Synergistic Effect of Beauveria bassiana and Trichoderma asperellum to Induce Maize (Zea mays L.) Defense against the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera, Crambidae) and Larval Immune Response

Abstract

Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

Keywords: Beauveria bassiana; Ostrinia furnacalis; Trichoderma asperellum; combine application; entomopathogenic fungi; transcriptomic response.

Figure 1

Mortality (%) of O. furnacalis larvae fed on B. bassiana OFDH1-5 (A) and T. asperellum GDFS1009 (B) incorporated diet at the first, third, and fifth day post treatment (DPT). Means followed by different lowercase letters above each bar indicates significant differences among the treatments (p < 0.05).

Figure 2

Mortality (%) of O. furnacalis larvae fed on diet incorporated with binary combination of B. bassiana OFDH1-5 + T. asperellum GDFS1009 in different proportions recorded at 1, 3, and 5 days post treatment (DPT). Means followed by different lowercase letters above each bar indicates significant differences among the treatments (p < 0.05).

Figure 3

Scanning electron micrograph of infected larva reveals spores and hyphae structures in body cavity; B. bassiana OFDH1-5 (A); T. asperellum GDFS1009 (B) and B. bassiana OFDH1-5 + T. asperellum GDFS1009 (C). Arrow indicates spores (S) and hyphae (Hy).

Figure 4

Scanning electron micrograph sowing cuticle proliferation (C-pro) due to hyphal penetration in infected larval body: B. bassiana OFDH1-5 (A); T. asperellum GDFS1009 (B) and B. bassiana OFDH1-5 + T. asperellum GDFS1009 (C). Arrow indicates cuticle proliferation (C-pro), spores (S) and hyphae (Hy).

Figure 5

Scanning electron micrograph of infected larval body fully covered with myceli and a closer view showing dense structure of fungal spores and hyphae growing on the body surface: B. bassiana OFDH1-5 (A); T. asperellum GDFS1009 (B) and B. bassiana OFDH1-5 + T. asperellum GDFS1009 (C). Arrow indicates spores (S) and hyphae (Hy).

Figure 5

Scanning electron micrograph of infected larval body fully covered with myceli and a closer view showing dense structure of fungal spores and hyphae growing on the body surface: B. bassiana OFDH1-5 (A); T. asperellum GDFS1009 (B) and B. bassiana OFDH1-5 + T. asperellum GDFS1009 (C). Arrow indicates spores (S) and hyphae (Hy).

Figure 6

Effect of entomopathogenic fungi on POD (A); SOD (B); proline (C); protease (D) and PPO (E) activity of plants at 0 h and 72 h of insect attack. Treatment detail: uninoculated and uninfested control (C); seed coating with B. bassiana OFDH1-5 (T1); soil drenching with B. bassiana OFDH1-5 (T2); seed coating with T. asperellum GDFS1009 (T3); soil drenching with T. asperellum GDFS1009 (T4); seed treatment with binary combination of B. bassiana OFDH1-5 + T. asperellum GDFS1009 (T5); soil drenching with binary combination of B. bassiana OFDH1-5 + T. asperellum GDFS1009 (T6); infested control (insect control) (T7). Means followed by different lowercase letters above each bar indicates significant differences among the treatments (p < 0.05).

Figure 7

Effect of entomopathogenic fungi on chlorophyll a (A); chlorophyll b (B); carotenoids (C) and total chlorophyll content (D) of plants, at 0 h and 72 h of insect attack. Treatment detail: uninoculated and uninfested control (C); seed coating with B. bassiana OFDH1-5 (T1); soil drenching with B. bassiana OFDH1-5 (T2); seed coating with T. asperellum GDFS1009 (T3); soil drenching with T. asperellum GDFS1009 (T4); seed treatment with binary combination of B. bassiana OFDH1-5 + T. asperellum GDFS1009 (T5); soil drenching with binary combination of B. bassiana OFDH1-5 + T. asperellum GDFS1009 (T6); infested control (insect control) (T7).2.4. Transcriptome analysis and identification of differentially expressed genes (DEGs) in ACB. Means followed by different lowercase letters above each bar indicates significant differences among the treatments (p < 0.05).

Figure 8

PCA analysis showing variability of data (A) and hierarchical cluster analysis showing expression level of DEGs in all treatments (B). Green color indicates genes with a higher expression and red color indicates lower expression.

Figure 9

Gene Ontology (GO) classifications of differentially expressed genes. The differentially expressed genes were grouped into three hierarchically stretched GO terms, biological process, cellular component and molecular functions. Control vs B. bassiana (Control vs. BB) (A); Control vs B. bassiana + T. asperellum (Control vs. BB+TH) (B) and Control vs T. asperellum (control vs. TH) (C).

Figure 10

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment scatter plot. The vertical axis represents the path name, and the horizontal axis represents the path factor corresponding to the Rich factor. The size of the q-value is represented by the color of the point. The smaller the q-value, the closer the color is to the red color. The number of differential genes included in each pathway are expressed by the size of the point. Control vs B. bassiana (Control vs. BB) (A); Control vs B. bassiana + T. asperellum (Control vs. BB+TH) (B) and Control vs T. asperellum (Control vs. TH) (C).

Figure 11

The qRT-PCR analysis of selected differentially expressed genes to confirm expression patterns indicated by RNA-sequencing. UDP-glucuronosyltransferase gene-LOC114350074 (A); larval/pupal rigid cuticle protein gene-LOC114366353 (B); larval/pupal rigid cuticle protein 2 gene-LOC114366367 (C); ABC transporter 1 gene-LOC114354308 (D); ABC transporter 2 gene-LOC114353130 (E); cadherin gene-LOC114362562 (F); HSP70 gene-LOC114356221 (G); HSP68 gene-LOC114363773 (H); cytochrome P450 gene-LOC114353087 (I); PRPB gene-LOC114352113 (J) and PRPS gene-LOC114352122 (K). Means followed by different lowercase letters above each bar indicates significant differences among the treatments (p < 0.05).