Genotypic and phenotypic characterization of the first Latin America isolates of Corynebacterium rouxii, a recently described member of the Corynebacterium diphtheriae complex reported in Europe.

The genus Corynebacterium is the largest genera among corynebacteria and has a range of species widely spread in ecological niches, some with epidemic potential and capable of causing fatal diseases. In recent years, due to the reclassifications and discoveries of new potentially toxin-producing species, microbiological identification and epidemiological control have been compromised, becoming possible only with sequencing techniques. Two bacterial strains isolated from a cat were identified by MALDI-TOF mass spectrometry as Corynebacterium diphtheriae and sent to the collaborating center of the Brazilian Ministry of Health for molecular identification and determination of toxigenicity potential, which were initially performed by multiplex PCR method. In addition, the antimicrobial susceptibility profile was determined according to BrCAST. Finally, for the final identification at the species level and effective epidemiological monitoring, the sequencing of the 16S rRNA and rpoB housekeeping genes was carried out. The isolates were identified as nontoxigenic C. diphtheriae strains by mPCR. Both strains were found susceptible to all antimicrobial agents. Although the identification at the species level was not possible through similarity analysis of  S rRNA and rpoB housekeeping genes, the phylogenetic analysis showed that the isolates belonged to the species Corynebacterium rouxii with a high value of reliability. This is the first report of the isolation of C. rouxii in Latin America. Molecular identification, whether by the MALDI-TOF mass spectrometry or PCR techniques, does not discriminate C. rouxii from C. diphtheriae, requiring gene sequencing and phylogenetic analysis for correct identification at the species level.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Identification, Characterization, and Antibacterial Evaluation of Five Endophytic Fungi from Psychotria poeppigiana Müll. Arg., an Amazon Plant.

Endophytic fungi, residing within plants without causing disease, are known for their ability to produce bioactive metabolites with diverse properties such as antibacterial, antioxidant, and antifungal activities, while also influencing plant defense mechanisms. In this study, five novel endophytic fungi species were isolated from the leaves of Psychotria poeppigiana Müll. Arg., a plant from the Rubiaceae family, collected in the tropical Amazon region of Bolivia. The endophytic fungi were identified as a Neopestalotiopsis sp., three Penicillium sp., and an Aspergillus sp. through 18S ribosomal RNA sequencing and NCBI-BLAST analysis. Chemical profiling revealed that their extracts obtained by ethyl acetate contained terpenes, flavonoids, and phenolic compounds. In a bioautography study, the terpenes showed high antimicrobial activity against Escherichia coli. Notably, extracts from the three Penicillium species exhibited potent antibacterial activity, with minimum inhibitory concentration (MIC) values ranging from 62.5 to 2000 µg/mL against all three pathogens: Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis (both Gram-positive and Gram-negative bacteria). These findings highlight the potential of these endophytic fungi, especially Penicillium species as valuable sources of secondary metabolites with significant antibacterial activities, suggesting promising applications in medicine, pharmaceuticals, agriculture, and environmental technologies.

New records of Trichinella patagoniensis from Argentina.

Trichinella spiralis was long considered the sole Trichinella species in Argentina. However, since 2004, various Trichinella species, including the encapsulated Trichinella patagoniensis and Trichinella britovi, as well as the unencapsulated Trichinella pseudospiralis, have been detected in the country. The present study aimed to identify Trichinella ML at the species level from cougars naturally infected from Argentina. To this end, muscle tissue samples from one cougar each from Córdoba, Neuquén, and Santa Cruz Provinces were individually analysed using the artificial digestion technique. DNA extraction and molecular identification of Trichinella species were conducted on individual muscle larvae by PCR amplification of the ESV region and subsequent PCR amplification and sequencing of the COI gene. Morphological analysis revealed muscle larvae with characteristics consistent with Trichinella genus. PCR revealed a single band of approximately 127 bp for each individual muscle larva. PCR amplification of the COI gene from each isolate generated a 309 bp band. Sequencing of the mitochondrial COI gene confirmed the identity of the parasite as T. patagoniensis. The present study documents new occurrences of T. patagoniensis in Puma concolor from Argentina, including the first detection of T. patagoniensis in Puma concolor from Córdoba and Neuquén Province. These findings expand the limited knowledge of T. patagoniensis distribution in Argentina.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.

A Genetic Tool to Identify Predators Responsible for Livestock Attacks in South America and Recommendations for Human-Wildlife Conflict Mitigation.

Livestock predation induces global human-wildlife conflict, triggering the retaliatory killing of large carnivores. Although domestic dogs (Canis familiaris) contribute to livestock depredation, blame primarily falls on wild predators. Dogs can also transmit pathogens between wildlife, domestic animals, and humans. Therefore, the presence of free-ranging dogs can have negative consequences for biodiversity conservation, smallholder economy, food supply, and public health, four of the United Nations' Sustainable Developed Goals (SDGs) for 2030. In Ecuador, where livestock sustains rural households, retaliatory poaching threatens Andean bear (Tremarctos ornatus), jaguar (Panthera onca), and puma (Puma concolor) populations. However, the role of dogs in these incidents remains underexplored. The present study evaluates the possibility of reliable molecular identification of predatory species from DNA traces in bite wounds. Our results revealed the presence of dog saliva on four out of six livestock carcasses presumably attacked by wild predators. These findings highlight the importance of rectifying misinformation about large carnivores in Ecuador and the need to control dog populations. We recommend that local administrations incorporate DNA analysis into livestock predation events to examine how common the problem is, and to use the analysis to develop conflict mitigation strategies which are essential for the conservation of large carnivores.

Molecular Techniques for Root-Knot Nematode Identification.

Among plant-parasitic nematodes, root-knot nematodes (RKN), Meloidogyne spp., are the most important parasite infecting economically important crops globally and causing severe losses in crop production. The use of efficient nematode control methods against these parasites depends upon their correct detection in roots and soil samples. Currently, the use of integrated identification methods, including biochemical, molecular, and morphological-based characters, is preferred. But the techniques using morphology and phylogenetic analysis are time-consuming and not suitable for routine analysis. They have only been used for studies of cryptic species, which were identified using integrative taxonomy. Here we describe the enzymatic and molecular-based methods that have successfully been used in Brazil for more than 25 years in the Nematology Lab at Embrapa Genetic Resources and Biotechnology for routine analysis. This technique is a combination of isozyme esterase profiling and molecular markers, with the aim of having a rapid and correct diagnosis of Meloidogyne spp. populations from field and greenhouse.

Detection of non-tuberculous mycobacteria in native wildlife species at conservation risk of Argentina.

Introduction: Non-tuberculous Mycobacteria (NTM) are mainly environmental but can cause opportunistic infections and diseases in humans and animals. Livestock and wild animals can be infected with NTM. In Argentina, there are native wild species facing conservation risks, and they are the focus of protection and reintroduction projects designed to preserve biodiversity in various ecoregions. The aim of this study was to report the presence of NTM in samples collected from four endangered native wild species from nine Argentine provinces, as part of their pre-release health assessment. Methods: A total of 165 samples from giant anteater, peccary, tapir and pampas deer were obtained, these included either bronchoalveolar or endotracheal lavages, or oropharyngeal, nasopharyngeal or tracheal swabs. Bacteriological culture followed by molecular identification and sequencing were performed. Results: A total of 27 NTM were detected, including Mycobacterium avium subsp. hominissuis, M. intracellulare, M. terrae, M. gordonense, M. kumamotonense, M. fortuitum, M. saskatchewanense, and M. genavense. Results revealed a 16,36% NTM recovery rate, with the giant anteater showing the highest prevalence among the mammals under study. Discussion: In Argentina, due to extensive production systems, the interaction between domestic and wild species sharing the same environment is frequent, increasing the exposure of all the species to these NTM. In this way, the transmission of infectious agents from one to another is feasible. Moreover, NTMs might interfere with the diagnosis of bovine tuberculosis and paratuberculosis. These findings emphasize the importance of active health surveillance in conservation programs. It highlights the need to address NTM epidemiology in wildlife and its impact on conservation and public health.

First report of fatal fungemia due Fusarium oxysporum in a patient with COVID-19 in Ecuador.

Filamentous fungal infections are an important cause of systemic infections in immunocompromised patients. Fusarium genus members potentially cause disseminated infections, especially in patients with catheters, due to the ability to adhere to these devices. We describe a case of fatal fungemia due to Fusarium oxysporum in a patient with COVID-19 in Ecuador. The genus identification was carried out with conventional techniques and species identification by molecular and phylogenetic techniques through sequencing of the ITS region.

Molecular identification of new Trypanosoma evansi type non-A/B isolates from buffaloes and cattle in Indonesia / Identificação molecular de novos isolados de Trypanosoma evansi tipo não A/B em búfalos e gado na Indonésia

Abstract Trypanosoma evansi is reportedly divided into two genotypes: types A and B. The type B is uncommon and reportedly limited to Africa: Kenya Sudan, and Ethiopia. In contrast, type A has been widely reported in Africa, South America, and Asia. However, Trypanosoma evansi type non-A/B has never been reported. Therefore, this study aims to determine the species and genotype of the Trypanozoon subgenus using a robust identification algorithm. Forty-three trypanosoma isolates from Indonesia were identified as Trypanosoma evansi using a molecular identification algorithm. Further identification showed that 39 isolates were type A and 4 isolates were possibly non-A/B types. The PML, AMN-SB1, and STENT3 isolates were likely non-A/B type Trypanosoma evansi isolated from buffalo, while the PDE isolates were isolated from cattle. Cladistic analysis revealed that Indonesian Trypanosoma evansi was divided into seven clusters based on the gRNA-kDNA minicircle gene. Clusters 6 and 7 are each divided into two sub-clusters. The areas with the highest genetic diversity are the provinces of Banten, Central Java (included Yogyakarta), and East Nusa Tenggara. The Central Java (including Yogyakarta) and East Nusa Tenggara provinces, each have four sub-clusters, while Banten has three.

Resumo Trypanosoma evansi é reportado como dividido em dois genótipos: tipos A e B. O tipo B é incomum e reportado como limitado à África: Quênia, Sudão e Etiópia. Em contraste, o tipo A tem sido amplamente relatado na África, América do Sul e Ásia. No entanto, Trypanosoma evansi tipo não-A/B nunca foi relatado. Portanto, este estudo tem como objetivo determinar a espécie e o genótipo do subgênero Trypanozoon, utilizando-se um algoritmo robusto de identificação. Quarenta e três isolados de tripanosoma da Indonésia foram identificados como Trypanosoma evansi, usando-se um algoritmo de identificação molecular. A identificação adicional mostrou que 39 isolados eram do tipo A e 4 isolados eram, possivelmente, do tipo não A/B. Os isolados PML, AMN-SB1 e STENT3 foram, provavelmente, Trypanosoma evansi do tipo não A/B isolado de búfalos, enquanto os isolados de PDE foram isolados de bovinos. A análise cladística revelou que o Trypanosoma evansi indonésio foi dividido em sete grupos baseados no gene do minicírculo gRNA-kDNA. Os clusters 6 e 7 foram divididos cada um em dois subclusters. As áreas com maior diversidade genética são as províncias de Banten, Java Central (incluindo Yogyakarta) e East Nusa Tenggara. As de Java Central (incluindo Yogyakarta) e East Nusa Tenggara têm, cada uma, quatro subgrupos, enquanto Banten tem três.

Acute, fatal Sarcocystis falcatula infection in rose-ringed parakeets (Psittacula krameri).

Sarcocystosis is an important avian disease that affects several intermediate host species. Birds not endemic from Americas, like Old World psittacine species, appear to be more susceptible to lethal infection than New World psittacine species. The aim of this study was to investigate the sudden death of rose-ringed parakeets (Psittacula krameri) in an exotic private parrot's aviary. Macroscopically, the most prevalent findings were severe lung congestion, slight superficial myocardial hemorrhagic lesions, enlarged liver and congestion of meningeal vessels. The initial diagnosis of sarcocystosis was made in all birds by microscopic observations of intravascular pulmonary schizonts, as well hepatitis, myocarditis, and nephritis. Immunohistochemistry for detection of Sarcocystis sp. antigen revealed an intense immunoreactivity in the lungs. Molecular identification of Sarcocystis falcatula were obtained by nested PCR and sequencing of amplified fragments of internal transcribed spacer 1 (ITS1) and three surface antigen-coding genes (SAG2, SAG3 and SAG4). SAG-based phylogenies showed a close relatedness of the isolate described here and S. falcatula previously detected in naturally infected native birds, which suggests that the isolates that affected ringnecks are a common isolate that circulates in Brazil.

First Report of Anthracnose Caused by Colletotrichum nymphaeae on Strawberry Fruits in Central Argentina.

Strawberry (Fragaria x ananassa) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 µm (avg.= 4.3 µm), and from 10.37 to 19.52 µm (avg.= 14.27 µm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of Colletotrichum nymphaeae. The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with C. nymphaeae, in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 µl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as C. nymphaeae by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with Colletotrichum acutatum, C. gloeosporioides and C. fragariae species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of C. nymphaeae causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.

Molecular confirmation of Coleosporium plumeriae Causing Rust of Plumeria rubra in Mexico.

Frangipani (Plumeria rubra L.; Apocynaceae.) is a deciduous ornamental shrub, native to tropical America and widely distributed in tropical and subtropical regions. In Mexico, P. rubra is also used in traditional medicine and religious ceremonies. In November 2018-2022, rust-diseased leaves of P. rubra were found in Yautepec (18°49'29"N; 99°05'46"W), Morelos, Mexico. Symptoms of the disease included small chlorotic spots on the adaxial surface of the infected leaves, which as the disease progressed turned into necrotic areas surrounded by a chlorotic halo. The chlorotic spots observed on the adaxial leaf surface coincided with numerous erumpent uredinia of bright orange color on the abaxial leaf surface. As a result of the infection, foliar necrosis and leaves abscission was observed. Of the 40 sampled trees, 95% showed symptoms of the disease. On microscopic examination of the fungus, bright orange, subepidermal uredinia were observed, which subsequently faded to white. Urediniospores were bright yellow-orange color. They were ellipsoid or globose, sometimes angular, echinulate, (21.5) 26.5 (33.0) × (16.0) 19.0 (23.0) µm in size. Morphological features of the fungus correspond with previous descriptions of Coleosporium plumeriae by Holcomb and Aime (2010) and Soares et al., (2019). A voucher specimen was deposited in the Herbarium of the Departmet of Plant-Insect Interactions at the Biotic Products Development Center of the National Polytechnic Institute under accession no. IPN 10.0113. Species identity was confirmed by amplifying the 5.8S subunit, the ITS 2 region, and part of the 28S region with rust-specific primer Rust2inv (Aime, 2006) and LR6 (Vilgalys and Hester 1990). The sequence was deposited in GenBank (OQ518406) and showed 100% sequence homology (1435/1477bp) with a reference sequence (MG907225) of C. plumeriae from Plumeria spp. (Aime et al. 2018). Pathogenicity was confirmed by spraying a urediniospores suspension of 2×104 spores ml-1 onto ten plants of P. rubra. Six plants were inoculated and sealed in plastic bags, while four noninoculated plants were applied with sterile distilled water. Plants were inoculated at 25°C and held for 48 h in a dew chamber, after this, the plants were transferred to greenhouse conditions (33/span>2°C). The experiment was performed twice. All inoculated plants developed rust symptoms after 14 days, whereas the non-inoculated plants remained symptomless. The recovered fungus was morphologically identical to that observed in the original diseased plants, thus fulfilling Koch's postulates. According to international databases (Crous 2004; Farr and Rossman 2023), C. plumeriae has not been officially reported in Mexico, despite being a prevalent disease. Diseased plants have been collected and deposited in herbaria, unfortunately, these reports lack important information such as geographic location of sampling, pathogenicity tests, or molecular evidence, which are essential for a comprehensive study of the disease in Mexico. To our knowledge, this is the molecular confirmation of Coleosporium plumeriae causing rust of Plumeria rubra in Mexico. Rust of P. rubra caused by C. plumeriae has been previously identified in India, Taiwan, Malaysia, and Indonesia by Baiswar et al. (2008), Chung et al. (2006), Holcomb and Aime (2010) and Soares et al., (2019). This disease causes important economic losses in nurseries, due to the defoliation of infected plants.

Genetic characterization and regional distribution of lymnaeid snails in northern Patagonia, Argentina.

Lymnaeid snails serve as intermediate hosts for Fasciola hepatica (Linnaeus, 1758), the etiological agent of fasciolosis, which is a widespread livestock disease in Argentina. Determining their geographic distribution and identifying the snail species involved in the transmission of fasciolosis can provide crucial information for designing strategic control programs. In this context, this work aimed at genetically characterizing the species of lymnaeid snails collected in different water bodies of northern Patagonia, Argentina. To this end, 689 snails were collected in 12 sites in the provinces of Neuquén, Río Negro and Chubut, in areas where fasciolosis is endemic. According to the morphological characteristics of their valves, they were identified as Galba spp. Twenty-three of these specimens were further identified using the nuclear sequences of the internal transcribed spacers ITS-1 and ITS-2 and 18S rRNA. The results confirmed the identity of all the analyzed snails as Galba viatrix and provided evidence that studying the variable region V2 of the 18S rRNA gene is not enough to differentiate closely related species, as observed in lymnaeid snails. Both the fact that G. viatrix was the only species identified in the endemic area surveyed and previous evidence of the high prevalence of F. hepatica infestation in grazing animals in the region suggest that this species is the main intermediate host of F. hepatica. The correct identification of lymnaeid snail species has great importance to determine risk zones and develop appropriate control measures to reduce transmission, according to the different ecological characteristics of each species.

DNA barcoding and new records of Ornithodoros yumatensis from Central Mexico.

Bats represent the second order of mammals with the highest number of species worldwide with over 1,616 species, and almost 10% of them are recorded in Mexico. These mammals have a great diversity of ectoparasites, in particular soft ticks of the genus Ornithodoros. Desmodus rotundus is one of the bat species that has scarcely been studied in terms of tick species richness in Mexico, with three tick species reported in five of the 32 Mexican states. For this reason, the aim of the present work was to identify ticks associated with D. rotundus from Central Mexico. Fieldwork was undertaken in the municipality El Marqués, Ejido Atongo A, Querétaro, Mexico. Bats were captured using mist nets and were visually inspected for tick presence. The ectoparasites were identified morphologically and molecularly with the use of mitochondrial markers 16SrDNA and cytochrome oxidase subunit I (COI). A total of 30 D. rotundus (1 female, 29 males) were captured, from which 20 larvae identified as Ornithodoros yumatensis were recovered. Molecular analysis confirmed the presence of this species with identity values of 99-100% with sequences of this species from the southwestern US, and the Yucatán Peninsula, Mexico. This is the first report of ticks associated with bats for the state of Querétaro, providing the first sequences of the COI gene from Mexican populations of O. yumatensis and shows an increase in the distribution of this soft tick across Central Mexico.

The DNA barcode reveals cryptic diversity and a new record for the genus Leporinus (Characiformes, Anostomidae) in the hydrographic basins of central northern Brazil.

Leporinus is one of the most speciose genera of the order Characiformes, with 81 valid species distributed throughout much of Central and South America. The considerable diversity of this genus has generated extensive debate on its classification and internal arrangement. In the present study, we investigated the species diversity of the genus Leporinus in central northern Brazil, and conclude that six valid species-Leporinus maculatus, Leporinus unitaeniatus, Leporinus affinis, Leporinus venerei, Leporinus cf. friderici, and Leporinus piau-are found in the hydrographic basins of the Brazilian states of Maranhão, Piauí, and Tocantins. We analyzed 182 sequences of the Cytochrome Oxidase subunit I gene, of which, 157 were obtained from Leporinus specimens collected from the basins of the Itapecuru, Mearim, Turiaçu, Pericumã, Periá, Preguiças, Parnaíba, and Tocantins rivers. The species delimitation analyses, based on the ABGD, ASAP, mPTP, bPTP, and GMYC methods, revealed the presence of four distinct molecular operational taxonomic units (MOTUs), identified as L. maculatus, L. unitaeniatus, L. affinis, and L. piau (from the Parnaíba River). The bPTP method restricted L. venerei to a single MOTU, and confirmed the occurrence of this species in the rivers of Maranhão for the first time. The separation of L. cf. friderici into two clades and the subsequent formation of different operational taxonomic units was consistent with polyphyly in this species, which indicates the existence of cryptic diversity. The arrangement of L. cf. friderici and L. piau in two different clades supports the conclusion that the L. piau specimens from Maranhão were misidentified, based on their morphological traits, reflecting the taxonomic inconsistencies that exist among morphologically similar species. Overall, then, the species delimitation methods employed in the present study indicated the presence of six MOTUs-L. maculatus, L. unitaenitus, L. affinis, L. cf. friderici, L. venerei, and L. piau. In the case of two other MOTUs identified in the present study, one (L. venerei) is a new record for the state of Maranhão, and we believe that the other represents a population of L. piau from the basin of the Parnaíba River.

Molecular identification and antimicrobial resistance pattern of Nocardia isolated from 14 diseased dogs and cats.

Nocardia are ubiquitous, saprophytic and opportunistic bacteria. They cause a set of pyogenic clinical infections in animals and humans, particularly immunocompromised patients, mostly affecting the skin and respiratory tract, with refractoriness to conventional therapy. The most descriptions of nocardial infections in companion animals involve case reports, and there are scarce case series studies focused on canine and feline nocardiosis in which diagnosis has been based on molecular techniques. We investigated epidemiological aspects, clinical findings, in vitro susceptibility profile, and molecular identification of Nocardia using PCR-based method targeted 16S rRNA gene in twelve dogs and two cats. Among dogs were observed cutaneous lesions (8/12 = 67%), pneumonia (3/12 = 25%), and encephalitis (2/12 = 17%), whereas cats developed cutaneous lesions and osteomyelitis. Nocardia and canine morbillivirus coinfection was described in six dogs (6/12 = 50%). A high mortality rate (6/8 = 75%) was seen among dogs. Three dogs (3/4 = 75%) and one cat (1/2 = 50%) with systemic signs (pneumonia, encephalitis, osteomyelitis), and 83% (5/6) of dogs with a history of concomitant morbillivirus infection died. N. nova (5/12 = 42%), N. cyriacigeorgica (3/12 = 25%), N. farcinica (2/12 = 17%), N. veterana (1/12 = 8%), and N. asteroides (1/12 = 8%) species were identified in dogs, whereas N. africana and N. veterana in cats. Among the isolates from dogs, cefuroxime (12/12 = 100%), amikacin (10/12 = 83%), gentamycin (10/12 = 83%), and imipenem (10/12 = 83%) were the most effective antimicrobials, whereas cefuroxime, cephalexin, amoxicillin/clavulanic acid, imipenem, and gentamycin were efficient against isolates from cats. Multidrug resistance was observed in 36% (5/14) of isolates. We describe a variety of Nocardia species infecting dogs and cats, multidrug-resistant ones, and a high mortality rate, highlighting a poor prognosis of nocardiosis in companion animals, particularly among animals systemically compromised or coinfected by canine morbillivirus. Our study contributes to species identification, in vitro antimicrobial susceptibility profile, clinical-epidemiological aspects, and outcome of natural Nocardia-acquired infections in dogs and cats.

Detection of Brasiliomyces malachrae Causing Powdery Mildew on Ornamental Cotton (Gossypium hirsutum) plants in Mexico.

Cotton (Gossypium L.; Malvaceae) is the most important fiber crop worldwide, also as a source of vegetable protein and edible oil. Cultivated species of cotton were apparently domesticated independently in four separate regions, in both the Old and the New World. Due to its economic importance, it is necessary to study the diseases that limit its production. During July of 2020-2022, symptoms of powdery mildew were observed on 80 ornamental cotton plants in a nursery located in Cuautla (18°52'38"N; 98°58'28"W), Morelos, Mexico. Disease incidence was 29%. Signs first appeared as small white colonies, which subsequently developed into abundant mycelial grown mainly on the upper leaf surface. White patches of mycelia were observed on leaves. In advanced stages of the disease, plants exhibited symptoms of yellowing, necrosis, and early defoliation. Microscopic analysis from 10 plant samples showed that mycelia were amphigenous, epiphyllous, in thin patches and evanescent. Hyphae were hyaline, thin walled and hyphal appressoria were simply lobed. Chasmothecia (n=50) were sub-aggregate, generally spherical to subglobose (46-61 µm in diameter), whitish, subhyaline, smooth, with a peridium of a single cell layer and appendages were absent. Three asci per chasmothecia, subspherical, 30-44 × 26-38 µm, with 4-6 ascospores per ascus. Ascospores were hyaline, ellipsoid to ovoid (16-23 × 10-18 µm). The asexual phase was not observed. The characteristics observed correspond to Brasiliomyces malachrae (Braun and Cook 2012; Cabrera et al. 2018). A voucher specimen was deposited in the Herbarium of the Department of Plant-Insect Interactions at the Biotic Products Development Center of the National Polytechnic Institute under accession no. IPN 10.0114. To confirm identification, DNA was recovered from the fungus and the internal transcribed spacer (ITS) from one sample was amplified by PCR, using the primers ITS1/ITS4 (White et al. 1990). The sequence was deposited in GenBank (OQ546720) and showed 100% sequence homology (647/1642bp) with the type sequence of B. malachrae (LC191217) from Malvastrum coromandelianum in Argentina (Cabrera et al. 2018). Pathogenicity was verified through inoculation by gently dusting conidia from infected leaves onto leaves of five healthy cotton plants. Five noninoculated plants served as controls. All plants were maintained in a greenhouse at temperatures from 28±2°C and relative humidity ranging from 80±5%. The experiment was performed twice. Inoculated plants developed powdery mildew symptoms after 14 days, whereas the control plants remained healthy. The fungus on the inoculated leaves was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Brasiliomyces malachrae causing powdery mildew on Gossypium hirsutum in Mexico and North America (Farr and Rossman 2023). Powdery mildew on G. hirsutum caused by B. malachrae has been previously identified in Venezuela by Hanlin and Tortolero (1984). This disease could be a primary source of inoculum of powdery mildew for commercial cotton plantations, derived from the free movement of ornamental plants.

In vitro evaluation of the antagonistic activity of native strains of Trichoderma spp. against Fusarium spp.

Five samples of agricultural soil and five samples of Aloe barbadensis (P. Mill., 1768) plants with symptoms of wilt and root necrosis were collected in five localities of the state of Tamaulipas, México. The aims of this study were the morphological identification, molecular identification and in vitro evaluation of the antagonistic activity of Trichoderma spp. on Fusarium spp. Four strains of Trichoderma asperellum, one strain of Trichoderma harzianum and five strains of Fusarium oxysporum were identified by morphological and molecular methods. The evaluation of the antagonistic activity of T. harzianum isolate (TP) showed the highest inhibition in Fusarium spp. (78.80%). The evaluation of the antagonistic activity of Trichoderma spp. extracts in Fusarium spp. did not show significant differences between treatments (P ≤ 0.05), with Trichoderma growth percentages that oscillated between 81.08 and 94.38%. The native isolate of T. harzianum (TP) showed significant competitive capability against the mycelial growth of F. oxysporum. Trichoderma species are promising agents of biological control in the central area of the State Tamaulipas, Mexico.

Molecular detection of tick-borne rickettsial pathogens in ticks collected from domestic animals from Cauca, Colombia.

Some hard ticks' species can act as vectors of a wide variety of pathogens of human and animal importance such as Anaplasma, Ehrlichia and Rickettsia spp. In Colombia, a total of forty-six tick species have been described, and some of them have been implicated as vectors of some infectious agents. The department of Cauca is one of the thirty-two departments of Colombia. Most of its population lives in rural areas and depends on agriculture as the main economic activity, favoring exposure to ticks and tick-borne pathogens. Thus, the present study aimed to determine the tick species and tick-borne pathogens circulating in this region. From August to November 2017, ticks were collected from dogs, horses and cattle from eight rural areas of four municipalities in the department of Cauca. All collected ticks were classified according to taxonomic keys and organized in pools. DNA was extracted from all tick pools for molecular confirmation of tick species and detection of Anaplasma, Ehrlichia and Rickettsia spp. A total of 2809 ticks were collected which were grouped in 602 pools. Ticks were morphologically identified as Amblyomma cajennense sensu lato, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato. The molecular identity of A. cajennense s.l. was confirmed as Amblyomma patinoi. A total of 95% of the pools scored positive for members of the Anaplasmataceae family, of which, 7.8% and 7.3% were positive to Anaplasma and Ehrlichia spp., respectively, being identified as Anaplasma marginale, Ehrlichia minasensis and Ehrlichia canis; and 16.1% were positive for Rickettsia spp. with high identity for Rickettsia asembonensis, Rickettsia felis and Candidatus Rickettsia senegalensis. This is the first report describing the natural infection of ticks with rickettsial pathogens and the occurrence of A. patinoi ticks in Cauca department, Colombia.