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 Record in Syria of Didymella fabae, the Teleomorph of Ascochyta fabae and Causal Organism of Faba Bean Blight.

Ascochyta blight, caused by Ascochyta fabae Speg., is a common and destructive disease of faba bean (Vicia faba L.) in the Middle East, Europe, Canada, New Zealand (4), and Australia. The main sources of inoculum are debris and seeds from which spores are air- and splashborne. The teleomorph of A. fabae has been reported previously only from England (2). The presence of the teleomorph supports the variability reported in the fungus populations from Canada (3) and Poland (1). Stems of faba bean plants, severely infected with A. fabae, were collected in July 1999 from Tel Hadya, Syria. The plants previously had been inoculated with a mixture of isolates of the pathogen, collected from the main faba bean-growing regions in Syria between 1996 and 1998, and kept under shade. The infested stems were used to inoculate the ICARDA Faba Bean Ascochyta Nursery planted on 29 November 1999. During late January 2000, symptoms appeared on the susceptible faba bean genotype. Stem pieces from debris used for inoculations were collected from the field and examined microscopically for the presence of ascomata. The maximum, minimum, and mean temperatures and rainfall at Tel Hadya during December 1999 were 16.5, 5.8, and 8.7°C and 22.4 mm, respectively. There were 16 nights when temperatures dropped below 0°C, and 10 nights when temperatures were between 0 and 5°C. Ascomata of A. fabae ranged from 76 to 209 µm wide (average 158 ± 3.9 µm) and 101 to 285 µm in length (average 178 ± 4.1 µm). Asci were 10 to 15 µm wide (average 14 ± 0.3 µm) and 51 to 96 µm long (average 63 ± 1.1 µm). Ascospores were 5 to 8 µm wide (average 7 ± 0.2 µm) and 15 to 20 µm in length (average 17 ± 0.3 µm). These measurements are comparable to those reported from England. Individual ascomata were dissected from stem tissue and fixed to the lids of petri dishes containing 2% water agar. After 24 h, the petri dishes were examined microscopically to locate ascospores on the surface of the medium. Germinating ascospores and developing colonies were transferred from water agar to faba bean dextrose agar. Colonies characteristic of A. fabae developed on the latter medium within 7 days of incubation at 20 ± 2°C. Pathogenicity tests of developing colonies were carried out on 3-week-old faba bean plants (Giza 4) using a spore suspension (2.5 × 105 spores per ml) of each of the isolates. Both inoculated seedlings and control seedlings inoculated with sterile water were covered with plastic bags for 48 h in a plastic house maintained at 18 ± 2°C. After removal of the plastic bags, seedlings were wetted four times per day by spraying with tap water to runoff. Inoculated plants showed characteristic symptoms of Ascochyta blight 15 days after inoculation. The fungus was reisolated from lesions that developed on leaflets of all inoculated seedlings, but not from any of the control seedlings. This is the first report of the occurrence of A. fabae, the sexual stage of Didymella fabae Jellis & Punithalingam in Syria, and indicates that the fungus could develop population variants. These findings have implications for breeding for resistance to Ascochyta blight. References: (1) A. Filipowicz. Faba Bean Abstr. 4:47, 1983. (2) G. J. Jellis and E. Punithalingam. Plant Pathol. 40:150, 1991. (3) P. D. Kharbanda and C. C. Bernier. Can. J. Plant. Pathol. 2:139, 1980. (4) K. Y. Rachid et al. Plant Dis. 75:852, 1991.