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Conventional and real-time PCR for the identification of Fusarium fujikuroi and Fusarium proliferatum from diseased rice tissues and seeds

Conventional and real-time PCR for the identification of Fusarium fujikuroi and Fusarium proliferatum from diseased rice tissues and seeds | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Fusarium fujikuroi is a species of the Gibberella fujikuroi species complex (GFSC) and the causal agent of bakanae disease on rice. Even if F. fujikuroi is the most abundant Fusarium species found on rice, other species can also be isolated from rice, such as F. proliferatum. Multiple alignment of translation elongation factor (TEF) gene sequences of different Fusarium spp., showed a deletion of six nucleotides in F. fujikuroi sequence and a two nucleotide polymorphism in the same region of F. proliferatum sequence. These elements of variability were used to develop a conventional and Real-Time PCR assay for diagnosis. The species specific primer pairs (Fuji1F/TEF1R and Proli1F/TEF1R) gave a product of 179 and 188 bp for F. fujikuroi and F. proliferatum respectively. Primer specificity was confirmed by analyzing the DNA of the most representative species of the GFSC and 298 strains of Fusarium spp. isolated from rice plants and seeds in Italy. The specific primers were also successfully used to detect fungal presence directly from infected rice tissues and seeds, providing a rapid tool for the early detection of pathogen contamination.

 

Maria Teresa Amatulli, Davide Spadaro, Maria Lodovica Gullino and Angelo Garibaldi

European Journal of Plant Pathology

October 2012, Volume 134, Issue 2, pp 401-408

DOI: 10.1007/s10658-012-9998-0

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Development of a quantitative real-time PCR assay for Phytophthora infestans and its applicability to leaf, tuber and soil samples

Development of a quantitative real-time PCR assay for Phytophthora infestans and its applicability to leaf, tuber and soil samples | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non-potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogensP. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestansoospore detection are explored and tested experimentally.

 

A. K. Lees, L. Sullivan, J. S. Lynott and D. W. Cullen
Plant Pathology
Volume 61, Issue 5, pages 867–876, October 2012

 

DOI: 10.1111/j.1365-3059.2011.02574.x

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A Nested PCR Assay for Detecting Valsa mali var. mali in Different Tissues of Apple Trees

A nested polymerase chain reaction (PCR) assay for detecting Valsa malivar. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. maliisolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsaspp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.


Rui Zang, Zhiyuan Yin, Xiwang Ke, Xiaojie Wang, Zhengli Li, Zhensheng Kang, and Lili Huang
Plant Disease
November 2012, Volume 96, Number 11, Pages 1645-1652


http://dx.doi.org/10.1094/PDIS-05-11-0387-RE


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Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units

Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Significance and Impact of the Study: Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthoracomplexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments.

 

A.M. Vettraino, P. Bonants, A. Tomassini, N. Bruni, A. Vannin

Letters in Applied Microbiology

Volume 55, Issue 5, pages 390–396, November 2012

 

DOI: 10.1111/j.1472-765x.2012.03310.x

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Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing

Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it
Aim: To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores ofLeptosphaeria maculans, a fungal pathogen of canola (Brassica napus). Methods and Results: The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90-bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble. Conclusion: This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops. Significance and Impact of the Study: Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white-nose syndrome has been detected.

 

A.P. Van de Wouw, B.J. Howlett

Journal of Applied Microbiology
Volume 113, Issue 5, pages 1145–1153, November 2012

 

DOI: 10.1111/j.1365-2672.2012.05413.x

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Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples

Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genusAspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20 pg of pure DNA/reaction for A. flavus, A. nomiusand A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry.


Jie Luo, Rudi F. Vogel, Ludwig Niessen

International Journal of Food Microbiology

Volume 159, Issue 3, 15 October 2012, Pages 214–224



http://dx.doi.org/10.1016/j.ijfoodmicro.2012.08.018

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Development and evaluation of a real-time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease

Development and evaluation of a real-time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi-selective medium and identifying it using morphological traits. This method is time-consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real-time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real-time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10−5 ng of F. circinatum DNA per PCR. The culture-based method detected Fusarium spp. in four of six of the same seed lots. The real-time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture-based method requires a minimum of 1–2 weeks. This new real-time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.

 

T. J. Dreaden, J. A. Smith, E. L. Barnard and G. Blakeslee

 

DOI: 10.1111/j.1439-0329.2012.00774.x

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The autors use also primers previously developed. Data from the original works will be loaded onto the database.

 

Schweigkofler, W.; O’Donnell, K.; Garbelotto, M., 2004: Detection and quantification of Fusarium circinatum, the casual agent of pine pitch canker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method. Appl. Environ. Microbiol.70, 3512–3520.

 

Ioos, R.; Fourrier, C.; Iancu, G.; Gordon, T.R., 2009: Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry. Phytopathol.99, 582–590.

 

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Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F. poae isolates. A total of 125 F. poae, four F. cerealis, two F. culmorum, oneF. langsethiae, one F. sporotrichioides and seven F. graminearum, plus F. austroamericanum,F. meridionale,F. graminearum sensu stricto andF. cortaderiae from the NRRL collection were analysed, and only F. poae isolates gave a positive result for the presence of a 296-bp partial tri7DNA fragment. Moreover, the primer set was tested from cereal seed samples where F. poae and other Fusarium species with a negative result for the specific reaction (F. graminearum,F. oxysporum,F. chlamydosporum,F. sporotrichioides,F. equiseti and F. acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F. poae isolates.


María I. Dinolfo, Germán G. Barros and Sebastián A. Stenglein
FEMS Microbiology Letters
Volume 332, Issue 2, pages 99–104, July 2012

 

DOI: 10.1111/j.1574-6968.2012.02581.x


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Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Apple scab, the most important disease of apple worldwide, is caused by Venturia inaequalis. Currently, evaluation of fungal pathogenicity and host resistance is based on a symptomatic disease rating. However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. In this study, a Venturia-specific real-time PCR assay was developed using primers designed around the specific internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene. Using SYBR® Green I technology, the assay can accurately quantify Venturia DNA over a concentration range of at least five orders of magnitude. Detection sensitivities were in the order of 100 fg. The method was used to quantify Venturia genomic DNA levels in leaves of three apple cultivars with different levels and types of scab resistance and artificially infected with V. inaequalis. The assay clearly discriminated between Venturia levels in monogenic resistant (‘Topaz’), polygenic resistant (‘Discovery’), and susceptible (‘Golden Delicious’) cultivars, and proved especially useful to quantify pathogen levels during the initial latent stage of infection. The real-time PCR data of ‘Golden Delicious’ were consistent with the observed evolution of the degree of sporulation during a time-course experiment. Although measurements were influenced by the presence of co-extracted PCR-inhibitors, the impact of these compounds was independent of the apple cultivar or the initial amount of fungal DNA present. In conclusion, real-time PCR amplification of the ITS2-5.8S rDNA of Venturia spp. is a faster, more objective and more sensitive method to monitor fungal growth and to evaluate host resistance than phenotypic disease rating scores.

 

Bruno Daniëls, Anke De landtsheer, Rozemarijn Dreesen, Mark W. Davey, Johan Keulemans

European Journal of Plant Pathology
December 2012, Volume 134, Issue 4, pp 821-833

 

DOI:10.1007/s10658-012-0058-6

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A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1 kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R⊃2; = 0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.

 

James W. Woodhall, Kathryn M. Webb, Patricia M. Giltrap, Ian P. Adams, Jeff C. Peters, Giles E. Budge, Neil Boonham

European Journal of Plant Pathology

November 2012, Volume 134, Issue 3, pp 467-473

 

DOI: 10.1007/s10658-012-0025-2

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Development of Real-Time PCR for in wood-detection of Ceratocystis platani, the agent of canker stain of Platanus spp.

Development of Real-Time PCR for in wood-detection of Ceratocystis platani, the agent of canker stain of Platanus spp. | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Ceratocystis platani is a quarantine fungal pathogen agent of canker stain, a destructive disease affecting Platanus. Despite its diagnosis being critical for disease control, there is still no effective diagnostic tool as all known mycological and biological detection assays are problematical. In this study we developed highly effective Real-Time PCR methods based on the use of an intercalating dye, EvaGreen, and on a Taqman probe. We designed primers and probe on the internal transcribed spacer (ITS) region of rDNA, and used them for the amplification and detection of a 95 bp C. platani amplicon. Inference of standard curves revealed that both Real-Time procedures have similar and high values of amplification efficiency when applied to a range of templates, e.g. genomic fungal DNA and DNA extracted from diseased wood. The methods were sensitive with a detection limit of 10 fg μl−1 C. platani genomic DNA. They were specific as they did not yield any detection signal when applied to non-target fungal taxa colonizers of Platanus wood. Reliability was demonstrated through the positive detection of a collection of C. platani isolates and of wood samples collected from naturally infected trees. Robustness was positively verified through detection on artificially infected trees, which were tested at different times after death, up to 27 months. Generating a standard curve with a target-amplicon-containing plasmid enabled an absolute quantification and a comparison between the discoloured wood of resistant and susceptible genotypes. The importance of the methods for studies on pathogen epidemiology and host resistance is also discussed.

 

 

Massimo Pilotti, Valentina Lumia, Giovanni Di Lernia, Angela Brunetti

European Journal of Plant Pathology
September 2012, Volume 134, Issue 1, pp 61-79

 

DOI: 10.1007/s10658-012-0022-5

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FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

Background

Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques.Methods

We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.

Results

We designed FungiQuant, a TaqMan(R) qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged fromConclusions

FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

 

Cindy M Liu, Sergey Kachur, Michael G Dwan, Alison G Abraham, Maliha Aziz, Po-Ren Hsueh, Yu-Tsung Huang, Joseph D Busch, Louis J Lamit, Catherine A Gehring, Paul Keim and Lance B Price

BMC Microbiology 2012, 12:255

doi:10.1186/1471-2180-12-255


 

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Early detection of gray mold in grape using conventional and molecular methods

Early detection of gray mold in grape using conventional and molecular methods | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Botrytis cinerea affects grape quality and yield, and can be difficult to manage due in part to non-symptomatic, quiescent infection in berry development. The aim of this study was to develop a dual system for the detection, isolation and quantification of B. cinerea. After three days of samples replication on the modified selective medium (mKERS), the results showed a significant infection effect on the majority of inflorescence samples, especially on the small berries which demonstrated Botrytisinfection in all tested plants and appeared to be highly susceptible to Botrytis infection prior to harvest. Moreover, infection variation was determined in almost all inflorescence samples taken from different plants. The real-time PCR assay was used to determine the DNA quantity of B. cinerea in each sample tested. A linear relationship was found in these two systems, conventional and molecular assays, to demonstrate the infection of different samples with B. cinerea. Although, the real-time PCR assay was highly expensive, it appeared to be more rapid and sensitive than the conventional selective medium assay, allowing both detection and quantification of B. cinerea within 3 h. However, conventional assay has an advantage of both detection and isolation of viable cells of B. cinerea, which resulted in making a wide collection of different isolates. Furthermore, this conventional assay is cheaper than molecular test, especially when we carry out a routine work. This dual method proved to be selective and sensitive assays and should be used to monitor Botrytis infection in the field.

 

Hala Abdel Wahab and Rania A. A. Younis

African Journal of Biotechnology Vol. 11(86), pp. 15251-15257

25 October 2012

DOI: 10.5897/AJB12.1079

 

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Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.


Via Alejandro Rojas, Guogen Yang
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Assessment of Infection by Fusarium pseudograminearum in Wheat Seedling Tissues Using Quantitative PCR and a Visual Discoloration Scale

Assessment among cereal genotypes of relative seedling resistance to the crown rot pathogen Fusarium pseudograminearum has been primarily based on visual discoloration of the leaf sheaths. This study is the first to investigate the relationship between the widely used visual rating of seedling leaf sheath discoloration and the degree of colonization of these tissues by the pathogen, based on quantitative polymerase chain reaction (qPCR) of fungal DNA using primers specific for the translation elongation factor α sequence. Fourteen-day-old seedlings of four hard white spring wheat genotypes which differ in their degree of resistance to the pathogen, based on the expression of visible symptoms, were inoculated using a droplet method and assessed weekly from 7 to 35 days after inoculation (dai) for both discoloration and fungal DNA content per unit of tissue weight. Both visual assessment of disease symptoms and qPCR of fungal biomass indicated significant differences between the partially resistant and susceptible wheat genotypes from 14 dai. Visual discoloration of leaf sheath tissues was strongly correlated with fungal biomass estimated by qPCR in all four genotypes; however, this correlation became weaker with increasing time after inoculation. Significant correlations between these parameters were indicated at 14, 21, and 28 dai whereas, by 35 dai, the correlation was not significant. Evaluation of plants at 14 dai provided a rapid test which gave clear discrimination between lines for both parameters and was the time point of closest correlation between fungal colonization and disease symptoms. Symptom expression at all times following inoculation was accompanied by tissue infection, and at no time was symptomless infection observed under this screening environment. These qPCR results confirm that visual assessments of disease symptoms reflect the extent of tissue colonization by the pathogen in recently colonized tissues and confirm the validity of visual assessments for disease rating in high-throughput screening of breeding materials.


Noel L. Knight, Mark W. Sutherland, Anke Martin, and Damian J. Herde
Plant Disease
November 2012, Volume 96, Number 11, Pages 1661-1669


http://dx.doi.org/10.1094/PDIS-12-11-1050-RE


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Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae

Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

In North America, one of the most important root diseases of Poa and Festuca turf is summer patch, caused by Magnaporthe poae. Detection and identification of M. poaein infected roots by conventional culture-based methods is difficult and time consuming, typically taking 3 wk or longer to accomplish. In this study, a culture-independent, TaqMan real-time PCR assay was developed for the detection of M. poae from the roots of fungicidetreated and non-treated Kentucky bluegrass (Poa pratensis) turf. The assay was validated with the target pathogen, closely related fungal species and a number of other microorganisms that inhabit the same host and soil environment. This assay was more sensitive (could detect as little as 3.88 pg genomic DNA of M. poae), rapid and accurate compared to direct microscopic observation and isolation on a selective medium. The real-time PCR detection results corresponded closely to visual assessments of disease severity in the field. Utilization of this assay in diagnostic laboratories will enable turfgrass managers to more quickly and effectively detect and potentially reduce fungicide usage through early and accurate identification of the pathogen.


Shuang Zhao, Bruce B. Clarke, Qirong Shen, Lisa Zhang, and Ning Zhang
Mycologia 2012 104:1250-1259

 

doi: 10.3852/11-365

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Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.


Masahiro Shishido, Itsuki Kubota, Tatsuya Ohashi, Toshiyuki Usami

Journal of General Plant Pathology

January 2013, Volume 79, Issue 1, pp 18-27


DOI: 10.1007/s10327-012-0415-5


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Quantitative PCR: An appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10 cells mL− 1. B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated.


Elize Willenburg, Benoit Divol
Quantitative PCR: An appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine
International Journal of Food Microbiology, 160 (2012), pp. 131–136

 

http://dx.doi.org/10.1016/j.ijfoodmicro.2012.09.012

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A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.


Jonathan Scauflaire, Marie Godet, Mélanie Gourgue, Charlotte Liénard, Françoise Munaut

Fungal Biology

Volume 116, Issue 10, October 2012, Pages 1073–1080

 

http://dx.doi.org/10.1016/j.funbio.2012.07.011


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Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae

Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae,Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL−1 of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields.

 

Ting-Ting Dai, Chen-Chen Lu, Jing Lu, SuoMeng Dong, WenWu Ye, YuanChao Wang and XiaoBo Zheng

FEMS Microbiology Letters
Volume 334, Issue 1, pages 27–34, September 2012

 

DOI: 10.1111/j.1574-6968.2012.02619.x

 

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A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4

The Fusarium genus causes devastating plant diseases worldwide, in which Fusarium oxysporum is the most serious crop pathogen. Disease monitoring is the basis of integrated pest management of any disease. The lack of rapid, accurate, and reliable device to detect and identify plant pathogens is one of the main limitations in integrated disease management. This study describes an efficient and quantifiable diagnosis method for the specific detection of F. oxysporum f. sp. cubense (Foc) race 4 in field-infected banana. With the optimized PCR parameters using the SCAR (sequence characterized amplified region) primers FocSc-1/FocSc-2 and a real-time PCR strategy, the developed method showed high reproducibility and was very sensitive to detect extremely low quantities of Foc genomic DNA (gDNA). We also found that Foc gDNA in severely symptomatic banana pseudostems and leaves were 6946-fold and 26.69-fold more than in those of mild-symptomatic banana, respectively.


Ying-Hong Lin, Ching-Chung Su, Chih-Ping Chao, Chi-Yu Chen, Chung-Jan Chang, Jenn-Wen Huang, Pi-Fang Linda Chang

European Journal of Plant Pathology
February 2013, Volume 135, Issue 2, pp 395-405

 

DOI: 10.1007/s10658-012-0096-0

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Development of specific markers for identification of Indian isolates of Fusarium oxysporum f.sp. ricini

Development of specific markers for identification of Indian isolates of Fusarium oxysporum f.sp. ricini | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Fusarium oxysporum f. sp. ricini (F. o. ricini) is a ubiquitous soil borne pathogen which causes wilt disease on castor (Ricinus communis L). Rapid and reliable detection of the pathogen is essential for undertaking appropriate and timely disease management measures. Identification based on cultural, morphological characteristics and pathogenicity tests are time-consuming and laborious. Traditional methods are now being increasingly replaced by molecular detection techniques, which are much faster and more specific. In this study we have identified two RAPD markers of 1100 bp and 1350 bp in size which can be amplified by OPJ-14 and OPK-12 primers respectively for detection of F. o. ricini. These two fragments were fully sequenced and two pairs of SCAR primers (For-J14 Fwd/Rev and For-K12 Fwd/Rev) were designed. The specific primer pairs amplified a single band from all F. o. ricini isolates and there was no amplification from another thirteen Fusarium species / subspecies tested. These results clearly demonstrate that the designed SCAR primer pairs can be used consistently to detect F. o. ricini isolates, isolated from the diseased samples or soil samples. To our knowledge this is the first report on generation of SCAR markers for identification of Indian F. o. ricini isolates.

 

 

J. Madhusudhana Reddy, M. A. Raoof, K. Ulaganathan

European Journal of Plant Pathology
December 2012, Volume 134, Issue 4, pp 713-719

DOI:10.1007/s10658-012-0047-9

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The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato

The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates ofStagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma andBoeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.

 

Johannes de Gruyter, Marga P. E. van Gent-Pelzer, Joyce H. C. Woudenberg, Patricia C. J. van Rijswick, Ellis T. M. Meekes, Pedro W. Crous, Peter J. M. Bonants

European Journal of Plant Pathology
October 2012, Volume 134, Issue 2, pp 301-313

 

DOI: 10.1007/s10658-012-9990-8

 

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Development of a quantum dots FRET-based biosensor for efficient detection of Polymyxa betae

Rhizomania is the most destructive disease in sugar beet throughout the world. The disease is caused byBeet necrotic yellow vein virus (BNYVV). Polymyxa betae (Keskin) is the only known vector of BNYVV, for transmission of the virus between the plants. Developing molecular diagnostic methods has a major impact on forecasting and epidemiological studies as well as screening sugar beet plants used in resistance breeding programmes. In the present study, quantum dots (QDs) were biofunctionalized with a specific antibody against P. betae. The glutathione-S-transferase protein's (GST) corresponding antibody (anti-GST) was effectively conjugated to Tioglicolicacid-modified Cadmium-Telluride QDs (CdTe-QDs) synthesized in an aqueous solution via electrostatic interaction. The dye (Rhodamine) molecules were attached to the GST. Donor–acceptor complexes (QDs-Ab-GST-Rhodamine) were then formed based on the antigen–antibody interaction. The mutual affinity of the antigen and the antibody brought the CdTeQDs and rhodamine together close enough to allow the resonance dipole–dipole coupling required for fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, acceptable stability and could be used for real sample detection with consistent results. To the best of our knowledge, this is the first report on designing a nano-based biosensing tool for the detection of plant pathogenic fungi.

 

Hossein Safarpour, Mohammad Reza Safarnejad, Meisam Tabatabaei, Afshin Mohsenifar, Fatemeh Rad, Marzieh Basirat, Fatemeh Shahryari & Fatemeh Hasanzadeh
Canadian Journal of Plant Pathology
Volume 34, Issue 4, 2012, pages 507-515

DOI:10.1080/07060661.2012.709885

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Accurate detection of chestnut ink disease causing Phytophthora katsurae by nested PCR

Accurate detection of chestnut ink disease causing Phytophthora katsurae by nested PCR | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

We designed two specific primer pairs, KatI 3F/KatI 5R and KatI 4F/KatI 5R to detect P. katsurae by PCR and each primer produced specific amplicons of 747 bp and 721 bp, respectively. To assess the specificity and sensitivity of primer, 10-fold serial dilutions of P. katsurae genomic DNA were made ranging in concentration from 1 mgml−1 to 1 pgml−1. Using nested PCR, the limits of detection for P. katsurae were 100 ngml−1 and 10 ngml−1 of DNA, respectively. To identify the limits of detection for P. katsurae zoospores, zoospore suspensions were serially diluted 10-fold from 10 × 105 to 10 × 101 before being extracted. The limit of detection for both primer sets was 10 × 101. We applied to test tissue and soil samples from chestnut stands that had been infested by P. katsurae in Gongju, Chungnam Province, and Hadong, Gyeongsangnam Province, and could detect specific P. katsurae amplicons from trunk tissue collected from sites containing infected trees in nested PCR. These results show that nested PCR may be a useful tool for the accurate and sensitive detection of P. katsurae infection.

 

Dong Hyeon Lee, Sun Keun Lee, Sang Hyun Lee, Sang Yong Lee, Jong Kyu Lee

Australasian Plant Pathology
Volume 41, Issue 5 , pp 535-539

DOI: 10.1007/s13313-012-0154-2

 

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A new section on validation data in the EPPO database on diagnostic expertise

A new section on validation data in the  EPPO database on diagnostic expertise | PCR PRIMERS FOR THE IDENTIFICATION OF PHYTOPATHOGENIC FUNGI | Scoop.it

A new section on validation data for diagnostic tests for regulated pest has just been made open access in the EPPO Database on Diagnostic Expertise


Via Petter Françoise
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