Multicomponent reactions in crop protection chemistry.

An overview is given of the significance of multicomponent reactions in the synthesis of agrochemicals. The most important applications of multicomponent condensations, such as the Biginelli reaction, Bucherer-Bergs reaction, Hantzsch dihydropyridine synthesis, Kabachnik-Fields reaction, Mannich reaction, Passerini reaction, Petasis reaction, Strecker reaction, Ugi reaction and Willgerodt-Kindler reaction, to the synthesis of herbicidally, fungicidally and insecticidally active compounds are presented. Also the mode of action and biological activity of these multicomponent reaction products are reported.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Antibacterial and antifungal activities of different polar extracts from Anoectochilus roxburghii.

This study evaluates the antibacterial and antifungal activities of petroleum ether, acetic ether, n-butanol and aqueous extracts from Anoectochilus roxburghii. The in vitro antibacterial and antifungal effects against three bacterial strains (Escherichia coli, Bacillus subtilis, Bacillus thuringiensis) and three fungal species (Exserohilum turcicum (Pass.) Leonard et Suggs, Botrytis cinerea Pers., Fusahum graminearum Sehw.) were assayed by the dilution and disc-diffusion methods. All of the polar extracts expressed dose-dependent antimicrobial activity against all tested microorganisms. The most active extract was aqueous extract, with a minimum inhibitory concentration below 0.625mg/ml in both bacteria and fungi. The results suggest that new chemical classes of natural antimicrobial substances (such as A. roxiburghii extracts) can be selectively exploited for the chemotherapy and control of infectious diseases.

Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1.

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.

My Life and Virus Research Journey.

My long career in virology has been a continuous learning exercise with a very modest start. Virology and related pertinent fields have changed significantly during my lifetime. Sometimes I wish that my career had just started and I could apply all available and state of the art technology to solving problems and explaining intriguing observations. I was always convinced that visiting growers' fields is essential for researchers to get firsthand observations and knowledge of virus disease problems under field conditions. I never thought I would pursue so many avenues of research, yet it is true that research never ends. I enjoyed dissecting strain diversity in a very important plant pathogen like bean pod mottle virus (BPMV) and using BPMV-based vectors to address fundamental virology questions. Lastly, solving the enigma of the transmissible disease of Helminthosporium victoriae and attempting to gain an understanding of the molecular basis of disease in a plant pathogenic fungus were thrilling.

Novel Trichoderma strains isolated from tree barks as potential biocontrol agents and biofertilizers for direct seeded rice.

This study is the first time report of utilization of Trichoderma spp. isolated from different tree bark from Odisha state of India for rice crop health management and higher productivity. Six isolates of Trichoderma spp. were identified based on the morphological characteristics and species determination was performed by molecular assays. One of the isolated strains determined as Trichoderma erinaceum outperformed others. Trichoderma erinaceum controlled three soil borne plant pathogens i.e. Rhizoctonia solani, Sclerotium rolfsii and Sclerotium oryzae effectively under controlled condition and R. solani and Helminthosporium oryzae under filed condition. Seed treatments with the formulated isolates improved the germination rate of rice and enhanced vigour. These parameters along with higher chlorophyll content could be related to higher yield observed in two rice varieties; Karuna and Sahabhagidhan. Among the six isolates tested, Trichoderma erinaceum treatment recorded highest yield. Significantly higher expression of some stress related enzymes was observed in Trichoderma treated plants which helped in better crop growth both under biotic and abiotic stresses. These isolates helped both the varieties to accumulate more nutrients. This study proves that Trichoderma erinaceum obtained from tree bark may be incorporated in integrated rice crop management both as biocontrol agent and biofertilizer.

Cyclohelminthols Y1-Y4 Metabolites Possessing Two Spirocyclopropanes in Their Structure.

Cyclohelminthols Y1-Y4 (1-4) were isolated from the culture broth of Helminthosporium velutinum yone96. These compounds are diastereomers to each other featuring 3-azabicyclo[3.1.0]hexane-6-spirocyclopentane linked with a cyclopentanespirocyclopropane framework. Their planar structures were established via the comparison of their spectra with the simpler analogue cyclohelminthol X as well as analysis of their HMBC spectra. Although the proton-deficient core frameworks of 1-4 prevented us from obtaining configurational information via conventional NMR analysis, their total structures involving the relative and absolute configurations were established using density functional theory (DFT)-based molecular modeling calculations. The present study demonstrates the effectiveness of the comparison between the theoretical and experimental δ13C values for stereochemical analysis by focusing on the carbons that show relatively large δ13C deviations among the isomers. The G-ring of these molecules most likely originates from the cyclopropanation of the C6C7 double bond with the carbene equivalent 6 derived from cyclohelminthol IV (7), which was isolated from the same producer fungus. Preliminary biological experiments revealed the potent cytotoxicity of the (6 S)-isomers against COLO201 cells, whereas the (6 R)-isomers exhibited weak activity. The antifungal assay with Cochiobolus miyabeanus showed a slightly different profile.

Heterologous expression and antimicrobial activity of OsGASR3 from rice (Oryza sativa L.).

According to an in silico analysis, OsGASR3 (LOC_Os03g55290) from rice (Oryza sativa L.) was predicted to be involved in plant defense mechanisms. A semi-quantitative reverse transcription polymerase chain reaction assay revealed that OsGASR3 is highly expressed in the inflorescences of Thai jasmine rice (O. sativa L. subsp. indica 'KDML 105'). To characterize the biological activity of OsGASR3, we produced an OsGASR3-glutathione S-transferase fusion protein in Escherichia coli Rosetta-gami (DE3) cells for a final purified recombinant OsGASR3 yield of 0.65 mg/L. The purified OsGASR3 inhibited the hyphal growth of Fusarium oxysporum f.sp. cubense and Helminthosporium oryzae at a relatively low concentration (7.5 µg/mL). Furthermore, OsGASR3 exhibited in planta inhibitory activity against Xanthomonas campestris, suggesting its involvement in defense mechanisms, in addition to its previously reported functions affecting growth and development. These observations indicate that recombinant OsGASR3 may be useful for protecting agriculturally important crops against pathogenic microbes.

A review of wheat diseases-a field perspective.

Wheat is one of the primary staple foods throughout the planet. Significant yield gains in wheat production over the past 40 years have resulted in a steady balance of supply versus demand. However, predicted global population growth rates and dietary changes mean that substantial yield gains over the next several decades will be needed to meet this escalating demand. A key component to meeting this challenge is better management of fungal incited diseases, which can be responsible for 15%-20% yield losses per annum. Prominent diseases of wheat that currently contribute to these losses include the rusts, blotches and head blight/scab. Other recently emerged or relatively unnoticed diseases, such as wheat blast and spot blotch, respectively, also threaten grain production. This review seeks to provide an overview of the impact, distribution and management strategies of these diseases. In addition, the biology of the pathogens and the molecular basis of their interaction with wheat are discussed.

Genetic control and combining ability of agronomic attributes and northern leaf blight-related attributes in popcorn.

The present study was conducted to investigate the genetic control and to estimate the general and specific combining abilities of popcorn for agronomic attributes and attributes related to resistance to northern leaf blight (NLB). The 56 hybrids (F1 and reciprocals), together with the eight parent lines and six controls, were evaluated in two harvests, in a randomized-block design with four replications. Dominance components were more expressive than the additive components for grain yield and expression of resistance, and hybridization was the most suitable option for obtaining resistant and productive genotypes. For grain yield, popping expansion, and resistance to NLB, there was no significance for reciprocal effects, which indicates that the direction in which the cross is performed does not interfere with the hybrid's performance. Then, the superior hybrids recommended for more profitable growth were P8 x L61, L61 x L76, and L61 x L77.

A Three-Way Transcriptomic Interaction Study of a Biocontrol Agent (Clonostachys rosea), a Fungal Pathogen (Helminthosporium solani), and a Potato Host (Solanum tuberosum).

Helminthosporium solani causes silver scurf, which affects the quality of potato. The biocontrol agent Clonostachys rosea greatly limited the severity of silver scurf symptoms and amount of H. solani genomic DNA in laboratory experiments. Transcriptomic analysis during interaction showed that H. solani gene expression was highly reduced when coinoculated with the biocontrol agent C. rosea, whereas gene expression of C. rosea was clearly boosted as a response to the pathogen. The most notable upregulated C. rosea genes were those encoding proteins involved in cellular response to oxidative stress, proteases, G-protein signaling, and the methyltransferase LaeA. The most notable potato response to both fungi was downregulation of defense-related genes and mitogen-activated protein kinase kinase kinases. At a later stage, this shifted, and most potato defense genes were turned on, especially those involved in terpenoid biosynthesis when H. solani was present. Some biocontrol-activated defense-related genes in potato were upregulated during early interaction with C. rosea alone that were not triggered by H. solani alone. Our results indicate that the reductions of silver scurf using C. rosea are probably due to a combination of mechanisms, including mycoparasitism, biocontrol-activated stimulation of plant defense mechanisms, microbial competition for nutrients, space, and antibiosis.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.

Direct Estimation of Local pH Change at Infection Sites of Fungi in Potato Tubers.

Fungi can modify the pH in or around the infected site via alkalization or acidification, and pH monitoring may provide valuable information on host-fungus interactions. The objective of the present study was to examine the ability of two fungi, Colletotrichum coccodes and Helminthosporium solani, to modify the pH of potato tubers during artificial inoculation in situ. Both fungi cause blemishes on potato tubers, which downgrades tuber quality and yield. Direct visualization and estimation of pH changes near the inoculation area were achieved using pH indicators and image analysis. The results showed that the pH of the area infected by either fungus increased from potato native pH of approximately 6.0 to 7.4 to 8.0. By performing simple analysis of the images, it was also possible to derive the growth curve of each fungus and estimate the lag phase of the radial growth: 10 days for C. coccodes and 17 days H. solani. In addition, a distinctive halo (an edge area with increased pH) was observed only during the lag phase of H. solani infection. pH modulation is a major factor in pathogen-host interaction and the proposed method offers a simple and rapid way to monitor these changes.

Design, synthesis and biological evaluation of novel nicotinamide derivatives bearing a substituted pyrazole moiety as potential SDH inhibitors.

BACKGROUND: Succinate dehydrogenase (SDH) plays an important role in the Krebs cycle, which is considered as an attractive target for development of succinate dehydrogenase inhibitors (SDHIs) based on antifungal agents. Thus, in order to discover novel molecules with high antifungal activities, SDH as the target for a series of novel nicotinamide derivatives bearing substituted pyrazole moieties were designed and synthesised via a one-pot reaction. RESULTS: The biological assay data showed that compound 3 l displayed the most potent antifungal activity with EC50 values of 33.5 and 21.4 µm against Helminthosporium maydis and Rhizoctonia cerealis, respectively. Moreover, 3 l exhibited the best inhibitory ability against SDH enzymes. The results of docking simulation showed that 3 l was deeply embedded into the SDH binding pocket, and the binding model was stabilised by a cation-π interaction with Arg 43, Tyr 58 and an H-bond with Trp 173. CONCLUSION: The study suggests that the pyrazole nicotinamide derivative 3 l may serve as a potential SDHI that can be used as a novel antifungal agent, and provides valuable clues for the further design and optimisation of SDH inhibitors. © 2016 Society of Chemical Industry.

Two novel antimicrobial defensins from rice identified by gene coexpression network analyses.

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63µg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities.

[Cross-reactivity in allergic fungal sinusitis. Case report]. / Reactividad cruzada en sinusitis alérgica fúngica. Reporte de caso.

BACKGROUND: The presence of allergic mucin in allergic fungal sinusitis (AFS) is a manifestation that identifies it as a hypersensitivity process. AFS has a phenomenon of cross-reactivity to IgE-bound proteins having at least two shared epitopes. CLINICAL REPORT: A 13-year-old male with nasal obstructive symptoms of three years of evolution. An obstructive mass was identified in the sinuses through physical examination and CT. In endoscopic surgery, the left nostril polyp was identified with the macroscopic appearance of allergic mucin; the polyp was resected. Final histopathological examination using periodic acid-Schiff and Grocott's methenamine silver staining indicated Aspergillus. Two weeks after surgery, percutaneous tests showed sensitization to Alternaria, Helminthosporium sativum, and Deramatophagoides farianae with negativity to Aspergillus fumigatus. CONCLUSIONS: The absence of significant titers of specific IgE antibodies to Aspergillus fumigatus was the evidence that the hypersensitivity response was triggered by a pathogen other than that isolated in histopathological study, which coupled with positive tests for other fungi may be explained by the cross-reactivity phenomenon in a phenomenon of likely hypersensitivity.

Antecedentes: La presencia de mucina alérgica en la rinosinusitis alérgica fúngica (RAF) es una manifestación que la identifica como un proceso de hipersensibilidad. En la RAF existe un fenómeno de reactividad cruzada entre proteínas unidas a IgE que tienen al menos dos epítopes compartidos. Caso clínico: Varón de 13 años de edad con síntomas obstructivos nasales de tres años de evolución. Por exploración física y tomografía se identificó masa obstructiva en los senos paranasales. En la cirugía endoscópica, en la fosa nasal izquierda se identificó pólipo con aspecto macroscópico de mucina alérgica; el pólipo fue resecado. El examen histopatológico final mediante tinciones con ácido peryódico de Schiff y metenamina plata de Grocott indicó Aspergillus. Dos semanas después de la cirugía, las pruebas percutáneas mostraron sensibilización a Alternaria alternata, Helminthosporium sativum y Deramatophagoides farianae, con negatividad a Aspergillus fumigatus. Conclusiones: La ausencia de títulos significativos de anticuerpos IgE específicos para Aspergillus fumigatus constituyó la evidencia de que la respuesta de hipersensibilidad fue desencadenada por un patógeno distinto del aislado en el estudio histopatológico, que aunada a las pruebas positivas para otros hongos puede explicarse por el fenómeno de reactividad cruzada en un probable fenómeno de hipersensibilidad.

Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

[Cultural Properties and Taxonomic Position of Helminthosporium-Like Fungal Isolates from the White Sea].

Morphological, cultural and physiological characteristics of 19 Helminthosporium-like hyphomycetes isolated from the White Sea were studied. Taxonomic status of the isolates was verified using molecular genetics techniques. One of the isolates was identified as Alternatia sp., while the rest of marine isolates belonged to the species Paradendriphiella salina (G.K. Sutherl.) Woudenb. & Crous. The specific features of the isolates studied were characterized as adaptive. Optimum salinity for their growth was 1-2% NaCl, which is lower than the value for the known open ocean isolates. This is probably due to relatively low salinity of White Sea (22-24 per thousand) as compared with the ocean water (35 per thousand). While the temperature optimum for growth was 22 per thousand, growth and sporulation occurred at 6 degrees C, which has not been reported for marine fungi isolated from warmer seawater. All isolates studied grew and sporulated efficiently on the medium supplied with the Fucus algae extract and in the sea water layer. Conidia of the isolates submerged in the sea water were propagated efficiently, unlike the soil-born fungi. Holoblastic conidiogenesis was demonstrated by light and scanning electron microscopy, confirming the separation of P. salina from the genus Scolecobasidium.

An RNA cassette from Helminthosporium victoriae virus 190S necessary and sufficient for stop/restart translation.

Prototype victorivirus HvV190S employs stop/restart translation to express its RdRp from the downstream ORF in its bicistronic mRNA. The signals for this activity appear to include a predicted RNA pseudoknot directly upstream of the CP stop and RdRp start codons, which overlap in the motif AUGA. Here we used a dual-fluorescence system to further define which HvV190S sequences are important for stop/restart translation and found that the AUGA motif plus 38 nt directly upstream are both necessary and sufficient for this activity. This RNA cassette encompasses the predicted pseudoknot, and indeed substitutions that disrupted the pseudoknot disrupted the activity whereas complementary substitutions that restored the pseudoknot restored the activity. Replacement of this RNA cassette with those from other victoriviruses with a predicted pseudoknot in comparable position also supported stop/restart translation. To our knowledge, this is the first example of stop/restart translation regulated by an RNA pseudoknot.

Acidophilic actinomycetes from rhizosphere soil: diversity and properties beneficial to plants.

Three hundred and fifty-one isolates of actinomycetes were recovered from 21 rhizospheric soil samples using acidified media of pH 5.5. They were evaluated for their antifungal, siderophore production and phosphate solubilization activities. The total count of actinomycetes growing on acidified starch casein agar and Gause no. 1 agar were below 2.48 × 10(4) CFU g(-1) soil. Two hundred and twelve isolates were assigned to acidophiles and the remaining 139 isolates were neutrophiles. Of these actinomycetes, 57.8, 32.5 and 50.4%, showed antagonistic activity against three rice pathogenic fungi; Fusarium moniliforme, Helminthosporium oryzae and Rhizoctonia solani, respectively. More than half of the isolates (68.1%) inhibited at least one tested pathogenic fungus, whereas 25.9% exhibited antifungal activities against all tested fungi. Three hundred and thirty-eight isolates (96.3%) produced siderophore and 266 isolates (75.8%) solubilized phosphate. A greater proportion of the acidophilic actinomycetes exhibited antifungal, siderophore production and phosphate solubilization activity compared with the neutrophiles. Three hundred and twenty-five isolates (92.6%) were classified as streptomycetes based on their morphological characteristics and the presence of the LL-isomeric form of diaminopimelic acid in whole-cell hydrolysates. The 16S ribosomal RNA (rRNA) gene analysis of representative non-streptomycete strains showed that the isolates belonged to seven genera, that is, Allokutzneria, Amycolatopsis, Mycobacterium, Nocardia, Nonomuraea, Saccharopolyspora and Verrucosispora. The potential antifungal acidophilic isolates, R9-4, R14-1, R14-5 and R20-5, showed close similarity to Streptomyces misionensis NBRC 13063(T) (AB184285) in terms of morphological characteristics and 16S rRNA gene sequences.