A User-Friendly Method to Isolate and Single Spore the Fungi Magnaporthe oryzae and Magnaporthe grisea Obtained from Diseased Field Samples

2009 ◽  
Vol 10 (1) ◽  
pp. 37 ◽  
Author(s):  
Yulin Jia
Keyword(s):  
Magnaporthe Oryzae ◽  
Magnaporthe Grisea ◽  
Single Spore ◽  
Field Samples ◽  
User Friendly

Although M. oryzae has been studied under extensively for several decades worldwide, descriptions of methods for efficient single spore isolation from diseased leaves from the field are not available. The ability to isolate the fungus from field samples is essential for specialists around the globe to study M. oryzae. The objective of this study was to develop a user-friendly method to isolate and evaluate M. oryzae from field samples. Accepted for publication 30 September 2009. Published 15 December 2009.

scholarly journals Development of the Automated Primer Design Workflow Uniqprimer and Diagnostic Primers for the Broad-Host-Range Plant Pathogen Dickeya dianthicola

Plant Disease ◽  
2019 ◽  
Vol 103 (11) ◽  
pp. 2893-2902 ◽  
Author(s):  
Shaista Karim ◽  
R. Ryan McNally ◽  
Afnan S. Nasaruddin ◽  
Alexis DeReeper ◽  
Ramil P. Mauleon ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Pathogen ◽  
The United States ◽  
Broad Host Range ◽  
Software Pipeline ◽  
Widespread Occurrence ◽  
Field Samples ◽  
Primer Sets ◽  
Genome Analyses ◽  
User Friendly

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.

scholarly journals Development of microsatellite markers for the genetic analysis of Magnaporthe grisea

2000 ◽  
Vol 23 (4) ◽  
pp. 753-762 ◽  
Author(s):  
Claudio Brondani ◽  
Rosana Pereira Vianello Brondani ◽  
Lucas da Ressurreição Garrido ◽  
Márcio Elias Ferreira
Keyword(s):  
Magnaporthe Grisea ◽  
Pcr Amplification ◽  
Polymorphism Analysis ◽  
Single Spore ◽  
Genetic Studies ◽  
Central Brazil ◽  
Dna Library ◽  
Ssr Loci ◽  
Genomic Dna Library ◽  
Microsatellite Alleles

An AG microsatellite-enriched genomic DNA library was constructed for Magnaporthe grisea (anamorph Pyricularia grisea), the causal agent of rice blast. Seventy-two DNA clones containing microsatellite repeats were isolated and sequenced in order to develop a series of new PCR-based molecular markers to be used in genetic studies of the fungus. Twenty-four of these clones were selected to design primer pairs for the PCR amplification of microsatellite alleles. Single spore cultures of M. grisea isolated from rice and wheat in Brazil, Colombia and China were genotyped at three microsatellite loci. Isolates from southern Brazil were predominantly monomorphic at the tested SSR loci, indicating a low level of genetic variability in these samples. However, seven alleles were observed at the MGM-1 locus in isolates from Central Brazil and at least nine alleles were detected at the same locus in a sample of Colombian isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of M. grisea isolates and a powerful tool for studying M. grisea genetics.

scholarly journals MGOS: Development of a Community Annotation Database for Magnaporthe oryzae

2012 ◽  
Vol 25 (3) ◽  
pp. 271-278 ◽  
Author(s):  
Anupreet Kour ◽  
Kevin Greer ◽  
Barbara Valent ◽  
Marc J. Orbach ◽  
Carol Soderlund
Keyword(s):  
Magnaporthe Oryzae ◽  
Magnaporthe Grisea ◽  
Rice Genome ◽  
Blast Disease ◽  
Biological Databases ◽  
Cultivated Rice ◽  
Annotation Database ◽  
Individual Gene ◽  
One Stop ◽  
Serious Disease

Magnaporthe oryzae causes rice blast disease, which is the most serious disease of cultivated rice worldwide. We previously developed the Magnaporthe grisea–Orzya sativa (MGOS) database as a repository for the M. oryzae and rice genome sequences together with a comprehensive set of functional interaction data generated by a major consortium of U.S. researchers. The MGOS database has now undergone a major redesign to include data from the international blast research community, accessible with a new intuitive, easy-to-use interface. Registered database users can manually annotate gene sequences and features as well as add mutant data and literature on individual gene pages. Over 900 genes have been manually curated based on various biological databases and the scientific literature. Gene names and descriptions, gene ontology annotations, published and unpublished information on mutants and their phenotypes, responses in diverse microarray analyses, and related literature have been incorporated. Thus far, 362 M. oryzae genes have associated information on mutants. MGOS is now poised to become a one-stop repository for all structural and functional data available on all genes of this critically important rice pathogen.

scholarly journals Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

2010 ◽  
Vol 76 (19) ◽  
pp. 6583-6590 ◽  
Author(s):  
Machiko Takahashi ◽  
Hideyuki Takahashi ◽  
Yuki Nakano ◽  
Teruko Konishi ◽  
Ryohei Terauchi ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Magnaporthe Grisea ◽  
Catalytic Domain ◽  
Cellulose Binding Domain ◽  
Enhanced Activity ◽  
Hydrolytic Activities ◽  
Cellulose Binding ◽  
Hydrolysis Of ◽  
Increased Activity

ABSTRACT Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

scholarly journals A Rapid PCR-Based Method for the Detection of Magnaporthe oryzae from Infected Perennial Ryegrass

Plant Disease ◽  
2003 ◽  
Vol 87 (9) ◽  
pp. 1072-1076 ◽  
Author(s):  
Philip F. Harmon ◽  
Larry D. Dunkle ◽  
Richard Latin
Keyword(s):  
Perennial Ryegrass ◽  
Magnaporthe Oryzae ◽  
Leaf Spot ◽  
Leaf Tissue ◽  
Gray Leaf Spot ◽  
Rapid Pcr ◽  
Field Samples ◽  
Multiple Copies ◽  
Serious Disease ◽  
Time Required

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass in the midwestern United States. Symptoms of gray leaf spot can be confused with those caused by other fungal diseases that also are common during periods of high temperatures and ample moisture. Because turf managers must select appropriate fungicides for remedial treatment, accurate and timely identification of the pathogen is essential for efficient and effective disease management. We developed and evaluated a polymerase chain reaction (PCR)-based method to detect M. oryzae in infected perennial ryegrass tissue. The method utilizes a commercially available kit that is used for isolation and amplification of plant DNA from leaf tissue. The kit protocol was modified and found to be reliable for the extraction of M. oryzae DNA from infected perennial ryegrass. Primers were designed to amplify a 687-bp fragment of the Pot2 transposon that is found in multiple copies in the genome of the pathogen. The protocol amplified amounts of purified DNA as low as 5 pg and consistently and specifically detected M. oryzae in single diseased leaf blades as well as in field samples of infected perennial ryegrass. The total time required for detection was approximately 4 to 8 h.

scholarly journals RNA Silencing in the Phytopathogenic Fungus Magnaporthe oryzae

2003 ◽  
Vol 16 (9) ◽  
pp. 769-776 ◽  
Author(s):  
Naoki Kadotani ◽  
Hitoshi Nakayashiki ◽  
Yukio Tosa ◽  
Shigeyuki Mayama
Keyword(s):  
Magnaporthe Oryzae ◽  
Rna Silencing ◽  
Magnaporthe Grisea ◽  
Green Fluorescence Protein ◽  
Phytopathogenic Fungus ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Egfp Gene ◽  
Systematic Analysis ◽  
Rna Molecules

Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.

scholarly journals Nonhost Resistance of Barley Is Successfully Manifested Against Magnaporthe grisea and a Closely Related Pennisetum-Infecting Lineage but Is Overcome by Magnaporthe oryzae

2006 ◽  
Vol 19 (9) ◽  
pp. 1014-1022 ◽  
Author(s):  
Nina Zellerhoff ◽  
Birgit Jarosch ◽  
Johannes Z. Groenewald ◽  
Pedro W. Crous ◽  
Ulrich Schaffrath
Keyword(s):  
Magnaporthe Oryzae ◽  
Magnaporthe Grisea ◽  
Oryza Sativa L ◽  
Triticum Aestivum L ◽  
Gene Trees ◽  
Hordeum Vulgare L ◽  
Host Interaction ◽  
Specific Host ◽  
Polymerase Chain ◽  
Compatible Interactions

Magnaporthe oryzae is a major pathogen of rice (Oryza sativa L.) but is also able to infect other grasses, including barley (Hordeum vulgare L.). Here, we report a study using Magnaporthe isolates collected from other host plant species to evaluate their capacity to infect barley. A nonhost type of resistance was detected in barley against isolates derived from genera Pennisetum (fontaingrass) or Digitaria (crabgrass), but no resistance occurred in response to isolates from rice, genus Eleusine (goosegrass), wheat (Triticum aestivum L.), or maize (Zea mays L.), respectively. Restriction of pathogen growth in the nonhost interaction was investigated microscopically and compared with compatible interactions. Real-time polymerase chain reaction was used to quantify fungal biomass in both types of interaction. The phylogenetic relationship among the Magnaporthe isolates used in this study was investigated by inferring gene trees for fragments of three genes, actin, calmodulin, and β-tubulin. Based on phylogenetic analysis, we could distinguish different species that were strictly correlated with the ability of the isolates to infect barley. We demonstrated that investigating specific host interaction phenotypes for a range of pathogen isolates can accurately highlight genetic diversity within a pathogen population.

scholarly journals First Report of Lasiodiplodia pseudotheobromae Causing Stem End Rot of Mango Fruit in Pakistan

Plant Disease ◽  
2021 ◽  
Author(s):  
Muhammad Waqar Alam ◽  
Arif Malik ◽  
Abdul Rehman ◽  
Mubeen Sarwar ◽  
Tahir Shafeeq ◽  
...  
Keyword(s):  
Disease Incidence ◽  
Mangifera Indica ◽  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
Postharvest Disease ◽  
Single Spore ◽  
Mango Fruit ◽  
First Report ◽  
Field Samples ◽  
Rot Disease

Mango (Mangifera indica L.) is considered a desirable fruit in international markets and is grown throughout tropical and sub-tropical countries around the world (Alemu, 2014). Stem end rot is the most damaging and complex postharvest disease of mango, resulting in losses of up to 40% in Pakistan, which is the leading producer and exporter (Alam et al. 2017). A field survey was conducted in June of 2017 and 2018 in the Rahim Yar Khan and Multan- major mango producing regions of Punjab Province. After mature but unripe mango fruit (cv. Samar Bahisht Chaunsa) were stored at 12°C for 2 weeks to permit ripening, water-soaked, dark brown to purplish black decay began to appear around the stem end portion. The decay gradually enlarged and covered the whole fruit after 7 days. Disease incidence was estimated at 30%. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected with 1% sodium hypochlorite for 2 min, rinsed three times in sterilized distilled water, air dried, and then placed aseptically onto potato dextrose agar (PDA) medium and incubated at 25°C under a 12-h light/dark photoperiod for 7 days. Twelve single-spore isolates with similar morphology were isolated from the infected tissues. Initially the fungus produced thick, fluffy and greyish-white aerial mycelium, that later turned into dark gray colonies. Conidia were unicellular, ellipsoidal, and initially hyaline, but with age became dark brown and developed a central septum. Conidia measured 24.5 to 31.5 × 11.4 to 15.7 µm (n = 60). Conidiophores were inflated at their base with one diaphragm which reduced to conidiogenous cells. Conidiogenous cells were hyaline and cylindrical. On the basis of morphological characteristics, the fungus was tentatively identified as Lasiodiplodia sp., a member of the family Botryosphaeriaceae (Alves et al. 2008). For molecular identification, genomic DNA was extracted from mycelium following the CTAB method. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn searches of sequences revealed 99% to 100% identity with the reference sequences of various Lasiodiplodia pseudotheobromae isolates (GenBank accession nos. MH057189 for ITS; MN638768 for TEF-1a). The sequences were deposited in GenBank (accession nos. MW439318, MW433883 for ITS; and MW463346, MW463347 for TEF-1a). To fulfill Koch’s postulates, a suspension of 105 conidia/ml from a 7-day-old culture of L. pseudotheobromae was used to inoculate fully mature but unripe mango fruit (cv. Samar Bahisht Chaunsa). Fruit were pricked with a sterilized needle to a depth of 4 mm at the stem end portion, injected with 50 μl of the prepared spore suspension (Awa et al. 2012), and stored at 12°C for 3 weeks under 70 to 80% RH. Twenty mango fruit were inoculated, and 10 were inoculated with sterile water only. After 15 days, most fruit showed typical symptoms at the stem end. Reisolations from symptomatic fruit following the procedures described above for isolating and identifying the fungal cultures from infected field samples, consistently yielded a fungus identical to L. pseudotheobromae. Control fruit remained disease-free. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), this is the first report of the pathogen causing stem end rot disease of mango in Pakistan. This report is important for the new studies aiming at management of stem end rot disease of mango caused by L. pseudotheobromae in Pakistan.

scholarly journals First Report of Black Leaf Spot of Banana Caused by Deightoniella torulosa in Georgia

Plant Disease ◽  
2008 ◽  
Vol 92 (10) ◽  
pp. 1470-1470 ◽  
Author(s):  
D. Koné ◽  
P. Ji ◽  
G. E. Fonsah ◽  
A. S. Csinos
Keyword(s):  
Morphological Characteristics ◽  
Black Spot ◽  
Central Vein ◽  
Leaf Margin ◽  
Single Spore ◽  
Potato Dextrose Agar Medium ◽  
Banana Production ◽  
Spot Disease ◽  
Field Samples ◽  
Black Spot Disease

Black spots were observed on the leaves of bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA in November 2007. Symptoms occurred on more than 60 plants, representing 16 of 34 cultivars of bananas investigated. Most lesions were less than 10 mm in diameter and tan to black. However, larger oval lesions more than 20 mm across with black borders and yellow halos also occurred. Lesions were more prevalent on older leaves. On young leaves, lesions first appeared along the leaf margin near the tip of the leaf on one side of the central vein. Lesions expanded to the entire leaf as the disease progressed, but were more prevalent along leaf margins. Thirty-two diseased leaf samples, two from each cultivar, were incubated at 25°C in the dark and conidia were produced on the lesions 2 days after incubation. Pure cultures of the fungus were obtained from five leaf samples by single-spore culturing on potato dextrose agar medium and identified on the basis of morphological characteristics. Conidia on V8 agar are straight or slightly curved, obpyriform to obclavate, and olive to brown with 3 to 13 septa. Conidiophores are brown and swollen at the apex. The fungus was identified as Deightoniella torulosa (Syd.) Ellis on the basis of morphological characteristics described previously (1,2). Pathogenicity studies to fulfill Koch's postulates were conducted on banana cvs. Dwarf Namwah and Dwarf Nino under greenhouse conditions (25 to 27°C). Six plants of each cultivar were used in one experiment and the experiment was repeated one more time. Banana leaves were inoculated by spraying with a suspension of conidia from a pure culture. Symptoms developed as small black lesions on the leaves of both cultivars within 1 week of inoculation. As the disease progressed, some of the small lesions expanded to form larger oval lesions. Symptoms were identical to those on the field samples and were identified as the black spot disease as described on abaca and banana (2). The fungus was reisolated from symptomatic leaves and the identity was confirmed. No symptoms were observed on noninoculated control plants. The black spot disease has been reported in Florida attacking banana and plantain (3). To our knowledge, this is the first description of the presence of the disease on field-grown banana in Georgia. In recent years, increasing efforts have been made in Georgia in the search of banana cultivars suitable of commercial production in the coastal and southern areas of the state. Black spot of banana is an important disease and its occurrence deserves consideration in evaluating banana cultivars and developing disease management approaches for banana production in Georgia. References: (1) M. B. Ellis. Mycol. Pap. No. 66. CAB International Mycological Institute, Wallingford, UK, 1957. (2) R. H. Stover. Banana, Plantain and Abaca Diseases. Commonw. Mycol. Inst., Kew, Surrey, UK, 1972. (3) C. Wehlburg et al. Bull. 11. Fla. Dep. Agric. Consum. Serv. Div. Plant Ind., 1975.

scholarly journals Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus

2019 ◽  
Author(s):  
Mostafa Rahnama ◽  
Olga Novikova ◽  
John Starnes ◽  
Li Chen ◽  
Shouan Zhang ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Tandem Repeats ◽  
In Planta ◽  
Single Spore ◽  
Telomere Repeat ◽  
Restriction Fragments ◽  
Molecular Alterations ◽  
Filamentous Ascomycete ◽  
Rdna Sequences ◽  
Chromosome Alterations

ABSTRACTMagnaporthe oryzae is a filamentous ascomycete fungus that causes devastating diseases of crops that include rice and wheat, and a variety of turf, forage and wild grasses. Strains from ryegrasses possess highly stable chromosome ends that undergo frequent rearrangements during vegetative growth in culture and in planta. Instability is associated with the presence of two related retrotransposons (Magnaporthe oryzaeTelomeric Retrotransposons - MoTeRs) inserted within the telomere repeat tracts. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, restriction mapping, and sequencing of both parental and novel telomeric restriction fragments, along with MinION sequencing of DNA from three single-spore cultures, allowed us to document the molecular alterations for 109 newly-formed telomeres. Rearrangement events included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by “plain” telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; expansion and contraction of subtelomeric tandem repeats; MoTeR truncations; duplication and terminalization of internal sequences; and breakage at long, interstitial telomeres generated during MoTeR insertion. Together, our data show that when MoTeRs invade the telomeres, they can dramatically perturb the integrity of chromosome ends, leading to the generation of unprotected DNA termini whose repair has the potential to generate chromosome alterations that extend well into the genome interior.

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