First Report of Anthracnose on Juglans regia Caused by Colletotrichum siamense in Korea.

Abstract

Walnut (Juglans regia) is one of the main tree crops cultivated for nut production in South Korea with an estimated production of about 1,189 tons per year (Korea Forest Service 2020). In August 2021, anthracnose symptoms, including dark, depressed, irregularly shaped lesions on fruits and leaves of walnut cv. Sinlyeong, were observed at three orchards in Nonsan (36°10'22.5"N 127°06'14.8"E) and Suwon (37°16'04.7"N 126°55'22.3"E and 37°15'10.6"N 126°57'35.6"E). This led to severe yield loss of walnut fruit with a disease incidence of approximately 70 to 80% in each orchard. Three samples, including infected fruits and leaves, were randomly collected per site. Fungal isolates were isolated either from acervuli filled with conidial masses on infected walnut tissues or from plant tissues that were surface-disinfested, followed by plating onto 2% PDA. Colonies were initially white, later became pale brownish to light gray with concentric rings of salmon sporodochia. White to gray aerial mycelia, reaching 65 mm diameter in 5 days, were abundantly produced on PDA at 25 °C. Appressoria were brown, ovoid, and in some cases, clavate, 5.1-8.7 μm in length, and 3.2-5.1 μm in width (n = 50). Conidia were single celled, hyaline, cylindrical with rounded ends and smooth walls, guttulate, 13.6-18.8 µm in length, and 4.4-6.3 µm in width (n = 50). Setae were absent. Three isolates, i.e., one per orchard, were retained and deposited in the culture collection (CDH) of National Institute of Forest Science, Korea (Accession No. CDH052-054). The internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) and a partial sequence of the actin (ACT) genes were amplified and sequenced for each of the isolates using the pair of primers, ITS1F/ITS4 (Gardes and Bruns 1993; White et al. 1990), T1/Bt2b (ODonnell and Cigelnik 1997; Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. A BLAST search in GenBank revealed that the sequences of ITS (OK631731-733), TUB2 (OK665927-929) and ACT (OK665930-932) showed sequence identities of 98.6 to 99.6% to Colletotrichum siamense sequences (FJ972613, FJ907423, FJ907438). A maximum likelihood tree, based on a combined dataset of ITS, ACT and TUB2 gene sequences for Colletotrichum spp., revealed that the three isolates were clustered with type specimens of C. siamense. To prepare larger quantities of inoculum for the pathogenicity, mycelial plugs bearing acervuli taken from 2% PDA were incubated in a conical flask containing 200 ml of 2% potato dextrose broth at 25°C on a rotary shaker at 150 rpm for two weeks. Spore concentration was adjusted to 1.0 × 104 ml-1 conidia of C. siamense (CDH054). A 10 to 15 ml of spore suspension was then sprayed on each leaf of 12 seedlings of 'Sinlyeong' walnut (three-year-old), while 7 seedlings were treated with sterile distilled water as a control. Each treated seedling was covered by a plastic bag to maintain moisture for one day. Inoculation trials were repeated twice, in August and September 2021. Symptoms identical to those observed in the field developed four to five days after the inoculations from which the inoculated pathogen was successfully re-isolated, fulfilling Koch's postulates. However, no symptoms were observed in the control. To our knowledge, this is the first report of anthracnose on J. regia caused by C. siamense in Korea. This indicates that disease occurrences must be further rigorously surveyed at the nation-wide scale to effectively control the disease in the country.