Vicia sativa, commonly known as common vetch, is one of the most economically important annual grain legumes in the world, used in animal feeding as forage or grain legume. Furthermore, vetch plays a vital role in agriculture due to its potential to significantly increase soil fertility, making it suitable for cultivation in sustainable agriculture as a cover crop, main crop, rainfed rotation component or in rotation with cereals (Ramírez-Parra and De la Rosa 2023). Vetch is now widespread throughout many parts of the world with a cultivated area of 322,715 ha as a major crop (FaoStat 2022). However, fungal diseases, insect, and parasitic weeds are the most important constraints for vetches production, causing quality and yield losses. (Ramírez-Parra and De la Rosa 2023). In 2022, symptoms suggestive of fusarium wilt were observed in vetch fields located in Constantine province, in Northeastern Algeria (36° 15′ 31.224″ north, 6° 38′ 37.58 ″ east), posing a potential threat to its cultivation. Affected plants showed yellowing, curling, and wilting of leaves, accompanied by vascular discoloration and death of some branches and plants in some cases (Fig. 1). Symptomatic adult plants were randomly sampled. Segments from roots and stems were surface sterilized in 1% sodium hypochlorite for 5 min, rinsed thrice with sterile distilled water, and cultured on potato dextrose agar amended with 0.1 mg/ml kanamycin monosulfate. After 4 days of incubation at 28 °C in the dark, seven isolates were recovered, single-spored, and examined through morphological and molecular analyses. Colonies on PDA formed white to pink flat aerial mycelium. Microconidia were abundant, oval, and mostly single-celled, they were formed in false heads on short monophialides. Three-septate macroconidia with short apical cells were also observed. Chlamydospores were formed after 4-5 weeks of incubation, they were spherical and either terminal or intercalary in hyphae (Fig. 2). According to Leslie and Summerell (2006), these cultural and morphological characteristics were similar to Fusarium oxysporum and Fusarium redolens.
Pathogenicity tests were conducted to fulfill Koch’s postulates. Ten-day-old seedlings of healthy vetch plants were inoculated by submerging their trimmed roots in a 5 × 106 conidia/ml suspension for 5 min, while seedlings dipped in sterile water were used as negative controls (Bani et al. 2012). All plants were maintained in a greenhouse at 26 ± 2 °C. Among the seven isolates tested, two isolates, FrV1 and FrV3, were identified as pathogenic on vetch plants and induced wilt symptoms similar to those observed in the field. Wilt symptoms started 15 days post inoculation (dpi) and progressed until the 25th to 30th dpi, where the majority of inoculated plants were dead, while control seedlings remained asymptomatic (Fig. 1b). The symptoms were accompanied by vascular discoloration, a common indicator of fusarium wilts (Fig. 1c).
The pathogenic isolates (FrV1 and FrV3) were studied by amplifying and sequencing their translation elongation factor 1-alpha (TEF-1α) using EF1/2 primers (Bani et al. 2014). PCR products were purified via ExoSAP-IT™ Express PCR Product Cleanup purification kit (Thermo Fisher Scientific Baltics, Lithuania). The two strands of each amplified DNA fragment were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Baltics, Lithuania) and the ABI 3500 Genetic Analyzer (Thermo Fisher Scientific, CA, USA). DNA sequences were edited and consensus sequences between forward and reverse strands were generated using the software BioEdit version 7.2. (Informer Technologies, Inc). These sequences were deposited in GenBank with the accession numbers PP968024 for FrV1 and PP968025 for FrV3. BLASTn analysis of the obtained sequences revealed 99 to 100% identity with the F. redolens sequences found in GenBank, with the reference sequence OM622423.1 showing the highest score of 100% match. The newly obtained sequences and the sequences of other Fusarium taxa retrieved from GenBank were aligned using ClustalW implemented in MEGA11. A phylogenetic tree was constructed using the neighbor-joining method with a bootstrap test of 1000 replications. The phylogenetic analysis clustered FrV1 and FrV3 consistently with reference F. redolens sequences (Fig. 3).
Fusarium redolens is known for its association with root rot disease in numerous plant species, particularly in temperate regions (Leslie and Summerell 2006). However, its association with wilt symptoms has been reported in two crops only, lentil (Chekali et al. 2022 ; Rafique et al. 2020 ; Riccioni et al. 2008) and chickpea (Bouhadida et al. 2017; Tekeoğlu et al. 2017). To our knowledge, the only existing reference on vetch fusarium wilt is the CABI Compendium (Samac 2020), which identifies Fusarium oxysporum f. sp. medicaginis as the causative agent of wilt symptoms in vetch. Therefore, our findings are considered the first report of Fusarium redolens as a pathogen on vetch, causing vascular wilt disease.