Investigating the cell and developmental biology of plant infection by the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast disease, the most widespread and serious disease of cultivated rice. Live cell imaging and quantitative 4D image analysis have provided new insight into the mechanisms by which the fungus infects host cells and spreads rapidly in plant tissue. In this video review article, we apply live cell imaging approaches to understanding the cell and developmental biology of rice blast disease. To gain entry to host plants, M. oryzae develops a specialised infection structure called an appressorium, a unicellular dome-shaped cell which generates enormous turgor, translated into mechanical force to rupture the leaf cuticle. Appressorium development is induced by perception of the hydrophobic leaf surface and nutrient deprivation. Cargo-independent autophagy in the three-celled conidium, controlled by cell cycle regulation, is essential for appressorium morphogenesis. Appressorium maturation involves turgor generation and melanin pigment deposition in the appressorial cell wall. Once a threshold of turgor has been reached, this triggers re-polarisation which requires regulated generation of reactive oxygen species, to facilitate septin GTPase-dependent cytoskeletal re-organisation and re-polarisation of the appressorium to form a narrow, rigid penetration peg. Infection of host tissue requires a further morphogenetic transition to a pseudohyphal-type of growth within colonised rice cells. At the same time the fungus secretes an arsenal of effector proteins to suppress plant immunity. Many effectors are secreted into host cells directly, which involves a specific secretory pathway and a specialised structure called the biotrophic interfacial complex. Cell-to-cell spread of the fungus then requires development of a specialised structure, the transpressorium, that is used to traverse pit field sites, allowing the fungus to maintain host cell membrane integrity as new living plant cells are invaded. Thereafter, the fungus rapidly moves through plant tissue and host cells begin to die, as the fungus switches to necrotrophic growth and disease symptoms develop. These morphogenetic transitions are reviewed in the context of live cell imaging studies.

Fungal oxylipins direct programmed developmental switches in filamentous fungi.

Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

Oligomycins inhibit Magnaporthe oryzae Triticum and suppress wheat blast disease.

Oligomycins are macrolide antibiotics, produced by Streptomyces spp. that show antagonistic effects against several microorganisms such as bacteria, fungi, nematodes and the oomycete Plasmopara viticola. Conidiogenesis, germination of conidia and formation of appressoria are determining factors pertaining to pathogenicity and successful diseases cycles of filamentous fungal phytopathogens. The goal of this research was to evaluate the in vitro suppressive effects of two oligomycins, oligomycin B and F along with a commercial fungicide Nativo® 75WG on hyphal growth, conidiogenesis, conidial germination, and appressorial formation of the wheat blast fungus, Magnaporthe oryzae Triticum (MoT) pathotype. We also determined the efficacy of these two oligomycins and the fungicide product in vivo in suppressing wheat blast with a detached leaf assay. Both oligomycins suppressed the growth of MoT mycelium in a dose dependent manner. Between the two natural products, oligomycin F provided higher inhibition of MoT hyphal growth compared to oligomycin B with a minimum inhibitory concentration of 0.005 and 0.05 µg/disk, respectively. The application of the compounds completely halted conidial formation of the MoT mycelium in agar medium. Further bioassays showed that these compounds significantly inhibited MoT conidia germination and induced lysis. The compounds also caused abnormal germ tube formation and suppressed appressorial formation of germinated spores. Interestingly, the application of these macrolides significantly inhibited wheat blast on detached leaves of wheat. This is the first report on the inhibition of mycelial growth, conidiogenesis, germination of conidia, deleterious morphological changes in germinated conidia, and suppression of blast disease of wheat by oligomycins from Streptomyces spp. Further study is needed to unravel the precise mode of action of these natural compounds and consider them as biopesticides for controlling wheat blast.

Isolation and characterization of nutrient dependent pyocyanin from Pseudomonas aeruginosa and its dye and agrochemical properties.

Pyocyanin is a blue green phenazine pigment produced in large quantities by active cultures of Pseudomonas aeruginosa, with advantageous applications in medicine, agriculture and for the environment. Hence, in the present study, a potent bacterium was isolated from agricultural soil and was identified morphologically and by 16S rRNA sequencing as P. aeruginosa (isolate KU_BIO2). When the influence of nutrient supplements in both King's A and Nutrient media as amended was investigated, an enhanced pyocyanin production of 2.56 µg ml-1 was achieved in King's A medium amended with soya bean followed by 1.702 µg ml-1 of pyocyanin from the nutrient medium amended with sweet potato. Purified pyocyanin was characterized by UV-Vis Spectrophotometer and Fourier-Transform Infrared spectroscopy (FTIR). Furthermore, Liquid Chromatography Mass Spectrum (LCMS) and Nuclear Magnetic Resonance (NMR) confirmed its mass value at 211 and as N-CH3 protons resonating at 3.363 ppm as a singlet respectively. The isolated pyocyanin displayed remarkable dye property by inducing color change in cotton cloth from white to pink. Lastly, the antifungal activity of test pyocyanin showed inhibition of growth of rice blast fungus, Magnaporthe grisea and bacterial blight of rice, Xanthomonas oryzae at concentrations of 150 and 200 ppm, respectively. Thus, this investigation provides evidence for diverse actions of pyocyanin which are nutrient dependent and are capable of acting on a large scale, by utilizing microbes existing in agriculture wastes, and thus could be used as an alternative source in the making of natural textile dyes with strong durability and a broad spectrum of ecofriendly agrochemicals.

Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1-6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice's defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Molecular and morphological characterization of rice phylloplane fungi and determination of the antagonistic activity against rice pathogens.

Cladosporium spp. is a cosmopolitan fungal genus. In the literature, it has been reported as a biological agent for controlling several plant diseases, but its mechanism of action has never been clarified. The present study aims to identify Cladosporium spp. based on the DNA phylogeny of nine isolates obtained from the phylloplane of rice and their potential antagonistic activity against the main fungal pathogens that affect rice crop. Nine isolates of Cladosporium spp. were identified based on DNA phylogeny, molecular and morphological characterization, and their antagonistic effects with the rice pathogens C. miyabeanus, M. oryzae, M. albescens and S. oryzae. Four isolates were selected to study lytic enzymes such as ß-1,3-glucanase, chitinase and protease, and only one isolate was selected for a conidial germination and appressoria formation assay. The nine isolates were identified as C. cladosporioides, C. tenuissimum and C. subuliforme. Four isolates, identified as C. cladosporioides, inhibited the mycelial growth of rice pathogens such as C1H (68.59%) of S. oryzae, C5 G (74.32%) of C. miyabeanus, C11 G (75.97%) of M. oryzae and C24 G (77.39%) of M. albescens. C24 G showed a high activity of lytic enzymes, was tested against C. miyabeanus and M. oryzae, and inhibited conidial germination and appressorium formation by more than 59.36%. The characterization of C. cladosporioides suggested this species as a potential bioagent for the management of several rice diseases, especially rice blast. This is the first time that a potential biological agent from the genus Cladosporium identified at the species level was isolated from the rice phylloplane, and some of its mechanisms of action were demonstrated, such as increasing lytic enzyme activity against rice pathogens.

MoFap7, a ribosome assembly factor, is required for fungal development and plant colonization of Magnaporthe oryzae.

Fap7, an important ribosome assembly factor, plays a vital role in pre-40S small ribosomal subunit synthesis in Saccharomyces cerevisiae via its ATPase activity. Currently, the biological functions of its homologs in filamentous fungi remain elusive. Here, MoFap7, a homologous protein of ScFap7, was identified in the rice blast fungus Magnaporthe oryzae, which is a devastating fungal pathogen in rice and threatens food security worldwide. ΔMofap7 mutants exhibited defects in growth and development, conidial morphology, appressorium formation and infection, and were sensitive to oxidative stress. In addition, site-directed mutagenesis analysis confirmed that the conserved Walker A motif and Walker B motif in MoFap7 are essential for the biological functions of M. oryzae. We further analyzed the regulation mechanism of MoFap7 in pathogenicity. MoFap7 was found to interact with MoMst50, a regulator functioning in the MAPK Pmk1 signaling pathway, that participates in modulating plant penetration and cell-to-cell invasion by regulating the phosphorylation of MoPmk1. Moreover, MoFap7 interacted with the GTPases MoCdc42 and MoRac1 to control growth and conidiogenesis. Taken together, the results of this study provide novel insights into MoFap7-mediated orchestration of the development and pathogenesis of filamentous fungi.

A histone deacetylase, MoHOS2 regulates asexual development and virulence in the rice blast fungus.

Histone acetylation/deacetylation represent a general and efficient epigenetic mechanism through which fungal cells control gene expression. Here we report developmental requirement of MoHOS2-mediated histone deacetylation (HDAC) for the rice blast fungus, Magnaporthe oryzae. Structural similarity and nuclear localization indicated that MoHOS2 is an ortholog of Saccharomyces cerevisiae Hos2, which is a member of class I histone deacetylases and subunit of Set3 complex. Deletion of MoHOS2 led to 25% reduction in HDAC activity, compared to the wild-type, confirming that it is a bona-fide HDAC. Lack of MoHOS2 caused decrease in radial growth and impinged dramatically on asexual sporulation. Such reduction in HDAC activity and phenotypic defects of ΔMohos2 were recapitulated by a single amino acid change in conserved motif that is known to be important for HDAC activity. Expression analysis revealed up-regulation of MoHOS2 and concomitant down-regulation of some of the key genes involved in asexual reproduction under sporulation-promoting condition. In addition, the deletion mutant exhibited defect in appressorium formation from both germ tube tip and hyphae. As a result, ΔMohos2 was not able to cause disease symptoms. Wound-inoculation showed that the mutant is compromised in its ability to grow inside host plants as well. We found that some of ROS detoxifying genes and known effector genes are de-regulated in the mutant. Taken together, our data suggest that MoHOS2-dependent histone deacetylation is pivotal for proper timing and induction of transcription of the genes that coordinate developmental changes and host infection in M. oryzae.

MoLEU1, MoLEU2, and MoLEU4 regulated by MoLEU3 are involved in leucine biosynthesis, fungal development, and pathogenicity in Magnaporthe oryzae.

Amino acids are vital components in cell metabolism. Leucine is a regulatory factor that generates significant impact on protein synthesis/turnover, modulates diverse cellular signalling pathways and participates in oxidative processes and immune responses. Here, we identified and characterized the functions of a leucine-associated Zn2 Cys6 -type transcription factor, MoLeu3. Disruption of MoLEU3 resulted in significantly reduced pathogenicity in barley and rice. Quantitative RT-PCR showed that the expression levels of the putative leucine biosynthesis-related genes, MoLEU1, MoLEU2 and MoLEU4 were downregulated in the ΔMoleu3 mutant. We used high-throughput gene knockout method to generate the null mutants of MoLEU1, MoLEU2 and MoLEU4 respectively. The ΔMoleu1, ΔMoleu2 and ΔMoleu4 mutants are leucine auxotroph and showed similar phenotypic characterizations, including reduced conidiation, delayed mobilization and degradation of glycogen and lipid droplets, limited appressorium-mediated penetration, and restricted invasive hyphae growth within host cells. Collectively, MoLEU1, MoLEU2, and MoLEU4 regulated by MoLEU3 play crucial roles in fungal development and infectious processes through modulation of leucine biosynthesis in Magnaporthe oryzae.

Two distinct nucleic acid binding surfaces of Cdc5 regulate development.

Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding.

Candidate screening of blast resistance donors for rice breeding.

Rice blast is one of the most serious diseases in the world. The use of resistant cultivars is the most preferred means to control this disease. Resistance often breaks down due to emergence of new races; hence identification of novel resistance donors is indispensable. In this study, a panel of 80 released varieties from National Rice Research Institute, Cuttack was genotyped with 36 molecular markers that were linked to 36 different blast resistance genes, to investigate the varietal genetic diversity and molecular marker-trait association with blast resistance. The polymorphism information content of 36 loci varied from 0.11 to 0.37 with an average of 0.34. The cluster analysis and population structure categorized the 80 National Rice Research Institute released varieties (NRVs) into three major genetic groups. The principal co-ordinate analysis displays the distribution of resistant and moderately resistant NRVs into different groups. Analysis of molecular variance result demonstrated maximum (97%) diversity within populations and minimum (3%) diversity between populations. Among tested markers, two markers (RM7364 and pi21_79-3) corresponding to the blast resistance genes (Pi56(t) and pi21) were significantly associated and explained a phenotypic variance of 4.9 to 5.1% with the blast resistance. These associated genes could be introgressed through marker-assisted to develop durable blast resistant rice varieties. The selected resistant NRVs could be good donors for the blast resistance in rice crop improvement research.

The isolation of the antagonistic strain Bacillus australimaris CQ07 and the exploration of the pathogenic inhibition mechanism of Magnaporthe oryzae.

Biological control as a promising method to combat plant disease has gained public attention in recent years. In the present study, we isolated 12 strains resistant to Magnaporthe oryzae from western Sichuan subalpine soil. Among them, CQ07 exhibited remarkable activity against M. oryzae. The result of 16S rRNA sequence analysis revealed that CQ07 is approximately 99% similar to Bacillus australimaris. The sterilized culture filtrate of CQ07 inhibited the growth of M. oryzae, which motivated us to deduce the influence of CQ07 on the pathogenicity of M. oryzae. As shown by experimentation, sterilized culture filtrate (10 µl/ml) of CQ07 can delay and even suppress the germination of conidia and prevent the formation of appressorium in vitro and in vivo. In addition, by simulative field tests, the spraying of conidia suspension diluted with sterilized culture filtrate of CQ07 reduced infection of rice blast. To better control rice blasts, understanding the infection mechanism of M. oryzae and inhibiting the mechanism of the antagonistic strain is of great importance.

A MYST family histone acetyltransferase, MoSAS3, is required for development and pathogenicity in the rice blast fungus.

Histone acetylation has been established as a principal epigenetic regulatory mechanism in eukaryotes. Sas3, a histone acetyltransferase belonging to the largest family of acetyltransferase, MYST, is the catalytic subunit of a conserved histone acetyltransferase complex. To date, the functions of Sas3 and its orthologues have been extensively studied in yeast, humans and flies in relation to global acetylation and transcriptional regulation. However, its precise impact on development and pathogenicity in fungal plant pathogens has yet to be elucidated. Considering the importance of Sas3 in H3K14 acetylation, here we investigate the roles of its orthologue in the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Unlike a previously reported Sas3 deletion in yeast, which led to no remarkable phenotypic changes, we found that MoSAS3 deletion alone had a profound effect on fungal growth and development, including asexual reproduction, germination and appressorium formation in M. oryzae. Such defects in pre-penetration development resulted in complete loss of pathogenicity in the deletion mutant. Furthermore, genetic analysis of MoSAS3 and MoGCN5 encoding a Gcn5-related N-acetyltransferase family histone acetyltransferase suggested that two conserved components of histone acetylation are integrated differently into epigenetic regulatory mechanisms in the yeast and a filamentous fungus. RNA-seq analysis of ΔMosas3 showed two general trends: many DNA repair and DNA damage response genes are up-regulated, while carbon and nitrogen metabolism genes are down-regulated in ΔMosas3. Our work demonstrates the importance of MYST family histone acetyltransferase as a developmental regulator and illuminates a degree of functional variation in conserved catalytic subunits among different fungal species.

The farnesyltransferase ß-subunit RAM1 regulates localization of RAS proteins and appressorium-mediated infection in Magnaporthe oryzae.

Post-translational farnesylation can regulate subcellular localization and protein-protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal infection. Here, through functional analyses of the farnesyltransferase ß-subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C-terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized RAS proteins. Our findings indicate that protein farnesylation regulates the RAS protein-mediated signaling pathways required for appressorium formation and host infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control.

Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe oryzae.

To develop an antimicrobial agent for preventing the devasting damage caused by rice blast, a novel peptide aptamer was identified to interact with calmodulin (CaM) for the inhibition of the spore development in the pathogen Magnaporthe oryzae. A peptide aptamer designated as SNP-D4, consisted of the scaffold protein Staphylococcus aureus nuclease (SN) and an exposed surface loop of 16 random amino acids, was screened from the constructed peptide aptamer libraries by bacterial two-hybrid system using CaM of M. oryzae as the bait. The preliminary inhibition in the sporulation development was observed after treating with the crude extracts expressing SNP-D4. The inhibition efficacies of the purified SNP-D4 were quantified at the stages of conidial germination, germ tube elongation, and appressorium formation in M. oryzae. The binding affinity analysis revealed that SNP-D4 interacted with CaM at a dissociation constant (Kd) of about 20 µM. Moreover, the N-terminus of CaM was identified as the key binding region.

Mitochondrial dynamics and mitophagy are necessary for proper invasive growth in rice blast.

Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology during Magnaporthe-rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.

Magnaporthe oryzae CK2 Accumulates in Nuclei, Nucleoli, at Septal Pores and Forms a Large Ring Structure in Appressoria, and Is Involved in Rice Blast Pathogenesis.

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores.

Induction of resistance in rice plants using bioproducts produced from Burkholderia pyrrocinia BRM 32113.

Leaf blast is the main rice disease in the world causing significant losses in productivity. Blast integrate management (BIM) requires the use of genetic resistance, cultural practices, and chemical control, although for sustainable BIM, the insertion of biological agents may be the fourth component for. The objective of this work was to test three formulations of Burkholderia pyrrocinia (BRM32113) previously selected and to verify the effectiveness in resistance induction and blast control in rice. Two experiments were carried out, in a completely randomized design with three replications, in the greenhouse (E1 and E2). E1 aimed to select the best treatment for suppressing leaf blast severity and activating plant defense mechanisms. It was composed of 8 treatments: (1) formulated 11+ B. pyrrocina × Magnaporthe oryzae; (2) formulated 17+ B. pyrrocina × M. oryzae; (3) formulated 32+ B. pyrrocina × M. oryzae; (4) formulated 11 × M. oryzae; (5) B. pyrrocinia 17 × M. oryzae; (6) formulated 32 × M. oryzae; (7) B. pyrrocina × M. oryzae; (8) M. oryzae; (9) control (water). E2 aimed to investigate the effect of the best treatments, for the promotion of plant growth and suppression of leaf blast by calculating AUDPC. It was composed of 6 treatments: (1) formulated 11+ B. pyrrocina × M. oryzae; (2) formulated 32+ B. pyrrocina × M. oryzae; (3) formulated 11 × M. oryzae; (4) formulated 32 × M. oryzae; (5) B. pyrrocina × M. oryzae; (6) water. And after, we did two assays aimed to localize this biological agent after application at seed, soil, and rice plant. In E1, formulated 11+ B. pyrrocinia and 32+ formulated and B. pyrrocina were the best, suppressing leaf blast by up to 97% and providing the significant increase of the enzymes ß-1,3-glucanase, chitinase, phenylalanine ammonia lyase, lipoxygenase, and salicylic acid at 24 h and 48 h after inoculation with M. oryzae. In E2, treatments formulated 11+ B. pyrrocinia, formulated 32+ B. pyrrocinia, and B. pyrrocina provided more significant increases in growth promotion and reduced area under disease progress curve. B. pyrrocinia was detected in the rice plant for 18 days, predominantly in the root system (internal and external). The use of B. pyrrocinia formulations based on sugarcane molasses and glycerol can be an essential strategy for sustainable management. Although all the benefits come from these sustainable formulations, the adoption by commercial biological segment depends on an established formulation process. It seems that all the results showed here by this research will be readily assimilated by startups of the organic segment.

The Methylcitrate Cycle is Required for Development and Virulence in the Rice Blast Fungus Pyricularia oryzae.

The methylcitrate cycle metabolizes propionyl-CoA, a toxic metabolite, into pyruvate. Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus that causes a destructive blast disease in rice and wheat. We characterized the essential roles of the methylcitrate cycle in the development and virulence of P. oryzae using functional genomics. In P. oryzae, the transcript levels of MCS1 and MCL1, which encode a 2-methylcitrate synthase and a 2-methylisocitrate lyase, respectively, were upregulated during appressorium formation and when grown on propionyl-CoA-producing carbon sources. We found that deletion of MCS1 and MCL1 inhibited fungal growth on media containing both glucose and propionate, and media using propionate or propionyl-CoA-producing amino acids (valine, isoleucine, methionine, and threonine) as the sole carbon or nitrogen sources. The Δmcs1 mutant formed sparse aerial hyphae and did not produce conidia on complete medium (CM), while the Δmcl1 mutant showed decreased conidiation. The aerial mycelium of Δmcs1 displayed a lowered NAD+/NADH ratio, reduced nitric oxide content, and downregulated transcription of hydrophobin genes. Δmcl1 showed reduced appressorium turgor, severely delayed plant penetration, and weakened virulence. Addition of acetate recovered the growth of the wild type and Δmcs1 on medium containing both glucose and propionate and recovered the conidiation of both Δmcs1 and Δmcl1 on CM by reducing propionyl-CoA formation. Deletion of MCL1 together with ICL1, an isocitrate lyase gene in the glyoxylate cycle, greatly reduced the mutant's virulence as compared with the single-gene deletion mutants (Δicl1 and Δmcl1). This experimental evidence provides important information about the role of the methylcitrate cycle in development and virulence of P. oryzae by detoxification of propionyl-CoA and 2-methylisocitrate.

The Secreted Protein MoHrip1 Is Necessary for the Virulence of Magnaporthe oryzae.

Secreted effectors from Magnaporthe oryzae play critical roles in the interaction with rice to facilitate fungal infection and disease development. M. oryzae-secreted protein MoHrip1 can improve plant defense as an elicitor in vitro, however, its biological function in fungal infection is not clear. In this study, we found that the expression of mohrip1 was significantly induced in the stages of fungal penetration and colonization. Although dispensable for the growth and conidiation, MoHrip1 was necessary for the full virulence of M. oryzae. Deletion of mohrip1 remarkably compromised fungal virulence on rice seedlings and even on rice leaves with wounds. Rice sheath inoculation assay further demonstrated the defects of mohrip1-deleted mutants on penetration and proliferation in rice cells. Additionally, compared with WT and complementation strain, the inoculation of mohrip1-deleted mutants induced a higher expression of specific defense related genes and a higher production of specific defensive compounds in rice leaves. These data collectively indicated that MoHrip1 is necessary for fungal penetration and invasive expansion, and further full virulence of rice blast fungus.