Tuf Gene Research Articles

First Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of India

HomePlant DiseaseVol. 104, No. 3First Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of IndiaSurabhi Mitra, Prasenjit Debnath, Amar Bahadur, Suken Chandra Das, Kiran Kirdat, Amit Yadav, and G. P. RaoSurabhi MitraDivision of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, IndiaSearch for more papers by this author, Prasenjit DebnathCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Amar BahadurCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Suken Chandra DasCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Kiran KirdatNational Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, IndiaSearch for more papers by this author, Amit Yadavhttp://orcid.org/0000-0002-4882-9075National Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, IndiaSearch for more papers by this author, and G. P. Rao†Corresponding author: G. P. Rao; E-mail Address: gprao_gor@rediffmail.comhttp://orcid.org/0000-0003-1057-7826Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, IndiaSearch for more papers by this author AffiliationsAuthors and Affiliations Surabhi Mitra1 Prasenjit Debnath2 Amar Bahadur2 Suken Chandra Das2 Kiran Kirdat3 Amit Yadav3 G. P. Rao1 † 1Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India 2College of Agriculture, Tripura, Lembucherra, West Tripura 799210, India 3National Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, India Published Online:30 Dec 2019https://doi.org/10.1094/PDIS-08-19-1715-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Cashew nut (Anacardium occidentale L.), an evergreen perennial tree, is an economically important cash crop extensively used in many Indian desserts and cuisines. Cashew nuts are used for their nutritional value and are a major source of protein, fats, vitamins, oleic acid, and palmitic acid. Cashew nut cultivation is undertaken in 17 states of India covering an area of 1.04 Mha. India is the second-largest cashew nut producer in the world, producing about 817,000 MT of raw cashew nuts per annum (https://cashew.icar.gov.in). During a survey in June 2018 of cashew nut orchards in Nagicherra (latitude N 23° 48′ 37.6″; longitude E 91° 19′ 29.7″), West Tripura, India, cashew nut plants were found exhibiting deformed nuts and inflorescence decline (CaID) with prevalence of 5.6% (in a 1-ha area), which were suggestive of phytoplasma infection. Three symptomatic and asymptomatic cashew nut samples were collected, and DNA was extracted from tender shoots using CTAB. To detect the association of phytoplasma with symptomatic cashew nut samples, the isolated genomic DNA samples were assayed for amplification of 16S rRNA region using phytoplasma-specific primer pairs, namely, P1/P7 followed by nested PCR primers R16F2n/R16R2. The 16S rRNA gene of phytoplasma was detected in all collected symptomatic CaID samples, but no amplification was observed in asymptomatic samples. The obtained nested PCR fragments of ∼1.25 kb were sequenced directly (MK076296, MK729150) which showed 99.8% sequence identity with ‘Candidatus Phytoplasma australasia’, strain ‘Carica papaya’ (Y10097). The virtual restriction fragment length polymorphism analysis of 16S rRNA gene sequences delineated the CaID strain to 16SrII-D subgroup with a similarity coefficient of 1.0 using iPhyClassifier. The association of CaID phytoplasma strain with symptomatic cashew nut samples was further established by amplifying phytoplasma-specific partial secA gene (Hodgetts et al. 2008) and tuf gene (Al-Subhi et al. 2018). The phylogenetic analysis of the obtained sequences of 16S rRNA, secA (MK726365, MK726366), and tuf (MK726367, MK726368) genes confirmed the close association of CaID phytoplasma isolates with ‘Ca. P. australasia’ 16SrII-D subgroup member strains. To the best of our knowledge, this is the first report of the association of ‘Ca. P. australasia’ (16SrII-D) with inflorescence decline symptoms of cashew nut in the world. This is significant because phytoplasma strains of 16SrII-C and D have widespread host range including several fruit trees all across India (Rao et al. 2017). These strains are also known to be associated with diseases in weeds, acting as alternate hosts, spreading phytoplasmas through polyphagous insect vectors (Thorat et al. 2016). Likewise, the cashew plants are intercropped in coconut plantations and with plants like maize, groundnut, cowpea, millets, pineapple, and cassava, which are known to be susceptible to phytoplasma-related diseases, posing a threat to cashew nut plantations (Rao et al. 2017; Visalakshi et al. 2015).The author(s) declare no conflict of interest.

Technical note: Development of multiplex PCR assays for the molecular characterization of Streptococcus uberis strains isolated from bovine mastitis

Streptococcus uberis is an important causative agent for clinical and subclinical mastitis in dairy cattle. The aim of this study was to develop 2 multiplex PCR assays (mPCR) for the simultaneous detection of virulence factors and housekeeping genes for use when investigating the genetic variability and distribution of Strep. uberis virulence factors. The tuf, cpn60, pauA, sodA, sua, oppF, and gapC genes were grouped in assay 1 (mPCR1) and the hasA, hasB, and hasC genes were included in assay 2 (mPCR2). The detection limits were 11.8 pg and 5.9 pg of DNA for mPCR1 and mPCR2, respectively. The 2 mPCR assays were validated with 56 Strep. uberis strains isolated from mastitis milk samples collected from different bovine herds in northern Italy. Results revealed that gapC and oppF were detected in 98.2% of the strains, whereas sua and hasC genes were detected in 94.6 and 89.2% of the strains, respectively. The most common pattern was gapC+, oppF+, cpn60+, sua+, sodA+, pauA+, tuf+, hasA+, hasB+, and hasC+, which appeared in 59% of the strains analyzed. The molecular assays developed in the present study represent a powerful tool for the evaluation of virulence pattern distribution in Strep. uberis strains associated with intramammary infections.

Epidemiological analysis of Trueperella abortisuis isolated from cases of pig abortion of a single farm.

The present study was designed to characterize six Trueperella (T.) abortisuis strains, cultured over a period of 5 months from fetus and abortion material of six pigs of a single farm in Mecklenburg-West Pomerania federal state, Germany. It was of interest to investigate the epidemiological relationships of the six strains among each other and whether a single bacterial clone was responsible for the abortion situation of the single farm. All six strains were identified phenotypically, by MALDI-TOF MS analysis and by phylogenetic analysis based on 16S rRNA gene and gap (encoding the glyceraldehyde 3-phosphate dehydrogenase) and tuf (encoding elongation factor tu) gene sequencing. Further genotypic comparison was performed using different genomic DNA fingerprint methods including BOX-PCR, (GTG)5-PCR, and three RAPD-PCRs. The sequence analysis of the genes gap and tuf and the genomic DNA fingerprinting results revealed, as noval findings, that the six T. abortisuis strains cultured from a single farm represent six different bacterial clones showing a genetic variability of this bacterial species in the pig population. All six T. abortisuis strains were isolated in mixed culture with several other bacterial species. However, the T. abortisuis strain, generally found in high numbers, seemed to be responsible for the abortion situation in the farm.

Multilocus gene-specific characterization of 'Candidatus Phytoplasma australasia' associated with shoot proliferation disease of small cardamom in India.

Symptoms of excessive shoot proliferation were observed in the Njallani cultivar of small cardamom accompanied by stunting of stalks with fewer degenerated capsules at Nedumkandam Panchayat of Idukki district of Kerala in 2017. Five symptomatic Elettaria cardamomum shoot proliferation (ECSP) plant samples were collected and processed for DNA extraction and PCR assays utilizing universal phytoplasma 16S ribosomal-specific primers pair, P1/P7 followed by R16F2n/R16R2. Sequence comparison analysis of the R16F2n/R16R2 region of 16SrRNA gene showed 100% sequence identity with the 'Candidatus Phytoplasma australasia'- related strain. Phylogeny and virtual RFLP analyses of 16S rRNA gene sequences confirmed the association of 'Ca. P. australasia' strain subgroup D with ECSP disease. The association of 16SrII group was further established and validated by amplifying phytoplasma-specific multilocus candidate genes by utilizing specific primers of secA, secY, SAP11, and tuf genes. The multilocus gene sequence comparison analysis again confirmed the association of 'Ca. P. australasia' with the ECSP phytoplasma isolate. This is the first report of phytoplasma association with small cardamom.

First Report of Strawberry (Fragaria × ananassa) as a Host of a ‘Candidatus Phytoplasma americanum’-Related Strain in the United States

In June 2018, while conducting a survey for possible presence of phytoplasma pathogens in Pennsylvania production fields, a strawberry plant (Fragaria × ananassa) was observed with symptoms of stunting and unseasonal reddening and distortion of leaves. Only one out of 500 strawberry plants expressed the described symptoms in a farm in Perry County, PA. Several leaves and stems were collected from the symptomatic plant as well as from a few asymptomatic ones for further work. Total DNA was extracted from 100 mg of leaf midveins with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). All DNA samples were initially tested using a phytoplasma universal real-time polymerase chain reaction (PCR) assay (Hodgetts et al. 2009). Positive amplification results were obtained only with samples derived from the symptomatic strawberry plant. To detect and identify the suspected phytoplasmal pathogen strain(s), the DNA samples were used as a template in conventional PCRs for amplification of phytoplasma 16S rRNA, secY, secA, and tuf gene sequences as previously described (Dickinson and Hodgetts 2013; Lee et al. 2004, 2010; Makarova et al. 2012). All of the obtained amplicons were of expected sizes, and no amplification was observed from the asymptomatic strawberry samples. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen) and sequenced. Nucleotide sequences of the PCR products were deposited in the GenBank database under accession numbers MN227133 to MN227136. The nucleotide sequences were analyzed using Geneious software (Biomatters, Auckland, NZ) for sequence alignments and using iPhyClassifier (Zhao et al. 2009) for group-subgroup and ‘Candidatus Phytoplasma’ species assignments. The classification into 16Sr groups and subgroups was established by virtual restriction fragment length polymorphism (RFLP) analysis of PCR products compared with reference strains. The phytoplasma, termed strain SRL1-PA (Strawberry Red Leaf1-PA), was found to share 99.9% similarity with that of the ‘Candidatus Phytoplasma americanum’ reference strain (GenBank accession DQ174122). The virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is most similar to the reference group XVIII, subgroup A (GenBank accession DQ174122), with a pattern similarity coefficient of 0.98. Another sample from the same strawberry plant was independently determined positive for ‘Ca. P. americanum’ by CPHST Beltsville Lab using nucleotide sequence analysis of 16S rRNA gene sequences amplified in end point PCR. Alignments and phylogenetic analyses of secY, secA, and tuf gene sequences confirmed the close relatedness of the Pennsylvania SRL1-PA strain with the reference strains of ‘Ca. P. americanum’. ‘Ca. P. americanum’ was previously reported (Lee et al. 2006) in Texas and Nebraska on potato and associated with potato purple top wilt disease. In 2019, the Pennsylvania Department of Agriculture team revisited the same farm and collected additional samples including some from potato but was not able to detect any phytoplasma. To our knowledge, this report provides the first evidence for the presence of a ‘Ca. P. americanum’-related strain in Pennsylvania and more importantly describes strawberry (Fragaria × ananassa) as a new host for this phytoplasma. With only one single positive strawberry plant in a single year, the true extent of disease spread on strawberries remains undetermined.

Molecular detection and identification of Brassica juncea phyllody and witches’ broom-associated phytoplasmas of group 16SrVI-A in Inner Mongolia, China

In September 2017, oilseed mustard (Brassica juncea) showing symptoms of phyllody and witches’-broom, indicative of phytoplasma infection, was observed in Ordos, Inner Mongolia, China. Using universal primers P1/P7, the fragment encompassing phytoplasmal 16S rRNA, 16S–23S internal transcribed spacer region and partial 5′ end of 23S rRNA genes was amplified; the ribosomal protein (rp) genes (rplV-rpsC region) and the partial elongation factor TU (tuf) gene of phytoplasmas were amplified using primer pairs rpF1C/rp(I)R1A and fTufu/rTufu, respectively. The 16S rRNA, rp and tuf phylogenies suggested that both the B. juncea phyllody and witches’-broom associated phytoplasmas are closely related to members of 16SrVI (clover proliferation) group. Based on in silico restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the detected phytoplasmas were classified into 16SrVI-A subgroup. The in silico RFLP pattern is consistent with the actual RFLP pattern of the 16S rRNA genes. This was the first record of 16SrVI-A phytoplasma infecting B. juncea in China, probably the first case of phytoplasma on oilseed mustard as well.

Rapid detection and identification of ‘Candidatus Phytoplasma pini’‐related strains based on genomic markers present in 16S rRNA and tuf genes

AbstractIn order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma.

Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.

Detection and identification of 16SrXIII-F and a novel 16SrXIII phytoplasma subgroups associated with strawberry phyllody in Chile

Since the year 2015, strawberry plants with symptoms of phyllody, achenes’ hypertrophy and leaf reddening have been observed in several fields in various regions of Chile. In recent surveys on five individual fields in Biobio and Araucania regions, samples were collected from symptomatic plants and DNA was extracted. Two genomic regions corresponding to the 16S rRNA and tuf genes were amplified by nested PCR using phytoplasma universal primers. Three individual clones of each amplicon from each sample were then sequenced. The RFLP analyses of the 16S rDNA amplicons showed that all of the symptomatic plants were infected by phytoplasmas enclosed in the 16SrXIII group. This result was confirmed by phylogenetic analysis with tuf amplicons. RFLP similarity coefficients indicated the presence of two phytoplasmas, one belonging to the 16SrXIII-F subgroup, while the other resulting not enclosed in any of the described subgroups and therefore it was assigned to the newly proposed 16SrXIII-K subgroup.

Molecular Typing of 'Candidatus Phytoplasma solani' in Iranian Vineyards.

Grapevine (Vitis vinifera L.) is one of the most important horticultural crops in Iran, with >200,000 ha of cultivated area. Recently, outbreaks of the grapevine yellows Bois noir that is associated with phytoplasma strains related to 'Candidatus Phytoplasma solani' were recorded in several Iranian regions. This has resulted in severe economic losses. We carried out a survey in 2015, followed by collection of leaf samples from symptomatic grapevines and weeds. Because no information is available on the molecular epidemiology of 'Ca. P. solani' in Iran, multiple gene analyses were carried out here according to molecular characterization of the tuf and vmp1 genes. From the molecular characterization, all of the samples (i.e., grapevines, weeds) were infected with tuf b type. Detailed molecular characterization of the vmp1 gene (i.e., PCR-restriction fragment length polymorphism, sequence analysis) defined five molecular types: V1, V4, V10, V15, and V20. The abundance of Convolvulus arvensis in vineyards and detection of the same 'Ca. P. solani' molecular types in grapevines and weeds suggest that C. arvensis has a major role in Bois noir epidemiology of Iranian vineyards. Therefore, control strategies should be developed to manage these host plants to reduce inoculum sources of the phytoplasma in vineyards.

First Report of ‘Candidatus Phytoplasma ulmi’ on Elm in Belgium

In April 2017, the project ‘Belgian Plant Sentinel Network’ started as part of the Euphresco-IPSN2 network. The botanical gardens and arboreta of the network contain diverse plant collections with both exotic and native taxa that can be used as sentinels by recording their sensitivity to either native or to (newly) introduced pests and diseases. One of the case studies focused on monitoring phytoplasma diseases in trees, especially on Ulmus spp. To monitor these species, a total of 117 root samples were taken from collection trees in five botanical gardens and arboreta in Flanders, Belgium. Total genomic DNA was extracted by a CTAB protocol (Doyle and Doyle 1990). The 16S rRNA gene was partially amplified using the phytoplasma universal primer pairs P1/P7 (Deng and Hiruki 1991) followed by nested PCR with primer pairs R16F2/R2 (Lee et al. 1995) as diagnostic test, and P1/Tint (Smart et al. 1996) on positive samples for sequencing reasons. Additionally, specific group V nPCR targeting the secY (primer sets FD9f2L/FD9r followed by FD9f3L/FD9r2L) gene, according to Arnaud et al. (2007), was done to yield a nested PCR product of 1,174 bp, and finally, a short fragment from the tuf gene was generated following the Q-Bank methodology for Q-phytoplasma (www.Q-bank.eu). In one location in Vlaams Brabant, one sample, roots from an Ulmus minor subsp. canescens tree (∼8 years old) tested positive for the presence of a phytoplasma. The sample was taken in March 2018 (early spring) and only consisted of roots. An amplified fragment of ∼1,600 bp (P1/Tint) was gel purified (SmartPure, Eurogentec) and directly sequenced (Macrogen, Amsterdam, the Netherlands). The obtained partial sequence of the 16S ribosomal RNA gene (1,516 bp) was submitted to GenBank (accession MH583020.2). This sequence revealed the closest homology (99%) with ‘Candidatus Phytoplasma ulmi’ strains from the U.S.A. (AF189214.1) and Czech Republic (EU184021). In addition, the secY and tuf fragment were directly bidirectionally sequenced and the sequences of the resulting contigs, 1,027 bp (MH727566) and 367 bp (MK491655), respectively, were also submitted to GenBank. Phylogenetic analysis was undertaken (MEGA version 7.0.26) on the 16S, secY, and tuf fragments, indicating the phytoplasma to be a member of ‘Ca. P. ulmi’, 16SrV subgroup A, and showing the genetic relationship with other ‘Ca. P. ulmi’ isolates previously reported in Europe. Virtual RFLP on the 16S fragment using iPhyClassifier (Zhao et al. 2009) confirmed this result. All other Ulmus trees from the five botanical gardens and arboreta, as well as related Zelkova trees (Z. carpinifolia and Z. serrata) and a Celtis aetnensis tree, tested negative. In addition, the phytoplasma survey on elm has expanded to the complete affected botanical garden (28 Ulmus or related trees) and to lane trees from the surrounding area. A second sampling of the same tree in May 2018 confirmed the presence of ‘Ca. P. ulmi’ on new root and leaf samples. From July onward, yellowing and partial branch decay could be observed on the affected tree. All additional samples from the botanical garden and surrounding area tested negative. To our knowledge, this is the first report of ‘Ca. P. ulmi’ on Ulmus in Belgium. Considering the other recent findings in Europe, we suspect that ‘Ca. P. ulmi’ is likely to be distributed throughout the region, but with few previous reports, rather than being a new emerging risk.

Identificación genotípica de Staphylococcus con fenotipo meticilino resistente aislados de muestras de humanos, animales y ambiente

El objetivo del estudio fue dentificar el gen mecA-1 de aislados de Staphylococcus con fenotipo meticilino resistente (SMR) obtenidos de muestras biológicas y superficies de una clínica veterinaria. Se seleccionaron nueve aislamientos con fenotipo SMR clasificado por el sistema automatizado VITEK. El género y la variabilidad se corroboraron por amplificación y secuenciación de los genes 16S rRNA y tuf. También se estableció la presencia del gen mecA-1 por PCR. En los nueve aislamientos con fenotipo SMR se identificaron los genes 16S rRNA y tuf, evidenciando un 99% de similitud con cepas de referencia; sin embargo, solo en cinco muestras se encontró el gen mecA-1. Los resultados demuestran una mayor identificación del genotipo MR en superficies intrahospitalarias, lo que evidencia la necesidad del control y seguimiento de estas cepas en hospitales veterinarios debido a las implicaciones que tienen en salud pública.

Mechanism of action of the moonlighting protein EfTu as a Substance P sensor in Bacillus cereus

The striking feature of the ubiquitous protein EfTu (Thermo unstable ribosomal Elongation factor) is its moonlighting (multifunctional) activity. Beyond its function at the ribosomal level it should be exported to the bacterial surface and act as an environmental sensor. In Bacillus cereus, and other cutaneous bacteria, it serves as a Substance P (SP) receptor and is essential for bacterial adaptation to the host. However, the modus operandi of EfTu as a bacterial sensor remains to be investigated. Studies realized by confocal and transmission electron microscopy revealed that, in the absence of an exogenous signal, EfTu is not exposed on the bacterial surface but is recruited under the effect of SP. In addition, SP acts as a transcriptional regulator of the tuf gene encoding for EfTu. As observed using gadolinium chloride, an inhibitor of membrane mechanosensitive channels (Msc), Msc control EfTu export and subsequently the bacterial response to SP both in terms of cytotoxicity and biofilm formation activity. Microscale thermophoresis revealed that in response to SP, EfTu can form homopolymers. This event should occur after EfTu export and, as shown by proteo-liposome reconstruction studies, SP appears to promote EfTu polymers association to the membrane, leading subsequently to the bacterial response. Molecular modeling suggests that this mechanism should involve EfTu unfolding and insertion into the bacterial cytoplasmic membrane, presumably through formation of homopolymers. This study is unraveling the original mechanism action of EfTu as a bacterial sensor but also reveals that this protein should have a broader role, including in eukaryotes.

Multilocus gene characterization of phytoplasmas in 16SrII-D subgroupassociated with Coreopsis grandiflora little leaf disease in India

In September 2018 symptoms of little leaf were observed in Coreopsis grandiflora located in flower nurseries at IARI campus, New Delhi. Little leaf and phyllody symptoms were also observed on nearby growing ornamental hedge plants belonging to Duranta genus and in a weed (Parthenium hysterophorus). Amplicons of ∼1.25 kb were consistently amplified with nested PCR assays using DNA extracted from the symptomatic Coreopsis, Duranta and Parthenimum samples, while no amplification was observed with any of the asymptomatic samples of the same species. Leafhoppers (Empoasca sp.) collected from the nurseries of Coreopsis were also found phytoplasma positive using the same nested PCR protocol. BLAST and pair wise sequence comparison of the R16F2n/R2 region of 16S rRNA gene of C. grandiflora little leaf (CGLL), Duranta sp. little leaf (DLL), P. hysterophorus phyllody (PHP) and Empoasca sp (Em) revealed 99.8% sequence identity with the ‘Candidatus Phytoplasma australasia’ sequence. Phylogeny and virtual RFLP analysis with iPhyclassifier confirmed the affiliation to ‘Ca. P. australasia’, subgroup 16SrII-D of all these phytoplasma strains. Amplicons of the expected size were also consistently amplified from these strains with primers specific to 16SrII group of SAP11, imp and tuf genes. All the amplified products were sequenced and confirmed the phytoplasma affiliation to 16SrII-D subgroup. This is the first report of 16SrII-D group phytoplasma presence in C. grandiflora showing little leaf disease.

First report of ‘Candidatus Phytoplasma rubi’ on blackberry in Belgium

In May 2018 stunted blackberry plants were observed on a small-scale organic farm producing several small fruit types in West Flanders, Belgium. Besides severe stunting, leaf yellowing and distortions could also be observed (Figs. 1-2). Only Rubus fruticosus plants of the cultivar Obsidian showed symptoms but disease incidence was estimated to be almost 100%, of which almost half the plants were stunted. Other cultivars did not seem affected. Seven symptom-bearing plants, of which four had severe stunting, and three only leaf symptoms, were sampled and tested. Additionally, three symptomless plants from neighbouring rows and belonging to different cultivars, were tested. All samples consisted of leaves and roots, and tests were conducted on a pooled leaf/root sample. Total genomic DNA was extracted by a CTAB protocol. The 16S rRNA gene was partially amplified using the phytoplasma universal primer pair P1/P7 (Deng & Hiruki, 2; Schneider et al., 5) followed by a nested PCR with R16F2/R2 primers (Lee et al., 3) as a diagnostic test, and P1/Tint primers (Smart et al., 6) on positive samples for sequencing. All of the diseased plants tested positive for phytoplasma in nested PCR tests. Additionally, specific phytoplasma group V primers targeting the tuf (FDTUF-F1/R1 & FDTUF-F2/R2; Malembic-Maher et al., 4), and secY (primer sets FD9f2L/FD9r & FD9f3L/FD9r2L; Arnaud et al., 1) genes were used in nested PCRs and yielded fragments of 998 and 1174 bp respectively. The obtained 16SrRNA (P1/Tint), tuf and secY fragments were gel purified (SmartPure, Eurogentec), and sequenced directly (Genewiz, Leipzig, Germany). BLAST analysis of the 16SrRNA sequence, revealed the highest identity (99.85%) with a ‘Rubus stunt phytoplasma’ strain from blackberry (R. fruticosus) in Italy (isolate RuS400; GenBank Accession No. AY197649). Partial sequence of the 16S ribosomal RNA (1607 bp), and tuf (835 bp) and secY (1053 bp) genes were submitted to GenBank (MH801133, MH809672 and MH809673, respectively). Phylogenetic analysis was undertaken on all three fragments, and the phytoplasma was identified as ‘Candidatus Phytoplasma rubi’, ‘elm yellows’ group 16SrV-E (Figs. 3-5). To our knowledge, this is the first report of ‘Ca. P. rubi’ on blackberry in Belgium. An additional nationwide survey is needed to assess the phytopathological impact of this outbreak. Although a growing number of reports indicate the increased importance of Rubus stunt, associated with ‘Ca. P. rubi’, unlike the related ‘Ca. P. ulmi’, associated with elm trees, no regulatory actions are currently taken against this pathogen.

First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L

HomePlant DiseaseVol. 103, No. 1First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/LA. Zwolińska, N. Borodynko-Filas, D. Nowaczyk, and B. Hasiów-JaroszewskaA. Zwolińska†Corresponding author: A. Zwolińska; E-mail: E-mail Address: a.zwolinska@iorpib.poznan.plhttp://orcid.org/0000-0003-2681-2915Search for more papers by this author, N. Borodynko-FilasSearch for more papers by this author, D. NowaczykSearch for more papers by this author, and B. Hasiów-JaroszewskaSearch for more papers by this authorAffiliationsAuthors and Affiliations A. Zwolińska † N. Borodynko-Filas D. Nowaczyk B. Hasiów-Jaroszewska , Institute of Plant Protection – National Research Institute, Virology and Bacteriology Department, Poznań, Poland. Published Online:31 Oct 2018https://doi.org/10.1094/PDIS-06-18-0963-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In July 2016 and 2017, diseased plum trees (Prunus domestica L.) were observed in the orchard localized in the Wielkopolska region of Poland. The symptoms of witches’ brooms with internode shortening, axillary bud growth, and reduced leaf size indicated a potential phytoplasma infection. Samples of shortened shoots were collected from five symptomatic and five asymptomatic plants, respectively. DNA samples were extracted from 2 g of leaf tissues using a modified cetyltrimethylammonium bromide procedure (Maixner et al. 1995) and subsequently used as templates in nested polymerase chain reaction (PCR) for the amplification of genes encoding 16S rRNA; ribosomal proteins (rp) S19, L22, and S3 and elongation factor Tu (tuf) with primers P1/P7 followed by R16F2n/R16R2; rpF1/rpR1 followed by rp(I)F1A/rp(I)R1A and fTuf1/rTuf1 followed by fTufAY/rTufAY, respectively (Duduk et al. 2013; Martini et al. 2007; Schneider et al. 1997). Amplicons of correct sizes (1.2, 1.2, and 0.9 kb) were obtained from all the diseased plants tested (samples Plum1 to Plum5), respectively. No PCR products were obtained from the asymptomatic samples. All PCR products were ligated into pGEM-T Easy Vector Systems (Promega), and plasmid DNA was sequenced by an external company (Genomed, Warsaw, Poland). The 16S rDNA, tuf, and rp genes coding sequences from Plum1 to Plum5 were identical, and therefore one representative sequence of each region was deposited in GenBank under accession numbers MH061193, MH061368, and MH061366, respectively. The R16F2n/R16R2 (MH061193) primed fragment was subjected to in silico restriction digestion using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The collective restriction fragment length polymorphism patterns indicated that diseased plants were infected by ‘Candidatus Phytoplasma asteris’ of subgroup 16SrI-L. Next, the 385-bp barcode fragments of the EF-Tu gene from representatives of different phytoplasma groups were retrieved from the GenBank database. The phylogenetic analysis was performed based on those sequences and the tuf sequence obtained in this study using the neighbor joining algorithm of MEGA 7 software (https://www.megasoftware.net/). The 1.2-kbp DNA segment containing rp genes was also analyzed. In the phylogenetic trees, the Polish isolate of plum witches’ broom phytoplasma clustered together with ‘Ca. P. asteris’ representatives of ribosomal subgroup 16SrI-B and 16SrI-B/L. Based on all the performed analyses, the plum witches’ broom phytoplasma (PlumWB) was classified as a member of group 16SrI, subgroup B/L, which consists of two heterogeneous 16Sr RNA genes (Jomantiene et al. 2010). To our knowledge, this is the first report of plums infected with ‘Ca. P. asteris’ representing subgroup I-B/L in Poland, suggesting that fruit trees can be perennial reservoirs of the phytoplasma, which has great significance in the pathogen’s epidemiology.

Reduction of hexavalent chromium by Bacillus spp. isolated from heavy metals-polluted soil

Hexavalent chromium, Cr(VI), one of the major pollutants from industrial facilities, is very toxic and harmful for human health and environmental quality. Due to the lack of conventional methods, bioremediation was recommended as an environmentally friendly and effective technique. The aim of this paper was the isolation, identification and selection of the microorganisms which are capable of Cr(VI) reduction in vitro. Heavy metal concentration, detected in four soil samples, within and around the former bicycle factory ?Rog? (Republic of Slovenia), was measured using the ICP-OES method. Bacteria were isolated and tested for chromium tolerance using LB agar supplemented with various Cr(VI) concentrations, whilst Cr(VI) reduction and bacterial growth was determined using the LB liquid medium. From 53 bacterial isolates, five of them showed a tolerance of 1000 mg/L of Cr(VI). Those five isolates showed the capability of growth under various Cr(VI) concentrations (50-1000 mg/L). Initial Cr(VI) concentrations ranging from 50 to 100 mg/L were completely reduced by four bacterial isolates, whilst 500 to 1000 mg/L by Bacillus safensis 342-9. Using 16S rDNA and tuf gene sequence analyses, isolates 270-9R and 342-9 were identified as Bacillus safensis, isolates 351-9 and 270-9C as Bacillus subtilis subsp. subtilis, and 212-9 as Bacillus thuringiensis. These results indicated that these bacteria may be promising tools for remediation of metal-polluted sites.

Biofilm Formation of Methicillin-resistant Coagulase-Negative Staphylococci Isolated from Clinical Samples in Northern Thailand.

Background:Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are multidrug-resistant bacteria that are difficult to treat because of their ability to form biofilms.Objectives:In the present study, we evaluated the antibiotic-resistant phenotypes, biofilm-forming ability, and biofilm associated genes of 55 clinical MR-CoNS isolates obtained from two hospitals in Thailand.Materials and Methods:MALDI-TOF-MS and tuf gene sequencing were performed to determine the species of all isolates. Biofilm production was determined using Congo red agar (CRA) and the microtiter plate (MTP) assay. Biofilm-associated genes were characterized using polymerase chain reaction (PCR).Results:Among the 55 MR-CoNS isolates, five species were identified as Staphylococcus haemolyticus (34.5%), Staphylococcus epidermidis (32.7%), Staphylococcus capitis (18.2%), Staphylococcus cohnii (9.1%), and Staphylococcus hominis (5.5%). The antimicrobial susceptibility pattern of MR-CoNS isolates indicated high resistance to cefoxitin (100%), penicillin (98.2%), erythromycin (96.4%), ciprofloxacin (67.3%), sulfamethoxazole/trimethoprim (67.3%), gentamicin (67.3%), and clindamycin (63.6%). All the isolates were susceptible to vancomycin and linezolid. The biofilm production was detected in 87.3% isolates through the CRA method and in 38.1% isolates through the MTP assay. The prevalence rates of icaAD, bap, fnbA, and cna were 18.2%, 12.7%, 47.3%, and 27.3%, respectively. There were significant differences in the presence of these biofilm-associated genes among the MR-CoNS isolates. Moreover, quantitative biofilm formation was significantly different among MR-CoNS species.Conclusion:The present study revealed that biofilm-associated genes are important for biofilm biomass in MR-CoNS isolates, and the findings of this study are essential for finding new strategies to control biofilm formation and prevent the spread of MR-CoNS infectious diseases.

Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis

ABSTRACT: We compared the potential of routine techniques used for the identification of Staphylococcus species, aiming to evaluate their accuracy in the detection of 43 Staphylococcus chromogenes strains isolated from bovine mastitis that, despite being a coagulase-negative species, are able to clot plasma. These strains could be mistakenly suspected to be S. aureus and lead to an unappropriated treatment of the disease. MALDI-TOF, PCR-RFLP of the chaperonine gene groEL, and sequencing of the 16S rRNA and elongation factor Tu gene tuf were employed. Results from the four methods were coincident for only half of the strains because of the low accuracy of the groEL PCR-RFLP (51.2% accuracy). Even though all the sequencing results were identical, the high accuracy of the MALDI-TOF results (97.7% accuracy, with only one strain misidentified) encourage the use of this technique, since it does not require laborious sample preparation, being fast and simple to perform.

Molecular detection of Escherichia coli in Wadi Hanifa, Saudi Arabia

Background: The threat of controlling water borne infectious diseases was always a challenge for health authorities because water related diseases are a major cause of morbidity and mortality worldwide. Faecal coliforms such as Esherichia coli (E. coli) are the indicators most commonly analysed to evaluate the level of faecal contamination in water. Objectives: The study aimed in the first part to compare different techniques for the best detection of E. coli from water. The chosen method was applied in the second part on samples from Wadi Hanifa and neighboring wells. Colonies considered as E. coli were subjected to single colony PCR targeting the genes hha (indicator of enteric bacteria) and tuf (indicator of E. coli), in order to determine if there is a correlation between positive results given with both genes, thus the gene hha could be used as an indicator of fecal contamination in water. Material and methods: 3 techniques of water concentration for the detection of E. coli in artificially contaminated tap water with K12 (E. coli) were compared: (i) The European directive ISO 9308-1 (ii) Ultrafiltration method (UF) and (iii) Membrane filtration followed by direct isolation of genomic DNA. The best technique was applied for E. coli detection in 156 samples collected monthly and during one year from Wadi Hanifa and neighboring wells in Riyadh. Presumptive E. coli strains were subjected to single colony PCR targeting the genes hha and tuf. Results: The comparison of the three techniques has shown that the ISO 9308-1 was more reliable, accurate and economic than the other two tested techniques. Ten (10) E. coli strains (6.41%) were found exclusively in the surface water of Wadi Hanifa whereas; no E. coli strain was detected in wells, fortunately. All E. coli strains tested positive for the gene tuf, were found also positive for the gene hha. Conclusion: The ISO 9308-1 was the technique of choice for the detection and enumeration of coliforms and E. coli in water samples. A good correlation was found between the two genes suggesting that the gene hha could be used as indicator of fecal contamination in water samples.