| Abstract |
Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also an important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County (32°42′N, 111°48′E), Henan Province, China, with an incidence of ∼30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5 × 5 mm) of diseased roots from six trees were surface sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, plated on potato dextrose agar, and then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval, or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5) × 3.8 to 8.0 (average 5.6) μm (n = 100). Macroconidia were sickle shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) μm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) μm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates (Fs1 and Fs3). The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using the primer pairs ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr and RPB2-7cf/RPB2-11ar (O’Donnell et al. 2022), respectively. BLAST analysis showed that the ITS, TEF-1α, and RPB2 sequences of the isolates Fs1 (GenBank accession nos. OK001765, OQ921726, and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727, and OQ928397) showed 99 to 100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711, and ON331935). The phylogenetic tree based on concatenated sequences of ITS, TEF-1α, and RPB2 using maximum-likelihood analysis revealed that both isolates (Fs1 and Fs3) were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1 × 10⁸ spores/ml) of the isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots 2 months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi reisolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on various plants around the world (Qiu et al. 2023; Xu et al. 2023). The results of pathogen identification are essential precursors to the development of management strategies for the effective control of this disease.
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