Less Virulent Leptosphaeria biglobosa Immunizes the Canola Plant to Resist Highly Virulent L. maculans, the Blackleg Pathogen

Abstract

Leptosphaeria biglobosa is a less virulent Leptosphaeria spp. that causes blackleg disease in canola. Previous studies from our lab have shown that inoculation with the less virulent L. biglobosa can boost the resistance of canola plants against the highly virulent L. maculans. The objective of this study was to confirm the effectiveness of L. biglobosa as a biocontrol agent against L. maculans utilizing morphology, fluorescence microscopy, gene quantification, and transcriptomic analysis. The in planta development of two Leptosphaeria species inoculated at different time points was assessed using fluorescent protein-tagged isolates which are GFP-tagged L. maculans and DsRed-tagged L. biglobosa. The growth inhibition of L. maculans by pre-and co-inoculated L. biglobosa was supported by no lesion development on cotyledons and no or weak fluorescence protein-tagged mycelia under the confocal microscope. The host defense-related genes, WRKY33, PR1, APX6, and CHI, were upregulated in L. biglobosa inoculated Westar cotyledons compared to L. maculans inoculated cotyledons. The quantification of each pathogen through qPCR assay and gene expressions analysis on host defense-related genes by RT-qPCR confirmed the potential of L. biglobosa "brassicae' in the management of the blackleg disease pathogen, L. maculans 'brassicae', in canola.

Keywords: Brassica napus; Leptosphaeria biglobosa; Leptosphaeria maculans; biological control.

Figure 1

Blackleg disease lesion development in Westar cotyledons at 14 days post-inoculation under controlled conditions with Leptosphaeria maculans ‘brassicae’ and L. biglobosa ‘brassicae’ inoculations. Seven-day-old cotyledons were wound inoculated and observed the lesion development in time course. Cotyledons inoculated with only GFP-tagged L. maculans (LmGFP) and cotyledons pre-inoculated with LmGFP showed significantly higher lesion development at 14 dpi. Control: distilled water treatment; Treatment 1: LbDsRed inoculation; Treatment 2: LmGFP inoculation; Treatment 3: LbDsRed and LmGFP co-inoculation; Treatment 4: LbDsRed followed by LmGFP inoculation (in 24 h intervals); Treatment 5: LmGFP followed by LbDsRed inoculation (in 24 h intervals). (a) Scanned images of disease lesion development in cotyledons; (b) Lesion size shown as a percentage of the total leaf area of both host types calculated by ImageJ. Different letters refer to significant differences between treatments (p ≤ 0.05).

Figure 2

Gel image of species-specific PCR products of Leptosphaeria maculans ‘brassicae’ and L. biglobosa ‘brassicae’ isolates in inoculated Westar cotyledons. PCR identification of L. biglobosa and L. maculans with species-specific primers, LmacF/LmacR and LbigF/LmacR in a multiplex PCR. L. biglobosa ‘brassicae’ and L. maculans ‘brassicae’ gave PCR bands of 444 bp and 331 bp, respectively. PCR products were resolved by electrophoresis through a 1% (w/v) agarose gel. Lane 1: L. maculans ‘brassicae’ inoculated cotyledon gDNA; Lane 2: L. biglobosa ‘brassicae’ inoculated cotyledon gDNA; Lane 3: both isolates co-inoculated cotyledon gDNA; M: 100 bp DNA ladder.

Figure 3

DsRed-tagged L. biglobosa (LbDsRed) and GFP-tagged L. maculans (LmGFP) in planta development of Westar cotyledons under controlled environment at 14 days post-inoculation. (a) Confocal microscopic observations of fluorescence protein-tagged Leptosphaeria isolates (10× magnification). (b) Quantification of each isolate in planta at 3-, 7-, 11-, and 14- dpi. LbDsRed and LmGFP mycelia shown in fluorescent red and green colors, respectively. In graphs, red and green colors represent LbDsRed and LmGFP DNA, respectively. (a,b) LbDsRed inoculation; (c,d) LmGFP inoculation; (e,f) LbDsRed and LmGFP co-inoculation; (g,h) LbDsRed followed by LmGFP inoculation with one day interval; (i,j) LmGFP followed by LbDsRed inoculation with one-day interval; (k) Chlorophyll autofluorescence of cellular chloroplasts of cotyledons in control.

Figure 3

DsRed-tagged L. biglobosa (LbDsRed) and GFP-tagged L. maculans (LmGFP) in planta development of Westar cotyledons under controlled environment at 14 days post-inoculation. (a) Confocal microscopic observations of fluorescence protein-tagged Leptosphaeria isolates (10× magnification). (b) Quantification of each isolate in planta at 3-, 7-, 11-, and 14- dpi. LbDsRed and LmGFP mycelia shown in fluorescent red and green colors, respectively. In graphs, red and green colors represent LbDsRed and LmGFP DNA, respectively. (a,b) LbDsRed inoculation; (c,d) LmGFP inoculation; (e,f) LbDsRed and LmGFP co-inoculation; (g,h) LbDsRed followed by LmGFP inoculation with one day interval; (i,j) LmGFP followed by LbDsRed inoculation with one-day interval; (k) Chlorophyll autofluorescence of cellular chloroplasts of cotyledons in control.

Figure 4

Relative transcript levels of several important genes involved in host defense signaling pathways were assessed by quantitative RT-PCR at 7 days post-inoculation. The expression of WRKY70 and PDF1.2 showed significantly higher expressions in Lm solely inoculated cotyledons. PR1, WRKY33, RbohD, APX6, and CHI showed significantly higher expression in cotyledons inoculated with only Lb. (a) Relative gene expression of WRKY70 and PR1; (b) Relative gene expression of WRKY33 and PDF1.2; (c) Relative gene expression of RbohD and APX6; (d) Relative gene expression of CHI; in Westar cotyledons inoculated with only DsRed-tagged L. biglobosa (Lb), with only GFP-tagged L. maculans (Lm) and co-inoculated with both isolates (Lm + Lb). Different letters refer to significant differences (p ≤ 0.05).

Figure 5

Expression profile of RbohD in Westar cotyledons infected with LmGFP, LbDsRed as sole and combined inocula. RbohD expression was upregulated at an exponential rate from 3- to 7- dpi. The observations were taken at 0-, 6-, 12- hpi and 1-, 3-, 7- dpi.

Figure 6

Schematic representation of the research design. Seven-day-old Westar cotyledons were wound inoculated with Control: distilled water treatment; Treatment 1: LbDsRed inoculation; Treatment 2: LmGFP inoculation; Treatment 3: LbDsRed and LmGFP co-inoculation; Treatment 4: LbDsRed followed by LmGFP inoculation (in 24-h intervals); Treatment 5: LmGFP followed by LbDsRed inoculation (in 24-h intervals). Inoculated cotyledons were collected at different time points to analyze the lesion development in planta mycelial development, pathogen quantification, and host-defense-related gene expressions.