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.

First Report of Chickpea Wilt Caused by Clonostachys rhizophaga in Syria.

In 2007 and 2008, disease symptoms were observed on four cultivars of chickpea (Cicer arietinum L.), including two of the most popular cultivars grown in Syria (Ghab 3 and Ghab 4), in a replicated on-farm trial conducted in the fertile Al Ghab Plains. Affected plants exhibited chlorosis of the foliage, vascular discoloration, and death. In both years, plant mortality reached 100% in plots of cvs. ICC 12004, Ghab 3, and Ghab 4, but only 60% in plots of cv. ILC 97-706. Five monosporic isolates obtained from surface-disinfested stems and roots were identified morphologically. All micromorphological characteristics indicated that the isolated fungi fit the description of Clonostachys rhizophaga Schroers (1). Wilting of chickpea was widespread in the area, and fungal isolations from a random sample of diseased plants in neighboring farmers' fields revealed the presence of C. rhizophaga. In culture, isolates formed dimorphic, Verticillium-like (primary) or penicillate (secondary) conidiophores and ovoidal to elongate, slightly curved or asymmetrical, 5 to 9 µm long and 2.5 to 3.5 µm wide conidia showing a slightly laterally displaced hilum. The identification of the five isolates as C. rhizophaga was supported by sequencing approximately 600 bp of the ß-tubulin gene (tub2). Two representative sequences have been deposited under GenBank, Accession No. FJ593882 for strain CBS 124507 and No. FJ593883 for CBS 124511. Both were 100% similar to the sequence of C. rhizophaga strain CBS 361.77 (GenBank Accession No. AF358158) but differed by a deletion of 2 nucleotides relative to the ex-type strain of C. rhizophaga, CBS 202.37 (GenBank Accession No. AF358156). Two methods were used to inoculate plants and complete Koch's postulates. Method 1 used a 10-mm-diameter mycelial plug to inoculate healthy 3-day-old seedlings grown on 40 ml of Hoagland nutrient agar medium in a glass tube (one seedling per tube). The plug was placed mycelial-side down on the surface of the medium, and the fungus subsequently colonized the medium and penetrated the plant roots. Method 2 involved mixing autoclaved seed that had been colonized by each isolate with sterilized soil (1:12 vol/vol) prior to transplanting healthy seedlings into the soil mix. Thirty plants of each cultivar were tested per isolate per method, and controls received sterile agar plugs or autoclaved chickpea seed only. Irrespective of inoculation method, all five isolates caused wilt and plant death of all cultivars within 15 days (method 1) or 2 months (method 2) postinoculation. Symptoms were similar to those originally observed in the field and controls remained healthy. C. rhizophaga was recovered from all affected plants. To our knowledge, this is the first report of C. rhizophaga as a pathogen of chickpea. In an earlier report, C. rhizophaga (as Verticillium rhizophagum Tehon & Jacobs, nom. invalid.) was identified as the causal agent of a disastrous disease of Ulmus americana in Ohio (2). C. rhizophaga has been reported from Chile, Ecuador, the United States, and Switzerland (1). References: (1) H.-J. Schroers. Stud. Mycol. 46:85, 2001. (2) L.-R. Tehon and H. L. Jacobs. Bull. Davey Tree Expert Company, Kent, OH. 6:3, 1936.

Effect of caffeine-coconut products interactions on induction of microsomal drug-metabolizing enzymes in Wistar albino rats.

Effect of caffeine-coconut products interactions on induction of drug-metabolizing enzyme in Wistar albino rats was studied. Twenty rats were randomly divided into four groups: The control group (1) received via oral route a placebo (4.0 ml of distilled water). Groups 2 to 4 were treated for a 14-day period with 50 mg/kg body weight of caffeine, 50 mg/kg body weight of caffeine and 50 mg/kg body weight of coconut water, and 50 mg/kg body weight of caffeine and 50 mg/kg body weight of coconut milk in 4.0 ml of the vehicle via gastric intubation respectively. One day after the final exposure, the animals were anaesthetized by inhalation of an overdose of chloroform. The blood of each rat was collected by cardiac puncture while the liver of each rat was harvested and processed to examine several biochemical parameters, i.e., total protein and RNA levels, protein/RNA ratios, and activities of alanine and aspartate amino transferase (ALT and AST, respectively). The results showed that while ingestion of coconut milk and coconut water increased the values of protein and protein/RNA ratios, it decreased alanine and aspartate amino transferase (ALT and AST) activities. These effects, in turn, enhanced the induction of the metabolizing enzymes and a resultant faster clearance and elimination of the caffeine from the body, there by reducing the toxic effect on the liver.

Effect of alcohol and kolanut interaction on brain sodium pump activity in Wistar albino rats.

Effect of alcohol-kolanut interaction on sodium pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4 ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0 ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities.

A genetic linkage map of water yam ( Dioscorea alata L.) based on AFLP markers and QTL analysis for anthracnose resistance.

A genetic linkage map of the tetraploid water yam ( Dioscorea alata L.) genome was constructed based on 469 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F(1) cross. The F(1) was obtained by crossing two improved breeding lines, TDa 95/00328 as female parent and TDa 87/01091 as male parent. Since the mapping population was an F(1) cross between presumed heterozygous parents, marker segregation data from both parents were initially split into maternal and paternal data sets, and separate genetic linkage maps were constructed. Later, data analysis showed that this was not necessary and thus the combined markers from both parents were used to construct a genetic linkage map. The 469 markers were mapped on 20 linkage groups with a total map length of 1,233 cM and a mean marker spacing of 2.62 cM. The markers segregated like a diploid cross-pollinator population suggesting that the water yam genome is allo-tetraploid (2n = 4 x = 40). QTL mapping revealed one AFLP marker E-14/M52-307 located on linkage group 2 that was associated with anthracnose resistance, explaining 10% of the total phenotypic variance. This map covers 65% of the yam genome and is the first linkage map reported for D. alata. The map provides a tool for further genetic analysis of traits of agronomic importance and for using marker-assisted selection in D. alata breeding programmes. QTL mapping opens new avenues for accumulating anthracnose resistance genes in preferred D. alata cultivars.