Occurrence of Alternaria alternata Causing Leaf Spot on Pyracantha fortuneana in China.

Abstract

Pyracantha fortuneana, commonly known as firethorn, is an evergreen shrub used as a landscape plant for decorative purposes. Its fruits have been traditionally used as a herbal remedy and functional food to treat various ailments in China. From September to November 2023, a leaf spot occurred in the Nanyang central park (33°0'3″ N, 112°32'16″ E), Henan Province, China. The incidence of this disease reached 47% (n = 100 plants). Initial symptoms included light brown spots that expanded into dark brown necrotic lesions. The lesions subsequently expanded to the entire leaves, forming distinct yellow halos, leading to yellowing, defoliation and a decline in plant aesthetics. Twenty diseased leaves were randomly collected and cut into small pieces at the margins between infected and healthy tissues. The tissues were sterilized in 75% ethanol and 1% NaClO solution for 30 seconds and 1 minute, respectively. After rinsing with sterile water, the fragments were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. A total of 35 Alternaria sp. strains were obtained, exhibiting comparable phenotypes. Three strains (WWY11, WWY12, WWY13) from different locations of the park were selected to be further characterised. The colony exhibited abundant white aerial mycelium with shades ranging dark-brown on the reverse side. To analyze the morphological characteristics of conidia, potato carrot agar (PCA) was used as the culture medium and incubated at 25°C with a 12-hour light/dark cycle. Conidia were club-shaped, catenulate, dark brown, with 2 to 3 transverse septa and 1 to 3 longitudinal septa, measuring 11.1 to 38.2 × 5.2 to 10.3 μm (n=100), with conical beak lengths ranging from 0 to 3.3 μm. These morphological features suggested that the pathogen belonged to the genus Alternaria. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and RNA polymerase II second largest subunit (rpb2) of three selected strains were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, GDF1/GDF2, and RPB2-5F/RPB2-7cR from genomic DNA. The ITS (PP993911 to PP993913), tef1 (PP997440 to PP997442), gapdh (PP997443 to PP997445), and rpb2 (PP997446 to PP997448) sequences were submitted to GenBank. A phylogenetic tree was constructed of combined sequences (ITS, tef1, gapdh, and rpb2) alongside sequences from ex-type strains using MEGA software (version 11) with the neighbor-joining method. The three strains formed a clade with Alternaria alternata strain DZ, which caused tobacco brown spot (Lu et al. 2023), distinct from other Alternaria spp. Morphological and molecular analyses confirmed the strains as A. alternata (Woudenberg et al. 2015). To test pathogenicity, a conidial suspension (10⁶ conidia ml^-1) was sprayed onto healthy leaves of five firethorn. Another group of five plants received sterile water as a control. All plants were incubated in a greenhouse at 28°C and 90% relative humidity. Ten days post-inoculation, lesions resembling natural symptoms were observed on inoculated plants, whereas control plants remained symptom-free. The reisolated strains were identified as A. alternata based on morphological and ITS sequence analyses, fulfilling Koch's postulates. Scab and dieback of Pyracantha coccinea (closely related species of P. fortuneana) caused by Spilocaea pyracanthae and Trichothecium roseum has been reported in Slovakia (Juhasova et al. 2004) and Iran (Firouzianbandpey et al. 2021). This is the first report of A. alternata causing leaf spot on P. fortuneana. This identification highlights the potential impact of the new disease on growers and the need for adjustments in landscape vegetation to mitigate its effects.