ITS Gene Research Articles

Identifying the hidden parasitic intruders: Molecular insights into the health of common Myna

Birds are known to act as the parasite reservoir and can transmit them to other organisms through food chains. This study aims to report the molecular prevalence and phylogenetic evaluation of various blood borne pathogens (Toxoplasma gondii, Isospora spp., Plasmodium spp., Haemoproteus spp., Leucocytozoan spp. and Neospora caninum) in blood samples of common Myna (Acridotheres tristis: N = 80) collected from four region (Jhang, Khanewal, Multan and Muzaffargarh) in Punjab Pakistan. Effect of pathogens on the complete blood count of the host was also determined. Results revealed by 2/80 Myna (2.5 %) amplified ITS-1 gene of Toxoplasma (T.) gondii (confirmed by DNA sequencing) while 2/80 (2.5 %) birds amplified 18S rDNA gene and Isospora spp. Phylogenetic analysis of both pathogens showed that Pakistani isolates were clustered together and were closely related to isolates that were reported from worldwide countries.Risk factor analysis revealed that prevalence of both pathogens was not restricted to a particular sampling site or a particular bird sex (P > 0.05). T. gondii infected birds had elevated red cell distribution width while Isospora sp. infected birds had elevated % monocytes and platelet distribution width while decreased mean cell hemoglobin, mean corpuscular hemoglobin concentration and platelets hematocrit than their respective uninfected birds. In conclusion, we are reporting the presence of T. gondii and Isospora sp. among Pakistani common Myna that had disturbed the complete blood count parameters that may have affected their normal physiology.

Influence of Varied Phosphorus Fertilizer Ratios on the Rhizosphere Soil Microbial Community in Idesia polycarpa Seedlings

Phosphorus (P) is crucial for tree growth and development, and it significantly influences the rhizosphere microbial community. However, the effects of phosphorus addition on the microbial communities in the rhizosphere soil of Idesia polycarpa remain understudied. In this study, two-year-old “Yuji” Idesia polycarpa seedlings were used to investigate the effects of phosphorus fertilization at four different levels of 0 g (control, CK), 0.92 g (low phosphorus, LP), 1.83 g (medium phosphorus, MP), and 2.75 g (high phosphorus, HP) per plant. The fertilizers were applied every 40 days over 120 days. MiSeq high-throughput sequencing of the 16S rRNA and ITS1 genes was employed to analyze the microbial community composition and diversity of bacteria and fungi in the rhizosphere soil under different phosphorus levels. The results showed that compared with CK treatment, the application of phosphorus fertilizer changed the physicochemical properties of the soil. The LP treatment significantly increased the soil pH, while the HP treatment group exhibited the highest soil-available phosphorus (AP) content. LP treatment significantly increased the number of microbial OTUs in the early and rapid growth stages and the richness and diversity of microbial communities. In addition, the bacterial community structure was significantly correlated with soil pH and AP, while the fungal community had no significant effect. The primary metabolic pathway function of bacteria in the rhizosphere soil of Idesia polycarpa seedlings is mainly metabolism, while fungi are mainly biosynthesis. Compared with CK treatment, 20 differential metabolic pathways were screened out in the bacterial community. Only two differential metabolic pathways were screened out in the fungal community by LP treatment at 120 days. In summary, applying low-level phosphate fertilizer is conducive to promoting the diversity of rhizosphere soil microorganisms. Therefore, potted planting of Idesia polycarpa seedlings is more suitable for applying low phosphorus levels.

Potential of Trichoderma strains to positively modulate plant growth processes and bulb yield in Rabi onion

The use of beneficial microbes is hitherto known and constantly increasing in agriculture due to their positive impact on crop growth and yield, and their minimal negative impact on the environment. The objective of this study was to evaluate the impact of eight Trichoderma strains of diverse origin on crop growth and yield of onion under field conditions. The identity of the strains used in the current study was confirmed by ITS and Tef1 gene sequencing. Field experiments were conducted in the Rabi season for 2 years (2020–21, and 2022–23) to evaluate the effect of the application of eight different Trichoderma strains that were applied individually and separately as eight different treatments (T1–T8) in experimental plots. In the plant growth promotion assay conducted in vitro, all strains showed the ability to produce IAA (indole-3-acetic acid), with levels ranging from 23.52 μg/mL (T6) to 45.54 μg/mL (T3). Our results revealed that Trichoderma treated experimental plots displayed better growth indices (plant height, pseudostem diameter), RWC (Relative water content), leaf chlorophyll content, and yield-attributing features like biomass (bulb and root dry mass), bulb diameter, and harvested bulb yield compared to the untreated control plants. In terms of yield, the T2 strain exhibited the highest bulb yield consistently for both the years (2020–21 and 2022–23) followed by T3 being statistically at par with T5. Among all the evaluated Trichoderma strains, the strain T2 (OGRDT2) and T3 (GRDT1), taxonomically identified as Trichoderma longibrachiatum, registered bulb yield of 32.24 t/ha and 30.76 t/ha, respectively while T5 (GRDT3), identified as Trichoderma asperellum, registered 30.55 t/ha average yield for 2 years compared to 24.08 t/ha average yield recorded for untreated control plants with an increase of 34, 28 and 27%, respectively. Based on our findings, it is concluded that the T. longibrachiatum strains OGRDT2 (T2) and GRDT1 (T3), T. asperellum strain GRDT3 (T5) are the best inducers of the onion crop growth and yield in the Rabi season and would be explored further for its commercial application in onion farming.

Diversity of Mycotoxin-Producing Fungi in Leafy and Fruit Vegetables Sold in Port Harcourt Metropolis, Nigeria

Mycotoxins are secondary metabolites produced by fungi and are capable of causing diseases and even death in both humans and other animals. This study investigated the diversity of mycotoxin-producing fungi from leafy (Telfairia occidentalis and Bassica oleracea) and fruit (Solanum lycopersicum and Cucumis sativus) vegetables sold in Port Harcourt metropolis, Nigeria using molecular techniques. The fungi were isolated from vegetable samples showing signs of disease using standard methods. DNA extraction and Polymerase chain reaction were carried out at the molecular laboratory of Regional Centre of Biotechnology and Bioresources research, University of Port Harcourt. The isolates were screened for the presence of mycotoxigenic genes (nor1 (aflatoxin), tri6 (trichothecene), otanps (Ochratoxin A), fum13 (fumonisin) and ZEA (zearalenone)) using PCR technique. The isolates containing mycotoxins were identified using ITS gene sequences. Ten out of the eighteen isolates were positive for four (nor1, tri6, otanps and fum13) out of the five mycotoxigenic genes screened. The nine isolates included fungi belong to six genera: Meyerozyma, Pithomyces, Fusarium, Trametes, Penicillium and Aspergillus. The ten isolates were classified as Meyerozyma carbbica RCBBR_Sf13, Pithomyces chartarum RCBBR_Sf5, Fusarium falciforme RCBBR_Sf10, Trametes duplexa RCBBR_Sf17, Trametes versicolor RCBBR_Sf2a, Trametes duplexa RCBBR_Mf1, Fusarium longifundum RCBBR_Mf4, Penicillium soosanum RCBBR_Mf7, Aspergillus aflatoxiformans RCBBR_Sf9, Fusarium circinatum RCBBR_Sf3 based on their ITS gene sequences. Their ITS gene sequences have been deposited in GenBank under the accession numbers OR816039-OR816047, with the exception of Fusarium circinatum RCBBR_Sf3 which could not accession. This study has demonstrated that mycotoxin-producing fungi are diverse and widespread in leafy and fruit vegetables sold within Port Harcourt Metropolis. This raises both public health and food security concerns.

MARKER REGIONS OF THE GENOME FOR MOLECULAR DIAGNOSTICS OF OPISTHORCHIIDAE FAMILY

Infectious diseases caused by representatives of the Opisthorhidae family rank 8th in terms of health concern in the world among all food parasites. According to FAO data, studies of representatives of the Opisthorhidae family rank first and range from 0.66 to 88.7%. When performing a PCR analysis, a molecular marker plays an important role. In our studies, ribosomal genome clusters and mitochondrial genes were used as molecular markers. In eukaryotic organisms, ribosomal genes are presented in the form of clusters located in groups on different chromosomes. Each cluster includes three ribosomal genes (18S, 5.8S and 28S), separated by spacer regions. The ribosomal gene cluster consists of a transcribed region, internal transcribed spacers (ITS - Internal Transcribed Spacer) and flanking external transcribed spacers (ETS - Eternal Transcribed Spacer) The ITS noncoding sequence differs greatly among closely related organisms and is therefore used to classify parasites at the genus, species, and subspecies level. The ribosomal nuclear gene 5.8S forms the complete transcribed internal spacer region together with the molecular markers ITS1 and ITS2. This region of the genome is used in species studies of 19 families of digenetic flukes. Mitochondrial genes are also one of the most commonly used mtDNA gene fragments in the molecular identification of the family Opisthorchiidae. The advantage of using COX in research is the high degree of mutation, which makes it possible to differentiate closely related species The use of these genome regions makes it possible to quickly and accurately identify pathogens of the family Opishorchiidae, even for such individuals with practically indistinguishable morphological stages of the life cycle, such as parasite eggs and capsules in fish muscles. Therefore, in our studies, primers were used for the nuclear regions of ribosomal DNA ITS1, ITS2 and mitochondrial genes COX1, COX3.

MARKER REGIONS OF THE GENOME FOR MOLECULAR DIAGNOSTICS OF OPISTHORCHIIDAE FAMILY

Infectious diseases caused by representatives of the Opisthorhidae family rank 8th in terms of health concern in the world among all food parasites. According to FAO data, studies of representatives of the Opisthorhidae family rank first and range from 0.66 to 88.7%. When performing a PCR analysis, a molecular marker plays an important role. In our studies, ribosomal genome clusters and mitochondrial genes were used as molecular markers. In eukaryotic organisms, ribosomal genes are presented in the form of clusters located in groups on different chromosomes. Each cluster includes three ribosomal genes (18S, 5.8S and 28S), separated by spacer regions. The ribosomal gene cluster consists of a transcribed region, internal transcribed spacers (ITS - Internal Transcribed Spacer) and flanking external transcribed spacers (ETS - Eternal Transcribed Spacer) The ITS noncoding sequence differs greatly among closely related organisms and is therefore used to classify parasites at the genus, species, and subspecies level. The ribosomal nuclear gene 5.8S forms the complete transcribed internal spacer region together with the molecular markers ITS1 and ITS2. This region of the genome is used in species studies of 19 families of digenetic flukes. Mitochondrial genes are also one of the most commonly used mtDNA gene fragments in the molecular identification of the family Opisthorchiidae. The advantage of using COX in research is the high degree of mutation, which makes it possible to differentiate closely related species The use of these genome regions makes it possible to quickly and accurately identify pathogens of the family Opishorchiidae, even for such individuals with practically indistinguishable morphological stages of the life cycle, such as parasite eggs and capsules in fish muscles. Therefore, in our studies, primers were used for the nuclear regions of ribosomal DNA ITS1, ITS2 and mitochondrial genes COX1, COX3.

Gastrointestinal nematodes in German outdoor-reared pigs based on faecal egg count and next-generation sequencing nemabiome data

BackgroundThere is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes.MethodsFLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity.ResultsOn 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08).ConclusionsInfections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections.

The impacts of ecological disturbances on the diversity of biosynthetic gene clusters in kauri (Agathis australis) soil

BackgroundThe ancient kauri (Agathis australis) dominated forests of Aotearoa New Zealand are under threat from a multitude of ecological disturbances such as forest fragmentation, biodiversity loss, climate change, and the spread of the virulent soil pathogen Phytophthora agathidicida. Taking a wider ecosystem-level approach, our research aimed to explore the impacts of forest disturbance and disease outbreaks on the biosynthetic potential and taxonomic diversity of the kauri soil microbiome. We explored the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in soils from a range of kauri forests that varied according to historical disturbance and dieback expression. To characterise the diversity of microbial BGCs, we targeted the non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene regions for sequencing using long-read PacBio® HiFi sequencing. Furthermore, the soil bacterial and fungal communities of each forest were characterized using 16 S rRNA and ITS gene region sequencing.ResultsWe identified a diverse array of naturally occurring microbial BGCs in the kauri forest soils, which may offer promising targets for the exploration of secondary metabolites with anti-microbial activity against P. agathidicida. We detected differences in the number and diversity of microbial BGCs according to forest disturbance history. Notably, soils associated with the most undisturbed kauri forest had a higher number and diversity of microbial NRPS-type BGCs, which may serve as a potential indicator of natural levels of microbiome resistance to pathogen invasion.ConclusionsBy linking patterns in microbial biosynthetic diversity to forest disturbance history, this research highlights the need for us to consider the influence of ecological disturbances in potentially predisposing forests to disease by impacting the wider health of forest soil ecosystems. Furthermore, by identifying the range of microbial BGCs present at a naturally high abundance in kauri soils, this research contributes to the future discovery of natural microbial compounds that may potentially enhance the disease resilience of kauri forests. The methodological approaches used in this study highlight the value of moving beyond a taxonomic lens when examining the response of microbial communities to ecosystem disturbance and the need to develop more functional measures of microbial community resilience to invasive plant pathogens.

Prevalence and species identification of trematode metacercariae in Qiqihar, Northeast China.

Fishborne trematode (FBT) is an important group of parasites that are endemic worldwide to a certain extent. However, despite the epidemiological significance, the species and phylogenetic evolution characteristics of FBT metacercariae have not been well studied. In this study, a total of 600 Pseudorasbora parva (P. parva) specimens were collected from Qiqihar, 61.8% (371/600) were found to be infected with trematode metacercariae. A total of three kinds of trematodes metacercariae were obtained, and they were identified as Clonorchis sinensis (C. sinensis), Metorchis orientalis (M. orientalis), and Metorchis taiwanensis (M. taiwanensis) by morphological and phylogenetic analysis with infection rates of 47.7% (286/600), 15.5% (93/600), and 23.7% (142/600), respectively. Meanwhile, a survey of the three trematodes metacercariae showed that the infection rate of C. sinensis metacercariae was the highest in September, up to 66% (66/100), and the lowest in June at 26% (26/100). The infection rate of M. orientalis metacercariae was the highest in October at 26% (26/100) and the lowest in June at 5% (5/100). The infection rate of M. taiwanensis metacercariae was at its peak in November at 36% (36/100) and the lowest in July at 15% (15/100). The co-infection of metacercariae of C. sinensis and M. taiwanensis was the most common and reached a peak in October, and their infection rate was higher in autumn than in summer. The peak of infection intensity of metacercariae for C. sinensis, M. orientalis, and M. taiwanensis were different: C. sinensis was 24/g in September, M. orientalis was 7/g in October, and M. taiwanensis was 10/g in November. From the above results, it was confirmed that three species of trematodes metacercaria played an important role in infection of second intermediate hosts in Qiqihar region. Studying the morphological characteristics and sequencing the ITS2 gene for a phylogenetic tree of them will be useful for future molecular evolution, biology, and ecology of trematode metacercariae.

Fusarium oxysporum causes the root rot disease of Polygonatum cyrtonema in China

Polygonatum cyrtonema, also known as Huangjing, belongs to the Liliaceae family and is a commonly used medicinal and edible plant in Asia. In recent years, P. cyrtonema root rot disease has frequently occurred in China, significantly impacting its yield and medicinal value. However, the pathogen of the disease has not yet been identified. In this study, we aimed to identify the pathogen causing the root rot disease of P. cyrtonema by applying morphological and molecular methods following Koch's postulates. Twelve fungal strains were isolated from nine diseased rhizomes of P. cyrtonema. Morphological analysis showed that the twelve isolates included eight colony types. Among that strain, HJ-5 has the typical characteristics of Fusarium oxysporum, including single-cell microconidia, chlamydospores, and sickle macroconidia. BLAST alignment and phylogenic tree based on the tef1, rpb2, β-tubulin, and ITS genes demonstrated that strain HJ-5 exhibits the highest similarity with Fusarium oxysporum. The strain HJ-5 is identified as F. oxysporum based on morphological and molecular characteristics. In addition, the pathogenicity test showed that among twelve isolates, only strain HJ-5 led to the rhizome softening and rotting on detached rhizomes and living plants of P. cyrtonema, consistent with symptoms observed in the field. To our knowledge, this is the first report on root rot disease in P. cyrtonema caused by F. oxysporum in China, which will provide a clear prevention and management target for the root rot disease of P. cyrtonema.

Molecular Characterization of Native Isolates of Metarhizium rileyi (Farlow) Samson (Ascomycetes: Hypocreales) from Major Cropping Ecosystems of Northeastern Karnataka, India

Aims: In India, commercial biopesticides have been recommended against different insect pests on different crops, but their application has shown only a reduced efficiency attributed to differences in susceptibility of target insect pests or non-adaptability to Indian agro-climatic conditions. Therefore, there is a need to isolate region-specific biopesticide strains to enhance the efficacy of such biopesticides for pest management. The current study aimed to explore the new isolates of the entomopathogenic fungus, Metarhizium rileyi (Farlow) Samson species across the major cropping ecosystems of kalyana Karnataka. M. rileyi is an important entomopathogenic fungus of more than 30 species of Lepidoptera larvae. The aim of this research was to characterize the isolate of M. rileyi from Northeastern Karnataka by using morphological and molecular approaches. Methodology: The fungus was isolated from the naturally infected cadavers collected from different cropping ecosystems and it was cultured on Sabouraud’s Maltose Yeast Extract Agar (SMAY) media for morphological characterization, SMY media used for molecular characterization. The vegetative hyphae of fungi were 2-3 µm in diameter and hyaline, septate. The conidia ellipsoidal, smooth, and pale green were observed under the optical microscope. The DNA was isolated using the CTAB method and an internal transcribed spacer (ITS1, ITS4) was used as the primers for the amplification. The amplified products of 650 bp were purified and sequenced at ClustaL W in both directions using the above primers. A consensus sequence was obtained by alignment of the forward and reverse sequences for this region and deposited in GenBank (UASRBC Mr-24, UASRBC Mr-3, UASRBC Mr-20, UASRBC Mr-29, UASRBC Mr-7, UASRBC Mr-18). Result: A BLAST search of the sequence in GenBank revealed a 50-100 percent identity with several strains of M. rileyi (Eg. JQ686668, MN907775 and KJ725726 etc.,). Conclusion: The phylogenetic relationship was derived from comparisons of the ITS gene sequences and a Dendrogram was constructed. The homology and variations based on amino acid sequences among the isolates showed 71 per cent (OK184897) and 51 per cent similarity with four isolates (OK184899, OK177835, OK178862 and OK184898).

Assessment of High-Resolution Melting Curve Analysis for Leishmania spp. Detection in Different Clinical Manifestations of Leishmaniasis in India.

The accurate diagnosis and identification of Leishmania species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for Leishmania species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using L. donovani, L. major, and L. tropica standard strain DNA. The assay was applied on 93 clinical samples with confirmed Leishmania infection, including VL (n = 30), PKDL (n = 50), and CL (n = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for L. major and up to 0.1 pg for L. donovani and L. tropica. The detection limit for the 7SL-RNA HRM-PCR was 1 pg for L. major and 10 pg for L. donovani and L. tropica. The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL (p = 0.007), PKDL (p = 0.0002), and CL (p = 0.03) samples. The ITS-1 HRM-PCR assay could identify Leishmania spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions.

Trichoderma harzianum TIND02 upregulates the expression of pathogenesis-related genes and enzymes and enhances gray blight resistance in tea

The gray blight incited by Pestalotiopsis and allied genera is a prevalent disease affecting tea cultivation, and managing it with Trichoderma spp. is an alternative to synthetic fungicides. Plants modify their arsenal system against pathogens when they are exposed to Trichoderma spp., which produces proteins and enzymes associated with pathogenesis. Understanding the expression pattern of defense-related markers will help in developing gray blight resistance tea cultivars. Thus, this study intended to induce resistance against gray blight in tea by Trichoderma harzianum TIND02. For this, a total of eight Trichoderma isolates originated from organic tea rhizospheres were characterized and evaluated for their efficacy. Dual culture test revealed isolate TIND02 as the most potential candidate with 74.6% inhibitory activity against gray blight pathogen Pseudopestalotiopsis theae. Molecular characterization based on ITS and tef-1 alpha genes confirmed isolate TIND02 as T. harzianum. Scanning electron microscopic study showed the mycoparasitic nature of T. harzianum TIND02 (TH-TIND02) to Ps. theae. The ethyl acetate extract of TH-TIND02 at 100 and 200 μg mL−1 showed potential inhibitory activity (>69.9%) against Ps. theae which confirmed the presence of higher volatile metabolites. Gas chromatography–Mass spectrometry study revealed that ethyl acetate extract of TH-TIND02 was composed of 21 major and minor volatile organic compounds with acetamide, 2, 2, 2-trifluoro-N, N-bis trimethyIsilyl–C (94.74%) as a major component. The isolate also produced chitinase, cellulase, β-1, 3 glucanase, and protease hydrolytic enzymes. Nursery experiments revealed that 2% and 5% doses (2 × 106 CFU mL−1) of TH-TIND02 significantly reduced respective 65.0% and 70.0% disease severity over control with improved plant growth. Besides, expressions of defense-related enzymes (chitinase, pHenolics, peroxidase, phenylalanine ammonia lyase, β-1, 3-glucanase, and polyphenol oxidase) and pathogenesis-related genes (chitinase and β-1, 3-glucanase) due to TH-TIND02 were determined. The secretion of defense-related enzymes was highly upregulated in plants applied with TH-TIND02 followed by Ps. theae inoculation compared to controls. The RT-qPCR analysis showed that the expression of both genes in co-inoculated plants was two-fold higher than in control after 21-day post incubation. These results suggest that TH-TIND02 application reduced gray blight severity by elevated enzyme activity and overexpressed pathogenesis-related genes in tea plants which offer for its eco-friendly and sustainable use as a bio-fungicide in tea gardens.

Multifaceted and dual-edged native Trichoderma strains from subabul rhizospheric soil to combat Fusarium wilt disease – a sustainable approach

ABSTRACT Fusarium wilt and gummosis disease is a major concern in Subabul plantations, resulting in quantitative and qualitative wood losses to the paper and pulp industry. Growers currently rely on agrochemicals, which are not environmentally safe. Therefore, developing a sustainable control strategy employing biocontrol agents is necessary. For this, the investigation was performed to identify native Trichoderma strains from Subabul rhizospheric soil having antagonistic, disease control efficiency, and PGPR attributes. Six antagonistic Trichoderma (TR1 to TR6) strains were isolated and their efficacy was tested against Fusarium in-vitro by dual plate technique and volatile metabolites. Among these, Trichoderma strains TR1 and TR2 were found efficient in counteracting the growth of F. equiseti (70–90% inhibition) compared to control. The effectiveness was primarily attributed to the increased production of volatile metabolites, siderophores, and the ability to solubilise the nutrients (P and K). Further, six potent Trichoderma strains were identified as T. asperellum (TR1, TR2, TR3, TR6), and T. hamatum (TR4, TR5) through rDNA ITS gene sequencing and phylogenetic analysis. These isolates were mass-multiplied, formulated using Talc powder followed by bio-priming in rooting media. The application of Trichoderma into rooting media reduced the wilt disease caused by artificial inoculation of F. equiseti. Further, TR1 and TR2 were found promising with conferred protection against wilt, enhanced survival rate (10–15%), and improved growth compared to untreated control. Application of adapted TR1 and TR2 antagonists in rooting media during clonal multiplication has the potential wilt control efficiency, enhanced survival rate, and plant growth promotion in Subabul.

First Sero-Molecular Diagnosis of Toxoplasma gondii and Toxocara spp. Infections in the Police Dogs and Their Trainers in Iran.

Toxoplasma gondii (T. gondii) and Toxocara spp. are two types of parasites that can infect humans and various animals, including dogs. Police dogs and their trainers have a vital role in law enforcement, and their health and well-being are crucial for them to effectively carry out their duties. No study has yet been conducted on the prevalence of T. gondii and Toxocara spp. infections among police dogs and their trainers in Iran. The objective of this study was to determine the sero-molecular prevalence of T. gondii and Toxocara spp. infections in police dogs and their trainers in Tehran, the capital of Iran. In Tehran province, the anti-narcotics police have nearly 200 well-trained police dogs. Each dog is assigned a dedicated trainer and upon completing missions, is housed separately in a designated area. In the present study, a total of 150 samples were gathered. These included 50 blood samples from randomly selected police dogs, 50 fecal samples from the same dogs, and 50 blood samples from their trainers. The Modified Agglutination Test (MAT) was performed to detect T. gondii antibodies in dog blood samples and the ELISA system was utilized to identify anti-Toxoplasma and anti-Toxocara antibodies in the sera of the dog trainers. A specific segment of the SAG2 and ITS genes were amplified via nested-PCR in order to molecularly detect T. gondii in human blood samples and Toxocara spp. in dog fecal samples. Regarding serological findings, the prevalence of T. gondii in dog and human blood samples was 4% (2/50) and 10% (5/50), respectively. According to reports, the seroprevalence of Toxocara spp. in human blood samples was 6% (3/50). No statistically significant association was found between the prevalence of the examined parasites and variables (age, sex, and breed) in dogs, as well as the age variable in military personnel. Molecular findings showed that out of the 50 dog fecal samples and 50 human blood samples, there was no presence of Toxocara spp. and T. gondii, respectively. Understanding the prevalence of parasitic infections helps public health officials assess the risk to human and animal populations. This information can guide the development of prevention and control measures to reduce the spread of these infections. Overall, the prevalence of parasitic infections, particularly T. gondii and Toxocara spp., in police dogs and their trainers remains uncertain and necessitates further in-depth research.

First Molecular and Phylogenetic Identification of Toxoplasma Gondii in Sheep Liver Intended for Human Consumption in Northern Tunisia.

The aim of this study was to estimate the molecular infection prevalence of Toxoplasma gondii in sheep liver tissues destined for human consumption. A total number of 224 liver tissues were collected from slaughtered sheep in Sejnane slaughterhouse (Northwest Tunisia). PCR was used to detect T. gondii DNA in liver tissues followed by phylogenetic analysis of amplicons. The phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences.The overall molecular prevalence of T. gondii in sheep livers was 25% (56/224). The highest molecular prevalence of T. gondii was recorded in sheep aged of less than one year old (27.3%; 52/190). Infection prevalence was significantly higher in Noire de Thibar breed (33%; 17/51) compared to other breeds (p = 0.023). There were no differences depicted according to sheep's gender. The T. gondii sequences obtained in the present study (GenBank accession numbers: OR509829 and OR509830) were 98.40-100% homologous to T. gondii sequences published in the GenBank. These results highlight a high level of T. gondii contamination of tissues destined for human consumption. Further studies are needed to improve our knowledge on different genotypes of T. gondii that infect Tunisian sheep population.

Assessing the Potential of Tortistilus (Hemiptera: Membracidae) from Northern California Vineyards as Vector Candidates of Grapevine Red Blotch Virus.

Ceresini treehoppers are present in northern California vineyard ecosystems, including the closely related Spissistilus and Tortistilus (Hemiptera: Membracidae). These membracids are not direct pests of wine grapes, but S. festinus is a vector of grapevine red blotch virus (GRBV). No information is available on the ability of Tortistilus spp. to transmit GRBV. In this study, Tortistilus were collected on yellow panel cards across 102 vineyard sites and surrounding areas in Napa Valley, California, USA in 2021-2023. Specimens were morphotyped, sexed and tested for GRBV ingestion and acquisition by multiplex PCR or qPCR. Phylogenetic analysis of the partial sequence of mt-COI and ITS gene fragments of a subset of 40 Tortistilus specimens revealed clustering in a monophyletic clade with T. wickhami with the former barcode sequence. Only 6% (48/758) of the T. wickhami tested positive for GRBV, but none of the heads with salivary glands (0%, 0/50) of the dissected specimens tested positive for GRBV, indicating no virus acquisition. In contrast, half of the dissected heads with salivary glands of S. festinus (52%, 12/23), from the same collection vineyard sites, tested positive for GRBV. Together, our findings confirmed the presence of T. wickhami in northern California vineyards and suggested a dubious role of this treehopper as a vector of GRBV.

Biochar combined with humic acid improves the soil environment and regulate microbial communities in apple replant soil

Apple replant disease (ARD) negatively affects plant growth and reduces yields in replanted orchards. In this study, biochar and humic acid were applied to apple replant soil. We aimed to investigate whether biochar and humic acid could promote plant growth and alleviate apple replant disease by reducing the growth of harmful soil microorganisms, changing soil microbial community structure, and improving the soil environment. This experiment included five treatments: apple replant soil (CK), apple replant soil with methyl bromide fumigation (FM), replant soil with biochar addition (2 %), replant soil with humic acid addition (1.5 ‰), and replant soil with biochar combined with humic acid. Seedling biomass, the activity of antioxidant enzymes in the leaves and roots, and soil environmental variables were measured. Microbial community composition and structure were analyzed using ITS gene sequencing. Biochar and humic acid significantly reduced the abundance of Fusarium and promoted the recovery of replant soil microbial communities. Biochar and humic acid also increased the soil enzymes activity (urease, invertase, neutral phosphatase, and catalase), the plant height, fresh weight, dry weight, the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), and root indexes of apple seedlings increased in replant soil. In sum, We can use biochar combined with humic acid to alleviate apple replant disease.

Human-water interactions associated to cercarial emergence pattern and their influences on urinary schistosomiasis transmission in two endemic areas in Mali

BackgroundMali is known to be a schistosomiasis-endemic country with a limited supply of clean water. This has forced many communities to rely on open freshwater bodies for many human-water contact (HWC) activities. However, the relationship between contact with these water systems and the level of schistosome infection is currently receiving limited attention. This study assessed human-water interactions including cercarial emergence pattern and their influences on urinary schistosomiasis transmission in two communities in the Kayes district of Mali.MethodsWe carried out a parasitological study first in children in September 2021, then a cross-sectional study of quantitative observations of human-water contact activities in the population, and finally a study of snail infectivity at contact points in September 2022. The study took place in two communities, Fangouné Bamanan and Diakalèl in the Kayes region of western Mali. The chronobiological study focused on cercarial release from naturally infected snails. Released cercariae were molecularly genotyped by targeting the cox1 region, and the ITS and 18S ribosmal DNA gene (18S rDNA) regions of the DNA. Links between sociodemographic parameters, human water-contact points and hematuria were established using multivariate statistical analysis or the logistic regression model.ResultsThe main factor predisposing the 97 participants to water contact was domestic activity (62.9%). Of the 378 snails collected at 14 sampling sites, 27 (7.1%) excreted schistosome cercariae, with 15.0% (19/126) at Fangouné Bamanan and 3.3% (8/252) at Diakalel. The release of Schistosoma cercariae shows three different patterns in Fangouné Bamanan: (i) an early release peak (6:00–8:00 AM), (ii) a mid-day release peak (10:00 AM–12:00 PM) and (iii) a double peak: (6:00–8:00 AM) and (6:00–8:00 PM) cercariae release; and two release patterns in Diakalel: early release (6:00–8:00 AM) and (ii) mid-day release (12:00–2:00 PM). All cercariae released during early diurnal (6:00–8:00 AM) or nocturnal emission patterns (6:00–8:00 PM) were hybrids parasite having an cox1 S. bovis or S. curassoni associated with an ITS and 18S rDNA of S. haematobium while the cercariae released during diurnal, or mid-day patterns (8:00 AM–6:00 PM) were pure S. haematobium.ConclusionsOur study showed that domestic activity is the main source of exposure in the Kayes region. Two and three cercariae emission patterns were observed at Diakalel and Fangouné Bamanan respectively. These results suggest that the parasite adapts to the human-water contact period in order to increase its infectivity.Graphical

Molecular diagnosis and phylogenetic insights of Eimeria species infecting buffaloes (Bubalus bubalis) in Meghalaya's subtropical hilly region, India

Coccidiosis is an intestinal infection caused by Eimeria spp. that results in economic losses owing to morbidity and mortality in young buffalo calves. This study aimed for molecular diagnosis and phylogenetic analysis of Eimeria spp. in buffaloes of Meghalaya's sub-tropical mountainous terrain. Fresh buffaloes' fecal samples were collected from buffalo farms of Umling, Umsning and Bhoirymbong blocks, Ri Bhoi, Meghalaya and screened for Eimeria oocysts using flotation and modified McMaster methods. Fecal sample examination revealed 27.44 % (87/317) infection in buffaloes. Age wise, 64.44 % (29/45), 25.35 % (36/142) and 16.92 % (22/130) infections were recorded in <6 months, 6 months to 1 year and 1–2 year old buffaloes, respectively. Morphological characterization of Eimeria spp. revealed E. bovis (21.83 %), E. bareillyi (18.39 %), E. zuernii (11.49 %), E. ellipsoidalis (3.44 %) and mixed infection (44.82 %). Amplification of ITS-1 gene confirmed Eimeria spp. (410 bp), E. bovis (238 bp) and E. zuernii (344 bp). Phylogenetic analysis of E. bovis Umiam isolate revealed that these were closely related to the E. bovis isolate from South Korea (MH245198.1), and Turkey (KU351711.1) and distantly related to the isolates from Jammu and Kashmir (OQ103422.1) and Uttar Pradesh, Mathura (OK486542.1). E. zuernii isolate from Umiam, Meghalaya was observed to be phylogenetically close to the isolates from South Korea (MH245202.1), Japan (LC171339.1) and Turkey (KU351715.1), whereas phylogenetic divergence was observed between, E. zuernii isolate from Umiam, Meghalaya with isolates of Andhra Pradesh, Tirupati (MN601278.1) and Jammu and Kashmir (OQ103424.1). Therefore, treatment and effective control strategies should be implemented immediately to prevent spread of infection in the buffaloes.