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Maydis leaf blight (MLB) caused by Helminthosporium maydis Y. Nisik. & C. Miyake is prevalent in almost all maize growing areas and is a major limiting factor in increasing yield. Knowing about the metabolic pathways that results in accumulation of enzymatic and non-enzymatic antioxidative biochemical compounds which constitutes a second line of defense, an in vitro experiment was conducted. Reactive oxygen species produced during stresses is one of the most instant defense reactions to arrest plant pathogen at the site of infection. Oxidative burst results in the production of ROS, mainly H2O2 and superoxide at the invading site. Production of secondary metabolites and induction of ISR and SAR mediate biochemical defenses in plant. In vitro study to observe the effect of different elicitors applied exogenously against H. maydis by investigating the biochemical parameters viz., Total phenols, Proline, Hydrogen peroxide (H2O2) and, enzymatic anti-oxidative biochemicals viz., Superoxide dismutase (SOD), Polyphenol oxidase (PPO), Peroxidase (POD) and, Phenylalanine ammoniase (PAL) activity is conducted. The activity of different biochemical parameters was observed on the 0th day (before inoculation), 3rd day, 6th day and 9th day of inoculation with the help of spectrophotometer. Accumulation of H2O2 concentration was recorded maximum (815.61 µmol g−1FW) at the 9th day after inoculation in 2,6-dichloroisonicotinic acid (INA)-treated plants. INA-treated plants showed highest proline accumulation (43.39 µmol g−1FW) and phenolics content (560.92 µg g−1FW) at the 6th and 3rd day after inoculation of the pathogen, respectively. In biochemical studies for elucidation of defence induction, biochemical markers reached maximum on the 3rd day after pathogen inoculation, and INA treatments were found most effective. [ABSTRACT FROM AUTHOR]

Biocontrol agents are organisms effective against plant pathogens. In present study, random mutations were made to Bacillus velezensis (accession number KY214239) by UV irradiation and ethyl methane sulfonate (EMS) to improvethe biocontrol capacity of B. velezensis strains. Mutant strain of B. velezensis (M3) showed increased biosurfactant production. The highest crude biosurfactant production were observed for the M3 mutant followed by the M5 mutant. Mutant M3 showed the highest antagonistic activity against Helminthosporium maydis with a significant decrease in the leaf blight severity index compared with the wild-type strain. Thus, the mutated strains of B. velezensis were significantly improved with respect to their bio-control capacity. This could increase the success of B. velezensis as biocontrol agent to control southern corn leaf blight of maize. Pot experiments showed significant improvement in the growth promotion of maize and inhibition of H. maydis. Maize shoot length (107.11 cm), root length (37.6 cm), shoot fresh weight (15.8 g), and root fresh weight (4.4 g) was recorded after 45 days of sowing and shows promise for the use of B. velezensis in disease suppression in maize. [ABSTRACT FROM AUTHOR]

Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases dephosphorylate key signaling proteins. Fungal mitogen activated protein kinases (MAPK), in contrast to their mammalian counterparts, often show detectable basal phosphorylation levels. Dephosphorylation, therefore, could act as a signal. In Cochliobolus heterostrophus, the Dothideomycete causing Southern corn leaf blight, ferulic acid (FA)—an abundant phenolic found in plant host cell walls—acts as a signal to rapidly dephosphorylate the stress-activated MAP kinase Hog1 (High Osmolarity Glycerol 1). In order to identify the protein phosphatases responsible, we constructed mutants in Hog1 phosphatases predicted from the genome by homology to yeast and other species. We found that Cochliobolus heterostrophus mutants lacking PtcB, a member of the PP2C family, exhibited altered growth, sporulation, and attenuated dephosphorylation in response to FA. The loss of the dual-specificity phosphatase CDC14 led to slow growth, decreased virulence, and attenuated dephosphorylation. Mutants in two predicted tyrosine phosphatase genes PTP1 and PTP2 showed normal development and virulence. Our results suggest that a network of phosphatases modulate Hog1’s dual phosphorylation levels. The mutants we constructed in this work provide a starting point to further unravel the signaling hierarchy by which exposure to FA leads to stress responses in the pathogen. [ABSTRACT FROM AUTHOR]

A rapid and sensitive polymerase chain reaction (PCR) method was established for detection of mating types of Cochliobolus heterostrophus, the causal agent of southern corn leaf blight (SCLB). Two pairs of mating type-specific primers (ChMAT01-1 for MAT1-1 and ChMAT02-1 for MAT1-2) were designed and tested on DNA extracted from pure cultures, conidial suspensions or naturally infected corn leaf samples with C. heterostrophus. The PCR-based method exhibited specificity in differentiating mating types of C. heterostrophus from the closely related species C. sativus, C. carbonum, C. lunata, C. eragrostidis and Cochliobolus sp., as well as 13 other fungal genera. Specifically, the method was capable of reliably detecting mating types of C. heterostrophus at 0.0001 ng DNA for MAT1-1 or 0.01 ng DNA for MAT1-2 from pure cultures or from 10 conidia or one lesion of C. heterostrophus on inoculated or infected corn leaf samples, even in the presence of as high as 20 ng corn genomic DNA. This PCR assay also successfully detected mating types of C. heterostrophus in naturally infected samples. [ABSTRACT FROM AUTHOR]

Cochliobolus heterostrophus is the causal pathogen of the southern corn leaf blight ( SCLB). There are three known races: race O, race C and race T. To determine which C. heterostrophus races comprise the field population in southern China and to assess diversity of these strains in terms of virulence, 200 isolates from diseased plants were collected in nine provinces/municipalities. All were race O, that is, no race T or race C isolates were found. Sixty race O isolates that sporulated well were chosen for further analysis. Virulence was measured using the integral optical density ( IOD) of leaf lesions on four maize inbred lines. UPGMA cluster analysis of AFLP markers was applied to the 60 race O isolates plus control race O, T and C strains. Phylogenetic distribution, geographic location and virulence were not correlated. These results can provide valuable information for guidance in early warning and disease control. [ABSTRACT FROM AUTHOR]

Southern leaf blight ([SLB], causal agent Cochliobolus heterostrophus) is an important fungal disease of maize (Zea mays L.). Teosinte (Z. mays ssp. parviglumis), the wild progenitor of maize, offers a novel source of resistance alleles that may have been lost during domestication. The aims of this study were to identify teosinte alleles that, when present in a temperate maize background, confer a significant level of resistance to SLB. Ten populations of BC4S2 near isogenic lines (NILs), developed by crossing 10 different teosinte accessions to the maize inbred B73, comprising 774 lines in total, were screened for SLB resistance. Quantitative trait locus (QTL) analysis identified four significant QTL associated with SLB in bins 2.04, 3.04, 3.05, and 8.05. Sixteen individual NILs which were significantly different to the susceptible recurrent parent, B73 and which were carrying at least one of the teosinte-derived resistance alleles were used to develop F2:3 populations by crossing each to B73 followed by two rounds of self-pollination. These F2:3 populations were evaluated for SLB resistance and genotyped at the loci of interest. In 13 of 19 cases single marker analysis validated allelic substitution effects predicted from the original NIL population analysis, while in five cases we were not able to validate the effects and in one case a significant effect was detected in the opposite to the predicted direction. An allele at the QTL in bin 2.04 was shown to confer resistance to both SLB and a second maize foliar disease, gray leaf spot (GLS). [ABSTRACT FROM AUTHOR]

Bipolaris maydis (anamorph: Cochliobolus heterostrophus ) is the causal agent of Southern Corn Leaf Blight (SCLB), leading to huge annually losses worldwide. Although fludioxonil, a phenylpyrrole fungicide with a broad spectrum of activity, was introduced in the 1990s, no baseline sensitivity has been established for B. maydis. One hundred field isolates were used to establish a baseline sensitivity of B. maydis against fludioxonil during 2015–2016. The results showed that the baseline sensitivity was distributed as a unimodal curve with a mean EC 50 value of 0.044 ± 0.022 μg mL − 1 . With repeated exposure to fludioxonil, a total of five fludioxonil-resistant mutants (RF > 100, RF = Resistance factor) were obtained in the laboratory. Compared with the parental isolates, the five fludioxonil-resistant mutants showed decreased fitness in sporulation and virulence, and exhibited different features of sensitivity to various stresses (oxidation and osmotic pressure, cell membrane and cell wall inhibitors), but not in mycelial growth on PDA without stress amendation. The five fludioxonil-resistant mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide procymidone, but not between fludioxonil and boscalid or fluazinam. All mutants exhibited stable resistance to fludioxonil after 10 transfers, as indicated by resistance factor values that ranged from 116.82 to 445.59. When treated with 1.0 M NaCl, all the fludioxonil-resistant mutants showed greater mycelial glycerol content than corresponding parental isolates. Sequencing alignment results of Bmos1 indicated that mutant R27-5 had a single point mutation (Z1125K), while the mutant R104 had a 34-bp deletion fragment between the codons of amino acid residues 1125 to 1236 and encodes a putative attenuated 1133-AA protein. The 34-bp deletion fragment led to not only a 11-AA deletion(DNAVNQKLAVR), but also the resulting frameshift mutation and early stop. The mutations of R27-5 and R104 were located in the Rec domain of the Bmos1 gene. No mutations at the Bmos1 were detected in the other three resistant mutants R27-1, R27-2 and R32. [ABSTRACT FROM AUTHOR]

The maize rhm1 mutant resists Bipolaris maydis, the causal agent of Southern corn leaf blight, by producing small necrotic lesions surrounded by chlorotic haloes. The rhm1 and wild-type lesions contain viable fungus in equal frequency, but fungal sporulation was markedly inhibited on rhm1. The levels of the pathogenesis-related (PR) proteins chitinase, PR1, and peroxidase differ little between rhm1 and wild type, with or without B. maydis inoculation. The global mRNA profiles surveyed revealed hundreds of cDNA fragments that were twofold or more induced or suppressed in rhm1 and wild-type plants following B. maydis inoculation. Nonetheless, between rhm1 and wild type, only 0.4 to 0.7% of the cDNA fragments were expressed differentially by twofold or more. Among the up-regulated genes in rhm1 was beta-glucosidase glu1, which prompted a test of whether rhm1 resistance depends upon the antimicrobial compound 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one or other hydroxamic acids whose glucosyl conjugates are preferred substrates for the Glu1 enzyme. Double mutants of rhm1 and bx1, a hydroxamic acid-deficient mutant, indicate that rhm1 resistance is hydroxamic acid independent. The rhm1 resistance presently appears to operate via a mechanism unlike those of previously described resistance genes.

Nonhost resistance (NHR) pertains to the most common form of plant resistance against pathogenic microorganisms of other species. Bipolaris maydis is a non-adapted pathogen affecting soybeans, particularly of maize/soybean intercropping systems. However, no experimental evidence has described the immune response of soybeans against B. maydis. To elucidate the molecular mechanism underlying NHR in soybeans, proteomics analysis based on two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to identify proteins involved in the soybean response to B. maydis. The spread of B. maydis spores across soybean leaves induced NHR throughout the plant, which mobilized almost all organelles and various metabolic processes in response to B. maydis. Some enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), mitochondrial processing peptidase (MPP), oxygen evolving enhancer (OEE), and nucleoside diphosphate kinase (NDKs), were found to be related to NHR in soybeans. These enzymes have been identified in previous studies, and STRING analysis showed that most of the protein functions related to major metabolic processes were induced as a response to B. maydis, which suggested an array of complex interactions between soybeans and B. maydis. These findings suggest a systematic NHR against non-adapted pathogens in soybeans. This response was characterized by an overlap between metabolic processes and response to stimulus. Several metabolic processes provide the soybean with innate immunity to the non-adapted pathogen, B. maydis. This research investigation on NHR in soybeans may foster a better understanding of plant innate immunity, as well as the interactions between plant and non-adapted pathogens in intercropping systems. [ABSTRACT FROM AUTHOR]

ToxA, the first discovered fungal proteinaceous host-selective toxin (HST), was originally identified in 1989 from the tan spot fungus Pyrenophora tritici - repentis ( Ptr ). About 25 years later, a homolog was identified in the leaf/glume blotch fungus Stagonospora nodorum ( Parastagonospora nodorum ), also a pathogen of wheat. Here we report the identification and function of a ToxA-like protein from the maize pathogen Cochliobolus heterostrophus ( Ch ) that possesses necrosis-inducing activity specifically against maize. Ch ToxA is encoded by a 535-bp open reading frame featuring a ToxA -specific intron with unusual splicing sites (5′-ATAAGT…TAC-3′) at conserved positions relative to PtrToxA . The protein shows 64% similarity to Ptr ToxA and is predicted to adopt a similar three-dimensional structure, although lacking the arginyl-glycyl-aspartic acid (RGD) motif reported to be required for internalization into sensitive wheat mesophyll cells. Reverse-transcriptase PCR revealed that the ChTOX A gene expression is up-regulated in planta , relative to axenic culture. Plant assays indicated that the recombinant Ch ToxA protein induces light-dependent leaf necrosis in a host-selective manner on maize inbred lines. Gene deletion experiments confirmed that ChtoxA mutants are reduced in virulence on specific Ch ToxA-sensitive maize lines, relative to virulence caused by wild-type strains. Database searches identified potential Ch ToxA homologues in other plant-pathogenic ascomycetes. Sequence and phylogenetic analyses revealed that the corresponding ToxA-like proteins include one member recently shown to be associated with formation of penetration hypha. These results provide the first evidence that C . heterostrophus is capable of producing proteinaceous HSTs as virulence factors in addition to well-known secondary metabolite-type toxins produced biosynthetically by polyketide synthase megaenzymes. Further studies on Ch ToxA may provide new insights into effector evolution in host–pathogen interactions. [ABSTRACT FROM AUTHOR]

The heterotrimeric G proteins and mitogen-activated protein kinases (MAPKs) conserved signaling pathways are involved in the development, reproduction and pathogenicity in filamentous fungi. The two-component histidine kinase, known also as the HOG MAPK pathway, regulates a similar complex set of responses and is known to mediate the phenylpyrrole fludioxonil fungicide response in fungi. We used Cochliobolus heterostrophus mutant strains deficient in G-protein α ( cga1) and/or β ( cgb1) subunits or MAPK ( chk1, mps1 and hog1) to uncover their role in the mediation of this fungicide's activity and resistance. The results revealed complex interactions between the G-protein subunits and the MAPK in response to osmotic/ionic and fludioxonil stresses. Under normal conditions, the Hog1 pathway restricts glycerol accumulation since its disruption leads to hyperosmosensitivity and very high cellular glycerol accumulation. The hog1 mutants were also relatively resistance to fludioxonil. Moreover, our results suggest that cgb1, chk1 and mps1 are also weak repressors of this response since mutation in these genes caused relatively high elevation in glycerol levels in the cells. Supporting this is the finding that these three strains exhibit resistance to KCl stress. In contrast, the cga1 strain has only moderate levels of cellular glycerol (higher than those of the wild type, but lower than those of the other mutants) that are little affected by KCl or fludioxonil stress. Indeed, these mutants are highly sensitive to KCl stress. This suggests that Cga1 is a moderate repressor of cellular glycerol under normal conditions and an enhancer of glycerol accumulation under osmotic/ionic stress conditions. Together these findings reveals that the sensitivity to fludioxonil is not only positively controlled by the Hog1 pathway, but also mediated by the Chk1, Mps1, Cga1 and Cgb1 pathways. This study provides insight into the roles of G-protein in mediating the anti-fungal fludioxonil response. A model is proposed for the interactions between the G-protein and MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

The tubers of Bletilla formosana were fermented with eight plant pathogen fungi, respectively, and antioxidant activities and total phenolic content (TPC) of the crude extracts of fermented products and non-fermented products were investigated. The antioxidant activities were evaluated in three different test systems [DPPH, ABTS radical-scavenging activity, and ferric reducing-antioxidant power (FRAP)]. It was found that the extract of Helminthosporium maydis fermented B. formosana (FBF) possessed the highest TPC and exhibited a significant antioxidant activity compared with non-fermented product and other fermented products. Correlation analysis between antioxidant activities and TPC was also investigated. The good correlation between antioxidant activities and TPC revealed that the phenolic compounds might be the major contributors for the high antioxidant activities of the fermented B. formosana . Two phenolic compounds, curvularin and dehydrocurvularin, were isolated from H. maydis FBF, which had never been reported from plant of orchidaceae or H. maydis . Curvularin exhibited significant antioxidant activities, and was also present at a high concentration (0.373 mg/mg extract sample), implying an important role for the antioxidant activity of H. maydis FBF. This study suggested that proper fermentation processing could improve TPC and antioxidant activities of B. formosana . [ABSTRACT FROM AUTHOR]

Natural products have been in focus as alternative, effective and safe materials against the phytopathogens. Investigations show Nepeta oils as effective in controlling the food crops decay. The inhibitory effects of essential oils derived from Nepeta leucophylla , Nepeta ciliaris , Nepeta clarkei and Calamintha umbrosa against five phytopthogenic fungi have been determined. In vitro antifungal activity varied with their constituents and target species. More active being the oils containing oxygenated terpenoids. Helminthosporium maydis was sensitive to the all oils, IC 50 values have 43.6–109.3 μg mL −1 . The N. leucophylla oil possessing oxygenated iridoids was more effective against H. maydis (IC 50 value of 43.6 μg mL −1 ) while N. ciliaris was more active against Fusarium oxysporum (IC 50 value of 219.2 μg mL −1 ). The oils were effective against the spore germination of all the tested plant pathogens. [ABSTRACT FROM AUTHOR]

Cochliobolus heterostrophus Vel2 and Vos1, members of the velvet family of proteins, play crucial roles in sexual and asexual development as reflected by deletion mutant and overexpression strain phenotypes. vel2 and vos1vel2 mutants are female sterile. Pseudothecia from vel2 or vos1 mutant crosses to an albino wild-type tester strain produce asci, however no full tetrads are found in these crosses, in contrast to crosses between wild-type strains which typically yield asci with a full complement of ascospores. In addition, none of the progeny from crosses of vel2 or vos1 mutants to wild-type mating testers is mutant, thus vos1 and vel2 ascospores are unable to survive meiosis. vos1vel2 double mutants are also female sterile like vel2 single mutants, however, asci in pseudothecia formed in crosses to wild-type testers are devoid of ascospores. Vel2 and Vos1 negatively regulate production of asexual spores, but positively regulate their morphology. vel2 and vos1 single mutant conidia vary in size, in septum number, septum position in the spore, and in germination rate, and are more sensitive to oxidative and thermal stresses compared to wild-type conidia. Trehalose amounts are decreased in single mutants, supporting previous findings that this disaccharide is required for conidium health. [ABSTRACT FROM AUTHOR]

Plant leaves are inhabited by a diverse group of microorganisms that are important contributors to optimal growth. Biotic and abiotic effects on plant growth are usually studied in controlled settings examining response to variation in single factors and in field settings with large numbers of variables. Multi-factor experiments with combinations of stresses bridge this gap, increasing our understanding of the genotype-environment-phenotype functional map for the host plant and the affiliated epiphytic community. The maize inbred B73 was exposed to single and combination abiotic and the biotic stress treatments: low nitrogen fertilizer and high levels of infection with southern leaf blight (causal agent Cochliobolus heterostrophus). Microbial epiphyte samples were collected at the vegetative early-season phase and species composition was determined using 16S ribosomal intergenic spacer analysis. Plant traits and level of southern leaf blight disease were measured late-season. Bacterial diversity was different among stress treatment groups (P < 0.001). Lower species richness-alpha diversity-was correlated with increased severity of southern leaf blight disease when disease pressure was high. Nitrogen fertilization intensified the decline in bacterial alpha diversity. While no single bacterial ribotype was consistently associated with disease severity, small sets of ribotypes were good predictors of disease levels. Difference in leaf bacterial-epiphyte diversity early in the season were correlated with plant disease severity, supporting further tests of microbial epiphyte-disease correlations for use in predicting disease progression. [ABSTRACT FROM AUTHOR]

Background Cochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ∼ 1.2-Mb of DNA encoding genes responsible for the production of Ttoxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species. Results A detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions. Conclusions New families of transposons were identified. In C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus. [ABSTRACT FROM AUTHOR]

Abstract: Three Himalayan Erigeron (Asteraceae) species viz Erigeron mucronatus, Erigeron annuus and Erigeron karwinskianus growing in sub-alpine region revealed occurrence of isomeric polyacetylenic constituents viz., matricaria and lachnophyllum esters which accounted for 83.3%, 69.3% and 30.1% of the essential oils from these species, respectively, in addition to mono- and sesquiterpenoids as minor constituents. The antifungal activity tested by poisoned food (PF) techniques against Fusarium oxysporum, Helminthosporium maydis, Rhizoctonia solani, Alternaria solani and Sclerotinia sclerotiorum demonstrated significant inhibition of the mycelial growth of all strains (p < 0.05). The oils (500 μg/mL) showed significant antifungal effect against tested fungi in the growth inhibition range of 37.6–85.5% with respective IC50 values ranging from 88.8 to 660.0 μg/mL as compared to standard fungicides (100% inhibition) with IC50 value in the range of 32.2–129.4 μg/mL. Significant inhibition of spore germination was noticed for F. oxysporum, Curvularia lunata and Albugo candida which were highly susceptible to E. annuus oil with their IC50 values 120.7, 253.5 and 300.4 μg/mL, respectively. Thus, the results obtained in this study demonstrate the potential of essential oils from Himalayan Erigeron species as non-toxic, eco-friendly and biodegradable natural fungicides. [Copyright &y& Elsevier]

The transcription factors Ch AP1 and Skn7 of the maize pathogen Cochliobolus heterostrophus are orthologs of Yap1 and Skn7 in yeast, where they are predicted to work together in a complex. Previous work showed that in C. heterostrophus, as in yeast, Ch AP1 accumulates in the nucleus in response to reactive oxygen species ( ROS). The expression of genes whose products counteract oxidative stress depends on Ch AP1, as shown by impaired ability of a Δ chap1 mutant to induce these 'antioxidant' genes. In this study, we found that under oxidative stress, antioxidant gene expression is also partially impaired in the Δ skn7 mutant but to a milder extent than in the Δ chap1 mutant, whereas in the double mutant - Δ chap1 -Δ skn7 - none of the tested genes was induced, with the exception of one catalase gene, CAT2. Both single mutants are capable of infecting the plant, showing similar virulence to the WT. The double mutant, however, showed clearly decreased virulence, pointing to additive contributions of Ch AP1 and Skn7. Possible mechanisms are discussed, including additive regulation of gene expression by oxidative stress. [ABSTRACT FROM AUTHOR]

The redox-sensitive transcription factor ChAP1 [ Cochliobolus heterostrophus YAP1 ( Yeast Activator Protein 1) orthologue] of C. heterostrophus is required for oxidative stress tolerance. It is not known, however, to what extent the intracellular redox state changes on exposure of the fungus to oxidants, and whether ChAP1 is involved in the return of the cell to redox homeostasis. In order to answer these questions, we expressed a ratiometric redox-sensitive fluorescent protein sensor, p Hyper, in C. heterostrophus. The fluorescence ratio was sensitive to extracellular hydrogen peroxide ( H2O2) concentrations that had been shown previously to inhibit the germination of conidia and growth of the pathogen in culture. chap1 mutants showed a slower return to redox homeostasis than the wild-type on exposure to H2O2. Plant extracts that mimic oxidants in their ability to promote nuclear retention of ChAP1 reduced, rather than oxidized, the fungal cells. This result is consistent with other data suggesting that ChAP1 responds to plant-derived signals other than oxidants. pHyper should be a useful reporter of the intracellular redox state in filamentous fungi. [ABSTRACT FROM AUTHOR]

Abstract: Filamentous fungi produce hydrophobins, small proteins localized on the outer surface of their cell walls and involved in growth and development. Hydrophobin gene expression depends on nutrient availability, light, and the activity of conserved signal-transduction pathways. We found four hydrophobins, one class I and three class II family members, in the Cochliobolus heterostrophus genome with high homology to other ascomycete hydrophobins, which present a typical conserved array of cysteines. The expression profile of a selected gene from each class was determined in a series of signaling-deficient mutants. Loss of either of two mitogen-activated protein kinase (MAPK) genes, CHK1 and MPS1, led to decreased hydrophobin class I (CHYD1) gene expression. Mutants in both MAPK genes had easily wettable colonies, but decreased CHYD1 expression was not the sole explanation for this phenotype. A significant elevation of hydrophobin class II (CHYD3) gene expression was measured in the chk1 mutant, suggesting a complex role for MAPK in controlling the expression of these hydrophobins. Similar but less marked tendencies were observed in G-protein α and β subunit loss-of-function mutants; however these showed no alteration in colony hydrophobicity. Overexpression of CHYD1 in the wild-type background caused a change in colony morphology and a small but significant increase in aerial growth. Thus G-protein and MAPK signal transduction influence hydrophobin gene expression and colony hydrophobicity. The connection between colony hydrophobicity and expression of the hydrophobin genes CHYD1 and CHYD3, however, is not one-to-one, indicating that additional factors determine colony-surface properties. The approach of using hydrophobin-overexpression mutants to investigate their role may be generalized to other hydrophobins and small secreted proteins. [Copyright &y& Elsevier]

Abstract: Heterotrimeric G proteins transduce extracellular signals to control development in eukaryotes, including filamentous fungi. Targeted disruption of their α- and β-subunit genes has shown that fungal G proteins play essential roles in sexual and asexual sporulation, hyphal growth pattern and virulence. The G-protein β-subunit gene CGB1 of Cochliobolus heterostrophus, the agent of Southern leaf blight, is essential for virulence and sporulation, while one of the α-subunit genes, CGA1, has roles in several developmental pathways. We constructed a strain with insertions/deletions at both CGA1 and CGB1. The double mutant, like the single mutants, was deficient in mating and appressorium formation and its hyphae followed a straight path rather than the typical meandering growth pattern on a hard surface. The two genes shared a combined role in determining pigmentation, surface hydrophobicity, and resistance to different stresses. However, in contrast to the single mutants, the double mutants developed white-gray to completely white colonies that are hydrophilic and form wet, autolytic-appearing patches. These phenotypes resemble some of those conferred by mutations in the MAPK gene CHK1, suggesting crosstalk between MAPK and G-protein pathways. Loss of signal-transduction functions, while reducing virulence, increases resistance to some stresses. A model is proposed for the interactions between the signaling pathways. [Copyright &y& Elsevier]

Abstract: A Lae1-like methyltransferase, Llm1, was identified in maize pathogen Cochliobolus heterostrophus which is renowned for production of the secondary metabolite host-selective toxin, T-toxin, and is a model for mechanisms of reproduction of heterothallic Dothideomycetes. Previously, we determined that C. heterostrophus mutants lacking Lae1 and Vel1 proteins were decreased in ability to produce T-toxin when the fungus was grown in the dark, demonstrating that these proteins are positive regulators of toxin production. We showed also that Lae1 and Vel1 regulate resistance to oxidative stress and both sexual and asexual reproduction. Here, it is demonstrated that Llm1, one of nine Lae1-like methyltransferases in the C. heterostrophus genome, acts as a negative regulator of T-toxin production and thus impacts virulence to the host. In vitro, in the dark, and in planta, llm1 mutants make more T-toxin than do wild-type (WT) strains, while overexpressing strains make less than WT. Virulence (amount of chlorosis) to maize, due to T-toxin, follows accordingly. Expression of nine genes involved in T-toxin production is elevated in llm1 mutants and reduced in overexpressing strains. llm1 mutations cannot rescue deficiencies in T-toxin production of lae1 or vel1 mutants indicating that Llm1 represses T-toxin biosynthesis, and that vel1 and lae1 mutations are epistatic to llm1 mutations. Thus, increased T-toxin production, and presumably gene expression, in the llm1 mutant is dependent on the presence of Vel1 and Lae1 proteins. There is no evidence that Llm1 has an effect on oxidative stress tolerance. llm1 mutants are fully fertile in crosses to WT mating testers, while LLM1 overexpressing strains and llm1lae1 and llm1vel1 double mutants are unable to act as females. Overexpression of LLM1 leads to de-repression of asexual sporulation during sexual development, and of asexual sporulation in the light and the dark during vegetative growth, as is the case for vel1, llm1vel1, and llm1lae1-deletion strains. llm1vel1 and llm1lae1 double mutants are similar to lae1 single mutants and accumulate more hyphal melanin in liquid medium than do llm1 or vel1 single mutants, implying Llm1 plays a redundant role in regulating pigmentation with Vel1, while Lae1 plays a major role. [Copyright &y& Elsevier]

A melanin biosynthetic gene, Sal1, in the southern corn leaf blight fungus Cochliobolus heterostrophus, was identified by degenerate and inverse PCR. Structural analysis revealed that the open reading frame is 555 bp in length, interrupted by one intron. Melanin deficiency of the sal1 mutant strain, which accumulates the intermediate scytalone, was successfully complemented by introducing the gene fragment. These results suggest that Sal1 is a scytalone dehydratase gene of C. heterostrophus. [ABSTRACT FROM AUTHOR]

Protein-protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein-protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein-protein interactions in filamentous ascomycetes. [ABSTRACT FROM AUTHOR]

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation. [ABSTRACT FROM AUTHOR]

A simple procedure is reported for transformation of the rice false smut fungus Villosiclava virens (anamorph: Ustilaginoidea virens) using electroporation of intact conidial cells. The transformation vector pCB1004eGFP was constructed with a green fluorescent protein (eGFP) gene under a constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of Cochliobolus heterostrophus. When a linearized vector was applied, eGFP-expressing transformants were successfully acquired. An inoculation test in rice plants showed that the eGFP-expressing transformants were able to form rice false smut balls. [ABSTRACT FROM AUTHOR]

Southern leaf blight (SLB) is a foliar necrotrophic disease of maize (Zea mays L.) caused by the ascomycete fungus Cochliobolus heterostrophus (Drechs.) Drechs. It is particularly important in warm humid parts of the world where maize is cultivated, such as the southern Atlantic coast area of the United States and parts of India, Africa, and Western Europe. Quantitative trait loci (QTL) for resistance to SLB disease caused by C. heterostrophus race O were identified in three maize recombinant inbred populations assessed in two environments: Clayton, NC, in the summer and Homestead, FL, in the winter. The three populations were derived from the crosses B73 × CML254, CML254 × B97, and B97 × Ki14. Each of these populations was derived from a cross between a temperate maize line (B73 or B97) and a tropical maize line (Ki14 or CML254). Quantitative trait loci were identified by separate analysis of each population and by joint connected and disconnected analyses of all the populations. The most significant QTL identified were on chromosomes 3, 8, 9, and 10. Joint analysis led to more precise position estimates than separate analysis in each case. Results are discussed in the context of previous SLB QTL analysis studies and a recent flowering time QTL study that used the same populations. The chromosome 8 and 9 QTL colocalized with previously identified flowering time QTL, which suggested that the perceived effect on SLB resistance at these QTL may have been mediated through an effect on flowering time. [ABSTRACT FROM AUTHOR]

Abstract: Sexual fungi can be self-sterile (heterothallic, requiring genetically distinct partners) or self-fertile (homothallic, no partner required). In most ascomycetes, a single mating type locus (MAT) controls the ability to reproduce sexually. In the genus Cochliobolus, all heterothallic species have either MAT1-1 or MAT1-2 (but never both) in different individuals whereas all homothallic species carry both MAT1-1 and MAT1-2 in the same nucleus of an individual. It has been demonstrated, previously, that a MAT gene from homothallic Cochliobolus luttrellii can confer self-mating ability on a mat-deleted strain of its heterothallic relative, Cochliobolus heterostrophus. In this reciprocal study, we expressed, separately, the heterothallic C. heterostrophus MAT1-1-1 and MAT1-2-1 genes in a mat-deleted homothallic C. luttrellii strain and asked if this converts homothallic C. luttrellii to heterothallism. We report that: (1) A C. luttrellii transgenic strain carrying C. heterostrophus MAT1-1-1 and a C. luttrellii transgenic strain carrying C. heterostrophus MAT1-2-1 can mate in a heterothallic manner and the fertility of the cross is similar to that of a wild type C. luttrellii self. Full tetrads are always found. (2) A C. luttrellii transgenic strain carrying C. heterostrophus MAT1-1-1 can mate with the parental wild type C. luttrellii MAT1-1;MAT1-2 strain, indicating the latter is able to outcross, a result which was expected but has not been demonstrated previously. (3) A C. luttrellii transgenic strain carrying C. heterostrophus MAT1-2-1 cannot mate with the parental wild type C. luttrellii MAT1-1;MAT1-2 strain, indicating outcrossing specificity. (4) Each transgenic C. luttrellii strain, carrying only a single C. heterostrophus MAT gene, is able to self, although all pseudothecia produced are smaller than those of wild type and fertility is low (about 4–15% of the number of wild type asci). These data support the argument that in Cochliobolus spp., the primary determinant of reproductive mode is MAT itself, and that a heterothallic strain can be made homothallic or a homothallic strain can be made heterothallic by exchange of MAT genes. The selfing ability of transgenic C. luttrellii strains also suggests that both MAT1-1-1 and MAT1-2-1 genes of C. heterostrophus carry equivalent transcription regulatory activities, each capable of promoting sexual development when alone, in a suitable genetic background. [Copyright &y& Elsevier]

Southern corn leaf blight (SCLB) is an important foliar disease of maize and its control depends on resistant cultivars or fungicides. Maneb is the recommended fungicide to control SCLB; however, application of maneb has been increasingly limited due to public concerns about the potential harmful effects of fungicide residues on human health and the environment. Therefore, an alternative approach was explored in this study using a plant activator, probenazole, to control SCLB. Application of probenazole provided effective control of SCLB under greenhouse conditions. For practical use, using probenazole at a practical rate (1.8 kg a.i./ha) was demonstrated to effectively suppress SCLB under field conditions. In addition, the effect of probenazole treatment on the growth of maize was examined. Compared with the untreated control, leaf length, leaf width, plant height, and the fresh and dry weights of probenazole-treated corn plants were not influenced; therefore, it is suggested that application of probenazole could be an alternative to control SCLB. [ABSTRACT FROM AUTHOR]

Nested association mapping (NAM) offers power to resolve complex, quantitative traits to their causal loci. The maize NAM population, consisting of 5,000 recombinant inbred lines (RILs) from 25 families representing the global diversity of maize, was evaluated for resistance to southern leaf blight (SLB) disease. Joint-linkage analysis identified 32 quantitative trait loci (QTLs) with predominantly small, additive effects on SLB resistance. Genome-wide association tests of maize HapMap SNPs were conducted by imputing founder SNP genotypes onto the NAM RILs. SNPs both within and outside of QTL intervals were associated with variation for SLB resistance. Many of these SNPs were within or near sequences homologous to genes previously shown to be involved in plant disease resistance. Limited linkage disequilibrium was observed around some SNPs associated with SLB resistance, indicating that the maize NAM population enables high-resolution mapping of some genome regions. [ABSTRACT FROM AUTHOR]

A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallo-oxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (∆ ChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that ∆ ChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization. [ABSTRACT FROM AUTHOR]

A 5443 Da peptide with sequence homology to defensins was purified from purple pole beans (Phaseolus vulgaris cv. 'Extra-long Purple Pole bean'). This peptide was isolated by adsorption on an affinity chromatographic medium Affi- Gel Blue gel and ion-exchange chromatographic media SP-Sepharose (sulfopropyl-Sepharose) and Mono S and by gel filtration on Superdex peptide. The peptide inhibited mycelial growth in Mycosphaerella arachidicola, Helminthosporium maydis, Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, Candida albicans and Setosphaeria turcica with an IC50 of 0.8, 0.9, 2.3, 3.2, 4.3, 4.8 and 9.8 μM respectively. Its antifungal potency was higher than that of the plant defensin coccinin (IC50 >50 μM). It induced membrane permeabilization in C. albicans as evidenced by SYTOX Green uptake, but did not affect erythrocyte membrane permeability. It inhibited growth in M. arachidicola by inducing chitin accumulation at hyphal tips as was shown by Congo Red staining. The antifungal activity was pH stable and thermostable. The peptide inhibited the proliferation of hepatoma (HepG2), breast cancer (MCF7), colon cancer (HT29) and cervical cancer (SiHa) cells but not that of human embryonic liver (WRL68) cells. Its anti- HepG2 activity (IC50 =4.1±0.8 μM, n=3) was higher than that of another plant defensin, gymnin (IC50 >50 μM). Its anti-MCF7 activity (IC50 =8.3±0.3 μM, n=3) was similar to that of other plant defensins. It reduced the activity of HIV-1 reverse transcriptase with an IC50 of 0.5±0.1 μM, n=3, much more potently than other plant defensins (IC50 >40 μM). There is the possibility of using the purple pole bean defensin for producing antifungal drugs and/or transgenic plants with fungal resistance. [ABSTRACT FROM AUTHOR]

Northern leaf blight [NLB; caused by Exserohilum turcicum (Pass) K.J. Leonard and E.G. Suggs] is an important fungal disease of maize (Zea mays L.) in the United States and worldwide. The IBM population, an advanced intercross recombinant inbred line population derived from a cross between the lines Mo17 and B73, was evaluated in three environments (Aurora, NY, in 2006 and 2007 and Clayton, NC in 2007) for two traits related to NLB resistance, weighted mean disease (WMD) and incubation period (IP), and for days to anthesis (DTA). Two WMD quantitative trait loci (QTL) in bins 2.00/2.01 and 4.08 were detected from the overall analysis; of these, only the QTL in bin 4.08 was detected in all three environments analyzed separately. Likewise, only one IP QTL, in bin 2.02, was detected in all three environments and from the overall analysis. Several environment-specific QTL for each trait were also detected. Several DTA QTL were detected with the strongest effect detected in bin 8.05. Correlations between disease resistance traits and days to anthesis were uniformly low. The results from this study were compared to those of previous studies that used the IBM population to identify QTL for two other maize foliar diseases, southern leaf blight {causal agent Cochliobolus heterostrophus (Drechs.) Drechs. [anamorph = Bipolaris maydis (Nisikado and Miyake) Shoemaker; synonym = Helminthosporium maydis (Nisikado and Miyake)]} and gray leaf spot [causal agent Cercospora zeae-maydis (Tehon and E.Y. Daniels)]. Although we did not find QTL conferring resistance to all three diseases, significant correlations between resistances to these diseases in the IBM population were identified, implying the existence of loci (and possibly genes) affecting resistance to all three diseases. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi. [Copyright &y& Elsevier]

Abstract: We performed a phylogenetic analysis of heavy-metal ATPases (HMAs) in fungi and found that HMAs can be divided into three groups, A, B, and C. Group A is predicted to deliver copper ions to copper-containing proteins, while Groups B and C are thought to function as cell-membrane copper-efflux pumps. Furthermore, Groups B and C consist of fungal-specific HMAs, while Group A consists of fungal orthologues that have been well conserved in eukaryotes. We also cloned and characterized a Group A-type HMA gene (i.e., ChCcc2) of the filamentous plant pathogen, Cochliobolus heterostrophus. Mutation of ChCcc2 severely affected growth, pigmentation, conidiation, and colonial morphology. Activity of the copper-containing protein, laccase, was also lost in ChCcc2 mutants, suggesting that ChCCC2 plays an important role in growth and morphology by activating various copper-containing proteins in C. heterostrophus. [Copyright &y& Elsevier]

The genetic architecture underlying resistance in maize to southern leaf blight (SLB) caused by Cochliobolus heterostrophus race O is not well understood. The objective of this study was to identity loci contributing to SLB resistance in two recombinant inbred line populations and to compare these to SLB resistance loci in other populations. The two populations used were derived from crosses between maize inbred lines H99 and B73 (HB population--142 lines) and between B73 and B52 (BB population--186 lines). They were evaluated for SLB resistance and for days from planting to anthesis (DTA) in 2005 and 2006. Two replications arranged as randomized complete blocks were assessed in each year for each population. Entry mean heritabilities for disease resistance were high for both populations (0.876 and 0.761, respectively). Quantitative trait loci (QTL) for SLB resistance were identified in bins 3.04 (two QTL), 6.01, and 8.05 in the HB population and in bin 2.07 in the BB population. No overlap of DTA and SLB resistance QTL was observed. nor was there any phenotypic correlation between the traits. A comparison of the results of all published SLB resistance QTL studies suggested that bins 3.04 and 6.01 are 'hotspots' for SLB resistance QTL. [ABSTRACT FROM AUTHOR]

Abstract: An exhaustive characterization of the set of non-ribosomal peptide synthetase (NRPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small molecule peptides produced by the enzymes they encode, has been undertaken to ascertain the role of the peptide metabolites in the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. In addition to structural diversity, non-ribosomal peptides have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. However, to suggest that inter-organismal activities is their primary function is likely incorrect; in fact, the physiological significance of these peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches and that some are conserved, but most are not. [Copyright &y& Elsevier]

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. NatI. Acad. Sci. USA 103:15997–16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection. [ABSTRACT FROM AUTHOR]

The intermated 873 X Mol7 (IBM) population, an advanced intercross recombinant inbred line population derived from a cross between the maize lines B73 (susceptible) and Mol7 (resistant), was evaluated in four environments for resistance to southern leaf blight (SLB) disease caused by Cochliobolus heterostrophus race O. Two environments were artificially inoculated, while two were not inoculated and consequently had substantially lower disease pressure. Four common SLB resistance quantitative trait loci (QTL) were identified in all environments, two in bin 3.04 and one each in bins 1.10 and 8.02/3. There was no significant correlation between disease resistance and days to anthesis. A direct comparison was made between SLB QTL detected in two populations, independently derived from the same parental cross: the IBM advanced intercross population and a conventional recombinant inbred line population. Several QTL for SLB resistance were detected in both populations, with the IBM providing between 5 and, in one case, 50 times greater mapping resolution. [ABSTRACT FROM AUTHOR]

A recombinant inbred line population derived from a cross between the maize lines NC300 (resistant) and B104 (susceptible) was evaluated for resistance to southern leaf blight (SLB) disease caused by Cochliobolus heterostrophus race 0 and for days to anthesis in four environments (Clayton, NC, and Tifton, GA, in both 2004 and 2005). Entry mean and average genetic correlations between disease ratings in different environments were high (0.78 to 0.89 and 0.9, respectively) and the overall entry mean heritability for SLB resistance was 0.89. When weighted mean disease ratings were fitted to a model using multiple interval mapping, seven potential quantitative trait loci (QTL) were identified, the two strongest being on chromosomes 3 (bin 3.04) and 9 (bin 9.03-9.04). These QTL explained a combined 80% of the phenotypic variation for SLB resistance. Some time-point-specific SLB resistance QTL were also identified. There was no significant correlation between disease resistance and days to anthesis. Six putative QTL for time to anthesis were identified, none of which coincided with any SLB resistance QTL. [ABSTRACT FROM AUTHOR]

NPS6, encoding a nonribosomal peptide synthetase, is a virulence determinant in the maize (Zea mays) pathogen Cochliobolus heterostrophus and is involved in tolerance to H2O2. Deletion of NPS6 orthologs in the rice (Oryza sativa) pathogen, Cochliobolus miyabeanus, the wheat (Triticum aestivum) pathogen, Fusarium graminearum, and the Arabidopsis thaliana pathogen, Alternaria brassicicola, resulted in reduced virulence and hypersensitivity to H2O2. Introduction of the NPS6 ortholog from the saprobe Neurospora crassa to the Δnps6 strain of C. heterostrophus restored wild-type virulence to maize and tolerance to H2O2, demonstrating functional conservation in filamentous ascomycete phytopathogens and saprobes. Increased sensitivity to iron depletion was identified as a conserved phenotype of Δnps6 strains. Exogenous application of iron enhanced the virulence of Δnps6 strains of C. heterostrophus, C. miyabeanus, F. graminearum, and A. brassicicola to each host. NPS6 is responsible for the biosynthesis of extracellular siderophores by C. heterostrophus, F. graminearum, and A. brassicicola. Application of the extracellular siderophore of A. brassicicola restored wild-type virulence of the Δnps6 strain to Arabidopsis. It is proposed that the role of extracellular siderophores in fungal virulence to plants is to supply an essential nutrient, iron, to their producers in planta and not to act as phytotoxins, depriving their hosts of iron. [ABSTRACT FROM AUTHOR]

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides. [ABSTRACT FROM AUTHOR]

Abstract: A 5586bp sequence (accession no. DQ278491), which includes the RNA polymerase II gene (RPB2) encoding the second largest protein subunit (RPB2), was obtained from the wheat biotype Phaeosphaeria nodorum (PN-w) by PCR amplification. The 3841bp full length RPB2 gene contains two exons and a 52bp intron, and encodes a complete 1262 amino acid protein. Similar to the C-terminals of the β subunits of prokaryotes and yeast RNA polymerases, the deduced RPB2 protein contained many structural features needed for gene transcription. Based on the phylogenetic analysis with the deduced RPB2 polypeptide sequences, the PN-w was closely related to the maize pathogen Cochliobolus heterostrophus. Size differences were found in the full length RPB2 gene of cereal Phaeosphaeria species, mainly due to differences in intron size. No nucleotide substitutions were found in homothallic P. avenaria f.sp. triticea (Pat1) and barley biotype P. nodorum (PN-b) isolates used in this study. The nucleotide and deduced amino acid sequences of the RPB2 gene in Pat1 were closely related to that in PN-w. [Copyright &y& Elsevier]