First Report of <i>Phakopsora cherimoliae</i> Causing Rust Disease on <i>Annona squamosa</i> in China

Annona squamosa L., one of the most economically significant fruit trees in southern China, is increasingly threatened by invasive pathogens. In May 2024, severe rust disease (60-70% incidence) was observed on Annona squamosa trees (Jifena, native variety) in a commercial orchard (1.2 ha, 117.354808°E, 23.905476°N) in Yunxiao County, Fujian Province, China. Affected trees exhibited leaf yellowing, premature defoliation, dense chlorotic lesions on the adaxial leaf surfaces, and brown rust-like powdery substances (later identified as uredinia) on the abaxial surfaces. Microscopic analysis revealed globose to ovoid urediniospores (26.0 ± 3.9 × 21.6 ± 3.3 μm, n=30) with echinulate walls; teliospores were absent. Genomic DNA of isolate PC001 was extracted (CTAB method, Allen et al. 2006) and amplified for the nuclear large subunit ribosomal DNA (LSU rDNA, primers LRust1R/LRust3) (Beenken 2014) and the mitochondrial cytochrome oxidase subunit 3 gene (CO3, primers CO3_F1/R1) (Beenken et al. 2012). Bidirectional sequencing yielded LSU (PV342396) and CO3 (PV351739), showing 99.35% (606/610 bp) and 99.77% (425/426 bp) identity to Phakopsora cherimoliae ex-type sequences KF528011 and KF528048 (BLASTn E-value <1×10-150). Phyogenetic reconstruction was performed on concatenated LSU-CO3 alignments using Uromyces viciae-fabae as the outgroup. Phylogenetic analyses using PhyloSuite (maximum likelihood [ML] with Tamura 3-parameter model in MEGA11; Bayesian inference [BI] with Bayes) demonstrated that isolate PC001 formed a fully supported clade (ML bootstrap = 100%, BI posterior probability = 1.0) with P. cherimoliae references, distinctly separated from other Phakopsora species and Batistopsora spp. Diseased specimens and DNA samples were deposited in the Fungal Herbarium of Fujian Agriculture and Forestry University (Voucher no. FAFUN54320). Pathogenicity was validated through Koch's postulates: Inoculation of whole healthy 2-month-old A. squamosa seedlings with surface-sterilized urediniospores (5 × 104 spores/mL in 0.01% Tween 20, sterilized in 0.5% NaOCl for 1 min, rinsed thrice with sterile water) under controlled conditions (25°C, 12-h photoperiod) induced chlorotic lesions and abaxial uredinia on leaves within 12 days. Urediniospores from inoculated plants were morphologically identical to field isolates. Combined morphological, molecular (multilocus phylogeny), and pathogenicity evidence conclusively identified the pathogen as P. cherimoliae, representing the first documented infection of this pathogen on A. squamosa in China. The lack of telia in field observations suggests that P. cherimoliae likely persists year-round via urediniospores in this region, aligning with the genus’ monophagy without alternate hosts (Ono et al. 1992). These findings directly inform rust disease management strategies critical to protecting China's A. squamosa industry.