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Cited by 53Plant-Microbe Interactions and ImmunityPowdery Mildew Fungal DiseasesPlant Disease Resistance and Genetics

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Trichoderma asperellum (T42) and Pseudomonas fluorescens (OKC)-Enhances Resistance of Pea against Erysiphe pisi through Enhanced ROS Generation and Lignifications

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Patel, Jai S., Kharwar, Ravindra N., Singh, Harikesh B., Upadhyay, Ram S., Sarma, Birinchi K.·2017·Frontiers in Microbiology
DOI 10.3389/fmicb.2017.00306PMC PMC5332396
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Abstract

Plant signalling mechanisms are not completely understood in plant-fungal biotrophic pathogen interactions. Further how such interactions are influenced by compatible rhizosphere microbes are also not well studied. Therefore, we explored the pea-Erysiphe pisi (obligate biotroph) system to understand the interaction and applied compatible rhizospheric bio-agents Trichoderma asperellum (T42) and Pseudomonas fluorescens (OKC) singly or in combination to assess their influence on the host while under the pathogen challenge. Transcript accumulation pattern of some vital genes in the lignin biosynthetic pathway in pea under E. pisi challenge indicated enhanced activation of the pathway. Interestingly, transcript accumulations were even higher in the bio-agent treated plants compared to untreated plants after pathogen inoculation particularly in co-inoculated treatments. Further, down regulation of the lignifications-associated ABC transporter gene in the pathogen challenged plants possibly is an indication of passive diffusion of monolignols across the membrane from symplast. Additionally, up regulation of NADPH oxidase gene revealed ROS generation in the challenged plants which was confirmed through spectrophotometric estimation of H2O2. Up regulation of laccase and peroxidase along with higher H2O2 generation points out their involvement in lignifications which was further confirmed through cross section analysis of pea stems that showed increased lignifications in pathogen challenged plants co-inoculated with the bioagents. Interestingly, pathogen responsive MAPK homologs MAPK3/MAPK6 and the enzyme serine threonine kinase (STK) that activates MAPKs were down regulated and the results possibly indicate non-participation of the MAPK cascade in this interaction. Therefore, it can be concluded that the microbial treatments enhanced pea resistance to E. pisi by generation of ROS and lignifications.

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FIGURE 1 Reactive oxygen species (ROS) generation and disease development in pea following inoculation with Erysiphe pisi. NADPH oxidase activity (A) after 48 h of pathogen inoculation which leads to production of ROS H2O2 (B). HR like responses (C) observed after 72 h of pathogen inoculation around the developing conidia (arrowhead). HR-like response was more in microbial treatments (OP, TP, and OTP) compared to non-microbial treatment (P). E. pisi development on pea leaves in different microbial treatments after 48 h and on intact plants after 10 days of pathogen inoculation (D). Germ tube development of E. pisi on pea leaves in different treatments after 48 h (E). Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars in (A,B,E) represent SD (standard deviation) from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).
FIGURE 1 Reactive oxygen species (ROS) generation and disease development in pea following inoculation with Erysiphe pisi. NADPH oxidase activity (A) after 48 h of pathogen inoculation which leads to production of ROS H2O2 (B). HR like responses (C) observed after 72 h of pathogen inoculation around the developing conidia (arrowhead). HR-like response was more in microbial treatments (OP, TP, and OTP) compared to non-microbial treatment (P). E. pisi development on pea leaves in different microbial treatments after 48 h and on intact plants after 10 days of pathogen inoculation (D). Germ tube development of E. pisi on pea leaves in different treatments after 48 h (E). Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars in (A,B,E) represent SD (standard deviation) from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central
FIGURE 2 Transcript accumulation pattern of MAP Kinases and their phosphorylating enzyme Serine/Threonine Kinase in pea leaves after 24 h of E. pisi inoculation. (A) STK (Serine/threonine kinase), (B) MAP kinase 3 and (C) MAP kinase 6. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).
FIGURE 2 Transcript accumulation pattern of MAP Kinases and their phosphorylating enzyme Serine/Threonine Kinase in pea leaves after 24 h of E. pisi inoculation. (A) STK (Serine/threonine kinase), (B) MAP kinase 3 and (C) MAP kinase 6. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central
FIGURE 3 Phenylpropanoid activities in pea leaves after 24 h of E. pisi inoculation. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).
FIGURE 3 Phenylpropanoid activities in pea leaves after 24 h of E. pisi inoculation. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central
FIGURE 4 Lignin monomer biosynthetic pathway demonstrating higher transcript accumulation of the key genes (shown in red colors). increase in color intensity showed level of expression of the genes in pea leaves.
FIGURE 4 Lignin monomer biosynthetic pathway demonstrating higher transcript accumulation of the key genes (shown in red colors). increase in color intensity showed level of expression of the genes in pea leaves.CC BY · PubMed Central
FIGURE 5 Transcript accumulation pattern of some key genes involved in biosynthesis of lignin monomers after 24 h of E. pisi inoculation on pea leaves. Transcript accumulation pattern of PAL (Phenylalanine ammonia lyase) (A), C4H (Cinnamate 4-hydroxylase) (B), COMT (Caffeic acid O-methyltransferase) (C), HCT (Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase) (D), CCoAOMT (Caffeoyl CoA O-methyltransferase) (E), F5H (Ferulate 5-hydroxylase) (F), Laccase (G), PO (Peroxidase) (H). Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).
FIGURE 5 Transcript accumulation pattern of some key genes involved in biosynthesis of lignin monomers after 24 h of E. pisi inoculation on pea leaves. Transcript accumulation pattern of PAL (Phenylalanine ammonia lyase) (A), C4H (Cinnamate 4-hydroxylase) (B), COMT (Caffeic acid O-methyltransferase) (C), HCT (Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase) (D), CCoAOMT (Caffeoyl CoA O-methyltransferase) (E), F5H (Ferulate 5-hydroxylase) (F), Laccase (G), PO (Peroxidase) (H). Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central
FIGURE 6 Transcript accumulation pattern of the ABC transporter gene in pea leaves. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).
FIGURE 6 Transcript accumulation pattern of the ABC transporter gene in pea leaves. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central
FIGURE 7 (A) Histochemical staining demonstrating lignin deposition in transverse sections of pea stem in different treatments after 15 days of E. pisi inoculation. Pink color represents lignin deposition. Intensity of color represents higher deposition of lignin. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. (B) Quantitative estimation of lignin in all treatments in leaf and stem. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments in the same plant part (P ≤ 0.05; Tukey’s test).
FIGURE 7 (A) Histochemical staining demonstrating lignin deposition in transverse sections of pea stem in different treatments after 15 days of E. pisi inoculation. Pink color represents lignin deposition. Intensity of color represents higher deposition of lignin. Where O = Pseudomonas fluorescens (OKC), T = Trichoderma asperellum (T42), P = pathogen (E. pisi), C = control. (B) Quantitative estimation of lignin in all treatments in leaf and stem. Error bars represent SD from means of three measurements. Different superscript letters indicate data significantly different from the other treatments in the same plant part (P ≤ 0.05; Tukey’s test).CC BY · PubMed Central

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Metadata

Authors
Patel, J. S., Kharwar, R. N., Singh, H. B., Upadhyay, R. S., & Sarma, B. K.
Affiliations
Banaras Hindu University (IN)
Corresponding author
Sarma, Birinchi K.
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2017-03-02
Volume
08
Pages
306-306
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2017.00306
PMC
PMC5332396
OpenAlex
W2593940733
Cited by
53
References
1
Citations by year
2026: 2 · 2025: 6 · 2024: 3 · 2023: 6 · 2022: 11 · 2021: 7 · 2020: 6 · 2019: 7 · 2018: 3 · 2017: 2
FWCI
4.85
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant-Microbe Interactions and Immunity
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Powdery Mildew Fungal Diseases
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Disease Resistance and Genetics
Keywords
Trichoderma asperellum, Pseudomonas fluorescens, pea, Erysiphe pisi, powdery mildew, plant disease resistance, lignification, lignin biosynthesis, reactive oxygen species, NADPH oxidase, hydrogen peroxide, laccase, peroxidase, ABC transporters, MAPK signaling, gene expression

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