Pathotype IV, a New and Highly Virulent Pathotype of Didymella rabiei, Causing Ascochyta Blight in Chickpea in Syria.

The causal agent of Ascochyta blight disease of chickpea (Cicer arietinum L.) is highly variable because of the presence of a sexual phase (Didymella rabiei). There is also selection pressure on the pathogen due to wide adoption of improved resistant chickpea cultivars in some countries. The pathogen is able to produce pathotypes with specific virulence on particular cultivars. Three pathotypes, I, II, and III, have been reported (3). In this study, we confirmed the presence of a new and highly virulent pathotype that we designate as pathotype IV. To test the pathogenicity of the isolates collected and maintained at ICARDA, 10 isolates representing a wide spectrum of pathogenic variation, including those classified by S. M. Udupa et al. (3) and a putatively identified more virulent type, which was collected from a chickpea production field in the Kaljebrine area, Syria, were inoculated onto a set of differential chickpea genotypes. The differential genotypes, ILC 1929, ILC 482, ILC 3279, and ICC 12004, were sown in individual 10-cm-diameter pots containing potting mix and arranged in a randomized block design with three replications in a plastic house maintained at 18 to 20°C. Each differential genotype was inoculated individually with the 10 isolates following the methodology of S. M. Udupa et al. (3). DNA was extracted from single-spored isolates to compare the genotypes of the isolates using three simple sequence repeat (SSR) markers (ArA03T, ArH05T, and ArH06T) (2) and to determine the frequency of mating types (MAT) through the use of MAT-specific PCR primers for MAT1-1 and MAT1-2 (1). Host genotype reactions were measured on a 1 to 9 rating scale (1 = resistant and 9 = plant death). On the basis of the pathogenicity tests, the isolates were classified into four pathotypes: I (least virulent, killed ILC 1929 but not ILC 482, ILC 3279, or ICC12004); II (virulent, killed ILC 1929 and ILC 482 but not ILC 3279 or ICC12004); III (more virulent, killed ILC 1929, ILC 482, and ILC 3279 but not ICC12004); and IV (highly virulent, killed all four host differentials). Of 10 single-spore isolates tested, four showed similar disease reactions unique to pathotype I, four revealed pathotype II reactions, and one isolate each behaved like pathotype III or pathotype IV. SSR fingerprinting of these isolates provided evidence for genetic diversity since SSR ArH05T was highly polymorphic and amplified five bands, including pathotypes III- and IV-specific bands, which need further investigation to discern if this locus has any role to play in the virulence. MAT-type analysis showed that seven isolates were MAT1-1 while the remaining three isolates were MAT1-2. Only pathotype I showed the profile of MAT1-2 and the other three pathotypes were MAT1-1. Initially, a number of chickpea wild relatives were screened to identify sources of resistance to pathotype IV, but none of the accessions tested showed resistance. However, efforts are underway to combine minor and major gene(s) available in the breeding program in addition to a further search of the wild gene pools to control pathotype IV. References: (1) M. P. Barve et al. Fungal Genet. Biol. 39:151, 2003. (2) J. Geistlinger et al. Mol. Ecol. 9:1939, 2000. (3) S.M. Udupa et al. Theor. Appl. Genet. 97:299, 1998.

Effect of Centella asiatica L (Umbelliferae) on normal and dexamethasone-suppressed wound healing in Wistar Albino rats.

Centella asiatica is a reputed medicinal plant used in the treatment of various skin diseases in the Indian system of medicine. The objective of the study presented in this article was to evaluate the wound-healing potential of the ethanolic extract of the plant in both normal and dexamethasone-suppressed wound healing. The study was done on Wistar albino rats using incision, excision, and dead space wounds models. The extract of C asiatica significantly increased the wound breaking strength in incision wound model compared to controls (P < .001). The extract-treated wounds were found to epithelize faster, and the rate of wound contraction was significantly increased as compared to control wounds (P < .001). Wet and dry granulation tissue weights, granulation tissue breaking strength, and hydroxyproline content in a dead space wound model also increased at statistically significant levels as shown. The extract of the leaves had the effect of attenuating the known effects of dexamethasone healing in all wound models (P < .001, P < .05). The results indicated that the leaf extract promotes wound healing significantly and is able to overcome the wound-healing suppressing action of dexamethasone in a rat model. These observations were supported by histology findings.

Effect of Ocimum sanctum Linn. on normal and dexamethasone suppressed wound healing.

Ethanolic extract of leaves of O. sanctum was investigated for normal wound healing and dexamethasone depressed healing using incision, excision and dead space wound models in albino rats. The extract of O. sanctum significantly increased the wound breaking strength in incision wound model. The extract treated wounds were found to epithelialize faster and the rate of wound contraction was significantly increased as compared to control wounds. Significant increase in wet and dry granulation tissue weight, granulation tissue breaking strength and hydroxyproline content in dead space wound model was observed. The extract significantly decreased the antihealing activities of dexamethasone in all the wound models. The results indicated that the leaf extract promotes wound healing significantly and able to overcome the wound healing suppressing action of dexamethasone. Histological examination of granulation tissue to determine the pattern of lay-down for collagen confirmed the results.

A genetic linkage map of Lens sp. based on microsatellite and AFLP markers and the localization of fusarium vascular wilt resistance.

Microsatellites have currently become the markers of choice for molecular mapping and marker-assisted selection for key traits such as disease resistance in many crop species. We report here on the mapping of microsatellites which had been identified from a genomic library of lentil (Lens culinaris Medik.). The majority of microsatellite-bearing clones contained imperfect di-nucleotide repeats. A total of 41 microsatellite and 45 amplified fragment length polymorphism (AFLP) markers were mapped on 86 recombinant inbred lines derived from the cross ILL 5588 x L 692-16-1(s), which had been previously used for the construction of a random amplified polymorphic DNA and AFLP linkage map. Since ILL 5588 was resistant to fusarium vascular wilt caused by the fungus Fusarium oxysporum Shlecht. Emend. Snyder & Hansen f.sp. lentis Vasud. & Srini., the recombinant inbreds were segregating for this character. The resulting map contained 283 markers covering about 751 cM, with an average marker distance of 2.6 cM. The fusarium vascular wilt resistance was localized on linkage group 6, and this resistance gene was flanked by microsatellite marker SSR59-2B and AFLP marker p17m30710 at distances of 8.0 cM and 3.5 cM, respectively. These markers are the most closely linked ones known to date for this agronomically important Fw gene. Using the information obtained in this investigation, the development and mapping of microsatellite markers in the existing map of lentil could be substantially increased, thereby providing the possibility for the future localization of various loci of agronomic interest.

Tightly linked di- and tri-nucleotide microsatellites do not evolve in complete independence: evidence from linked (TA)n and (TAA)n microsatellites of chickpea (Cicer arietinum L.).

In order to understand the dynamics of microsatellite evolution, we have studied allelic variation at a closely linked (TA)(n) and (TAA)(n) microsatellite loci in 114 land races of chickpea ( Cicer arietinum L.), sampled worldwide. These two loci are separated by 27 bp. The two loci showed a very high degree of polymorphism and hence the combined length with the genetic diversity of 0.93, 0.90 and 0.98 for (TAA)(n), (TA)(n) and the combined length, respectively. Using the variation data at the linked loci, a standardized index of linkage disequilibrium was also computed ( I(S)(A)=0.092), which tests the null hypothesis of no linkage and was significant, indicating the presence of linkage disequilibrium. Furthermore, the dynamics of allelic variation showed that there is a threshold combined length, below which both (TAA)(n) and (TA)(n) loci evolve independently, and above which, if one locus increase in size, the other closely linked locus has a tendency to decrease its size and vice versa, without change in the overall ratio of (TAA)(n) and (TA)(n) allele sizes at the region. This result indicates that there are processes in the cell, which 'read' the combined size of the two loci both for proportion and length and determine the direction of tightly linked di- and tri-nucleotide repeat evolution.

Wound healing studies of Aristolochia bracteolata Lam. with supportive action of antioxidant enzymes.

Aristolochia bracteolata is a perennial herb, the leaves of which are used by the native tribals and villagers of the Chittoor District of Andhra Pradesh in India for the rapid healing of cuts and wounds. The ethanol extract of the shade-dried leaves of Aristolochia bracteolata Lam. was studied for its effect on wound healing in rats, using incision, excision and dead-space wound models, at two different dose levels of 400 and 800 mg/kg/body wt./day. The plant showed a definite, positive effect on wound healing, with a significant increase of the level of two powerful antioxidant enzymes, super oxide dismutase and catalase, in the granuloma tissue.

Genetic dissection of pathotype-specific resistance to ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatellite markers.

Ascochyta blight is an economically important disease of chickpea caused by the fungus Ascochyta rabiei. The fungus shows considerable variation for pathogenicity in nature. However, studies on the genetics of pathotype-specific resistance are not available for this plant-pathosystem. The chickpea landrace ILC 3279 has resistance to pathotype I and II of the pathogen. In order to understand the inheritance of pathotype-specific resistance in this crop, both Mendelian and quantitative trait loci analyses were performed using a set of intraspecific, recombinant inbred lines derived from a cross between the susceptible accession ILC 1272 and the resistant ILC 3279, and microsatellite markers. We identified and mapped a major locus (ar1, mapped on linkage group 2), which confers resistance to pathotype I, and two independent recessive major loci (ar2a, mapped on linkage group 2 and ar2b, mapped on linkage group 4), with complementary gene action conferring resistance to pathotype II. Out of two pathotype II-specific resistance loci, one (ar2a) linked very closely with the pathotype I-specific resistance locus, indicating a clustering of resistance genes in that region of the chickpea genome.

Wound healing property of ethanolic extract of leaves of Hyptis suaveolens with supportive role of antioxidant enzymes.

Ethanolic extract of leaves of Hyptis suaveolens was evaluated for its wound healing activity in ether-anaesthetized Wistar rats at two different doses (400 and 800 mg/kg) using incision, excision, and dead space wound model. Significant increase in skin breaking strength, granuloma breaking strength, wound contraction, hydroxyproline content and dry granuloma weight and decrease in epithelization period was observed. A supportive study made on granuloma tissue to estimate the levels of catalase and superoxide dismutase recorded a significant increase in the level of these antioxidant enzymes. Granuloma tissue was subjected to histopathological examination to determine the pattern of lay-down for collagen using Van Gieson and Masson Trichrome stains. Enhanced wound healing activity may be due to free radical scavenging action of the plant and enhanced level of antioxidant enzymes in granuloma tissue. Better collagenation may be because of improved antioxidant studies.

High mutation rate and mutational bias at (TAA)n microsatellite loci in chickpea (Cicer arietinum L.).

Microsatellites, very short tandemly repeated DNA sequences, are being extensively used in evolutionary genetics and molecular breeding of crop plants, because of their high degree of allelic variability, which is presumably caused by a high rate of mutation that changes microsatellite array length. In humans and various animals, mutation rates vary greatly and fall within the range of 10(-3) to 10(-6). In plants, the mutation rate at microsatellite loci seems to be higher than in animals, but no experimental estimates are available yet. Here, we report high spontaneous mutation rates (micro) and mutational bias at fifteen perfect (TAA)n microsatellite loci in inbred populations of chickpea. We show a significantly higher mutation rate, averaged across all loci, in the long-lived variety Ghab 2 (mu = 1.0 x 10(-2); detected in 16,050 allele-generations) compared to the variety Syrian Local (mu = 3.9 x 10(-3); detected in 15,600 allele-generations), which has a short life-span, with the majority of mutants (96.9%) in both varieties differing by < or = 1 repeat unit. Compared to animals, higher mutation rates in chickpea are likely to be due to the presence of long (TAA)n microsatellite repeat arrays and the larger number of DNA replications that meristematic initials of the plants undergo before reaching the reproductive phase. Thus, the long-lived variety undergoes more DNA replications, resulting in an accumulation of more mutations than in the variety with the shorter life-span.

Indigenous drugs and atherosclerosis.

Coronary artery disease (CAD) is a major health problem worldwide and its increasing prevalence in younger age groups is of serious concern. Precipitating factors include changes in lifestyles, food fads and stress, as well as the impact of longevity resulting from the control and prevention of communicable diseases. Dietary practices can often lead to increased levels of lipids in blood or trigger an underlying disposition to atherosclerosis. Hence, dietary modification has been an important method for the management of CAD, with the aim of lowering lipid levels. However, long-term trials have shown that this approach reduces cholesterol and triglycerides only moderately. Drug therapy with clofibrate, cholestyramine, etc., has been successful but some disadvantages limit their long-term use. An alternative approach to the treatment of atherosclerosis has been the use of indigenous drugs. This article reviews some of the drugs of the traditional Indian system of medicine, Ayurveda, which have been shown to possess antiatherogenic properties. (c) 2001 Prous Science. All rights reserved.

Genetic variation among populations of the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae) in Morocco and Syria.

The RAPD-PCR technique was used to study genetic variation within and among geographical populations of the Hessian fly, Mayetiola destructor (Say), from Morocco and Syria, associated with the fly's ability to overcome resistance in three wheat cultivars containing H5, H13 and H22 resistance genes. Variation was detected both for the level of susceptibility of the cultivars and RAPD profiles of M. destructor populations. By the use of RAPD-PCR, high genetic variability was detected among individuals and populations of M. destructor within and between areas separated geographically. The DNA fingerprints of populations of M. destructor were area-specific with Nei's measures of genetic distance ranging from 0.156 (between Abda and Beni Mellal, Morocco) to 1.977 (between Marchouch, Morocco and Lattakia, Syria). Cluster analysis of the genetic distances among the populations, identified the Syrian population as an outlier. A highly significant correlation (r = 0.81) observed between the genetic and geographic distances among the populations, provided genetic support for dispersal of the fly from its presumed origin in West Asia to Morocco.

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome.

A size-selected genomic library comprising 280,000 colonies and representing approximately 18% of the chickpea genome, was screened for (GA)n, (GAA)n and (TAA)n microsatellite-containing clones, of which 389 were sequenced. The majority (approximately 75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%-9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P > or = 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.

Allelic variation at (TAA)n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm.

A set of 12 randomly selected (TAA)n microsatellite loci of the cultivated chickpea (Cicer arietinum L.) were screened in a worldwide sample comprising 72 landraces, four improved cultivars and two wild species of the primary gene pool (C. reticulatum and C. echinosperum) to determine the level and pattern of polymorphism in these populations. A single fragment was amplified from all the accessions with each of 12 sequence-tagged microsatellite site markers, except for one locus where no fragment was obtained from either of the two wild species. There was a high degree of intraspecific polymorphism at these microsatellite loci, although isozymes, conventional RFLPs and RAPDs show very little or no polymorphism. Overall, the repeat number at a locus (excluding null alleles) ranged from 7 to 42. The average number of alleles per locus was 14.1 and the average genetic diversity was 0.86. Based on the estimates obtained, 11 out of the 12 frequency distributions of alleles at the loci tested can be considered to be non-normal. A significant positive correlation between the average number of repeats (size of the locus) and the amount of variation was observed, indicating that replication slippage may be the molecular mechanism involved in generation of variability at the loci. A comparison between the infinite allele and stepwise mutation models revealed that for 11 out of the 12 loci the number of alleles observed fell in between the values predicted by the two models. Phylogenetic analysis of microsatellite polymorphism in C. arietinum showed no relationship between accession and geographic origin, which is compatible with the recent expansion of this crop throughout the world.

Hypolipidemic activity of three indigenous drugs in experimentally induced atherosclerosis.

The effect of orally administered indigenous drugs Terminalia arjuna, T. belerica and T. chebula were investigated on experimental atherosclerosis. Rabbits were fed a cholesterol-rich diet to induce atherosclerosis. The three drugs were fed along with cholesterol. At the end of the experimental period the animals were killed and their plasma and tissue lipid components estimated. Atherosclerotic lesions of the aorta were examined histologically. T. arjuna was found to be the most potent hypolipidemic agent and induced partial inhibition of rabbit atheroma. The results indicate that T. arjuna may play an anti-atherogenic role.

Inhibition of lysyl oxidase by isoniazid and its effect on wound healing.

Isoniazid, an antitubercular drug, is known to be a potent inhibitor of monoamine oxidases. Effects of this drug, on lysyl oxidase (also a monoamine oxidase) and other wound healing parameters were studied in albino rats, in presence and absence of pyridoxal phosphate, using a dead space wound model. Tensile strength, collagen and glycosaminoglycan contents as well as lysyl oxidase activity were estimated in the granuloma tissue harvested from 10 day old dead space wounds. Isoniazid inhibited lysyl oxidase activity and a decrease in tensile strength as well as collagen content were observed. The effects were reversed on administration of a stoichiometric amount of pyridoxal phosphate. Hexosamine level was increased and hexuronic acid level decreased in the drug treated animals. Therefore, isoniazid may decrease the mechanical strength of collagen by inhibiting lysyl oxidase, by competiting for its obligatory cofactor pyridoxal phosphate, as well as by interfering in electrostatic interactions between collagen and the ground substance.

Preventive actions of Terminalia belerica in experimentally induced atherosclerosis.

Hypercholesterolaemia and atherosclerosis were induced experimentally in rabbits by cholesterol feeding. The effect of an indigenous drug, T. belerica, was evaluated in these hypercholesterolemic rabbits. T. belerica reduced the levels of lipids in hypercholesterolemic animals. There was also a significant decrease in liver lipids and heart lipids (P < 0.05) in the drug-treated animals.

The possible site of anti-asthmatic action of Tylophora asthmatica on pituitary-adrenal axis in albino rats.

The effects of the alcoholic extract, the petroleum ether fraction, and the aqueous fraction of the alcoholic extract of Tylophora asthmatica on weight of the adrenal gland and its functional activities (as evidenced by its cholesterol and vitamin C content, plasma steroid, hydroxyproline content in skin) and pituitary-adrenal axis were studied using normal, unilaterally adrenalectomised, dexamethasone-treated (steroid suppression of ACTH), and stereotaxically hypophysectomised male albino rats. The extracts showed similar actions (i.e. stimulation of adrenals as indicated by increase in weight and decrease in cholesterol and vitamin C contents). The plasma steroid level was increased but skin hydroxyproline level findings were not conclusive. T. asthmatica was found to antagonise dexamethasone/hypophysectomy-induced suppression of pituitary on activity of the adrenals. It may be concluded that T. asthmatica may act by a direct stimulation of the adrenal cortex.

Influence of Tridax procumbens on lysyl oxidase activity and wound healing.

The effects of an indigenous drug, Tridax procumbens L. (Compositae), on developing granulation tissue in rats were studied. Subcutaneously harvested granuloma tissue formed on dead space wound was removed at 4 day intervals up to 32 days of wounding. Lysyl oxidase activity, protein content, specific activity, and breaking strength were all increased in drug-treated animals as compared to controls. A fall in the lysyl oxidase activity was observed in drug-treated animals after day 8. The drug may be having a dual role: one a stimulatory (direct) effect in the initial phase of wound healing and the other a depressant (indirect) effect in the later stage.

The effect of etch-time on the bond strength of a sealant and on the etch-pattern in primary and permanent enamel: an evaluation.

This study was undertaken to establish laboratory evidence of minimum etch-time for primary enamel, for effective retention of occlusal sealants. The effect on enamel of contamination by oral fluid for different exposure times was also evaluated. Here it is shown that a short etch-time of 15 sec is satisfactory for primary enamel, and is also sufficient to produce the required etch-pattern for the strong binding of sealants.