Pigeon pea (Cajanus cajan (L. ) Millsp. ) is a grain legume, widely cultivated and distributed in tropical and subtropical regions. The plant has high agricultural potential due to its use as food, fodder, medicine, and fuel (Gargi et al. 2022). However, wilt caused by Fusarium spp. (Pushpa et al. 2020) represents a significant constraint on production. In October 2020, pigeon pea seedlings of cultivar BRS Mandarim from the 2018 seed crop in the municipality of Brejo Alegre, São Paulo, Brazil (21º 9’ 57” S; 50º 11’ 6” W), showed stunting, chlorosis and leaf necrosis; with reduced roots, stem cracking, and leaf drop; when grown in a greenhouse of the CCA/UFPB (Areia, Paraíba, Brazil, 6º 58’ 12” S; 35º 42’ 1” W). The disease incidence was 25% in 200 sampled seedlings. The surface of symptomatic leaf tissues was disinfected with 70% ethanol for 30 seconds, followed by 1% sodium hypochlorite solution for 1 minute. The tissues were then rinsed two times with sterilized distilled water (SDW), air-dried on sterile filter paper, and subsequently plated on potato dextrose agar (PDA). They were incubated at 25 °C for 7 days. Mycelia were transferred to SNA (synthetic nutrient deficient agar) and incubated at 25 °C for 7 to 15 days to obtain pure cultures. Only one isolate was obtained from symptomatic leaf samples. Monosporic cultures were obtained by transferring a single germinated conidium to SNA. Colonies grown on PDA exhibited white mycelium, with a yellowish pigmentation on the reverse side. Colonies on SNA exhibited hyaline microconidia, oval to kidney-shaped, zero to one septate, measuring 7. 2 to 20. 2 × 2. 7 to 7. 8 µm (n = 40), forming in monophialides. Macroconidia were absent. Chlamydospores were globose to subglobose, produced singly and in pairs. The morphological characteristics are typical of the Fusarium solani complex (Leslie and Summerell, 2006). For molecular characterization, DNA of isolate CMA064 was extracted using the Wizard Genomic DNA Purification kit (Promega Corporation, Fitchburg, USA). For molecular identification, the elongation factor 1-α region (EF1-α) and the RNA polymerase II second largest subunit (RPB2) were chosen, as they are the main genomic regions used for the identification of fusarioid fungi. Amplification was done with the primers EF1 and EF2 (O'Donnell et al. 1998) for EF1-α and the primers RPB2-5F2 (Reeb et al. 2004) and RPB2-7cR (Liu et al. 1999) for RPB2. The EF1-α and RPB2 sequences of the isolate CMA064 were deposited in GenBank (OR545373 and OR542858 for EF1-α and RPB2, respectively), and compared to other Fusarium species found at Fusarium-ID and GenBank databases. A phylogenetic analysis was inferred using the maximum likelihood method, using concatenated sequences. BLAST analysis found that the EF1-α sequence had 99. 70% match (666/668 bp) with F. falciforme strain FSSC₃+4SF19 (MG272436) and RPB2 had 100% match (865/865 bp) with F. falciforme strain NC20775 (OP913399). Isolate CMA064 clustered with F. falciforme (CBS475. 67) with 84% support. To confirm pathogenicity, the isolate was cultured on PDA at 25 ± 2 °C for seven days. A conidial suspension (10⁵ conidia/mL) was prepared and 200 pigeon pea seeds were soaked in the suspension for five minutes. The negative control consisted of 200 seeds immersed in SDW. The seeds were sown on a sterile substrate in a greenhouse. After 20 days, chlorosis, stunting and leaf necrosis were observed on 33% of the emerged plants when compared to negative controls (healthy plants), same symptoms originally observed. Fusarium falciforme was successfully reisolated from the inoculated tissues (using the morphological characterization methodology mentioned above), thereby completing Koch’s postulates. The test was performed twice. This study represents the first report of F. falciforme causing wilt in pigeon pea in Brazil. Previously reported species associated with this disease include F. udum in India (Pande et al 2013; Pushpa et al. 2020). Fusarium wilt is one of the major constraints in the production and productivity of pigeon pea (Pushpa et al. 2020; Reddy et al. 2024). Given the results of this study, proper strategies are needed to mitigate seed infection manage this disease and to be compatible with sustainable agricultural production.
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