Bakanae disease caused by Fusarium fujikuroi is an important fungal disease in rice. Among the seven strains isolated from symptomatic rice grains in this study, one strain, FfB14, triggered severe root growth inhibition and decay in the crown and root of rice seedlings. The remaining six strains caused typical Bakanae symptoms such as etiolation and abnormal succulent rice growth. To reveal the relationship between mycelial growth in the infected tissues and Bakanae disease progression, we have established a reliable quantification method using real time PCR that employs a primer pair and dual-labeled probe specific to a unigene encoding F. fujikuroi PNG1 (FfPNG1), which is located upstream of the fumonisin biosynthesis gene cluster. Plotting the crossing point (CP) values from the infected tissue DNAs on a standard curve revealed the active fungal growth of FfB14 in the root and crown of rice seedlings, while the growth rate of FfB20 in rice was more than 4 times lower than FfB14. Massive infective mycelial growth of FfB14 was evident in rice stems and crown; however, FfB20 did not exhibit vigorous growth. Our quantitative evaluation system is applicable for the identification of fungal virulence factors other than gibberellin.

No abstract

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.

Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method's sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman real-time PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R(2)>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.