First report of post harvest blue mold disease in 'Cuimi' kumquat fruit caused by Penicillium italicum in China.

The 'Cuimi' kumquat (Citrus japonica), a geographical indication product in Rong'an County, China, is highly susceptible to post-harvest pathogenic infections, leading to soft decay. In spring 2024, fruits demonstrated symptoms of soft decay, displaying white mycelium and blue conidia on surfaces, with lesions later transitioning to a blue hue. The soft decay incidence averaged approximately 18.3%, based on assessments involving ten trees per orchard and fifty fruits per tree, across four orchards in Rongan County. To isolate the pathogen, six partially rotted fruits from each orchard, representing about 30% of the symptomatic fruits, were collected, totaling 24 fruit samples. Tissue sections excised near lesions, measuring 3 mm3, were sterilized using 75.0% ethanol for 30 seconds and 2.0% sodium hypochlorite (NaClO) for 3 min, followed by two rinses with sterile distilled water. These sections were cultured on potato dextrose agar (PDA) with 30.0 µg/ml of chloramphenicol and incubated at 25 °C in the dark for 3 days to facilitate sporulation. Four consistent strains were purified using the single-spore technique and displayed white mycelium and blue conidia on PDA with the agar's reverse being dark pink. The strains displayed a mean mycelial growth rate of 3.58 ± 0.27 mm per day on PDA. The conidia were globose to subglobose, smooth-walled, and measured 3.83 ± 0.24 μm (n= 60), consistent with Penicillium italicum species traits (Cavalcanti et al., 2020) . For molecular characterization, the internal transcribed spacer (ITS) region and the calmodulin (cmd) gene were sequenced using ITS1/ITS4 (White et al., 1990) and CF1/CF4 (Peterson, 2004) primers, respectively. The representative sequences were deposited in GenBank (Accession nos. PQ443772 for ITS and PQ351750 for cmd). Blastn analysis revealed 100% sequence similarity with two strains of P. italicum (Accession nos. ON082769.1 and MT872093.1). The phylogenetic analysis was conducted using a neighbor-joining algorithm based on the ITS and cmd gene sequences. The isolates were clustered with P. italicum clade. In a controlled laboratory experiment to verify Koch' s postulates, ten ripe, healthy 'Cuimi' kumquat fruits were immediately harvested and surface sterilized (using the previously described method), after then, fruits were wounded with a sterile stainless-steel rod before inoculation. Fruits were inoculated with a P. italicum RAKQ-1 spore suspension (1 × 105 spores/ml, 20 µl per fruit). Controls received sterile water. Fruits were incubated at 25°C with 90% relative humidity in the dark. After four days, all treated fruits exhibited soft decay symptoms, while controls remained symptom-free. The pathogen was re-isolated from symptomatic specimens via the aforementioned method and confirmed as P. italicum through morphological and genetic assessments of ITS and cmd sequences. P. italicum has previously been documented as a pathogen of oranges (Palou et al., 2003; Archer et al., 2021), causing post-harvest citrus decay (He et al., 2022), and lemon blue mold disease (Hernández-Montiel and Ochoa, 2007). However, this study represents the first documentation of P. italicum inducing rot in 'Cuimi' kumquat fruits. These observations underscore the imperative for researchers and agriculturalists to devise efficient management strategies for controlling P. italicum infections in harvested citrus fruits.