Trichoderma harzianum elicits induced resistance in sunflower challenged by Rhizoctonia solani.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Trichoderma harzianum elicits induced resistance in sunflower challenged by Rhizoctonia solani B.N. Singh1, A. Singh2, B.R. Singh3 and H.B. Singh1 1 Department of Mycology & Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India 2 Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi, India 3 Department of Applied Physics, Z.H. College of Engg. & Tech., Centre of Excellence in Materials Science (Nanomaterials), Aligarh Muslim University, Aligarh, India

Keywords defence responses, lignin, phenolics, Rhizoctonia, sunflower, Trichoderma harzianum NBRI-1055. Correspondence Harikesh B. Singh, Department of Mycology & Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi– 221 005, Uttar Pradesh, India. E-mail: [email protected] 2013/1769: received 29 August 2013, revised 6 November 2013 and accepted 6 November 2013 doi:10.1111/jam.12387

Abstract Aims: To investigate the efficacy of Trichoderma harzianum NBRI-1055 (denoted as ‘T-1055’) in suppression of seedling blight of sunflower caused by Rhizoctonia solani K€ uhn and their impact on host defence responses. Methods and Results: T-1055 was applied as seed treatment, soil application and combined application (seed treatment + soil application). Higher protection afforded by combined application of T-1055 was associated with the marked induction of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (PO) and cinnamyl alcohol dehydrogenase (CAD) activities. The activities of PAL and PPO reached maximum at 10 days after sowing (DAS), while PO and CAD levels reached maximum at 12 DAS. This was further supported by the accumulation of total phenolic content that showed an increase up to threefold at 14 DAS. In addition, HPLC analysis revealed that the contents of ferulic and p-coumaric acids increased by 63 and 46 times, respectively, at 14 DAS. Amount of gallic acid was also little more than double. Lignin deposition in sunflower root increased by 27, 34 and 37 times through combined application of T-1055 at 16, 18 and 20 DAS, respectively. Combined application also increased the accumulation of PR-2 and PR-3 proteins by 33 and 39 times, respectively, at 12 DAS in followed by seed treatment alone. Conclusions: The combined application of T-1055 triggered defence responses in an enhanced level in sunflower than the soil and seed alone and provided better protection against Rhizoctonia seedling blight. Significance and Impact of the Study: Rhizospheric fungal bioagent ‘T-1055’ can enhance protection in sunflower against the R. solani pathogen through augmented elicitation of host defence responses.

Introduction Rhizoctonia solani K€ uhn, (teleomorph: Thanatephorus cucumeris) is among the most diverse and widely dispersed soil-borne plant pathogenic fungus, causing economically important blights, root rots and wilts (Wilson et al. 2008; Ahvenniemi et al. 2009). It has a remarkably broad host range, infecting both monocotyledonous and dicotyledonous plants. Seedling blight caused by R. solani is one of the most devastating postemergence diseases of 654

sunflower (Helianthus annuus L.) infects usually root portion of seedlings by rotting. Emergence of new races of the pathogen has drastically reduced the effectiveness of the classical control measures. Besides, application of pesticides has raised widespread concern for damaging the sustainability of the nature. In recent years, efforts have concentrated on the biological control of phytopathogens using bacterial and fungal antagonists (Heil and Silva Bueno 2007; Singh et al. 2007; Lorito et al. 2010; Shoresh and Harman 2010; Shoresh et al. 2010; Beaulieu et al.

Journal of Applied Microbiology 116, 654--666 © 2013 The Society for Applied Microbiology

Biological control of Rhizoctonia seedling blight

B.N. Singh et al.

2011). These biological control agents (BCAs) represent promising alternatives to minimize the impact of chemicals on the environment by replacing these chemicals or reducing their rate of application for management of plant diseases (De Meyer et al. 1998; Yedidia et al., 1999; Vinale et al. 2008). Trichoderma species are among the most studied fungal BCAs and widely used as commercial biofungicides of the phytopathogens control (Verma et al. 2007; Dubey et al. 2011). These fungi are opportunistic, avirulent plant symbionts and function as parasites and antagonists of many phytopathogenic fungi, thus protecting plants from diseases (Vinale et al. 2008; Wilson et al. 2008; Lorito et al. 2010). In addition to directly affecting plant pathogens through antibiosis and mycoparasitism, Trichoderma spp. can colonize roots and trigger defence responses against bacterial and fungal pathogens (Yedidia et al. 2003; Lorito et al. 2010; Shoresh et al. 2010; Singh et al. 2011). These responses may include an accumulation of PR proteins, phenolics, lignin and defence enzymes involved in phenylpropanoid pathway (Harman et al. 2004; Fobert and Despres 2005). The first clear demonstration of induced resistance by Trichoderma was reported by De Meyer et al. (1998) and authors observed that T. harzianum T39 soil treatment reduced disease symptoms of Botrytis cinerea by inducing phenolics accumulation. Treatment of cucumber root with T. harzianum T-203 also found that the induction of defence-related proteins including chitinase, b-1,3-glucanase, peroxidase (PO) as well as esterification of phenylpropanoids (Yedidia et al., 1999; Mandal and Mitra 2007). Moreover, Trichoderma treatment elicited the accumulation of terpenoids, phytoalexins, fungitoxic phenolics, phenylalanine ammonialyase (PAL), polyphenol oxidase (PPO) and cinnamyl alcohol dehydrogenase (CAD) in cotton, tomato and coconut seedlings, thus providing protection against wide range of pathogens (Howell et al. 2000; Viterbo et al. 2002; Harman 2006). The beneficial effects of Trichoderma harzianum to control R. solani have been reported in different studies (Vinale et al. 2008). Recently, Gallou et al. (2009) investigated the dynamics of R. solani on potato plants in the presence of T. harzianum Rifai MUCL 29707. These authors noted an induction of pathogenesis-related proteins, glutathione-S-transferase 1 gene and other defencerelated genes in the plants inoculated with both organisms at early stage. Indeed, there are a few extensive evidences that Trichoderma species are able to up-regulate the defence systems of a number of plants (De Meyer et al. 1998; Yedidia et al., 1999; Shoresh et al. 2010). This evidence was consolidated by studies on the effect of Trichoderma inoculations in plant proteome/gene expression by 2D electrophoresis and high-density oligo

microarrays (Marra et al. 2006; Alfano et al. 2007; Segarra et al. 2007). In the recent decade, a growing body of evidence from various studies indicates that increased resistance by Trichoderma may be associated in part with marked metabolic changes in the host, including enhanced accumulation of hydrolases, such as chitinases and b-1,3-glucanases, with antimicrobial potential (Karthikeyan et al. 2006); production of defence proteins; synthesis of fungitoxic phenolics (Yedidia et al. 2003); and phytoalexins (Howell et al. 2000). If one considers that the increased production of enzymes involved in phenylpropanoid pathway and phenolic compounds may be of key importance in the resistance process (Yedidia et al. 2003) and that the accumulation of structural substances may increase the mechanical strength of the host cell walls, the induction of such defence mechanisms by Trichoderma would likely inhibit or at least restrict pathogen invasion (Mandal and Mitra 2007). However, at present, the situation is not clearly defined, and additional research is needed to confirm the effective stimulation of the plant defence system upon infection by nonpathogenic fungi. Nevertheless, these earlier observations, together with the finding that T. harzianum NBRI-1055 (denoted as ‘T-1055’) was able to promote plant growth (Singh et al. 2007), raise key question: Is T-1055 capable of stimulating the plant to defend itself through the accumulation of defence-related chemicals and proteins to inhibit pathogen invasion (Mandal and Mitra 2007)? In an attempt to answer this important question, our goals were to (i) determine the bioefficacy of T-1055, for their effectiveness to control seedling blight of sunflower caused by a highly virulent R. solani isolate and to (ii) quantify elicitation of some defence-related responses in sunflower roots against the pathogen. In this study, we presented the conclusive evidence that T-1055 stimulates the accumulation of defence-related compounds and proteins in sunflower roots against seedling blight caused by R. solani. Materials and methods Isolation of Trichoderma isolates and chemicals One hundred and nine isolates of Trichoderma were isolated from different agricultural fields and forest soils of Uttar Pradesh, India, using the modified Trichoderma selective medium Singh et al. (2007). The colonies were determined to be Trichoderma species (Castle et al. 1998). The isolates of Trichoderma were belonging to different species viz., T. harzianum, T. viride, T. koningii and T. pseudokoningii. Of 109 isolates, one isolate appointed as T. harzianum and NBRI-1055 (denoted as ‘T-1055’) was found to be a stress tolerant. This isolate grew on up


655


B.N. Singh et al.

to 10% of NaCl, pH range up to 3–12 and temperature up to 53°C (data not shown). T-1055 was also found to be a most effective for in vitro antagonistic activity against R. solani (data not shown). Therefore, T-1055 (GenBank Accession No. NRRL30595) was selected for further study, grown and maintained on potato dextrose agar (PDA) medium (HiMedia M096) (Singh et al. 2010). Analytical grade solvents and deionized water for all the procedures were obtained from E. Merck, Mumbai, India. All authentic phenolic standards were procured from Sigma-Aldrich, St. Louis, MO, USA. Mass production and preparation of talc-based formulation of T-1055 For mass production of T-1055, maize spent (var. Malaviya-2) was used as solid substrate. Maize spent was chopped, dried and dipped in tap water for 3 h. Two hundred grams of moist maize spent (moisture 30% w/v) was filled in 20 kg capacity polypropylene bags and autoclaved twice at 15 psi. for 20 min. Spore suspension of T-1055 was prepared by harvesting the spores from 7-day-old culture in sterilized distilled water (SDW). Five millilitres of spore suspension containing 2 9 1010 colony-forming units (CFU ml 1) was injected into autoclaved bags with the help of a sterilized syringe and incubated at 25 2°C for 10 days. Colonized maize spent with Trichoderma was dried under aseptic conditions and grinded to fine powder (technical) in a controlled cool temperature jacket grinder (Complab, India) at 30°C. The talc-based formulation of T-1055 was prepared by following the method described by Karthikeyan et al. (2006). Briefly, 10 kg of talc powder was taken in a sterilized metal tray, and its pH was adjusted to neutral by adding calcium carbonate at the rate of 15 g kg 1. Ten grams of carboxymethyl cellulose was added to 10 kg of talc and mixed well. Then, the mixture was autoclaved for 30 min on each of the two consecutive days. Two grams of technical was mixed with sterilized talc mixture under aseptic conditions, and the spore concentration was adjusted to 2 9 106 CFU g 1.

with 15 kg per pot. Talc-based formulation of T-1055 was used as seed treatment, soil application and combined application (seed treatment + soil application). For seed treatment, seeds of sunflower were coated with 15% (w/v) gum arabic (HiMedia, RM682) as an adhesive and uniformly coated with talc-based formulation of T-1055 at 5 g kg 1 seed and kept for air-drying for 2 h under a stream of sterile air (Singh et al. 2007). For soil application, formulation of T-1055 was mixed in the pot mixture at the rate 10 g kg 1 soil mixture. For combined application, seed treatment and soil application was performed as described above. Sunflower seeds without T-1055 and pathogen were also maintained as control (healthy), whereas pathogen-inoculated was used as pathogen-treated. A sterile cornmeal sand medium (240 g of clean quartz sand, 60 g of yellow cornmeal and 75 ml of SDW) was used for mass culture of R. solani. Mixture was inoculated with sclerotia of R. solani and incubated for 2 weeks at 25°C. Inoculum of R. solani was added in the pot mixture at the rate of 50 g per pot. After 1 week of inoculum application, sunflower seeds treated as above were placed about 30 cm deep in the potting soil and 50 g of the same soil mixture was placed above the seeds. After sowing, each pot received 250 ml of tap water. The seedlings were thinned to two plants/pot on 7 days after sowing (DAS). The pots were maintained in a greenhouse at 21 2°C. All the pots were irrigated at 1-day intervals until harvest. Disease incidence was recorded at 25 DAS using the following formula: Per cent disease index = number of infected plants/ total number of plants 9 100 There were six replications, and the pots were arranged in a randomized manner. Sampling for biochemical analysis The roots of treated and untreated control plants were collected at 10, 12, 14, 16, 18 and 20 DAS. The roots were washed in running tap water and stored in a deep freezer ( 80°C) for biochemical analysis at a later date. HPLC analysis of phenylpropanoid derivatives

Modes of application and greenhouse experiment Sunflower (var. NFSH-9) seeds were surface sterilized with 02% mercuric chloride (HgCl2) solution for 5 min, followed by washing with SDW to remove HgCl2. The moistened seeds were stored at room temperature (25 2°C) for 24 h to promote germination. Plastic pots (15 9 10 cm) were used for sterile soil assay (SSA). Soil mixture containing sandy soil, vermicompost and farm yard manure (1 : 1 : 2) was sterilized by autoclaving at 15 psi. for 30 min on three consecutive days and filled 656

For HPLC analysis, 10 g of fresh tissue was extracted with 50% methanol (10 ml). The solvent was removed under reduced pressure on rota evaporator (Eyela N–N series, Tokyo, Japan). The residue was dissolved in HPLC grade methanol and subjected to HPLC analysis for specific phenolics composition. The HPLC system Shimadzu LC-10A (Japan) was equipped with dual pump LC-10A binary system, UV detector SPD-10A and C18 column (RP-Hydro, 4 lm, 250 9 46 mm) (Phenomenex, Torrance, CA, USA). Separation of compounds was achieved



B.N. Singh et al.

with acetonitrile/water (1 : 1 v/v) containing 1% acetic acid in a linear gradient programme, started with 18% acetonitrile, changing to 32% in 15 min and finally to 50% in 40 min (Singh et al. 2009a). The solvent flow rate was 10 ml min 1. Results (lg g 1 fresh wt.) were obtained by comparing the peak areas (kmax 254 nm) of the samples with those of standards. The data were integrated by Shimadzu Class VP series software. MS/MS analysis An API 2000 triple quadripolar mass spectrometer (Applied Biosystems, Ontario and Canada) was used for identification of phenolic compounds. Analysis were performed on a Turbo ions spray source in negative mode using settings, nebuliser gas (N2) 16, curtain gas (N2) 12, collision gas (N2) 1–2 (arbitrary units), focusing potential – 400V, entrance potential – 10, declustering potential (DP) 25–60 and collision energy (CE) 15–35. Full-scan acquisition was performed by scanning from m/z 100–1000 U at a cycle time of 2 s. MS/MS product ions were produced by collision-associated dissociation (CAD) of the selected precursor ions in collision cell. In all the experiments, quadrupole (Q1) was operated at resolution unit. Production scan of selected molecules was carried out to confirm structures of the compounds (Singh et al. 2009b). Assessment of defence-related chemicals, enzymes and PR proteins Approximately 500 mg of fresh root samples were placed in 50 ml of 95% ethanol, kept at 0°C for 48 h and centrifuged at 5000 g for 10 min. The supernatant was used for the estimation of total phenolic content (TPC) (Singh et al. 2010), and absorbance values were converted to mg gallic acid equivalent (GAE) g 1 fresh weight (FW). The activity of defence-related enzymes and PR proteins was assessed in the bioagents-treated and bioagents-untreated control plants. The fresh roots were homogenized in liquid nitrogen, and 05 g of powdered sample was extracted with 20 ml of 05 mol l 1 sodium borate buffer (pH 70). The mixture was centrifuged at 16 000 g for 15 min at 4°C, and the extract was used for the estimation of phenylalanine ammonia-lyase (PAL) (Dickerson et al. 1984). The samples extracted with 01 mol l 1 phosphate buffer, and pH 70 was used for assaying of activities of peroxidase (PO) (Hammerschmidt et al. 1982) and polyphenol oxidase (PPO) (Gauillard et al. 1993). For the determination of cinnamyl alcohol dehydrogenase (CAD) activity, the plant material was homogenized and extracted with 01 mol l 1 Tris-HCl, pH 75 (Wyrambik and Grisebach 1975). Lignin was extracted

according to the method of Bruce and West (1989) and assayed quantitatively in the alcohol insoluble residue (AIR) as thioglycolic acid (TGA) derivatives following alkali hydrolysis (Campbell and Ellis 1992), and results were expressed as the increase in A280 g 1 of AIR FW. The plant samples (05 g) were extracted with 005 mol l 1 sodium acetate buffer, pH 50 and used for assaying of PR-2 (Pan et al. 1991) and PR-3 (Trudel and Asselin 1989) proteins. The protein content was measured by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. Statistical analysis Values from different experiments shown in tables and figures were mean standard error (SE) of at least twelve repeated analyses in two experiments. Results were subjected to the analysis of variance (ANOVA) test and critical difference (CD) at P < 005. Results Reduction in plant mortality All the treatments of T-1055 protected sunflower plants from R. solani infection with maximum protection offered by combined application, as only 90% plant mortality was recorded, compared with untreated control (560%). Seed treatment and application also reduced plant mortality significantly by 190 and 270%, respectively (Fig. 1a,b). Effect of T-1055 on TPC and specific phenolic profile Most pronounced increase in TPC accumulation was observed by combined application of T-1005 challenged with the pathogen which was 31 times higher, compared with untreated control. TPC reached maximum level at 14 DAS (Fig. 2a). Seed treatment and soil application challenged with pathogen also accumulated 26 and 26 times more TPC, respectively. Although, in case of seed treatment, TPC reached maximum at 14 DAS, while the maximum level of TPC was observed at 16 DAS by soil application. Moreover, the TPC of all the treatments was less compared with combined application treated plants challenged with the pathogen. The concentration of TPC in T-1055-treated plants was significantly higher than the untreated control plants. Three major phenolic compounds such as gallic acid (GA), ferulic acid (FA) and p-coumaric acid (p-CA) were identified through HPLC analysis in untreated control and pathogen-infected plants (Table 1). However, in T-1055-treated plants challenged with the pathogen, five


657


(a)

T1

T2

T3

B.N. Singh et al.

by seed treatment at 14 DAS. A gradual decrease in the level of these compounds with the passage of time was observed, but CHA, t-CA and FA levels never returned to the levels of untreated control plants in respect to treatment by T-1055. Interestingly, untreated control and pathogen-treated plants were not able to accumulate CHA and t-CA. There was recorded significant change in plants treated with T-1055 alone during the time course of experimental period, and level of phenolics remained higher compared with the untreated control. The identification of these compounds was further substantiated by MS/MS analysis (Table 2 and Fig. 2c) that showed deprotonated molecules [M-H] . Loss of CO2 was observed for GA and FA giving the [M-H-44] as a characteristic ion. FA also showed the loss of methyl group, providing [M-15] . CHA showed the deprotonated molecule at m/z 353. In the UV spectra, p-CA and t-CA with symmetrical chemical structures, and without a hydroxyl group, showed a single absorption peak, while FA and CHA with nonsymmetrical chemical structures had a major absorption peak. The reason for this discrepancy may be the substitution of hydroxyl or methoxyl groups of the cinnamic type which caused hypsochromic shifts.

T4

(b) 70

Plant mortality (%)

60 50 40 30 20

Effect of T-1055 on PAL and PPO activities

10 0 T1

T2

T3

T4

Treatments Figure 1 Effect of T-1055 on Rhizoctonia solani-infected sunflower plants on (a) plant shoot and root phenotype and (b) plant mortality, grown in greenhouse conditions for 25 days. Rhizoctonia solani -infected roots are shown by the arrow. Key to the treatments: T1, plant grown with R. solani alone was used as an infected control; T2, plants co-inoculated with seed treatment of T-1055 and R. solani; T3, plants co-inoculated with soil application of T-1055 and R. solani; T4, plants co-inoculated with combined application of T-1055 and R. solani. Vertical bars indicate standard error (SE) of at least twelve repeated analysis in two experiments.

phenolics include GA, FA, p-CA, chlorogenic acid (CHA) and trans-cinnamic acid (t-CA) were detected. All the treatments of T-1055 challenged with pathogen increased the levels of phenol in varying degrees right from 10 DAS compared with the untreated controls. Among all the treatments of T-1055, most effective treatment was combined application when challenged with the pathogen (Fig. 2b). Most profound effects were observed on FA and p-CA among the phenolic compounds and increased 63- and 46-folds at 14 DAS by combined application, respectively. The level of GA was little more than double 658

In this experiment, it was observed that the combined application of T-1055 stimulated the expressions of PAL and PPO, which were stronger than other treatments (Fig. 3a,b). The expression of PAL and PPO was markedly increased by 32 and 30 times higher, respectively, than untreated controls, challenged with the pathogen at 10 DAS. After that, there was a sharp decrease in the level of PAL, whereas gradual decrease was examined with PPO level. Seed treatment also induced the expressions of PAL and PPO by 22 and 26 times, respectively, at 10 DAS, while on the other hand, 18 and 21 times more induction were observed by soil application at the same time of interval. Plants infected with the pathogen alone showed increased levels of PAL and PPO at 10 DAS, subsequently, declined drastically at 20 DAS and returned almost to the level of untreated controls. The plants inoculated with T-1055 alone had significantly higher activities of PAL and PPO, compared with untreated controls, but these were less to T-1055-treated plants challenged with the pathogen. Effect of T-1055 on PO and CAD activities The activities of PO and CAD remained at low levels in the untreated control plants. In contrast, their activities were stimulated strongly through combined application



B.N. Singh et al.

(a)

(b)

Figure 2 Effect of T-1055 on (a) accumulation of total phenolic content (TPC, mg gallic acid equivalent (GAE) g 1 fresh weight) in healthy and Rhizoctonia solaniinfected sunflower roots. Vertical bars indicate SE of at least twelve repeated analysis in two experiments. (b) Representative HPLC chromatograms of T-1055-treated plants (combined application) challenged by R. solani at 14 DAS and identification of compounds were obtained by comparison of peak (kmax 254 nm) of the sample with that of the standards. 1, GA; 2, t-CA; 3; CHA; 4, FA; 5, p-CA. (c) Mass spectra for MS/MS of the phenolic compounds from T-1055-treated plants (combined application) challenged by R. solani at 14 DAS. ( ) Untreaterd control; ( ) infected (pathogen); ( ) combined application; ( ) infected + seed treatment; ( ) infected + soil application and ( ) infected + combined application.

(c)

▪

of T-1055 challenged with the pathogen followed by seed treatment and soil application (Fig. 3c,d). Combined application enhanced the activities of PO and CAD registered an increase by 38 and 36 times, respectively, when compared to the untreated controls. Similarly, levels of PO and CAD were found to increase by 23 and 24 times, respectively, by seed treatment. Maximum activities were reached at 12 DAS. Application of T-1055 alone also showed a significant increase to the levels of PO and CAD during the experimental period when compared to untreated controls, but both the enzymatic activities were

lower compared with T-1055-treated plants challenged with the pathogen. Effect of T-1055 on the lignin content The plants inoculated with the pathogen alone showed an increased deposition of lignin, but deposition started at 10 DAS and subsequently declined drastically after 16 DAS and returned almost to the level of untreated control. Most pronounced increase in lignin deposition was caused by combined application of T-1055 challenged


659


B.N. Singh et al.

Table 1 Effect of treatment with T-1055 on phenolic content in healthy or Rhizoctonia solani-infected sunflower roots Phenolic content (lg g 1)* at 10 DASa Treatment

GA‡

Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5%

175 202 187 221 122 259 44 DAS† 212 298 241 273 199 302 54 DAS† 142 234 285 383 222 246 59 DAS† 97 127 165 198 153 391 616 DAS† 91 74 125 114 102 312 54 DAS† 88 47 75 095 082 304 92

12 Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5% 14 Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5% 16 Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5% 18 Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5% 20 Control (neither infected nor treated) Infected Treated (seed- and soil-treated) Infected + seed-treated Infected + soil-treated Infected + seed- and soil-treated CD at 5%

t-CA‡

CHA‡

08 07 12 06 02 10

ND ND ND 52 02 32 01 101 06 29

ND ND ND ND ND 177 06 21

141 176 203 397 193 765 79

07 08 17 09 08 17

182 274 206 156 148 735 38

10 15 18 05 08 26

10 21 16 09 06 12

ND ND 12 72 22 153 32

ND ND ND 95 02 23 01 251 10 59

323 244 192 393 231 795 62

12 15 16 12 15 19

117 321 219 253 279 432 58

01 22 14 09 07 16

07 15 23 11 11 12

ND ND 32 81 02 44 02 232 12 35

ND ND ND 153 06 137 01 324 11 69

166 221 227 448 273 1048 113

08 18 16 13 12 31

98 215 284 383 258 452 71

03 13 25 12 11 21

01 08 08 07 03 10

ND ND 21 56 02 32 01 168 05 274

ND ND ND 128 01 42 01 292 09 514

142 143 194 341 212 882 277

08 11 14 09 08 24

83 93 174 337 232 383 592

01 07 12 08 08 13

01 04 03 01 05 08

ND ND 18 43 27 102 28

ND ND ND 106 01 41 02 288 09 41

123 103 143 257 161 791 97

02 06 11 10 05 12

84 105 124 283 133 272 55

02 06 09 11 09 07

02 03 04 02 03 11

ND ND 12 35 23 83 36

ND ND ND 82 02 24 01 253 13 48

57 42 123 195 143 701 69

01 02 07 09 08 20

75 52 92 154 119 185 56

02 04 05 02 02 04

01 01 01 03

02 02 01 03

01 01 02 02

FA‡

p-CA‡

*Data represent mean standard error (SE) of twelve repeated analysis in two experiments; †DAS, days after sowing; ND, not detected; ‡GA, gallic acid; ‡t-CA, trans-cinnamic acid; ‡CHA, chlorogenic acid; ‡FA, ferulic acid and bp-CA, p-coumaric acid. CD, critical difference (P ≤ 005) for the different between the treatments.

with the pathogen followed by seed treatment and soil application (Fig. 4). Lignin deposition increased by 27, 34 and 37 times at 16, 18 and 20 DAS, respectively. 660

Maximum lignin deposition was recorded under influence by seed treatment and soil application at 20 DAS. There was recorded marked change in plants treated with



B.N. Singh et al.

Table 2 Identification of phenolic compounds by LC-UV-MS/MS analysis Ion full-scan MS Phenol

Molecular weight

[M-H]

Fragments

MS/MS approach product ion scan

UV band (nm)

GA CHA p-CA FA t-CA

170 354 164 194 148

169 353 163 193 147

125 191 119 178, 149, 134 147

169 353 163 193 147

272 326 (max) 300 310 324 (max), 296 278

GA, gallic acid; CHA, chlorogenic acid; p-CA, p-coumaric acid; FA, ferulic acid; t-CA, trans-cinnamic acid.

(a)

(c)

(b)

(d)

Figure 3 Effect of T-1055 on expressions of defence-related enzymes in sunflower. (a) phenylalanine ammonia-lyase (PAL, lmol t-CA min 1 mg 1 protein); (b) polyphenol oxidase (PPO, nkat mg 1 protein); (c) peroxidases (PO, nkat mg 1 protein); (d) cinnamyl alcohol dehy) Untreaterd control; drogenase (CAD, nkat mg 1 protein). Vertical bars indicate SE of at least twelve repeated analysis in two experiments. ( ( ) infected (pathogen); ( ) combined application; ( ) infected + seed treatment; ( ) infected + soil application and ( ) infected + combined application.

T-1055 alone during the time course of experimental period and the deposition of lignin remained lower compared with combined application, but content was higher when compared to the plants treated with the pathogen alone. The content of lignin was remained at low level in untreated control plants.

Effect of T-1055 on PR proteins The levels of PR-2 and PR-3 proteins were recorded maximum under influence by combined application challenged with the pathogen (Fig. 5a,b). The level of PR-2 protein registered an increase of 28, 33 and 34 times at


661


B.N. Singh et al.

(a)

Figure 4 Effect of T-1055 on deposition of lignin in sunflower expressed as thioglycolic acid derivatives [TGA g 1 alcohol insoluble residue (AIR)]. Vertical bars indicate SE of at least twelve repeated analysis in two experiments. ( ) Untreaterd control; ( ) infected (pathogen); ( ) combined application; ( ) infected + seed treatment; ( ) infected + soil application and ( ) infected + combined application.

10, 12 and 14 DAS, respectively, compared with the controls. While a markedly increased level of PR-3 protein 39 times higher was recorded at 12 DAS. Similarly, seed treatment with the pathogen only was found to induce maximum level of both proteins at 12 DAS, while on the other hand, maximum induction of PR-2 and PR-3 was recorded at 14 and 12 DAS, respectively, through soil application. The infected plants with the pathogen alone, and the levels of these proteins increased initially, but later declined drastically. Although the plants treated with T-1055 alone also had significantly higher activities of PR-2 and PR-3 proteins, the induction were observed at 10 and 12 DAS, respectively, thereafter it declined drastically. However, the levels of the proteins were remained higher compared with controls. Discussion Inducing the plants own defence mechanisms against pathogen through postapplication of BCAs and their products is a novel technique in plant protection (Singh et al. 2007; Vinale et al. 2008). Attempts to exploit fungal antagonists such as Trichoderma species as potential BCAs have recently led to the proposal that besides their recognized antifungal properties. Such organisms could also act as elicitors of plant defence reactions, thereby promoting the expression of plant defence-related products (Alfano et al. 2007; Segarra et al. 2007; Lorito et al. 2010). Biological control of R. solani with T. harzianum has been reported in many studies over the last few years (Shoresh and Harman 2008; Gallou et al. 2009; Singh 662

(b)

Figure 5 Effect of T-1055 on expression of PR-2 and PR-3 proteins in sunflower against. (a) PR-2 (nkat min 1 g 1 protein) and (b) PR-3 activities (nkat min 1 g 1 protein). Vertical bars indicate SE of at least twelve repeated analysis in two experiments. ( ) Untreaterd control; ( ) infected (pathogen); ( ) combined application; ( ) infected + seed treatment; ( ) infected + soil application and ( ) infected + combined application.

et al. 2011; Roatti et al. 2013). However, no study has been investigated on induction of defence substances in sunflower roots by T. harzianum against seedling blight caused by R. solani. This is crucial to understand the mechanism of bioprotection deliberated by T. harzianum and to develop innovative strategies to control the plant diseases. Our results demonstrate the beneficial effect of T-1055 by repressing R. solani ingress in the root of sunflower. It was through induction of phenolic, lignin deposition and activities of PAL, PPO, PO, CAD, PR-2 and PR-3. A close relationship between TPC in the roots and percentage of plant mortality as observed in the present investigation is in conformity with some earlier reports including those in chickpea against tomato wilt (Mandal and Mitra 2007), coconut palm against Ganoderma (Karthikeyan et al. 2006), cotton against R. solani



B.N. Singh et al.

(Howell et al. 2000) and chickpea against collar rot (Singh et al. 2007). The abilities of T. virens strains to induce phytoalexins were strongly correlated with disease control in the cotton seedlings challenged by R. solani (Howell et al. 2000). Furthermore, it has also been observed that T. viride enhanced the TPC in chickpea plants that lead to induced resistance against Machrophomina phaseolina, when applied as combined application. Therefore, suppression of seedling blight of sunflower might be due to highest accumulation of TPC. Several phenols viz. CHA, FA and protocatechuic acid have been reported as strongly antifungal, while others like GA which itself not being antimicrobial, is biotransformed into potent antimicrobial gallotannins (Sarma et al. 2002). Similarly, t-CA is a key product of phenylpropanoid pathway, synthesized from phenylalanine through catalysis by PAL and plays a key role in host resistance under pathogenic stress (Howell et al. 2000; Karthikeyan et al. 2006; Verma et al. 2007). In present investigation, significantly high accumulation of FA in the T-1055-treated plants was observed. Colonization of BCAs can be resulted in the accumulation of substantial amount of antimicrobial FA as well as increased esterification of phenylpropanoids of the cell wall (Mandal and Mitra 2007). Fry observed that a common host response is the esterification of FA to the host cell wall, and it has been suggested that cross-linking of FA leads to the formation of lignin-like polymers (Fry 1986). Similarly, synthesis of high amount of CHA and t-CA in the wall of the sunflower root cells by T-1055, compared with controls, suggesting their role in inducing resistance against R. solani. It is worth to note that no data on the toxicity of these phenols to R. solani were available, but it was very probable because their fungitoxicity was universal. The rapid accumulation of phenols may be a result of sensing of elicitor produced by T-1055 during root colonization at informational level by sunflower roots. Phenylalanine ammonia-lyase has been shown to regulate the production of several secondary compounds, including phytoalexins (Gauillard et al. 1993). Application of BCAs and their effectors molecules elevates the level of the flux through phenylpropanoid pathway, thereby supplying the carbon skeletons for phenolics which were the precursor molecules of lignin (De Ascensao and Dubery 2000). Coconut and cucumber plants colonized by BCAs had rapid and transient increase in PAL activity (Gallou et al. 2009). Similarly, recent study has shown that treatment of cucumber by T. asperellum T-203 lead to marked induction of PAL in the stems and roots (Yedidia et al. 2003). Gene expression analysis revealed an induction of PAL at early hours of postinoculation of potato plants treated with T. harzianum against R. solani (Gallou et al. 2009).

Trichoderma-activated PAL activity in bean roots and increased the level of signalling molecule: salicylic acid in the leaves (Harman et al. 2004). In our study, the rapid and transient increase in the level of PAL in the treated roots is in agreement with earlier reports (Yedidia et al., 1999; Yedidia et al. 2003; Verma et al. 2007). Invasion of root tissues by the pathogen might have been resulted in decreased activity of PAL, whereas increased activity of PAL due to T-1055 treatment might have prevented the fungal invasion, and thus, the activity maintained at higher levels during the experimental period. The last step in the synthesis of lignin and suberin has been proposed to be catalysed by PO, although other proteins may also be involved (Marra et al. 2006). Lignin is highly resistant to attack by invaders, and lignified cell walls are an effective barrier to pathogen entrance and spread (Quiroga et al. 2000). The products of this defencerelated chemical in the presence of a hydrogen peroxide and hydrogen donor have potent antimicrobial and antiviral activities (van Loon et al. 2008). In bean, rhizospheric colonization by various BCAs induced PO activity (Quiroga et al. 2000). Yedidia et al. (1999) also showed that the root inoculation of T. harzianum induced increased PO activity in Cucumis sativus seedlings. Results of PO activity indicate that the sunflower plants responded actively to T-1055 application through the rapid induction or activation of PO which might have contributed to induced resistance against invasion by R. solani. The importance of PPO activity in disease resistance is because of its property to oxidize phenolic compounds to quinines, which are more toxic to microorganisms that the original phenols (Vinale et al. 2008). It is reasonable to assume that increased activity of PPO is inversely proportional to the accumulation of toxic products of oxidation and therefore greater degree of resistance to pathogen infection. The possible involvement of PPO in defence of sunflower was suggested by the remarkably increased PPO activity after T-1055 treatment. In addition to PAL, PO and PPO, CAD is also an indicator of lignification because of its specific role at the end of the monolignol biosynthetic pathway (Moerschbacher et al., 1990). Recently, it has been shown that elicitors made from Trichoderma mycelium extract induced a rapid stimulation of the monolignol pathway in tomato roots, as confirmed by the increase in CAD activity (Mandal and Mitra 2007). The prominent increase in CAD activity in the roots of sunflower suggests that this enzyme plays an important role in the lignification response and correlates with the lignin determined concentration. PR proteins are host-coded proteins that are induced only in pathological or related situations (Yedidia et al. 2003). Some of


663


B.N. Singh et al.

the PR proteins such as PR-2 and PR-3 have potential to hydrolyse b-1,3-glucan and chitin, respectively, which are components of fungal cell walls, leading to direct growth inhibition of several fungi (Legrand et al. 1987). In addition, PR-2 and PR-3 stimulate plant defence by releasing oligosaccharides from fungal cell wall by their enzymatic action which act as elicitors or inducers of several defence genes (van Loon et al. 2008). In the present investigation, PR-2 and PR-3 levels significantly increased in the T-1055-treated sunflower plants. In Trichoderma-treated cucumber seedlings upon pathogen challenge, increased levels of defence-related plant enzymes such as chitinases and ß-1, 3-glucanases have been recorded (Shoresh et al. 2005). This potentiating of gene expression enables Trichoderma-treated plants to be more resistant to subsequent pathogen infection. Colonization of cucumber roots by T. harzianum strain T-203 was correlated with induction of PR proteins resulting in induction of systemic resistance against a broad range of pathogens (Yedidia et al. 2003). Yedidia et al. (1999) reported that the root inoculation of T. harzianum induced chitinase activity in leaves of cucumber seedlings. There has been a spate of publications over the past two decades on the induction of glucanases and chitinases by species of Trichoderma involved in disease resistance in the host plants (Harman et al. 2004). In summary, this study contributes to a better physiological and biochemical characterization of induced resistance by T. harzianum against R. solani in sunflower roots and can be useful for the plant disease management. Our results demonstrate that the combined application of T-1055 elicits a series of defence responses such as accumulation of phenolics, production of enzymes involved in phenylpropanoid pathway, deposition of lignin and PR proteins against the pathogen, and thus, it may be recommended for use as a promising alternate to minimize the impact of chemical fungicides on the environment. Further studies, designed to assess the molecular mechanisms of T-1055 in the induction of resistance against R. solani, are in progress. Acknowledgements Brahma N. Singh is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, for providing financial support. This research was also supported by a grant from the Ministry of Agriculture & Cooperation, Government of India under Macromode Project. Conflict of interest The authors declare that there are no conflict of interests. 664

References Ahvenniemi, P., Wolf, M., Lehtonen, M.J., Wilson, P., German-Kinnari, M. and Valkonen, J.P. (2009) Evolutionary diversification indicated by compensatory base changes in ITS2 secondary structures in a complex fungal species, Rhizoctonia solani. J Mol Evol 69, 150–163. Alfano, G., Lewis Ivey, M.L., Cakir, C., Bos, J.I., Miller, S.A., Madden, L.V., Kamoun, S. and Hoitink, H.A. (2007) Systemic modulation of gene expression in tomato by Trichoderma hamatum 382. Phytopathology 97, 429–437. Beaulieu, R., Lopez-Mondejar, R., Tittarelli, F., Ros, M. and Pascual, J.A. (2011) qRT-PCR quantification of the biological control agent Trichoderma harzianum in peat and compost-based growing media. Bioresour Technol 102, 2793–2798. Bruce, R.J. and West, C.A. (1989) Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. Plant Physiol 91, 889–897. Campbell, M.M. and Ellis, B.E. (1992) Fungal elicitormediated responses in pine cell cultures: iii. purification and characterization of phenylalanine ammonia-lyase. Plant Physiol 98, 62–70. Castle, A., Speranzini, D., Rghei, N., Alm, G., Rinker, D. and Bissett, J. (1998) Morphological and molecular identification of Trichoderma isolates on North American mushroom farms. Appl Environ Microbiol 64, 133–137. De Ascensao, A.R. and Dubery, I.A. (2000) Panama disease: cell wall reinforcement in banana roots in response to elicitors from Fusarium oxysporum f. sp. cubense Race Four. Phytopathology 90, 1173–1180. De Meyer, G., Bigirimana, J., Elad, Y. and H€ ofte, M. (1998) Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea. Eur J Plant Pathol 104, 279–286. Dickerson, D.P., Pascholati, S.F., Hagerman, A.E., Butler, L.G. and Nicholson, R.L. (1984) Phenylalanine ammonia-lyase and hydroxycinnamate: CoA ligase in maize mesocotyls inoculated with Helminthosporium maydis or H. carbonum. Physiol Plant Pathol 25, 111–123. Dubey, S.C., Bhavani, R. and Singh, B. (2011) Integration of soil application and seed treatment formulations of Trichoderma species for management of wet root rot of mungbean caused by Rhizoctonia solani. Pest Manag Sci 67, 1163–1168. Fobert, P.R. and Despres, C. (2005) Redox control of systemic acquired resistance. Curr Opin Plant Biol 8, 378–382. Fry, S.C. (1986) Cross-linking of matrix polymers in the growing cell walls of angiosperms. Ann Rev Plant Physiol 37, 165–186. Gallou, A., Cranenbrouck, S. and Declerck, S. (2009) Trichoderma harzianum elicits defense response genes in roots of potato plantlets challenged by Rhizoctonia solani. Eur J Plant Pathol 124, 219–230.



B.N. Singh et al.

Gauillard, F., Richard-Forget, F. and Nicolas, J. (1993) New spectrophotometric assay for polyphenol oxidase activity. Anal Biochem 215, 59–65. Hammerschmidt, R., Nuckles, E.M. and Kuc, J. (1982) Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol Plant Pathol 20, 73–82. Harman, G.E. (2006) Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96, 190–194. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I. and Lorito, M. (2004) Trichoderma species — opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2, 43–56. Heil, M. and Silva Bueno, J.C. (2007) Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense in nature. Proc Natl Acad Sci USA 104, 5467–5472. Howell, C.R., Hanson, L.E., Stipanovic, R.D. and Puckhaber, L.S. (2000) Induction of terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed treatment with Trichoderma virens. Phytopathology 90, 248–252. Karthikeyan, M., Radhika, K., Mathiyazhagan, S., Bhaskaran, R., Samiyappan, R. and Velazhahan, R. (2006) Induction of phenolics and defense-related enzymes in coconut (Cocos nucifera L.) roots treated with biocontrol agents. Bra J Plant Physiol 18, 367–377. Legrand, M., Kauffmann, S., Geoffroy, P. and Fritig, B. (1987) Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases. Proc Natl Acad Sci USA 84, 6750–6754. van Loon, L.C., Bakker, P.A., van der Heijdt, W.H., Wendehenne, D. and Pugin, A. (2008) Early responses of tobacco suspension cells to rhizobacterial elicitors of induced systemic resistance. Mol Plant Microbe Interact 21, 1609–1621. Lorito, M., Woo, S.L., Harman, G.E. and Monte, E. (2010) Translational research on Trichoderma:from ‘omics to the field. Annu Rev Phytopathol 48, 395–417. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. Mandal, S. and Mitra, A. (2007) Reinforcement of cell wall in roots of Lycopersicon esculentum through induction of phenolic compounds and lignin by elicitors. Physiol Mol Plant Pathol 71, 201–209. Marra, R., Ambrosino, P., Carbone, V., Vinale, F., Woo, S.L., Ruocco, M., Ciliento, R., Lanzuise, S. et al. (2006) Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Curr Genet 50, 307–321. Moerschbacher, B.M., Flott, B.E., Noll, B.E. and Reisener, H.J. (1990) On the specificity of an elicitor preparation from stem rust which induces lignification in wheat leaves. Plant Physiol Biochem 27, 305–315. Pan, S.Q., Ye, X.S. and Kuc, J. (1991) Association of b-1, 3-glucanase activity and isoform pattern with systemic

resistance to blue mould in tobacco induced by stem injection with Peronospora tabacina or leaf inoculation with tobacco mosaic virus. Physiol Mol Plant Pathol 39, 25–39. Quiroga, M., Guerrero, C., Botella, M.A., Barcel o, A., Amaya, I., Medina, M.I., Alonso, F.J., de Forchetti, S.M. et al. (2000) A tomato peroxidase involved in the synthesis of lignin and suberin. Plant Physiol 122, 1119–1128.. Roatti, B., Perazzolli, M., Gessler, C. and Pertot, I. (2013) Abiotic stresses affect Trichoderma harzianum T39-induced resistance to downy mildew in grapevine. Phytopathology 103, 1227–1234. Sarma, B.K., Singh, D.P., Mehta, S., Singh, H.B. and Singh, U.P. (2002) Plant growth-promoting rhizobacteria-elicited alterations in phenolic profile of chickpea (Cicer arietinum) infected by Sclerotium rolfsii. J Phytopathol 150, 277–282. Segarra, G., Casanova, E., Bellido, D., Odena, M.A., Oliveira, E. and Trillas, I. (2007) Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34. Proteomics 7, 3943–3952. Shoresh, M. and Harman, G.E. (2008) The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach. Plant Physiol 147, 2147–2163. Shoresh, M. and Harman, G.E. (2010) Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity. BMC Plant Biol 10, 136. Shoresh, M., Yedidia, I. and Chet, I. (2005) Involvement of jasmonic acid/ethylene signaling pathway in the systemic resistance induced in cucumber by Trichoderma asperellum T203. Phytopathology 95, 76–84. Shoresh, M., Harman, G.E. and Mastouri, F. (2010) Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol 48, 21–43. Singh, A., Srivastava, S. and Singh, H.B. (2007) Effect of substrates on growth and shelf life of Trichoderma harzianum and its use in biocontrol of diseases. Bioresour Technol, 9, 470–473. Singh, B.N., Singh, B.R., Singh, R.L., Prakash, D., Dhakarey, R., Upadhyay, G. and Singh, H.B. (2009a) Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera. Food Chem Toxicol 47, 1109–1116. Singh, B.N., Singh, B.R., Singh, R.L., Prakash, D., Sarma, B.K. and Singh, H.B. (2009b) Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L. Food Chem Toxicol 47, 778–786. Singh, H.B., Singh, B.N., Singh, S.P. and Nautiyal, C.S. (2010) Solid-state cultivation of Trichoderma harzianum NBRI1055 for modulating natural antioxidants in soybean seed matrix. Bioresour Technol 101, 6444–6453.


665


B.N. Singh et al.

Singh, B.N., Singh, A., Singh, S.P. and Singh, H.B. (2011) Trichoderma harzianum-mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defense against Rhizoctonia solani. Eur J Plant Pathol 131, 121–134. Trudel, J. and Asselin, A. (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178, 362–366. Verma, M., Brar, S.K., Tyagi, R.D., Surampalli, R.Y. and Valero, J.R. (2007) Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochem Eng J 37, 1–20. Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Marra, R., Woo, S.L. and Lorito, M. (2008) Trichoderma– plant–pathogen interactions. Soil Biol Biochem 40, 1–10. Viterbo, A., Montero, M., Ramot, O., Friesem, D., Monte, E., Llobell, A. and Chet, I. (2002) Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203). Curr Genet 42, 114–122.

666

Wilson, P.S., Ketola, E.O., Ahvenniemi, P.M., Lehtonen, M.J. and Valkonen, J.P.T. (2008) Dynamics of soilborne Rhizoctonia solani in the presence of Trichoderma harzianum: effects on stem canker, black scurf and progeny tubers of potato. Plant Pathol 57, 152–161. Wyrambik, D. and Grisebach, H. (1975) Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Eur J Biochem 59, 9–15. Yedidia, I.I., Benhamou, N. and Chet, I.I. (1999) Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 65, 1061–1070. Yedidia, I., Shoresh, M., Kerem, Z., Benhamou, N., Kapulnik, Y. and Chet, I. (2003) Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Appl Environ Microbiol 69, 7343–7353.


Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani.

Analysis of Phaseolus vulgaris response to its association with Trichoderma harzianum (ALL-42) in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani.

Trichoderma harzianum T-22 Induces Systemic Resistance in Tomato Infected by Cucumber mosaic virus.

Plant Phenotypic and Transcriptional Changes Induced by Volatiles from the Fungal Root Pathogen Rhizoctonia solani.

Transcriptome profile of Trichoderma harzianum IOC-3844 induced by sugarcane bagasse.

Optimization of Fungal Enzyme Production by Trichoderma harzianum KUC1716 through Surfactant-Induced Morphological Changes.

Nitrogen Metabolism and Growth Enhancement in Tomato Plants Challenged with Trichoderma harzianum Expressing the Aspergillus nidulans Acetamidase amdS Gene.

Molecular characterization and screening for sheath blight resistance using Malaysian isolates of Rhizoctonia solani.

Host Delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani).

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

Systemic Resistance to Powdery Mildew in Brassica napus (AACC) and Raphanus alboglabra (RRCC) by Trichoderma harzianum TH12.

Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum - Rhizoctonia solani Interaction.

Stem Rot on Adzuki Bean (Vigna angularis) Caused by Rhizoctonia solani AG 4 HGI in China.

Enhancement of Shikalkin Production in Arnebia euchroma Callus by a Fungal Elicitor, Rhizoctonia solani.

Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing.

Production and preliminary characterization of RNA-depolymerase from Trichoderma harzianum.

New C13 lipids from the marine-derived fungus Trichoderma harzianum.

Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani).

Statistical culture-based strategies to enhance chlamydospore production by Trichoderma harzianum SH2303 in liquid fermentation.

Integrated options for the management of black root rot of strawberry caused by Rhizoctonia solani Kuhn.

Proteomic response of Rhizoctonia solani GD118 suppressed by Paenibacillus kribbensis PS04.

Trichoderma hamatum: Its hyphal interactions withRhizoctonia solani andPythium spp.

Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana.