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.

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 differences 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 different 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 differences 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 different 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.

Silver Nanoparticles on Nanoscale Silica Platelets (AgNP/NSP) and Nanoscale Silica Platelets (NSP) Inhibit the Development of Fusarium oxysporum f. sp. niveum.

Nanotechnology has attracted much attention recently because of its agricultural applications. In this study, we analyzed the ability of two potential nanomaterials (NMs), nanoscale silica platelets (NSP) and silver nanoparticles on nanoscale silica platelets (AgNP/NSP), to control Fusarium wilt [caused by Fusarium oxysporum f. sp. niveum (Fon)] disease in watermelon. Both AgNP/NSP and NSP significantly reduced Fon mycelial growth and spore viability. In addition, AgNP/NSP decreased the mycelium viability at concentrations of 150 and 200 ppm. Scanning and transmission electron microscopy showed significant morphological effects on Fon cells, such as increased roughness and interior hollowing after AgNP/NSP and NSP treatments. Further, fluorescence staining experiments showed that a concomitant increase in membrane permeability occurred after treatment with NMs. The biochemical effects of NM treatment included a significant reduction in secreted cellulase activity. Interestingly, the addition of cysteine as a reducing agent decreased effects of NSP on Fon spores, suggesting suppression of Fon spore development attributable to oxidative stress. Taken together, these results indicate that AgNP/NSP and NSP may potentially serve as nanofungicides for future control of Fusarium wilt and other fungal diseases.

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.

Sequence diversity of beta-tubulin (tubA) gene in Phaeosphaeria nodorum and P. avenaria.

Full-length coding sequences of the beta-tubulin gene (tubA) were PCR-amplified and sequenced from 42 Phaeosphaeria isolates, including 16 P. nodorum and 23 P. avenaria species from cereals, two Polish isolates from rye (Secale cereale L.), and one isolate from dallis grass (Paspalum dilatatum Poir). A tubA gene of size 1556bp was identified in wheat- and barley-biotype P. nodorum (PN-w and PN-b), P. avenaria f. sp. avenaria (Paa), homothallic P. avenaria f. sp. triticea (P.a.t.) (Pat1) and the P.a.t. isolate (Pat3) from the State of Washington. The tubA gene length polymorphisms were detected in two P.a.t. isolates (Pat2) from foxtail barley (Hordeum jubatum L.), one from dallis grass and two Polish isolates from rye. These size differences were due to the variation of intron lengths among these three Phaeosphaeria species. All Phaeosphaeria isolates have identical 1344bp exons that can be translated into a 447 amino acid beta-tubulin. Like glyceraldehyde-3-phosphate dehydrogenase, the beta-tubulin amino acid sequence was identical in all Phaeosphaeria species used in this study, with the exception of the two Pat2 isolates. Six amino acid differences were evident in the beta-tubulin of these Pat2 isolates.

Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F. oxysporum f. sp. niveum in Taiwan.

Fusarium wilt, caused by Fusarium oxysporum (Fo), is one of the most important fungal diseases worldwide. Like other plant pathogens, Fo displays specialized forms in association with its hosts. For example, F. oxysporum f. sp. niveum (Fon) is the damaging pathogen causing Fusarium wilt disease on watermelon, whereas F. oxysporum f. sp. cubense is the pathogen that infects banana. A rapid and reliable pathogen identification or disease diagnosis is essential for the integrated disease management practices in many crops. In this study, two new primer sets, Fon-1/Fon-2 and FnSc-1/FnSc-2, were developed to differentiate Fon and Fo, respectively. The PCR method using the novel primer sets has high sensitivity to detect Fon when the DNA concentration was as low as 0.01 pg or when the conidia number was as few as 5. In comparison with the published primer set, the Fon-1/Fon-2 primer set, derived from the sequence of OP-M12 random primer-amplified fragment, produced a 174 bp DNA fragment, and was more specific to Fon in Taiwan. In addition, with optimized PCR parameters, the molecular method using the Fon-1/Fon-2 primer set could directly detect Fon even when watermelon samples were collected in its early stage of disease development.

Diversity of the trifunctional histidine biosynthesis gene (his) in cereal Phaeosphaeria species.

Phaeosphaeria species are important causal agents of Stagonospora leaf blotch diseases in cereals. In this study, the nucleotide sequence and deduced polypeptide of the trifunctional histidine biosynthesis gene (his) are used to investigate the phylogenetic relationships and provide molecular identification among cereal Phaeosphaeria species. The full-length sequences of the his gene were obtained by PCR amplification and compared among cereal Phaeosphaeria species. The coding sequence of the his gene in wheat-biotype P. nodorum (PN-w) was 2697 bp. The his genes in barley-biotype P. nodorum (PN-b), two P. avenaria f. sp. triticea isolates (homothallic Pat1 and Pat3), and Phaeosphaeria species from Polish rye and dallis grass were 2694 bp. The his gene in heterothallic isolate Pat2, however, was 2693 bp because the intron had one fewer base. In P. avenaria f. sp. avenaria (Paa), the his gene was only 2670 bp long. The differences in the size of the his gene contributed to the variation in amino acid sequences in the gap region located between the phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase sub-domains. Based on nucleotide and deduced amino acid sequences of the his gene, Pat1 was not closely related to either PN-w or the Paa clade. It appears that rates of evolution of the his gene were fast in cereal Phaeosphaeria species. The possible involvement of meiotic recombination in genetic diversity of the his gene in P. nodorum is discussed.

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 beta 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.