Epidemiological investigations in representative chickpea (Cicer arietinum L.) fields in southern Italy (Larino, Campobasso, 41°50′45″ N, 14°55′28″ E) identified severe withering (25 to 51%) of plants during flowering. Diseased plants showed a reduced total root biomass associated with less vigorous and chlorotic foliage. Browning and necrosis of subcortical and xylematic tissues of the crown and main roots were observed in affected plants. Symptomatic root and stem portions from 50 plants were sampled, surface disinfected with a sodium hypochlorite water solution (2% v/v for 2 min), rinsed with sterile distilled water, and placed in petri dishes containing potato dextrose agar with streptomycin sulfate (200 mg/l) and incubated at 25°C for 10 days. The most frequent fungal colony isolated showed macro- and microscopic characters specific of the genus Fusarium (3), with falcate and three-septate macroconidia (24.0 to 43.8 μm long) and microconidia (6.8 to 10.4 μm long) with zero or one septa. The ribosomal DNA of the fungal isolate processed by PCR using the ITS1F/ITS4 primers (2) produced an amplicon of 545 bp (ENA, Accession No. HG423346). A BLAST search with the amplified sequence in the database of the International Mycological Association (www.mycobank.org) revealed 99% identity with F. oxysporum sequences. Additional molecular analysis using the specific primers Foc0-12/Foc0-12rf for F. oxysporum f.sp. ciceris (Foc) produced an amplicon only in the chickpea virulent strain Foc-7952, race 0 (1) used as control; furthermore, PCR amplification for the Pisatin Demetylase gene by using the specific primers PDAF2a and PDAR3a (4) yielded the expected amplicon only for the new isolate, whereas no amplification was obtained with the control strain Foc-7952. Pathogenicity assays were carried out to complete Koch's postulates. To this aim, horticultural peat was infested with a conidial suspension (1 × 104 conidia/g of soil) from the new fungal pathogen, dispensed in plastic pots, and sown with surface sterilized seeds of chickpea (cv. Real, ISEA, Italy). Uncontaminated peat was used as control. For both treatments, 3 replicates of 10 seeds were used and experiments repeated twice. The plastic pots were kept in a growth chamber (28°C; 70% RH; 15/9 h light/dark) where the first disease symptoms on plants appeared 20 days after sowing. At the end of the experiments, all plants inoculated with the new isolate showed a high disease severity (98%), whereas non-inoculated plants remained healthy. The seedlings from infested soil demonstrated the same symptoms previously observed in the field, and after re-isolation, the causal agent demonstrated the same morphological features of the isolate used for inoculation. Pathogenicity tests were performed on pea, faba bean, melon, and tomato by using three cultivars for each crop. The results demonstrated high virulence of the new isolate of F. oxysporum f.sp. pisi (Fop) on both chickpea and pea with seed germination reduction, rot on main and secondary roots and cotyledonary leaves, and root biomass reduction and foliage chlorosis. No symptoms were observed on other inoculated vegetal species. Collectively, data of our investigation allow us to affirm that this is the first report of Fop as a new pathogen of chickpea. This result has great economic importance since it enables specific monitoring and management plans for this new disease caused by Fop on chickpea, a key crop for human and animal nutrition.
References: (1) M. M. Jiménez-Gasco and R. M. Jiménez-Díaz, Phytopathology 93:201, 2003. (2) I. Larena et al. J. Biotechnol. 75:187, 1999. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) N. A. Milani et al. Fungal Genet. Biol. 933:942, 2012.
