Molecular Identification of Mycobacterium Strains Responsible of Bovine Tuberculosis Cases in Bobo-Dioulasso Slaughterhouse, Burkina Faso

Bovine tuberculosis (bTB) is an endemic zoonosis significantly affects animal health in Burkina Faso. The primary causative agent is Mycobacterium tuberculosis (M. tuberculosis) complex, mainly M. bovis. Cattle are considered as natural reservoir of M. bovis. However, in Burkina Faso, the circulation of these strains remains poorly understood and documented. This study aimed to identify and characterize Mycobacterium strains from suspected carcasses during routine meat inspection at Bobo-Dioulasso refrigerated slaughterhouse. A prospective cross-sectional study was conducted from January 2021 to December 2022 on cases of seizures linked to suspected bovine tuberculosis. Microbiological and molecular analyzes were used for mycobacterial strain isolation and characterization. Out of 50 samples, 24% tested positive by microscopy and 12% by culture. Molecular analysis identified 6 strains of Mycobacteria, exclusively Mycobacterium bovis specifically the subspecies bovis (Mycobacterium bovis subsp bovis). In conclusion, M. bovis subsp bovis is the primary agent responsible for bovine tuberculosis in Bobo-Dioulasso. Continuous monitoring of mycobacterial strains is therefore necessary for the effective control of this pathology in the local cattle population.

Morphological and Molecular Identification of Fungi Associated with Sesame Diseased Plants of the Three Agroclimatic Zones of Burkina Faso

Sesame is Burkina Faso’s second essential agricultural export after cotton. It’s consequently a supply of income for producers and foreign exchange for the country. However, sesame production is characterized by low average yields of about 538 kg·ha-1 at the farmer’s field as compared to the potential yield of the improved varieties (1500 - 2000 kg·ha-1). Fungal diseases are some of the major constraints to sesame production in Burkina Faso. The present study contributes to the development of means to control pathogenic fungi of this crop, which are responsible for significant losses. The objective is to identify the fungi associated with diseased sesame plant samples. To this end, 149 samples of diseased sesame plants were collected from different production sites located in three agro-climatic zones of the country. The analysis of the samples according to the blotting paper method, based on the morphological characteristics of the fungi, allowed the identification of 18 genera with prevalence rates from 2.68% to 97.98%. The most frequently identified genera were Macrophomina (97.98%), Cercospora (86.57%), Fusarium (85.23%), Phoma (62.41%) and Colletotrichum (61.07%). The results also showed a variable distribution of fungi according to the agro-climatic zone with the predominance of Macrophomina in all three zones. Molecular identification by DNA sequencing of 120 isolates belonging to the different fungi detected allowed the identification of 25 species of which the most representative were Macrophomina phaseolina, Cercospora sesami, Corynespora cassiicola, Alternaria simsimi, Alternaria porri, Fusarium oxysporum, F. fujikuroi, F. equiseti, Colletotrichum capsici, and C. gloesporiodes. The present study showed that diseased sesame plants collected from different production sites in Burkina Faso housed several species of fungi. The fungi presence in diseased plants indicates the need to inform and raise the stakeholders’ awareness about the phytosanitary problems of sesame, but also to develop effective and appropriate control methods against these crop pathogens in Burkina Faso.

Morphology and Molecular Identification of Dry Fish Fungus Cunninghamella blakesleeana from Small Indigenous Fish “Kachki” Corica soborna (Hamilton 1822) in Bangladesh

The present experiment was conducted to investigate a dry fish fungus, Cunnighamella blakesleeana, which was identified from the infected part of the Corica soborna, locally named as Kachki fish. Mycelium was hyaline, often with granular content, and conidiophores were erected, with verticillate or solitary branches. Zygospores were globose, tuberculate, suspensors equal, smooth, hyaline and heterothallic. Using ITS4 and ITS5 primers, the 740 bp-long ITS region was amplified and sequenced. The ITS region sequences had reciprocal homologies of 98% to 100%. The findings showed that several species of C. blakesleeana fall into the same cluster. It has been determined by molecular data that the fungus we had studied was C. blakesleeana. The maximum mycelial growth (95.33 mm) was observed in the PDA medium, followed by the PSA medium, and the lowest growth (65.50 mm) was measured in the HPA medium in the study of the impact of culture media on the mycelial growth of C. blakesleeana. The influence of temperature on the radial mycelial growth of C. blakesleeana on PDA medium was investigated through five different temperatures. Although pH is a crucial factor in understanding the ecology of spoilage fungus, the highest mycelial growth of C. blakesleeana (88.25 mm) was seen at pH 7, followed by pH 8 and pH 6, while pH 9 was revealed to have the lowest mycelial growth. The outcome suggested that C. blakesleeana thrived in neutral environments.

Molecular Identification of Alternaria alternate (Fr.) Keissl. from the Leaf Blight Disease of Centella asiatica L.

Centella asiatica (L.), frequently known as Thankuni, is an important ethnobotanical plant in Bangladesh. This study was conducted to evaluate the morphological characteristics, cultural factors and molecular identification of the causal agent of Alternaria leaf blight disease of C. asiatica. The potato dextrose agar (PDA) medium recorded the maximum mycelial growth (69 mm), followed by the yeast extract agar (YEA) medium, while the honey peptone agar (HPA) medium recorded the lowest growth (27 mm). The optimal pH and temperature for mycelial growth of Alternaria alternata were 6 and 30°C, respectively. Internal transcribed spacer (ITS) region of Alternaria alternata PCR products measured 558 bp and blast search showed 99% sequence similarity with Alternaria alternata species complex. To the best of our knowledge, Alternaria leaf blight disease caused by Alternaria alternata is the first record in Bangladesh.

The Utility of Specific Markers Based on ITS2 Sequences for Molecular Identification and Detection of Trichogramma spp.

The technology based on specific PCR amplification using internal transcribed spacer 2 of nuclear ribosomal DNA for molecular identification and detection of Trichogramma species was studied. Firstly the ITS2s of six Trichogramma species were cloned and sequenced, and the interspecific sequence variation was analyzed. Secondly the ITS2 regions of six geographical populations of T. dendrolimi were cloned and sequenced, and the intraspecific sequence identity was analyzed. The results show that the interspecific variation and intraspecific similarity of ITS2 sequences are very suitable for designation of specific primers at species-level. Screening of specific primers for T. dendrolimi leads to final sensitive and stable diagnostic primers. This system lets non-specialists can not only identify adults (males and females), but also identify eggs in parasitized hosts rapidly and accurately, which is impossible by conventional methods. Further development of this protocol can create a complete set of specific primers for different species of the whole genus Trichogramma .

Pathogenicity Assay and Molecular Identification of Fungi and Bacteria Associated with Diseases of Tomato in Malaysia

This study was conducted in order to determine the fungi and bacteria associated with tomato plants at Cameron Highlands Malaysia. The fungi which have been isolated and detected from tomato plants were: Fusarium oxysporum, F. solani, F. acuminatum, Rhizoctonia solani, Colletotrichum boninense, C. acutatum and Phoma destructiva. The bacteria which have been isolated and detected from tomato plants were: Ralstonia solanacearum, Xanthomonas vesicatoria, X. gardneri and Pseudomonas syringae. While the most pathogenic fungi were C. boninense, P. destructive and F. oxysporum with the disease incidence (89.6%, 86.6%, 85.6%) respectively, the most pathogenic bacteria were X. vesicatoria and R. solanacearum with the disease incidence (96.6% and 87.6%) respectively.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective:To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok,Nonthaburi,and Pathum Thani provinces of Central Thailand,and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage.Methods:The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method.The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2(ITS2).Results:A total of 1921 snail specimens were classified into five families and seven species.The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes,with an overall prevalence of infection of 2.45%(47/1921).The infected snail specimens included five groups of the cercarial type:cercariaeum cercariae,echinostome cercaria,megalurous cercaria,parapleurolophocercous cercaria,and xiphidiocercariae.This is particularly true of xiphidiocercariae,which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%.With regard to molecular identification,the phylogenetic tree was reconstructed using the neighbor-joining method with 10000 bootstraps and separated the trematodes into three clades:Echinostomatoidea,Microphalloidea and Opisthorchioidea.Conclusions:The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok,Nonthaburi and Pathum Thani provinces.In addition,the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

Development of A High-throughput Molecular Identification Method Suitable for Fungi

Fungi play a significant role in biology-related domains, and with the molecular biology technology advancing, identification of fungi at molecular level and verification of genetic transformant has become the necessary step of test. By far, however, there is no ideal high-throughput molecular identification method. In this paper, a high-throughput device was designed, and a novel PCR-mediated molecular identification method suitable for the device was developed. Through cloning of ccllulase encoding genes and regulatory genes on the genome of Trichoderma reesei, cloning of promoter of phesphoglycerate kinase gene 1 and xylanase encoding genes on the genome and expression vector of Saccharomyces cerevisiae, and ITS sequences cloning and RAPD analysis of T. reesei, S. cerevisiae, Penicil- lium oxalicum, Rhizopus stolonifer, Aspergillus niger, A. carbonarius and A. japonicas, the method turned out to be an effective one with wide application potential. The establishment of the method has worked out the bottleneck of high-throughput molecular identification.

Molecular Identification of D. nuttalli and D.marginatus

This study was to establish a molecular identification method to distinguish Dermacentor nuttalli(D. nuttalli) and Dermacentor marginatus(D. marginatus),and to study their phylogenetic relationship. The ticks were collected from domestic animals in Xinjiang Uygur Autonomous Region for morphological identification,then their genomic DNAs were isolated for amplifying mitochondrial 16 S rRNA and cytochrome oxidase subunit I gene(COI). The phylogenetic tree was constructed based on the sequencing results of PCR products by employing Mega 5. 0 and Mrbayes 3. 2 for homology analysis. Clustering analysis PCR product for 16 S rRNA from both D. marginatus and D. nuttalli was clustered together with their respective 16 S rRNA sequences previously accessed in GenBank,and that for COI gene as well. These are basically identical with morphological identification results. Our results indicate that morphological identification,combined with molecular markers,would be a simple and accurate method for distinguishing D. nuttalli and D. marginatus.

Rapid Molecular Identification of Tribolium destructor

In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the results, （ 1 ） with PCR method, specific primers were designed based on CO1 gene of T. destructor for PCR amplification, and electrophoresis detection confirmed that PCR method could be used to rapidly and accurately identify T. destructor; （2） with PCR-RFLP method, two pairs of degenerate primers were used to amplify CO1 gene of Tribolium species, PCR products were digested with HindIII and detected by electrophoresis, results indicated that PCR-RFLP method could also be used for rapid identification of T. destructor in quarantine practice.

Mophological and Molecular Identification of Potato Cyst Nematode from Sichuan,China

[Objectives]The infection symptoms of cyst nematodes were found in Yuexi, Sichuan. In order to identify the pathogen, the isolated nematodes were identified by morphology and molecular biology. [Methods] The potato roots and soil around the roots were collected, the nematodes in the roots were stained and observed, and the cysts were separated by the simple floating method. The second-stage juveniles(J2 s), females and cysts were found, and they were photographed and morphologically measured. The DNA of cysts and J2 s were extracted and identified by species-specific PCR. The DNA sequences of 18 S gene, 28 S D2-D3 region and ITS region in ribosomal DNA were obtained. Sequences of some cyst nematode species were downloaded from GenBank for sequence alignment, and MrBayes 3.2.3 software was used to construct a Bayesian phylogenetic tree. [Results] The nematodes could invade hosts’ root system. The basal knobs of J2 s was nearly round and inclined backward;the average length of the stylet was shorter than 23 μm;the Granek ratio of cysts was greater than or equal to 3, which is highly consistent with that of Globodera rostochiensis. The DNA templates of three cysts and four J2 s were amplified by species-specific PCR, and a band of about 430 bp was obtained. After further sequencing, the length was 434 bp, which is consistent with G. rostochiensis. The evolutionary analysis of rDNA 18 S and 28 S showed that the cyst nematode population was a Globodera species, and further evolutionary analysis of ITS gene confirmed that the population was G. rostochiensis. [Conclusions] The nematodes are G. rostochiensis, which is a quarantine species of great concern to both domestic and import quarantine. Once introduced and colonized, it is hard to eradicate. It is necessary to establish a monitoring system as soon as possible, strengthen the quarantine, supervision and management, increase investment in G. rostochiensis research and development, and develop detection and control technologies, so as to escort the healthy and sustainable development of China’s potato industry.

Vegetative Growth and Molecular Identification of Fusarium equiseti Isolated from Wilt Disease of Centella asiatica L. in Bangladesh

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30°C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% - 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.

Molecular identification of plants regenerated from somatic hybridization between rice and apomictic Panicum

We attempted to introduce apomictic gene(s)into rice via somatic hybridization by usingapomictic Panicum maximum Jacq.as thedonor of apomictic gene(s).Protoplasts of rice derived from suspen-sion cells were inactivated with indoacetamide(IOA)and protoplasts of Panicum maximum

Purification and Molecular Identification of an Antifungal Peptide from the Hemolymph of Musca domestica （housefly）

Antibacterial and antifungal peptides found in houseflies （Musca domestica） in large number are indispensable components of its immune defense mechanism. In this study the anterior tip of the larvae of housefly was cut off with a pair of fine scissors and hemolymph was collected and exuded in an ice-cold test tube. From the hemolymph an antifungal substance was isolated by solid-phase extraction combined with reverse phase-high performance liquid chromotography （RP-HPLC） and named as Musca domestica antifungal peptide-1 （MAF-1）. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE） showed its molecular weight was 17 kDa. UV absorption spectra revealed that this antifungal substance possessed the characteristics of protein peptides. Analysis by fingerprint-identification and tandem mass spectrometry suggested MAF-I was an unknown protein. Edman degradation identified the sequence of 30 amino acids of its N-terminal which matched no peptide in the MASCOT search database, indicating MAF-1 was a novel insect antifungal peptide. Mass spectrometry showed the precise molecular weight of MAF-1 was 17203.384 Da. Its isoelectric point was acidic.

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.

Commercialized non-Camellia tea:traditional function and molecular identification

Non-Camellia tea is a part of the colorful Chinese tea culture,and is also widely used as beverage and medicine in folk for disease prevention and treatment.In this study,37 samples were collected,including 33 kinds of non-Camellia teas and 4 kinds of teas(Camellia).Traditional functions of non-Camellia teas were investigated.Furthermore,non-Camellia teas of original plants were characterized and identified by molecular methods.Four candidate regions(rbcL,matK,ITS2,psbA-trnH)were amplified by polymerase chain reaction.In addition,DNA barcodes were used for the first time to discriminate the commercial non-Camellia tea and their adulterants,and to evaluate their safety.This study showed that BLASTN and the relevant phylogenetic tree are efficient tools for identification of the commercial non-Camellia tea and their adulterants.However,some sequences from original plants have not been found and there is a limitation of sequence number of original plants in GenBank.Submitting more original plant sequences to the GenBank will be helpful for evaluating the safety of non-Camellia teas.

Molecular-based Integrated Identification of Bacterial Blight(Xanthomonas axonopodis pv. dieffenbachiae) in Anthurium and Detection of Latent Infection

Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae （Xad）, is the most destructive disease of anthurium worldwide, and no effective control technique has been developed currently. The comprehensive survey and precise detection of the pathogen is essential for evaluating disease progress and strengthening management to avoid a serious epidemic. In this study, a total of 253 blight-suspected samples of anthurum and other Araceae species were collected across the country, and 166 potential pathogenic bacteria strains were isolated and purified, after combined analysis on the characteristics of morphology, pathoge- nicity, 16S rDNA sequences and amplicans of Xad-specific SCAR markers. Finally, 93 of which were considered as X. axonopod/s pv. dieffenbachiae. In addition, by using a nested-PCR in repeated detections, 17 out of 21 prevalent anthurium cultivars without blight symptom exhibited latent infection even in young leaves. The results indicated that the anthurium bacterial blight distributed commonly in growing areas in China, and most of the commercial cuhivars had no strong resistance. The identification of Xad infection （latent） would be beneficial for the disease forecasting and management improving in anthttrium production.

Selection and Molecular Biological Identification of a Strain of Bacillus sp. Inhibiting the Growth of Saprolegnia ferax

Based on the theory of biological control of Saprolegnia ferax,antagonism test of nine strains of Bacillus sp. to S. ferax JL was carried out. Bacillus sp.BA1 was screened to have significantly inhibitory effects on the growth of S. ferax JL( P 【 0. 05). Then,the effects of Bacillus sp. BA1 on different sources of S. ferax were carried out. Results showed that BA1 also had significantly inhibitory effects on S. ferax 6#,10# and S2( P 【 0. 05). Sequence of 16 S r DNA of BA1 was analyzed; and homologous alignment analysis showed that BA1 had more than 99% similarity with Bacillus cereus. Therefore,it could be concluded that strain BA1 was B. cereus,which significantly inhibited the growth of S. ferax and could be used as the biological control agent for S. ferax diseases in aquaculture.

Systematic Evaluation of Pharmacognostic Identification of Polygonum capitatum

[Objectives] To investigate the systematic evaluation of pharmacognostic identification of Polygonum capitatum . [Methods] 10 batches of P. capitatum cultivated in Guizhou were chosen for plant samples. Macroscopical identification was conducted on plant roots, stems, leaves, flowers and fruits. The P. capitatum powder was processed for physical and chemical distinction by FeCl 3 chromogenic reaction, hydrochloric acid magnesium powder reaction, AlCl 3 color development reaction and thin-layer chromatography.Microscope identification was carried out on the powder. Plant genome DNeasy Plant Kit was adopted for DNA molecular marker identification. [Results] The results showed that the stem of P. capitatum was tufted, the leaves were oval, 2 to 5 cm long, and 1 to 2 cm wide;the leaf apex was acute and cuneate at the base, the inflorescence was capitate, paired or solitary;the raceme was erect and nearly spherical, and the perianth was light red. Furthermore, for the chromogenic reaction of FeCl 3 ethanol extract of P. capitatum , appeared blue and turned to dark blue after long time storing at room temperature. For the reaction of hydrochloric acid magnesium powder, the alcohol extract of P. capitatum , exhibited deep red. In the color reaction of AlCl 3, the alcohol extract revealed yellow fluorescence under 360 nm UV lamp. Microscope identification of the powder displayed pollen grains, crystal sheath fibers, cellulose, vessels, starch grains, cork cells, and other characteristic fragments. In addition, DNA barcoding electrophoresis results showed that P. capitatum showed a clear and bright single band near 500 bp, and further sequencing results showed that the sequence differences were mainly concentrated in ITS1 and ITS2 region. [Conclusions] Systematic evaluation for the identification of P. capitatum is established, which combines with macroscopic identification, physicochemical identification, powder microscope identification, and DNA molecular identification. Finally, the original medicinal material is identified as P. capitatum Buch.-Ham. ex D. Don.

Identification of Sphaeroma terebrans via morphology and the mitochondrial cytochrome c oxidase subunit I (COI) gene

Sphaeroma terebrans, a wood-boring isopoda, is distributed worldwide in tropical and subtropical mangroves. The taxonomy of S. terebrans is usually based on morphological characteristics, with its molecular identification still poorly understood. The number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod are considered as the major morphological characteristics in S. terebrans, which can cause difficulty in regards to accurate identification. In this study, we identified S. terebrans via molecular and morphological data. Furthermore, the validity of the mitochondrial cytochrome c oxidase subunit I （COl） gene as a DNA barcode for the identification of genus Sphaeroma, including species S. terebrans, S. retrolaeve, and S. serratum, was examined. The mitochondrial COl gene sequences of all specimens were sequenced and analysed. The interspecific Kimura 2-parameter distances were higher than intraspecific distances and no intraspecificinterspecific distance overlaps were observed. In addition, genetic distance and nucleotide diversity （TT） exhibited no differences within S. terebrans. Our results revealed that the mitochondrial COl gene can serve as a valid DNA barcode for the identification of S. terebrans. Furthermore, the number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod were found to be unreliable taxonomic characteristics for S. terebrans.