Biocontrol of Orchid-pathogenic Mold, Phytophthora palmivora, by Antifungal Proteins from Pseudomonas aeruginosa RS1

Abstract

Black rot disease in orchids is caused by the water mold Phytophthora palmivora. To gain better biocontrol performance, several factors affecting growth and antifungal substance production by Pseudomonas aeruginosa RS1 were verified. These factors include type and pH of media, temperature, and time for antifungal production. The results showed that the best conditions for P. aeruginosa RS1 to produce the active compounds was cultivating the bacteria in Luria-Bertani medium at pH 7.0 for 21 h at 37 °C. The culture filtrate was subjected to stepwise ammonium sulfate precipitation. The precipitated proteins from the 40% to 80% fraction showed antifungal activity and were further purified by column chromatography. The eluted proteins from fractions 9-10 and 33-34 had the highest antifungal activity at about 75% and 82% inhibition, respectively. SDS-PAGE revealed that the 9-10 fraction contained mixed proteins with molecular weights of 54 kDa, 32 kDa, and 20 kDa, while the 33-34 fraction contained mixed proteins with molecular weights of 40 kDa, 32 kDa, and 29 kDa. Each band of the proteins was analyzed by LC/MS to identify the protein. The result from Spectrum Modeler indicated that these proteins were closed similarly to three groups of the following proteins; catalase, chitin binding protein, and protease. Morphological study under scanning electron microscopy demonstrated that the partially purified proteins from P. aeruginosa RS1 caused abnormal growth and hypha elongation in P. palmivora. The bacteria and/or these proteins may be useful for controlling black rot disease caused by P. palmivora in orchid orchards.

Keywords: Biocontrol; Phytophthora palmivora; Pseudomonas aeruginosa; orchid.

Figure 1.

Inhibition of P. palmivora by P. aeruginosa RS1 on a V8 agar plate (A) and control growth of the mold without the bacteria (B). Microscopic observation (400×) of the hyphae of P. palmivora growing with (C) and without (D) P. aeruginosa RS1.

Figure 2.

Inhibition percentage against P. palmivora and absorbance at OD_660 nm of P. aeruginosa RS1 in LB medium (pH 7) at 37 °C, at time points measured every 3 h, 24 h.

Figure 3.

Chromatogram from DEAE-cellulose column chromatography indicates the absorbance at OD_280 nm of the proteins eluting from the column using gradient concentrations of NaCl.

Figure 4.

Inhibition of P. palmivora by eluted proteins from DEAE-cellulose column chromatography. P. palmivora grew on a V8 agar plate: control without protein (A), with protein fraction 9–10 (B), and with protein fraction 33–34.

Figure 5.

SDS-PAGE of partial purification of the antifungal proteins from the ammonium sulfate precipitation and anion exchange chromatography steps. The labels 9–10 and 33–34 refer to the proteins from DEAE fractions 9–10 and 33–34, respectively.

Figure 6.

Morphology of P. palmivora under scanning electron microscope (2000×) after incubation for 2 days without (A) and with the purified antifungal protein from DEAE-cellulose column chromatography at fractions 9–10 (B) and 33–34 (C).