Taxonomy and Broad-Spectrum Antifungal Activity of Streptomyces sp. SCA3-4 Isolated From Rhizosphere Soil of Opuntia stricta

Abstract

Actinobacteria are important producers of bioactive compounds. Extreme ecosystems cause evolution of novel secondary metabolic pathways of Actinobacteria and increase the possible discovery of new biological functions of bioactive compounds. Here, we isolated 65 Actinobacteria from rhizosphere soil samples of Opuntia stricta. An Actinobacteria strain (named SCA3-4) was screened against Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4, ATCC 76255). The strain produced pink-white aerial mycelia and brown substrate mycelium on Gause No. 1 agar. Biverticillate chains of cylindrical spores were observed by scanning electron microscopy (SEM). Based on alignment of 16S rRNA sequences, a constructed phylogenetic tree showed that strain SCA3-4 shared a 99.54% similarity with Streptomyces lilacinus NRRL B-1968T. The morphological, biochemical, physiological, and molecular characteristics further indicated that strain SCA3-4 belongs to the Streptomyces sp. It can grow well on medium with the following antibiotics chloramphenicol, streptomycin, penicillin-G, gentamicin, erythromycin, nystatin or neomycin sulfate. The polymerase chain reaction (PCR) amplification of types I and II polyketide synthase genes (PKS-I and PKS-II) suggested its bioactive potential. Under treatment with 100 μg/ml of ethyl acetate extracts isolated from Streptomyces sp. SCA3-4, growth of Foc TR4 was inhibited and cell membrane was destroyed. Crude extracts also showed a broad-spectrum antifungal activity against 13 phytopathogenic fungi including Foc TR4 and displayed the lowest minimum inhibitory concentration (MIC) (0.781 μg/ml) against Colletotrichum fragariae (ATCC 58718). A total of 21 different compounds identified by gas chromatography-mass spectrometry (GC-MS) were composed of phenolic compound, pyrrolizidine, hydrocarbons, esters, and acids. Besides the known active compounds, Streptomyces sp. SCA3-4 possesses antimicrobial or other biological activities. Further attention will be paid on other compounds with no functional annotation, aiming at the discovery of new bioactive substances.

Keywords: Actinobacteria; GC-MS; Streptomyces lilacinus; antifungal activity; banana Fusarium wilt; biosynthetic genes.

FIGURE 1

Inhibition of strain SCA3-4 on the mycelium growth of Foc TR4. Control, plate inoculated only with Foc TR4; Treatment, plate inoculated with Foc TR4 and strain SCA3-4.

FIGURE 2

Morphologic characteristics of mycelium (A) as well as spore chain and spore (B) of strain SCA3-4.

FIGURE 3

Construction of phylogenetic tree based on the 16S rRNA sequences from strain SCA3-4 and other selected strains. The phylogenetic tree was constructed using the neighbor-joining method. Numbers on the branches are bootstrap values calculated from 1,000 replicates. Bar, 0.002 substitutions per nucleotide position.

FIGURE 4

Effects of the crude extracts of Streptomyces sp. SCA3-4 on spore germination of Foc TR4. Control, treatment with 10% DMSO; Treatment, treatment with the crude extracts (100 μg/ml) of Streptomyces sp. SCA3-4.

FIGURE 5

Transmission electron microscopy micrographs of Foc TR4 treated with crude extracts (100 μg/ml) of Streptomyces sp. SCA3-4. (A–C) Treatment with 10% DMSO. (D–F) Treatment with the crude extracts.

FIGURE 6

Chemical structures of the identified compounds from crude extracts of Streptomyces sp. SCA3-4.