotyle (Brassica napus, Brassica oleracea subsp. capitata, Brassica oleracea subsp. bullata, Lepidium sativum, Vicia villosa, Sinapis alba, Cucumis sativus, Linum usitatissimum, Helianthus annuus, Agrostemma githago, Raphanus sativus, Convolvulus tricolor) und 3 weitere monocotyle (Hordeum distichon, Secale cereale, Triticum aestivum) Pflanzenarten.Die phototrope Empfindlichkeit ist für Avena-Coleoptilen und Lens-Epicotyle im Bereich der ersten positiven Reaktion gleich groß, im Bereich der zweiten positiven Krümmung sind Avena-Coleoptilen empfindlicher. Das Reizmengengesetz ist für Lens-Epicotyle (wie für Avena-Coleoptilen) in der ersten positiven Krümmung gültig, in der zweiten positiven nicht.

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The dose response relations in geotropic and phototropic stimulation: Comparison of mono-with dicotyledonous plants

Summary The increase in the geotropic curvatures of Avena coleoptiles and Lens epicotyls is linear and proportional to the time of stimulation. With low stimulation the sensitivities of both objects are equal; with longer stimulation the curvature of Avena coleoptiles is somewhat greater.To increased phototropic irradiance Lens epicotyls react with a first and a second positive curvature. Between the two ranges of curvature (from about 10,000 up to 1,000,000 lx · sec) they are phototropically insensitive; negative curvatures never occur. The same reaction is shown by 12 other dicotyledonous plants (Brassica napus, Brassica oleracea subsp. capitata, Brassica oleracea subsp. bullata, Lepidium sativum, Vicia villosa, Sinapis alba, Cucumis sativus, Linum usitatissimum, Helianthus annuus, Agrostemma githago, Raphanus sativus, Convolvulus tricolor) and by 3 other monocotyledonous plants (Hordeum distichon, Secale cereale, Triticum aestivum). In the first positive reaction the phototropic sensitivities of Avena coleoptiles and Lens epicotyls are equal, in the second positive reaction Avena coleoptiles are more sensitive. For Lens epicotyls (for Avena coleoptiles, too) the reciprocity law is valid in the first positive reaction, but not in the second positive reaction.

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Aus einer Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Rostock (Steyer, 1964).     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

 Lens includes L. culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples. Received May 8, 2000 Accepted October 26, 2001