Production of porphyrin intermediates in Escherichia coli carrying soil metagenomic genes.

Tetrapyrrole pigments are important components of many biological processes, and many of them are produced primarily by microorganisms. We constructed a soil metagenomic library using rice paddy soil consisting of 107 000 fosmid clones with an average DNA insert size of 35 kb. We isolated a clone carrying genes in the porphyrin biosynthetic pathway based on function-driven screening of the library. Through subcloning and mutagenesis analysis, we showed that two genes from soil metagenome, gtrA and hemC, were responsible for pigmentation in Escherichia coli. HPLC and LC-MS analysis of the purified pigments from E. coli carrying pSY143 identified coproporphyrin III without metal as a major compound as well as some other minor porphyrin intermediates. As gtrA and hemC encode glutamyl-tRNA reductase and porphobilinogen deaminase, respectively, which are enzymes involved in the C5 biosynthetic pathway for porphyrin intermediates, our results suggest that hemL, hemB, hemD, and hemE should be provided by the E. coli chromosome to generate a hybrid biosynthetic pathway for production of porphyrin intermediates using E. coli and metagenomic genes.

Biological control of strawberry gray mold caused by Botrytis cinerea using Bacillus licheniformis N1 formulation.

Bacillus licheniformis N1 is a biological control agent to control gray mold diseases caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were generated and evaluated for the activity to control strawberry gray mold. The wettable powder type formulation N1E was selected in pot experiments with remarkable disease control activity on both strawberry leaves and flowers. The N1E formulation contained 400 g of corn starch, 50 ml of olive oil, and 50 g of sucrose per a liter of bacterial fermentation culture. Optimum dilution of N1E to appropriately control the strawberry gray mold appeared to be 100-fold dilution in plastic house artificial infection experiments. The significant reduction of symptom development in the senescent leaves was apparent by the treatment of N1E at 100-fold dilution when N1E was applied before Bo. cinerea inoculation, but not after the inoculation. Both artificial infection experiments in a plastic house and natural infection experiments in the farm plastic house under production conditions revealed that the disease severity of gray mold on strawberry leaves and flowers was significantly reduced by N1E treatment. The disease control value of N1E on strawberry leaves was 81% under production conditions, as compared with the 61.5% conferred by a chemical fungicide, iprodione. This study suggests that our previously generated formulation of B. licheniformis N1 will be effective to control strawberry gray mold by its preventive activity.

Characteristics of Hypovirulent Strains of Chestnut Blight Fungus, Cryphonectria parasitica, Isolated in Korea.

Chestnut blight disease caused by Cryphonectria parasitica is widely distributed throughout chestnut tree plantations in Korea. We surveyed 65 sites located at 9 provinces in South Korea, and isolated 248 virulent and 3 hypovirulent strains of chestnut blight fungus. Hypovirulent strains had dsRNA virus in the cytoplasm, which is one of the typical characteristics of hypovirulent strains. In addition, they showed more characteristics of hypovirulent strains, i.e., suppressed conidiation, reduced pigmentation in colony color, and reduced phenol oxidase activity as well as reduced pathogenicity. Hypovirulent strains, KCPH-22, KCPH-135 and KCPH-136, had a genomic dsRNA band with the molecular weight of 12.7 kb, which is the L-dsRNA of CHV1. They also had a 2.7 kb defective dsRNA band. Single conidia isolated from hypovirulent strains were cultured and various phenotypes and absence of dsRNA bands were obtained from single conidial cultures, which means that hypovirulence transmission is unstable in asexual reproduction and variations in viral heredity by asexual reproduction. Biocontrol trial using hypovirulent strains was also carried out in the chestnut tree plantations, and canker expansion in the treated trees was stopped and healed by callus formation at the margin of the canker. These results show the potentials in successful biocontrol of chestnut blight if the vegetatively compatible hypovirulent strains could be directly used around the canker formed by compatible virulent strains.