Restriction Fragment Length Polymorphism Profiles Research Articles

Antimicrobial growth promoters approved in food-producing animals in South Africa induce shiga toxin-converting bacteriophages from Escherichia coli O157:H7.

In this study, four antimicrobial growth promoters, including virginiamycin, josamycin, flavophospholipol, poly 2-propenal 2-propenoic acid and ultraviolet light, were tested for their capacity to induce stx-bacteriophages in 47 Shiga toxin-producing E. coli O157:H7 isolates. Induced bacteriophages were characterized for shiga toxin subtypes and structural genes by PCR, DNA restriction fragment length polymorphisms (RFLP) and morphological features by electron microscopy. Bacteriophages were induced from 72.3% (34/47) of the STEC O157:H7 isolates tested. Bacteriophage induction rates per induction method were as follows: ultraviolet light, 53.2% (25/47); poly 2-propenal 2-propenoic acid, 42.6% (20/47); virginiamycin, 34.0% (16/47); josamycin, 34.0% (16/47); and flavophospholipol, 29.8% (14/47). A total of 98 bacteriophages were isolated, but only 59 were digestible by NdeI, revealing 40 RFLP profiles which could be subdivided in 12 phylogenetic subgroups. Among the 98 bacteriophages, stx2a, stx2c and stx2d were present in 85.7%, 94.9% and 36.7% of bacteriophages, respectively. The Q, P, CIII, N1, N2 and IS1203 genes were found in 96.9%, 82.7%, 69.4%, 40.8%, 60.2% and 73.5% of the samples, respectively. Electron microscopy revealed four main representative morphologies which included three bacteriophages which all had long tails but different head morphologies: long hexagonal head, oval/oblong head and oval/circular head, and one bacteriophage with an icosahedral/hexagonal head with a short thick contractile tail. This study demonstrated that virginiamycin, josamycin, flavophospholipol and poly 2-propenal 2-propenoic acid induce genetically and morphologically diverse free stx-converting bacteriophages from STEC O157:H7. The possibility that these antimicrobial growth promoters may induce bacteriophages in vivo in animals and human hosts is a public health concern. Policies aimed at minimizing or banning the use of antimicrobial growth promoters should be promoted and implemented in countries where these compounds are still in use in animal agriculture.

Detection and Practical Differentiation of Phytoplasmas from Several Host Plants Using PCR-RFLP

Phytoplasma as a phytopathogenic prokaryote with a wide host range is a pathogen that needs more attention in Indonesia. This pathogen is relatively difficult to detect and identify due to its complicated biological properties. This study involved detection of phytoplasmas by polymerase chain reaction (PCR) technique with P1/P7 primers from seven symptomatic plants, i.e. Bermuda grass white leaf, bamboo yellows, witches’ broom of peanut, soybean, yard long bean, and cactus, and sweet potato little leaf. The phytoplasma DNA of the 16S rRNA gene resulting from PCR amplification was examined by digestion reaction using three endonuclease enzymes AluI, RSaI, and MSeI to generate restriction fragment length polymorphism (RFLP) profile. The seven diseased plants were confirmed positive to be associated with phytoplasma as indicated by the PCR product of 1800 bp. Based on the RFLP profiles of the three enzymes, the phytoplasmas were divided into two groups, namely group I (Bermuda grass and bamboo) and group II (peanuts, soybeans, yard long beans, cactus, and sweet potatoes). Cactus phytoplasma is a sub-group (strain) because it has a slightly different fragment of MSeI RFLP profile.

Molecular characterization of PMQR carrying bacteriophages in effluent discharge samples of Azerbaijan hospitals.

Quinolones are one of the most widely used drugs in medicine. Resistance to this agent has been increased significantly among the nosocomial isolates. The objective of this research was to study generalized transduction, as a potential mechanism for plasmid-mediated quinolone resistance (PMQR) genes acquisition among hospital effluent isolates. Discharge samples from hospital effluent were taken from four medical centers in Azerbaijan. Resident phages were enriched against resident enterobacterial hosts using standard phage enrichment protocols. Polymerase chain reaction (PCR) was used to examine phage stocks and bacterial isolates for the presence of PMQR determinants. All positive bacterial isolates for target genes were subjected to transduction assays. Restriction fragment length polymorphism (RFLP) profiles were determined for cluster analysis. A total of 55 pure phage stocks were prepared from 42 effluents. A total of 95 non-duplicated Gram-negative bacteria were isolated. Thirty-two EcoRV-RFLP profiles were determined for the 40 Escherichia coli phage stocks. Twenty-six of 40 (65%) E. coli phages were positive for qnrB (n = 15), qnrD (n = 7), qnrA (n = 3), and qnrC (n = 2) genes. A total of 34 (35.7%) bacterial isolates were recognized to have any PMQR genes including qnrB (n = 23), qnrD (n = 8), qnrA (n = 5), and qnrC (n = 3) genes. Present research provided a strong evidence for potential role of generalized transduction in persistence and circulation of PMQR genes in health care settings of Azerbaijan.

Molecular Characterization of Fall Armyworm (Spodoptera frugiperda, J.E. Smith) Feeding on Rice (Oryza sativa L.) in Tamil Nadu

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is an invasive insect pest of India native to the Western Hemisphere. The pest damages more than 350 plant species. FAW is genetically divided into two strains viz. corn (C) strain which feeds mostly on maize, sorghum, etc. and the other is a rice (R) strain that prefers rice and other small grasses. In the present study, fall armyworm samples were collected from rice crop in two different places of Tamil Nadu, India during 2019-20 and strain identity was confirmed. Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP) profile of mitochondrial cytochrome oxidase subunit I fragment confirmed the presence of both ‘R’ and ‘C’ strains of FAW in rice crop. Nucleotide variations were observed from PBS, TNAU isolate at five places and two places in Killikulam isolate. Phylogenetic analysis revealed that PBS, TNAU isolate ON247930 (C strain) has completely diverged from Killikulam isolate OM491244 (R strain).

Diversity analysis of BmNPV Isolates Infecting Mulberry Silkworm (Bombyx mori) using Restriction Endonuclease Digestion Profiling

Understanding the genetic diversity of BmNPV isolates causing grasserie viral disease of mulberry silkworm Bombyx mori L. is essential for adoption of management strategies including biotechnological tools. The present study was aimed at the use of restriction fragment length polymorphism (RFLP) profiling for studying the molecular diversity analysis of six BmNPV isolates collected from Devanahalli, Kolar, Shidlaghatta, Hosakote, Tumakuru, and Ramanagara areas in Karnataka, India (BmNPV-Ko, BmNPV-Ho, BmNPV-SG, BmNPV-DH, BmNPV-TM, BmNPV-Ram respectively). DNA was extracted from each of these isolates and subjected to digestion with different restriction enzymes EcoR1, BamH1, Sma1, Nco1, and a combination of BamH1+Nco1 and electrophoresed in 0.8% w/v agarose gel to visualize restriction enzyme profile. The analysis revealed that all the six BmNPV isolates had similar Nco1 and Sma1 restriction patterns, although there was variation in low molecular weight fragments. The EcoR1 and BamH1 restriction patterns were nearly the same for all the isolates except for the presence of an approximately 4kb and an additional 1.5kb polymorphic band only in BmNPV-TM and BmNPV-Ram isolates. BamH1+Nco1 digestion of the DNA from each isolate yielded numerous fragments, which was different in BmNPV-Ram isolate. Molecular diversity analysis can helps in understanding the evolution and phylogeny of the virus, enhance the knowledge on its pathogenicity and can help to develop and adopt suitable measures to combat and diagnose the disease to reduce crop loss and increase income generating ability of the farmer.

The Role of Bacteriophages as Important Reservoirs of Extended-Spectrum Beta-Lactamase Genes in Azerbaijan Hospitals.

Aims: The aim of this study was to investigate the role of resident bacteriophages in hospital effluents, as a potential reservoir of extended-spectrum beta-lactamase (ESBL) genes. Methods: Effluent samples were collected from four major medical centers in Azerbaijan. Phage enrichments were prepared and purified using standard subculturing, amplification, and phage purification protocols. DNA materials from phage stocks and bacterial isolates were examined for the presence of ESBL genes using polymerase chain reaction. Restriction fragment length polymorphism (RFLP) profiles were used for the construction of a dendrogram and cluster analysis. Results: A total of 112 phage enrichments were obtained from 48 effluent samples against resident bacterial hosts. A total of 95 nonduplicate Gram-negative isolates were recovered from effluent samples. The most common isolate was Escherichia coli (n = 48), followed by Klebsiella pneumoniae (n = 18), Pseudomonas spp. (n = 9), and Enterobacter cloacae (n = 6). Thirty-two EcoRV-RFLP profiles consisting of ∼4 to 20 bands were obtained for the 40 E. coli phage enrichments. ESBL genes were detected in 23 of 40 (57.5%) E. coli phage enrichments, including blaCTX-M (n = 15), blaTEM (n = 14), and blaSHV (n = 6). Detected genes in phage enrichments against resident hosts other than E. coli include blaTEM (n = 4), blaCTX-M (n = 3), and blaSHV (n = 1). A total of 63 (66.3%) bacterial isolates were positive for tested genes, including blaCTX-M (n = 32), blaTEM (n = 61), and blaSHV (n = 12). The present research provides a strong evidence for the possible role of bacteriophages in antimicrobial resistance genes circulation in Azerbaijan clinical settings through generalized transduction. Conclusions: Our results showed a remarkable occurrence of ESBL genes in bacteriophage and bacterial population of effluent discharge, which clearly indicates that bacteriophages are an important factor in ESBL genes exchange among bacterial population through generalized transduction.

Equine Herpesvirus Type 4 (EHV-4) Outbreak in Germany: Virological, Serological, and Molecular Investigations.

Equine herpesvirus type 4 (EHV-4) is enzootic in equine populations throughout the world. A large outbreak of EHV-4 respiratory infection occurred at a Standardbred horse-breeding farm in northern Germany in 2017. Respiratory illness was observed in a group of in-housed foals and mares, which subsequently resulted in disease outbreak. Out of 84 horses in the stud, 76 were tested and 41 horses were affected, including 20 foals, 10 stallions, and 11 mares. Virological investigations revealed the involvement of EHV-4 in all cases of respiratory illness, as confirmed by virus isolation, qPCR, and/or serological follow-up using virus neutralization test and peptide-specific ELISA. Among infected mares, 73% (8 out of 11) and their corresponding foals shed the virus at the same time. EHV-4 was successfully isolated from four animals (including one stallion and three foals), and molecular studies revealed a different restriction fragment length polymorphism (RFLP) profile in all four isolates. We determined the complete 144 kbp genome sequence of EHV-4 isolated from infected horses by next-generation sequencing and de novo assembly. Hence, EHV-4 is genetically stable in nature, different RFLP profiles, and genome sequences of the isolates, suggesting the involvement of more than one animal as a source of infection due to either true infection or reactivation from a latent state. In addition, epidemiological investigation revealed that stress caused by seasonal changes, management practices, routine equestrian activities, and exercises contributed as a multifactorial causation for disease outbreak. This study shows the importance of implementing stress alleviating measures and management practices in breeding farms in order to avoid immunosuppression and occurrence of disease.

Whole-Genome Analysis of Mycobacterium avium subsp. paratuberculosis IS900 Insertions Reveals Strain Type-Specific Modalities.

Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of Johne’s disease in ruminants. The IS900 insertion sequence (IS) has been used widely as an epidemiological marker and target for PCR diagnosis. Updated DNA sequencing technologies have led to a rapid increase in available Map genomes, which makes it possible to analyze the distribution of IS900 in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS900 element and how it affects genomic evolution and gene function of Map. A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS900. Complete genomes from the major phylogenetic lineages known as C-type and S-type (including subtypes I and III), were chosen to represent the genetic diversity of Map. IS900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the BstEII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS900 and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The program developed for this study allowed automated localization of IS900 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS900 copies ranged from 16 in the C-type isolate to 22 in the S-type subtype I isolate. A loci-by-loci sequence alignment of all IS900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. Preferential insertion motif sequences were identified for IS900 along with genes bordering all IS900 insertions. Rarely did IS900 insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS900 distribution in Map genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding Map evolution and for studying the biological implications of IS900 insertions.

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera).

Background:Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma.Methods:Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs).Results:Rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species.Conclusion:The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.

Breeding and fruiting evaluation of new strains by the interspecific mating of the high commercial potential mushroom Pleurotus tuoliensis with its closely related P. eryngii

Pleurotus tuoliensis (Bailinggu) is a precious mushroom species cultivated industrially in eastern Asia. The long cropping cycle, high deformity rate, and low yield are the major bottlenecks for its widespread cultivation. There is thus a need for improved P. tuoliensis cultivars. In the present study, interspecific crossing of monokaryons between P. tuoliensis and P. eryngii was carried out. Six out of nine pairings were observed to result in clamp connections. Dikaryotized regions within a mating Petri dish varied considerably with the compatible pairings. A total of nine hybrids were isolated within the junction zone and on the periphery at either sides of the paired colonies from three selected crosses. The internal transcribed spacer (ITS) restriction fragment length polymorphism (ITS-RFLP) profiles, together with ITS sequencing revealed significant genetic differences between the nine selected hybrids with parental strains. The polymerase chain reaction (PCR) amplification revealed that the inheritance of mitochondrial DNA was uniparental between these two species. Of nine crossbred strains, six were selected for fruiting test. Compared with their parental P. tuoliensis strains, all the six tested hybrids fruited faster, and three hybrids showed improved biological efficiency compared to their parents, either P. tuoliensis or P. eryngii. Two hybrids (Ht-26 and He-26) formed a white-colored fruit body, with improved biological efficiency (62.5 %–64.2 %) compared to parents P. tuoliensis (41.8 %) and P. eryngii (52.1 %), and thus hold great potential as new cultivars for the commercial production of P. tuoliensis.

Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

First detection of the kdr mutation (L1014F) in the plague vector Xenopsylla cheopis (Siphonaptera: Pulicidae)

BackgroundThe oriental rat flea, Xenopsylla cheopis, is the most efficient vector of the plague. Pyrethroid insecticides such as cypermethrin, cyhalothrin and deltamethrin have been often used to limit plague transmission via controlling the vector during outbreaks. However, this strategy is threatened by the development of insecticide resistance. Understanding the mechanisms underlying pyrethroid resistance is the prerequisite for successful flea control.MethodsPartial DNA sequences of X. cheopis voltage gated sodium channel (VGSC) gene were amplified from a total of 111 individuals, collected from a natural plague epidemic foci in Baise City, Guangxi Zhuang Autonomous Region of China. These DNA fragments were sequenced. The frequency and distribution of kdr mutations were assessed in four X. cheopis populations. The origin of kdr mutations was investigated by phylogenetic and network analysis.ResultsThe classical knockdown resistance (kdr) mutation (L1014F) was detected in four field populations at frequencies ranging between 0.021–0.241. The mutant homozygote was observed only in one of the four populations. Seven haplotypes were identified, with two of them carrying the resistance L1014F mutation. Phylogenetic tree and network analysis indicated that the L1014F allele was not singly originated. Based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) profiling, an easy-to-use and accurate molecular assay for screening individual fleas for the L1014F mutation was developed.ConclusionsTo our knowledge, this work represents the first report of the L1014F mutation in the plague vector X. cheopis. The incidence of the L1014F allele highlights the need of further studies on the phenotypic effect of this mutation in this plague vector. Early detection and monitoring of insecticide resistance is suggested in order to make effective control strategies in case of plague outbreaks in this region.

Effects of earthworms and arbuscular mycorrhizal fungi on preventing Fusarium oxysporum infection in the strawberry plant

Fusarium wilt is a devastating fungal disease in strawberries caused by Fusarium spp. We aimed to determine the role of earthworms and arbuscular mycorrhizal (AM) fungi in preventing Fusarium oxysporum (Fof) infection in strawberry plants. AM fungi, Fof and the copy number of Actinomyces genes were determined by a quantitative real-time polymerase chain reaction. The Shannon–Wiener index for microbial communities was determined by their terminal restriction fragment length polymorphism profiles. Structural equation modelling was used to establish the relationships between the disease index and abiotic/biotic variables. Earthworms and AM fungi could individually or interactively prevent the infection of strawberry plants by Fof. Earthworms significantly decreased the copy number of Fof in the soil. The AM fungi increased the copy number of Actinomycetes and bacterial diversity and decreased the disease index of Fusarium wilt. Correlation analysis indicated that the root Ca and shoot Mg contents and the number of AM gene copies in plant roots had a significant negative correlation with the disease index of Fusarium wilt and the number of gene copies of Fof in plant roots. The addition of earthworms and AM fungi to soil is a promising biological control method for the prevention of Fusarium wilt in strawberries and acts via an increase in the soil organic matter content, regulation of the soil environment, and improved root (P, Ca, Mg and Fe) and shoot (N, P, K, Ca and Mg) nutrient contents.

Effects of biochar on the growth of apple seedlings, soil enzyme activities and fungal communities in replant disease soil

Apple replant disease (ARD) is a soil-borne disease that negatively effacts tree growth and reduces the yields in replanted orchards. We aimed to determine whether the addition of biochar to replant soil could promote plant growth by improving the soil environment. Biochar was added to apple replant soil at 0, 5, 20, or 80 g kg−1, and its effects on Malus hupehensis Rehd. seedling growth and on the soil environment were determined. Biochar significantly increased seedling height, dry weight, root length, root volume, and respiration rate. Biochar increased the activities of soil enzymes (invertase, urease, proteinase, neutral phosphatase, catalase, and polyphenol oxidase). The terminal restriction fragment length polymorphism (T-RFLP) profiles of fungal communities differed between the 80 g kg-1 biochar treatment and the other treatments. Shannon’s diversity index was highest in the 80 g kg-1 biochar treatment. Biochar decreased the abundance of the soil-borne pathogen Fusarium solani. These effects resulted in enhanced plant growth.

First Report of ‘Candidatus Phytoplasma trifolii’ Associated with a Witches’ Broom Disease in Cannabis sativa in Nevada, U.S.A.

In September 2018, symptoms including leaf curling, mottling, chlorosis, witches’ broom, stunting, and node shortening were detected in Cannabis sativa L. plants at two growing sites in Central and Southern Nevada, U.S.A., respectively. Incidence of the disease varied between 5 and 20% at the growing site in Central Nevada, and 30% of plants were affected at the growing site in Southern Nevada. Symptomatic leaves showed “green islands” that were constrained by the veins on the upper leaf surface, and interveinal discoloration on the lower surface, forming a pale “pustule-like” appearance. One infected plant with 80% of leaves exhibiting symptoms was sampled from the Central Nevada site. One infected plant with 50% of leaves exhibiting symptoms was sampled from the Southern Nevada site. Two nonsymptomatic plants were provided by a local grower in California. Total DNA was extracted from the petioles of the most symptomatic leaves. DNA extracted from nonsymptomatic plants was used as the negative control. A diagnostic TaqMan real-time PCR test was conducted using the universal primer pair JH-F/JH-R (Hodgetts et al. 2009) targeting the 23S rRNA gene. Positive amplification signals were generated only from the symptomatic plants indicating the presence of Phytoplasma species. To identify the species, the 16S ribosomal RNA gene was amplified with the universal primer pairs P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), P1/Tint (Smart et al. 1996), followed by nested PCR primer pair R16F2/R2 (Lee et al. 1993). The anticipated amplicons (1.8, 1.6, and 1.2 kb, respectively) were gel purified and subjected to Sanger sequencing (UC Davis DNA Sequencing Facility). The sequence data were deposited in GenBank (accession no. MK377248). BLASTn results suggested a 99% sequence similarity with ‘Candidatus Phytoplasma trifolii’ (accession no. KF178706.1), a member of 16SrVI group associated with alfalfa witches’ broom disease. iPhyClassifier analysis (Zhao et al. 2013) was conducted to generate a restriction fragment length polymorphism profile using the amplified 16S rRNA gene sequence, revealing the most similarity (98.8%) to ‘C. P. trifolii’. Phytoplasmas of elm yellows group (16SrV) (Zhao et al. 2007) and aster yellows group (16SrI) (Raj et al. 2008), both associated with witches’ broom disease on Cannabis sp., have been reported in China and India, respectively. To our knowledge, this is the first report of 16SrVI group ‘Ca. P. trifolii’ infecting C. sativa in the United States. The identified pathogen may pose a significant threat to the production of C. sativa in the United States.

Identification of Forensically Important Flesh Flies Using the Cytochrome C Oxidase Subunits I and II Genes.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Vitis vinifera leaf yellowing and reddening in the west of Iran

Leaf yellowing and reddening symptoms on Vitis vinifera vines were observed in different grapevine nurseries in the province of Kurdistan in the west of Iran during 2016–2017. Total DNA was extracted from 27 symptomatic and four symptomless plants and tested for the presence of phytoplasma DNA by nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 primer pairs. PCR amplicons of about 1.8 and 1.2 kb in size were obtained from all symptomatic V. vinifera but not from symptomless plants. Restriction fragment length polymorphism (RFLP) profiles of R16F2n/R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII and Sau3AI restriction enzymes were identical to each other and to those obtained from 16SrI phytoplasmas, indicating that the phytoplasmas associated with grapevine yellowing and reddening were members of the 16SrI group. A R16F2n/R16R2 16S rDNA sequence of Kurdistan V. vinifera yellowing and reddening strain (KVvY) was submitted to GenBank as accession number MH638316. The Blast analysis of KVvY showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade closer to the Onion yellows mild strain (OY-M), a 16SrI-B phytoplasma. Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY were identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession AP006628).

First Report of Phytoplasma Infecting Pterocarpus indicus in China

Pterocarpus indicus Willd., an adaptable and fast-growing landscape tree, is commonly cultivated in landscaping in southern China. In April 2018, a P. indicus tree with symptoms of flat branches and typical fasciation, which had the same symptoms of phytoplasma infection, was observed in Haikou, Hainan province, China. To determine the presence of phytoplasma, stem samples of symptomatic and asymptomatic P. indicus were subjected to total DNA extraction using the plant Genomic DNA Kit (Tiangen, Beijing, China), followed by nested polymerase chain reaction (PCR) detection of phytoplasma (Jomantiene et al. 1998; Lee et al. 1998). Briefly, the universal primer pair R16mF2/R16mR1 was employed for the initial PCR amplification of 16SrDNA fragment of phytoplasma and primer set R16F2n/R16R2 (Lee et al. 1998) for nested PCR amplification. As expected, the PCR products were generated from the samples of the symptomatic plant but not from the symptomless control samples. The anticipated amplicon was approximately 1.2 kb in size. The PCR products were gel purified using TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned into pMD-18T vector (Takara Bio, Dalian, China). Three independent positive clones were subjected to Sanger sequencing. The obtained sequence was deposited into NCBI GenBank database with GenBank accession no. MH727702. A BLASTn search against the nonredundant nucleotide database revealed high sequence identities with the corresponding regions of phytoplasmas of aster yellows phytoplasma group (16SrI). A maximum of 99.7% nucleotide sequence identity with Malvastrum coromandelianum phyllody phytoplasma (GenBank accession no. MF490802) was observed. Moreover, an iPhyClassifier (Zhao et al. 2013) operation was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequence derived from the symptomatic P. indicus sample, which showed the most similarity with that of the reference strain (GenBank accession no. AP006628) belonging to 16SrI group. However, this strain may represent a new subgroup because the similarity coefficient was only 0.91, which is within the values set for a new subgroup (Biswas et al. 2013). To our best knowledge, this is the first report of phytoplasma infecting P. indicus in China.

Changes in soil bacterial community structure and microbial function caused by straw retention in the North China Plain

ABSTRACTThe effects of straw retention on soil bacterial community structure, microbial function, and biochemical properties were assessed. Terminal restriction fragment length polymorphism (T-RFLP) and community-level physiological profile (CLPP) assays were used to assess the bacteria community structure and microbial function respectively. Treatments included straw removal with conventional tillage (CT), straw retention with conventional tillage (SRCT) and straw retention with no tillage (SRNT). SRCT and SRNT significantly (p < 0.05) increased soil organic carbon by 8.9% and 9.7%, and microbial biomass carbon by 44.7% and 330.8%, respectively, compared with CT. T-RFLP analysis indicated that straw retention had no favourable effect on soil bacterial diversity, and SRCT significantly (p < 0.05) decreased bacterial diversity compared to CT. Among the three treatments, SRNT had the highest activity of urease, invertase, cellulase, and β-glucosidase. SRCT significantly (p < 0.05) increased the activity of invertase and β-glucosidase compared to CT treatment. CLPP analysis showed that microbial functional diversity was significantly (p < 0.05) increased by straw retention. Enzyme activity and microbial functional diversity were not correlated with bacterial diversity. Therefore, according to this study, SRNT is a better farming practice because it improves soil fertility and biological quality.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.