Morphological and Molecular Characterization of Bacterial Pathogens Associated with Leaf Mottle of Sunflower in Northern Kazakhstan.

Leaf mottle is a serious disease in the common sunflower (Helianthus annuus L.), which affects plant growth and development and seed quality and yield. Over the past few years, the North Kazakhstan region, a sunflower-producing area in Kazakhstan, has been seriously affected by leaf mottle. Since 2021, symptomatic leaves have been collected from production areas of this base to determine the pathogens causing sunflower foliar diseases. One hundred bacterial strains were isolated, and two genera and five species were identified based on morphological characteristics, molecular genetics, and phylogenetic analysis (16S gene region). The genus Bacillus was represented by four species: Bacillus subtilis, B. megaterium, B. amyloliquefaciens, and B. flexus. The genus Paenibacillus was represented by one species, P. peoriae. Pathogenicity experiments showed that B. subtilis, B. megaterium, B. flexus, and P. peoriae could cause leaf mottle disease symptoms. However, disease symptoms caused by B. flexus were highly similar to those observed on infected leaves under natural conditions in the field. Therefore, these bacterial isolates were found to be the primary pathogens causing sunflower leaf mottle, and B. flexus was the most common and virulent pathogen in this study. In addition, this is the first report of B. megaterium, B. flexus, and P. peoriae as pathogens associated with sunflower leaf mottle in Kazakhstan.

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.

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

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.

Epizootiological monitoring of wolf helminths in Northern and Central Kazakhstan.

Background and Aim: Wolves (Canis lupus) play a role in nature, including the regulation of the number of ungulates and the use of dead animals. In addition, wolves are a natural link and carrier for the spread of many parasitic invasions. Hence, the main task in preventing the spread of parasitic invasions is to regulate the wolf population. This study aimed to monitor the endoparasitological fauna of wild wolves inhabiting Northern and Central Kazakhstan. Materials and Methods: Overall, 81 wolves were investigated for parasitic worms using the K. I. Scriabin method. Wolf intestinal materials were collected from the following six regions: North Kazakhstan, Pavlodar, Kostanay, Akmola, Ulytau, and Karaganda. The genetic diversity of the parasites was identified using a polymerase chain reaction with specific primers. After data collection, a comprehensive statistical analysis was performed. Results: Several helminth types were identified in wolves, including Echinococcus granulosus, Taenia hydatigena, Mesocestoides spp., Toxascaris leonina, Trichinella nativa, Alaria alata, and Dirofilaria repens. Based on the results of this study, young male wolves aged 1-4 years were the most vulnerable to helminthiasis. Wolves living in steppe and semi-desert regions are often exposed to helminth infections. The prevalence of T. nativa in the wolves was 20.4%. This study also revealed the presence of echinococcosis among wolf populations in Karaganda and Kostanay, with prevalence rates of 4.1% and 4.7%, respectively. The overall prevalence of tapeworms in wolves was 54.3%. Conclusion: This study highlights the significance of understanding the potential risks associated with helminth infections in wild carnivores because helminths can act as disease reservoirs and pose a threat to humans, livestock, and other wild carnivores. These results can contribute to the development of effective control and management strategies for helminth infections in wolves, which can infect humans and livestock.

Genetic diversity and haplotype analysis of cattle hydatid cyst isolates using mitochondrial markers in Kazakhstan.

Background and Aim: In Kazakhstan, the study of Echinococcus infection among farm animals is crucial to monitor the invasion among livestock and map the data obtained. Unfortunately, there are only partial data on the study of Echinococcus among cattle's in Kazakhstan, which makes it difficult to conduct a comparative analysis of the epidemiological situation among livestock animals. The present study aimed to molecularly identify the species and haplotypes of the E. granulosus complex infecting cattle in Kazakhstan and investigate their genetic variation relative to mitochondrial (mt) targets. Materials and Methods: Individual cyst isolates (n = 700) were collected from infected cattle lungs and livers after slaughter from the slaughterhouse. Total DNA was extracted from the germinal layers of the cyst from each isolate. This DNA sequenced partial mt genes of cytochrome c oxidase 1 (450 bp) and NADH dehydrogenase 1 (1200 bp). Results: We determined that all the sequences were detected as E. granulosus s.s., of which 69 (94.5%) samples belonged to G1, and only 4 (5.4%) samples belonged to the G3 genotype. After bioinformatic analysis, 38 haplotypes were identified. Conclusion: Our findings revealed that the G1 genotype of E. granulosus s.s. is the predominant cattle genotype in Kazakhstan. However, only one region showed the presence of two genotypes G1 and G3, in the sequence, which suggests that further research is needed to investigate the epidemiology of Echinococcus infection in cattle in Kazakhstan.

Molecular identification of Baylisascaris melis (Gedoelst, 1920) from the Eurasian badger (Meles meles) and ascarids from other wild carnivores in Kazakhstan.

Introduction: The presence of gastrointestinal nematodes, including zoonotic ascarids, in wild canids, felids and mustelids as definitive hosts in Central Asian countries has been documented in many studies based on traditional morphological methods. In contrast, relevant data for the badger are scarce. The aim of this study was the molecular identification of ascarid nematodes from five wild carnivore species in different regions of Kazakhstan. Methods: A total of 211 adult ascarids were collected from gray wolves (Canis lupus, 8 of 83 infected with 2-6 Toxascaris leonina), red foxes (Vulpes vulpes, 26 of 53, with 2-8 Toxascaris leonina), corsac foxes (Vulpes corsac, 6 of 11, 3-6 Toxascaris leonina), lynx (Lynx lynx, 2 of 3, with 2-5 Toxocara cati) and badgers (Meles meles, 2 of 4, with 2-7 Baylisascaris melis). Genomic DNA was extracted from the worms and ribosomal DNA, including the first and second internal transcribed spacer genes, was amplified by polymerase chain reaction using specific oligonucleotide primers and then sequenced. Results: Toxascaris leonina, but not Toxocara canis, was molecularly identified in the wild canids, Toxocara cati in the lynx and Baylisascaris melis in the badger. The maximum likelihood phylogenetic tree showed three distinct clades: the canid Toxascaris leonina was placed in one clade, Toxocara cati in another and Baylisascaris melis in a third. Discussion: The study provides the world's first molecular data and phylogenetic analysis of Baylisascaris melis, identified for the second time since its description over 100 years ago. This species was shown to be genetically distinct from other Baylisascaris spp. (B. columnaris, B. procyonis, B. transfuga, B. devosi). The possible zoonotic significance of ascarids from wild carnivores is discussed in the light of conditions in Central Asia.

Genetic identification of Trichinella species found in wild carnivores from the territory of Kazakhstan.

Trichinellosis, also called trichinosis, is a foodborne parasitic disease caused by eating raw or undercooked meat from animals infected with Trichinella spp. larvae and affects both animals and humans. Although on the territory of Kazakhstan, the species characteristics and prevalence of this helminth were studied back in the 90s, the data have not been updated since then. Given the above, our study was aimed at identifying Trichinella spp. using parasitological and molecular genetics methods. In our work, we studied 160 samples of muscle tissue of wild animals living in the natural zones of steppes and semi-deserts. Of the animals examined, 32 were positive for Trichinella spp., including 1 lynx (Lynx lynx), 17 wolves (Canis lupus), 11 foxes (Vulpes vulpes), 1 jackal (Canis aureus) and 2 corsac foxes (Vulpes corsac). Helminths were extracted using the digestion method. DNA was extracted using a Gene Jet commercial kit (Thermo Fisher Scientific, United Kingdom). For species identification a multiplex PCR, amplification of ESV, ITS1, and ITS2 genes regions was performed. After that, uniplex PCR was performed on the 5S rDNA and ITS1 genes region for sequencing analysis. The resulting sequences were subsequently used to construct a phylogenetic tree and the studied samples were identified as Trichinella nativa and Trichinella britovi. Thus, we can conclude that there is a circulation of two species of Trichinella in Kazakhstan, highlighting that constant control and monitoring of wild animals are necessary to prevent transmission and protect the health of people.

Genetic diversity of Echinococcus spp. in wild carnivorous animals in Kazakhstan.

Background and Aim: The study of Echinococcus infection among farm animals in Kazakhstan was carried out to monitor the invasion among livestock and map the data obtained. Unfortunately, there are only partial data on the study of echinococcosis among wild carnivores in Kazakhstan, which makes it difficult to conduct a comparative analysis of the epidemiological situation among wild animals. The present study aimed to estimate the genetic diversity of Echinococcus spp. (Leuckart, 1863) in Kazakhstan based on sequence analysis of cytochrome c oxidase subunit 1 (cox1) and dehydrogenase subunit 1 (nad1) of worms isolated from wild carnivorous animals wolf (Canis lupus), red fox (Vulpes vulpes) and corsac (Vulpes corsac). Materials and Methods: DNA from parasite tissue was used as a template for the amplification of the two mitochondrial genes cox1 and nad1. Sequencing was performed according to the manual for the Seq Studio Genetic Analyzer. The multiple alignments of obtained sequences were performed using the ClustalW algorithm in Mega (v.11) software. Alignments were exported as a Nexus extension and used as input for TCS v1.21 for the identification of haplotypes. The phylogenetic analysis was constructed according to the neighbor-joining method using Mega (v.11) software. Results: Analysis of the extensiveness of echinococcosis invasion showed that 6.3% were wolves, 18.2% were corsacs, and 85% were foxes. In total, 159 adults of Echinococcus spp. from the three species of animals in different parts of Kazakhstan were analyzed, and 17 individual biological samples were successfully sequenced. Sequence analysis of cox1 and nad1 genes revealed two types of echinococcosis - Echinococcus granulosus in red foxes and wolves, and Echinococcus multilocularis in corsacs. Sequencing of a portion of the mitochondrial genome made it possible to determine seven haplotypes of the pathogen in the studied samples of E. granulosus. Molecular analysis of cox1 and nad1 genes of E. multilocularis revealed three new haplotypes, which have significant variability compared with other studied Asian haplotypes. Conclusion: This study made it possible to fill the gaps in understanding the localization of the foci of the spread of the echinococcosis pathogen among the main wild carnivores and to determine the species reservoir of the pathogen in the greater territory of Kazakhstan.