Restriction Fragment Length Polymorphism Patterns Research Articles

First Report of a 'Candidatus Phytoplasma sacchari'-Related Strain Associated with Yellowing and Decline of Silver Bluestem in Texas, U.S.A.

Silver bluestem [Bothriochloa laguroides (DC.) Herter] is a warm-season grass native to Texas. This perennial grass plays a crucial role in maintaining ecological balance and supporting wildlife in the region. In September 2022, while investigating the ecological impact of invasive grass species on a grassland located near Pipe Creek (TX), B. laguroides plants were observed showing symptoms that included yellowing of the blades and occasionally brown discoloration of the midveins and stems (Fig. S1). Disease incidence was estimated as 2% of silver bluestem plants in the 2 hectares surveyed. To investigate the possibility of a phytoplasma association with the symptoms, four symptomatic and four asymptomatic leaf samples were collected for further study. Total DNA was extracted from leaf midribs using a DNeasy Plant Mini Kit (Qiagen). The DNA extracts were tested using a phytoplasma-specific quantitative PCR assay (Hodgetts et al. 2009), which identified two out of the four symptomatic B. laguroides samples as positive for phytoplasmas. A semi-nested PCR assay for amplification of the 16S rRNA gene fragment was then performed on these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004), and two additional housekeeping genes (tuf and secA) were amplified as previously described (Makarova et al. 2012; Hodgetts et al. 2008; Bekele et al. 2011). All amplicons of the expected size, 1.5 kb (16S rRNA), 0.4 kb (tuf) and 0.6 kb (secA), were purified and bi-directionally sequenced using primers from each gene second round PCR amplification. Analysis of the sequences derived from the three gene fragments revealed no variation between the two plant samples and confirmed they originated from a phytoplasma, termed strain TXSB-2 (Texas Silver Bluestem). Sequences from a single B. laguroides plant DNA extract were deposited in GenBank with accession numbers OR711913 (16S rRNA), OR709687 (tuf) and OR709688 (secA). A BLAST search of the 16S rRNA gene sequence from TXSB-2 against the NCBI nucleotide database, showed 99.58% sequence identity with an unclassified phytoplasma clone 139-1 from a leafhopper collected in Australia (MW281491) (Fig. S2). The partial nucleotide sequence of the tuf and secA genes showed 90.60% and 89.78% similarity, respectively, to the corresponding genes in 'Ca. P. sacchari' strain SCWL1 (CP115156) associated with sugarcane in China. The iPhyClassifier, an interactive online tool for phytoplasma identification and classification (Zhao et al. 2009), was used to determine the 'Candidatus Phytoplasma' species affiliation and group/subgroup classification status of this phytoplasma strain. The result showed that the TXSB-2 16S rDNA shared 98.94% sequence identity with that of the 'Ca. P. sacchari' reference strain (GenBank accession: MN889545), indicating TXSB-2 is a 'Ca. P. sacchari'-related strain. The result from virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA F2nR2 fragment revealed that TXSB-2 possessed a collective RFLP pattern that is distinct from the reference patterns of all established phytoplasma ribosomal subgroups and is proposed as the representative strain of a new subgroup designated as 16SrXI-H. 'Candidatus Phytoplasma sacchari' has been reported associated with sugarcane grassy shoot disease, which is considered among the most damaging diseases of sugarcane across parts of Southeast Asia and India (Kirdat et al. 2021). The same phytoplasma was recently confirmed infecting sorghum in India (Nithya et al. 2024). To our knowledge, this is the first report of a 'Ca. P. sacchari'-related strain infecting B. laguroides in the United States. Moreover, B. laguroides is a new host for strains related to 'Ca. P. sacchari'. Further investigation is required to elucidate the prevalence of this disease in the area, its natural vectors, and the potential consequences arising from this novel phytoplasma strain within its ecosystem in Texas.

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

Association Analysis of MMP-13 (rs2252070) Gene Polymorphism and the Susceptibility to Chronic Periodontitis.

Chronic periodontitis is a multifactorial inflammatory condition influenced by genetic factors. Matrix metalloproteinase(MMP)-13, serving as a crucial enzyme involved in extracellular matrix remodeling, is associated with the degradation of periodontal tissues. Therefore, this study assesses the genetic link between the MMP-13 (rs2252070) genetic variation and chronic periodontitis in a Southern Indian demographic. The study was conducted at Saveetha Dental Collegein Chennai, India. It involved a total of 100 subjects,50 individualsaffected with periodontitis (classified as stage II and above, American Association of Periodontology 2018 criteria) and 50 individuals who were periodontally healthy or were diagnosed as having mild gingivitis. We isolated DNAfrom the blood samples obtained from the participants. Specific primers that flank the BsrI region of the MMP-13 receptor gene were used in the process of DNA amplification. Subsequently, a restriction fragment length analysis using the BsrI enzyme was carried out for genotyping of the amplicon. Based on the restriction fragment length polymorphism pattern, we obtained certain genotypes. These were further recorded and followed by statistical analysis. We conducted a chi-square test to draw a comparison in terms of their genotype and allele frequencies. We calculated the odds ratio, along with 95% confidence intervals. The frequency of genotypes and distribution of MMP-13 polymorphism did not exhibit a statistically significant difference at χ2 degrees of freedom (P = 0.913). We inferred from our study that there was no significant difference between the groups concerning homozygous and heterozygous mutant genotypes (AA vs. AG + GG), with a P-value of 0.6871. The observed frequencies of GG (47% vs. 43%) and AG+AA (41% vs. 42%) genotypes did not indicate a significant difference between the groups. Similarly, there was no noteworthydistinction between the A allele (62% vs. 65%) and G allele (38% vs. 35%) in the case and control groups. Thefindings of the study reveal that there is no correlation between MMP-13 (rs2252070)gene polymorphism and periodontitis.

Contrasting PRRSV temporal lineage patterns at the individual farm, production system, and regional levels in Ohio and neighboring states from 2017 to 2021

Porcine reproductive and respiratory virus (PRRSV), one of the most significant viruses in the swine industry, has been challenging to control due to its high mutation and recombination rates and complexity. This retrospective study aimed to describe and compare the distribution of PRRSV lineages obtained at the individual farm, production system, and regional levels. PRRSV-2 (type 2) sequences (n = 482) identified between 2017 – 2021 were provided by a regional state laboratory (Ohio Department of Agriculture, Animal Disease Diagnostic Center (ODA-ADDL)) collected from swine farms in Ohio and neighboring states, including Indiana, Michigan, Pennsylvania, and West Virginia. Additional sequences (n = 138) were provided by one collaborating swine production system. The MUSCLE algorithm on Geneious Prime® was used to align the ORF5 region of PRRSV-2 sequences along with PRRSV live attenuated vaccine strains (n = 6) and lineage anchors (n = 169). Sequenced PRRSV-2 were assigned to the most identical lineage anchors/vaccine strains. Among all sequences (n = 620), 29.8% (185/620) were ≥ 98.0% identity with the vaccine strains, where 93.5% (173/185) and 6.5% (12/185) were identical with the L5 Ingelvac PRRS® MLV and L8 Fostera® PRRS vaccine strains, respectively, and excluded from the analysis. At the regional level across five years, the top five most identified lineages included L1A, L5, L1H, L1C, and L8. Among non-vaccine sequences with production system known, L1A sequences were mostly identified (64.3% – 100.0%) in five systems, followed by L1H (0.0% - 28.6%), L1C (0.0% – 10.5%), L5 (0.0% – 14.4%), L8 (0.0% – 1.3%), and L1F (0.0% – 0.5%). Furthermore, among non-vaccine sequences with the premise identification available (n = 262), the majority of sequences from five individual farms were either classified into L1A or L5. L1A and L5 sequences coexisted in three farms, while samples submitted by one farm contained L1A, L1H, and L5 sequences. Additionally, the lineage classification results of non-vaccine sequences were associated with their restriction fragment length polymorphism (RFLP) patterns (Fisher’s exact test, p < 0.05). Overall, our results show that individual farm and production system-level PRRSV-2 lineage patterns do not necessarily correspond to regional-level patterns, highlighting the influence of individual farms and systems in shaping PRRSV occurrence within those levels, and highlighting the crucial goal of within-farm and system monitoring and early detection for accurate knowledge on PRRSV-2 lineage occurrence and emergence.

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

Prevalence of permethrin-resistant kdr mutation in head lice (Pediculus humanus capitis) from elementary school students in Jeddah, Saudi Arabia.

Head lice (Pediculus humanus capitis) are a major global concern, and there is growing evidence of an increase in head lice prevalence among Saudi schoolchildren. The purpose of this study is to investigate the prevalence of an insecticidal resistance mutation in head lice collected from schoolchildren. A polymerase chain reaction (PCR) was used to amplify a segment of the voltage-gated sodium channel gene subunit to assess the prevalence and distribution of the kdr T917I mutation in head lice. Subsequently, the restriction fragment length polymorphism (RFLP) patterns revealed two genotypic forms: homozygous-susceptible (SS) and homozygous-resistant (RR). The results showed that 17 (37.80%) of the 45 samples were SS, whereas 28 (62.2%) were RR and T917I and L920F point mutations were found in the nucleotide and amino acid sequences of RR. Compared to other nations, the frequency of permethrin resistance mutation in the head louse population in Saudi Arabia was low. This study provides the first evidence of permethrin resistance mutation in human head lice in Saudi Arabia. The findings of this study will highlight the rising incidence of the kdr mutation in head lice in Saudi Arabia.

First Report of 'Candidatus Phytoplasma solani' (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India.

Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of 'Ca. P. solani' was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of 'Ca. P. solani' was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani' (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560.

Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

Fasciola species are a member of flatworms belonging to the trematodes (flukes), commonly known as liver fluke, they are extremely pathogenic parasites that affect the liver of humans and animals, nowadays, most laboratories and research facilities use molecular-based techniques for identifying and describing Fasciola species. The molecular diagnostic markers such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-RFLP methods are accurate and more specific than the immunological and microscopical methods. The identification of the species of liver flukes will give a new clue for the treatment and control of fascioliasis. The aim, of this study, is to find the molecular characterization of Fasciola spp. isolated from humans and animals in Sulaimani city. The flukes were isolated from humans using endoscopic techniques and from slaughtered livestock at the new slaughterhouse of Sulaimani, 48 liver flukes were collected from different hosts; human (n = 3), cattle (n = 20), sheep (n = 20), and goats (n = 5) from October 2021 to April 2022. The uinversal primers ribosomal Deoxy Ribo Nuclic Acid (rDNA) were used, then the PCR products were subjected to restriction fragment polymorphism (RFLP) assay and The PCR Product was digested with restriction enzymes DraII, also the DNA sequencing was used for the PCR product of the primer Cytochrome Oxidase subuint 1 (COX1). The results of the PCR-RFLP of the 28s rDNA show the genetic polymorphisms among the flukes and two patterns of RFLP were observed F. hepatica, and F. gigantica, also the sequence analysis of the partial gene of the COX1 showed the isolated flukes belonged to F. hepatica and F. gigantica with some genetic variation, and the result of the sequences was deposited in the Gene Bank under the following Accession numbers; F. gigantica (OP718780 and OP718781) and F. hepatica (OP718782, OP718783, and OP718784). The present study concludes thatF. hepatica and F. gigantica are both responsible for human and animal Fasciolasis in Kurdistan-Iraq, Therefore, RFLP techniques and DNA sequencing are a reliable, and differential method for species and genotyping identification of liver fluke.

Molecular Ecology of Freshwater Turtles and Future directions

The distribution and diversity of turtles now reflect the lengthy and complex evolution of the taxonomy, which represents an old group of tetrapod vertebrates in terms of evolutionary history. Freshwater turtles represent the majority of the 365 species, and they mostly live in tropical and subtropical regions. Emydidae diversity hotspots can be found in Southeast North America, as can Geoemydidae and Trionychidae in the Indo-Malayan area. While Pelomedusidae are mostly found in Africa, Chelidae are primarily found in the Neotropics and Australia. Most species of the genus are endemic to a particular region or even to a single location. The majority of freshwater turtles suffer varied degrees of threat, mostly from habitat changes and collection. With the use of morphological and molecular data, the majority of phylogenetic trees for different turtle species have been generated using DNA techniques and procedures. The complete mitochondrial DNA (mtDNA), dehydrogenase subunit 4 (ND4), cytochrome b (Cyt b), carapacial ridge (CR), and cytochrome c oxidase subunit I (CO I) genes of freshwater turtles were sequenced by using universal PCR and long-PCR methods. Along with CR sequences of freshwater turtles, the composition and structure of the control region of diverse species were compared and analysed. Functional domains in the regulatory area, as well as their conserved sequences, were determined based on sequence similarities to other turtles. The mitochondrial regulatory regions and flanking sequences of diverse freshwater turtle species were recovered using Long-PCR and gene-specific primers. To clarify the genetic links between the fresh water turtle species that share the same habitat type, a tree was created based on Cytochrome b sequencing data and the PCR- Restriction fragment length polymorphism (RFLP) pattern. Keywords: Complex evolution, Phylogenetics, Phylogenomics, Tetrapod vertebrates

Application of 16S rRNA virtual RFLP for the discrimination of some closely taxonomic-related lactobacilli species

BackgroundSeveral species in Lactobacillaceae family were recognized as potential probiotic bacteria. In this group of lactic acid bacteria, species are taxonomically closed and usually share similar 16S rRNA gene, thus, instead of so their identification and discrimination are too difficult. MethodIn the present study, virtual restriction fragment length polymorphism (RFLP) is instead of was used as a tool to discriminate between the closely related species Lactiplantibacillus plantarum (L plantarum), Lactiplantibacillus paraplantarum (L paraplantarum), and Lactiplantibacillus pentosus (L pentosus); Latilactobacillus sakei (L sakei), Latilactobacillus curvatus(L curvatus), and Latilactobacillus graminis (L graminis); Lacticaseibacillus casei (L casei), Lacticaseibacillus paracasei (L paracasei), Lacticaseibacillus zeae, and Lacticaseibacillus rhamnosus; Lactobacillus gasseri (L gasseri) and Lactobacillus johnsonii (L johnsonii). In silico comparative analysis of 16S rRNA sequences digested by 280 restriction enzymes was performed in order to search the key enzymes which gives different profiles. ResultsResults revealed that L casei, L paracasei, L zeae, and Lb rhamnosus could be separated from each other on the basis of AlwI, BpuEI, BsgI, BsrDI, BstYI, EarI, MluCI, and NsPI RFLP. Results showed also that different RFLP patterns were obtained from L sakei, L graminis and L curvatus by using both AflI and NspI endonucleases (in separated restriction) and L plantarum, L paraplantarum, and L pentosus were distinguished each one from the other by MucI, NspI, and TspDTI PCR-RFLP. Lb gasseri and L johnsonii were also separated on the basis of Mse I, Taq I, and Dra I RFLP. ConclusionIn this study, we proved that too closely related species could be separated in virtual analysis on basis of their 16S rRNA RFLP patterns using key restriction enzymes method.

First Report of 'Candidatus Phytoplasma asteris' (16SrI group) Associated with Murraya exotica Witches'-Broom Disease in Taiwan.

Murraya exotica L., commonly known as orange jasmine, is an evergreen shrub belonging to the Rutaceae family. It has long been used as traditional Chinese medicine for treating abdominal pain, toothache, scabies, and other disorders (Liu et al. 2018). M. exotica is widely grown as a garden bush in Taiwan. A prokaryotic pathogen, 'Candidatus Liberibacter asiaticus' (Damsteegt et al. 2010), reportedly could infect M. exotica, but there is no reported phytoplasma disease in M. exotica. In June 2020, M. exotica plants exhibiting witches'-broom (WB), leaf yellowing, and small leaves (Fig. s1) were observed in a horticultural landscaping field in Taichung City, Taiwan. It was estimated that more than 70% of M. exotica plants within a single area were affected. DNA was extracted separately from petioles of five symptomatic and one asymptomatic plants using a modified CTAB method (Echevarría-Machado et al. 2005) and used for nested PCR with two universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995) followed by R16F2n/R16R2 (Gundersen and Lee 1996) to amplify a 1.2-kb 16S rRNA fragment. PCR was also conducted by primers, rp(I)F1A/rp(I)R1A to amplify a partial ribosomal protein S3 and L22 (rplV-rpsC) fragment (Lee et al. 2004). Expected 1.2-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no bands were amplified from that of the asymptomatic plant. The amplicons were cloned, sequenced with an ABI 3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) in Biotechnology Centre DNA-sequencing facility at National Chung Hsing University (NCHU) and deposited in GenBank. BLAST analysis revealed that 16S rDNA sequences (MZ373297 and MZ373298) shared 100% identity to each other and both shared 99.4% identity with those of several phytoplasma strains, e.g., rapeseed phyllody phytoplasma (CP055264), Brassica sp. phyllody phytoplasma (MN877914), Plumbago auriculata leaf yellowing phytoplasma (MN239504), and aster yellows phytoplasma (MK992774), which all belonging to the 16SrI group, by using the CLUSTAL W Methods of MegAlign program (DNASTAR, Inc., Madison, WI, USA). Further analysis using iPhyClassifier tool (https://plantpathology.ba.ars.usda.gov) indicated that the virtual restriction fragment length polymorphism (RFLP) patterns derived from the 16S rDNA F2nR2 fragment of the M. exotica WB phytoplasma was most similar to the reference pattern of the 16SrI-B subgroup, with a pattern similarity coefficient of 0.97 and shared 99.3% sequence identity to 'Candidatus Phytoplasma asteris' (M30790). The partial rplV-rpsC gene sequence (OM275408) showed 99.7% of sequence identities to those of rapeseed phyllody phytoplasma (CP055264), plum witches'-broom phytoplasma (MH061366) and oilseed rape phytoplasma (KX551965), by using the CLUSTAL W Methods of MegAlign program. Taken together, we concluded that the phytoplasma strain associated with M. exotica WB disease was a strain belonging to a 16SrI. To the best of our knowledge, this is the first report of M. exotica being infected by a phytoplasma in the aster yellows group, and M. exotica may also serve as an intermediate reservoir host to other plants, e.g., wax apple, periwinkle and roselle, of 16SrI phytoplasma.

Multilocus sequence typing of diverse phytoplasmas using hybridization probe-based sequence capture provides high resolution strain differentiation.

Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus ‘Candidatus Phytoplasma’, which includes 49 ‘Candidatus’ species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting cpn60, tuf, secA, secY, and nusA genes, as well as 16S and rp operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including ‘Ca. P. asteris’ (16SrI-B), ‘Ca. P. pruni’ (16SrIII-A),‘Ca. P. prunorum’ (16SrX-B), ‘Ca. P. pyri’ (16SrX-C), ‘Ca. P. mali’ (16SrX-A), and ‘Ca. P. solani’ (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and cpn60 gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding ‘Ca. Phytoplasma’ species/strain differentiation.

Isolation of Nine Bacteriophages Shown Effective against Erwinia amylovora in Korea.

Erwinia amylovora is a devastating bacterial plant pathogen that infects Rosaceae including apple and pear and causes fire blight. Bacteriophages have been considered as a biological control agent for preventing bacterial infections of plants. In this study, nine bacteriophages (ΦFifi011, ΦFifi044, ΦFifi051, ΦFifi067, ΦFifi106, ΦFifi287, ΦFifi318, ΦFifi450, and ΦFifi451) were isolated from soil and water samples in seven orchards with fire blight in Korea. The genetic diversity of bacteriophage isolates was confirmed through restriction fragment length polymorphism pattern analysis. Host range of the nine phages was tested against 45 E. amylovora strains and 14 E. pyrifoliae strains and nine other bacterial strains. Among the nine phages, ΦFifi044 and ΦFifi451 infected and lysed E. amylovora only. And the remaining seven phages infected both E. amylovora and E. pyrifoliae. The results suggest that the isolated phages were different from each other and effective to control E. amylovora, providing a basis to develop biological agents and utilizing phage cocktails.

Association of SRXN1 Receptor Gene Polymorphism with Susceptibility to Periodontitis.

Emerging evidence suggests that oxidative stress forms a key component in the etiopathogenesis of periodontitis. Literature evidence have shown potential antioxidants responsible for combating the pro-oxidants which stress the periodontium, but the peroxiredoxin-sulfiredoxin system is explored very minimally in periodontal disease. Thus, the present study was aimed to evaluate the genetic association of SRXN1 receptor gene polymorphism (rs6053666). A total of 100 subjects were recruited for this study, which included 50 Periodontitis patients (Stage II and above based on the criteria of American Association of Periodontology-2018) and 50 periodontally healthy or mild gingivitis. Genomic DNA was extracted from the whole blood collected from the subjects. DNA was amplified using specific primers flanking the BtgI region of the SRXN1 receptor gene. The amplicon was further subjected to genotyping using restriction fragment length using BtgI enzyme. The genotype obtained based on the restriction fragment length polymorphism pattern was recorded and used for statistical analysis. The distribution of genotypes and allele frequencies in the periodontitis and control groups were compared using the Chi-square test. The risk associated with individual alleles or genotypes was calculated as the odds ratio with 95% confidence intervals. Statistical significance in all tests was determined at P < 0.05. The genotype frequency and distributions of SRXN1 receptor BtgI polymorphism did not differ significantly at ꭕ2df (P = 0.557). Our study results showed that homozygous and heterozygous mutant genotypes had no significant difference (CC vs. CT + TT) between the periodontitis patients and control group with a P = 0.4266. The detected frequency of CT (38% vs. 34%) and TT (42% vs. 52%) genotype showed no significant difference between control and test group. There was no significant difference in C allele (39% vs. 31%) and T allele (61% vs. 69%) between the test and control group. The present study denotes that SRXN1 receptor gene polymorphism is not associated with periodontitis in the study group analyzed.

First Detection of NADC34-like PRRSV as a Main Epidemic Strain on a Large Farm in China.

The newly emerged sublineage 1.5 (NADC34-like) porcine reproductive and respiratory syndrome virus (PRRSV) has posed a direct threat to the Chinese pig industry since 2018. However, the prevalence and impact of NADC34-like PRRSV on Chinese pig farms is unclear. In the present study, we continuously monitored pathogens—including PRRSV, African swine fever virus (ASFV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2)—on a fattening pig farm with strict biosecurity practices located in Heilongjiang Province, China, from 2020 to 2021. The results showed that multiple types of PRRSV coexisted on a single pig farm. NADC30-like and NADC34-like PRRSVs were the predominant strains on this pig farm. Importantly, NADC34-like PRRSV—detected during the period of peak mortality—was one of the predominant strains on this pig farm. Sequence alignment suggested that these strains shared the same 100 aa deletion in the NSP2 protein as IA/2014/NADC34 isolated from the United States (U.S.) in 2014. Phylogenetic analysis based on open reading frame 5 (ORF5) showed that the genetic diversity of NADC34-like PRRSV on this farm was relatively singular, but it had a relatively high rate of evolution. Restriction fragment length polymorphism (RFLP) pattern analysis showed that almost all ORF5 RFLPs were 1-7-4, with one 1-4-4. In addition, two complete genomes of NADC34-like PRRSVs were sequenced. Recombination analysis and sequence alignment demonstrated that both viruses, with 98.9% nucleotide similarity, were non-recombinant viruses. This study reports the prevalence and characteristics of NADC34-like PRRSVs on a large-scale breeding farm in northern China for the first time. These results will help to reveal the impact of NADC34-like PRRSVs on Chinese pig farms, and provide a reference for the detection and further prevention and control of NADC34-like PRRSVs.

Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

Identification of a 16SrIX‐B phytoplasma strain associated with Daphne mucronata phyllody in Iran

AbstractDuring surveys from 2017 to 2018, a phyllody disease was observed in Daphne mucronata plants located in Lordegan and Ahmadliveh areas in Chaharmahal and Bakhtiari province (west of Iran). The main symptoms were yellowing, little leaf, phyllody and flower sterility. Total DNA was extracted from midrib tissue of fresh leaves from 30 symptomatic and five asymptomatic plants. DNA samples were subjected to nested PCR, using primer pairs P1/P7 and R16F2n/R16R2. PCR amplicons of 1.8 and 1.25 kb, respectively, were obtained from all samples from symptomatic plants whilst no amplification was observed in samples from asymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using MseI, HhaI, AluI, HaeIII, RsaI, HpaII, TaqI, KpnI, ThaI and BfaI enzymes showed identical RFLP patterns in all the samples that were also identical to the profiles of the 16SrIX‐B phytoplasma subgroup. Sanger sequencing of five randomly selected amplicons confirmed that they were 100% identical to each other and had &gt;99% identity with ‘Candidatus Phytoplasma phoenicium’‐related strains as also confirmed by the clustering in phylogenetic analysis. This is the first report of 16SrIX‐B phytoplasma presence in a non‐Prunus species, D. mucronata, also highlighting its possible role as reservoir species for this phytoplasma that is severely affecting stone fruits in the Middle East regions.

Clustered Regularly Interspaced Short Palindromic Repeat Analysis of Clonal Complex 17 Serotype III Group B Streptococcus Strains Causing Neonatal Invasive Diseases.

Group B Streptococcus (GBS) is an important pathogen of neonatal infections, and the clonal complex (CC)-17/serotype III GBS strain has emerged as the dominant strain. The clinical manifestations of CC17/III GBS sepsis may vary greatly but have not been well-investigated. A total of 103 CC17/III GBS isolates that caused neonatal invasive diseases were studied using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) loci and restriction fragment length polymorphism (RFLP) analyses. All spacers of CRISPR loci were sequenced and analyzed with the clinical presentations. After CRISPR-RFLP analyses, a total of 11 different patterns were observed among the 103 CRISPR-positive GBS isolates. GBS isolates with the same RFLP patterns were found to have highly comparable spacer contents. Comparative sequence analysis of the CRISPR1 spacer content revealed that it is highly diverse and consistent with the dynamics of this system. A total of 29 of 43 (67.4%) spacers displayed homology to reported phage and plasmid DNA sequences. In addition, all CC17/III GBS isolates could be categorized into three subgroups based on the CRISPR-RFLP patterns and eBURST analysis. The CC17/III GBS isolates with a specific CRISPR-RFLP pattern were more significantly associated with occurrences of severe sepsis (57.1% vs. 29.3%, p = 0.012) and meningitis (50.0% vs. 20.8%, p = 0.009) than GBS isolates with RFLP lengths between 1000 and 1300 bp. Whole-genome sequencing was also performed to verify the differences between CC17/III GBS isolates with different CRISPR-RFLP patterns. We concluded that the CRISPR-RFLP analysis is potentially applicable to categorizing CC17/III GBS isolates, and a specific CRISPR-RFLP pattern could be used as a new biomarker to predict meningitis and illness severity after further verification.

Plant hosts may influence arbuscular mycorrhizal fungal community composition in mangrove estuaries

We investigated the role of plant host and soil variables in determining arbuscular mycorrhizal fungi (AMF) community composition in plant roots of two spatially separated mangrove estuaries on the rivers Aghanashini (14° 30' 30″ N-74° 22' 44″ E) and Gangavali (14° 35' 26″ N-74° 17' 51″ E) on the west coast of India. Both mangrove estuaries had similar plant species composition but differed in soil chemistries.We amplified a 550-bp portion of 18S small subunit (SSU) rDNA from mangrove plant roots and analysed it by restriction fragment length polymorphism (RFLP). Clones representing unique RFLP patterns were sequenced. A total of 736 clones were obtained from roots of seven and five plant species sampled at Aghanashini and Gangavali, respectively. AMF phylotype numbers in plant roots at Aghanashini (12) were higher than at Gangavali (9) indicating quantitative differences in the AMF community composition in plant roots at the two mangrove estuaries. Because both estuaries had similar plant species composition, the quantitative difference in AMF communities between the estuaries could be an attribute of the differences in rhizospheric chemistry between the two sites.Non-metric multidimensional scaling (NMDS) revealed overlap in the AMF communities of the two sites. Three and two AMF phylotypes had significant indicator value indices with specific hosts at Aghanashini and Gangavali, respectively. Environmental vector fitting to NMDS ordination did not reveal a significant effect of any soil variable on AMF composition at the two sites. However, significant effects of both plant hosts and sites were observed on rhizospheric P. Our results indicate that root AMF community composition may be an outcome of plant response to rhizospheric variables. This suggests that plant identity may have a primary role in shaping AMF communities in mangroves.

​Identification of Single Nucleotide Polymorphism (SNP) C-2161G in the Promoter Region of Prolactin Gene and its Association with Egg Production in Tellicherry Native Chicken

Background: Prolactin is a physiological candidate gene which has significant effects on egg production in poultry. Also, it plays a major role on incubation behaviour (broodiness) in birds. The main objective of the present study was identification of single nucleotide polymorphism (SNP) C-2161G in the promoter region of prolactin gene and its association with egg production in Tellicherry native chicken population. Methods: A total of 200 blood samples were collected from the randomly selected birds of Tellicherry native chicken in All India Co-ordinated Research Project on Poultry improvement (AICRP) farm, Mannuthy, Thrissur, Kerala. Isolation of Genomic DNA was done and the isolated samples were subjected to polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis to identify the SNP C-2161G of prolactin gene. Result: On the basis of RFLP patterns, birds were designated with three different genotypes namely CC, CG and GG. The allelic and genotypic frequency was calculated. The observed genotypic frequency at the SNP site C-2161G was CC (0.61), CG (0.160) and GG (0.23) and the frequency of allele was 0.69 for C and 0.31 for G. The egg production was shown to be statistically similar for the genotypes of SNP C-2161G.