Plant growth promotion and differential expression of defense genes in chilli pepper against Colletotrichum truncatum induced by Trichoderma asperellum and T. harzianum

Abstract

Background: Trichoderma asperellum and T. harzianum were assessed in this study as a potential biological control against Colletotrichum truncatum. C. truncatum is a hemibiotrophic fungus that causes anthracnose disease in chilli thereby affecting plant growth and fruit yield. Scanning electron microscope (SEM) technique showed the beneficial interaction between chilli root-Trichoderma spp. inducing the plant growth promotion, mechanical barrier, and defense network under C. truncatum challenged conditions.

Methods: Seeds bio-primed with T. asperellum, T. harzianum, and T. asperellum + T. harzianum promoted the plant growth parameters and strengthening of physical barrier via lignification on the wall of vascular tissues. Seed primed with bioagents were used for exploring the molecular mechanism of defense response in pepper against anthracnose to assess the temporal expression of six defense genes in the Surajmukhi variety of Capsicum annuum. QRT-PCR demonstrated induction of defense responsive genes in chilli pepper bioprimed with Trichoderma spp. such as plant defensin 1.2 (CaPDF1.2), superoxide dismutase (SOD), ascorbate peroxidase (APx), guaiacol peroxidase (GPx), pathogenesis related proteins PR-2 and PR-5.

Results: The results showed that bioprimed seeds were assessed for T. asperellum, T. harzianum, and T. asperellum + T. harzianum-chilli root colonization interaction under in vivo conditions. The results of the scanning electron microscope revealed that T. asperellum, T. harzianum and T. asperellum + T. harzianum interact with chilli roots directly via the development of plant-Trichoderma interaction system. Seeds bio-primed with bioagents promoted the plant growth parameters, fresh and dry weight of shoot and root, plant height, leaf area index, number of leaves, stem diameter and strengthening of physical barrier via lignification on the wall of vascular tissues and expression of six defense related genes in pepper against anthracnose.

Conclusions: Application of T. asperellum and T. harzianum and in combination of treatments enhanced the plant growth. Further, as seeds bioprimed with T. asperellum, T. harzianum and in combination with treatment of T. asperellum + T. harzianum induced the strengthening of the cell wall by lignification and expression of six defense related genes CaPDF1.2, SOD, APx, GPx, PR-2 and PR-5 in pepper against C. truncatum. Our study contributed for better disease management through biopriming with T. asperellum, T. harzianum and T. asperellum + T. harzianum. The biopriming possess enormous potential to promote plant growth, modulate the physical barrier, and induced the defense related genes in chilli pepper against anthracnose.

Keywords: Bio-priming; Capsicum annuum; Defense related genes; Electron microscopy; Growth promotion; Lignification; Real-time PCR.

Fig. 1

SEM images of chilli root colonized by Trichderma spp. A Uncolonized roots, B Colonized by T. asperellum, C Colonized by T. harzianum, D Colonized by T. asperellum + T. harzianum. The arrows indicate conidia of Trichoderma spp.: valleys (V), root surface (RS)

Fig. 2

(I) Morphological growth characteristics of T. asperellum, T. harzianum and T. asperellum + T. harzianum bioprimed and untreated chilli plants at different intervals of 30 and 60 days. Seeds bioprimed samples were found to have increased the root length, profuse growth and thickness. A Image showing the unprimed (control) plant after 30 and 60 days, B T. asperellum bioprimed seeds after 30 and 60 days, C T. harzianum bioprimed seeds after 30 and 60 days, D T. asperellum + T. harzianum bioprimed seeds after 30 and 60 days. (II) Bioprimed seeds with T. asperellum, T. harzianum and T. asperellum + T. harzianum showed their growth parameters, increased in height, number and size of leaves, branches, flowering buds, stem diameter of plants. A Unprimed (control) plants, B Seeds were primed T. asperellum, C Seeds were primed with T. harzianum, and D Seeds were primed with T. asperellum + T. harzianum. Scale bar represents 10 cm

Fig. 3

Effect of T. asperellum, T. harzianum and T. asperellum + T. harzianum bioprimed seeds on plant growth parameters of chilli under glasshouse conditions at 30 days. Each data indicated the mean of triplicates value, and the vertical bars give out the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The bar denotes the SE of the mean

Fig. 4

Effect of T. asperellum, T. harzianum and T. asperellum + T. harzianum bioprimed seeds on plant growth parameters of chilli under glasshouse conditions at 60 days. Each data indicated the mean of triplicates value, and the vertical bars give out the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The bar denotes the SE of the mean

Fig. 5

Assessment of plant mechanical barrier in the form of lignin deposition. The image indicates transverse section of chilli stem tissues with lignification in the wall of xylem strands collected from 4 different samples (unprimed, T. asperellum, T. harzianum and T. asperellum + T. harzianum bioprimed seeds) at time intervals 30 and 60 days. Intensity of red-pink color indicates the deposition of lignin. The lignified vascular bundles were seen on primary (protoxylem) and secondary xylem (metaxylem). A-B Microscopic view of transverse section of unprimed (control) plant samples at 30 days intervals. C-D Microscopic view of transverse section of T. asperellum bioprimed plant samples at 30 days. E–F Stem sections were taken from T. harzianum samples at 30 days. G-H Stem sections were taken of T. asperellum + T. harzianum samples at 30 days. I-J T. S of stem sections were taken from unprimed samples at 60 days. K-L Stem sections were taken from T. asperellum samples at 60 days. M–N T. S. of stem sections were taken from T. harzianum samples at 60 days. O-P T. S. of stem sections were taken from T. asperellum + T. harzianum samples at 60 days intervals. All the sections were observed at 4 × and 10 × magnification. Px, primary xylem; Sx, secondary xylem, Pi, pith; Ph, phloem; C, cambium; V, vessel; E, endodermis. Scale bar represents 250 µm

Fig. 6

Morphological analysis of suppression of anthracnose lesion development on matured fruits of Surajmukhi variety of Capsicum annuum induced by Trichoderma asperellum and T. harzianum and in combination of treatment of T. asperellum + T. harzianum upon C. truncatum challenged condition and C. truncatum inoculated fruits. The bioprimed seeds were grown in greenhouse condition and the fruits were infected with acervuli of C. truncatum under laboratory conditions. Seeds bioprimed with T. asperellum, T. harzianum and T. asperellum + T. harzianum under challenged conditions showed no lesion compared to C. truncatum inoculated fruits. Unprimed and unchallenged (control) showed no development of anthracnose lesion. Photographs were taken at 0 h, 2- and 4-days post inoculation (dpi)

Fig. 7

The expression of plant defensin (CaPDF1.2), antioxidative (SOD, APx, and GPx) and pathogenesis related protein (PR-2 and PR-5) genes in pepper bioprimed with T. asperellum, T. harzianum and T. asperellum + T. harzianum upon challenged, C. truncatum inoculated and control samples. Each data indicated the mean of triplicates value, and the vertical bars give out the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The Error bars indicate SD, and the asterisk indicates a significant difference. (Student’s t-test; ⁎ p < 0.005; ⁎⁎ p < 0.01; ⁎⁎⁎ p < 0.001)