Streptomyces Strains Induce Resistance to Fusarium oxysporum f. sp. lycopersici Race 3 in Tomato Through Different Molecular Mechanisms

Abstract

Plant growth promoting rhizobacteria (PGPR) are potential natural alternatives to chemical fungicides in greenhouse production via inducing plant immune system against biotic stresses. In this research, 126 Streptomyces isolates were recovered from rhizosphere soils of 13 different commercial vegetable greenhouses in Iran. Streptomyces isolates were screened for in vitro Plant growth promoting (PGP) traits and ability to antagonize Fusarium oxysporum f. sp. lycopersici race 3 (FOL), the causal agent of Fusarium wilt of tomato (FWT). Six isolates with the highest antagonistic activity and at least three PGP traits were selected and compared with chemical fungicide Carbendazim^® in a greenhouse experiment. All bacterial treatments mitigated FWT disease symptoms like chlorosis, stunting and wilting at the same level or better than Carbendazim^®. Strains IC10 and Y28 increased shoot length and shoot fresh and dry weight compared to not inoculated control plants. Phenotypic characterization and 16S rRNA gene sequencing showed, strains IC10 and Y28 were closely related to S. enissocaesilis and S. rochei, respectively. The ability of the superior biocontrol strains to induce antioxidant enzymes activity and systemic resistance (ISR) was investigated. Increased activity of catalase (CAT) in plant treated with both strains as well as an increase in peroxidase (POX) activity in plants treated with Y28 pointed to a strain specific-induced systemic resistance (ss-ISR) in tomato against FOL. The differential induced expression of WRKY70 and ERF1 (two transcription factors involved in plant defense) and LOX and TPX by the analyzed Streptomyces strains, especially after inoculation with FOL, suggests that ss-ISR is triggered at the molecular level.

Keywords: Fusarium wilt; Streptomyces; induced systemic resistance; siderophore production; tomato growth promoting.

FIGURE 1

The percent (%) of colonies isolated from Isfahan, Yazd, and Kerman provinces: colonies (A) with Streptomyces appearance (B) growing in N-free medium, (C) phosphate solubilizing, and (D) siderophore producing.

FIGURE 2

Phylogenetic tree based on almost complete 16S rRNA gene sequences of S. enissocaesilis strain IC10 and S. rochei strain Y28. Tree was calculated using neighbor-joining method illustrating the taxonomic position of the strains with related species. Accession numbers of the sequences are given in parentheses. The sequences of Alloactinosynnema album 03-9939T (EU438907) was used as outgroup. Bootstrap values are based on 1000 resampling and shown at the branching points. Bar indicates 0.01 substitutions per nucleotide position.

FIGURE 3

Biocontrol of tomato wilt and stunt caused by FOL using S. enissocaesilis strain IC10 and S. rochei strain Y28 in greenhouse conditions. Data recorded 60 days after bacterial treatment. Stunting is seen in infected plants. Arrows showing wilting symptoms (chlorosis and dried leaves) in FOL inoculated plants.

FIGURE 4

Effect of biological (S. enissocaesilis strain IC10 and S. rochei strain Y28) and chemical (SA and MeJA) treatments on the induction of peroxidase (POX) activity in tomato leaves non-inoculated (left) and inoculated (right) with FOL at different time intervals of inoculation (df = 29; F = 2.90; P < 0.01). Data represent the mean values ± SE of three biological replicates. Negative control: untreated and non-inoculated. In each treatment, the values marked with an asterisk are significantly (P < 0.05) different from negative control at time point 0.

FIGURE 5

Effect of biological (S. enissocaesilis strain IC10 and S. rochei strain Y28) and chemical (SA and MeJA) treatments on the induction of catalase (CAT) activity in tomato leaves non-inoculated (left) and inoculated (right) with FOL at different time intervals of inoculation (df = 29; F = 30.73; P < 0.01). Data represent the mean values ± SE of three biological replicates. Negative control: untreated and non-inoculated. In each treatment, the values marked with an asterisk are significantly (P < 0.05) different from negative control at time point 0.

FIGURE 6

The relative level of gene expression (fold) determined by qRT-PCR of seven target genes including UDP (A), PAL (B), WRKY70 (C), TPX (D), LOX (E), PR1 (F), and ERF1 (G) versus reference control (Tubulin gene) in tomato “Riogrande” treated with chemical (MeJA and SA) or biological agents (S. enissocaesilis strain IC10 and S. rochei strain Y28) 48 h after inoculation with FOL/ distilled water. Untreated and non-inoculated plants were considered as control (C) and as reference sample. Standard error represents for three biological replicates. Positive values of fold change indicate up-regulation while negative values indicate down-regulation. The values marked with one and two asterisk are significantly different from control at P < 0.05 and P < 0.01, respectively.