Species-specific Primers Research Articles

PCR-based detection of Botryosphaeria canker pathogens in fig trees

Canker and dieback, caused by members of the Botryosphaeriaceae family, pose significant threats to plant productivity, food security, and natural ecosystems, particularly in economically important woody crops including fig trees. Detecting and identifying these pathogens is challenging due to their latent infections and the presence of multiple pathogens within the same host. In our study, we developed a PCR assay using three carefully selected primer pairs based on sequence differences in two protein-coding genes, β-tubulin and RNA polymerase II. The species-specific primers TUB-Bd1 (specific for Botryosphaeria dothidea), TUB-Np1 (specific for Neofusicoccum parvum), and RPB-Nd2 (specific for Neoscytalidium dimidiatum) effectively amplified target gene sequences in pure cultures and infected tissue samples using PCR and nested PCR conditions. The efficiency test results demonstrated that TUB-Bd1, TUB-Np1, and RPB-Nd2 primer pairs could detect specific DNA fragments at very low concentrations in nested PCR. Furthermore, we collected 180 symptomatic and asymptomatic fig samples from different regions of Fars Province, Iran. Applying the species-specific primer pair RPB-Nd2 in direct PCR led to the detection of N. dimidiatum in 25.5% of symptomatic woody samples and 13% of asymptomatic one-year-old branch samples of fig trees. This represents a significant improvement compared to the mere 6.67% detected using traditional culturing methods on PDA. Interestingly, neither B. dothidea nor N. parvum were detected in the 180 samples using nested PCR. Moreover, multiplex PCR enabled simultaneous DNA detection of the target Botryosphaeriaceae pathogens. Our findings emphasize the importance of molecular techniques for early detection of evident and latent infections caused by these three pathogens in fig trees.

P-252. Culture-based Viability PCR: Strategies to Harness its Sensitivity and Minimize False Positives

Abstract Background qPCR is a highly sensitive method for identifying the presence of potential pathogens. However, its utility as a tool to detect environmental contamination is limited by its inability to differentiate between viable and non-viable target cells.Table 1.Detection of target organisms via qPCR and Culture Methods We completed a prospective microbiological analysis of patient bed footboard samples at a tertiary care center using foam sponges and the stomacher method to process samples. Target species included E. coli (EC), S. aureus (SA), and C. difficile (CD). Sponge homogenates were split into three paths: 1) T0: 500uL was added to 4.5ml of species-specific (SS) broth; 500uL of the resulting mixture underwent DNA extraction and qPCR with SS primers, 2) T1: 500uL was added to 4.5mL of SS broth, and 3) Growth negative control (GNC): 500uL was added to 4.5mL of 8.25% sodium hypochlorite, incubated for 10 minutes, centrifuged for 15 minutes at 3100 RPM, then decanted and added to 5mL of SS broth after 2 PBS washes. T1 and GNC samples were then incubated at SS conditions (24 hours at 37C for EC and SA, and 48 hours for CD). After incubation, 500uL from T1 and GNC samples underwent DNA extraction and qPCR. All samples were also cultured on SS agar. A sample was considered viable for each species if 1) it was detected at T0, and the CT decreased by at least 1.0 at T1 compared to GNC or 2) it was undetected at T0, detected at T1, and undetected for GNC, or 3) grew on standard culture agar. Table 2: Viability of target organisms via qPCR and Culture Results 468 samples from 26 patient rooms were analyzed, including 156 for each species. Of the 26 original samples, 24 (92%), 11 (42%), and 2 (8%), had detectable levels of EC, SA, and CD via qPCR at T0 or T1, respectively, and could be assessed for viability. Of those, 3 (13%), 8 (73%), and 0 (0%) contained viable cells of EC, SA and, CD via qPCR, respectively. Notably, 5 (19%) of SA samples were culturable at T1, indicating broth enrichment enhances culture sensitivity; however, all were determined viable via qPCR as well. Conclusion Culture-based viability PCR outperformed traditional culture methods in detecting viable pathogens with improved specificity compared to qPCR, highlighting its potential as a tool for assessing environmental contamination. Further large-scale studies are needed to confirm these results across different species. Disclosures All Authors: No reported disclosures

Survey and first report of the soybean cyst nematode (Heterodera glycines Ichinohe) in soybean fields in the province of Manitoba, Canada

The Soybean Cyst Nematode (SCN), Heterodera glycines (Ichinohe 1952), is a major soybean (Glycine max (L.) Merr.) pest worldwide. In Canada, SCN is present in Ontario and Quebec. Past surveys conducted in Manitoba failed to report the pest. Nevertheless, the nematode is expected to be present in Manitoba as it has spread rapidly northwards in North Dakota and Minnesota. Thus, the objective of this study was to survey soybean fields in Manitoba for the presence of SCN. In October 2017, soil samples were collected from 30 commercial soybean fields in Manitoba. A modified Fenwick elutriator was used to recover cysts. Cysts were identified based on morphological characters, PCR with species-specific primers for H. glycines (COXIII and SCAR), and DNA sequencing of four regions of the genome. Four fields were positive for SCN and had 2.9, 0.95, 3.9, and 1.81 eggs per 100 g soil. In 2021, an additional soybean field showing stunting and yellowing symptoms and swollen nematode females on roots was sampled. The SCN population density of this symptomatic headland area centre was 7,797 eggs per 100 cm3 soil. The SCN population from this field was of HG type 7 (race 3). Three positive fields were located along the Red River border crossing in the rural municipalities of Rhineland, Emerson-Franklin, and Montcalm. One field was in the rural municipality of Norfolk-Treherne, and the symptomatic field was in Thompson municipality, away from the Red River border. This study reports the first finding of H. glycines and SCN disease in Manitoba.

Molecular diagnostics of the hop cyst nematode, Heterodera humuli Filipjev, 1934, using real-time PCR

Summary The hop cyst nematode, Heterodera humuli, is a pest of hop with the potential to substantially limit yields worldwide. To enable molecular-based diagnostics, a real-time PCR assay for the identification of H. humuli was designed. Polymorphism in the COIII gene fragments was used to design species-specific primers and a TaqMan probe. The specificity of the primer and probe set was tested against different populations of H. humuli and non-target cyst nematodes in multiplex reactions. In multiplex real-time PCR experiments with specific and universal primer and probe sets, fluorescent signals were simultaneously monitored for COIII and D3 of 28S rRNA target genes. Assays with species-specific primers and probe were able to detect DNA extracted from 0.0016 of a H. humuli second-stage juvenile per reaction while showing no reaction with non-target species.

Exploring Competitive Relationship Between Haemophilus parainfluenzae and Mitis Streptococci via Co-Culture-Based Molecular Diagnosis and Metabolomic Assay

Various bacterial strains with nitrate-reducing capacity (NRC), such as Haemophilus, Actinomyces, and Neisseria, are known to promote NH3 production, control pH in the oral cavity, and inhibit the growth of aciduric bacteria. However, experimental evidence on various estimated bacterial networks within the salivary microbiome is insufficient. This study aims to explore potential bacterial compositional competition observed within saliva samples from dental caries patients through a co-culture assay of mitis Streptococci, which is a primary colonizer in the salivary microbiome, and nitrate-reducing bacteria Haemophilus parainfluenzae. We investigated bacterial growth efficiency change by co-culture time using the qRT-PCR method. In addition, we applied LC/Q-TOF-based metabolites screening to confirm metabolic interactions between oral bacterial species and their association with dental caries from a metabolomics perspective. As a result, we first found that the nitrate reduction ability of H. parainfluenzae is maintained even in a co-culture environment with the mitis Streptococci group through a nitrate reduction test. However, nitrate reduction efficiency was hindered when compared with monoculture-based nitrate reduction test results. Next, we designed species-specific primers, and we confirmed by qRT-PCR that there is an obvious competitive relationship in growth efficiency between H. parainfluenzae and two mitis Streptococci (S. australis and S. sanguinis). Furthermore, although direct effects of nitrate reduction on competition have not been identified, we have potentially confirmed through LC/Q-TOF-based metabolite screening analysis that the interaction of various metabolic compounds synthesized from mitis Streptococci is driving inter-strain competition. In particular, we constructed a basic reference core-metabolites list to understand the metabolic network between each target bacterial species (H. parainfluenzae and mitis Streptococci) within the salivary microbiome, which still lacks accumulated research data. Ultimately, we suggest that our data have potential value to be referenced in further metagenomics and metabolomics-based studies related to oral health care.

PCR-based detection technique and gamma irradiation strategies for managing Ralstonia solanacearum-induced brown rot of potato

Purpose The study focused on developing a rapid PCR-based detection method and employing gamma irradiation techniques to manage Ralstonia solanacearum, aiming to produce brown rot-free export-quality potatoes. This initiative seeks to enhance potato exports from Bangladesh. Materials and Methods Samples of potato tubers and soil were collected from various commercially significant potato-growing areas, resulting in a total of 168 Ralstonia solanacearum isolates from potato tubers and soil across 12 regions. The detection of R. solanacearum in the enriched tuber extract and soil were conducted using the primer pairs (PS-1, PS-2) and (759, 760). For the gamma irradiation experiment, petri dishes containing R. solanacearum cultures were subjected to different doses of gamma rays at the Bangladesh Institute of Nuclear Agriculture using a 60Co source. The irradiation doses applied to the samples were 0–6.0KGy. Results Morphological identification based on pink/light red colonies on TTC medium was confirmed R. solanacearum in 148 isolates. PCR using species-specific primers (PS-1/PS-2) and (759, 760) verified 26 isolates (14 tubers, 12 soil), producing 553 bp and 281 bp fragments in latently infected tubers and soil samples respectively. Gamma irradiation at 2.5 kGy damaged R. solanacearum’s DNA and cells, preventing brown rot, while higher doses eliminated it entirely. This offers a promising strategy to enhance safety of stored potatoes, potentially mitigating economic losses from this quarantine pathogen. Conclusion The study developed a PCR detection method and gamma irradiation techniques to manage R. solanacearum, enhancing the export quality of potatoes.

Challenges in cellular agriculture: lessons from Pacific white shrimp, Litopenaeus vannamei.

The overall goal of this research was to develop an embryonic stem cell (ESC) line from the Pacific white shrimp, Litopenaeus vannamei, to support production of cell-based cultivated seafood products towards meeting a growing global demand for sustainable seafood. It was hypothesized that characteristics of ESCs, such as high proliferation and pluripotency, would facilitate development of a continuous cell line that could be triggered to differentiate into a muscle cell phenotype. The targeted approach was based on collection of ESCs from fertilized shrimp eggs at the blastomere stage. Various media, supplements, growth factors, and plate coatings were tested to achieve growth of the shrimp ESCs. Although successful in early culture, this manuscript describes substantial challenges encountered as cultures grew over time. The cell cultures were initially dominated by shrimp as indicated by 18S rDNA community analysis, but after multiple passages, thraustochytrids, a common contaminant of invertebrate cell culture, became the predominant cell type. Presence of shrimp cells was confirmed through species-specific primers for the cytochrome C oxidase subunit 1 gene. Presence of thraustochytrids was also confirmed using species-specific primers, morphological features, growth properties, and acriflavine staining. Unsuccessful attempts to eradicate thraustochytrid contamination prevented shrimp cells from thriving. The future of shrimp cell culture depends on eliminating culture contaminants while encouraging growth of shrimp ESCs.

Isolation and Molecular Detection of Antimicrobial Resistance Genes in Escherichia coli and Staphylococcus Species from Joint Ill Cases of Calves in Puducherry

Background: Joint ill is a septicemic polyarthritis condition due to the localization of pathogenic bacteria within the joints of young calves. The emergence of antibiotic resistant pathogen in such cases may lead to further complication in treatment. Thus, the present study was aimed to isolate and identify the bacterial pathogens and its antimicrobial resistant genes from joint ill cases in calves. Methods: A total of 31 pus swabs were collected from joint ill cases from calves and subjected for bacterial isolation and identification by phenotypic and genotypic method followed by determination of its antimicrobial resistant genes (tet and mecA gene) and antibiogram. Result: Based on colony characters, microscopic observation, biochemical tests, 17/31 (54.83%) Escherichia coli and 14/31 (45.16%) Staphylococcus aureus were isolated and identified phenotypically. All the isolates were further confirmed by polymerase chain reaction using E. coli and S. aureus species specific primers. The antimicrobial resistant genes carrying E. coli and S. aureus isolates were also detected, in which 4/17 (23.5%) isolates were positive for tet gene and 4/14 (28.57%) were positive for the mecA gene. The antibiotic susceptibility test showed that the isolates were highly sensitive to Enrofloxacin (100%) and Gentamicin (77%), but were resistant to Amoxyclav (70%) and tetracycline (50%). On conclusion, E. coli and S. aureus are the most common bacterial pathogens identified from this study. The presence of antimicrobial resistant genes (tet and mecA) and antibiogram pattern of the bacterial isolates indicating the possible treatment failure and serious public health risks in future.

Detection of Hymenoscyphus fraxineus in Leaf Rachises from European Ash (Fraxinus excelsior) in Germany

The ash dieback disease caused by Hymenoscyphus fraxineus is widespread in Germany and is the subject of intensive research efforts. The fungus identification is based on the genomic internal transcribed spacer (ITS) region, which can also be the site of intragenomic variability. In Germany, despite intense research efforts, only a few numbers of H. fraxineus sequence data are recorded. Therefore, this study aims to characterize H. fraxineus isolates obtained from diseased ash leaves in Brandenburg (Germany). Fungal isolates from infected ash leaf tissue were analyzed molecularly using species-specific primers and based on sequencing the ITS region of rDNA. The analysis of the two identified sequences revealed two base substitutionscompared to the reference sequences. Thus, they show an identity of 98.8%–100% to the reference sequences and support the assumption that H. fraxineus has multiple copies of the ITS region. The phylogenetic grouping with reference sequences did not show a distinct cluster for the European, particularly the German, sequences. This indicates that the evolvement of the genetic variability of the ITS region is still an ongoing process.

Development of a Duplex PCR-NALFIA Assay for the Simultaneous Detection of Macrophomina phaseolina and Verticillium dahliae Causal Agents of Crown and Root Rot of Strawberry

Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp) and Verticillium dahliae (Vd). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious, time-consuming, and slow in providing results. In this work, we developed a duplex PCR-NALFIA assay using two pairs of species-specific primers labeled at the 5′ end with different molecules for the simultaneous identification of Mp and Vd. For the NALFIA assay, a lateral flow device (LFD) for the detection of two analytes was used. The method was developed by single and duplex PCR (Mp, Vd, Mp + Vd) using increasingly complex biological systems: (i) DNA from pure cultures of the pathogens; (ii) DNA from artificially inoculated cut melon stems; and (iii) DNA from artificially inoculated strawberry plants cv. Aromas. The duplex PCR protocol was effective in detecting the two pathogens within melon tissues and provided good results with strawberry crown tissues only when the DNA samples were purified by removing the PCR inhibitors. The amplicons were used for both agarose gel electrophoresis (AGE) and NALFIA assays and demonstrated the greater sensitivity of the NALFIA assay (10 pg) for simultaneous detection of the two pathogens.

Antibacterial activity of Nigella sativa against multi-drug resistant bacteria

Background: Multidrug Resistant (MDR) bacteria are considered as the one of the most dangerous pathogens causing a high mortality rate globally. Medicines from the natural source might be the best therapeutic option in current emerging trends of resistance. Methods: We determined the antibacterial activity of Nigella sativa seeds extracts against the clinical isolates of MDR Staphylococcus aureus and Escherichia coli. The collection of different isolates was done from the different hospitals’ settings in Faisalabad. Identifications of these isolates were done phenotypically and through the conventional PCR via genus and species-specific primers. The isolates were then tested for antimicrobial susceptibility using agar well diffusion technique. The activity of N. sativa seed oil was also evaluated in DMSO at concentrations (25%, 50% and 75%). Results: Against S. aureus, 50% oil concentration in DMSO exhibited highest inhibitory zone of 15 mm followed by 90% ethanol extract showed clear zone of 13 mm and 12 mm zone of inhibition showed by the methanol-based extract. Against E. coli 50% oil concentration in Dimethyl sulfoxide (DMSO) and 90% ethanol extract both showed clear zone of 12 mm and 11 mm zone of inhibition showed by the methanol-based extract. However, Minimum inhibitory concentration (MIC) of 50% oil concentration is 0.4 μl followed by 90% ethanol extract showed 0.4 mg and 0.8 mg showed by the methanol-based extract 90% on S. aureus. The oil concentration MIC value of 50% is 0.4 μl then followed by the ethanol extract at 90% and revealed 0.4mg and methanol was followed by 90% extract showed 0.4 mg to the E. coli. Conclusion: We concluded that N. sativa can be used as a pharmaceutical and new therapy for action against MDR bacteria.

Isolation and Molecular Detection of Bacteria from Frequently Touched Objects of Various Public Places at Sadar Upazila of Mymensingh District

The current research was conducted to investigate the magnitude of bacterial contamination in public buses, different wards of hospitals, and public toilets of bus stations and hospitals in Sadar Upazila, Mymensingh, Bangladesh. A total of 90 swab samples were collected aseptically from the most frequently touched surfaces of the sample area. Identification of the isolated bacteria was done by staining and biochemical tests, followed by molecular detection by PCR using genus or speciesspecific primers. Isolated organisms were then subjected to an antibiotic sensitivity test using disk diffusion techniques using 13 commonly available antibiotics. Among the samples, 76.67% (n=69/90) were positive for E. coli, 80% (n=72/90) were positive for Klebsiella spp., and 68% (n=61/90) were positive for Staphylococcus spp. Positive tetA and stx-1 genes were found in 40 and 19 E. coli isolates, respectively. 23 positive mecA genes, or MRSA, were found in Staphylococcus aureus isolates that pose a threat to public health. Toilets of the bus stations were the most contaminated place by the selected bacteria, with the prevalence of E. coli,Klebsiella spp., and Staphylococcus aureus being 81.48% (n=22/27), 77.78% (n=21/27), and 81.48% (n=22/27), respectively. In an antibiogram study, E. coli isolates showed 100% resistance against amoxicillin, azithromycin, tetracycline, and co-trimoxazole. Klebsiella spp. was revealed to be 100% resistant to amoxicillin, followed by colistin sulfate (60%). Staphylococcus aureus isolates were 100% resistant to methicillin, cefoxitin, and cefixime. The findings can be used to raise public awareness about the possible threat, hence preventing the spread of infectious disease in public places. J. of Sci. and Tech. Res. 6(1): 125-137, 2024

A method evaluating ectomycorrhizal fungal colonization in vitro by using relative quantitative PCR

ABSTRACT Ectomycorrhiza (ECM) significantly influences the establishment and stability of forest ecosystems. While evaluating ECM colonization is important for ECM symbiosis research, conventional methods using microscopes prove both time-intensive and subjective. To overcome this, we developed a high throughput and objective method for evaluating ECM colonization in a target tree under laboratory conditions by using quantitative PCR (qPCR). Primers specific for Populus tomentosa, Cenococcum geophilum (Cg), and Laccaria japonica (Lj) were designed and validated. After separately inoculating Cg and Lj, as well as co-inoculating Cg and Lj to P. tomentosa, the root samples were harvested at three different time points; 10, 20, and 40 days post-inoculation. ECM colonization was assessed by conventional microscopic method and qPCR with the designed primers. The designed primers showed a high specificity and quantitativity for each species, indicating their practical use in quantifying individual ECM species in tripartite conditions. Both ectomycorrhizal species showed a positive correlation between the conventional method and the qPCR method: Cg (Spearman correlation = 0.917, p < 0.001), Lj (Spearman correlation = 0.690, p < 0.01). Therefore, by using highly species-specific primers, we established a method to evaluate ECM colonization in vitro by qPCR, which can be an alternative and solution to the conventional method.

ANALYSIS OF SALIVARY ORAL BACTERIA ASSOCIATED WITH SEVERE EARLY CHILDHOOD CARIES: A CROSS-SECTIONAL STUDY

Background: Severe early childhood caries is a rapidly progressing form of dental caries that affects young children and is strongly associated with microbial factors. Understanding the salivary microbial profile in children with S-ECC is critical for developing targeted preventive and therapeutic strategies. Objectives: This study aims to analyze the presence of S. mutans, S. sobrinus and S. wiggsiae in saliva samples obtained from children with severe-ECC. Materials and Methods: This cross-sectional study included children aged 3 to 5 years diagnosed with S-ECC, from January 2020 to May 2020. Ethical approval was obtained from Marmara University Clinical Research Ethical Committee and written informed consent was obtained from parents. Oral examinations were conducted under WHO criteria (1997), recording dmft and ICDAS II scores alongside demographic and feeding habits. Unstimulated saliva samples were collected using a saliva ejector and stored for analysis. q-PCR was performed to detect saliva S. mutans, S. wiggsiae, and S. sobrinus using species-specific 16S rRNA primers. Results: A total of 54 children (40.7% female, 59.3% male) with a mean age of 4.2 ± 0.8 years and a mean dmft score of 13.2 ± 3.2 participated in the study. All children were classified within the ICDAS code 5–6 group. Saliva samples from 52 children were analyzed via qPCR, revealing S. mutans in 96.2%, S. wiggsiae in 67.3%, and S. sobrinus in 26.9%. Co-detection rates were 66.0% for S. mutans and S. wiggsiae, 26.9% for S. mutans and S. sobrinus, and 9.6% for S. sobrinus and S. wiggsiae. No significant associations were found between S. mutans levels and the duration of bottle feeding or breastfeeding (p=0.207 and p=0.184, respectively). Conclusions: This study suggests that S. mutans and S. wiggsiae are prevalent in children with S-ECC, but feeding practices showed no significant impact on the quantity of S. mutans.

A Novel Polymerase Chain Reaction (PCR)-Based Method for the Rapid Identification of Chrysodeixis includens and Rachiplusia nu.

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers.

Development and validation of a species-specific environmental DNA (eDNA) primer for endangered Asian arowana, Scleropages formosus (Teleostei: Osteoglossidae) for law enforcement and wild population monitoring.

The Asian Arowana, Scleropages formosus (Müller and Schlegel, 1844) is a large majestic freshwater teleost, crowned as the king of aquariums with its bright charismatic appearance and magnificent swimming performance. The most expensive Asian arowana is the Golden Blue-based Malayan Arowana which is endemic to the Bukit Merah Lake and Kerian River Basin, Perak, Malaysia. S. formosus has been listed as endangered by the IUCN (International Union for Conservation of Nature), regulated under Appendix 1 of the Convention of International Trade on Endangered Species (CITES) for commercial trade. Environmental DNA (eDNA) analysis has become widely accepted in biodiversity monitoring for the detection of rare and endangered species without harming any ecosystem or threatened species. Hence, the application of eDNA as wild population monitoring of S. formosus is possible for conservation and CITES enforcement program.•The species-specific primer of S. formosus was designed based on selected sequences obtained from GenBank•This report presents the potential application of eDNA in the management of the Malaysian 686 CITES Act for conservation monitoring of the Asian Arowana•The detection and wild population monitoring is possible through the eDNA method as complementary tools.

Rapid and visual detection of Pyricularia oryzae using coupled recombinase polymerase amplification-lateral flow dipstick assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on target gene MGG_15975. The optimized reaction temperature and time were set at 37 °C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated highly sensitivity, capable of detecting as low as 10-2 ng/µL of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields, and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.

Comparative proteomic and genomic analysis of Lactococcus garvieae and Lactococcus petauri isolates from Turkey

Piscine lactococcosis, a bacterial disease caused by Lactococcus garvieae, L. petauri and L. formosensis, induces mass mortalities in multiple farmed fish species globally. Following the identification of L. petauri as one of the emerging etiological agents of piscine lactococcosis, it was revealed that the majority of isolates previously recognized as L. garvieae actually belong to L. petauri. In this study, a total of 134 Lactococcus sp. isolates, including L. garvieae (n = 40) and L. petauri (n = 94) were analyzed. Differential identification of L. garvieae and L. petauri isolates was performed through basic sucrose fermentation and end-point PCR analysis using species-specific primer sequences. Comparative analysis of capsule formation in both species was conducted for the first time using transmission electron microscopy, whole-cell protein profile via 1D SDS-PAGE, antigenic protein characterization using Western blot analysis, and serological profile description by slide and microagglutination assays. Moreover, comprehensive genomic comparisons were made, deciphering overall genomic relatedness indices, virulence genes, and antimicrobial resistance patterns among these closely related species.Analysis showed microcapsule formation in some isolates of L. garvieae and L. petauri for the first time. While a high similarity rate was observed in the whole-cell protein profiles of these species, significant differences were identified among antigenic proteins. Variations between species were observed in slide agglutination and quantitative agglutination tests of L. garvieae and L. petauri field isolates. Capsule-deficient L. garvieae reference and field strain antisera did not agglutinate with each other; however, L. garvieae field strain antisera agglutinated with all L. garvieae field strains and L. petauri strains. These results indicated distinct serological characteristics of capsule-deficient L. garvieae isolates from Turkey compared to previously reported serological classifications.Genomic analyses revealed that none of the investigated L. garvieae and L. petauri isolates harbored the capsule gene cluster. In addition, Turkish isolates of L. garvieae and L. petauri clustered separately from isolates of other geographical origins but together with each other. This study aims to address the problem of lactococcosis, commonly referred to as “Summer Mortality Outbreak and Vaccine-Preventable Diseases,” in a wide variety of cultured fish species through the selected L. garvieae and L. petauri isolates as potential vaccine strains.

Molecular methods for the detection and identification of parasitoids within larval wheat midges

Three species of cecidomyiid midges (Diptera: Cecidomyiidae) cause significant yield losses on wheat in Europe: Sitodiplosis mosellana (Géhin), Contarinia tritici (Kirby) and Haplodiplosis marginata (von Roser). Eggs and young larvae may be parasitised by a complex of hymenopteran parasitoids belonging to the Pteromalidae and Platygastridae families which contributes to natural pest control. We have developed molecular tools for detecting and identifying seven parasitoid species previously encountered in Belgium inside individual wheat midge larvae. Barcode DNA sequences from COI, 18S and 28S genes were obtained from the midges and parasitoid species. Each of the three genes allowed all the species to be distinguished although 18S was the only one displaying a barcoding gap, both between parasitoids and midges, and at the species level. Based on the 18S gene, we developed a TaqMan assay to assess parasitism in midge larvae, regardless of the midge and parasitoid species. Next, two group-specific PCR primer pairs were generated, allowing the separate amplification of midge DNA or parasitoid DNA in parasitised individuals and subsequent identification by Sanger sequencing. Finally, species-specific primers were designed to identify six parasitoid species by simple PCR amplification. These tools were successfully applied to assess the parasitism rate of S. mosellana larvae in seven Belgian fields.

Mycotoxigenic Fusarium species and zearalenone concentration in commercial maize kernels in northern Ghana.

The fungal genus Fusarium contains many toxigenic pathogens of maize with associated yield losses, reduction of grain quality, and accumulation of mycotoxins in harvested grains. To determine zearalenone (ZEN) concentration and identify the various Fusarium species in commercial maize grains, a survey of 75 maize samples, collected from 11 market centers in the five regions in northern Ghana was identified based on morphological characteristics, sequence analysis of the internal transcribed spacer region, and polymerase chain reaction using species-specific primers. ZEN levels were determined using HPLC. ZEN contamination was recorded in 33.3% of the maize samples, with concentrations ranging from 0.61 to 3.05µg/kg. Based on VERT1/2 and TEF 1-α sequencing, F. verticillioides was the most prevalent species in the studied samples: 40.35% from the Upper East Region, 28.07% from the North East Region, 19.30% from the Upper West Region, 10.53% from the Savannah Region, and 1.75% for the Northern Region. Other fungal species found were F. equiseti and F. solani. A higher number of the Fusarium isolates were found in white maize (609 isolates from 27 samples) compared to yellow maize (225 isolates from 23 samples).