scholarly journals IDENTIFICATION OF SUNFLOWER PATHOGENIC FUNGUS PLENODOMUS LINDQUISTII USING PCR WITH SPECIES-SPECIFIC OLIGONUCLEOTIDE PRIMERS

2020 ◽  
Vol 103 (3) ◽  
pp. 207-210
Author(s):  
М. М. Gomzhina ◽  
Ph. B. Gannibal
Keyword(s):  
Causative Agent ◽  
Pathogenic Fungus ◽  
Its Region ◽  
Fungal Species ◽  
Cross Reaction ◽  
Pcr Assay ◽  
Selective Amplification ◽  
Oligonucleotide Primers ◽  
Pure Cultures ◽  
Species Specific

Plenodomus lindquistii causes Phoma black stem of sunflower which is the most common stem disease of this crop in Russia. The diagnostics of both field specimens and pure cultures of P. lindquistii is troublesome. Molecular methods involving the use of the PCR are rapid diagnostic express tests that can precisely identify and detect fungal species. The aim of this study was to develop species-specific oligonucleotide primers for selective amplification of P. lindquistii DNA. The primers LepliF2/LepliR2 were designed on the basis of ITS region analysis and showed stable amplification of the target fungus DNA with no cross-reaction with other fungal species. The primers are recommended for express detection of the causative agent of Phoma black stem of sunflower. This is the first PCR assay that could be used to rapidly reveal and identify this pathogen.

scholarly journals Molecular Detection of Apiosporina morbosa, Causal Agent of Black Knot in Prunus virginiana

Plant Disease ◽  
2005 ◽  
Vol 89 (8) ◽  
pp. 815-821 ◽  
Author(s):  
J. X. Zhang ◽  
W. G. D. Fernando ◽  
W. R. Remphrey
Keyword(s):  
Pcr Amplification ◽  
High Sensitivity ◽  
Its Region ◽  
Fungal Species ◽  
Causal Agent ◽  
Diseased Plant ◽  
Pcr Assay ◽  
Single Spore ◽  
Prunus Virginiana ◽  
Pure Cultures

A specific and sensitive polymerase chain reaction (PCR) assay was developed to detect Apiosporina morbosa, the causal agent of black knot disease on chokecherry, Prunus virginiana (including the cultivar ‘Shubert Select’). A pair of A. morbosa-specific forward and reverse primers (AMF and AMR) was designed from the internal transcribed spacer (ITS) regions of A. morbosa, preamplified by universal ITS primers ITS1 and ITS4, and compared with the ITS region sequences of Fusarium, Alternaria, Phoma, and Cladosporium species associated with black knots. The primers were tested for their specificity to A. morbosa detection in the PCR assays using DNA derived from 64 pure cultures, including 42 single-spore isolates of A. morbosa and 22 isolates of other fungi, as well as healthy and diseased plant branches collected from the field. A product of ~400 bp was amplified from DNA of all isolates belonging to A. morbosa. No product was amplified from DNA of other fungal species, confirming the specificity of the newly designed primers. Within plant tissues, the pathogen was detected at further distances from the edges of knots on thicker branches bearing larger knots compared with thinner branches bearing smaller knots. The PCR assay has shown high sensitivity, needing only 100 fg of the A. morbosa DNA for a reliable PCR amplification with the AMF and AMR primers.

scholarly journals A PCR-Based Assay for Detection of Puccinia striiformis f. sp. tritici in Wheat

Plant Disease ◽  
2007 ◽  
Vol 91 (12) ◽  
pp. 1669-1674 ◽  
Author(s):  
J. Zhao ◽  
X. J. Wang ◽  
C. Q. Chen ◽  
L. L. Huang ◽  
Z. S. Kang
Keyword(s):  
Stripe Rust ◽  
Puccinia Striiformis ◽  
Pathogenic Fungus ◽  
Its Region ◽  
Bipolaris Sorokiniana ◽  
Fungal Species ◽  
Universal Primers ◽  
Effective Control ◽  
Pcr Assay ◽  
Wheat Leaves

Monitoring the pathogenic fungus of wheat stripe rust, Puccinia striiformis f. sp. tritici, plays a key role in effective control of the disease. In the present study, we developed a specific and sensitive polymerase chain reaction (PCR) assay for detecting the pathogen in wheat (Triticum aestivum) leaves. A pair of primers (PSF and PSR) was designed based on the internal transcribed spacer (ITS) region sequence of P. striiformis f. sp. tritici. PCR products that were amplified with universal primers ITS1 and ITS4 were cloned into pGEM-T Easy vectors and sequenced. The ITS sequence was compared with those of P. striiformis f. sp. tritici, P. triticina, P. graminis f. sp. tritici, Blumeria graminis f. sp. tritici, Fusarium graminearum, Rhizoctonia cerealis, and Bipolaris sorokiniana, which are associated with early symptoms of foliar diseases on wheat. Specificity of the primers was tested in the PCR assays using DNA extracted from all tested P. striiformis f. sp. tritici isolates, other fungal species, and healthy and infected wheat leaves sampled around stripe rust foci in wheat fields, different days after inoculation with P. striiformis f. sp. tritici, as well as asymptomatic wheat leaves sampled around stripe rust foci in the fields. A PCR product of 169 bp was amplified from DNA of all P. striiformis f. sp. tritici isolates. The primers did not amplify DNA from the other tested fungal species. The pathogen was detected from asymptomatic wheat leaves inoculated with P. striiformis f. sp. tritici under greenhouse conditions, as well as leaves sampled around stripe rust foci in wheat fields. Under optimum conditions, the PCR assay was highly sensitive and required only 0.1 pg of the target DNA for a detectable and reliable amplification with the PSF and PSR primers.

scholarly journals A PCR-based Technique for Identification of Fusicoccum sp. from Pistachio and Various Other Hosts in California

Plant Disease ◽  
2002 ◽  
Vol 86 (5) ◽  
pp. 515-520 ◽  
Author(s):  
Zhonghua Ma ◽  
Themis J. Michailides
Keyword(s):  
Host Plants ◽  
Extraction Procedure ◽  
Its Region ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Fungal Species ◽  
Pcr Assay ◽  
Shoot Blight ◽  
Dna Fragment ◽  
Polymerase Chain

Botryosphaeria panicle and shoot blight of pistachio, caused by Fusicoccum sp. is a destructive disease in California. In this study, a pair of group-specific polymerase chain reaction (PCR) primers BDI and BDII, was developed for identification of Fusicoccum sp. from pistachio and other hosts in California based on the sequences of the rDNA internal transcribed spacer (ITS) region. The primers amplified a 356-bp DNA fragment for all 73 tested isolates of Fusicoccum sp. collected from pistachio and other hosts throughout California in different years, but not for the other 33 fungal species isolated from pistachio and the eight isolates of Fusicoccum sp. obtained from pistachio trees in Greece. The PCR assay using this pair of primers was sensitive enough to detect 5 pg of genomic DNA of Fusicoccum sp. A simple DNA extraction procedure was developed that led to the rapid identification of Fusicoccum sp. from pistachio and other host plants in California.

scholarly journals Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1932 ◽  
Author(s):  
Byeong Moon ◽  
Wook Kim ◽  
Inkyu Park ◽  
Gi-Ho Sung ◽  
Pureum Noh
Keyword(s):  
Dietary Supplements ◽  
Real Time ◽  
Real Time Pcr ◽  
Herbal Medicines ◽  
Fungal Species ◽  
Scar Markers ◽  
Pcr Assay ◽  
Food Ingredients ◽  
The Real ◽  
Species Specific

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

scholarly journals Development of a Method for Detection of the Biocontrol Agent Penicillium oxalicum Strain 212 by Combining PCR and a Selective Medium

Plant Disease ◽  
2009 ◽  
Vol 93 (9) ◽  
pp. 919-928 ◽  
Author(s):  
I. Larena ◽  
P. Melgarejo
Keyword(s):  
Its Region ◽  
Fungal Species ◽  
Penicillium Oxalicum ◽  
Selective Medium ◽  
Nucleotide Sequences ◽  
Tomato Plants ◽  
Its Regions ◽  
Specific Pair ◽  
Species Specific ◽  
Biocontrol Strain

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Penicillium oxalicum strain 212 (PO212) is being developed for the control of tomato pathogens. In this study, we demonstrated that PO212 was more effective for controlling Fusarium oxysporum f. sp. lycopersici in tomato plants than 13 other P. oxalicum strains. A new semiselective medium was developed as a preliminary screen for P. oxalicum from soil. This semiselective medium was a modified Fusarium selective medium that contained 0.006 g of nystatin per liter. The growth of P. oxalicum strain 212 was not inhibited on this medium, but it did inhibit the growth of 11 fungal species. Specific identification of the biocontrol strain and its quantification were achieved using a polymerase chain reaction with a strain-specific pair of primers (POITS1F/POITS2R1) and dilution plating. This primer set differentiated the biocontrol strain from 13 other strains of P. oxalicum. There were differences in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA of 25 strains of P. oxalicum and those of PO212. Based on the differences in the nucleotide sequences of the ITS regions in rDNA of PO212 and other P. oxalicum strains, a relationship between the nucleotide sequences in the ITS region and biocontrol efficacy is postulated.

scholarly journals A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 599-608 ◽  
Author(s):  
Martin I. Chilvers ◽  
Lindsey J. du Toit ◽  
Hajime Akamatsu ◽  
Tobin L. Peever
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
Fungal Species ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Onion Seed ◽  
Pure Cultures ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

scholarly journals Shoot Blight Caused by Diplodia pinea on Afghan and Austrian Pines in Texas

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1056-1056 ◽  
Author(s):  
K. Ong ◽  
S. Hill ◽  
D. R. Smith ◽  
G. R. Stanosz
Keyword(s):  
Gulf Coast ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Diplodia Pinea ◽  
Pine Seedlings ◽  
Pure Cultures ◽  
Shoot Blight ◽  
Specific Pcr Assay ◽  
Species Specific ◽  
Hyphal Tip

Shoot blight was observed on ornamental Afghan (Pinus eldarica) and Austrian pines (P. nigra) at several sites in metropolitan Dallas, TX in the summer of 2005. Shoots were stunted, cankered, often resinous, sometimes curled or crooked at the tips, and bore brown needles that often had been killed before full elongation. Pycnidia on necrotic needles, stems, and cones of each host species yielded conidia characteristic of the fungus Diplodia pinea. Individual conidia and hyphal tip transfers produced pure cultures confirmed as D. pinea using a species-specific PCR assay (1), which allows differentiation from the similar pine shoot blight pathogen D. scrobiculata. Five isolates (three from Afghan pine and two from Austrian pine) were tested for pathogenicity by inoculation of potted 1-year-old Afghan pine seedlings obtained from the Texas Forest Service Nursery. Elongating terminal shoots were wounded by removing a needle pair approximately 2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing culture on water agar (WA) was placed fungus side down on the wound. Noncolonized WA plugs were placed on similarly wounded control seedlings. Nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed 2 weeks later. Each treatment was applied to four seedlings. Five weeks after inoculation, 9 of the 20 inoculated seedlings (including at least one inoculated with each isolate) exhibited dieback of shoot tips. One wounded control seedling exhibited slight tip dieback, no other nonwounded or wounded control seedlings developed symptoms. Segments of shoots were harvested, surface disinfested, and incubated on WA to determine the presence of the pathogen. The pathogen was reisolated from 11 of the 20 inoculated seedlings but not from any control seedlings. To our knowledge, this is the first report of D. pinea as a cause of shoot blight of Afghan pine and the first substantiated report of the occurrence of D. pinea in Texas. Although widely distributed in much of eastern North America, reports of the presence of D. pinea in the other southern gulf coast states of Alabama, Louisiana, and Mississippi, as well as the western states of Colorado, New Mexico, and Utah, are lacking. Reference: (1) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

scholarly journals A New Application Using a Chromogenic Assay in a Plant Pathogen DNA Macroarray Detection System

Plant Disease ◽  
2012 ◽  
Vol 96 (9) ◽  
pp. 1365-1371 ◽  
Author(s):  
Mui-Yun Wong ◽  
Christine D. Smart
Keyword(s):  
Plant Pathogens ◽  
Detection System ◽  
Phytophthora Capsici ◽  
Its Region ◽  
Nuclear Ribosomal Dna ◽  
5.8S Rdna ◽  
Chromogenic Assay ◽  
Chemiluminescent Detection ◽  
Pure Cultures ◽  
Species Specific

A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.

scholarly journals Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

2020 ◽  
Vol 55 (1) ◽  
Author(s):  
Anna Baturo-Cieśniewska ◽  
Wojciech Pusz ◽  
Katarzyna Patejuk
Keyword(s):  
Species Identification ◽  
Its Region ◽  
Fungal Species ◽  
Plant Diseases ◽  
Its Sequences ◽  
Local Alignment ◽  
Correct Identification ◽  
Rdna Its ◽  
Its Sequence Analysis ◽  
Species Specific

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

scholarly journals Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Pathogenicity Test ◽  
Kashmir Valley ◽  
Fusarium Equiseti ◽  
Infected Root ◽  
Fusarium Sp ◽  
High Degree

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

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