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|a Peach (Prunus persica) is an important stone fruit crop cultivated on 14,350 hectares in temperate areas of Pakistan, where post-harvest fungal rots cause major economic losses for growers. During April-August 2019, rotted peaches were observed in fruit markets of Rawalpindi district (33° 37' 33.8052'' N and 73° 4' 17.1912'' E), Punjab Province, Pakistan. Symptomatic fruit were surface sterilized for 3 min in 0.5% NaClO and 1 min in 70% ethanol, then rinsed three times in sterilized distilled water and air-dried for 30 minutes on filter paper. Tissue segments were excised from the margins of rot lesions and placed on potato dextrose agar (PDA), followed by incubation for one week at 25 +1o C. Furthermore, to maintain original sporodochial morphology and conidial description tissue segments were also grown on carnation leaf agar (CLA) and synthetic nutrient agar (SNA) (Sever et al., 2012). A total of 22 isolates were obtained from different fruit markets; colonies were fast-growing with pinkish-white, fluffy mycelia. Microconidia were abundant, 7.5 to 11.3 × 2.1 to 4.1 μm, oval to spindle-shaped, thin-walled, and hyaline whereas, macroconidia were thick-walled, sickle-shaped, 5- to 7-septate, hyaline, and 23.5 to 39.7 × 3.7 to 7.0 μm on carnation leaf agar. Virulence intensity of fungal isolates was confirmed after pathogenicity. Based on morphological characteristics, isolates were identified provisionally as Fusarium sporotrichioides (Leslie and Summerell 2006). For molecular identification, three target gene regions (ITS, elongation factor (EF), and β-tubulin benA) were amplified for highly pathogenic fungal isolates (FUS21K, FUS9SM, and FUS63KP) (O'Donnell et al. 2015). BLAST searches of the obtained sequences indicated 100% homology with GenBank accession number MN452643, and 99.56% homology with MT635298 for the ITS region, similarly 100% identity with GenBank accession numbers MN555016, MN555126, and MN555037 for the EF gene, and 100% homology with GenBank accession numbers MK435577, MH791363, and GQ915448 for β-tubulin benA of the F. sporotrichioides species complex (O'Donnell et al. 2015). Sequences were deposited in GenBank; accession numbers for each gene were as follows: ITS, ON180670, ON180671, and ON180672; EF, ON312093, ON312094, and ON312095; and β-tubulin benA, ON312090, ON312091, and ON312092. Maximum likelihood analysis on RPB1 and RPB2 nucleotide sequences of various fusaria was conducted which revealed that F. sporotrichioides formed a monophyletic relationship to terminal fusarium clade (TFC) comprising 22 strongly supported species complexes and 10 monotypic lineages, which were provisionally recognized as Fusarium. To complete Koch's postulates, 10μl aliquots of spore suspensions (106 spores/ml) of each FUS21K, FUS9SM and FUS63KP were pipetted individually onto six ripe, asymptomatic peaches. Sterile distilled water was applied as a negative control on four fruit. Fruit were incubated at 25 ± 1°C for one week in a sterile moist chamber, and the trial was conducted three times. Rot symptoms associated with pinkish-white fluffy mycelia were observed on inoculated fruits after 72 hours, whereas no mycelium was observed on the negative controls. The cultures on PDA, CLA and SNA from each of the inoculated fruit were morphologically identical to the original culture. The pathogen was confirmed as a member of F. sporotrichioides species complex based on pinkish-white mycelium, with red color on the underside of petri plates and the manner in which conidia are born on conidiophores. This is the first report of F. sporotrichioides on peaches from Pakistan, providing an important foundation for peach growers, agricultural advisers, plant pathologists, and plant breeders to improve the management of this economically damaging disease
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