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Mucormycosis is a type of opportunistic fungal infection caused by the Mucorales order of Zygomycetes. The study's goal was to characterize Lichtheimia and other fungal agents in Tekirdağ province of Türkiye by morphological and molecular methods. Head hair and skin scrapings of 13 cattle with mucormycosis lesions inoculated onto Rose Bengal Agar, Potato Dextrose, and Malt Extract Agar. After the incubation at 25°C and 27°C, pure colonies were evaluated morphologically and microscopically. For molecular identification, DNA isolation and PCR studies were followed by sequence analysis and the results were compared with the data in GeneBank using the nBLAST tool. ITS1/ITS4 primers used in PCR study. Fungal species were identified with data verified after morphological and molecular identification. The sequence analyses revealed that 12 samples had L. ramosa HBF570, 7 samples contained A. niger HBF572 and P. crustosum HBF571, 2 samples contained A. chevalieri HBF573 and A. flavus HBF576, and one sample contained A. pseudoglaucus HBF577 and Aspergillus sp. HBF570. The study's causative agents emerged were environmental fungus species. In conclusion, because of the fungal diversity in the environment, hygiene investigations must be conducted and implemented for the protection of mucormycosis. [ABSTRACT FROM AUTHOR]

Potato viruses PLRV, PVY, PVM, PVA, PVX and PVS can cause up to 90% loss of potato harvest. Therefore, they are monitored by law in many countries using DAS-ELISA or conventional real-time RT-qPCR. Previously, we developed a multiplex real-time DiRT-PCR (Direct reverse transcript – polymerase chain reaction), which works directly on diluted tuber sap and thus saves time and chemical processing for RNA extraction or time and space in the glasshouse. So far, this method only ran on sap of single tubers which is not practical for routine testing. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR on pooled samples of ten tubers. Here we show that there is an "almost perfect" agreement (Gwet's AC1 index) comparing this multiplex real-time DiRT-PCR on pooled samples with DAS-ELISA and a commercial RT-qPCR kit with a rapid extraction method. The multiplex real-time DiRT-PCR is now ready to be used for routine testing. [ABSTRACT FROM AUTHOR]

Copyright of Jurnal Fitopatologi Indonesia is the property of IPB University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes. [ABSTRACT FROM AUTHOR]

The emergence of human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses significant challenges to global public health. Despite the extensive efforts of researchers worldwide, there remains considerable opportunities for improvement in timely diagnosis, specific treatment, and effective vaccines for SARS-CoV-2. This is due, in part, to the large number of asymptomatic carriers, rapid virus mutations, inconsistent confinement policies, untimely diagnosis and limited clear treatment plans. The emerging of nanozymes offers a promising approach for combating SARS-CoV-2 due to their stable physicochemical properties and high surface areas, which enable easier and multiple nano-bio interactions in vivo. Nanozymes inspire the development of sensitive and economic nanosensors for rapid detection, facilitate the development of specific medicines with minimal side effects for targeted therapy, trigger defensive mechanisms in the form of vaccines, and eliminate SARS-CoV-2 in the environment for prevention. In this review, we briefly present the limitations of existing countermeasures against coronavirus disease 2019 (COVID-19). We then reviewed the applications of nanozyme-based platforms in the fields of diagnostics, therapeutics and the prevention in COVID-19. Finally, we propose opportunities and challenges for the further development of nanozyme-based platforms for COVID-19. We expect that our review will provide valuable insights into the new emerging and re-emerging infectious pandemic from the perspective of nanozymes. Highlights: Nanozyme-based platforms have demonstrated improved sensitivity for SARS-CoV-2 antigen detection, reduced cost, and facilitated rapid diagnosis, making them a promising tool for COVID-19 diagnostics. Nanozyme-based platforms offer potential benefits in targeted therapy for COVID-19 due to their ability to specifically counteract and eliminate SARS-CoV-2 with potentially fewer side effects. Nanozyme-based platforms have been shown to act as immunostimulatory molecules, activating the defense response of the immune system against SARS-CoV-2, potentially providing a therapeutic avenue for COVID-19 treatment. [ABSTRACT FROM AUTHOR]

The genus Cercospora contains many devastating plant pathogens linked to leaf spot diseases afflicting various plants. Identification of Cercospora species based on morphology or host plant association has proven unreliable due to simple morphology and wide host range in many cases; hence, multi-gene DNA sequence data are essential for accurate species identification. Considering the complexity and cost involved in application of multi-locus DNA phylogenetic approaches for species delineation in Cercospora; rapid and cost-effective methods are urgently needed for species recognition. In this study, we applied rep-PCR (repetitive-sequence based polymerase chain reaction) fingerprinting methods referred to as BOX-PCR to differentiate species of Cercospora. Cluster analysis of the banding patterns of 52 Cercospora strains indicated the ability of BOX-PCR technique using BOXA1R primer to generate species-specific DNA fingerprints from all the tested strains. Since this technique was able to discriminate between all the 20 examined Cercospora species during this study, which corresponded well to the species identified based on multi-gene DNA sequence data, our findings revealed the efficiency of BOX-PCR system as a suitable complementary method for molecular identification of the genus Cercospora at species level. [ABSTRACT FROM AUTHOR]

First Report of Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4) Causing Banana Wilt in the Island of Mayotte. MAP: Fig 1 Uncontained spread of Fusarium wilt in banana caused by Fusarium odoratissimum TR4.(A) Banana is a major food crop in tropical and subtropical regions, especially in sub-Saharan Africa. Banana is the most popular fruit worldwide [[1]] and a major staple food in tropical and subtropical regions where the majority of bananas is produced ( B Fig 1 b ) [[2]]. First detection of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) on Cavendish banana in India. [Extracted from the article]

Topmouth gudgeon is a freshwater fish species native to East Asia. Nowadays, P. parva is spread throughout Europe which is of concern because besides being considered one of the worst aquatic Invasive Alien Species (IAS) in Europe it is also a known vector of Spherotecum destruens, the rosette-like parasite lethal to other fish species. The present study describes the development and validation of a new species-specific assay based on hydrolysis probe chemistry to detect P. parva environmental DNA (eDNA) in water samples collected in a northern region of Italy (Friuli Venezia Giulia). Water samples were collected from 55 sites in an area where partial information on the occurrence of the species is available. eDNA was isolated from all samples and the presence of P. parva eDNA was tested by means of qPCR (quantitative PCR) and microfluidic qdPCR (quantitative digital PCR) techniques. Field results for both qPCR and qdPCR were largely in agreement in terms of detection (presence/absence). Thus, we judged the presence/absence by combining the results from the two methods and found that nine sites showed "strong positive" signal of P. parva eDNA (at least 2 positive replicates), 3 showed "suspected" (only 1 positive replicate), and 42 showed "absent". The current study shows the strong potential of the newly developed eDNA approach to be a valuable addition to the monitoring of the highly invasive topmouth gudgeon in freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Rice brown spot caused by Bipolaris oryzae (Breda de Haan) is the predominant disease in rice, infecting millions of hectares of cultivated rice, and greatly reduces yields. The various available diagnostic methods for B. oryzae disease detection are time consuming and require sophisticated instruments. Thus, an affordable, rapid and highly specific assay is needed. Here, we report a loop-mediated isothermal amplification (LAMP) assay protocol for specifically detecting B. oryzae by targeting a gene encoding a glycoside hydrolase (GH) family 13 protein. A set of six specific, sensitive LAMP primers were designed, allowing the effective LAMP detection of pathogens after incubation at 62°C for 60 min. Visual detection was performed by the addition of hydroxynapthol blue dye. In a positive reaction, the color changed to intense sky blue, while in a negative reaction, the color remained violet. The developed LAMP assay was highly sensitive, with a detection limit of 100 femtograms. The high specificity of the assay for the detection of B. oryzae was validated. Thus, the LAMP assay protocol provides a rapid, reliable tool for the early detection of B. oryzae under laboratory conditions. [ABSTRACT FROM AUTHOR]

Testing for potato viruses is globally very important to prevent a critical shortage of potato supply. In most countries, testing is obligated by law. In Germany, seed potatoes are monitored for six viruses: PLRV, PVY, PVM, PVA, PVX and PVS. They can cause up to 90% loss of potato tubers in the field. Common methods currently used for testing are ELISA and conventional real-time PCR, but both are very time-consuming, and the former needs a high capacity of green houses and human resources, the latter elaborate RNA extraction steps. Recently, we proposed a new method called real-time DiRT-PCR which enables us to test for PLRV, PVY and PVS along with an internal control in three duplex real-time PCR reactions directly on diluted tuber sap. In this study, we describe the first TaqMan® assay for PVM published so far and embed it into a multiplex system to detect the remaining viruses. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR without RNA purification. [ABSTRACT FROM AUTHOR]

Bulb crops are attacked by various soil-dwelling pests and pathogens. Entomopathogenic (EPFs) and mycoparasitic fungi (MPFs) which are distributed in natural and agricultural soils worldwide can play an important role as natural enemies of bulb pests. The species richness and density of these fungi in onion and garlic fields have not been investigated. The aim of this study was to determine the occurrence of EPFs and MPFs in soils where these crops were grown and compared the data from sites of the Czech Republic and Israel. Methods of fungi isolation and quantification were based on elution of soil samples by water and cultivation using selective media with dodine for EPFs and cultivation using potato dextrose agar with chloramphenicol for MPFs. Entomopathogenic fungi Beauveria spp., Isaria spp., Lecanicillium spp., Metarhizium spp., Purpureocillium spp. and mycoparasitic fungi Trichoderma spp. were isolated from soil samples in both countries. The highest density was observed in the genus Metarhizium in both countries. Metarhizium spp. were most abundant in the site Mlýn Podhora in the Czech Republic. The average density of colony-forming units (CFU) per 1 mL of soil sample was 1.47 × 104. The lowest density was observed in the genus Beauveria in both countries, up to 5.93 × 102 CFU per 1 mL of soil sample. Soils in the Czech Republic contained about ten times higher number of EPFs compared to Israel. Rather higher prevalence of MPFs was also found in the Czech Republic. Possible reasons for within and between countries variability in EPFs and MPFs occurrence are discussed. [ABSTRACT FROM AUTHOR]

A partial sequence of the putative polymerase (L) protein of Strawberry crinkle virus (SCV), genus Cytorhabdovirus, is described. The virus protein was found to be distantly related to the L protein of the rhabdoviruses Northern cereal mosaic virus, Rice yellow stunt virus and Sonchus yellow net virus. Moreover, a tentative grouping of SCV isolates is described, based on phylogenetic analysis of a region enclosing the GDN-motif within the RNA-dependent RNA polymerase gene. A sequence homology of 98% was found for each tentative group, and heterogeneity of at least 11% was observed between both groups. The tentative grouping did not appear to be related to symptomatology or geographical origin of the isolates. Nevertheless, the use of several reverse-transcription (RT)-PCR primer sets, all directed to different regions within the SCV genome, confirmed the presumptive classification into two groups, namely group I (isolates 1554, KG and Post), and group II (isolates 1553, Hb-A1, 37-1 and 37-2). Additionally, the detection of SCV isolates from herbaceous hosts and strawberry plant material was possible through use of a newly developed gel-based RT-PCR and a gel-free AmpliDet RNA assay. Both methods have the potential to provide rapid, sensitive and specific detection of SCV in in vitro propagation material. [ABSTRACT FROM AUTHOR]

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications. [ABSTRACT FROM AUTHOR]

The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/μL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2–4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/μL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. [ABSTRACT FROM AUTHOR]

Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. Methods: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. Results: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment. [ABSTRACT FROM AUTHOR]

Fire blight remains a serious threat to commercial apple production in the USA and worldwide. Other diseases and spray damage can result in fire blight-like symptoms that can lead to misdiagnosis and affect disease management strategies. Accurate and timely detection of the fire blight pathogen, Erwinia amylovora, is extremely important to deploy appropriate and timely measures to reduce fire blight epidemics in commercial apple orchards. We tested two commercial lateral flow immunoassays (AgriStrip®, and Pocket Diagnostics kit), Loop mediated isothermal amplification (LAMP), and quantitative PCR (qPCR) to diagnose E. amylovora infected samples in lab and field settings. The AgriStrip® and Pocket Diagnostics kits were able to detect actively growing bacteria up to ×106 cfu/ml bacterial concentration. Pocket Diagnostics kit had less specificity and showed positive tests for E. pyrifolia in addition to E. amylovora. The LAMP assay showed high specificity for E. amylovora and was able to detect up to ×103 cfu/ml bacterial concentrations. The qPCR assay was also able to detect bacterial cells up to ×10−3 cfu/ml bacterial concentration with highly specific E. amylovora detection. Grower surveys and comparative cost-benefit analysis indicated that immunoassay kits are less expensive, easier to use, and require less technical expertise for on-site fire blight diagnosis than LAMP and qPCR. However, the choice of a specific diagnostic assay depends on the time, sensitivity, and specificity required for the detection of fire blight and its management. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Plant Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Recombination leads to the generation of new viral progeny which remain undetected by routine testing procedures and may be a threat to the infected host. Here, we have characterised the complete genome sequences of two isolates of Apple stem pitting virus from apple cv. Red Chief (Palampur) and cv. Gold Spur (N) with distinct serological reactivities. The viral genomes consisted of 9267 nucleotides for isolate Palampur and 9254 nucleotides for isolate N, excluding the poly (A) tail and contained 5five open reading frames (ORFs). Isolate N shared 80.8% sequence identity with ASPV apple isolate GA2 from China, while isolate Palampur shared 81.4% sequence identity with ASPV apple isolate PB66 from the United Kingdom. The serological difference of isolates N and Palampur along with their low sequence identity indicated the existence of two distinct virus genotypes which was corroborated by evolutionary and genetic differentiation analyses. Recombination events were detected in the RdRp and CP sequences of Palampur isolate thereby suggesting the role of recombination in the evolution of distinct virus genotypes. [ABSTRACT FROM AUTHOR]

Aspergillus flavus and its main secondary metabolite AFB1 pose a serious threat to several important crops worldwide. Recently, it has been reported that some PHD family transcription factors are involved in the morphogenesis and AFB1 biological synthesis in A. flavus, but the role of Cti6, a PHD domain containing protein in A. flavus, is totally unknown. The study was designed to reveal the biological function of Cti6 in the fungus by deletion of cti6, and its two domains (PHD and Atrophin-1) through homologous recombination, respectively. The results showed that Cti6 might up-regulate the mycelium growth, conidiation, sclerotia formation and AFB1 biological synthesis of A. flavus by its PHD domain, while Atrophin-1 also improved the conidiation of the fungus. The qRT-PCR analysis showed that Cti6 increased the conidiation of the fungus through AbaA and BrlA mediated conidiation pathway, triggered the formation of sclerotia by orthodox sclerotia formation pathway, and improved the production of AFB1 by orthodox AFB1 synthesis pathway. Crops models analysis showed that A. flavus Cti6 plays vital role in colonization and the production of AFB1 on the host grains mainly via PHD domain. Bioinformatics analysis showed Cti6 is conservative in Aspergillus spp., and mCherry mediated subcellular localization showed that most Cti6 accumulated in the nuclei, which reflected that Cti6 performed its important biological function in the nuclei in Aspergillus spp.. The results of the current study elucidate the roles of PHD domain containing proteins in the mechanism of the infection of crops by A. flavus, and provided a novel target for effectively controlling the contamination of Aspergillus spp. to crops. [ABSTRACT FROM AUTHOR]

Apples are produced using a high-density plantation system using M.9 rootstock in Korea. However, the virus infection rate of apples tends to be high because the rootstocks produced from virus-infected mother stocks were widely used and vegetative propagation based on the grafting is common with scion cultivars. The most effective way to control plant virus and viroid depends on conducting a massively rapid and precise diagnostic inspection. Therefore, this study aims to develop a real-time PCR assay that can be precisely and inexpensively tested for ACLSV and ASSVd. To expand the detection range, we developed a singleplex real-time PCR method targeting the conserved region of ACLSV and ASSVd, respectively. The detection methods using two kinds of fluorescent chemical dye, SYBR Green I and TaqMan probe, have been developed in this study. Comparing these methods developed with two dyes, the assay using SYBR Green I was efficient for detection as much as the method using TaqMan probe and faster. The field test demonstrated real-time detecting methods developed using SYBR Green I dye were applicable to reliable diagnosis and quantification for virus and viroid in quarantine fields. [ABSTRACT FROM AUTHOR]

Globally, viral diseases cause huge economic losses in crops and their management is a big challenge to growers as well as researchers. Mixed infection is the existence of more than one virus in single plant, which results in varied symptoms at the same time. The presence of more than one virus always leads to difficulty in understanding the etiology of disease. Most of the viral diseases go unnoticed either due to the latent nature of infection of virus(es) or due to low severity of symptoms. But this might be true in case of single infection of the host by the concerned virus. When such viruses are seen causing infection in combination with other viruses at particular time, more severe disease symptoms can be observed. For any successful management of viral disease especially during mixed infection, detection and identification of plant viruses causative of the disease are of foremost importance. Several approaches like cocktail ELISA, multiplex PCR for known viruses and next-generation sequencing for both known and unknown viruses have been developed for detection of mixed infection of viruses. During mixed infection, several kinds of interaction commonly referred to as synergistic or antagonistic interactions are going on between and among the viruses, which aggravate the disease with more severe symptoms than with single infections. Here, we review the mixed infection of viruses, methods of detection, factors influencing, interactions and impact on plant during mixed infection. [ABSTRACT FROM AUTHOR]

We sequenced the entire genome of isolates SMoV T1 and T2 of strawberry mottle virus (SMoV) from strawberry cultivar UC-5 grown in Tochigi, Japan. Homology and molecular phylogenetic analyses revealed that SMoV T1 and T2 are close homologs of previously reported Canadian and Czech strains. This is the first report describing the complete genome sequences of SMoV isolated in Japan and the construction of infectious SMoV clones. The stability of infectious clones in strawberry is discussed. [ABSTRACT FROM AUTHOR]

Background: Various databases on genetically modified organisms (GMOs) exist, all with their specific focus to facilitate access to information needed for, e. g., the assistance in risk assessment, the development of detection and identification strategies or inspection and control activities. Each database has its unique approach towards the subject. Often these databases use different terminology to describe the GMOs. For adequate GMO addressing and identification and exchange of GMO-related information it is necessary to use commonly agreed upon concepts and terminology. Result: A hierarchically structured controlled vocabulary describing the genetic elements inserted into conventional GMOs, and GMOs developed by the use of gen(om)e-editing is presented: the GMO genetic element thesaurus (GMO-GET). GMO-GET can be used for GMO-related documentation, including GMO-related databases. It has initially been developed on the basis of two GMO databases, i.e. the Biosafety Clearing-House and the EUginius database. Conclusion: The use of GMO-GET will enable consistent and compatible information (harmonisation), also allowing an accurate exchange of information between the different data systems and thereby facilitating their interoperability. GMO-GET can also be used to describe genetic elements that are altered in organisms obtained through current targeted genome-editing techniques. [ABSTRACT FROM AUTHOR]

A novel method referred to as hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD) (HRCA-LFD) here was developed for specific, sensitive, rapid, and simple detection of Prorocentrum minimum. HRCA-LFD relies on a padlock probe (PLP) consisting of a common ligation sequence, two terminal sequences that complement the target DNA, and a manually designed detection probe (LFD probe). The two terminal sequences of the PLP were designed against the species-specific sites of the large subunit ribosomal DNA (LSU rDNA) D1-D2 region of P. minimum. The optimum parameters for HRCA were as follows: PLP concentration of 20 pM, ligation time of 30 min, ligation temperature of 59 °C, enzymic digestion time of 105 min, amplification time of 45 min, and amplification temperature of 58 °C. The HRCA-LFD displaying high specificity could accurately distinguish P. minimum from other microalgae. The detection limit of HRCA-LFD was as low as 1.42 × 10−7 ng μL−1 for genomic DNA, 1.03 × 10−7 ng μL−1 (approximately 27 copies) for recombinant plasmid containing the inserted LSU rDNA D1-D2, and 0.17 cells for crude DNA extract of P. minimum, which was consistently 100 times more sensitive than regular PCR. Interfering test suggested that the performance of HRCA-LFD is stable and would not be affected by other non-target species. The HRCA-LFD results of field samples that are comparable with microscopic examination confirmed that the developed method is competent for detection of target cells in field samples. In conclusion, the developed HRCA-LFD exhibiting stable performance is specific, sensitive, and rapid, which provides a good alternative to traditional microscopic examination for the detection of P. minimum cells in field samples. [ABSTRACT FROM AUTHOR]

Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. Key points: • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Plant Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Loop-mediated amplification (LAMP) is an isothermal amplification technique favored in diagnostics and point-of-care work due to its high sensitivity and ability to run in isothermal conditions. In addition, a visual readout by lateral flow strips (LFS) can be used in conjunction with LAMP, making the assay accessible at the point-of-care. However, the amplicons resulting from a LAMP reaction varied in length and shape, making them undiscernible on a double-stranded DNA intercalating dye stained gel. Standard characterization techniques also do not identify which amplicons specifically bind to the LFS, which generate the visual readout. We aimed to standardize our characterization of LAMP products during assay development by using fluorescein amidite (FAM) and biotin-tagged loop forward and backward primers during assay development. A pvuII restriction enzyme digest is applied to the LAMP products. FAM-tagged bands are directly correlated with the LFS visual readout. We applied this assay development workflow for an HPV 16 assay using both plasmid DNA and clinical samples to demonstrate proof of concept for generalized assay development work. [ABSTRACT FROM AUTHOR]

Background: Ericoid mycorrhizal fungi (EMF) play important roles in mineral cycling and plant nutrient acquisition, and they increase plant survival in nutrient-poor environments. In this study, we detected the colonization of EMF using a green fluorescent protein (GFP) expression method and in situ PCR. Results: Genetic transformants of Cryptosporiopsis ericae and Sordariomycetes sp. expressing GFP were obtained via Agrobacterium tumefaciens-mediated transformation. GFP transformants were able to infect Vaccinium uliginosum, and their fluorescence was visible in the hair roots. Both in situ PCR and the GFP-expressing method indicated that EMF could colonize the hair roots of V. uliginosum 2 weeks after inoculation. Conclusions: This research represents the first attempt to detect ericoid mycorrhizal colonization using in situ PCR. A GFP-expressing method is an excellent system for detecting the colonization of EMF, but it is dependent on the successful transformation and expression of the gfp gene. In situ PCR and the GFP expression may be developed as new tools to study the interactions of EMF both with ericaceous plants and with the environment. [ABSTRACT FROM AUTHOR]

Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10−3–10−4 ng/μL and a LOD of 10−4–10−5 ng/μL. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent. [ABSTRACT FROM AUTHOR]

Summary: Emerging and reemerging infectious diseases are global public concerns. With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS‐CoV‐2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS‐CoV‐2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS‐CoV‐2 are basically similar to SARS‐CoV and MERS‐CoV, the other two beta‐CoVs of medical importance. During the SARS‐CoV and MERS‐CoV epidemics, a variety of molecular and serological diagnostic assays were established and should be referred to for SARS‐CoV‐2. In this review, by summarizing the articles and guidelines about specimen collection, nucleic acid tests (NAT) and serological tests for SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, several suggestions are put forward to improve the laboratory testing of SARS‐CoV‐2. In summary, for NAT: collecting stool and blood samples at later periods of illness to improve the positive rate if lower respiratory tract specimens are unavailable; increasing template volume to raise the sensitivity of detection; putting samples in reagents containing guanidine salt to inactivate virus as well as protect RNA; setting proper positive, negative and inhibition controls to ensure high‐quality results; simultaneously amplifying human RNase P gene to avoid false‐negative results. For antibody test, diverse assays targeting different antigens, and collecting paired samples are needed. [ABSTRACT FROM AUTHOR]

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL−1. The potential application of the method was verified by analyzing spiked food samples. [ABSTRACT FROM AUTHOR]

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are "Satsumavirus" (one species: Satsuma dwarf virus), "Stramovirus" (two species: Strawberry mottle virus and Black raspberry necrosis virus) and "Cholivirus" (two species: Chocolate lily virus A and Dioscorea mosaic associated virus). [ABSTRACT FROM AUTHOR]

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care. [ABSTRACT FROM AUTHOR]

Aims: Candida species are normal flora in the human body and are the main cause of hospital infections in people with underlying disease. The purpose of the present study was comparison of the conventional and molecular laboratory methods in identifying different species of Candida to find the optimal method. Materials & Methods: 60 Candida isolates were obtained from oropharyngeal candidiasis patients and recognized using laboratory routine methods including germ tube production, culture on CHROMagar medium, API 20 C AUX kit and molecular ITS sequencing method. The concordances between the methods compared to the molecular method were measured by the kappa coefficient using SPSS 16.0 software. Findings: Out of 60 Candida isolates, 10 isolates (16.6%) in germ tube formation test, 8 isolates (13.33%) in the API test, and 9 isolates (15%) in the culture on CHROMagar medium have different results in comparison to the ITS sequencing method. Germ tube (k= 0.90) and culture on CHROMagar medium methods (k= 0.66) showed the highest and the lowest agreement with the molecular method in the identification of Candida albicans species respectively. Conclusion: The results showed that conventional methods alone cannot diagnose all Candida species and molecular methods such as ITS sequencing can be used to confirm the identification. [ABSTRACT FROM AUTHOR]

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Harmful algal blooms (HABs) caused by microalgae pose a great threat to human health, marine ecosystems, tourism, and aquaculture worldwide. Efficient, sensitive, and simple methods for the detection of harmful microalgal species must be developed to reduce the economic losses and damages caused by HABs. This study successfully established a novel method for the detection of the globally distributed HAB-forming species Karlodinium veneficum. The developed method was based on exponential rolling circle amplification (E-RCA) coupled with lateral flow dipstick (LFD) strips. A special oligonucleotide sequence-padlock probe (PLP) was designed on basis of the molecular cloning and subsequent sequencing of the D1–D2 region of the large subunit ribosomal DNA (LSU rDNA) of K. veneficum. The E-RCA reaction system was then established. E-RCA products could be visually analyzed through 2% agarose gel electrophoresis and the LFD assay. The optimized E-RCA conditions were as follows: PLP concentration of 20 pM, ligation cycle number of 12, ligation temperature of 56 °C, amplification duration of 45 min, and amplification temperature of 61 °C. The developed E-RCA-LFD assay was specific for K. veneficum and did not display cross-reactivity with other microalgal species. The E-RCA-LFD assay was 100-fold more sensitive than conventional polymerase chain reaction (PCR) and had detection limits of 8.0 × 101 ag μL−1 for the genomic DNA of K. veneficum and 5.0 × 101 ag μL−1 (approximately 13 copies μL−1) for recombinant plasmids containing the LSU rDNA D1–D2 regions of K. veneficum. Simulative tests indicated that the E-RCA-LFD assay demonstrated considerably higher sensitivity than conventional PCR and a detection limit of 0.01 cell mL−1. The practicability of the E-RCA-LFD assay was confirmed through tests with field samples. In conclusion, the highly sensitive, specific, and facile E-RCA-LFD assay established in this work may enable the field monitoring of natural samples containing K. veneficum. [ABSTRACT FROM AUTHOR]

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease. [ABSTRACT FROM AUTHOR]

Karenia mikimotoi is a globally distributed, toxic, bloom-forming dinoflagellate. The development of rapid, precise and sensitive detection methods is essential for the field monitoring of this harmful alga. In this study, exponential rolling circle amplification (E-RCA) and double-ligation E-RCA (dlE-RCA) were established for the detection of K. mikimotoi. The partial large subunit rDNA (D1-D2) of K. mikimotoi was PCR amplified, cloned and then sequenced. The resultant sequence was used to perform alignment analysis for species-specific regions and consequently design padlock probes and primers for E-RCA and dlE-RCA. Both E-RCA and dlE-RCA detection protocols were established and their parameters were optimized. dlE-RCA can avoid self-cyclization of PLP compared with E-RCA. The optimized parameters were as follows: ligation temperature, 61 °C; ligation time, 60 min (E-RCA)/30 min (dlE-RCA); amplification temperature, 61 °C (E-RCA)/64 °C (dlE-RCA); and amplification time, 30 min (E-RCA)/40 min (dlE-RCA). Specificity tests showed that both E-RCA and dlE-RCA were specific for K. mikimotoi. Sensitivity comparison indicated that E-RCA was 10-fold more sensitive than PCR and the sensitivity of dlE-RCA was comparable with that of PCR. Tests with simulated field samples suggested that the developed E-RCA and dlE-RCA obtained detection limits of 1 and 10 cells, respectively. Positive E-RCA and dlE-RCA could be confirmed by visual observation of coloration reaction with the addition of fluorescent SYBR Green I dye to the reaction tube. The developed E-RCA and dlE-RCA were also efficient for field samples with target cell densities ranging from 1 cell mL−1 to 1000 cells mL−1. These results suggest that the established E-RCA and dlE-RCA detection protocols show promising applications in the field monitoring of K. mikimotoi. [ABSTRACT FROM AUTHOR]

Phytophthora cactorum, P. cinnamomi and P. lateralis were reported to be pathogenic on kiwifruit trees in the main planting areas of China. We attempted to simultaneously detect the three pathogens using a multiplex polymerase chain reaction (PCR) and to survey their occurrence in the main production areas. Because of the need to combine different primer pairs for the multiplex PCR and the low specificity of published specific primers for P. cactorum, P. cinnamomi and P. lateralis, new species-specific primers for the three species were designed based on the ras-related protein gene, Ypt1. The specificity of the designed primers was demonstrated using 52 isolates, including 44 Phytophthora species, three Pythium species, and three other soil-borne pathogens. A multiplex PCR method for the simultaneous detection of P. cactorum, P. cinnamomi and P. lateralis was established, and the three pathogens were detected in artificially and naturally infested soils, indicating that these markers can be used in the diagnosis of kiwifruit Phytophthora diseases. In a survey of these pathogens in the main kiwifruit planting areas of China, 99 soil samples were collected at different locations and in different seasons and subjected to the new method, and the distribution of the three pathogens in the main kiwifruit planting areas of China was determined. [ABSTRACT FROM AUTHOR]

Background: Oligonucleotide signatures (signatures) have been widely used for studying microbial diversity and function in wet-lab settings, but using them for accurate in silico identification of organisms from high-throughput sequencing (HTS) data is only a proof of concept. Existing signature design programs for sequence signatures (signatures matching exactly one sequence) or clade signatures (signatures matching every sequence in a phylogenetic clade) are not able to identify all possible polymorphic sites for sequences with high similarity and perform poorly when handling large genome sequencing datasets. Results: We introduce cluster signatures: subsequences that match perfectly and exclusively any group of sequences in a data set. Cluster signatures provide complete recall for primer/probe design and increased discrimination between sequences beyond that of clade signatures. Using cluster signatures for in silico identification of HTS targets achieves good precision/recall and running time performance. This method has been implemented into an open source tool, the Automated Oligonucleotide Design Pipeline (adop), included in supplementary material and available at: https://bitbucket.org/wenchen_aafc/aodp_v2.0_release. Conclusions: Cluster signatures provide a rapid and universal analysis tool to identify all possible short diagnostic DNA markers and variants from any DNA sequencing dataset. They are particularly useful in discriminating genetic material from closely related organisms and in detecting deleterious mutations in highly or perfectly conserved genomic sites. [ABSTRACT FROM AUTHOR]

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State - UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli. [ABSTRACT FROM AUTHOR]

Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost-benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used. [ABSTRACT FROM AUTHOR]

Pectinolytic bacteria were isolated from 48 potato plants showing the symptoms of blackleg and collected in different fields of commercial potato production areas at Samsun, Amasya, Corum and Yozgat provinces in Turkey in 2015. The survey resulted in the isolation of 26 pectinolytic strains that belonged to P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. parmentieri species based on molecular identification with species-specific PCR and phenotypic characterization. The identified strains indicated typical biochemical characteristics of the assigned species. For 16 representative Pectobacterium isolates 10 unique rep-PCR band patterns were obtained. The 16S rRNA and recA and gapA gene fragment sequencing confirmed the species identity of the isolates. The phenotypic characterization of the strains revealed that for all assays but one (cellulase, protease activity, swimming but not swarming), the tested Pectobacterium species were significantly different from each other proving the diversity of the strains belonging to these genera. Recent outbreaks of blackleg and/or soft rot in potato production areas in Turkey may pose a threat on other crops, as tomato, pepper, cucumber, onion, cabbage, broccoli and sugar beet are cultivated in the same provinces. [ABSTRACT FROM AUTHOR]

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Čačanská raná. [ABSTRACT FROM AUTHOR]

Aphids are common herbivores in the strawberry crop that can reduce plant vigor and fruit quality and also transmit viruses. Aphid species prefer diverse plant organs, which represent particular habitats of different quality for aphids and for the development of natural enemies’ populations. Different habitat units (young leaves, mature leaves, buds, flowers) of strawberry were sampled fortnightly during all seasons. We identified seven aphid species, namely Chaetosiphon fragaefolii, Aphis gossypii, and Macrosiphum euphorbiae, the most abundant. During the autumn, C. fragaefolli and M. euphorbiae were scarce and A. gossypii was denser on mature leaves, while during summer M. euphorbiae was absent. During the winter, C. fragaefolii predominated on buds and young leaves, A. gossypii on flowers, and both species on mature leaves. During the spring, C. fragaefolii was even more abundant on buds, A. gossypii predominated on mature leaves, and the three species were equally abundant on flowers and young leaves. Parasitoids emerged from A. gossypii, M. euphorbiae and Myzus persicae, but not from C. fragaefolii. Three Aphidius and two Aphelinus species were recovered. All primary parasitoid species emerged from A. gossypii, and secondary parasitoids emerged only from this aphid. Aphis gossypii parasitism on mature leaves was markedly higher in winter and summer than in autumn and spring. Parasitism of A. gossypii was independent of its density, and the number of parasitized aphids was never higher than six. Our results contribute to define the most appropriate sample unit to estimate aphid density of different species and provide information about seasonal natural parasitism. [ABSTRACT FROM AUTHOR]

Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA68) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65o C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in gel electrophoresis. Our assay is significantly faster than the conventional methods used in the identification of P. triticina. The assay developed in the study shall be very much useful in the development of diagnostic kit for monitoring disease, creation of prediction model and efficient management of disease. [ABSTRACT FROM AUTHOR]

A risk assessment for imidacloprid applied on strawberries under different conditions was performed after residue determination using the quick, cheap, effective, rugged, and safe (QuEChERS) method. The application conditions were varied according to the applied dosage, addition of a plant oil or organosilicon surfactant, water volume, and sprayer type. The degradation dynamics of imidacloprid on strawberries followed first-order kinetics. At applied doses of 30–60 g a.i. ha−1, the half-lives of imidacloprid were 2.89–3.46, 1.98–3.65, and 2.57–2.77 days after application without a surfactant or with a plant oil or organosilicon surfactant, respectively. For water volumes of 112.5, 225, 450, 675, and 900 L ha−1, the half-lives of imidacloprid applied in the presence of the plant oil surfactant were 3.30, 7.70, 5.33, 7.70, and 6.30 days, respectively. The half-lives after application with a knapsack mist duster, electric sprayer, and manual sprayer were 2.16, 5.77, and 7.70 days, respectively. The health risk assessment revealed risk quotients less than 1 in all cases, indicating that the application of imidacloprid poses a low health risk to humans after a pre-harvest interval of 10 days under our application conditions. The risk assessment results can provide reference data for setting a reasonable maximum residue limit for imidacloprid on strawberries in China. [ABSTRACT FROM AUTHOR]