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.

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.