Molecular Identification and Genetic Characterization of Macrophomina phaseolina Strains Causing Pathogenicity on Sunflower and Chickpea.
ABSTRACT: Macrophomina phaseolina is the most devastating pathogen which causes charcoal rot and root rot diseases in various economically important crops. Three strains M. phaseolina 1156, M. phaseolina 1160, and M. phaseolina PCMC/F1 were tested for their virulence on sunflower (Helianthus annuus L.) and chickpea (Cicer arietinum L.). The strains showed high virulence on both hosts with a disease score of 2 on chickpea and sunflower. The strains also increased the hydrogen per oxide (H2O2) content by 1.4- to 1.6-fold in root as well as shoot of chickpea and sunflower. A significant increase in antioxidant enzymes was observed in fungal infected plants which indicated prevalence of oxidative stress during pathogen propagation. The M. phaseolina strains also produced hydrolytic enzymes such as lipase, amylase, and protease with solubilization zone of 5-43 mm, 5-45 mm, and 12-35 mm, respectively. The M. phaseolina strains were identified by 18S rRNA and analyzed for genetic diversity by using random amplified polymorphic DNA (RAPD) markers. The findings based on RAPD markers and 18S rRNA sequence analysis clearly indicate genetic variation among the strains collected from different hosts. The genetically diverse strains were found to be pathogenic to sunflower and chickpea.
Project description:The worldwide demand for natural bast fibers is met aptly by the long, golden and silky fibers of jute. This highest bast fiber producing crop is of great applicability and is extensively used in paper and textile industry. Macrophomina phaseolina (Tassi) Goid is a severely devastating necrotrophic fungal pathogen causing stem rot, root rot, and charcoal rot diseases in both the cultivated species of jute - Corchorus capsularis and Corchorus olitorius. Another major problem faced in jute cultivation is profuse weed infestation in the fields. Huge losses in quality fiber production is caused by this pathogenic fungi and cultivation cost increases as well due to weed management expenditure during cropping season. To solve these long persisting jute cultivation challenges, the chitinase (chi11) gene (to provide fungus resistance) and the bar gene (to provide herbicide tolerance) have been incorporated in C. capsularis JRC-321 via Agrobacterium transformation and analyzed up to T2 generation. Stable integration and expression of these two genes in the jute genome was confirmed upon extensive analyses. Transgenic plants showed higher chitinase expression and chitin degrading activity than non-transgenic control plants. Antifungal activity significantly increased in transgenic plants as confirmed by detached leaf and whole plant M. phaseolina bioassay. Herbicide tolerance was analyzed by growing transgenic plants in 10 mg/l glufosinate ammonium containing media and by spraying 0.25% (v/v) glufosinate herbicide Basta® on them. Assessment of residual phytotoxicity effects of Basta® on soil confirmed no negative impact on growth of indicator plants corn and cucumber. Transgenic jute plants were at par with non-transgenic (control) jute plants in all phenotypic aspects. Non-transgenic (control) jute plants suffered significant losses in fiber yield and quality due to M. phaseolina infection whereas the transgenic lines maintained the quality of fiber even after the infection.
Project description:M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.
Project description:Macrophomina phaseolina is one of the deadliest necrotrophic fungal pathogens that infect more than 500 plant species including major food, fiber, and oil crops all throughout the globe. It secretes a cocktail of ligninolytic enzymes along with other hydrolytic enzymes for degrading the woody lignocellulosic plant cell wall and penetrating into the host tissue. Among them, lignin peroxidase has been reported only in Phanerochaete chrysosporium so far. But interestingly, a recent study has revealed a second occurrence of lignin peroxidase in M. phaseolina. However, lignin peroxidases are of much significance biotechnologically because of their potential applications in bio-remedial waste treatment and in catalyzing difficult chemical transformations. Besides, this enzyme also possesses agricultural and environmental importance on account of their role in lignin biodegradation. In the present work, different properties of the lignin peroxidase of M. phaseolina along with predicting the 3-D structure and its active sites were investigated by the use of various computational tools. The data from this study will pave the way for more detailed exploration of this enzyme in wet lab and thereby facilitating the strategies to be designed against such deadly weapons of Macrophomina phaseolina. Furthermore, the insight of such a ligninolytic enzyme will contribute to the assessment of its potentiality as a bioremediation tool.
Project description:We report here the data of transcriptome sequencing of control and infected sesame genotypes. Sesame is an emerging oilseed crop . The destructive soil-borne fungi Macrophomina phaseolina Tassi (Goid) causes charcoal rot of sesame, leading to high (>50%) yield loss. Most of the high-yielding sesame cultivars (Sesamum indicum) of India are susceptible to charcoal rot. Wild sesame, Sesamum mulayanum shows a high degree of tolerance against many pathogens . We have earlier developed an interspecific hybrid between Indian cultivated sesame and S. mulayanum. The parents and the F6 recombinant constitute the three experimental genotypes in the present report. The seedlings were infected with M. phaseolina. The data of the infected and control (mock-inoculated) transcriptome is presented. The RNA-seq by Illumina NovaSeq 6000 technology generated 2.9?×?108 paired-end reads. We deposited the data in NCBI sequence read archive (SRA) with accession number PRJNA642699. The de novo assembly of clean reads generated 106,295 unigenes with an average length of 1,342 bp covering 1.42?×?108 nucleotides. The screening of 106,295 unigenes with MISA and SAMtools software resulted in the identification of 26,880 simple sequence repeats (SSRs), 90,181 single nucleotide polymorphisms (SNPs), and 25,063 insertion deletions (InDels). Apart from mono-base repeats, di-nucleotides repeats (42.51%) were found to be the most abundant, followed by tri-nucleotides (14.28%) among the SSRs. Subsequently, we have designed 22,494 pairs of primers based on perfect di and tri-nucleotide SSRs. Transitions (Ts, 60%) were the most abundant substitution type among the SNPs followed by transversions type (Tv, 40%), with a Ts/Tv ratio of 1.48. The development of genic-SSR markers and SNP information will pave the way for molecular marker-assisted breeding of sesame for tolerance against charcoal rot.
Project description:A halotolerant actinobacterial strain isolated from salinity affected soil of Eastern Indo-Gangetic plains (IGP), Uttar Pradesh, India, was characterised for its antagonistic potential against Macrophomina phaseolina by dual-culture assay. It was shown to effectively inhibit the growth of M. phaseolina with an inhibition zone of 27 ± 1.33 mm. Further the actinobacterial strain was evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz. amylase, protease, cellulase, chitinase, gelatinase and urease. The results revealed that the actinobacterial strain had PGP potential along with positive assay for amylase, chitinase and urease. The interaction study between antagonist strain and fungal pathogen, performed by scanning electron microscopy technique revealed that the actinobacterium was able to damage fungal mycelia may be due to chitinase, establishing its role as a potential antagonist against M. phaseolina. The actinobacterial isolate was characterised by 16S rDNA gene sequencing, and was identified as Streptomyces genera. The identified gene sequence was deposited to NCBI GenBank with an accession number KP331758.
Project description:Two of the most widely and intensively cultivated jute species, Corchorus capsularis and Corchorus olitorius, suffer severely from a stem rot disease caused by the fungus Macrophomina phaseolina. Wild jute species, C. trilocularis, shows resistance to this pathogenic fungus. In this study, the technique of differential display was applied to identify genes which are differentially expressed, under both infected and un-infected conditions, between C. trilocularis and C. olitorius var O-72. Two xyloglucan endotransglycosylase/hydrolase (XTH) genes designated CoXTH1 (from Corchorus olitorius) and CtXTH1 (from C.trilocularis) were identified from each of the two species which show different expression patterns upon fungal infection. A steady rise in the expression of CtXTH1 in response to infection was observed by quantitative real time PCR whereas the expression of CoXTH1 was found to be downregulated. Full length sequences of these two genes were determined using primer based gene walking and RACE PCR. This study confirms the involvement of XTH in molecular interactions between M. phaseolina and jute. However, it remains to be explored whether XTH is an essential component of the signaling pathway involved in plant-fungal interaction.
Project description:The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.
Project description:In this study, declining pistachio rootstocks were detected in newly planted commercial pistachio orchards in Kern County, California. Symptoms were characterized by wilted foliage combined with crown rot in the rootstock. From diseased trees, 42 isolates were obtained, and all had similar cultural and morphological characteristics of Macrophomina phaseolina. Analyses of nucleotide sequences of three gene fragments, the internal transcribed spacer region (ITS1-5.8S-ITS2), partial sequences of ?-tubulin, and translation elongation factor 1-? (TEF1) confirmed this identification, and 20 representative isolates are presented in the phylogenetic study. Testing of Koch's postulates showed that M. phaseolina, when inoculated to stems and roots of the pistachio rootstocks using mycelial plugs or a microsclerotial suspension, is indeed pathogenic to this host. The widely used clonal University of California Berkeley I (UCBI) rootstock appeared highly susceptible to M. phaseolina, suggesting that this pathogen is an emerging threat to the production of pistachio in California. This study confirmed the association of M. phaseolina with the decline of pistachio trees and represents the first description of this fungus as a crown rot-causing agent of pistachio in California.
Project description:<h4>Background</h4>Macrophomina phaseolina is one of the most destructive necrotrophic fungal pathogens that infect more than 500 plant species throughout the world. It can grow rapidly in infected plants and subsequently produces a large amount of sclerotia that plugs the vessels, resulting in wilting of the plant.<h4>Results</h4>We sequenced and assembled ~49 Mb into 15 super-scaffolds covering 92.83% of the M. phaseolina genome. We predict 14,249 open reading frames (ORFs) of which 9,934 are validated by the transcriptome. This phytopathogen has an abundance of secreted oxidases, peroxidases, and hydrolytic enzymes for degrading cell wall polysaccharides and lignocelluloses to penetrate into the host tissue. To overcome the host plant defense response, M. phaseolina encodes a significant number of P450s, MFS type membrane transporters, glycosidases, transposases, and secondary metabolites in comparison to all sequenced ascomycete species. A strikingly distinct set of carbohydrate esterases (CE) are present in M. phaseolina, with the CE9 and CE10 families remarkably higher than any other fungi. The phenotypic microarray data indicates that M. phaseolina can adapt to a wide range of osmotic and pH environments. As a broad host range pathogen, M. phaseolina possesses a large number of pathogen-host interaction genes including those for adhesion, signal transduction, cell wall breakdown, purine biosynthesis, and potent mycotoxin patulin.<h4>Conclusions</h4>The M. phaseolina genome provides a framework of the infection process at the cytological and molecular level which uses a diverse arsenal of enzymatic and toxin tools to destroy the host plants. Further understanding of the M. phaseolina genome-based plant-pathogen interactions will be instrumental in designing rational strategies for disease control, essential to ensuring global agricultural crop production and security.
Project description:A 70 year old female patient presented with complaints of pain, watering and swelling in the right eye. She gave a history of fall, as she was walking in the paddy field of her farm. Ophthalmological and Microbiological investigation revealed a fungal keratitis with an unusual fungus Macrophomina phaseolina which is primarily a plant pathogen, with a potential to cause human infections especially in immuno-compromised patients. The patient responded well to the antifungal treatment with Oral Voriconazole with absence of recurrence and dissemination.