QUANTIFICATION OF 2,4-DIACETYLPHLOROGLUCINOL PRODUCED BY FLUORESCENT PSEUDOMONAS SPP IN-VITRO AND IN THE RHIZOSPHERE OF WHEAT

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a ma jor determinant in the biological control of a wide range of plant dis eases by fluorescent Pseudomonas spp, A protocol was developed to read ily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants, Extraction,vith ethyl acetate at a n acidic pH was suitable for both in vitro and in situ sources of Phl, For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar org anic soil components, such as humic and fulvic acids, which can interf ere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 5 4.6%, and a second extraction added another 6.1%. These yields were su bstantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil, For the f irst time Phl was isolated from the rhizosphere environment in raw soi l, Following application of Pseudomonas fluorescens Q2-87 and the Phl- overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2. 4 mu g of Phl/g of root plus rhizosphere soil, respectively, were isol ated from wheat grown in a Ritzville silt loam; 0.37 and 1.3 mu g of P hl/g of root plus rhizosphere soil, respectively, were isolated from w heat grown in a Shano silt loam, However, when the amount of Phl was c alculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.