Systemic Resistance in Chilli Pepper against Anthracnose (Caused by Colletotrichum truncatum) Induced by Trichoderma harzianum, Trichoderma asperellum and Paenibacillus dendritiformis

Abstract

In the present study, Paenibacillus dendritiformis, Trichoderma harzianum, and Trichoderma asperellum were appraised as potential biocontrol agents that induce resistance in chilli (Capsicum annuum) against the devastating pathogen Colletotrichum truncatum, which causes anthracnose. Bright-field and scanning electron micrographs showed the hyphal degradation, lysis, and abnormal swelling in C. truncatum against P. dendritiformis in a dual plate assay. Under greenhouse conditions, chilli seeds pretreated with P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum by soil soak method inflicted an induced systemic resistance (ISR) in chilli against a C. truncatum-challenged condition. In chilli, the disease index percentage was significantly reduced in the T. asperellum + T. harzianum-treated seeds, followed by the T. harzianum-, T. asperellum-, and P. dendritiformis-treated seeds as compared to the untreated and challenged, respectively. Chilli seeds were primed with T. asperellum + T. harzianum (78.67%), which revealed maximum disease protection under the challenged condition, followed by T. harzianum (70%), T. asperellum (64%), and P. dendritiformis (56%) as compared to untreated and C. truncatum-challenged (6%) condition served as control. The seeds that were pretreated with biocontrol agents (BCAs) inflicted ISR against C. truncatum by enhancing the activity of defence-related enzymes (superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and phenylalanine ammonia-lyase (PAL)), accumulating phenolic compounds, and increasing the relative chlorophyll content in chilli. Nitroblue tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAB) stains were used to detect the accumulation of superoxide anion and hydrogen peroxide that appeared nearby the fungal infection sites. The accumulation of reactive oxygen species (O₂^- and H₂O₂) in the pathogen-inoculated leaves was a maximum of 48 hpi, followed by P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum treated tissue upon C. truncatum-challenged condition as compared to the control. Overall, our results showed the potential of T. harzianum, T. asperellum, and P. dendritiformis as biocontrol agents that prevent infection by C. truncatum and inflict an induced systemic resistance in chilli by enhancing the biosynthesis of phenolic compounds, defence and antioxidative enzymes, and reducing the lesion development and reactive oxygen species accumulation. This is the first report of induced systemic resistance against anthracnose in chilli obtained by application of T. harzianum, T. asperellum and P. dendritiformis, through seed priming.

Keywords: Capsicum annuum; Colletotrichum truncatum; biocontrol agents; biopriming; plant defence; reactive oxygen species.

Figure 1

(A) Chilli fruit associated with anthracnose disease. (B) Pure colony C. truncatum after 5 days on PDA medium. (C) Acervuli on chilli fruit. (D) Conidia on conidiophore and setae. (E) Conidia of C. truncatum at 10×. (F) Sickle-shaped conidia of C. truncatum at 40×.

Figure 2

Pathogenicity test on chilli fruit and leaf. (A) Water-treated control showing no symptoms on the fruit of Capsicum annuum (cv. Surajmukhi). (B) Fruit challenged with the conidial suspension of C. truncatum showing anthracnose symptoms after 7 days of inoculation. (C) Healthy plant served as a control. (D) Leaves sprayed with the conidial suspension showing symptoms after 7 days of inoculation.

Figure 3

The disease index of C. truncatum-inoculated chilli fruit. Each value is expressed as mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 4

The effects of T. harzianum, T. asperellum, and P. dendritiformis on the inhibition of radial growth of C. truncatum. (A) Culture plate of C. truncatum serving as control; (B) Antagonistic activity by T. harzianum after 6 days post-inoculation; (C) T. asperellum after 6 DPI; (D) P. dendritiformis after 6 DPI.

Figure 5

Effects of T. harzianum, T. asperellum, and P. dendritiformis on the inhibition of hyphal growth of C. truncatum. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 6

Antagonistic activity of P. dendritiformis in a dual culture plate. (I) Light microscope; (A) normal hyphae, (B) P. dendritiformis in a dual culture plate showing hyphal degradation and lysis. (II) Bright field; (C) normal hyphae, (D) hyphal swelling, disintegration, and leakage of cytoplasmic contents. (III) Scanning electron microscope; (E) normal hyphae, (F) lysis, degradation, and disintegration of hyphae.

Figure 7

Induced systemic resistance by suppression of anthracnose disease development in chilli fruits treated by P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum in challenged and C. truncatum-infected samples under greenhouse conditions. The disease lesion development was recorded after ten days post-inoculation of C. truncatum. Treatment with bioagents on inoculation of C. truncatum showing reduced lesion development as compared to C. truncatum inoculation and control.

Figure 8

The treatment of chilli with T. harzianum, T. asperellum, and P. dendritiformis showing protection against anthracnose disease in the greenhouse experiment. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 9

The effect on total chlorophyll, chlorophyll a, and chlorophyll b content in chilli fruits treated with C. truncatum and bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) under a challenged condition and C. truncatum-infected samples after 48 h. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 10

The effect of bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) on the accumulation of the defence enzymes (POX and PAL) either individually or in the combination of treatment in chilli seeds under a challenged condition and C. truncatum-infected samples. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 11

The effect of bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) on the accumulation of the defence enzyme (PPO) either individually or in the combination of treatment in chilli seeds under a challenged condition and C. truncatum-infected samples. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 12

The effect of bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) on the accumulation of antioxidative enzymes (SOD and CAT) either individually or in the combination of treatment in chilli seeds under a challenged condition and C. truncatum-infected samples. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 13

The effect of bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) on the accumulation of antioxidative enzymes (APx and GPx) either individually or in the combination of treatment in chilli seeds under a challenged condition and C. truncatum-infected samples. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 14

The effect of bioagents (Pdt = P. dendritiformis, Tas = T. asperellum, Th = T. harzianum, and Tas+Th = T. asperellum + T. harzianum) on the production of total phenols either individually or in the combination of treatment in chilli seeds under a challenged condition and C. truncatum-infected samples. Each value is expressed as the mean of triplicates, and the bars sharing the same alphabetical letters are not significantly different (p ≤ 0.05) using Duncan’s multiple range test. The vertical bar designates the standard error.

Figure 15

(I) Accumulation of H₂O₂ in chilli leaves as visualized by 3,3′-diaminobenzidine staining at 48 hpi. (A,B) Untreated and unchallenged (control); (C,D) challenged with a pathogen; (E,F) Pre-treated with P. dendritiformis; (G,H) Pre-treated with T. asperellum; (I,J) Pre-treated with T. harzianum; (K,L) Pre-treated with T. asperellum + T. harzianum. (II) Superoxide anion production in chilli leaves as visualized by nitroblue tetrazolium staining at 48 hpi. (M,N) Untreated and unchallenged (control); (O,P) challenged with a pathogen; (Q,R) Pre-treated with P. dendritiformis; (S,T) Pre-treated with T. asperellum; (U,V) Pre-treated with T. harzianum; (W,X) Pre-treated with T. asperellum + T. harzianum.

Figure 16

Principal component analysis (PCA) of chilli fruits treated with bioagents under pathogen-challenged, untreated and unchallenged as negative control, and untreated and pathogen-challenged condition as positive control were harvested at different time intervals like 0, 12, 24, 48, 72, and 96 hpi.