First Record of Alternaria alternata causing necrosis of Thuja (Thuja occidentalis) in Kazakhstan.

Abstract

Thuja is one of the ornamental plants used for landscaping parks and health resorts. The plant is distinguished by a pyramidal and conical crown shape and the presence of many thin branches with scale-shaped needles, green all year round. In addition, this plant has a number of antimicrobial properties, which affects the popularity of the plant in landscaping the health resort territory (Bakht et al. 2020, Chindyaeva et al. 2020). In January 2020, symptomatic Thuja plants were observed in Southern Kazakhstan. Symptoms included distortion of the crown. External examination of the trees revelaed the presence of white fluffy mycelium on Thuja branches. The branches acquired a yellow color with a necrotic lesion developing below the affected area. Samples of infected branches from different Thuja trees (n = 13) were collected. The infected branches were cut into small pieces (5 × 5 mm), washed in 70% ethanol for 30 min, and then rinsed three times with sterile distilled water. Later, these pieces were placed on Sabouraud's medium (Laboratorios Conda S.A., Spain) and incubated at 28°C for 7 days. Yellow-green colonies grew from the pieces of wood. The colonies had a light gray-whitish aerial mycelium. Conidia (n = 35) were pale to dark brown in color, irregular and ellipsoid to ovoid conical in shape. The size of the conidia varied from 5 to 25 µm × 6 to 12 µm (n = 40) with longitudinal and transverse septations. These morphological characters were previously described and corresponded to the Alternaria alternata (Simmons et al. 2007). Genomic DNA was extracted from mycelium using the liquid nitrogen and phenol-chloroform extraction method (Butler 2012). A 568 bp product of the Alt a1 gene and 472 bp product of the calmodulin protein-coding gene was amplified using following primer pairs Alt-for/Alt-rev (Hong et al. 2005) and CALDF1/CALDR1, respectively (Lawrence et al. 2013) (Integrated DNA Technologies, Inc., Coralville, IA, USA). The PCR reaction was done in a SimpliAmp thermal cycler (Applied biosystems, Waltham, MA, USA) under the following conditions: initial denaturation at 94 °C for 1 min, 35 cycles at 94°C for 30 s to denature, 57°C for 1 min for annealing, and 72°C for 1 min for extension. A final extension step at 72°C for 10 min was also included. The sequencing was done using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession no. OL542696 calmodulin). These sequences were compared with other sequences in the GenBank by using the BLAST analysis (MZ222274.1 and MN473132.1). The phylogenetic analysis was carried out with MEGA 6 software (The Pennsylvania State University, University Park, PA, USA). To confirm the pathogenicity, 10 thuja branches from healthy trees from another area without visible pathologies were inoculated with a suspension of conidia (100 conidia/ml; obtained from 2-week-old cultures). Control samples were inoculated with sterile distilled water. The inoculated branches were placed in sterile plastic containers to maintain high humidity and incubated for 10 days at 28°C. After 7 days, irregular shaped lesions and fungal growth was observed at the site of inoculation. The affected area gradually increased in size with simioar symptomatology to that described above. Re-isolation of the pathogen and identification based on morphological features and sequencing confirmed the presence of the A. alternata pathogen. To our knowledge, this is the first report of A. alternata causing branches of thuja in Kazakhstan. Thuja is a rare plant species for this region; the cost and care are expensive. This case will allow timely diagnosis of the disease caused by Alternaria spp. in the future. It is necessary to develop preventive measures and a protocol for the treatment of thuja from a fungal infection. Bakht, J., 2020. Antibacterial activity of the crude extracts from medicinally important Thuja occidentalis. Pak J Pharm Sci. 33(2): 627-630. PMID: 32276908. Butler, J.M., 2012. Chapter 2 - DNA extraction methods. pp. 29-47 in Butler JM (Ed) Advanced topics in forensic DNA typing: Methodology. San Diego, Academic Press. doi: 10.1016/C2011-0-04189-3. Chindyaeva., L.N., et al. 2020. Comparative assessment of the phytoncidity of woody plants in the selection of species for landscaping: the possibility of use in sanatorium-and-spa practice. Vopr Kurortol Fizioter Lech Fiz Kult. 97(4): 44-51. Russian. doi: 10.17116/kurort20209704144. Hong, S.G. et al. 2005. Alt a1 allergen homologs from Alternaria and related taxa: analysis of phylogenetic content and secondary structure. Fungal Genet Biol 42:119-129. doi:10.1016/j.fgb.2004.10.009 Lawrence, D.P. et al. 2013. The sections of Alternaria: formalizing species-group concepts. Mycologia 105: 530-546. DOI: 10.3852/12-249. Simmons, E. G., 2007. Alternaria: An Identification Manual. CBS, Fungal Biodiversity Center, Utrecht, Netherlands.