Search

Adenium (Adenium obesum) and avocado (Persea americana) are commonly grown as exotic houseplants in city apartments of Kazakhstan. In April-May 2020, the wilting symptom was observed on the young stems of five 2-year-old A. obesum plants in a city apartment in Saryarqa District, Astana, Kazakhstan (71°25′E, 51°11′N). Leaves turned yellow and then dried up. Plants were completely wilted within 10 days (Fig. 1A). Similar symptoms were observed in newly grown A. obesum plants in November, 2021. At the same time, lesions were found on the leaves of three 3-month-old P. americana plants. Infected leaves displayed dry, dark-brown lesions and fell off easily (Fig. 2A). Both plants were cultivated side by side. The incidence of affected A. obesum was 80% out of 5 plants and P. americana was 100% out of 3 plants. To isolate the causal agent, the infected tissues from different leaves and stems of A. obesum and P. americana plants were cut into small pieces (5 × 5 mm), washed in 70% ethanol for 5 min, and then rinsed three times with sterile distilled water. Cut pieces were placed on potato dextrose agar (PDA) (Laboratorios Conda S.A., Spain) and incubated at 28°C for 7 days. Ten isolates were obtained from leaves and stems of the A. obesum and P. americana symptomatic samples. All fungal colonies were white initially, turned black gradually, reverse side light yellow (Fig. 1B and Fig. 2B), conidiophores biseriate with globose vesicles, conidia were spherical vesicles, light tan to black color, smooth-walled to roughened, and sizes ranged from 3.0 to 3.5 μm (n = 15) (Fig. 1C and Fig. 2C). These observations indicated that all the isolates resembled Aspergillus spp. (Bryan and Fennell 1965). DNA was extracted using the liquid nitrogen and phenol-chloroform extraction method (Butler 2012). A 526 bp product of the ITS region on rDNA and 568 bp product of the calmodulin protein-coding gene was amplified using following primer pairs ITS4/ITS5 (Abliz et al. 2003) and cmd5/cmd6, respectively (Hong et al. 2005). The PCR reaction was done under the following conditions: initial denaturation at 94°C for 5 min, 35 cycles at 95°C for 30 s to denature, 52°C for 40 s for annealing, and 72°C for 50 s for extension. A final extension step at 72°C for 7 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 nos. ON519078 (A. obesum ITS), ON519079 (P. americana ITS), OQ358173 (A. obesum calmodulin) and OQ358174 (P. americana calmodulin). These sequences were compared with other sequences of A. niger in GenBank using BLAST analysis (MG569619.1, MT588793.1, MH478660.1, MZ787576.1 and MW086485.1). The results showed that the sequences of ten isolates were identical and had 98-100% identity with those of Aspergillus niger (Fig. 3). The phylogenetic analysis was carried out with MEGA 11 (Tamura et al. 2021). To confirm the pathogenicity, three asymptomatic plants of each were inoculated with a suspension of conidia via pin-prick inoculation (1.0×106 conidia/ml; obtained from 2-week-old cultures). Control plants were inoculated with sterile distilled water. The inoculated plants were placed in climate chamber (BINDER, Germany) and incubated for 10 days at 28°C. Symptoms were developed in leaves of inoculated plants after 2 days in P. americana and after 5 days in A. obesum. Affected leaves turned yellow and their stems started drying. Symptoms of leaves were similar to those observed on naturally infected plants, while control plants remained asymptomatic. Re-isolation of the pathogen confirmed the presence of the A. niger pathogen. To our knowledge, this is the first report of A. niger causing stem rot of A. obesum and leaf spot of P. americana in Kazakhstan. Since different ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of A. niger among them. This finding provides a foundation to further investigate the biology and epidemiology of this disease so the developmentf diagnostic tools and management measures against it.

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.