- Open Access
Evaluation of biological efficacy of Trichoderma asperellum against tomato bacterial wilt caused by Ralstonia solanacearum
© The Author(s) 2018
- Received: 24 April 2018
- Accepted: 23 July 2018
- Published: 31 July 2018
Bacterial wilt, caused by soilborne bacterium Ralstonia solanacearum, is one of the most severe diseases of tomato worldwide, and no successful control measures are available to date. In the present study, a sustainable alternative tool such as use of fungi from tomato rhizosphere is being utilized to combat the pathogen attack. The application of Trichoderma asperellum (T4 and T8) isolates delayed wilt development, effectively decreased the disease incidence, increased fruit yield, and improved plant growth promotion under field conditions. The T. asperellum treatment decreased the disease incidence by 51.06% (RS + T4) and 52.75% (RS + T8) in Bhoomishettihalli (BH) and 47.21% (RS + T4) and 46.83% (RS + T8) in Madanahalli (MH) plots, respectively when compared with the pathogen-treated plot in year 2014. Correspondent decreases in year 2015 were 50.69% (RS + T4) and 52.38% (RS + T8) in BH and 48.18% (RS + T4) and 49.22% (RS + T8) in MH plots. In year 2014, T. asperellum (T4 and T8) treatment enhanced the yield with 5.45 t/ha and 5.50 t/ha in BH plot and 6.66 t/ha and 6.93 t/ha in MH plot, respectively, when compared with infected plots. In year 2015, T. asperellum (T4 and T8) treatment enhanced the yield with 5.29 t/ha and 5.51 t/ha in BH plot and 5.82 t/ha and 5.66 t/ha in MH plot, respectively, when compared with infected plots. The disease control and yield enhancement were highest at T8, followed by T4. Increase in the level of peroxidase (POX), phenylalanine ammonium lyase (PAL), polyphenol oxidase (PPO), β-1,3-glucanase and total phenol activities at 12th, 10th, 14th, 12th, and 10th days, respectively, after pathogen inoculation was observed. This indicates the induction of plant resistance mechanism by T. asperellum against R. solanacearum in tomato plants under field conditions.
- Ralstonia solanacearum
- Induced systemic resistance (ISR)
- Trichoderma asperellum
- Tomato yield
- Plant growth promoters
Tomato (Lycopersicon esculentum) is one of the most widely cultivated vegetable crops worldwide. Vegetable crops are extremely prone to soilborne and root diseases causing huge losses in yield and its quality (Sharma et al. 2004). The main constraint to tomato production in many parts of the world is several plant diseases. Bacterial wilt is a destructive and prevalent soilborne disease that limits tomato production in the tropics, subtropics, and warm temperate regions of the world (Ramesh et al. 2014). Ralstonia solanacearum is one of the most severe quarantine important diseases of tomato worldwide. Its host range contains solanaceous species, leguminous species, a small number of monocotyledons, trees, shrubs, and certain ecotypes of the model plant Arabidopsis thaliana. The pathogen persists in soils, water, or reservoir plants for several years to form latent infections within native weeds contributing to the hard eradication of the bacterium (Avinash et al. 2016).
Management of R. solanacearum, including use of resistant or tolerant varieties, cultural practices, chemical control, and biological control, are commonly employed methods to control bacterial wilt disease (Dalal et al. 1999). There are various beneficial microbes which have been completely implemented as biocontrol agents for inhibition of R. solanacearum under laboratory and/or greenhouse conditions, including Pseudomonas putida, P. fluorescens, Trichoderma spp., Bacteriophages, Streptomyces spp., Acinetobacter spp., Enterobacter spp., Bacillus spp. and Paenibacillus macerans (Vanitha et al. 2009 and Ling et al. 2010). Trichoderma isolates have strong antagonistic and mycoparasitic effects against phytopathogens and are therefore able to reduce disease severity in plants (Elsharkawy et al. 2012). The beneficial microorganisms have gained considerable attention as an ecofriendly and cost-effective platform for the stimulation of disease resistance through induced systemic resistance (ISR) and for the promotion of growth in plants for sustainable crop production (Abdelrahman et al. 2016). Trichoderma spp. induce plant growth by direct and indirect mechanisms (Zachow et al. 2016). Trichoderma spp. induce plant resistance against several phytopathogens, promote plant growth, and enhance photosynthetic activity of plants (Li et al. 2017). Presently, various reports indicate that Trichoderma induces systemic resistance by releasing not only proteins, but also secondary metabolites (Keswani et al. 2016).
Schonfeld et al. (2003) recorded a decrease in the R. solanacearum population in soil amended with decomposed organic fertilizer. The decomposed organic fertilizer or manure provides nutrients to the microbes, thus increases the biocontrol agent’s ability and makes them extra competitive in the rhizosphere soil and on roots (Liu et al. 2012). Root colonization by biocontrol agents is considered a prerequisite and is directly connected to their effectiveness in controlling soilborne infections (Ji et al. 2008). The earlier studies have described that Trichoderma spp., Bacillus spp., and Klebsiella spp. enhanced colonize plant roots and rhizosphere, if they are applied to the soil with nutrient carrier, such as decomposed organic fertilizer or manure (Huang et al. 2011). Trichoderma spp. are now the greatest common fungal biocontrol agents that have been broadly studied and deployed throughout the world (Alka et al. 2017).
The objectives of the present study were to investigate biochemical responses in terms of defense enzymes and to evaluate the effectiveness of T. asperellum to induce systemic resistance against bacterial wilt in tomato plants, as well its effectiveness on bacterial wilt control or suppression of Ralstonia wilt under field conditions.
Isolation and identification of microorganisms
Infected plant material and rhizosphere soil samples were collected from the wilted fields of tomato-growing areas of Karnataka. Ten virulent R. solanacearum strains were isolated from rhizosphere soil and shoot samples. The molecular identification of R. solanacearum isolates were confirmed based on 16S rRNA sequencing (Narasimha Murthy et al. 2016). Trichoderma spp. were isolated from healthy tomato plants’ rhizosphere, using the soil dilution plate technique. Identification of Trichoderma spp. was further confirmed by National Fungal Culture Collection of India (NFCCI), Agharkar Research Institute and Pune. Among ten Trichoderma spp. two (T4 and T8) strains were selected for field experiments based on antibacterial and greenhouse studies against R. solanacearum (Narasimha Murthy et al. 2013).
Preparation of bacterial inoculum
One milliliter of R. solanacearum stock suspension was added to casamino acid peptone glucose (CPG) broth (1-g casamino acid, 10-g peptone, 5-g glucose per liter) and incubated at 28 °C for 48 h on rotary shaker at 150 rpm (Kelman 1954). Culture broth was centrifuged at 12,000 rpm for 10 min at 10 °C. The bacterial pellet was resuspended in sterile distilled water and final concentration of suspension was set to 1 × 108 cfu/ml, by spectrophotometrically adjusting to O.D 600 nm = 0.1 (Ran et al. 2005).
Preparation of talc-based formulation
The suspension of each Trichoderma spp. was prepared from 7-day-old culture on potato dextrose agar (PDA), using sterile distilled water. The fungal inoculum was prepared by flooding the culture with sterile distilled water and then rubbing its surface with a bent sterile glass rod. The suspension was filtered through four layers gauze bandage to separate the spores from the mycelia. The fungal concentration in each suspension was estimated by counting with the help of hemocytometer and was adjusted to 5 × 108 spores/ml (Rojo et al. 2007). One kilogram of talc powder was taken in a sterilized metal tray, and its pH was adjusted to neutral as above. Ten grams of carboxy methyl cellulose (CMC) was added to 1 kg of talc, mixed well, and the mixture was autoclaved for 30 min at 121 °C and 15 lbs pressure, each on two consecutive days. Five hundred milliliters of spore suspension were mixed with sterilized talc powder under aseptic conditions, and the spore concentration was adjusted to 5 × 108 spores g−1 with a sterile talc powder. After shade drying overnight, the formulation was packed in a polypropylene bag and sealed. The formulations were mixed with 50 kg of farmyard manure and incubated for 30 days before applying to each plot.
Based on the previous in vitro and in vivo studies, under laboratory and greenhouse conditions (Narasimha Murthy and Srinivas 2012, and Narasimha Murthy et al. 2013), the most promising two T. asperellum isolates were selected for trial against the R. solanacearum under field conditions (Satish and Abhay 2016). The field experiment was conducted at the farmer’s agricultural plots located in Bhoomishettihalli (BH) (13° 28′ 05.7″ N, 78° 04′ 57.7″ E) and Madanahalli (MH) (13° 16′ 50.7″ N, 78° 05′ 52.6″ E) near Chintamani, Karnataka, India, during tomato growing season of March–June in 2014 and 2015. The experimental fields had been selected based on cultivation of tomatoes for several years and were naturally infested with R. solanacearum. Seeds of wilt susceptible tomato variety Arka Meghali were procured from Indian Institute of Horticultural Research (IIHR) Bangalore, India. Four-week-old tomato seedlings were uprooted from portrays and transplanted to experimental plots and treated with T. asperellum (T4 and T8) farmyard manure mixture (5 g/seedling) with spacing of 60 × 90 cm. The treatments were as shown as follows: (1) control (untreated seedlings), (2) T. asperellum alone (T4 and T8), (3) R. solanacearum alone, and (4) T. asperellum + R. solanacearum. The selected individual experimental plot area was of 25 m2 containing 14 rows with 80 or 100 plants per row, and the distance between rows was 50 cm (Narasimha Murthy et al. 2016). Buffer zones of 2 m without tomato seedlings were maintained between plots. Three replications were maintained for each treatment with 100 plants/ replication. The experiment was repeated thrice simultaneously in three different experimental plots of the fields. Seedlings were watered daily by drip irrigation, fertilized once with NPK fertilizer, farmyard manure (FYM) at 2.8 kg/m2 and vermicompost at 0.5 kg/m2. The NPK fertilizers consist of chemical fertilizers at the N:P:K ratio of 15:7:12; urea containing 46.5 N was applied, P applied as 7% mono superphosphate Ca(H2PO4)2, and K as potassium sulfate containing 41.7% (K2SO4). After 2 weeks of seedling transplantation, they were challenge inoculated by 48-h-old R. solanacearum suspension, 5 ml per plant by soil drenching method. The completely wilted tomato plants in each treatment were observed, 1 week after challenge inoculation up to 90 days. Disease incidence was calculated as the percentage of plants that had completely wilted. Fruits per plant, fresh weight, dry weight, plant height, stem growth, and tomato yield was calculated tons per hectare (t/ha) in each treatment, and total tomato yield was recorded at the end of the season. A total of four harvests were made at weekly intervals. The wilt incidence was evaluated when the infection emerged and calculated as the percentage of infected plants compared with the total number of growing plants in each plot. The percentage (%) of disease incidence was calculated by using the following formula: % of disease incidence = no. of wilted plants in a plot/total no. of plants in a plot × 100.
Sample collection for biochemical analysis
The leaf tissues of treated and untreated tomato plants were collected at different time intervals (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 days) after pathogen inoculation and stored in a deep freezer (− 80 °C) until used for biochemical analysis (Narasimha Murthy et al. 2016). Leaf tissues were homogenized by liquid nitrogen in a pre-chilled mortar and pestle. One gram of tomato leaf tissues was homogenized by 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) at 4 °C, and the homogenate was centrifuged for 20 min at 12,000 rpm. The supernatant was used as a crude extract for analyzing peroxidase (POX) (Hammerschmidt et al. 1982), polyphenol oxidase (PPO) (Mayer et al. 1965), and phenylalanine ammonia lyase (PAL) (Dickerson et al. 1984). As for the estimation of β-1,3-glucanase, 1 g of tomato leaf tissues was homogenized by 2 ml of 0.1 M sodium citrate buffer (pH 5.0) in a pre-chilled mortar and pestle, centrifuged, and supernatant was used for the estimation (Pan et al. 1991). The total phenol content was estimated as per the procedure given by Zieslin and Ben-Zaken (1993). The following treatments were included in the experiments: untreated control plants (C), plants inoculated with R. solanacearum alone (T1), plants inoculated with T. asperellum alone (T2), and plants inoculated with T. asperellum and challenge inoculated with R. solanacearum (T3).
Protein estimations of all the enzyme extracts were carried out by Lowry’s method (Lowry et al. 1951) using bovine serum albumin as a standard.
Native poly acrylamide gel electrophoresis analysis
The isoforms profiles of peroxidase and polyphenol oxidase were estimated by discontinuous native polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970). The protein extracts were prepared by homogenizing 1.0 g of leaf tissues with 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) and centrifuged at 20,000 rpm for 15 min at 4 °C. Samples (80-μg protein) were loaded into 8% polyacrylamide gels (Sigma, USA). After electrophoresis, POX isoforms were observed by soaking the gels in staining solution containing 0.05% benzidine (Sigma–Aldrich, Mumbai, India) and 0.03% H2O2 in acetate buffer (20 mM, pH 4.2) for 30 min in the dark, after which drops of 30% H2O2 were added with constant shaking ti1 the bands appeared (Nadolny and Sequeira 1980). For assessing the PPO isoform, the gels were equilibrated for 30 min in 0.1% 1,3-dihydroxyphenylalanine (DOPA) in 0.1 M potassium phosphate buffer (pH 7.0), followed by the addition of 10 mM catechol in the same buffer. The addition of catechol was followed by gentle shaking, resulted in appearance of dark brown discrete protein bands indicative of PPO isozymes appeared in the gel (Jayaraman et al. 1987).
All data of field trial experiments were statistically analyzed, using Microsoft Excel ™ and SPSS (version 20.0). The data were subjected to analysis of variance (ANOVA), and the means were analyzed, using Duncan’s new multiple range post test at p ≤ 0.05.
Field experiment for assessment of T. asperellum isolates to control of bacterial wilt
Influence of T. asperellum on tomato plant growth, yield, and control of bacterial wilt under field conditions during the year 2014
Plant height (cm)
Fresh weight (g)
Dry weight (g)
Stem growth (cm)
No. of fruits/plant
Disease incidence (%)
RS + T4
RS + T8
Influence of T. asperellum on tomato plant growth, yield, and control of bacterial wilt under field conditions during the year 2015
Plant height (cm)
Fresh weight (g)
Dry weight (g)
Stem growth (cm)
No. of fruits/plant
Disease incidence (%)
RS + T4
RS + T8
The treatment of T. asperellum (T4 and T8) on the pathogen-infected plants showed significant increase in overall plant growth, including plant height, fresh weight, dry weight stem growth, and fruits per plant (Tables 1 and 2). The treatment of T. asperellum T4 increased plant height, fresh weight, dry weight, stem growth, and fruits per plant by 14.69 cm, 225.79 g, 18.83 g, 0.77 cm, and 28.37 fruits/plant, respectively in BH plot as compared with the diseased control and by 11.7 cm, 225.78 g, 20.79 g, 0.64 cm, and 28.67 fruits/plant respectively in MH plot as compared with the diseased control. The treatment of T. asperellum T8 increased plant height, fresh weight, dry weight, stem growth, and fruits per plant by 15.47 cm, 231.28 g, 19.46 g, 0.79 cm, and 29.29 fruits/plant, respectively, in BH plot as compared with diseased control and by 11.33 cm, 232 g, 20.43 g, 0.66 cm, and 29.35 fruits/plant respectively in MH plot as compared with diseased control (Table 1). In year 2015, the disease incidence in untreated control plots was nil in both plots. In R. solanacearum-infected BH and MH plots, it ranged from 86.43 to 89.48% and 85.34 to 90.43%, respectively. T. asperellum-treated plots showed an average of 36.43 and 39.19% disease incidence in BH and MH plots, respectively. The T. asperellum treatment decreased the disease incidence by 50.69% (RS + T4) and 52.38% (RS + T8) in BH and 48.18% (RS + T4) and 49.22% (RS + T8) in MH plots, respectively. The tomato yield in untreated control plot of BH was 7.16 t/ha and 6.87 t/ha in MH plot. The tomato yield in R. solanacearum-infected BH plot was about 1.38 to 1.66 t/ha and 1.32 to 1.76 t/ha in MH plot. T. asperellum isolate (T4 and T8)-treated plots yielded an average of 7.39 and 7.53 t/ha in BH and of 7.89 and 8.12 t/ha of tomatoes in MH, respectively. Thus, tomato yield increased by an average of 3.22 and 5.16% in BH and 14.85 and 18.20% in MH at T4 and T8 treatments, respectively as compared with the untreated control plots (Fig. 1). Plots challenge inoculated with R. solanacearum and treated with T4 and T8 yielded an average of 6.67 and 6.89 t/ha in BH and an average of 7.14 and 6.98 t/ha in MH plots, respectively. T. asperellum (T4 and T8) treatment enhanced the tomato yield by 5.29 t/ha and 5.51 t/ha in BH and 5.82 t/ha and 5.66 t/ha in MH, respectively when compared with R. solanacearum-treated plots (Tables 1 and 2).
The treatment of T. asperellum T4 increased plant height, fresh weight, dry weight, stem growth, and fruits per plant by 14.3 cm, 238.9 g, 20.34 g, 0.71 cm, and 29.47 fruits/plant in BH plot as compared with diseased control and by 14.19 cm, 230.24 g, 17.84 g, 0.73 cm, and 24.34 fruits/plant in MH plot, respectively. The treatment of T. asperellum T8 increased plant height, fresh weight, dry weight, stem growth, and fruits per plant by 16.32 cm, 244.71 g, 21.62 g, 0.73 cm, and 29.33 fruits/plant in BH plot and by 13.07 cm, 233.36 g, 19.19 g, 0.71 cm, and 26.09 fruits/plant in MH plot, respectively, as compared to diseased control (Table 2).
Obtained results showed the induction of plant growth, increased tomato yield and reduced wilt incidence under field conditions upon soil treatment with T. asperellum. This outcome supports the report of Watanabe et al. (2007) who reported the management of disease by T. asperellum. The root colonization is a successful major requirement for the useful effects of Trichoderma spp. on plants not only concerning antagonistic behavior and increase in plant growth but also for inducing systemic resistance (Rubio et al. 2014). Trichoderma spp. have been previously demonstrated as efficient T. asperellum for the control of M. phaseolina in melon, corn, eggplant, sorghum, and chickpea (Manjunatha et al. 2013) and for the control of F. solani in beans, chili, and peanuts (Qualhato et al. 2013). Different studies on applications of Trichoderma spp. in farming practices as biological control agents, biofertilizers, and soil amendments for the control of plant pathogens and crop development in several crop plants have been well established. Trichoderma is accomplished of colonizing farmyard manure, and therefore, application of colonized FYM to the soil is more suitable and helpful. This is the mainly successful method of application of Trichoderma, particularly for the control of soilborne diseases (Hamed et al. 2015).
PPO activity in tomato plants treated with T. asperellum was significantly increased upon challenged with pathogen and reached at 14th day and declined thereafter in all the treatments (Fig. 3). It is copper containing enzymes that catalyze oxidation of hydroxy phenols to their quinone derivatives, which have antimicrobial activity (Chunhua et al. 2001). Oxidative enzymes such as POX and PPO can catalyze the formation of lignin and other oxidative phenols and contribute in the formation of defense barriers by changing the cell structure defense system that gets actuated against pathogens (Li and Steffens 2002). Several potentials of PPO including general toxicity of PPO-generated quinones to pathogens and plant cells, accelerating cell death, alkylation, and reduced bioavailability of cellular proteins to the pathogen, crosslinking of quinones with protein or other phenolics, forming a physical barrier to pathogens in the cell wall and quinone redox cycling leading to H2O2 and other reactive oxygen species. In the present experiment, PPO activity was significantly enhanced by T. asperellum-treated tomato plants. Also, PPO activity level increased at 14th day after challenge inoculation and helps in disease resistance as it oxidizes the phenolic level increase during this stage to toxic molecules such as quinones leads to invasion of pathogen (Vinale et al. 2008). Activity of PAL in tomato plants treated with T. asperellum was significantly increased in tomato plants inoculated with R. solanacearum. The PAL activity reached maximum at 10th day after challenge inoculation with the pathogen and declined thereafter in all the treatments. Activity of PAL in tomato plants treated with T. asperellum was induced upon inoculation with pathogen (Fig. 4). Induction of defense enzymes like PAL is one of the responses of the host for treatment with Trichoderma agents. PAL is the key enzyme that is responsible for linking primary metabolism of aromatic amino acids with secondary metabolic products (Macdonald and Dcunha 2007). It is the first enzyme in phenyl propanoid metabolism and synthesis of various phenolic compounds as well as anthocyanin, biosynthesis of lignin providing mechanical strength to the plant cell wall and phytoalexins which are responsible for prevention of establishment of plant pathogens (Karthikeyan et al. 2005). In the present study, increased PAL activity and the accumulation of phenolic content was recorded in T. asperellum isolates treated tomato plants infected with the R. solanacearum, apparently due to prevention of pathogen attack. Also, the T. asperellum treatment resulted in a significant increase in the PAL activity on 10th day after pathogen inoculation in tomato plants.
The β-1,3-glucanase activity reached the maximum at 12th days after inoculation with R. solanacearum and declined thereafter in all the treatments (Fig. 5). Improved level of pathogenesis-related PR protein such as β-1,3-glucanase activity was observed in T. asperellum-treated tomato plants and leads to disease resistance against R. solanacearum. It is a member of the PR protein family, known to directly destroy pathogen cell walls. Improved β-1,3-glucanase activity was observed by T. asperellum level up to the 12th day after pathogen inoculation, and thereafter, it starts decreasing leading to disease resistance in tomato plants against R. solanacearum. Similar outcomes were previously approved by Saksirirat et al. (2009) who demonstrated that the increase in PR proteins, like chitinase and β-1,3-glucanase level up to 14th day of X. campestris pv. vesicatoria inoculation on tomato plants, led to the leaf spot.
Accumulation of phenolics in plants pre-treated with T. asperellum was induced upon challenge inoculation with R. solanacearum. Its accumulation significantly increased on 10th days after inoculation with the pathogen and declined thereafter in all the treatments. Maximum accumulation of phenol was noticed in T. asperellum (T8) inoculated with R. solanacearum at 10 days when compared with the plants inoculated with the pathogen alone (Fig. 6). Minimum amounts of phenolic compounds were observed in untreated control. The development of production of phenolics, known as defense molecules of plants against plant pathogens and insects, is indicated by an increase in PAL activity in wounded plant tissues (Bi and Felton 1995). In the present study, a high quantity of phenolic compounds was observed in the plant as compared with treated and untreated controls when tomato seedlings were treated with T. asperellum. Investigation data accumulated in the past few years have produced a completely novel understanding of the way by which these fungi interact with plants. Lopes et al. (2012) described a positive correlation between the lytic enzyme activities and the antagonism capacity of T. asperellum against S. sclerotiorum. The presence of T. asperellum in cucumber roots triggers the SA and JA pathways in the plant and increased peroxidase activity, hence conferring protection to cucumber plants against foliar pathogens (Segarra et al. 2007).
Native PAGE analysis POX and PPO
The present study showed that biocontrol capacity and biochemical characterization of induced systemic resistance by T. asperellum against R. solanacearum in tomato and comprehends the role of defense enzymes in developing disease resistance under field conditions. The two T. asperellum isolates recorded efficient inhibition against R. solanacearum and increased yield of tomatoes under field experiments. The role of T. asperellum is as BCA in the induction of a series of defense responses such as accumulation of phenols and induction of POX, PPO, and PAL enzymes involved in phenylpropanoid metabolism and of PR protein (β-1,3-glucanase) in response to treatment with the biocontrol agent. In this regard, it is recommended the use of T. asperellum assessment to the actions within disease management framework is reasonable, provided that long term induced resistance and should be developed as a sustainable and environmental friendly approach.
The authors are thankful to the University Grants Commission, Government of India, New Delhi, for providing UGC-BSR Meritorious fellowship for the first author. The authors also like to thank the Chairman, Department of Microbiology and Biotechnology, Bangalore University, Bangalore, for providing the facilities for this investigation.
This study received funding from the University Grants Commission, Government of India, New Delhi.
Availability of data and materials
All datasets on which conclusions of the study have been drawn are presented in the main manuscript.
NK conceived the idea, suggested the point of research, designed the experimental work, conducted the experiments, and wrote the manuscript. SK worked on the management of the article, statistical analysis of data, and critical revision. SCN, SRN, and SC participated in the experiments’ design and coordination. All authors read and approved the final manuscript.
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