ERF Transcription Factor OsBIERF3 Positively Contributes to Immunity against Fungal and Bacterial Diseases but Negatively Regulates Cold Tolerance in Rice.

We previously showed that overexpression of the rice ERF transcription factor gene OsBIERF3 in tobacco increased resistance against different pathogens. Here, we report the function of OsBIERF3 in rice immunity and abiotic stress tolerance. Expression of OsBIERF3 was induced by Xanthomonas oryzae pv. oryzae, hormones (e.g., salicylic acid, methyl jasmonate, 1-aminocyclopropane-1-carboxylic acid, and abscisic acid), and abiotic stress (e.g., drought, salt and cold stress). OsBIERF3 has transcriptional activation activity that depends on its C-terminal region. The OsBIERF3-overexpressing (OsBIERF3-OE) plants exhibited increased resistance while OsBIERF3-suppressed (OsBIERF3-Ri) plants displayed decreased resistance to Magnaporthe oryzae and X. oryzae pv. oryzae. A set of genes including those for PRs and MAPK kinases were up-regulated in OsBIERF3-OE plants. Cell wall biosynthetic enzyme genes were up-regulated in OsBIERF3-OE plants but down-regulated in OsBIERF3-Ri plants; accordingly, cell walls became thicker in OsBIERF3-OE plants but thinner in OsBIERF3-Ri plants than WT plants. The OsBIERF3-OE plants attenuated while OsBIERF3-Ri plants enhanced cold tolerance, accompanied by altered expression of cold-responsive genes and proline accumulation. Exogenous abscisic acid and 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, restored the attenuated cold tolerance in OsBIERF3-OE plants while exogenous AgNO3, an inhibitor of ethylene action, significantly suppressed the enhanced cold tolerance in OsBIERF3-Ri plants. These data demonstrate that OsBIERF3 positively contributes to immunity against M. oryzae and X. oryzae pv. oryzae but negatively regulates cold stress tolerance in rice.

Evidence of Pyricularia oryzae adaptability to tricyclazole.

Pyricularia oryzae is the etiological agent of rice blast, the most destructive disease in rice crops and chemical control based on fungicide is the main method used in its management. The aim of this study was characterize pathogenicity and identify P. oryzae isolates adapted to tricyclazole. P. oryzae monosporic isolates were collected in the state of Tocantins and inoculated in international differentiating series of rice cultivars for determination of pathotypes. After, the same isolates were inoculated in the rice cultivar IRGA 424 to evaluate resistance to fungicide Bim® 750 BR (Tricyclazole - 250 g/ha) that was applied 24 and 48 hours after pathogen inoculation (hai). Leaf blast severity and infection efficiency were evaluated 9 days after inoculation (dai), latency period (2 dai) and sporulation intensity (7 dai). Nine different pathotypes were identified, predominantly as IA group. The latent period of isolates occurred between from 48 to 120 h. The application of tricyclazole, 24 hai reduced disease severity with the exception of the isolate Py 7.1. The great variability of the pathogen allowed for adaptation to this molecule and can increase its aggressiveness and should be considered to guide the integrated management of the disease.

A novel ß-1,3-glucanase Gns6 from rice possesses antifungal activity against Magnaporthe oryzae.

As members of the pathogenesis-related protein (PR)-2 family, ß-1,3-glucanases play pivotal roles in plant defense. Previous study showed that the rice genome contains 16 genes encoding putative ß-1,3-glucanases, and the ß-1,3-glucanases in subfamily A were deduced to be involved in plant defense. However, there was limited direct evidence. In this study, the expression of rice ß-1,3-glucanases Gns2-Gns6 belonging to subfamily A in rice plant infection with Magnaporthe oryzae was investigated, and the enhanced expression of Gns6 during infection confirmed its crucial role in the defense of rice seedlings. Enzymological characterization revealed that Gns6 preferentially hydrolyzed laminarin, pachymaran, and yeast glucan. The ß-1,3; 1,6-glucanase Gns6 exhibited a specific activity of 1.2 U/mg with laminarin as the substrate. In addition, Gns6 could hydrolyze laminarin via an endo-type mechanism, yielding a series of oligosaccharides with various degrees of polymerization that are known immune elicitors in plants. Moreover, Gns6 exhibited a significant inhibitory effect against the formation of the germ tubes and appressoria, with potential applications in plant protection. Taken together, this study shows that Gns6 is an essential effector in the defensive response of rice against pathogenic fungi.

Preparation and characterization of a novel green tea essential oil nanoemulsion and its antifungal mechanism of action against Magnaporthae oryzae.

Blast is one of the most devastating fungal diseases of rice caused by Magnaporthe oryzae. Plant essential oil (EO) can function as antifungal agents and are regarded as a safe and acceptable method for plant disease control. However, EOs are unstable and hydrophobic, which limits its use. In the present study, we aimed for the preparation and characterization of a nanoemulsion (NE) from green tea essential oil (GTO) by ultrasonication method and determined the antifungal activity of NE onM. oryzae. The particle size and zeta potential of the NE were 86.98 nm and -15.1 mV, respectively. The chemical composition and functional groups of GTO and NE were studied by using GC-MS analysis, portable Raman spectroscopy, and FTIR coupled with chemometric analysis. GC-MS analysis showed the major components in GTO and NE were n-Hexyl cinnamaldehyde and L-α-Terpineol. Both GTO and NE showed good antioxidant activity and total phenol content. Moreover, the NE showed good antifungal activity againstM. oryzae which was further confirmed by scanning electron microscopy (SEM) examination. Also, confocal Raman micro-spectroscopy (CRM) revealed the antifungal mechanism of GTO and NE on M. oryzae which proves the cell damage. To the best of our knowledge, this is the first study on the antifungal activity of GTO and NE against M. oryzae and also the use of CRM for the evaluation of the chemical changes in single fungal hyphae in a holistic approach. This study suggests that the prepared NE could be a potential candidate for use as a substitute for synthetic fungicides.

Identification of Pyruvate Dehydrogenase E1 as a Potential Target against Magnaporthe oryzae through Experimental and Theoretical Investigation.

Magnaporthe oryzae (M. oryzae) is a typical cause of rice blast in agricultural production. Isobavachalcone (IBC), an active ingredient of Psoralea corylifolia L. extract, is an effective fungicide against rice blast. To determine the mechanism of IBC against M. oryzae, the effect of IBC on the metabolic pathway of M. oryzae was explored by transcriptome profiling. In M. oryzae, the expression of pyruvate dehydrogenase E1 (PDHE1), part of the tricarboxylic acid (TCA cycle), was significantly decreased in response to treatment with IBC, which was verified by qPCR and testing of enzyme activity. To further elucidate the interactions between IBC and PDHE1, the 3D structure model of the PDHE1 from M. oryzae was established based on homology modeling. The model was utilized to analyze the molecular interactions through molecular docking and molecular dynamics simulation, revealing that IBC has π-π stacking interactions with residue TYR139 and undergoes hydrogen bonding with residue ASP217 of PDHE1. Additionally, the nonpolar residues PHE111, MET174, ILE 187, VAL188, and MET250 form strong hydrophobic interactions with IBC. The above results reveal that PDHE1 is a potential target for antifungal agents, which will be of great significance for guiding the design of new fungicides. This research clarified the mechanism of IBC against M. oryzae at the molecular level, which will underpin further studies of the inhibitory mechanism of flavonoids and the discovery of new targets. It also provides theoretical guidance for the field application of IBC.

Structural and Functional Insights into Iturin W, a Novel Lipopeptide Produced by the Deep-Sea Bacterium Bacillus sp. Strain wsm-1.

In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ß-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.

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.

Discovery of luotonin A analogues as potent fungicides and insecticides: Design, synthesis and biological evaluation inspired by natural alkaloid.

The prevention and control of plant diseases and insect pests is the most crucial issue facing crop protection. To discover novel pesticide candidates with diverse chemical structures from natural products, a series of luotonin A analogues were designed, synthesized and evaluated for their antifungal and insecticidal activities. Most of these compounds exhibited potent activity against Botrytis cinerea, Magnaporthe oryzae and Aphis craccivora. Among them, the antifungal activity of compound 10s against B. cinerea was comparable to azoxystrobin (EC50 = 0.09 mM) and against M. oryzae (EC50 = 0.19 mM) was slightly weaker than that of azoxystrobin (EC50 = 0.17 mM). Compounds 10k and 10o are the most active compounds against A. craccivora having identical mortality value of 42.05% at 50 µg/mL, respectively, which were slightly lower than pymetrozine (51.14%) at the same concentration. Revealed morphological changes of the fungal cell surface by scanning electron microscopy indicated that luotonin A analogues might exert their antifungal activity by destroying fungal cell membrane and cell wall. Furthermore, the results of the in vivo protective and curative activities of the compound 10s against S. sclerotiorum and B. cinerea showed that the curative effect was stronger than its protective effect and the curative effects reached 67.17% and 73.82% at 80 µg/mL respectively. The above results further demonstrated the potential of luotonin A analogues as novel fungicides and insecticides.

Rapid adaptation of signaling networks in the fungal pathogen Magnaporthe oryzae.

BACKGROUND: One fundamental question in biology is how the evolution of eukaryotic signaling networks has taken place. "Loss of function" (lof) mutants from components of the high osmolarity glycerol (HOG) signaling pathway in the filamentous fungus Magnaporthe oryzae are viable, but impaired in osmoregulation. RESULTS: After long-term cultivation upon high osmolarity, stable individuals with reestablished osmoregulation capacity arise independently from each of the mutants with inactivated HOG pathway. This phenomenon is extremely reproducible and occurs only in osmosensitive mutants related to the HOG pathway - not in other osmosensitive Magnaporthe mutants. The major compatible solute produced by these adapted strains to cope with high osmolarity is glycerol, whereas it is arabitol in the wildtype strain. Genome and transcriptome analysis resulted in candidate genes related to glycerol metabolism, perhaps responsible for an epigenetic induced reestablishment of osmoregulation, since these genes do not show structural variations within the coding or promotor sequences. CONCLUSION: This is the first report of a stable adaptation in eukaryotes by producing different metabolites and opens a door for the scientific community since the HOG pathway is worked on intensively in many eukaryotic model organisms.

The antifungal action mode of the rice endophyte Streptomyces hygroscopicus OsiSh-2 as a potential biocontrol agent against the rice blast pathogen.

Microbial antagonists and their bioactive metabolites provide one of the best alternatives to chemical pesticides to control crop disease for sustainable agriculture and global food security. The rice endophyte Streptomyces hygroscopicus OsiSh-2, with remarkable antagonistic activity towards the rice blast fungus Magnaporthe oryzae, was reported in our previous study. The present study deciphered the possible direct interaction mode of OsiSh-2 against M. oryzae. An in vitro antibiotic assay for OsiSh-2 culture filtrate revealed strong suppression of mycelial growth, conidial germination and appressorial formation of M. oryzae. Meanwhile, severe morphological and internal abnormalities in M. oryzae hyphae were observed under a scanning electron microscope and transmission electron microscope. Foliar treatment of rice seedlings by OsiSh-2 culture filtrate in the greenhouse and in the field showed 23.5% and 28.3% disease reduction, respectively. Correspondingly, OsiSh-2 culture filtrate could induce disorganized chitin deposition in the cell wall and lowered ergosterol content in the cell membrane of M. oryzae. Additionally, cell wall integrity pathway activation, large cell electrolytes release, reactive oxygen species accumulation and tricarboxylic acid cycle-related enzyme activity changes were found in M. oryzae. All these results suggested that the direct antagonistic activity of OsiSh-2 against M. oryzae may be attributed to damaging the integrity of the cell wall and membrane and disrupting mitochondrial function in the pathogen.

Chloramphenicol inhibits eukaryotic Ser/Thr phosphatase and infection-specific cell differentiation in the rice blast fungus.

Chloramphenicol (Cm) is a broad-spectrum classic antibiotic active against prokaryotic organisms. However, Cm has severe side effects in eukaryotes of which the cause remains unknown. The plant pathogenic fungus Magnaporthe oryzae, which causes rice blast, forms an appressorium to infect the host cell via single-cell differentiation. Chloramphenicol specifically inhibits appressorium formation, which indicates that Cm has a novel molecular target (or targets) in the rice blast fungus. Application of the T7 phage display method inferred that MoDullard, a Ser/Thr-protein phosphatase, may be a target of Cm. In animals Dullard functions in cell differentiation and protein synthesis, but in fungi its role is poorly understood. In vivo and in vitro analyses showed that MoDullard is required for appressorium formation, and that Cm can bind to and inhibit MoDullard function. Given that human phosphatase CTDSP1 complemented the MoDullard function during appressorium formation by M. oryzae, CTDSP1 may be a novel molecular target of Cm in eukaryotes.

Discovery of New Triterpenoid Saponins Isolated from Maesa japonica with Antifungal Activity against Rice Blast Fungus Magnaporthe oryzae.

While searching for new antifungal compounds, we revealed that a methanol extract of plant species Maesa japonica has a potent antifungal activity in vivo against rice blast fungus Magnaporthe oryzae. To identify the antifungal substances, the methanol extract of M. japonica was extracted by organic solvents, and consequently, six active compounds were isolated from the n-butanol layer. The isolated compounds were five new acylated triterpenoid saponins including maejaposide I (1), maejaposides C-1, C-2, and C-3 (2-4), and maejaposide A-1 (5), along with a known one, maejaposide A (6). These chemical structures were determined by NMR and a comparison of their NMR and MS data with those reported in the literature. Based on the in vitro antifungal bioassay, the five compounds (2-6) exhibited strong antifungal activity against M. oryzae with MIC values ranging from 4 to 32 µg/mL, except for maejaposide I (1) (MIC > 250 µg/mL). When the compounds were evaluated at concentrations of 125, 250, and 500 µg/mL for an in vivo antifungal activity against rice blast, compounds 2-6 strongly reduced the development of blast by at least 85% to 98% compared to the untreated control. However, compound 1 did not show any in vivo antifungal activity up to a concentration of 500 µg/mL. Taken together, our results suggest that the methanol extract of M. japonica and the new acylated triterpenoid saponins can be used as a source for the development of natural fungicides.

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.

The inhibitor of apoptosis protein MoBir1 is involved in the suppression of hydrogen peroxide-induced fungal cell death, reactive oxygen species generation, and pathogenicity of rice blast fungus.

The inhibitor of apoptosis protein (IAP) family has been identified in a variety of organisms. All IAPs contain one to three baculoviral IAP repeat (BIR) domains, which are required for anti-apoptotic activity. Here, we identified a type II BIR domain-containing protein, MoBir1, in the rice blast fungus Magnaporthe oryzae. Expression of the MoBIR1 gene in Saccharomyces cerevisiae suppressed hydrogen peroxide-induced cell death and delayed yeast cell chronological aging. Delayed aging was found to require the carboxyl terminus of MoBir1. M. oryzae transformants overexpressing the MoBIR1 gene demonstrated increased growth rate and biomass, delayed mycelial aging, and enhanced resistance to hydrogen peroxide but reduced reactive oxygen species generation and virulence. Moreover, MoBIR1-overexpressing transformants exhibited anti-apoptotic activity. However, MoBIR1 silencing resulted in no obvious phenotypic changes, compared with the wild-type M. oryzae strain Guy11. Our findings broaden the knowledge on fungal type II BIR domain-containing proteins.

Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis.

The evolutionarily conserved octameric exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.

The biological activities of prothioconazole enantiomers and their toxicity assessment on aquatic organisms.

Chiral fungicide prothioconazole has a wide range of antifungal spectrum; however, little research has been conducted to evaluate prothioconazole on an enantiomeric level. Five target pathogens and three common aquatic organisms were tested for the enantioselective bioactivity and toxicity of prothioconazole in this work. The antifungal activity of the enantiomers against wheat phytoalexin, rice blast fungus, exserohilum turcicum, Alternaria triticina, and Fusarium avenaceum was determined, and it was found that (-)-prothioconazole were 85 to 2768 times more active than (+)-prothioconazole toward these target organisms. In order to reflect the risk to aquatic ecosystem, the acute toxicity of the enantiomers to Daphnia magna, Chlorella pyrenoidosa, and Lemna minor L. was assessed. It was observed that the toxicity of (-)-prothioconazole to D. magna was 2.2 times higher than (+)-prothioconazole, but it was lower to C. pyrenoidosa and L. minor L. The toxicities of (+)-enantiomer and (-)-enantiomer to D. magna and C. pyrenoidosa were synergy, indicating that the racemate had higher threat to the organisms. It could be concluded that the effects of prothioconazole on target organisms and the acute toxicity to nontarget species were enantioselective with (-)-enantiomer possessing higher efficiency and lower toxicity. Such enantiomeric differences should be taken into consideration when assessing the performance of prothioconazole.

Characterization of Antifungal Lipopeptide Biosurfactants Produced by Marine Bacterium Bacillus sp. CS30.

This study was initiated to screen for marine bacterial agents to biocontrol Magnaporthe grisea, a serious fungal pathogen of cereal crops. A bacterial strain, isolated from the cold seep in deep sea, exhibited strong growth inhibition against M. grisea, and the strain was identified and designated as Bacillus sp. CS30. The corresponding antifungal agents were purified by acidic precipitation, sequential methanol extraction, Sephadex LH-20 chromatography, and reversed phase high-performance liquid chromatography (RP-HPLC), and two antifungal peaks were obtained at the final purification step. After analysis by mass spectrometry (MS) and tandem MS, two purified antifungal agents were deduced to belong to the surfactin family, and designated as surfactin CS30-1 and surfactin CS30-2. Further investigation showed that although the antifungal activity of surfactin CS30-1 is higher than that of surfactin CS30-2, both of them induced the increased generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm, thus leading to the cell death of M. grisea. Our results also show the differences of the antifungal activity and antifungal mechanism of the different surfactin homologs surfactin CS30-1 and surfactin CS30-2, and highlight them as potential promising agents to biocontrol plant diseases caused by M. grisea.

Synthesis and Fungicidal Activity of (E)-Methyl 2-(2-((1-cyano-2-hydrocarbylidenehydrazinyl)methyl)phenyl)-2-(methoxyimino)acetates.

BACKGROUND: Strobilurin fungicides are some of the most potent and successful agrochemicals. However, continued use of traditional strobilurins has led to the emergence of fungicide-resistant biotypes. Thus, a supply of new strobilurin fungicides is highly valuable. In this study, a series of novel methoxyacrylate analogs containing a cyano-substituted hydrazine moiety as the side chain was synthesized and evaluated for their anti-plant pathogenic activities. RESULTS: Compounds 2-04, 2-05, 2-07 and 2-14 exhibited a relatively broad range of fungicidal activity. Compounds 2-04, 2-13 and 2-14 exhibited good fungicidal activity against Sclerotinia sclerotiorum with median effective concentrations (EC50 ) of 3.84, 3.50 and 3.80 µg mL-1 , respectively. Most of these compounds showed excellent inhibition of spore germination in Magnaporthe grisea at 25 µg mL-1 . Moreover, in an in vivo test, compounds 2-02, 2-04, 2-07 and 2-13 exhibited potent fungicidal activities against the tested plant diseases at 400 µg mL-1 . Notably, compound 2-07 showed comparable or better activity than the commercially positive controls, azoxystrobin and procloraz, against powdery mildew of cucumber and rice blast fungus in the field trails at the same application dosages. CONCLUSON: This study indicated that methoxyacrylate analogs containing a cyano-substituted hydrazone side chain can serve as potential fungicidal candidates for crop protection. © 2019 Society of Chemical Industry.

Determination of the absolute configurations of the stereogenic centers of ustilaginoidins by studying the biosynthetic monomers from a gene knockout mutant of Villosiclava virens.

Ustilaginoidins are a kind of mycotoxins with 9,9'-linked bis-naphtho-γ-pyrones structures produced by the rice false smut pathogen Villosiclava virens. These metabolites displayed a wide range of bioactivities, such as teratogenic, cytotoxic, phytotoxic, and antibacterial activities. So far 26 ustilaginoidins have been isolated from V. virens, among which 18 compounds contained stereogenic center(s), however, most of them were unknown for the absolute configurations, except that of ustilaginoidin D. In this study, the absolute structures of these ustilaginoidins were constructed for the first time by analysis of the biosynthetic monomers obtained from a gene knockout mutant (ΔUV_2091) of V. virens. The gene UV_2091 was predicted to encode an enzyme that dimerized the monomeric naphtho-γ-pyrones in V. virens. Knockout of this gene led to the accumulation of three monomers, namely hemiustilaginoidin F (1), epihemiustilaginoidin D (2), and hemiustilaginoidin D (3), but the production of ustilaginoidins was completely blocked. The structures of the monomers were deduced by spectroscopic analysis, in combination with TDDFT ECD calculations for determining the absolute configurations. These compounds were tested for their phytotoxic, cytotoxic, antibacterial, and antifungal activities. Compounds 1 and 3 showed inhibition against the radicle and plumule elongation of rice and lettuce seeds at the tested concentrations. Compound 1 was active against the tested five human cancer cells, with half maximal inhibitory concentrations (IC50s) of 13.2~37.3 µM. Compounds 1~3 inhibited the growth of the tested pathogenic bacteria with minimum inhibitory concentrations of 8~32 µg/mL, while compound 3 exhibited antifungal activity against Magnaporthe oryzae (IC50, 5.21 µg/mL). A comparison of these data with those of the ustilaginoidins provided insights into the structure-bioactivity relationships.