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Eight new trichothecene derivatives, trichodermarins G-N (1-8), and two new cuparene derivatives, trichocuparins A (9) and B (10), as well as six known trichothecenes (11-16) were isolated from the fungal strain Trichoderma brevicompactum A-DL-9-2 obtained from the inner tissue of the marine red alga Chondria tenuissima. The structures and relative configurations of 1-10 were assigned by NMR and MS data, and the absolute configurations of 1, 2, and 9 were established by X-ray diffraction. Compound 8 features an aminosugar unit bond to the trichothecene framework for the first time, while 9 and 10 represent the first occurrence of cuparene sesquiterpenes in Trichoderma. All the isolates were assayed for growth inhibition of five phytopathogenic fungi (Botrytis cinerea, Cochliobolus miyabeanus, Fusarium oxysporum f. sp. cucumerium, Fusarium oxysporum f. sp. niveum, and Phomopsis asparagi) and four marine phytoplankton species (Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense). Several of them exhibited significant inhibitory activities against the fungi and phytoplankton tested of which trichodermin (12) showed the highest antifungal and antimicroalgal activities with MIC and IC50 values being 4.0 and 0.82 μg/mL, respectively.

A strain named as Pseudomonas aeruginosa 2016NX1, which could produce phenazine and cereusitin, was isolated from the root of Millettia specisoa. Phenazines were extracted, isolated and purified by chloroform, thin-layer chromatography, column chromatography and high-performance liquid chromatography. Then the purified materials were identified by analysis of nuclear magnetic resonance. The major yellow component is 1-hydroxyphenazine and the minor blue component is cereusitin A. The tests of antimicrobial activity of yellow component showed that the growth of several common plant pathogenic fungi and bacteria (such as Cochliobolus miyabeanus, Diaporthe citri, Salmonella sp., Klebsiella oxytoca) could be strongly inhibited. This study suggested that Pseudomonas aeruginosa strain 2016NX1 had a significant potential for biological control of phytopathogenic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, one bioactive substance from Pseudomonas aeruginosa 2016NX1 was identified and its antimicrobial activity was verified. This study demonstrated that one bioactive substance from P. aeruginosa can strongly inhibit the growth of plant pathogenic fungi and bacteria. This study suggested that P. aeruginosa strain 2016NX1 has a significant potential for biological control of phytopathogenic fungi.

Chelerythrine (CHE) possesses broad pharmacological activities. In this study, the extract of Chelidonium majus L. were characterized by high performance liquid chromatography (HPLC), infrared radiation (IR) spectroscopy and nuclear magnetic resonance (NMR). It was proved that the extract was CHE. The antifungal activity of CHE against five fungal pathogens of rice was researched in vitro, revealing that CHE inhibited Ustilaginoidea virens (U. virens) and Cochliobolus miyabeanus (C. miyabeanus) with 50% effective concentrations (EC50) of 6.53 × 10−3 mg/mL and 5.62 × 10−3 mg/mL, respectively. When the concentration of CHE was 7.5 × 10−3 mg/mL, the inhibition rate of U. virens reached 56.1%. Moreover, CHE (4 × 10−3 mg/mL) exhibited the greatest efficacy in inhibiting spore of U. virens growth with an inhibition rate as high as 86.7%. CHE displayed the best inhibitory activity against U. virens at the concentration of 7.5 × 10−3 mg/mL, compared with the other two isoquinoline alkaloids and commercial fungicide validamycin. After treating U. virens mycelia with CHE, twisted and atrophied mycelia were observed by optical microscopy. SEM results demonstrated narrow and locally fractured mycelium. TEM observations showed that the cell wall had become thin and broken, and most organelles were difficult to recognize. Furthermore, membrane of mycelia was destroyed and reactive oxygen species (ROS) of spores was accumulated, which induced apoptosis of pathogenic fungi. From these results, our understanding of the mechanisms of antifungal activity of CHE against U. virens was enriched and this research is relevant for developing novel pesticides.

Agriculture is a source of living in many areas and acts a backbone for the Indian economy. Agriculture crops are affected by fungus and bacteria to cause diseases in the plant. Brown spot is a serious hazard that takes place on leaves of the rice plant. The presence of fungus ‘Cochliobolus miyabeanus’ is the main factor for brown spot disease. The severity of the disease depends on growth of the fungus. The development of fungus depends on several factors such as temperature, humidity and rainfall. Whenever the disease occurs on a plant, level of the infection plays an importance in crop yield. Most of the farmer follow books or use their experience to detect the disease in rice plants which is a time-consuming process and requires lot of attention to produce more rice yield is a challenging task. Fuzzy logic is identified as powerful tool for disease detection. In this paper, fuzzy logic system is proposed to determine the level of presence of Cochliobolus miyabeanus and alert the farmer to take the primitive steps.

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (Magnaporthe oryzae resistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.

The novel trichothecene 12-deoxytrichodermin (3) was isolated from the fungus Trichoderma sp. 1212-03, and included with other known natural trichothecenes in a structure-activity relationship investigation against a human colon cancer cell line (COLO201) and filamentous fungus Cochliobolus miyabeanus. This revealed that the 12-epoxide functionality is critical for the cytotoxicity of simple trichothecenes trichodermin (4) and deoxynivalenol (2), while not critical for the cytotoxicity of roridin J (6) and epiisororidin E (8). In contrast, 12-epoxide is essential for the antifungal activity.

Waitea circinata (Warcup & Talbot) is an orchid antagonist mycorrhizal fungus with biocontrol potential against rice pathogens. This study aimed to optimize the extraction method, obtain a new extract and evaluate its efficiency against rice pathogens in vitro and in vivo, as well as to compare it with other extraction methods and W. circinata. The extracts were obtained and screened for in vitro growth inhibition against the pathogens Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae, using the following extracts: mycelial, crude, lyophilized and mycelial mass. An additional in vitro assay was performed with the principal rice pathogen (Magnaporthe oryzae), in order to evaluate the conidial germination and appressorium formation. Based on this evaluation, the lyophilized and mycelial mass extracts were tested in vivo against rice blast (M. oryzae) and compared to the W. circinata mycelial suspension, in different application forms (simultaneous and previous). The mycelial mass extract inhibited all the pathogens, and the crude and lyophilized extracts inhibited C. miyabeanus and M. albescens, respectively. The mycelial mass extract inhibited the M. oryzae conidial germination and appressorium formation by 80 %, and the simultaneous and previous applications suppressed the rice blast by 94 %. These results indicate that the new extract can be used to control rice pathogens. RESUMO Waitea circinata (Warcup & Talbot) é um fungo antagonista micorrízico de orquídea, com potencial de biocontrole contra patógenos de arroz. Objetivou-se otimizar o método de extração, obter um novo extrato e avaliar sua eficiência contra patógenos de arroz in vitro e in vivo, bem como compará-lo com outros métodos de extração e W. circinata. Os extratos foram obtidos e testados para inibição de crescimento in vitro contra os patógenos Cochliobolus miyabeanus, Monographella albescens e Sarocladium oryzae, utilizando-se os seguintes extratos: micelial, bruto, liofilizado e de massa micelial. Um ensaio adicional in vitro foi realizado com o principal patógeno do arroz (Magnaporthe oryzae), para avaliar a germinação de conídios e a formação de apressórios. Com base nessa avaliação, os extratos liofilizado e de massa micelial foram testados in vivo contra a brusone do arroz (M. oryzae) e comparados com a suspensão micelial de W. circinata, em diferentes formas de aplicação (simultânea e prévia). O extrato de massa micelial inibiu todos os patógenos, e os extratos bruto e liofilizado inibiram C. miyabeanus e M. albescens, respectivamente. O extrato de massa micelial inibiu a germinação de conídios e a formação de apressórios de M. oryzae em 80 %, e as aplicações simultâneas e prévias suprimiram a brusone em 94 %. Os resultados indicam que o novo extrato pode ser usado para controlar patógenos de arroz.

Pathogens utilize multiple types of effectors to modulate plant immunity. Although many apoplastic and cytoplasmic effectors have been reported, nuclear effectors have not been well characterized in fungal pathogens. Here, we characterize two nuclear effectors of the rice blast pathogen Magnaporthe oryzae. Both nuclear effectors are secreted via the biotrophic interfacial complex, translocated into the nuclei of initially penetrated and surrounding cells, and reprogram the expression of immunity-associated genes by binding on effector binding elements in rice. Their expression in transgenic rice causes ambivalent immunity: increased susceptibility to M. oryzae and Xanthomonas oryzae pv. oryzae, hemibiotrophic pathogens, but enhanced resistance to Cochliobolus miyabeanus, a necrotrophic pathogen. Our findings help remedy a significant knowledge deficiency in the mechanism of M. oryzae–rice interactions and underscore how effector-mediated manipulation of plant immunity by one pathogen may also affect the disease severity by other pathogens., Plant pathogens secrete various effectors to manipulate host immunity. Here, Kim et al. describe two Magnaporthe oryzae effectors that translocate into the nuclei of infected rice cells and reprogram expression of immunity-associated genes, increasing susceptibility to hemibiotrophic pathogens.

The panicle branch, which is the key node for transport of photosynthesis products from source to sink, is vulnerable to many diseases caused by fungal pathogens, such as Magnaporthe oryzae, Cochliobolus miyabeanus. Among these diseases, rice blast is the most important one which causes devastating losses in many regions. In 2019 and 2020, panicle branch rot of rice with a symptom which could be mistaken with rice blast was observed in a paddy field, where is not traditional epidemical region of rice blast, in Fuyang, Zhejiang province. In 2020, similar symptom was also observed in Hubei and Anhui Province. In a paddy field in Fuyang, the symptom appeared on more than 30% investigated panicles. Diseased panicle exhibited brown to black lesions on primary or secondary branches as well as pedicels, however the grain and the neck of spike could not be infected which is the most obvious difference with rice blast. Obviously, the disease can't destroy the entire function of branch and blank grain was rarely observed, so its damage is not comparable with neck blast. Normally, it caused incomplete grain filing commonly leading to 5% - 25% grain weight loss. During the booting stage of rice, local solar irradiation time and temperature were fewer and lower than common years which may be responsible for losses caused by this disease. After surface sterilized, lesion parts cut from infected branches from 25 panicle samples were cultured on 2% water agar at 28℃ for 24-28 h, and fungi were isolated and purified by mycelial tip transferring. Among 31 isolates, 26 showed similar cultural characters. The wool-like mycelia were luxuriant and grew rapidly on PDA spreading the whole 9 cm petri dish in less than a week at 28 ℃. The mycelia were white to ashen at beginning and gradually turned black from center of the plate after 5 days culture at 28 ℃. Hyphae were smooth, branched, septate, hyaline or pale brown. Conidia were single-celled, black, spherical to subspherical, and 10.2 to 14.6 × 12.2 to 15.7 μm (n=50) in dimension and born on tip of hyaline and ampulliform conidiophores. The fungus showed similar morphological characteristics with Nigrospora oryzae (1). ITS sequences of 6 representative strains of the fungus were amplified, sequenced with primer pair, ITS1/ITS4 (2), and submitted into GenBank with an accession number, MW228165. Phylogenetic analysis was conducted with sequences of reference strains (3). The result showed that the fungus obtained in this study was fallen into the same group with N.oryzae. In view of above both morphological and molecular analysis, the strains were finally identified as N. oryzae. Pathogenicity tests were conducted in triplicate with rice panicles in initial heading stage. Fifty panicles were wounded on branches with needles and inoculated by spreading the conidia suspension (10μl, 1 × 106 conidia ml-1) on the wounds. The panicles used as control were treated in same way with 10μl of sterile water. The inoculated and control plants were kept in dark, 25 ℃ and relative humidity of more than 85% for 24 h in culture chamber. Symptoms appeared on 44 of 50 inoculated panicles which were basically similar with those observed in paddy field, while negative controls remained symptomless. The fungi re-isolated from inoculated panicles were also confirmed as N. oryzae by both morphological and molecular analysis. To the best of our knowledge, this is the first report of N. oryzae causing panicle branch rot disease on Oryza sativa (rice). This disease not only cause yield losses and lower milling quality, but could also be mistaken as rice blast incurring unnecessary fungicides spray.

A series of cyclohumulanoids, i.e., tricocerapicanols A–C (1a–1c), tricoprotoilludenes A (2a) and B (3), tricosterpurol (4), and tricoilludins A–C (5–7) were isolated along with known violascensol (2b) and omphadiol (8) from the culture broth of Daedaleopsis tricolor, an inedible but not toxic mushroom. The structures were fully elucidated on the basis of NMR spectroscopic analysis, and the suggested relative structures were confirmed via density functional theory (DFT)-based chemical shift calculations involving a DP4 probability analysis. In the present study, the 1H chemical shifts were more informative than the 13C chemical shifts to distinguish the diastereomers at C-11. The absolute configurations of 1–5 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. For 6 and 7, the same chirality was assigned according to their biosynthetic similarities with the other compounds. The successful assignment of some Cotton effects was achieved by utilizing DFT calculations using simple model compounds. The plausible biosynthesis of 1–7 was also discussed on the basis of the structural commonality and general cyclohumulanoid biosynthesis. Compounds 2a and 5 were found to simultaneously induce hyphal swelling and branching at 5.0 μg/mL against a test fungus Cochliobolus miyabeanus.

Bipolaris oryzae causes rice brown spot (RBS) in common rice (Oryza sativa) inflicting substantial grain yield losses worldwide. Knowledge of the population structure, genetic diversity, and sexual recombination of the fungal pathogen can help to implement effective disease management strategies. In this study, B. oryzae isolates sampled from Iran, The Philippines, and Japan were analyzed with 12 simple-sequence repeat (SSR) markers newly developed from the genome sequence of the fungus. Among the 288 B. oryzae isolates genotyped, 278 unique haplotypes were identified. High genotype numbers (richness) with even distribution (evenness) were found within the collection sites. Both mating types, MAT1-1 and MAT1-2, were present in each collection area, and the sexual state was induced under controlled conditions with production of viable ascospores. However, the tests of linkage disequilibrium did allow rejection of the hypothesis of random mating. Discriminant analysis of principal components (DAPC) revealed that the B. oryzae collection formed three clusters each consisting of isolates from different collection sites. Analysis of molecular variance (AMOVA) showed that genetic variation among clusters was 18.7%, with the rest distributed within clusters (Rst = 0.187, P < 0.001). Statistically significant pairwise-genetic differentiation was found between the clusters. These results show that Asian B. oryzae isolates are genetically diverse, and overall distributed in three groups. Those findings will be helpful in managing the disease and guide the use of representative set of isolates needed for selection of RBS resistant rice varieties. This article is protected by copyright. All rights reserved.

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non-host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant's defence repertoire. We found that applied phytotoxin-containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.

Rice (Oryza sativa L.) is an important cereal crop in Venezuela and is planted on approximately 200,000 ha. Fungal diseases caused by the Helminthosporium complex of fungi are considered a major constraint to rice production. In 2005, a blotter method was used to identify fungi associated with rice seed. Seeds (150) of cv. INIA-017 were placed on a plate with moistened filter paper and incubated at 26 ± 2°C with a 12-h light/12-h dark cycle for 5 days until fungal sporulation. Single spores were transferred to 2% water agar (WA). Germinated spores were then transferred to potato dextrose agar (PDA), and resultant colonies were preserved until used. To induce sporulation, 4-mm-diameter discs were cut from 72-h-old cultures, transferred to WA, and incubated at 26 ± 2°C for 48 h (modified technique of Alcorn [1]). Discolored seed yielded isolates of Bipolaris oryzae (Berda de Haan) Shoemaker and Exserohilum rostratum (Drechs.) Leonard and Suggs (2). Pathogenicity tests were performed on 60-day-old rice plants of cv. Blue Bonnet 50. Four plants per pot were used for each isolate. Before inoculation, plants were placed in humidity chambers for 48 h. Spores were harvested from discs into a beaker containing 50 mL of a dilute gelatin solution (Gelatin Difco; 5 g/100 mL of sterile distilled water [SDW]). Spore suspensions were filtered through cheesecloth and adjusted to 7 × 104 spores per ml. The inoculated plants were placed in humidity chambers for 72 h. Control plants were sprayed with SDW. Inoculated plants were removed from humidity chambers and placed on a greenhouse bench. Plants inoculated with B. oryzae were symptomatic 5 days after inoculation; resultant lesions were red-brown with chlorotic borders. Lesions subsequently turned gray with reddish borders and were rhombic in shape. Plants inoculated with E. rotratum were symptomatic 7 days after inoculation with elliptical lesions that were orange-brown along venation. These lesions turned gray with reddish borders. Both fungi were reisolated from symptomatic leaf tissue. To our knowledge, this is the first report of E. rostratum from rice in Venezuela. References: (1) J. L. Alcorn. Mycotaxon. 17:1, 1983. (2) A. Sivanesam. Mycol. Pap. 158, 1987.

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10 billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.

Broad-spectrum disease resistance against two or more types of pathogen species is desirable for crop improvement. In rice, Xanthomonas oryzae pv. oryzae (Xoo), the causal bacteria of rice leaf blight, and Magnaporthe oryzae, the fungal pathogen causing rice blast, are two of the most devastating pathogens. We identified the rice BROAD-SPECTRUM RESISTANCE 1 (BSR1) gene for a BIK1-like receptor-like cytoplasmic kinase using the FOX hunting system, and demonstrated that BSR1-overexpressing (OX) rice showed strong resistance to the bacterial pathogen, Xoo and the fungal pathogen, M. oryzae. Here, we report that BSR1-OX rice showed extended resistance against two other different races of Xoo, and to at least one other race of M. oryzae. In addition, the rice showed resistance to another bacterial species, Burkholderia glumae, which causes bacterial seedling rot and bacterial grain rot, and to Cochliobolus miyabeanus, another fungal species causing brown spot. Furthermore, BSR1-OX rice showed slight resistance to rice stripe disease, a major viral disease caused by rice stripe virus. Thus, we demonstrated that BSR1-OX rice shows remarkable broad-spectrum resistance to at least two major bacterial species and two major fungal species, and slight resistance to one viral pathogen.

Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.

Rice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanus and Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanus and X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice. Rice plant accumulates phenylamides in response to infection, wounding and treatments with jasmonic acid, salycilic acid, cytokinin, and ethylene.

Cochliobolus miyabeanus is known as a significant causal agent in relation to brown spot disease of rice and causes significant yield losses. In the present study, the complete mitochondrial genome was determined using next-generation sequencing technology. This complete mitogenome is a circular molecule of 124,887 bp in length. It contains 13 conserved protein-coding genes, 21 transfer RNA genes, 2 ribosomal RNA genes and 9 open reading frames. The overall base composition of C. miyabeanus is 35.4% A, 34.4% T, 14.4% C, 15.8% G, with a CG content of 30.2%. Phylogenetic analysis based on concatenated protein genes from 15 taxa within Pezizomycotina showed that C. miyabeanus is closely related to Bipolaris cookei in the family Pleosporaceae (Dothideomycetes, Pleosporales). This work would facilitate the understanding of systematics and evolutionary biology of phytopathogenic fungi.

epi -Cochlioquinone D ( 1 ) and its 12-α-hydroxyderivative ( 2 ) were isolated from Helminthosporium velutinum TS28 based on antifungal screening against Cochliobolus miyabeanus . Relative configurations were established by the NMR analyses except for C5 locating far from the other asymmetric centers. ECD discussions involving spectral comparison with those of other related molecules as well as those based on theoretical calculations disclosed not only the C5 configuration but also the chirality for C12–C22 polycyclic moiety. H. velutinum TS28 also afforded precochlioquinol D ( 3 ), just the side chain component, which led us to discuss their biosynthetic pathway. Although 1 and 2 inhibit C. miyabeanus effectively, the C-14 epimeric cochlioquinones scarcely inhibited in spite of very close structures. Present study further revealed that C. miyabeanus tightly recognizes both the polycyclic moiety and the side chain. In contrast, both cochlioquinones and epi -cochlioquinones exhibited potent cytotoxicity against human colon adenocarcinoma (COLO 201) cells regardless of the C14 configuration as well as type of side chains.

Summary Over recent decades, a multitude of studies have shown the ability of silicon (Si) to protect various plants against a range of microbial pathogens exhibiting different lifestyles and infection strategies. Despite this relative wealth of knowledge, an understanding of the action mechanism of Si is still in its infancy, which hinders its widespread application for agricultural purposes. In an attempt to further elucidate the molecular underpinnings of Si-induced disease resistance, we studied the transcriptome of control and Si-treated rice plants infected with the necrotrophic brown spot fungus Cochliobolus miyabeanus. Analysis of brown spot-infected control plants suggested that C. miyabeanus represses plant photosynthetic processes and nitrate reduction in order to trigger premature senescence and cause disease. In Si-treated plants, however, these pathogen-induced metabolic alterations are strongly impaired, suggesting that Si alleviates stress imposed by the pathogen. Interestingly, Si also significantly increased photorespiration rates in brown spot-infected plants. Although photorespiration is often considered as a wasteful process, recent studies have indicated that this metabolic bypass also enhances resistance during abiotic stress and pathogen attack by protecting the plant's photosynthetic machinery. In view of these findings, our results favour a scenario in which Si enhances brown spot resistance by counteracting C. miyabeanus-induced senescence and cell death via increased photorespiration. Moreover, our results shed light onto the mechanistic basis of Si-induced disease control and support the view that, in addition to activating plant immune responses, Si can also reduce disease severity by interfering with pathogen virulence strategies.

The Pseudomonas- derived cyclic lipopeptide orfamide can induce resistance to Cochliobolus miyabeanus but not to Magnaporthe oryzae in rice. Abscisic acid signaling is involved in the induced systemic resistance response triggered by orfamide. Diverse natural products produced by beneficial Pseudomonas species have the potential to trigger induced systemic resistance (ISR) in plants, and thus may contribute to control of diseases in crops. Some beneficial Pseudomonas spp. can produce cyclic lipopeptides (CLPs), amphiphilic molecules composed of a fatty acid tail linked to an oligopeptide which is cyclized. CLPs can have versatile biological functions, but the capacity of Pseudomonas-derived CLPs in triggering ISR responses has barely been studied. Pseudomonas protegens and related species can produce orfamide-type CLPs. Here we show that in rice, orfamides can act as ISR elicitors against the necrotrophic fungus Cochliobolus miyabeanus, the causal agent of brown spot disease, but are not active against the blast fungus Magnaporthe oryzae. Orfamide A can trigger early defensive events and activate transcripts of defense-related genes in rice cell suspension cultures, but does not cause cell death. Further testing in rice cell suspension cultures and rice plants showed that abscisic acid signaling, the transcriptional activator OsWRKY4 and pathogenesis-related protein PR1b are triggered by orfamide A and may play a role in the ISR response against C. miyabeanus.

The integrated management of rice leaf blast (Magnaporthe oryzae) is carried out mainly with the adoption of chemical control. However, the search for alternative practices has grown in recent years. Thus, the variability of 28 Sarocladium oryzae isolates was evaluated for the cerulenin production, as well as its potential for reducing the severity of rice leaf blast, quantifying the activity of enzymes linked to the plant defense mechanisms. More than 55 % of the S. oryzae isolates were antagonistic to the pathogens M. oryzae, Cochliobolus miyabeanus, Thanatephorus cucumeris and Monographella albescens, and 60 % of the isolates produced cerulenin at detectable levels. Both BRM 6461 (296.0 µg mL-1) and BRM 6493 (undetectable cerulenin) inhibited the formation of M. oryzae appressoria in 89.5 % and 85 %, respectively. The BRM 6461 isolate, applied as conidial suspension and filtered, reduced the severity of rice leaf blast in 68.8 % and 75.5 %, respectively. The enzymatic activity in the presence of M. oryzae was higher for lipoxygenase at 5 h (filtered) and at 24 h and 72 h (conidial suspension) after the pathogen inoculation. For phenylalanine ammonia lyase, the highest expression was at 5 h (filtered) and 72 h (conidial suspension). The enzymes chitinase, β-1,3-glucanase and peroxidase and the salicylic acid phytohormone presented no differences, in relation to the controls (water and M. oryzae). The filtered from the BRM 6461 isolate, basically constituted by cerulenin, reduced the severity of rice leaf blast and possibly activated the defense mechanisms of the rice plants against M. oryzae. RESUMO O manejo integrado de brusone (Magnaporthe oryzae) em arroz é realizado, principalmente, com a adoção de controle químico. No entanto, a busca por práticas alternativas tem crescido nos últimos anos. Assim, avaliou-se a variabilidade de 28 isolados de Sarocladium oryzae, quanto à produção de cerulenina, e o seu potencial na redução da severidade de brusone foliar em arroz, quantificando-se a atividade de enzimas ligadas ao mecanismo de defesa das plantas. Mais de 55 % dos isolados de S. oryzae foram antagônicos aos patógenos M. oryzae, Cochliobolus miyabeanus, Thanatephorus cucumeris e Monographella albescens, e 60 % dos isolados produziram cerulenina em níveis detectáveis. Tanto BRM 6461 (296,0 µg mL-1) quanto BRM 6493 (cerulenina não detectável) inibiram a formação de apressórios de M. oryzae em 89,5 % e 85 %, respectivamente. O isolado BRM 6461, aplicado na forma de suspensão de conídios e filtrado, reduziu a severidade da brusone em 68,8 % e 75,5 %, respectivamente. A atividade enzimática, na presença de M. oryzae, foi maior para lipoxigenase após 5 h (filtrado) e 24 h e 72 h (suspensão de conídios) da inoculação do patógeno. Para fenilalanina-amônia liase, a maior expressão ocorreu após 5 h (filtrado) e 72 h (suspensão de conídios). As enzimas quitinase, β-1,3-glucanase e peroxidase e o fitohormônio ácido salicílico não apresentaram diferenças em relação aos controles (água e M. oryzae). O filtrado do isolado BRM 6461, constituído basicamente por cerulenina, reduziu a severidade da brusone e, possivelmente, ativou os mecanismos de defesa da planta de arroz contra M. oryzae.

Rice cultivation in Europe is restricted to a few southern European countries. In 2015, the rice-growing acreage of Italy and Spain together comprised around 75 % of a total area of around half a million hectares. The milled rice equivalents in the EU amounted to 0.4 % of the global rice production. Japonica rice varieties are dominating in Europe. Rice is planted in spring and harvested in autumn. All rice fields in Europe are irrigated. Most rice seed is drilled. In some Spanish areas, pregerminated rice is sown by air. Average yields per hectare range between 4 and 8 tons. In some regions 10 tons can easily be achieved. Monocot weeds are prevailing with wild rice, Echinochloa, Cyperus and Heteranthera species being the most frequent representatives. Hydrellia griseola, Chilo suppressalis, Eysarcoris inconspicuus and Lissorhoptrus oryzophilus have to be regarded as the most serious insect problems. Magnaporthe grisea, Cochliobolus miyabeanus and Gibberella fujikuroi are the most widespread disease-causing organisms. A wide range of chemical and biological products is registered for rice protection. For some countries, however, costs for the registration of new products are too high compared with the low acreage so that lacking product innovation becomes a major problem. The acreage of Clearfield rice is continuously growing primarily due to wild rice as an increasing problem. Water shortage is a problem in a few areas in Spain primarily. Irrigated rice provides a habitat for a great number of organisms such as migratory birds and deserves special attention as far as biodiversity is concerned. Greenhouse gas emission and heavy metal concentration in rice fields have been a problem in some areas in the past. New cultivation methods with a reduced water consumption and new rice varieties should, however, contribute to the reduction of these problems.

Neomacrophorins I (1), II (2), and III (3) were isolated from the culture broth of Trichoderma sp. 1212-03, which was collected at Shirakami Mountainous area in Japan. Structural analyses disclosed that these resemble known macrophorins but possess axial-hydroxy group at C3 as well as different side chains at C7′. These are diastereomeric forms of macrophorins for 5′,6′-epoxide functionality. The NMR analyses suggested their relative configurational relationship between the C1–C15 drimene and C1′–C7′ epoxyquinone moieties. ECD spectral discussions verified them particularly for C5′,C6′-epoxyquinone (1), C5′,C6′-epoxysemiquinone (2 and 3), and 2″,3″-dihydroxybutanoate moiety in 1 and 2. The configuration of C3″-stereocenter of 3 was determined by chiral GC–MS after converting into methyl (S)-3″-hydroxybutanoate by basic of 3 methanolysis. Biological assays disclosed that 1 induces hyphal branching of Cochliobolus miyabeanus as well as cytotoxicity against human colorectal cancer COLO 201.

The effects of essential oils (EO), cold water (CWE), hot water (HWE) and ethanol (ETHE) extracts of Callistemon citrinus L. and Cymbopogon citratus (DC) Stapf on the radial growth of Alternaria padwickii (Ganguly) M.B. Ellis and Bipolaris oryzae (Breda de Haan) Shoemaker, the control of brown spot disease, the tillering, the number of panicles and the yield increase in rice were evaluated under laboratory and field conditions. In vitro, the growth of both fungi was completely inhibited by the EO of C. citrinus and C. citratus at 4,520 μg/ml and 452 μg/ml, respectively. For solvent extracts, the ETHE of C. citrinus was the most active and inhibited 80–85 % of the fungal growth followed by the CWE of C. citratus with 77 % and 36 % diameter reduction against B. oryzae and A. padwickii, respectively at 10 000 μg/ml. Under laboratory conditions, seed treatment with the EO of C. citrinus reduced the incidence of B. oryzae in seeds by 85–100 % compared to the non-treated controls. Similarly, the seed treatment increased the germination of an irrigated rice cultivar by 10.6 %, whereas the percentage of germinated seeds of upland rice was not significantly affected. The highest germination (85–94 %) was found in the non-treated and treated samples with a low incidence (0–4 %) of B. oryzae. Under field conditions, the combined use of the essential oil of C. citrinus as a seed treatment and spraying the plants with 2 % ethanol followed by 2 % (w/v) aqueous extracts of C. citrinus or C. citratus increased the emergence, tillering, panicles/plant and the grain yield by 25–55 % of the irrigated rice. In addition, the brown spot severity was reduced by 36–42 %. For the upland rice, the treatments led to similar results with the grain yield increase of 54–137 % and 20–80 % reduction in the brown spot severity. From our results, we concluded that the EO and solvent extracts of C. citrinus and C. citratus have potential as control agents against brown spot and other seed-borne fungal diseases in rice under both conventional and organic farming.

The genotypic diversity of a collection of 352 isolates of Bipolaris oryzae obtained from 11 locations in the Philippines was estimated. The isolates could be divided into 50 haplotypes based on variation in microsatellite DNA with a moderately high genotypic diversity value of 0.88. Thirty nine haplotypes were represented by three or fewer isolates, whereas 80 % of the isolates belonged to only eight haplotypes, each containing 10 to 88 isolates indicating the prevalence of clonality. AMOVA revealed that the greatest variation was associated with the brown spot isolates collected within provinces (50.81 %), among varieties within provinces (48.17 %) and within ecosystems (49.33 %). Intensive sampling from a single field showed that the population was mostly clonal with about 98 % of the isolates belonging to a single VNTR haplotype. However, isolates within this haplotype exhibited a continuous range of aggressiveness when inoculated onto susceptible rice variety IR72. Several types of lesions were observed in the field during sampling, but the isolates obtained from each type of lesion produced a range of different lesion types when inoculated onto leaves of IR72, indicating that the type of lesion observed in the field was not related to the genotype of the pathogen. These results show that rice fields across the Philippines may contain B. oryzae isolates with considerable genotypic diversity, but an individual field may have both clonal and unique genotypes.