First report of stem rot of Adenium obesum and leaf spot of Persea americana caused by Aspergillus niger in Kazakhstan

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.