First Report of Aspergillus flavus Causing Fruit Rot on Kiwifruit in China.

Abstract

In October 2020, fruit rot symptoms were detected on kiwifruit (Actinidia chinensis var. deliciosa 'Xuxiang') in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Mature kiwifruit, during the harvest period, exhibited soft rot and brown lesions. The symptoms were similar than those reported for Alternaria alternata, Colletotrichum spp., Fusarium avenaceum and Rhizopus oryzae causing fruit rot on kiwifruit (Feng et al. 2019; Li et al. 2017; Kim et al. 2018; Zhao et al. 2020). The symptoms were observed in approximately 15% of the fruit in 6 kiwifruit orchards (31 ha in total). Ten samples of symptomatic tissue, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 30 seconds and washed twice with sterilized water. The pathogen was isolated from all collected samples via culturing on PDA medium, containing 50 µg/mL chloramphenicol, at 28 ºC. Green powdery-like colonies were detected after 5 days (Figure 1). A total of 12 isolates were obtained via single spore isolation. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and RNA polymerase II subunit (RPB2) genes were amplified using ITS5/ITS4, A_EF1_F/A_EF1_R and RPB2-5F/RPB2-7cR (NJC03), or RPB2-7cF/RPB2-11aR (NJC04), primers, respectively. Eleven isolates shared the same sequences (NJC03), MZ801787 (ITS), MZ701709 (EF1-α) and MZ701707 (RPB2), while one of the isolates provided different sequences (NJC04), OK618459 (ITS), OK634020 (EF1-α) and OL331017 (RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from A. flavus KU20018.4 (MT487825), the EF1-α sequences shared 100% homology to the EF1-α gene from A. flavus clinical2342 (KP054370) and the RPB2 sequences shared >99% homology to the RPB2 genes from A. flavus PW3170 (LC000581) and A. flavus NRRL3357 (XM_041293948). Molecular phylogenetic tree was constructed using MEGA7 with reference Aspergillus strains (Figure 2). Microscope observations of all isolates showed the presence of septate mycelium, circular unicellular conidia (2-4 µm diameter) and conidiophores, and agree with the morphology of A. flavus (Horn 2005). The pathogenicity of all isolates was screened using intact and wounded 'Xuxiang' kiwifruits (ten kiwifruits were used for each combination with 3 replicates), which were purchased from a local market. A 1 × 106 spores/mL (10 µL) solution of the isolates was used for the inoculation. Sterilized water was used in the control experiment. Inoculated kiwifruits were storage at 26 °C and 60% relative humidity for 10 days. Rot lesions in the wounded kiwifruits were totally covered by green mycelia, while the lesions on the intact kiwifruits were similar to the symptoms observed in the field. The pathogen was recovered and its identity was confirmed by sequence analysis of ITS, EF1-α and RPB2, fulfilling Koch's postulates. A. flavus is known to be an important fungal pathogen of corn, cotton and peanuts (Zhang et al. 2020). During recent years, A. flavus was reported to cause fruit rot on grapes (Ghuffar et al. 2020), and was identified on almond, fig, organic spelt and pistachio (Krulj et al. 2017; Ortega-Beltran et al. 2019). The presence of A. flavus in food products is an issue of global concern due to A. flavus is able to produce carcinogenic aflatoxin (Maxwell et al. 2021). As far as we know, this is the first report of A. flavus causing fruit rot on kiwifruit. This report will help to understand the distribution of A. flavus in crops and the food safety hazards that are present in China.