Sequence Analysis Of Region Research Articles

Molecular Identification of Mycoplasma agassizii in Confiscated Tortoises in Madagascar.

Mycoplasmosis is of concern for chelonian conservation. We detected a Mycoplasma sp. in confiscated radiated tortoises (Astrochelys radiata) and spider tortoises (Pyxis arachnoides). Sequence analysis of the 16S-23S ribosomal RNA intergenic spacer region was consistent with Mycoplasma agassizii. Mycoplasma spp. have not been previously reported in native tortoises in Madagascar.

Osteoporosis in postmenopausal women is associated with disturbances in gut microbiota and migration of peripheral immune cells

BackgroundPostmenopausal osteoporosis (PMO) results from a reduction in bone mass and microarchitectural deterioration in bone tissue due to estrogen deficiency, which may increase the incidence of fragility fractures. In recent years, the “gut-immune response-bone” axis has been proposed as a novel potential approach in the prevention and treatment of PMO. Studies on ovariectomized murine model indicated the reciprocal role of Th17 cells and Treg cells in the aetiology of osteoporosis. However, the relationship among gut microbiota, immune cells and bone metabolic indexes remains unknown in PMO.MethodsA total of 77 postmenopausal women were recruited for the study and divided into control (n = 30), osteopenia (n = 19), and osteoporosis (n = 28) groups based on their T score. The frequency of Treg and Th17 cells in lymphocytes were analyzed by flow cytometry. The serum levels of interleukin (IL)-10, 17 A, 1β, 6, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) were determined via enzyme-linked immunosorbent assay. Additionally, 16S rRNA gene V3-V4 region sequencing analysis was performed to investigate the gut microbiota of the participants.ResultsThe results demonstrated decreased bacterial richness and diversed intestinal composition in PMO. In addition, significant differences of relative abundance of the gut microbial community in phylum and genus levels were found, mainly including increased Bacteroidota, Proteobacteria, and Campylobacterota, as well as reduced Firmicutes, Butyricicoccus, and Faecalibacterium. Intriugingly, in the osteoporosis group, the concentration of Treg cells and associated IL-10 in peripheral circulation was negatively regulated, while other chronic systemic proinflammatory cytokines and Th17 cells showed opposite trends. Moreover, significantly elevated plasma lipopolysaccharide (LPS) in patients with osteoporosis indicated that disrupted intestinal integrity and permeability. A correlation analysis showed close relationships between gut bacteria and inflammation.ConclusionsCollectively, these observations will lead to a better understanding of the relationship among bone homeostasis, the microbiota, and circulating immune cells in PMO. The elevated LPS levels of osteoporosis patients which not only indicate a breach in intestinal integrity but also suggest a novel biomarker for assessing osteoporosis risk linked to gut health.

Microscopy and molecular survey of Hepatozoon spp. in domestic cats and their ticks: First report of H. silvestris from Türkiye

Hepatozoonosis is a tick-borne infection caused by protozoan species of the Hepatozoon genus in the family Hepatozoidae. This study aimed to determine the presence and prevalence of Hepatozoon spp. in domestic cats and their ticks, to characterize the genotypes, to reveal phylogenetic relationships, and to assess the risk factors associated with infection. Blood samples were collected in EDTA tubes from 311 cats that were brought to the Veterinary Faculty, Animal Hospital for treatment or routine control purposes. Smears were prepared from the buffy coat and examined microscopically for the presence of Hepatozoon spp. gamonts. Ticks were collected during the clinical examination of the cats and the species were identified. Genomic DNA obtained from buffy coats and ticks were analyzed using PCR analyses targeting the 18S rRNA gene region of Hepatozoon spp. The results showed that Hepatozoon spp. gamonts in neutrophils were determined in five samples as 1.6 % (5/311). A total of 58 (18.64 %, 58/311) were positive for the PCR analyses. Twelve ticks were collected from the cats, and all were identified as Rhipicephalus turanicus. Hepatozoon spp. were found in seven (58.3 %) ticks by molecular analyses. Sequence analysis of the 18S rRNA gene region of 58 positive isolates revealed the presence of H. felis (n = 25, 43.1 %) and H. silvestris (n = 33, 56.9 %) species in cats and H. felis in Rh. turanicus (n = 7). This study also presents the first report of H. silvestris in domestic cats from Türkiye. Phylogenetic analyses showed that our H. felis isolates clustered in H. felis genotype I and our H. silvestris were stated in the same cluster with Italian isolates.

First Report of Rhizoctonia solaniAG‐4 HGI Causing Corky Cracks on Potato Tubers in Mauritius

ABSTRACTPotato (Solanum tuberosum L.) is of great economic importance for Mauritius. During a disease survey carried out in a field in one of the major potato‐growing regions of Mauritius, corky cracked lesions with halo and mycelial growth were observed on the skin surfaces of around 45% of tubers. The suspected fungus from the affected areas was isolated on amended potato dextrose agar and after 7 days, several fast‐growing, pale brown Rhizoctonia‐like colonies with a few sclerotia were observed. Additionally, microscopic analysis revealed some cells being multinucleate, forming hyphal branching at right angles with slight constriction together with septum near the branch base. Pathogenicity tests confirmed development of the same symptoms on potato tubers and the causal agent was reisolated successfully. Sequence analysis of the ITS region of representative isolate P114 amplified using ITS1F/ITS4 primers showed that it was identical to several isolates of multinucleate Rhizoctonia solani AG‐4 HGI, and neighbour‐joining phylogenetic analyses confirmed the identification. To our knowledge, this is the first report of R. solani AG‐4 HGI‐infecting potato tubers and causing corky cracks in Mauritius.

Genetic Diversity of Bluefish Population in Türkiye Coastal

Bluefish (Pomatomus saltatrix) is a commercially important, highly migratory predatory fish species distributed along the coasts of Türkiye. Conservation and sustainability of bluefish populations requires knowledge of the genetic structure of the population, but There are no genetic studies on bluefish in Türkiye. In this study, the population structure of Bluefish in the coastal regions of Türkiye was investigated by sequence analysis of mitochondrial DNA gene regions (Cytochrome b and Displacement-loop) and nuclear gene region (Rhodopsin). We tried to identify bluefish samples collected from 14 regions in the coastal region of Türkiye by analyzing two mtDNA gene sequences (Cyt b and D-loop) and nuclear gene region (Rhodopsin). While a total of 35 haplotypes were found in the Cytb gene region, a total of 132 haplotypes were found in the D-loop (control region) gene region. A single haplotype was detected in all stations for Rhodopsin. Also, the maximum likelihood trees based on Cyt b and D-loop sequences suggested relationship of Bluefish populations from Türkiye and the world. Results showed a low level of genetic differentiation between Bluefish populations in the study area, but Aegean and Mediterranean populations are more diverse than other populations due to migratory trait of this species.

Fungal endophytes Fusarium solani SGGF14 and Alternaria tenuissima SGGF21 enhance the glycyrrhizin production by modulating its key biosynthetic genes in licorice (Glycyrrhiza glabra L.).

To identify promising fungal endophytes that are able to produce glycyrrhizin and enhance it in licorice and the mechanisms involved. Fifteen fungal endophytes were isolated from Glycyrrhiza glabra L. rhizomes among which SGGF14 and SGGF21 isolates were found to produce glycyrrhizin by 4.29 and 2.58µg g-1 dry weight in the first generation of their culture. These isolates were identified as Fusarium solani and Alternaria tenuissima, respectively, based on morphological characteristics and sequence analysis of internal transcribed spacer, TEF1, ATPase, and CAL regions. Subsequently, G. glabra plants were inoculated with these fungal isolates to examine their effect on glycyrrhizin production, plant growth parameters and the expression of key genes involved in glycyrrhizin pathway: SQS1, SQS2, bAS, CAS, LUS, CYP88D6, and CYP72A154. Endophytes were able to enhance glycyrrhizin content by 133%-171% in the plants. Natural control (NC) plants, harboring all natural endophytes, had better growth compared to SGGF14- and SGGF21-inoculated and endophyte-free (EF) plants. Expression of SQS1, SQS2, CYP88D6, and CYP72A154 was upregulated by inoculation with endophytes. LUS and CAS were downregulated after endophyte inoculation. Expression of bAS was higher in SGGF21-inoculated plants when compared with NC, EF, and SGGF14-inoculated plants. Two selected fungal endophytes of G. glabra can produce glycyrrhizin and enhance glycyrrhizin content in planta by modulating the expression of key genes in glycyrrhizin biosynthetic pathway.

An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China.

Chickpea (Cicer arietinum L.) is an important legume crop worldwide. An emerging disease, basal stem rot with obvious wilt symptoms, was observed in the upper part of chickpea plants during the disease survey in Qiubei County of Yunnan Province. Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue of chickpea plants collected from the field. Those isolates were morphologically found to be similar to Diaporthe aspalathi. Molecular sequence analyses of multiple gene regions (ITS, tef1, tub2, cal, and his3) indicated that the three isolates showed a high identity with D. aspalathi. Pathogenicity and host range tests of the isolates were performed on the original host chickpea and eight other legume crops. The isolates were strongly pathogenic to chickpea and appeared highly pathogenic to soybean, cowpea, and mung bean; moderated or mild pathogenic to adzuki bean and common bean; however, the isolates did not cause symptoms on grass pea (Lathyrus sativus). Diaporthe aspalathi was previously reported as a main pathogen causing the southern stem canker in soybean. To our knowledge, this is the first report of D. aspalathi inducing basal stem rot on chickpea worldwide.

Molecular Identification of Encephalitazoon intestinalis and the Prevalence of Renal Microsporidiosis in Renal Transplant Recipients in Türkiye.

In patients with end-stage kidney disease, kidney transplantation is the kidney replacement therapy option that provides the most successful survival. However, immunosuppression agents administered after kidney transplantation can increase the risk of opportunistic infections. Microsporidia are obligate intracellular pathogens that can be fatal in immunosuppressed patients. The present study aimed to determine the prevalence of microsporidia in kidney transplantation recipients and the molecular characterization of the detected species. To evaluate the prevalence of renal microsporidiosis in kidney transplant recipients, the urine samples from a total of 325 patients were analyzed by real-time and nested polymerase chain reaction for Encephalitozoon spp. and Enterocytozoon bieneusi. Only one (0.4%) sample from the adult patient was positive for the Encephalitozoon species, while no positivity was found in pediatric patients. It was determined as Encephalitozoon intestinalis by ITS rRNA gene region sequence analysis. A microsporidia species obtained from humans in Türkiye has been characterized for the first time and registered in GenBank. Our epidemiological results show that the prevalence of renal microsporidiosis in kidney transplant recipients is very low. In addition, as a result of the phylogenetic analysis of the detected isolate, it was observed that it was 100% identical to the isolates reported from dogs in Kayseri, Türkiye. This situation provided essential data regarding the zoonotic transmission dynamics of microsporidia.

Phytophthora polonica and Phytophthora hydropathica from Clade 9 Associated with Alder Decline in Bulgaria.

A number of Phytophthora species have been identified as destructive plant pathogens and invasive species. They have the potential to affect a wide range of host plants and cause diseases in agricultural and forest ecosystems. Two Phytophthora species from rhizosphere soil samples collected from declining Alnus glutinosa in Bulgaria were isolated in the autumn of 2022. They were identified as Phytophthora polonica and Phytophthora hydropathica according to the DNA sequence analysis of the ITS region, as well as their morphological and physiological characteristics. The pathogenicity of both species to common and gray alder was evaluated by the inoculation of detached leaves and cuttings. Experimental data proved that P. polonica and P. hydropathica are able to cause leaf necrosis not only on A. glutinosa from which they were derived, but also on A. incana. No significant deference in the aggressiveness of the studied isolates from both Phytophthora species against the two tested plants was observed. Therefore, P. polonica and P. hydropathica were determined as potential threats for alder ecosystems in the country. This is the first report for the isolation of P. polonica in Bulgaria and represents the most southeastern point of the species distribution in Europe.

Diversity of unique, nonmycorrhizal endophytic fungi in cultivated Phalaenopsis orchids: A pilot study.

Orchids comprise one of the largest, most diverse, and most broadly distributed families of flowering plants and contribute significantly to habitat biodiversity. One key aspect of orchid growth and development is the formation of mycorrhizal symbioses with compatible endophytic fungi, which are maintained throughout the life of the plant. Substantial efforts to identify the fungi that form mycorrhizal symbioses across a range of orchid species have often also uncovered numerous nonmycorrhizal, endophytic fungi. These fungi could also have significant effects on orchid growth and development and are beginning to be analyzed more closely, particularly in wild species. The role of endophytic fungi in the production, distribution, and continued growth by the hobbyist of orchids is not known. As an initial step toward characterizing nonmycorrhizal endophytic fungi associated with cultivated orchids, we undertook a survey of fungi residing within roots of Phalaenopsis plants growing in home environments. Sequence analysis of ITS regions amplified from total DNA isolated from roots allowed rapid identification of endophytic fungi to the class level and may offer a useful initial screening method for beneficial species, for example, in horticultural settings. ITS-PCR sequences subsequently obtained from individual fungi cultured from surface-sterilized orchid roots corroborated the findings of the initial screen, while also providing a more complete characterization of the array of fungal taxa that were present. Although lower in diversity than has been reported for orchids growing in the wild, these endophytes have the potential to substantially enhance the growth and disease resistance of horticultural orchids.

Genetic and pathogenic characterization of Rhizoctonia solani AG-4 isolates obtained from common bean

Common bean (Phaseolus vulgaris L.) is the most important grain legume. Crop production of common bean is affected by a number of diseases, such as Rhizoctonia root rot, being detected worldwide. Herein, we aimed to genetically and pathologically characterize Rhizoctonia solani isolates obtained from different provinces in Turkey. Anastomosis groups (AG) and subgroups of isolates were identified based on the sequence analysis of the rDNA-ITS region. The most prevalent subgroup was AG-4 HGIII, according to BLAST analysis and the phylogeny of resultant sequences, followed by AG-4 HGI and AG-4 HGII, respectively. iPBS retrotransposons highly supported the phylogenetic tree and provided a high level of genetic variability among isolates to discriminate AG subgroups. The results indicated that the iPBS DNA marker system based on retrotransposons could be used to discriminate Rhizoctonia isolates relying on AG grouping. The virulence of the pathogen isolates changed from 3.67 to 5 based on the agar-plate assay on the susceptible cv. Gina. The reactions of thirty common bean cultivars were also evaluated against the most aggressive isolate in each subgroup. The reaction assay showed significant differences among both isolates and cultivars; the highest disease severity among cultivars was observed to AG-4 HGI, followed by AG-4 HGIII and AG-4 HGII, respectively. None of the cultivars showed resistance to all AG subgroups. Screening of AGs among the isolates and selecting cultivars are critical to managing Rhizoctonia disease due to the range of host suitability according to AG subgroups.

Clinicopathological characteristics and gene mutations in 11 patients with lipoprotein glomerulopathy

Objective Lipoprotein glomerulopathy (LPG) is a rare disorder characterized by the development of glomerular lipoprotein thrombosis. LPG exhibits familial aggregation, with mutations in the apolipoprotein E (APOE) gene identified as the leading cause of this disease. This study aimed to investigate APOE gene mutations and the clinicopathological features in eleven LPG patients. Methods Clinicopathological and follow-up data were obtained by extracting DNA, followed by APOE coding region sequencing analysis. This study analyzed clinical and pathological manifestations, gene mutations, treatment and prognosis. Results The mean age of the eleven patients was 33.82 years. Among them, five had a positive family history for LPG, ten presented with proteinuria, four exhibited nephrotic syndrome, and six presented with microscopic hematuria. Dyslipidemia was identified in ten patients. In all renal specimens, there was evident dilation of glomerular capillary lumens containing lipoprotein thrombi, and positive oil red O staining was observed in frozen sections of all samples. APOE gene testing revealed that one patient had no mutations, while the remaining ten patients exhibited mutations in the APOE gene, with three patients presenting with multiple mutations simultaneously. Following the confirmation of LPG diagnosis, treatment with angiotensin-converting enzyme inhibitor (ACEI)/angiotensin receptor blocker (ARB) was initiated, and the disease progressed slowly. Conclusion LPG is histologically characterized by lamellated lipoprotein thrombi in glomeruli, and kidney biopsy is essential for diagnosis. Mutations in the APOE gene are the leading cause of LPG. This study revealed clinicopathological characteristics and APOE gene mutations in patients with LPG, which helps us better understand the disease.

Evolution of the enhancer-rich regulatory region of the gene for the cell-type specific transcription factor POU1F1

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5′ to 3′). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth.

Abstract 6265: FLCN variants and their role in parathyroid cancer and atypical parathyroid tumors

Abstract Parathyroid carcinoma (PC) is rare, poses challenges in definitive diagnosis and treatment, and carries a high rate of recurrence and mortality. Atypical parathyroid tumors (APT) have histopathologic features of malignancy but lack unequivocal evidence of invasion and are of uncertain malignant potential. Analysis of genetic alterations that may play a role in PC development, particularly those that might serve as markers of malignant potential, is important for further understanding this disease. FLCN is a gene found on chromosome 17 that encodes folliculin, a ubiquitously expressed protein with roles in multiple cellular processes such as apoptosis and cell signaling. Inactivating FLCN mutations cause Birt-Hogg-Dubé syndrome (BHD), an autosomal dominant condition characterized by benign fibrofolliculomas, pulmonary cysts and spontaneous pneumothorax, and increased risk of renal cancer. Benign parathyroid tumors had previously been reported in a few BHD patients and recently one study has reported a link between FLCN mutations and parathyroid carcinoma. Germline frameshift mutations in FLCN, accompanied by somatic loss of the normal allele, were reported in two unrelated PC patients, both with clinical features suggestive of BHD, but no established diagnosis of BHD. An identical somatic frameshift FLCN mutation was reported in one PC and one APT. Interestingly, the authors noted low coverage of FLCN on whole exome sequencing, resulting in the failure to detect variants identified by Sanger sequencing, thus raising the possibility that prior studies may have missed FLCN variants. To better understand the frequency of germline or somatic FLCN mutations in PC and APT, we performed Sanger sequence analysis of the entire coding region of the FLCN gene on available tumor DNA from 11 PCs and 15 APTs. We identified no inactivating FLCN mutations in any of the PC or APT samples examined. A germline missense variant, resulting in a p.Gly325Val change, predicted to be benign/tolerated by in silico analyses, was seen in one PC and a synonymous variant (c.1233G>A) was seen in one APT. The absence of pathogenic mutations in our series parathyroid carcinoma and atypical parathyroid tumors suggests that FLCN mutations are rare in these parathyroid tumors. Nevertheless, germline FLCN testing and/or additional screening for BHD-related lesions in parathyroid carcinoma patients merits further consideration and study. Citation Format: Callie Burke, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. FLCN variants and their role in parathyroid cancer and atypical parathyroid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6265.

In silico analysis to understand genetic variability, phylogenetic and phylogeographic relationships between Phytophthora capsici isolates infecting various crops

Phytophthora capsici is a major oomycete-based pathogen causing enormous damage to the productivity of peppers and other major crops from different families worldwide by causing fruit rot, root rot and foliar blight diseases. This study utilized molecular signatures present within ITS1-5.8S rRNA-ITS2 and Ras-related GTP binding protein (Ypt1) genes to evaluate haplotype variability and phylogenetic relationships among P. capsici isolates with significant epidemiological implications. Twelve different evolutionary lineages specific to particular hosts and/or geographical regions and differed by SNPs, and 30 haplotypes with maximum diversity in India and Pakistan, were identified by sequence analysis of the ITS regions. It includes country-specific lineages Pc-I and Pc-II in India with multiple host ranges; capsicum-specific lineages Pc-IV, Pc-V and Pc-VIII from Pakistan; papaya-specific lineages Pc-VI from China, and watermelon-specific lineages Pc-VII and Pc-XI from USA. Many lineages showed cross-border infections, such as Pc-III and Pc-X in India and Pakistan, Cocoa-specific lineage Pc-IX in Brazil and Cote d’Ivoire, and Capsicum-specific multi-continental lineage Pc-XII. Similarly, 11 different SNP lineages and 32 haplotypes with maximum diversity in China were identified for the Ypt1 gene. These findings would be very useful for epidemiological surveillance of P. capsici strains worldwide, understanding population and evolutionary dynamics, developing lineage- and haplotype-specific diagnostic assays and disease-resistant varieties.

Torque teno sus virus k2a (TTSuVk2a) in wild boars from northeastern Patagonia, Argentina.

Torque teno sus virus k2a (TTSuVk2a) is a member of the family Anelloviridae that can establish persistent infections in both domestic pigs and wild boars. Its association with diseases has not been precisely elucidated, and it is often considered only as a commensal virus. This infectious agent has been reported in herds throughout the world. In this study, we investigated the detection rate and diversity of TTSuVk2a in free-living wild boars from northeastern Patagonia, Argentina. Total DNA was extracted from tonsil samples of 50 animals, nested PCR assays were carried out, and infection was verified in 60% of the cases. Sequence analysis of the viral non-coding region revealed distinct phylogenetic groups. These clusters showed contrasting patterns of spatial distribution, which presented statistically significant differences when evaluating spatial aggregation. In turn, the sequences were compared with those available in the database to find that the clusters were distinguished by having similarity with TTSuVk2a variants of different geographic origin. The results suggested that Patagonian wild boar populations are bearers of diverse viral strains of Asian, European, and South American provenance.

Sequence Analysis of Coding Region of Prolactin Gene in Milking Beetal Goats

Sequence Analysis of Coding Region of Prolactin Gene in Milking Beetal Goats

Identificação de isolados de leveduras do gênero Trichosporon de infecções superficiais em pacientes do sexo masculino do Brasil Central: uma abordagem sobre a diversidade de infecções causadas por esse fungo basidiomiceto

Abstract The genus Trichosporon are currently recognized as opportunistic pathogens capable of causing superficial "white piedra" infections and potentially fatal invasive diseases (Trichosporonosis). In this work, determine the agent Trichosporon spp. isolated from the skin and appendages of a male population group in the Central-West region of Brazil. The isolates were analyzed by phenotypic, biochemical and molecular methods. Twenty-five strains of Trichosporon were isolated: T. asahii (18; 72%), followed by T. inkin (4; 16%) and T. faecale (3; 12%). Skin infections were the most affected (16; 64%) and the genitocrural region (13; 52%) was the most affected. The highest rate of isolation occurred between the ages of 21 and 30 years (9; 36%), with black men (African descent) (13; 52%) being the most affected by this type of superficial infection. After the advent of molecular techniques, more than 50 subspecies and about 16 different strains have been reported to cause human disease. In this series, three species of the genus Trichosporon of medical importance were highlighted, colonizing the genital and perigenital region of the studied population. For the identifications, classical phenotypic methods associated with genotypic identification were carried out, using molecular techniques based on the study of DNA; using sequence analysis of the DNA intergenic spacer region 1 (IGS1).

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 μm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 μl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

Genetic characterization of parvoviruses identified in stray cats in Nigeria

Parvoviruses are a major cause of haemorrhagic gastroenteritis, leukopenia and high mortality in cats and dogs. In this study, the presence and genetic characteristics of parvoviruses circulating among cats in Nigeria are reported. Faecal samples of stray cats from live animal markets in southwestern (Oyo and Osun States) and north-central (Kwara State) Nigeria were screened for the presence of parvoviral DNA using a qPCR. Positive samples were further characterized using a qPCR based on minor groove binder probes. Overall, 85/102 (83.3 %) stray cats tested positive for feline panleukopenia virus (FPV) DNA and one cat was co-infected with canine parvovirus-2 type a. Sequence analysis of the complete capsid region of 15 Nigerian FPV strains revealed that they were up to 99.9 % similar to the American reference strain FPV-b at the nucleotide level, and three of them presented amino acid mutations in key capsid residues. This is the first report of identification and molecular characterization of FPV strains in cats in Nigeria. The high prevalence of the virus emphasizes the need for constant surveillance of the circulation of parvoviruses in Nigeria and underscores the need to deploy an effective vaccination strategy.