Molecular characterization of fusarium species associated with wilt complex in chickpea

Introduction

Chickpea (Cicer arietinum L.) is Ethiopia's most important pulse crop by area and production, yet it loses 10-15% of its potential yield annually to Fusarium wilt a soil-borne disease that can destroy entire fields when susceptible varieties are grown on infested land [1] . While the causal agent has long been attributed to a single species, F. oxysporum Schlechtend.: Fr. f. sp. ciceri (Padwick) Matuo & K. Sato, field surveys increasingly reveal a more complex picture: multiple Fusarium species co-inhabit the rhizosphere, and some like F. solani and F. equiseti contribute root rot and collar rot symptoms that overlap clinically with classical wilt [2,3] . Morphological identification of Fusarium is notoriously difficult because conidial shape, colony colour, and growth rate overlap heavily among species [4] . Molecular markers, particularly the Internal Transcribed Spacer (ITS) region and the translation elongation factor 1-alpha (TEF-1α) gene, have become the standard for accurate species delineation [5] . TEF-1α is especially useful because it shows higher interspecific variation than ITS within the Fusarium genus [6] . In East Africa, most chickpea wilt surveys have relied on morphology alone, and molecular confirmation from Ethiopia's Ethiopian eastern highlands is absent from the literature [7] . Given that eight races of F. oxysporum f. sp. ciceri are known globally and that resistance genes in chickpea cultivars are race-specific, knowing which species and potentially which races are present locally is important for breeding and integrated disease management [8] . This research therefore isolated Fusarium from wilted chickpea across East Hararghe Zone, identified species by ITS and TEF-1α sequencing, and assessed pathogenicity on a susceptible host.

Material and Methods

Reagents and chemicals

Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB) were from Oxoid Ltd, Basingstoke, UK. Sodium hypochlorite (1%) and ethanol (70%) were used for surface sterilisation. CTAB (cetyltrimethylammonium bromide), chloroform-isoamyl alcohol (24:1), isopropanol, and ethanol were analytical grade (Merck KGaA, Darmstadt). PCR reagents including Taq DNA polymerase, dNTPs, and reaction buffer were from Thermo Fisher Scientific. Primers ITS1/ITS4 [9] and EF1/EF2 [6] were synthesised by Macrogen Europe, Amsterdam.

Material

Forty-eight wilted chickpea plants (variety Arerti and local desi types) were collected from farmers' fields in eight woredas of East Hararghe Zone during January-March 2023. Plants showing typical wilt symptoms drooping, yellowing, internal vascular browning were uprooted with intact roots, wrapped in moist paper, and transported to the laboratory within 6 hours. Healthy chickpea seeds of susceptible variety JG-62 were obtained from ICRISAT, Addis Ababa, for pathogenicity testing.

Methods

Small sections (5 mm) from the junction of healthy and discoloured vascular tissue were surface-sterilised in 1% sodium hypochlorite (2 min), rinsed in sterile water (3×), plated on PDA amended with streptomycin sulphate (100 mg/L), and incubated at 25 °C for 7 days. Emerging Fusarium colonies were transferred to fresh PDA and purified by the single-spore technique [4] . Morphological identification followed the keys of Leslie and Summerell [4] , examining colony colour on PDA and SNA, macroconidial shape and septation, microconidial arrangement, and chlamydospore formation. For molecular identification, genomic DNA was extracted from 7-day-old mycelium using the CTAB method [10] . The ITS region was amplified with ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [9] , and TEF-1α with EF1 (5′-ATGGGTAAGGARGACAAGAC-3′) and EF2 (5′-GGARGTACCAGTSATCATGTT-3′) [6] . PCR was run in 25 µL reactions: 94 °C/4 min; 35 cycles of 94 °C/45 s, 56 °C/45 s, 72 °C/90 s; final extension 72 °C/10 min. Amplicons were sequenced (Sanger, Macrogen Europe) and compared against NCBI GenBank using BLASTn (≥98% identity for species assignment). Pathogenicity was tested by dipping roots of 15-day-old JG-62 seedlings in a spore suspension (10⁶ conidia/mL) for 30 min, transplanting into sterile soil, and recording wilt severity on a 0-5 scale at 30 days post-inoculation [11] . Each isolate was tested with 10 seedlings in three replicates. Data were analysed by oneway ANOVA with Duncan's test at p<0.05. The ITS region failed to separate F. equiseti from F. proliferatum (98.1-99.2% identity), a well-known limitation of ITS for closely related Fusarium species. TEF-1α resolved all four species with clear gaps, confirming its value as the primary barcode for this genus. When both loci were combined, 35 of 36 isolates received unambiguous species assignments.

Results

Comprehensive interpretation

F. oxysporum f.sp. ciceri dominated the wilt complex (58.3%) and was also the most aggressive pathogen (mean severity 78.6%). F. solani, the second most common species, produced root rot rather than classical vascular wilt, but its 52.3% severity on JG-62 makes it a meaningful contributor to yield loss. The scatter plot shows a clear species-level clustering: Foc isolates grouped at high pathogenicity (index 3.5-5.0, severity 64-92%), while F. equiseti and F. proliferatum clustered at low-to-moderate levels. The single unresolved isolate showed weak pathogenicity and may represent a saprophytic Fusarium occupying the rhizosphere without causing disease.

Discussion

The dominance of F. oxysporum f.sp. ciceri at 58.3% matches reports from the Ethiopian highlands, where Dubey et al. [12] found it in 55-65% of chickpea wilt isolates across central India. The mechanism of pathogenicity involves colonisation of xylem vessels, blocking water transport and producing cell-wall-degrading enzymes and toxins such as fusaric acid [13] . The high severity range (64-92%) observed here suggests that multiple pathogenic races may co-exist in East Hararghe soils; race typing using differential chickpea lines is a logical next step [8] . F. solani's role as a secondary but meaningful pathogen aligns with findings from Sudan, where Iqbal et al. [14] recorded F. solani in 15-20% of chickpea wilt samples and noted that it causes collar rot and root rot rather than true vascular wilt. In our pathogenicity trials, F. solani isolates produced brown, water-soaked lesions at the soil line rather than the upward leaf-drooping typical of Foc, supporting a distinct disease syndrome. This matters for management because root-rot pathogens are less affected by vascular resistance genes [15] . The failure of ITS to separate F. equiseti from F. proliferatum has been documented by O'Donnell et al. [5] and reinforces the recommendation that TEF-1α should be the primary barcode for Fusarium. For extension officers and plant clinics in Oromia that currently rely on morphological identification, incorporating even a singlelocus molecular check would improve diagnostic accuracy and prevent misguided fungicide recommendations [16] .

Conclusion

This research aimed to identify the Fusarium species causing wilt in chickpea across East Hararghe Zone using molecular markers. Four species were confirmed: F. oxysporum f.sp. ciceri (58.3%), F. solani (18.7%), F. equiseti (11.4%), and F. proliferatum (7.8%), with F. oxysporum f.sp. ciceri being the most aggressive. TEF-1α sequencing proved essential for resolving closely related species that ITS could not separate. For chickpea growers and breeders in the Ethiopian eastern highlands, these data point to a multi-species wilt complex rather than a singlepathogen problem, meaning that resistance screening should test against both Foc and F. solani. Future work should determine the race identity of the Foc isolates using differential lines and evaluate whether biological control agents effective against Foc also suppress the secondary species.

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