Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908.
A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon. A bacterial strain, MET0908, showing a potent antifungal activity against C. lagenarium, was isolated from soil. An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography. The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE. The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi. Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C. lagenarium. The purified antifungal protein exhibited beta-1,3-glucanase activity. The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala. A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.
Kim
Pyoung Il
PI
Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA. pkim@nctr.fda.gov
Chung
Ki-Chul
KC
D000645:Ammonium Sulfate / Q000737:chemistry; D000898:Antibiosis; D000935:Antifungal Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001407:Bacillus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002473:Cell Wall / Q000187:drug effects; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D029773:Citrullus / Q000382:microbiology; D020231:Colletotrichum / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification*; D005638:Fruit / Q000382:microbiology; D043326:Glucan 1,3-beta-Glucosidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010572:Pest Control, Biological / Q000379:methods; D010935:Plant Diseases / Q000382:microbiology; D018515:Plant Leaves / Q000382:microbiology; D018547:Plant Stems / Q000382:microbiology; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology; D014462:Ultrafiltration
D000935:Antifungal Agents; D043326:Glucan 1,3-beta-Glucosidase; D000645:Ammonium Sulfate