Entomopathogenic fungus Beauveria bassiana-based bioinsecticide suppresses severity of powdery mildews of vegetables by inducing the plant defense responses

Abstract

The entomopathogenic fungus Beauveria bassiana is used commercially as a microbial insecticides against a wide range of agricultural insect pests. Some strains of B. bassiana protect the plants from pathogens, but the underlying mechanisms are largely unknown. Here, we found that prophylactic sprays of commercial bioinsecticide Botanigard on cucumber, tomato, and strawberry plants suppressed the severity of economically damaging powdery mildews. On leaf surfaces, hyphal elongation and spore germination of cucumber powdery mildew, Podosphaera xanthii, were inhibited, but B. bassiana strain GHA, the active ingredient isolated from Botanigard, only inhibited hyphal elongation but had no effect on spore germination of P. xanthii. In addition, strain GHA suppressed powdery mildew symptoms locally, not systemically. Treatment with Botanigard and strain GHA induced a hypersensitive response (HR)-like cell death in epidermal cells of the cucumber leaves in a concentration-dependent manner and inhibited penetration by P. xanthii. Transcriptome analysis and mass spectrometry revealed that GHA induced expression of salicylic acid (SA)-related genes, and treatment with Botanigard and GHA increased the SA level in the cucumber leaves. In NahG-transgenic tomato plants, which do not accumulate SA, the biocontrol effect of tomato powdery mildew by GHA was significantly reduced. These results suggested that B. bassiana GHA induces SA accumulation, leading to the induction of HR-like cell death against powdery mildew and subsequent suppression of fungal penetration. Thus, Botanigard has the potential to control both insect pests and plant diseases.

Keywords: Podosphaera xanthii; biocontrol agent; biofungicide; dual control; endophyte; induced resistance; plant-microbe interaction; salicylic acid.

Figure 1

Biocontrol effect of bioinsecticide Botanigard against powdery mildews of vegetables and whitefly in the greenhouse. (A) Cucumber leaves were treated with distilled water (Mock) or a 1,000-fold dilution of Botanigard and, were inoculated with a spore suspension of powdery mildew P. xanthii. Photograph was taken 10 days after inoculation. (B) Mean relative disease severity (± SD) on the cucumber leaves that were treated with a 1,000-fold dilution of Botanigard from 3 days before to 3 days after inoculation with a spore suspension of P. xanthii. Disease severity was assessed 12 days after inoculation with mean disease severity on mock plants set as 100. (C) Spore germination and hyphal growth on leaves stained with lactophenol aniline blue were measured using a microscope. Hyphae of P. xanthii are thick, whereas those of B. bassiana are thin. Arrow heads indicate hyphal interaction. Bars = 100 µm. Asterisks indicate a significant difference compared to the mock treatment (*p < 0.01) in the Mann–Whitney U-test. (D) Cucumber, tomato, and strawberry plants in the greenhouse were treated with 1,000-fold dilution of Botanigard or not (Untreated). The mean disease severity on the mock plants was set as 100, and the relative disease severity on treated plants was determined. Field trials were replicated at different locations as described in Supplemental Table 1 (N = 6 for cucumbers and N = 3 for tomatoes and strawberries, three replications in each trial). The whitefly larvae and adults were counted and means were compared between treatments.

Figure 2

Suppressive effect of cucumber powdery mildew and induction of plant resistance by Beauveria bassiana strain GHA. (A) One-half of each cucumber leaf was treated with a spore suspension of B. bassiana GHA (1 × 10⁹ spores/mL) and the other half with water (Mock), and, then, the entire leaf was inoculated with a spore suspension of powdery mildew Podosphaera xanthii. A representative leaf at 10 days after inoculation is shown. (B, C) Leaves of cucumber treated with distilled water (Mock), 1,000-fold dilution of Botanigard or a spore suspension of B. bassiana (1 × 10⁷, 1 × 10⁸, or 1 × 10⁹ spores/mL) and then inoculated with a spore suspension of P. xanthii. (B) Relative disease severity at 12 days after inoculation based on average severity on mock-treated plants as 100. (C) Hypersensitive response (HR)–like cell death in epidermal cells under germ tubes (gt) and spore (s), and spore germination of P. xanthii at 24 h. Bar = 20 µm. Asterisks indicate a significant difference compared to the mock treatment (*p < 0.005 and **p < 0.001) in the Mann–Whitney U-test.

Figure 3

Transcriptome and Gene Ontology (GO) enrichment analysis of the cucumber leaves treated with Beauveria bassiana strain GHA. Leaves were treated with a spore suspension of GHA or mock and then inoculated with a spore suspension of powdery mildew Podosphaera xanthii. (A) Volcano plot of differentially expressed genes (DEGs) between GHA- and mock-treated cucumber leaves. Red and blue points represent upregulated and downregulated DEGs, respectively (p < 0.05). (B) GO enrichment analysis of upregulated DEGs. GO enrichment were classified by GO terms for molecular function (orange) and biological process (green) (p < 0.05).

Figure 4

Effects of pretreatment of cucumber seedlings with Beauveria bassiana GHA on phytohormone levels at 24 h after inoculation. Seedlings were sprayed with distilled water (Mock), a 1,000-fold dilution of Botaniguard or a spore suspension of B. bassiana (1 × 10⁷, 1 × 10⁸, or 1 × 10⁹ spores/mL), and inoculated with a spore suspension of powdery mildew Podosphaera xanthii. Data are means ± SD of three independent measurements. Asterisks indicate a significant difference compared to the mock treatment at p < 0.05 in the Mann–Whitney U-test.

Figure 5

Effect of Beauveria bassiana GHA on NahG tomato plants against tomato powdery mildew. Leaf disks of cv. Moneymaker (MM) and Moneymaker-NahG (MM-NahG) were sprayed with distilled water (Mock) or a spore suspension of B. bassiana (1 × 10⁹ spores/mL) and then inoculated with tomato powdery mildew Pseudoidium neolycopersici. Fungal biomass in tomato leaves was quantified by a quantitative real-time PCR on the basis of amplification of the alpha-tubulin gene of P. neolycopersici compared with the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene in tomato. Asterisks indicate a significant difference (*p < 0.05 and **p < 0.005) in the Mann–Whitney U-test.