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Thuja is an ornamental plant 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 evergreen, scale-shaped needles. In addition, this plant has a number of
antimicrobial properties, which affect the popularity of the plant in landscaping health resort
grounds (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 revealed 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 rinsed three times with sterile distilled water. 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, and irregular and ellipsoid to ovoid conical. The
conidia varied from 5 to 25 × 6 to 12 μm (n = 40) with longitudinal and transverse septations.
These morphological characters were previously described and corresponded to Alternaria
alternata (Simmons 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 were amplified using the primer
pairs Alt-for/Alt-rev (Hong et al. 2005) and CALDF1/CALDR1, respectively (Lawrence et al.
2013) (Integrated DNA Technologies, Coralville, IA, U.S.A.). The PCR reaction was done in a
SimpliAmp thermal cycler (Applied Biosystems, Waltham, MA, U.S.A.) 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. Sequencing was done using a BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession
nos. OL542696 (Alt al) and OM471502 (calmodulin). These sequences were compared with other
sequences in GenBank using BLAST analysis (MZ222274.1 and MN473132.1). The phylogenetic
analysis was carried out with MEGA 6 software (Pennsylvania State University, University Park,
PA, U.S.A.). To confirm 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
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distinguished by a pyramidal and conical crown shape and the presence of many thin
branches with evergreen, scale-shaped needles. In addition, this plant has a number of
antimicrobial properties, which affect the popularity of the plant in landscaping health resort
grounds (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 revealed 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 rinsed three times with sterile distilled water. 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, and irregular and ellipsoid to ovoid conical. The
conidia varied from 5 to 25 × 6 to 12 μm (n = 40) with longitudinal and transverse septations.
These morphological characters were previously described and corresponded to Alternaria
alternata (Simmons 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 were amplified using the primer
pairs Alt-for/Alt-rev (Hong et al. 2005) and CALDF1/CALDR1, respectively (Lawrence et al.
2013) (Integrated DNA Technologies, Coralville, IA, U.S.A.). The PCR reaction was done in a
SimpliAmp thermal cycler (Applied Biosystems, Waltham, MA, U.S.A.) 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. Sequencing was done using a BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession
nos. OL542696 (Alt al) and OM471502 (calmodulin). These sequences were compared with other
sequences in GenBank using BLAST analysis (MZ222274.1 and MN473132.1). The phylogenetic
analysis was carried out with MEGA 6 software (Pennsylvania State University, University Park,
PA, U.S.A.). To confirm 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
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