First report of Alternaria alternariacida causing potato leaf blight in the Far East, Russia.

Abstract

Early blight of potato (Solanum tuberosum) is caused by Alternaria species and occurs annually in major potato producing regions of Russia. Diseased potato leaves displaying early blight symptoms were collected in July 2016 from a commercial field in Primorsky Krai, Russia (43.8242° N, 131.6219° E). The disease incidence was 30 to 40%. The initial symptoms appeared as typical early blight symptoms with a dark brown margin and diffused chlorosis on the leaf blade. Symptomatic leaves from different plants were randomly collected to isolate axenic cultures of the causal agents. Infected leaves were placed in wet chambers (moist filter papers in Petri dishes), and incubated at 25°C, 16 h/8 h dark/light photoperiod for 2-4 d. Single conidia were transferred to potato dextrose agar (PDA, Crous et al. 2009) in Petri dishes and incubated at 25°C for 7 d in the dark. Colonies were white-olivaceous, reverse side - olivaceous. Isolates were transferred onto potato carrot agar (PCA, Crous et al. 2009) and incubated at 22°C under a 16 h/8 h dark/light photoperiod for 7 d to stimulate sporulation. Most isolates (85%) were identified as A. protenta according to the morphological characteristics and molecular data, although one isolate showed sporulation that was somewhat atypical, having a smaller (especially narrower or more slender) conidia. Conidiophores were long, erect, and 65 to 100 µm × 5 to 6 µm in size. Conidia were solitary, long-ovoid in body with six to eight transverse septa, and 85 to 100 µm× 6 to 10 µm in size. Conidial beaks were filamentous, 110 to 200 µm × 2 to 5 µm in size. Genomic DNA was extracted from cultured isolates using the CTAB-chloroform extraction method (Griffith & Shaw 1998), and five gene regions including the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a 1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with the primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-5F2/FRPB2-7cR, Alt-for/Alt-rev, and gpd1/gpd2 respectively (Woudenberg et al. 2014). PCR products were Sanger sequenced. All sequences for isolate A16PrPL21 were identical to isolate CBS 105.51; (accession nos.: ITS, KJ718105; tef1, KJ718454; rpb2, KJ718279; Alt a 1, KJ718625; gpd KJ717959) of A. alternariacida Woudenb. & Crous. ITS, tef1, rpb2, Alt a 1 and gpd sequences were deposited in GenBank under the accessions OM348531, MN580518, MN580529, MN562258 and MN544404 respectively. Based on morphological and molecular data, the isolate was identified as Alternaria alternariacida Woudenb. & Crous. A. alternariacida is closely related to A. silybi, which is also found in the Far East (Woudenberg et al. 2014). Phylogenetic distances between these strains are based on differences at the ITS, TEF1 and Alt a 1 gene regions. A pathogenicity test was carried out with isolate A16PrPL21 on nine 5-week-old healthy potato plants (cv. Nevsky) grown in a greenhouse at 23 ± 2 °C. Seed tubers were grown in the greenhouse to obtain the seedlings. Inoculation was performed by spraying a conidial suspension (105 spores/ml) prepared from 10-day-old A. alternariacida culture grown on PCA at 23° C with a 12-h photoperiod. Nine negative control plants were treated with sterile distilled water. The inoculated plants were then maintained in a greenhouse at 25 °C with high humidity and 12-h light period. All test plants were covered with plastic bags for 24 h to maintain high relative humidity and incubated at 24 to 28°C. Leaf spot symptoms, brown lesions with chlorotic halos, similar to those previously observed in naturally infected plants, appeared 5 d post inoculation for all inoculated plants. After 7 d, the spots reached 18 to 25 mm in size. The symptoms were similar to the original symptoms that occurred in the field. Negative control leaves were symptomless. Koch's postulates were fulfilled by reisolating the pathogen from inoculated leaves and identified as A. alternaricida by rpb2 gene sequence and morphological characteristics. To our knowledge, this is the first report of disease caused by A. alternaricida on potato plants. Early blight, caused by large-spored Alternaria, is a widespread disease on potato. A. alternaricida is among a group of species that cause early blight, according to current research. Studies of the Alternaria species' biology and analyses of their distribution are important for improving potato protection from early blight.