Fungal Pathogen Magnaporthe Grisea Research Articles

GC-MS analysis of metabolites from blast pathogen infected from foxtail millet

Foxtail millet blast caused by the fungal pathogen Magnaporthe grisea (Anamorph: Pyricularia grisea) is an important and most destructive disease. M. grisea is a ubiquitous plant pathogen with a wide host range. To understand the host-pathogen interaction system, a knowledge about the role of toxins in disease development is paramount. This may helpful in development of a new resistant cultivar screening procedures. The crude toxin extracted from Magnaporthe grisea (TNFxM1) isolate was determined through Gas chromatography–mass spectrometry (GC-MS) technique. The main constituents were Glycerol and 1, 6 Dihydroxynaphthalene.

Genome Resource of Sphingomonas carotinifaciens L9-754T, an Endophyte Isolated From Leaf Tissues of Jatropha curcas.

Sphingomonas carotinifaciens strain L9-754T (DSM 27347) is a gram-negative, chemoheterotrophic, and rod-shaped endophyte isolated from the stem tissues of Jatropha curcas L. This strain has putative in vitro antagonistic ability against the plant pathogenic fungus Magnaporthe grisea. A draft genome of L9-754T was obtained using the PacBio SMRT cell platform. By analyzing the genome of strain L9-754T, a gene cluster (GQR91_18700 - GQR91_18715) related to an antioxidant enzyme was identified in the obtained draft genome. The information obtained from the draft genome is expected to reveal the putative properties helpful in biocontrol applications.

RNA-Seq revealed that infection with white tip nematodes could downregulate rice photosynthetic genes.

White-tip nematode, Aphelenchoides besseyi is a kind of widely distributed migratory parasitic nematode that can infect plant shoots. Transcriptome sequencing of plant parasitic nematodes and their host plants is helpful for understanding their interaction relationship. This study first reported expression patterns of defense-related genes in rice, and rice transcriptomes at different periods after infection with A. besseyi. The result showed that the defense response pathways of rice changed obviously in the early stage of A. besseyi infection, including upregulated salicylic acid and jasmonate pathways and a downregulated ethylene pathway. Transcriptome analysis results suggested that A. besseyi infection was associated with the downregulation of multiple genes related to photosynthesis with possible suppression of the photosynthetic activity. It suggested that the photosynthesis system of rice could be suppressed by infections of migratory nematodes, including A. besseyi and Hirschmanniella oryzae, but was stimulated by that of a sedentary nematode, Meloidogyne graminicola, by comparing our study with the reported transcriptome. OS09G0417800 (OsWRKY62) might play an important role in the interaction of migratory nematodes and rice. It also indicated that the infection strategy of both A. besseyi and the reported migratory nematode H. oryzae was similar to that of the fungal pathogen Magnaporthe grisea. These results provided an interesting starting point to elucidate the mechanism of the interaction between rice and A. besseyi, as well as the host and migratory plant nematodes.

Diterpenoids with herbicidal and antifungal activities from hulls of rice (Oryza sativa)

Diterpenoids are the main secondary metabolites of plants and with a range of biological activities. In the present study, 7 compounds were isolated from the hulls of rice (Oryza sativa L.). Among them, 3 diterpenoids are new namely, 3,20-epoxy-3α-hydroxy- 8,11,13-abietatrie-7-one (1), 4,6-epoxy-3β-hydroxy-9β-pimara-7,15-diene (2) and 2-((E)-3- (4-hydroxy-3-methoxyphenyl) allylidene) momilactone A (3). While, 4 terpenoids are known, namely momilactone A (4), momilactone B (5), ent-7-oxo-kaur-15-en-18-oic acid (6) and orizaterpenoid (7). The structures of these diterpenoids were elucidated using 1D and 2D NMR in combination with ESI-MS and HR-EI-MS. Furthermore, all isolated compounds displayed antifungal activities against four crop pathogenic fungi Magnaporthe grisea, Rhizoctonia solani, Blumeria graminearum and Fusarium oxysporum, and phytotoxicity against paddy weed Echinochloa crusgalli. The results suggested that rice could produce plenty of secondary metabolites to defense against weeds and pathogens.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

Lack of a Cytoplasmic RLK, Required for ROS Homeostasis, Induces Strong Resistance to Bacterial Leaf Blight in Rice.

Many scientific findings have been reported on the beneficial function of reactive oxygen species (ROS) in various cellular processes, showing that they are not just toxic byproducts. The double-edged role of ROS shows the importance of the regulation of ROS level. We report a gene, rrsRLK (required for ROS-scavenging receptor-like kinase), that encodes a cytoplasmic RLK belonging to the non-RD kinase family. The gene was identified by screening rice RLK mutant lines infected with Xanthomonas oryzae pv. oryzae (Xoo), an agent of bacterial leaf blight of rice. The mutant (ΔrrsRLK) lacking the Os01g02290 gene was strongly resistant to many Xoo strains, but not to the fungal pathogen Magnaporthe grisea. ΔrrsRLK showed significantly higher expression of OsPR1a, OsPR1b, OsLOX, RBBTI4, and jasmonic acid-related genes than wild type. We showed that rrsRLK protein interacts with OsVOZ1 (vascular one zinc-finger 1) and OsPEX11 (peroxisomal biogenesis factor 11). In the further experiments, abnormal biogenesis of peroxisomes, hydrogen peroxide (H2O2) accumulation, and reduction of activity of ROS-scavenging enzymes were investigated in ΔrrsRLK. These results suggest that the enhanced resistance in ΔrrsRLK is due to H2O2 accumulation caused by irregular ROS-scavenging mechanism, and rrsRLK is most likely a key regulator required for ROS homeostasis in rice.

Ferreting out blast disease resistance in advanced breeding lines of rice (Oryza sativa) by phenotypic evaluation and microsatellite markers

Rice (Oryza sativa L.) is the second most important cereal crop of developing countries and the staple food of about 65% of the world's population. Rice blast is a serious disease affecting rice, caused by a fungal pathogen Magnaporthe grisea (anamorph - Pyricularia grisea). In this endeavor, it is important to identify durable resistance sources. Forty two elite advanced breeding lines containing local and IRRI cultivars in their parentage were screened for the blast disease in uniform blast nursery during wet season of 2012 and 2013 using local land race Pankhali-203 as an inoculum spreader. Four elite lines, viz. GR-7/CRMAS-2231-36(8-4-1-1-1), GR-7/DDR-8(1-5-1-1), IR-28/IET-16804(4-1-1-1-1,2) and Jaya/IR-64(52-2-3-2) and GR-7, a released cultivar, were found highly resistant. Screening of these lines was also done using gene specific eighteen SSR markers. All markers amplified a total of 757 bands with an average of 42.10 bands per marker. The SSR marker RM535 for Pib gene and RM28050 for Pi20 gene produced maximum number (19) of alleles. Most of the breeding lines were found to have all the resistance genes with a diversity of allele range. The diversity analysis revealed the clustering pattern of these genotypes based on their parentage and not the genes present. The highest similarity index value of 0.53 was found between GR-7/NWGR-2002(3-1-1-1-1) and GR-7/NWGR-2002(2-3-1-2-1). Thus, present study revealed the resistance source for rice blast disease from advanced breeding lines simultaneously with the resistance genes present in them.

A new phenylpropane-pimarane heterodimer and a new ent-kaurene diterpene from the husks of Oryza sativa

A new phenylpropane-pimarane heterodimer, 2-[(E)-3-(4-hydroxy-3-methoxyphenyl) allylidene] momilactone A (1), and a new ent-kaurene diterpene, ent-3α,16α-dihydroxy-kauran-19-oic acid (2), were isolated from the rice husks of Oryza sative L., along with a known ent-kaurene diterpenoid ent-7-oxo-kaur-15-en-19-oic acid (3). Their structures were determined by spectroscopic means, including nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques. Compound 3 was isolated as a natural product for the first time. These compounds were tested for their in vitro antifungal activity against rice pathogenic fungi Magnaporthe grisea and Rhizoctonia solani. Compounds 1 and 3 were found to show anti-fungal activity against M. grisea and R. solani with MIC values ranging from 6.25 to 25 μg/mL. No inhibitory activity was detected for compound 2 in this bioassay.

Comparative Analysis of Pathogenicity, Phylogenetic Relationship and Host Fungal Rice Blast Pathogen Magnaporthe grisea

Magnaporthe grisea is the most devastating pathogen of rice in all over the world and the principal model organism for elucidating the molecular source of fungal disease of plants This study we report the draft sequence of the M grisea genome Examination of the gene set facilitates an insight into the adaptations essential by a fungus to source disease The genome encodes a huge and varied set of secreted proteins with those defined by remarkable carbohydrate binding domains This fungus also possesses a prolonged family of G protein coupled receptors numerous novel virulence associated genes and huge suites of enzymes occupied in secondary metabolism Consistent with a function in fungal pathogenesis the expression of a number of these genes up regulated during the early stages of infection related development The M grisea genome has been subject to invasion and proliferation of active transposable elements reflecting the colonel nature of this fungus imposed by widespread rice cultivation The study will be helpful to describe phylogenetic relations pathogenicity and genome analysis drug design against diseases caused by fungi and cDNA synthesis and amplification to desired targets

Registration of ‘Titan’ Southern Medium‐Grain Rice

‘Titan’ (Reg. No. CV‐151, PI 680613), is an early‐maturing and short‐statured medium‐grain rice cultivar (Oryza sativa L.) developed at the University of Arkansas System Division of Agriculture's Rice Research and Extension Center (RREC), near Stuttgart, AR. It was approved for release in February 2016 by the University of Arkansas System Division of Agriculture Experiment Station. Titan was derived from the three‐way cross (‘M‐206’//‘Bengal’/‘Lafitte’) F1/‘Jupiter’ made at the RREC in 2006 and initiated as a F4:5 bulk of a single panicle row STG09PR‐81‐087 in the Puerto Rico winter nursery in spring 2010. In 45 statewide and multistate yield trials conducted from 2012 to 2015, Titan had a 4% yield advantage (statistically insignificant) over the predominant commercial cultivar Jupiter; however, it matured about 6 d earlier than the latter. Titan possesses both Pi‐z and Pi‐ks blast resistance genes compared with Jupiter, which only has the Pi‐ks gene, and Titan has a better resistance to both leaf and neck blast, which are caused by the fungal pathogen Magnaporthe grisea (T.T. Hebert). Titan has a typical southern medium‐grain rice quality with an apparent amylose content of 15.0% and gelatinization temperature of 62.8°C, both very similar to that of Jupiter; however, Titan has a much larger kernel size than Jupiter. Titan is well adapted to the medium‐grain rice growing regions in the US Mid‐South. The release of the medium‐grain cultivar Titan will provide rice producers with an improved medium‐grain cultivar for rice production in Arkansas and the Mid‐South.

Antifungal Activities of Dimeric Sesquiterpenes, Shizukaols C and F, Isolated from Chloranthus japonicus Sieb.

Two dimeric sesquiterpenes were separated from Chloranthus japonicus Sieb. and identified as shizukaols C and F. They exhibited potent antifungal activities (MICs = 4-16 μg/ml) in vitro against various plant pathogenic fungi (Pythium ultimum, Phytophthora infestans, Botrytis cinerea, Colletotrichum lagenarium, Alternaria kikuchiana, and Magnaporthe grisea). Shizukaol C showed 88% and 91% protective activities in the greenhouse against Puccinia recondita (wheat leaf rust) and Phytophthora infestans (tomato late blight), respectively, at 100 μg/ml; shizukaol F exhibited 93% antifungal activity against Puccinia recondita at the same concentration. Therefore, these compounds might serve as interesting candidates for effective antifungal agents.

Pigeonpea Hybrid-Proline-Rich Protein (CcHyPRP) Confers Biotic and Abiotic Stress Tolerance in Transgenic Rice.

In this study, we report the overexpression of Cajanus cajan hybrid-proline-rich protein encoding gene (CcHyPRP) in rice which resulted in increased tolerance to both abiotic and biotic stresses. Compared to the control plants, the transgenic rice lines, expressing CcHyPRP, exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity, and heat, as evidenced by increased biomass, chlorophyll content, survival rate, root, and shoot growth. Further, transgenic rice lines showed increased panicle size and grain number compared to the control plants under different stress conditions. The CcHyPRP transgenics, as compared to the control, revealed enhanced activities of catalase and superoxide dismutase (SOD) enzymes and reduced malondialdehyde (MDA) levels. Expression pattern of CcHyPRP::GFP fusion-protein confirmed its predominant localization in cell walls. Moreover, the CcHyPRP transgenics, as compared to the control, exhibited increased resistance to the fungal pathogen Magnaporthe grisea which causes blast disease in rice. Higher levels of bZIP and endochitinase transcripts as well as endochitinase activity were observed in transgenic rice compared to the control plants. The overall results demonstrate the intrinsic role of CcHyPRP in conferring multiple stress tolerance at the whole-plant level. The multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants for enhanced tolerance/resistance to different stress factors.

Evaluation of DNA markers linked to blast resistant genes, Pikh, Pit (p), and Pita, for parental selection in Sri Lankan rice breeding

Rice blast, is a serious disease affecting rice, caused by a fungal pathogen Magnaporthe grisea (anarmorph - Pyricularia grisea). This paper summarizes the progress and the achievements made in detection of blast resistance genes in phenotypically evaluated, selected rice cultivars in Sri Lanka based on simple sequence repeats (SSR) DNA markers. In the present study blast pathogen was identified by morphological characters, molecular techniques which confirmed the identity as M. grisea. Total of 34 rice varieties including several Sri Lankan rice varieties and IRRI recommended rice varieties were evaluated for blast resistance. Total of 17 varieties were susceptible to the fungal infection and 17 varieties remained resistant. Tetep, Bg359, Bg304, IR64, Bg352, Bg357 were found to be highly resistant to the rice blast disease, while LD125, Bg94/1, Bw267-3, Bw451, Bw400, H7, Bg34-6, H10, IRRI119 were highly susceptible. In marker genotyping, most of the highly resistant and resistant varieties demonstrated the presence of blast resistant Pita gene of variety Tetep and Pita gene was absent in the highly susceptible varieties. But Pita gene was absent in some of the resistant varieties like Bg300, Bg348 and Ptb33 and present in susceptible varieties H10, Bg276-5 and IR07F291. For polymorphism of Pikh gene most of the resistant varieties depicted the band lengths of 161-171bp. Susceptible varieties gave the band length of 151bp. According to results of marker genotyping with Pit(p) blast resistant gene, the resistant varieties gave a band length of 117bp while susceptible varieties produced the band length of 111bp. Hence, resulted polymorphism findings can be used in future studies to differentiate the resistant and susceptible varieties. Tropical Agricultural Research Vol. 26 (1): 82 – 93 (2014)

Naturally produced citral can significantly inhibit normal physiology and induce cytotoxicity on Magnaporthe grisea

Given the importance of finding alternatives to synthetic fungicides, the antifungal effects of natural product citral on six plant pathogenic fungi (Magnaporthe grisea, Gibberella zeae, Fusarium oxysporum, Valsa mali, Botrytis cinerea, and Rhizoctonia solani) were determined. Mycelial growth rate results showed that citral possessed high antifungal activities on those test fungi with EC50 values ranging from 39.52 to 193.00 µg/mL, which had the highest inhibition rates against M. grisea. Further action mechanism of citral on M. grisea was carried out. Citral treatment was found to alter the morphology of M. grisea hyphae by causing a loss of cytoplasm and distortion of mycelia. Moreover, citral was able to induce an increase in chitinase activity in M. grisea, indicating disruption of the cell wall. These results indicate that citral may act by disrupting cell wall integrity and membrane permeability, thus resulting in physiology changes and causing cytotoxicity. Importantly, the inhibitory effect of citral on M. grisea appears to be associated with its effects on mycelia reducing sugar, soluble protein, chitinase activity, pyruvate content, and malondialdehyde content.

The Hordeum vulgare signalling protein MAP kinase 4 is a regulator of biotic and abiotic stress responses

Mitogen activated protein kinase (MAP kinase) signal transduction pathways are important eukaryotic mechanisms for regulating cellular responses to stress. The objective of this work was to investigate the role of the barley MAP kinase HvMPK4 (a homologue of the Arabidopsis MAP kinase AtMPK1) in the plant response to biotic and abiotic stress. Transgenic barley plants bearing antisense or overexpression constructs for HvMPK4 were produced, and RNA blot analysis showed that HvMPK4 gene expression was much reduced in the antisense lines and approximately double in the overexpression lines. Three independent lines of each construct were tested for their response to a fungal pathogen and to salt treatment. The antisense lines were more resistant to the hemibiotrophic fungal pathogen Magnaporthe grisea, and showed enhanced levels of salicylic acid (SA) and of hydrogen peroxide following infection; HvMPK4 is thus a negative regulator of SA production post infection. The overexpression lines had constitutively higher levels of jasmonic acid and enhanced levels of ethylene following infection but were not more resistant to the pathogen. However the overexpression lines showed greater tolerance to abiotic stress, as following 2 weeks of salt treatment these lines showed less reduction in fresh and dry weight, accumulated less salt in the leaves and contained enhanced levels of the osmoprotectant amino acid, proline.

Fungicidal Interference during Infection Related Developmental Stages in Magnaporthe grisea

Rice blast, a serious epidemic disease that limits grain yield worldwide is caused by fungal pathogen Magnaporthe grisea. The present investigation was carried out to identify the probable avenues of interference by different fungicides during the critical stages of infection related morphogenesis of M. grisea. Effect of six fungicides at different stages of infection related morphogenesis showed variable results like interference in conidial germination, distortion of surface structure of the spores, interference in the germ tube elongation, interference in the transfer of the cell contents from spore to appresorrium, deformity in appressorial dome, interference in the melanin deposition. We speculate the critical stages at which these fungicides may interfere. The activity of immunosuppressive drug cyclosporin A (CsA) which is a potential antifungal agent was equated with all the fungicides used. We hypothesize that the exposure of the M. grisea spore to the fungicide may lead to the formation of a cyclophilin CYP1-fungicide complex, which inactivates calcineurin and prevents calcium/ calmodulin-dependent protein phosphatase signaling and is therefore one of the target of fungicidal interference. An understanding of how fungal pathogens break the protective barrier that comprise the surface of the host plant as well as precise identification of avenues of fungicidal interference during infection related development in M. grisea will lead to novel approach for controlling plant diseases.

Screening Thai landrace rice for blast resistance gene Pi9, Pi36, Pigm(t) using DNA markers

Rice blast caused by the fungal pathogenMagnaporthe grisea, is one of the most devastatingdiseases in rice production worldwide. Informationon rice blast resistant gene is important for ricecultivar development. The objective of this studywas to identify rice blast disease resistant genes in203 landrace Thai rice cultivars including 40 cultivars from the South, 19 cultivars from the North,144 cultivars from the Northeast of Thailandcomprised of 45 floating rice cultivars and 99lowland rice cultivars using gene specific markersfor blast resistant gene Pi9, Pi36 and Pigm(t). Theresults showed that all 203 cultivars have at leastone blast resistant gene and 42 cultivars have all thethree blast resistant genes. The outcome of thisresearch will be very useful for development of newblast resistance elite rice cultivars in the future.

Substitution mapping of QTLs for blast resistance with SSSLs in rice (Oryza sativa L.)

Rice blast, caused by fungal pathogen Magnaporthe grisea Barr., is one of the most devastating rice diseases worldwide. It has greatly affected the rice production and quality. Development of resistant cultivars is the most effective and economical way for controlling this disease in rice. In this study, 114 of the single-segment substitution lines (SSSLs) in rice were inoculated at seedling stage by 16 rice blast isolates. The substituted segments in the 114 SSSLs distributed on 12 chromosomes with coverage of 57.32% of rice genome. Fifteen of the SSSLs were different in blast resistance from the HJX74 recipient. The SSSL W23-7-6-5-2-2 showed 100% of resistance frequency. A total of 11 QTLs for blast resistance were detected on chromosomes 1, 2, 3, 6, 10, 11 and 12 in rice. They were mapped at chromosomal intervals of 2.2–46.2 cM, of which 6 QTLs were mapped at less than 10.0 cM. Six of the 11 QTLs were first reported in this paper.

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily. Expression of Pib was low under non-challenged conditions, but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wild-type plants. Accordingly, the blast disease-resistance was compromised in the 5'-azaC-treated plants relative to wild-type. In contrast, the disease susceptibility was not affected by the 5'-azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.

Progress on avirulence genes of the rice blast fungus <I>Mag-naporthe grisea</I>

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases of rice. The specific interaction between rice and M. grisea is an important model system for studying the host-pathogen interaction mechanisms. In this article, we summarized recent research progresses on avirulence genes, which are the most important effectors in M. grisea with the focus on chromosome mapping, cloning method, functional analysis, and evolution study of avirulence genes, and the possible hotspot of the research on avirulence genes in the future was also been discussed. This knowledge may shed a light on the molecular mechanism underlying avirulence genes function and the possible interaction relationship between the host and the pathogen.