First Report of Alternaria tenuissimain Causing Leaf Spot of Celery (Apium graveolens) in China.

Abstract

Celery (Apium graveolens) is one of the most widely grown vegetables in the world. A survey in Anding District of Gansu Province in 2019 showed that the incidence of celery leaf spot was 25%-45%. The disease mainly occurs in late June and July. The leaf spot is conducive to the onset at high temperature and humidity environment. The initial symptoms were many small light brown, irregular-shaped on the leaves. The lesions gradually enlarged in the later stage of the disease, and multiple lesions coalesced to form large irregular brown spots, eventually the whole leaves died. A 3~4mm leaf tissue was cut from the junction of the diseased leaf and the healthy area, the leaf tisse was surface-sterilized in 1.5% NaClO for 1 min and washed with sterile water. Then, it was incubated on potato dextrose agar (PDA) and obtained the pure culture (Q1). After 5 days of cultivation at 25°C, the fungal colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. The conidia were obclavate or ellipsoid, pale brown, with 3~4 longitudinal septa and 2~7 transverse septa, and measured 20.0 to 50.0 × 3.5 to 14.0μm (n=50). Conidiophores were septate, arising singly, and measured 3.5 to 40.0 × 2.5 to 4.5 μm (n=50). Based on morphological characteristics, the fungus was preliminarily identified as A.tenuissima (Simmons 2007). To further confirm the identification, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), endopolygalacturonase gene (endoPG), anonymous gene region (OPA10-2) and glyceraldehyde 3-phos-phatedehydrogenase (GAPDH) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), RPB2-5F2/RPB2-5R (Sung et al. 2007), Alt-for/Alt-rev (Hong et al. 2005), EPG-specific/EPG-3b (Peever et al. 2004), OPA10-2R/OPA10-2L (Peever et al. 2004) and Gpd1/Gpd2 (Berbee et al. 1999) (GenBank accession no.MN046364, MW016001, MW016002, MW016003, MW016004, MW016005, MW016006). DNA sequences of TEF, RPB2, endoPG, OPA10-2 and GAPDH were 100% identical to those of A. tenuissima (MN256108, MK605866, KP789503, JQ859829 and MK683802), but ITS and Alt a 1 were 100% similarity with A. tenuissima (MN615420, JQ282277) and A. alternate (MT626589, KP123847). The ITS and Alt a 1 sequence did not distinguish A. tenuissima from the A. alternate complex. Maximum likelihood phylogenetic analyses were performed for the combined data set with TEF, RPB2, and endoPG using MEGA6 under the Tamura-Nei model (Kumar et al. 2016). The isolate Q1 clustered with type strain A. tenuissima CBS 918.96. The 20 celery plants of 4-7 leaf age were used test the pathogenicity of Q1, the ten plants were sprayed with 20ml of spore suspension (1×105 spores/ml), the control was sprayed with 20mL sterile water, which were placed in a growth chamber (25℃, a 14h light and 10h dark period, RH > 80%). Eight days after inoculation, 40% of the leaves formed lesions, which were consistent with the field observation，the control group was asymptomatic. The pathogen was reisolated from infected leaves to fulfill Koch's postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot on celery in China.