Biocontrol Activity of Bacillus amyloliquefaciens CNU114001 against Fungal Plant Diseases

Abstract

A total of 62 bacterial isolates were obtained from Gomsohang mud flat, Mohang mud flat, and Jeju Island, Republic of Korea. Among them, the isolate CNU114001 showed significant antagonistic activity against pathogenic fungi by dual culture method. The isolate CNU114001 was identified as Bacillus amyloliquefaciens by morphological observation and molecular data analysis, including 16SrDNA and gyraseA (gyrA) gene sequences. Antifungal substances of the isolate were extracted and purified by silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. The heat and UV ray stable compound was identified as iturin, a lipopeptide (LP). The isolate CNU114001 showed broad spectrum activity against 12 phytopathogenic fungi by dual culture method. The semi purified compound significantly inhibits the mycelial growth of pathogenic fungi (Alternaria panax, Botrytis cinera, Colletotrichum orbiculare, Penicillium digitatum, Pyricularia grisea and Sclerotinia sclerotiorum) at 200 ppm concentration. Spore germ tube elongation of Botrytis cinerea was inhibited by culture filtrate of the isolate. Crude antifungal substance showed antagonistic activity against cucumber scleotiorum rot in laboratory, and showed antagonistic activity against tomato gray mold, cucumber, and pumpkin powdery mildew in greenhouse condition.

Keywords: Antifungal substance; Bacillus amyloliquefaciens; Fungal plant pathogen; Iturin.

Fig. 1

Phylogenetic tree of the isolate CNU 114001 and other Bacillus species from Gene Bank based on 16S rRNA gene. Phylogenetic tree was constructed by the MEGA 5.0 program using maximum likelihood method. The boostrap analysis was performed with 1,000 replications. The present isolate is shown in bold (T, type strain).

Fig. 2

Antifungal activity of crude substance of CNU114001 against four pathogenic fungi. A, Botrytis cinera; B, Colletotrichum acutatum; C, Fusarium sp.; D, Penicillium digitatum (a, hexane; b, chloroform; c, ethyl acetate; d, buthatnol; e, water).

Fig. 3

Antifungal activity of different fractions against Botrytis cinerea (A). Fractions from silica gel column chromatography on thin layer chromatography plate eluted with CHCl₃ :MeOH : water = 65 : 25 : 4 v/v. B, 254 nm UV light; C, 365 nm UV light; D, Sprayed with water.

Fig. 4

High performance liquid chromatography profiles of isolated compounds from Bacillus amyloliquefaciens CNU114001 compares with iturin and fengycin.

Fig. 5

Morphological changes of Botrytis cinerea by dual culture. A, Normal mycelia; B, Mycelia from co-inoculated with CNU114001. The arrows indicate the swollen balloon-like structure (scale bars: A, B = 50 µm).

Fig. 6

Antifungal activity of the compound against 8 pathogenic fungi in potato dextrose agar. Ap, Alternaria panax; Bc, Botrytis cinerea; Cc, Corynespora cassicola; Co, Colletotrichum orbiculrae; Fo, F. oxysporum; Pd, Penicillium digitatum; Pg, Pyricularia grisea; Ss, Sclerotinia sclerotiorum.

Fig. 7

Effect of crude antifungal substance to control sclerotinia rot of cucumber fruit. A, 10 ppm; B, 50 ppm, C, 100 ppm.