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Waiteacircinata var. zeae, a pathogen with a relatively narrow host range, has recently been detected in cabbage and oilseed rape in Europe and worldwide. In this study, we developed specific conventional and real-time PCR protocols for direct detection of W.circinata var. zeae from mycelium and diseased plant tissue. The newly developed primer pair zeaefor1/zeaerew1, used in PCR protocols, specifically amplified only target isolates of W.circinata var. zeae when tested against isolates of 11 different binucleate and multinucleate anastomosis groups of Rhizoctonia spp. including AG-A, AG-G, AG-F, AG-U, AG-2-1, AG-2-2, AG-3, AG-4 HGI, AG-4 HGII, AG-4 HGIII, and AG-6 and common soil-borne pathogens. Total of nine previously published primer pairs designed for the detection of various Rhizoctonia spp. were also tested and did not amplify target isolates of W.circinata var. zeae. The detection limit of conventional and real-time PCR protocols was 10–2 and 10–5 (with starting concentration 9.5 ng/µl), respectively, and both methods are the first available tools for direct detection and identification of W.circinata var. zeae from mycelium and diseased oilseed rape seedlings. Both conventional and SYBR-Green-based real-time PCR protocols are cost-effective and provide a solid basis for further investigations of W.circinata var. zeae, particularly in relation to distribution, host range, and epidemiology. [ABSTRACT FROM AUTHOR]

Simple Summary: We developed a new test to detect Babesia aktasi, a parasite that infects goats, using a method called semi-nested PCR. This method focuses on a specific part of the parasite's DNA to ensure accuracy. We checked the test against several other similar parasites to make sure it only detected B. aktasi, which it did successfully. To see how sensitive our test is, we used blood samples with known amounts of the parasite and found that our test could detect even very low levels of infection. Our results show that this new test is both highly accurate and sensitive, making it a valuable tool for identifying B. aktasi infections in goats. This new PCR method provides a reliable tool for detecting B. aktasi in goats, which is crucial for managing and preventing the spread of this infection, ultimately protecting goat health and improving agricultural productivity. We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

This study developed a real-time polymerase chain reaction (real-time PCR) method for the detection of Lactobacillus kefiranofaciens subsp. kefiranofaciens. Based on the 16S rDNA gene sequence and whole genome sequence of the type strain of L. kefiranofaciens subsp. kefiranofaciens ZW3, specific primers were designed and screened. The fluorescent dye SYBR Green I was used in the real-time PCR method. Its specificity, sensitivity and repeatability were evaluated, and this method was applied to detect several strains of this species and its mixtures with other lactic acid bacteria. The results showed that the proposed method was highly specific, sensitive and repeatable. The standard curve was linear with a determination coefficient (R2) of 0.965. Moreover, this method could specifically detect L. kefiranofaciens subsp. kefiranofaciens from its mixture with other lactic acid bacteria. In summary, the real-time PCR method could quickly and accurately detect L. kefiranofaciens subsp. kefiranofaciens, providing a new method for the specific qualitative and quantitative detection of L. kefiranofaciens subsp. kefiranofaciens.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department 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.)

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

Introduction: Saffron with the scientific name Crocus sativus L belongs to the lily family (Iridaceae) and is one of the important economic and strategic products of Iran, which is important to investigate its limiting factors. In order to detect and identify Common bean mosaic virus as one of the limiting factors in saffron farms, sampling of 705 samples of saffron in Khorasan Razavi province, Khaf city was carried out during the spring of 1402. The saffron samples had mosaic symptoms, dwarfism, plant deformity and zigzagging, which were transferred to the laboratory under cool conditions. In the RT-PCR test, using specific primers of the nucleoprotein gene, in three saffron samples, a fragment of 343 pairs was found. They were reproduced and sent to Macrogen Korea for sequencing. The results confirmed the infection of three saffron samples with the mentioned virus, and in the genealogy studies of three saffron isolates in the nucleoprotein coding region of the mentioned virus, compared to the world isolates available in the gene bank, which were related to different geographical regions, five separate groups were formed, from three Iranian isolate related to saffron, two isolates (B1, B2) were placed in group 4 close to isolates from Brazil and one isolate (B3) was placed in group 5 close to two isolates from Iran (Ahvaz and Zanjan). The most similarity of the isolates in this research was between two Iranian isolates B1 and B2 with three isolates MW534342, MW534343 and MW534344 from the country of Zambia from the African continent with a similarity percentage of 99% and also the least similarity of the Iranian isolates B1, B2 and B3 with isolate KC702888 from Iran in Lorestan with a similarity level of 71%. Material and Methods: In order to detect and identify Bean common mosaic virus as one of the limiting factors in saffron farms, sampling of 705 samples of saffron in Khorasan Razavi province, Khaf city was carried out during the spring of 1402. The saffron samples had mosaic symptoms, dwarfism, plant deformity and zigzagging, which were transferred to the laboratory under cool conditions. Their total RNA was extracted and then RT-PCR test, using specific primers of the coat protein gene was carried out, in three saffron samples, a fragment of 343 base pairs was amplified. The amplified fragments were reproduced, then extracted from gel and sent to Macrogen Korea for sequencing. Results and Discussion: The results confirmed the infection of three saffron samples with the mentioned virus, and in the phylogeny studies of three saffron isolates in the nucleoprotein coding region of the mentioned virus, compared to the world isolates available in the gene bank, which were related to different geographical regions, five separate groups were formed, from three Iranian isolate related to saffron, two isolates (B1, B2) were placed in group 4 close to isolates from Brazil and one isolate (B3) was placed in group 5 close to two isolates from Iran (Ahvaz and Zanjan). The most similarity of the isolates in this research was between two Iranian isolates B1 and B2 with three isolates MW534342, MW534343 and MW534344 from the country of Brazil from the African continent with a similarity percentage of 100% and also the least similarity of the Iranian isolates B1, B2 and B3 with isolate KC702888 from Iran in Lorestan with a similarity level of 71%. Conclusion: This is the first survey of this virus in Razavi Khorasan that showed the infection of saffron field of Khafe city as one of the major areas of production of this product. Phylogenetic analysis of this virus showed that khaf isolates were located in two different groups that could be the result of different sources for entering of this virus in this area. Conflict of Interest: Authors declared no conflict of interest. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries 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.)

Tropaeolum majus (garden nasturtium) and Rudbeckia hirta are common ornamental plants that can be susceptible to plant virus infections, leading to symptoms that adversely affect their ornamental appeal and commercial production. In this research, severe leaf chlorosis was observed on T. majus and R. hirta plants in green spaces in Shiraz and Bushehr, Iran (2023). Total RNA was extracted from five symptomatic leaf samples of each species, as well as one asymptomatic sample as a negative control. RT-PCR with alfalfa mosaic virus (AMV)-specific primers successfully amplified the expected 780 bp fragment in all symptomatic samples, but not in the asymptomatic control. Nucleotide sequencing confirmed the 780 bp size of the amplicon, and analysis revealed that the AMV isolates infecting T. majus (IR-GN01-Shiraz and IR-GN02-Bushehr) were most similar to an Iranian AMV isolate (Acc. No. MW_014931.1), while those infecting R. hirta (IR-AN04-Shiraz and IR-AN03-Bushehr) were the most similar to Chinese AMV isolates. This study is the first report of AMV infecting T. majus and R. hirta in Iran, a noteworthy finding, considering the extensive cultivation of these ornamental plants and the potential impact of AMV on their visual and economic value. [ABSTRACT FROM AUTHOR]

Different genotypes of infectious bronchitis virus (IBV) in poultry are circulating worldwide. The most efficient classification system for IBV genotypes is based on the complete sequencing of S1 gene, and the target organ can be trachea, kidney, oviduct, lung, esophagus, proventriculus, intestine, liver, spleen, bursa, cecal tonsils, cloaca, and testis. In Iran, IBV genotyping is usually performed by partial sequencing of S1 gene, and the trachea is often selected for sampling. This study applies a different genotyping method, and compares the results with the routine method of genotyping. Samples were collected from the trachea and kidneys of 50 broiler flocks with respiratory symptoms. The presence of IBV was confirmed by a real-time RT-PCR analysis targeting the 5’UTR region of the genome. Genotype of positive samples was determined by two methods of partial S1 gene sequencing and genotype-specific primers. In the real-time RT-PCR test, 88% and 90% of tracheal and kidney samples showed positive results, respectively. When the S gene was sequenced, Variant 2 (GI-23) (68.18%), 793/B (GI-13) (22.73%), Massachusetts (GI-1) (6.82%), and QX (GI-19) (2.27%) were detected in the tracheal samples, whereas QX (GI-19) and Massachusetts (GI-1) were not diagnosed in the kidney samples. In the genotypespecific method, IBV genotypes Variant 2, 793/B, Massachusetts, and QX were detected in both tracheal and kidney samples. The results of genotype-specific method also demonstrated that 70% of tracheal samples and 62% of kidney samples were infected with a single IBV genotype, while 30% of tracheal samples and 38% of kidney samples were coinfected with different IBV types. When comparing two genotyping methods, the use of genotype-specific primers was superior to partial S1 gene sequencing, not only in detecting different IBV types, but also in efficiently applying them in both trachea and kidney. [ABSTRACT FROM AUTHOR]

Background and purpose: There are various methods for molecular detection of SARS-CoV2 genome among which, PCR-based methods are the most reliable for making diagnosis. The majority of approved PCR kits for detection of Coronavirus are based on TaqMan real-time PCR which is expensive due to incorporating fluorescent and quencher-harboring probe. The aim of this study was to design a simple and affordable method for detection of SARS-CoV2 based on a more affordable SYBR green qPCR method. Materials and methods: Specific primers were designed for the virus nucleocapsid gene and the human control gene using Oligo software. The specificities of the primers were examined by Primer-BLAST capability according to NCBI database. Nasal swab samples were obtained from 10 PCR-confirmed COVID-19 patients and 10 healthy control people. Results: The designed reactions were performed in full compliance with the kit approved by the Ministry of Health to discriminate healthy individuals from COVID-19 patients. The Ct cut-off values calculated for N1 and N2 reactions were 39.72 and 39.69, respectively. Conclusion: The designed SYBR green-based reaction showed a potential role for detection of SARS-CoV2 genome in clinical samples and this method can be considered as a less-expensive alternative to TaqMan real time PCR if the primers and standard reaction conditions are properly designed.

Nerium oleander is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new pair of oleander-specific primers, JZT-BF/BR, was designed and validated. Then, a qPCR method was developed using the primers, and its detective sensitivity was examined. The results showed that JZT-BF/BR could specifically identify oleander in forage and food mixtures, and qPCR was capable of accurate authentication even at a low DNA concentration of 0.001 ng/μL. This method was further applied to the analysis of SFS containing different ratios of N. oleander. The method was confirmed to be applicable to digested samples, and the detection limit reached 0.1% (w/w) oleander in mixture systems. Thus, this study undoubtedly provides strong support for the detection of highly toxic oleander and the diagnosis of food poisoning in humans and animals. [ABSTRACT FROM AUTHOR]

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays. [ABSTRACT FROM AUTHOR]

Abstract Background The dry body of the Tokay Gecko (Gekko gecko) is the source of a valuable traditional Chinese medicine, it is therefore listed as a Class II protected animal species in China. Due to increasing market demand and a declining supply of the species, a considerable number of adulterants have emerged in the market. Thus, it is necessary to establish an accurate and rapid method of identification for distinguishing G. gecko from its adulterants and for separating it from highly processed products. Methods A total of 274 COI sequences were analyzed by using MEGA 5.0 software. Several specific primers were designed to amplify mini-barcode regions and identify G. gecko from its counterfeits and products. Results 274 COI sequences of G. gecko and 15 adulterants species were analyzed. G. gecko could be distinguished from its adulterants through BLAST analysis, intra- and inter-specific distance analyses, and an NJ tree based on COI sequences. Two pairs of specific primers designed for this study, COISF2/COISR2 and COISF3/COISR3, amplified 200- and 133-bp fragments of the COI region, respectively, both of which were suitable for the identification of G. gecko and its adulterants. Furthermore, COISF3/COISR3 detected G. gecko in 15 batches of products. Conclusion Therefore, the specific DNA mini-barcoding method developed here may be a powerful tool for the identification of G. gecko and counterfeits, and may also be used to distinguish G. gecko from its highly processed by-products.

The article focuses on real-time PCR detection and quantification of Fusarium spp., potential producer of class B fumonisin mycotoxins in mature corn kernels using primers designed to genes FUM1 and FUM6, involved in mycotoxin biosynthesis. Research have shown that the real-time PCR assay can be used for detecting and quantifying potentially mycotoxigenic Fusarium in the maize kernels by the genes involved in mycotoxin synthesis. Three pairs of primers specific to genes involved in the synthesis of mycotoxin FB known to contaminate maize and maize products were developed and a pair of primers specific to a conserved Fusarium beta-tubulin gene, aimed at detection of most Fusarium species. There has been applied quantitative PCR assay for quantification of total Fusarium spp. and potentially mycotoxigenic Fusarium verticillioides.

Background and purpose: There are various methods for molecular detection of SARS-CoV2 genome among which, PCR-based methods are the most reliable for making diagnosis. The majority of approved PCR kits for detection of Coronavirus are based on TaqMan real-time PCR which is expensive due to incorporating fluorescent and quencher-harboring probe. The aim of this study was to design a simple and affordable method for detection of SARS-CoV2 based on a more affordable SYBR green qPCR method. Materials and methods: Specific primers were designed for the virus nucleocapsid gene and the human control gene using Oligo software. The specificities of the primers were examined by Primer- BLAST capability according to NCBI database. Nasal swab samples were obtained from 10 PCRconfirmed COVID-19 patients and 10 healthy control people. Results: The designed reactions were performed in full compliance with the kit approved by the Ministry of Health to discriminate healthy individuals from COVID-19 patients. The Ct cut-off values calculated for N1 and N2 reactions were 39.72 and 39.69, respectively. Conclusion: The designed SYBR green-based reaction showed a potential role for detection of SARS-CoV2 genome in clinical samples and this method can be considered as a less-expensive alternative to TaqMan real time PCR if the primers and standard reaction conditions are properly designed. [ABSTRACT FROM AUTHOR]

Soybean is threatened by many pathogens that negatively affect this crop's yield and quality, such as various Fusarium species that cause wilting and root rot diseases. Fusarium root rot (FRR) in soybean can be caused by F. graminearum and other Fusarium spp. that are associated with Fusarium head blight (FHB) in cereals. Therefore, it was important to inquire whether Fusarium pathogens from soybean can cause disease in wheat and vice versa. Here, we investigated the FRR complex in Manitoba (Canada) from symptomatic plants, using both culture- and molecular-based methods. We developed a molecular diagnostic toolkit to detect and differentiate between several Fusarium spp. involved in FHB and FRR, then we evaluated cross-pathogenicity of selected Fusarium isolates collected from soybean and wheat, and the results indicate that isolates recovered from one host can infect the other host. Trichothecene production by selected Fusarium spp. was also analyzed chemically via liquid chromatography mass spectrometry in both soybean (root) and wheat (spike) tissues. Trichothecenes were also analyzed in soybean seeds from plants with FRR to check the potentiality of trichothecene translocation from infected roots to the seeds. All of the tested Fusarium isolates were capable of producing trichothecenes in wheat spikes and soybean roots, but no trichothecenes were detected in soybean seeds. This study provided evidence, for the first time, that trichothecenes were produced by several Fusarium spp. (F. cerealis, F. culmorum, and F. sporotrichioides) during FRR development in soybean. [ABSTRACT FROM AUTHOR]

Stachybotrys (S.) chartarum had been related to dangerous health problems in animals and humans that take place when exposure to S. chartarum toxins. S. chartarum had been isolated from various substrates, ranging from inappropriately stored feed and culinary herbs to damp buildings. To evaluate the pathogenic potential of isolates, it is essential to identify them with different methods. The occurrence and genetic diversity of S. chartarum isolates from faba beans dust during threshing in Upper Egypt were investigated. Low counts of Stachybotrys were found (six isolates) and identified morphologically by single-spore isolation and molecularly by the amplification of the specific internal transcribed spacer (ITS) region and glyceraldehydes-3-phosphate dehydrogenase (gpd). The genetic diversity of the collected isolates was studied by specific genes random primer polymerase chain reaction (SGRP-PCR). The phylogenetic analysis of S. chartarum showed that the specific primers IT51 and StacR3 used by commercial laboratories for detecting S. chartarum were not able to differentiate species of S. chartarum from S. chlorohalonata and unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of SGRP fragments confirmed this result. The six isolates of S. chartarum were analyzed for the presence of trichodiene synthase (Tri5) gene, which needed in the early stage of the trichothecene synthesis path. All the tested isolates were positive for the Tri5 gene. Further study on the taxonomic status of the epithet S. chartarum is necessary and presence of sub species to S. chartarum might be acceptable depending on the variations of morphological characteristics which were confirmed by molecular techniques. [ABSTRACT FROM AUTHOR]

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department 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.)

BACKGROUND Apple snails from the genus Pomacea have spread widely in paddy fields and other wetlands of southern China since their introduction in the 1980s. Pomacea spp. are commonly identified using mitochondrial COI sequences. However, sequencing the nuclear elongation factor 1‐alpha (EF1α) gene revealed genetic introgression between field populations of P. canaliculata and P. maculata, which produce surviving hybrids in laboratory crossbreeding experiments. RESULTS: In this study, we sequenced 1054 EF1α clones to design specific primers and established a fast and accurate multiplex polymerase chain reaction (PCR) method for genotyping EF1α. Combined with genotyping P. canaliculata and P. maculata based on mitochondrial COI and nuclear EF1α, we revealed the genetic introgression patterns of 30 apple snail populations in China. Purebred and hybrid individuals of P. canaliculata were widely distributed, while pure maculata‐EF1α type was detected only in a few individuals identified as P. canaliculata based on COI sequences. Each egg clutch had one to three genetic patterns, indicating multiple paternity or segregation in the progeny of hybrids. The higher percentages of hybrids in both wild populations and progeny than the homozygotes indicated a potential heterosis in the apple snail populations. Additionally, egg size and clutch size of the apple snails became homogeneous among the non‐native populations exhibiting introgression hybridization. CONCLUSION: Our findings emphasize the value of apple snails as a model to study the mechanisms and impacts of introgressive hybridization on fitness traits. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The current study is conducted to detect bacterial contamination of groundwater in Al-Dour district within Salah al-Din Governorate for a distance extending about 35 km by eight wells with depths ranging between (65-90) meters during the study period extending from spring 2018 to winter 2019. The study includes isolation and identification of the most important species of bacteria contaminating well water and investigating its virulence factors with molecular diagnosis. The Isolates were detected as being able to produce some virulence factors, which included hemolycin, lipase and protease, lecithinase, biofilm, urease, and beta-lactamases, is detected. The results show that Klebsiella pneumoniae produces biofilm, hemolysin, lipase and lecithinase by 100%, while beta-lactamase and both urease and protease enzymes are not produced. As for Enterobacter cloacae, it produced hemolysin, protease and lipase by 100%, while it produced the enzymes beta-lactamase and lecithinase by 50%, and did not form or produce the biofilm and the urease enzyme. Concerning Aeromonas veronii, it produced beta-lactamase, lipase, biofilm, protease and lecithinase by 100% and hemolysin by 50% and did not produce urease enzyme. Enterococcous cecorum produced beta-lactamase, urease, protease and biofilm by 100%, and produced hemolysin and lipase enzymes by 66.7% and lecithinase enzyme by 33.3%, while Enterococcous columbae produced beta-lactamases, urease and biofilm by 100% but unable to produce lipase, lecithinase, hemolysin and protease. Granulicatella elegans were shown to be producing beta-lactamase, lecithinase, biofilm and protease by 100%, but did not produce hemolysin, urease and lipase enzymes. As for Aeromonas hydrophila, they appeared to be producing lipase, urease and hemolysin by 100%, but produced protease, lecithinase and beta-lactamase by 50% but did not produce the biofilm. The results of Polymerase Chain Reaction (PCR) show the use of specialized primers of some genes of virulence factors in the isolated bacteria. The protease gene encoded ProA and beta-lactamases gene encoded MOXM are found in Enterobacter cloacae, the hemolysin gene encoded HemK and the biofilm gene encoded LuxS found in Klebsiella pneumonia, the urease gene encoded UreA and lipase gene encoded lip found in Aeromonas hydrophila. As for A.veronii, it has been detected holding the protease gene encoded ProA and the lipase gene encoded lip the. With respect to the genera Enterococcus sp., Tuf biofilm encoded is found in both Enterococcous columbae and Enterococcous cecorum. In Granulicatella elegans, the presence of the RpoB gene is detected. [ABSTRACT FROM AUTHOR]

Abstract The gypsy moth—Lymantria dispar (Linnaeus)—is a worldwide forest defoliator and is of two types: the European gypsy moth and the Asian gypsy moth. Because of multiple invasions of the Asian gypsy moth, the North American Plant Protection Organization officially approved Regional Standards for Phytosanitary Measures No. 33. Accordingly, special quarantine measures have been implemented for 30 special focused ports in the epidemic areas of the Asian gypsy moth, including China, which has imposed great inconvenience on export trade. The Asian gypsy moth and its related species (i.e., Lymantria monocha and Lymantria xylina) intercepted at ports are usually at different life stages, making their identification difficult. Furthermore, Port quarantine requires speedy clearance. As such, it is difficult to identify the Asian gypsy moth and its related species only by their morphological characteristics in a speedy measure. Therefore, this study aimed to use molecular biology technology to rapidly identify the Asian gypsy moth and its related species based on the consistency of mitochondrial DNA in different life stages. We designed 10 pairs of specific primers from different fragments of the Asian gypsy moth and its related species, and their detection sensitivity met the need for rapid identification. In addition, we determined the optimal polymerase chain reaction amplification temperature of the 10 pairs of specific primers, including three pairs of specific primers for the Asian gypsy moth (L. dispar asiatic), four pairs of specific primers for the nun moth (L. monocha), and three pairs of specific primers for the casuarina moth (L. xylina). In conclusion, using our designed primers, direct rapid identification of the Asian gypsy moth and its related species is possible, and this advancement can help improve export trade in China.

Introduction: Canola is one of the important oil crops in Iran and blackleg disease caused by Leptosphaeriamaculans is an economically important disease of rapeseed especially in the northern provinces of Iran. At the beginning of the season in early autumn, L. maculansareinitiatesby air-borne ascospores released from infected stubbles of previous crops. These ascospores germinate and produce leaf lesions. The fungus then grows systemically from the leaf lesions to stem where cankers are producedwhich can result in major yield loss. Blacklegiscaused by a complex of at least two species of Leptosphaeria: L. maculans and L. biglobosa. Of these two species whichL. maculanswasis much more prevalent and virulent than L. biglobosa and was reported from almost all oilseed rape growing regions of the world.A number ofmethods has been developed to differentiate isolates of thesetwo groupsthrough morphological, physiological, genetic and molecular markers. Among these virulence markers of group pathogenicity are most widely used for characterizing the pathogen population. The purpose of this research work was to investigate isolates identification based on colony morphology and molecular methods. Moreover, for management strategies to be effectiveit is important to recognize the population structure based on pathogenicity groups and an understanding of possible shift in fungus population in thenorth of Iran. Materials and Methods: Infected plants of oilseed rape were collected from the northeast Iranduring 2013-2016. Diseased plant organs with clearly defined symptoms of the disease were used for the isolation of the fungi. All isolates were analyzed using a combination of morphological and physiological. In order to confirm the identification of L. maculans from oilseed rape isolates, DNA was prepared using the standard method described previously. The ITS region of genome of all isolates was amplified using polymerase chain reaction with specific primers pair LmF, lmR. Some isolates were sequenced with ITS1 and ITS2 along with the 5.8S rRNAregion and then sequence data were compared with that of available L. maculans isolates in Gene Bank. Aggressive isolates of L. maculans collected in widely separated geographic regions were further divided into pathogenicity groups based on virulence of three differentBrassica napuscultivars. Results and Discussion: Morphological and physiological and molecular characterizations of 72 isolates were performed. Isolates grewslowly, the pycnidia of the fungus were black, globose to subglobose in shape and conidia were single-celled, hyaline. The most of isolates formed yellow and pigment formation was scored in six groups in PDB at 18°C in the dark on the shaker. The PCR detection showed that all isolates were amplified by L. maculans-specific primer pair and a 334-bp PCR product was reliably amplified from L. maculans. The sequence analysis of the ITS region revealed that the sequences had 99.7% sequence similarity with the ITS sequences of known L. maculansisolates in GenBank by NCBI Blast search. This isolates were registered with accession number KX649997 and KX792142 in gene bank and this confirmed that the pathogen exists in Brassica napus samples. The role of morphological identification in virulence is complex and the production of pigment and mycelial growth is not always correlated with virulence. Therefore, based on amplification with type specific primers the amplified product fragment specific for group A was found in all isolates. It is important to know which pathotype is present, because the pathotypes of L. maculans differ in the amount of damage they cause. Based on the reaction of isolates on differential cultivars all four group pathogenicity PG-2, PG-3, PG-4 and PGT were observed in aggressive isolates. The most isolates were classified to be a pathogenicity PG-4 virulent on three differentcultivars and for the first time wasidentified in Iran. Conclusions: In order to determine whether isolates belonging to the canker L. maculans are present in the north of Iran, initial species identification based on colony morphology was confirmed using molecular methods. Asthe pathogen causes considerable losses, thefast identification and pathotype determination are important for agriculture and successful management of blackleg disease. Our results showed that an understanding of possible shift in fungus populations of PG2 to PG4 will be of value in developing strategies for successful management of blackleg disease.

Eucalyptus wilt disease was one of the most important stem disease in Eucalyptus plantation. Fusarium solani was one of the main pathogen. A specific fragment was detected from F. solani based on the RAPD-PCR technique. It was then cloned and sequenced. Specific SCAR primers F9/R9 were designed according to the sequencing results. The amplification conditions were optimized and the primer sensitivity was detected. With the SCAR primer F9/R9, a clear and bright fragment of about 200 bp was amplified only from F. solani, but no fragment from other pathogen. The primer could detect the specific fragment from 4×10-7 ng·μL-1 of genomic DNA and individual spore. F. solani from Eucalyptus infection stem and soil could be detected by these SCAR primer. Thus, the SCAR primer F9/R9 could be used for early diagnosis and quarantine of Eucalyptus wilt disease. This method was simple and of high specificity. [ABSTRACT FROM AUTHOR]

The invasive Crepidula fornicata caused major problems along the European Atlantic coast, especially in France and Netherlands where high densities leads on changes in the habitat, disturb native marine wildlife as well as it originates competition for space and food. Despite its dangerous invasive nature, regular monitoring to alert about its presence in risk areas, like the south Bay of Biscay (Spain and south France), is not done yet. Here, we developed a species-specific marker to detect the presence of C. fornicata in environmental samples (eDNA) of seawater. The novel C. fornicata specific primers amplified a region of 239 bp within the COI gen. We employed this tool to check its presence in 6 estuaries of the Cantabrian Sea, an area comprised between the Spanish and French limits of the previously reported presence of this limpet in the south Bay of Biscay. The presence of C. fornicata was confirmed in A Coruña (Galicia, Spain), Eo and Villaviciosa estuaries (Asturias, Spain) while it was not detected in Santander, Bilbao (Spain), and Bayonne (France). This new method to detect C. fornicata could be easily implemented in regular monitoring to prevent and manage future invasions of this species. • The first highly sensitive C. fornicata specific marker was designed to detect this invasive limpet in environmental samples. • Six Spanish and French estuaries in the South Bay of Biscay were monitorized to detect Crepidula fornicata employing eDNA. • The results confirmed the presence of this invasive mollusc in Galicia and Asturias, not in Santander, Bilbao and Bayonne. • Spreading of the invasive C. fornicata in European coasts can be regularly monitored using this new molecular tool. [ABSTRACT FROM AUTHOR]

Basal rot is the main and economically soil-borne disease of onion that caused by various Fusarium species worldwide. To identify the prevailing Fusarium species, 140 Fusarium isolates were obtained from red onion bulbs farms in 10 regions of East and West Azarbaijan provinces in 2015. By inoculating 80 selected isolates, 40 of them were pathogenic on onion. These 40 isolates were identified as F. oxysporum with 43.62%, F. subglutinans with 44%, F. culmorum with 50.66%, F. avenaceum with 51%, F. solani with 42.41%, F. crookwellens with 55%, F. proliferatum with 47.16% and F. redolens with 55.5% virulence. Their frequency were 20%, 2.5%, 7.5%, 5%, 42.5%, 2.5%, 15% and 5%, respectively. Forty studied isolates demonstrating that, 14.2% were highly virulent, 26.1% virulent, 40.3% moderately virulent and 19.4% weakly virulent. This is the first report of F. avenaceum and F. crookwellens as the causal agents of red onion basal rot in Iran. [ABSTRACT FROM AUTHOR]

A quantitative PCR (qPCR)-assay was developed to detect airborne inoculum of Nothopassalora personata, causal agent of late leaf spot (LLS) on peanut, collected with a modified impaction spore trap. The qPCR assay was able to consistently detect as few as 10 spores with purified DNA and 25 spores based on crude DNA extraction from rods. In 2019, two spore traps were placed in two peanut fields with a history of LLS. Sampling units were replaced every 2 to 4 days and tested with the developed qPCR assay, while plots were monitored for symptom development. The system detected inoculum 35 to 56 days before visual symptoms developed in the field, with detection related to environmental parameters affecting pathogen life-cycle and disease development. This study develops the framework of the qPCR spore trap system and represents the initial steps towards validation of the performance of the system for use as a decision support tool to complement integrated management of LLS.

Abstract Dengue virus (DENV) is one of the life-threatening viruses to the human. In this study, we have designed specific novel primers for rapid discriminative detection of DENV-1, DENV-2, and DENV-4 by real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction. The effect of parameters such as reaction temperature and magnesium sulfate was investigated on the RT-LAMP reaction for detection of DENV RNA. Under the optimal conditions, this method is able to differentiate and to detect DENV within 25 min, exhibiting detection limit of 3.5 copies/μL. Importantly, the novel specific primers-based RT-LAMP assay did not react with other viruses, suggesting the selectivity of the method towards DENV RNA. The RT-LAMP reaction products are easily visualized with naked-eye when irradiated them under UV light at 365 nm. Amplification products could be visualized directly for color changes. This method provides a facile, and accurate molecular amplication technique for the rapid discriminative detection of dengue viruses. The RT-LAMP platform can be used as a promissing diagnostic tool for discriminative detection of DENV without aid of complicated protocols or sophisticated equipment. Graphical abstract fx1 Highlights • RT-LAMP assay by newly designed primers for Dengue virus detection. • Rapid platform for discriminative detection of Dengue virus. • Detection limit of 3.5 copies/μL within only 25 min. • Four primers used in RT-LAMP assay for each molecular detection. [ABSTRACT FROM AUTHOR]

Abstract: A method is proposed for the rapid identification of the taxa of five industrially valuable yeast groups: Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Kluyveromyces lactis. The specific primers selected based on nucleotide sequences of genes encoding rRNA were used in PCR. The method was effective in the identification of either pure yeast cultures or a mixture of deposited yeast strain cultures. Thus, this method was shown to be potentially viable for the identification of the studied yeasts in microbial preparations at the stages of the construction of producing strains and in industrial fermentations. [ABSTRACT FROM AUTHOR]

Several Bacillus strains have been well studied for their ability to control soil-borne plant diseases. This property is linked to the production of several families of lipopeptides. Depending of their structure, these compounds show antifungal and/or plant systemic resistance inducing activities. In this work, the biodiversity of lipopeptides produced by different antifungal Bacillus strains isolated from seeds, rhizospheric, and non-rhizospheric soils in Algeria was analyzed. Sixteen active strains were characterized by PCR for their content in genes involved in lipopeptide biosynthesis and by MALDI-ToF for their lipopeptide production, revealing a high biodiversity of products. The difficulty to detect kurstakin genes led us to design two new sets of specific primers. An interesting potential of antifungal activity and the synthesis of two forms of fengycins differing in the eighth amino acid (Gln/Glu) were found from the strain 8. Investigation of its genome led to the finding of an adenylation domain of the fengycin synthetase predicted to activate the glutamate residue instead of the glutamine one. According to the comparison of both the results of MALDI-ToF-MS and genome analysis, it was concluded that this adenylation domain could activate both residues at the same time. This study highlighted that the richness of the Algerian ecosystems in Bacillus strains is able to produce: surfactin, pumilacidin, lichenysin, kurstakin, and different types of fengycins. [ABSTRACT FROM AUTHOR]

Opsariichthys uncirostris uncirostris is a subspecies of Amur three-lips Opsariichthys uncirostris (Temminck and Schlegel, 1846) and endemic to Japan. This only piscivorous fish among Cyprinidae in Japan, hereafter three-lips, is in a unique ecological situation, that is, the species is endangered in the native habitats such as Lake Biwa water system and Lake Mikata, but on the other hand, three-lips is threatening the other native species as an intra-nation invasive species in introduced habitats. In this study, a species-specific primer-probe set was developed for detecting three-lips using environmental DNA (eDNA) analysis for monitoring the species distribution. Ten out of 11 sites in Futatsugawa River system, Kyusyu, and 15 out of 28 sites in Lake Biwa tested positive. In the Futatsugawa, three-lips was confirmed to occupy both of the lotic sites in the main stem of the river and the lentic sites in the inner part of small ditches. In Lake Biwa, positives were biased to the sites in north basin of the lake than smaller south basin. The PCR amplicons in the positive samples were confirmed to have DNA sequences of the target species, suggesting the accuracy of this species-specific analysis. The eDNA detection system developed in this study will contribute to future follow-up monitoring by providing a cost-effective and less labor-intensive method for determining the distribution of three-lips with dual identities as a threatened and invasive species. [ABSTRACT FROM AUTHOR]

Red leaf blotch of almond, caused by the fungus Polystigma amygdalinum, results in early defoliation of trees and decreases fruit production in many Mediterranean and Middle Eastern countries. A latent period of 30-40 d has been reported for this pathogen before disease symptoms are expressed, which makes targeted fungicide control difficult. A quantitative real-time PCR detection method was developed to detect and quantify the fungus in biological samples, to assist early detection. A primer pair was designed based on the ITS region of the fungal rDNA, and this was highly specific and sensitive, enabling detection of a minimum of 12 pg of P. amygdalinum DNA and seven ascospores in artificially-prepared ascospore suspensions. A protocol was also developed to quantify ascospores on plastic tapes which are commonly used in volumetric air samplers. The detection limit for these samples was the same as for ascospore quantification in aqueous suspensions. The pathogen was also quantified in naturally infected leaves showing different stages of disease development, including early stages of leaf infection with doubtful visual identifications. Future practical applications of the method developed here are discussed in view of improving the management of red leaf blotch of almond. [ABSTRACT FROM AUTHOR]

Giardia duodenalis is one of the six Giardia species and it is the most common, cosmopolitan flagellate that infects humans and many species of animals. This species exhibits considerable genetic diversity; to date, eight assemblages (A-H) have been defined. These assemblages differ in host specificity: assemblages A and B have been found in both humans and in many animal species. Mixed infections with Giardia (A and B) assemblages have been reported in humans and in animals. Many molecular techniques are effective and rapid for the detection of G. duodenalis and also for the determination of genetic variability of isolates in clinical and environmental samples. In this context, the aim of this study was to design new assemblage-specific primers for rapid detection and identification of G. duodenalis assemblages A and B and both of these assemblages simultaneously using quantitative real-time polymerase chain reaction (qPCR). Fragments of glutamate dehydrogenase and triose phosphate isomerase were used as targets in the design of primers. In conclusion, the use of G. duodenalis assemblage-specific primers designed in this study allows quick identification of human infectious G. duodenalis assemblages A and B as well as mixed AB assemblages in a sample without further sequencing of the amplification products, which reduces the cost of study and the waiting time for the results. [ABSTRACT FROM AUTHOR]