Induction of pathogenesis-related genes and pathogen resistance in tobacco by fungal elicitors and plant growth regulators

Salicylic acid, methyl-jasmonate and ethylene have been suggested as signals for activation of plant defense responses against pathogen infections. Fungal elicitors have been shown to induce defense-related genes. However, it is not known how pathogenesis-related (PR) genes are regulated by fungal elicitors. Arachidonic acid (AA) and cellulases, which are fungal elicitors, have been used to study the induction of osmotin gene expression. The combination of AA and cellulases resulted in more than an additive effect of AA and cellulases alone on activating the osmotin gene. Inhibitor studies suggested that ethylene is involved in the induction of osmotin by these two elicitors. Heat-released elicitors from mycelial walls of pathogenic (Phytophthora parasitica var. nicotianae, race 0) and non-pathogenic (Trichoderma longibrachiatum) fungi have been isolated and used to induce PR-1 and osmotin genes by these elicitors. The non-pathogenic elicitor induced PR gene expression to a much higher level than did the pathogenic elicitor. The expression patterns of these two PR genes and the accumulations of their encoded proteins were dramatically affected by salicylic acid, methyl-jasmonate, and ethylene and combinations of these plant growth regulators. Ethylene, jasmonate, and salicylic acid are involved in the induction of PR-1 and osmotin by mycelial wall elicitor of non-pathogenic fungus. The non-pathogenic elicitor, but not the pathogenic elicitor, induced resistance of tobacco seedlings to P. parasitica var. nicotianae (race 0), a pathogen which causes black shank disease in tobacco. The protection induced by the non-pathogenic elicitor was dramatic and lasted for more than 3 weeks compared to salicylic acid or the combination of salicylic acid and methyl-jasmonate which could only induce resistance for a few days

Protective function of the recombinant Oshsp18.0-CII protein, a class II small heat shock protein of rice, in Escherichia coli

All plants synthesize multiple families of small heat shock proteins (sHSP). A cDNA clone, Oshsp18.0-CII, encoding an 18.0 kDa class II sHSP was isolated from rice previously. The function of the rice class II sHSP was studied by overproduction of the Oshsp18.0-CII fusion protein in transformed Escherichia coli cells. The results suggest that heterologous expression of the Oshsp18.0-CII fusion protein increases thermotolerance of E. coli cells in vivo and provided thermoprotection to E. coli soluble proteins in vitro. The survival rate of Oshsp 18.0-CII fusion protein-accumulating cells treated at 50°C for 1 h was almost 1000- fold higher than that of the control cells transformed with pET32a expression vector. Overproduction of the Oshsp18.0-CII fusion protein in E. coli also confers tolerance of E. coli cells to ultraviolet (UV) irradiation. The post-UV survival of the Oshsp18.0-CII fusion protein-accumulating cells is about 7.29-fold over that of the control cells transformed with pET32a expression vector when exposed to 1700 μJ of UV. There is almost no post-UV survival (≦0.4%) in the untransformed cells after exposing to 900 μJ UV light

Fumigant Activity of Bacterial Volatile Organic Compounds against the Nematodes Caenorhabditis elegans and Meloidogyne incognita

Plant-parasitic nematodes infect a diversity of crops, resulting in severe economic losses in agriculture. Microbial volatile organic compounds (VOCs) are potential agents to control plant-parasitic nematodes and other pests. In this study, VOCs emitted by a dozen bacterial strains were analyzed using solid-phase microextraction followed by gas chromatography–mass spectrometry. Fumigant toxicity of selected VOCs, including dimethyl disulfide (DMDS), 2-butanone, 2-pentanone, 2-nonanone, 2-undecanone, anisole, 2,5-dimethylfuran, glyoxylic acid, and S-methyl thioacetate (MTA) was then tested against Caenorhabditis elegans. DMDS and MTA exhibited much stronger fumigant toxicity than the others. Probit analysis suggested that the values of LC50 were 8.57 and 1.43 μg/cm3 air for DMDS and MTA, respectively. MTA also showed stronger fumigant toxicity than DMDS against the root-knot nematode Meloidogyne incognita, suggesting the application potential of MTA

RNA polymerase II gene (RPB2) encoding the second largest protein subunit in Phaeosphaeria nodorum and P. avenaria

A 5586 bp 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 3841 bp full length RPB2 gene contains two exons and a 52 bp intron, and encodes a complete 1262 amino acid protein. Similar to the C-terminals of the b 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

Degenerated Virulence and Irregular Development of Fusarium oxysporum f. sp. niveum Induced by Successive Subculture

Successive cultivation of fungi on artificial media has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, forms degenerated sectors after successive cultivation. In the present research, we demonstrated that subculture with aged mycelia increased the incidence of degenerations. To further investigate the di erences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelial type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results in variants (PT2, PT3, PT11, and MT6) were di erent from ST with mycelia growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculation with Fon variants and Fon ST. In root dipping methods, Fon variants showed no significant di erences in disease progress compared with ST. Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrasting results of two inoculation methods suggest that the degenerative changes due to repeated successive cultivation may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of di erent variants were analyzed. All Fon degenerated variants demonstrated significant decreases of CWDEs activities compared with ST. Additionally, transcript levels of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1, and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related gene transcripts except for fmk1, xlnR, and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelia. The degeneration showed significant impacts on virulence to watermelon, which was correlated with the reduction of CWDEs activities and declining expression of a set of virulence-related genes

Multibranched flower-like ZnO particles from eco-friendly hydrothermal synthesis as green antimicrobials in agriculture

Owing to the exponential growth of the human population and the rising food demand, identifying sustainable antimicrobials for higher agricultural productivity becomes essential. In this study, hydrothermal precipitation for synthesizing versatile nanostructured zinc oxide particles (nZnOs), including nanorods (1D), nanoplatelets (2D), and multibranched flower-like particles (3D), was achieved by controlling pH values of source precursors and growth time. Hydrothermal synthesis is an eco-friendly process without the requirement of hazardous organic solvents. The antimicrobial properties of these nZnO particles were evaluated on different soil-borne plant pathogens. These pathogens are serious limiting factors in agriculture. Results indicate that multibranched flower-like ZnO (3D nZnO) showed more remarkable, reliable, and stable antifungal activity compared with other nZnOs. The scanning electron microscopy results also demonstrated significant mechanical damage to pathogens that caused by the branches of flower-like particles from 3D nZnO. Moreover, results of photodegradation and environmental resistance of the synthesized nZnOs revealed their potentials to reduce hazardous chemical pesticides and the eco-friendliness. This work is the first to compare the antimicrobial (i.e., antifungal) properties of various nZnOs and their applications in agriculture. Our results suggest that multibranched flower-like ZnO can be served as promising candidates of green antimicrobials with multiple modes of action in sustainable agriculture

Development of a molecular method for rapid differentiation of watermelon lines resistant to Fusarium oxysporum f. sp niveum

The vascular wilt disease of watermelon caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major factors limiting watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) production worldwide. Breeding highly resistant lines is one of the best ways to control the disease. In order to accelerate the breeding programs, a reliable PCR (polymerase chain reaction) technique was developed to rapidly differentiate the disease-resistant watermelons. The G05-SCAR (sequence characterized amplified region) primer set GsF5/GsR5, derived from the OP-G05 random primer-amplified fragment sequence, produced an 898 bp DNA fragment which was specific to watermelon lines resistant or tolerant to Fon. With optimized PCR assay, the Molecular method could easily differentiate Fon-resistant and -susceptible hybrid watermelon populations

Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method

A molecular diagnosis method using real-time PCRfor quantification and detection of Fusarium oxysporumf. sp. cubense race 4

The Fusarium genus causes devastatingplant diseases worldwide, in which Fusarium oxysporumis the most serious crop pathogen. Disease monitoringis the basis of integrated pest management ofany disease. The lack of rapid, accurate, and reliabledevice to detect and identify plant pathogens is one ofthe main limitations in integrated disease management.This study describes an efficient and quantifiable diagnosismethod for the specific detection of F. oxysporumf. sp. cubense (Foc) race 4 in field-infected banana.With the optimized PCR parameters using the SCAR(sequence characterized amplified region) primersFocSc-1/FocSc-2 and a real-time PCR strategy, thedeveloped method showed high reproducibility andwas very sensitive to detect extremely low quantitiesof Foc genomic DNA (gDNA).We also found that FocgDNA in severely symptomatic banana pseudostemsand leaves were 6946-fold and 26.69-fold more than inthose of mild-symptomatic banana, respectively