154 ACTA BOT. CROAT. 76 (2), 2017 Acta Bot. Croat. 76 (2), 154–162, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0054 ISSN 0365-0588 eISSN 1847-8476 Physiological responses of crop plants against Trichoderma harzianum in saline environment Roomana Yasmeen, Zamin Shaheed Siddiqui* Stress Physiology Phenomics Centre, Department of Botany, University of Karachi, Karachi 75270, Pakistan Abstract – The physiological response of crop plants against Trichoderma harzianum (Th-6) in a saline habi- tat was studied. Trichoderma harzianum (Th-6) is an endophytic fungus that shows salt tolerance and estab- lishes a symbiotic relationship with a host plant. To evaluate the role of Trichoderma harzianum (Th-6) in mitigating the consequences of salinity stress on crop plants, seeds of maize and rice were coated with Trich- oderma before sowing and salt treatment. Later, after germination, twenty-one day old seedlings were sub- jected to NaCl concentrations (50, 100 and 150 mM). Salinity negatively affected all investigated physiologi- cal parameters in both crops. Treatment of seeds with Trichoderma improved plant growth and Th-treated plants exhibited substantial physiological adjustment in a saline environment compared to Th-untreated plants. The Th-treated plants under salt stress showed higher relative water content and stomatal conductance, better photosynthetic performance and higher pigment concentrations, as well as higher catalase and superox- ide dismutase activities. Moreover, proline content in salt stress environment was higher in Th-treated plants, while H2O2 content declined. The physiological role of Trichoderma harzianum in mitigating the salt related consequences of both crop plants is discussed. Keywords: antioxidant enzymes activity, maize, physiological performance, rice, salinity, Trichoderma har- zianum * Corresponding author, e-mail: zaminss@uok.edu.pk Introduction Plants are often subjected to unfavorable changes in their environment. Abiotic stresses play a major role in re- ducing crop production around the globe (Bybordi 2012). Among them, salinity is one of the most important abiotic stresses that are widely distributed in both irrigated and non-irrigated areas of the world. Research on salinity in plants has produced a vast litera- ture showing its negative influence on crop plant productiv- ity (Mahajan and Tuteja 2005, Oliveira et al. 2013). Most salt sensitive crops cannot tolerate a high concentration of NaCl, especially in the soil (Prasad et al. 2000). This results in poor germination, growth and biomass allocation (Neu- mann 2008, Ahmad and Prasad 2012). Studies have shown that plants at vegetative and reproductive phases are more sensitive to soil salinity (Hu and Schmidhalter 2005, Lauch- li and Grattan 2007, Siddiqui et al. 2014). Prolonged expo- sure to salinity causes specific ion toxicity, nutritional and hormonal imbalance, reduced water potential and so on (Ahmad et al. 2010, Siddiqui and Khan 2013) Trichoderma sp are beneficial endophytic plant symbi- onts that are widely used as biocontrol agents against fun- gal diseases in crop plants (Harman 2011, Afzal et al. 2013). However, some studies have reported that Trichoderma in- duces tolerance to biotic and abiotic stresses in plants (Monnet et al. 2001, Evelin et al. 2009, Mastouri et al. 2010, Shoresh et al. 2010, Estrada et al. 2013). Treatment of seed with Trichoderma spp in many cereals and vegeta- ble crops has a positive impact on plant growth, improving hormone performance (Howell 2003, Harman 2006), which could enhance tolerance to salinity stress (Gachomo and Kotchoni 2008, Rawat et al. 2011, Hashem et al. 2014). However, the role of Trichoderma harzianum in crop plant tolerance to abiotic stress like salinity and drought and the physiological mechanism involved needs to be closely monitored. Therefore, the present study was designed to ex- amine the physiological responses of two crops plants, maize and rice, in a saline environment following seed treatment with Trichoderma harzianum (Th-6). Some phys- iological attributes of salt tolerance were selected for this study. PHYSIOLOGICAL RESPONSES OF CROPS AGAINST ENDOPHYTES ACTA BOT. CROAT. 76 (2), 2017 155 Materials and methods Seed selection Seeds of maize (Zea mays L.) var. NT6621 and rice (Ory- za sativa L.) var. Kernel were obtained from the Depart- ment of Plant Protection, Karachi, Pakistan. Seeds were sur- face sterilized in 10% sodium hypochlorite solution for 3 minutes and rinsed thoroughly with distilled water then air dried. Culture collection and treatment The pure strain of Trichoderma harzianum (Th-6) was obtained from the Plant Pathology Laboratory, Department of Botany. First, an experiment was conducted in petriplates containing potato dextrose agar at different NaCl concen- trations (25, 50, 100, 150, 200 mM) together with a Tri- choderma harzianum (Th-6) disc for 8 days. The salt con- centration was selected according to the optimum growth achieved by Trichoderma harzianum in saline media (Fig. 1). Seeds of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel were treated with Trichoder- ma harzianum using 2% gum arabic as sticker. The colony forming unit (cfu) was 67.3 conidia 10–3 of Trichoderma. Later, seeds were sown on a pot filled with 500 g soil each (one plant per pot). Autoclaved (1 hour at 80 °C) soil was used for the experiment with the following composition: sand particles; 80.5, silt; 7.1, clay; 8.1, organic carbon; 0.20, nitrogen. pH 7.5 and EC 1.8 ds m–1 were recorded ac- cording to Dahnke and Whitney (1988) by a CMD 500 WPA conductivity meter, Linton Cambridge U.K). Maize and rice crops were allowed to grow at an average day- night temperature (26±4 °C and 18±3 °C). Salt stress was applied to twenty-one-day old seedlings each day by using 25 mM NaCl to achieve the desired level (Gorham et al. 1987) and moisture contents were maintained with tap wa-and moisture contents were maintained with tap wa- ter. Three NaCl concentrations (50, 100 and 150 mM NaCl) were applied. Plant treated with tap water served as control. Each treatment and control was replicated four times. Root, shoot length, biomass, physiological parameters and anti- oxidant enzyme activities were examined. Relative water content Four leaf strips of 4×2 cm2 from the mid-veins and the edge section of leaves were cut with scissors from each treatment of rice and 4 1.2 cm2 discs of maize were excised and fresh weights (FW) were determined. For the measure- ment of turgid weight (TW), leaves were left in distilled water for 24 h under low irradiance condition. Samples were then dried at 80 °C for 48 h in oven and dry weight (DW) was determined. Relative water content (RWC) was calculated by the fresh leaf sample method described by Barrs and Weatherley (1962) and modified as: RWC=(FW–DW/TW–DW)×100 Quantum yield PSII and stomatal conductance Measurements of chlorophyll fluorescence emission from the 20 randomly selected leaves were monitored with a fluorescence monitoring system (Handy PEA) in the pulse amplitude modulation mode. A leaf adapted to dark condi- tions for 30 min using leaf clips, was initially exposed to the modulated measuring beam of far-red light (LED source with typical peak at wavelength 735 nm). The original (F0) and maximum (Fm) fluorescence yields were measured un- der weak modulated red light (0.5 μmol m–2 s–1) with 1.6 s pulses of saturating light (6.8 μmol m–2 s–1 PAR). The vari- able fluorescence yield (Fv) was calculated by the equation Fm–F0. The ratio of the variable to maximum fluorescence (Fv/Fm) was calculated as the dark-adapted quantum yield of PSII photochemistry and performance index and non-pho- tochemical quenching were calculated as described by Maxwell and Johnson (2000). Likewise, the stomatal con- ductance (gs) of 20 randomly selected leaves of each treated and control plant was examined using a leaf porometer (Model SC-1, Decagon). Photosynthetic pigment extraction and estimation Leaf samples (0.5 g) were ground in 10 mL of 96% methanol and then centrifuged at 4000 rpm for 10 min. To- tal chlorophyll [Chl(a+b)], chlorophyll a (Ca), and chlorophyll b (Cb) contents were determined according to Lichtenthaler (1987). The supernatant was separated and the absorbances were read at 666, 653 and 470 nm on spectrophotometer. The amount of these pigments was calculated according to the following formulas: Ca=15.65×A666–7.340×A653 Cb=27.05×A653–11.21×A666 Cx+c=1000×A470–2.860×Ca–129.2×Cb/245 where, Ca – Chlorophyll a, Cb – Chlorophyll b, Cx+c – total carotenoids Chlorophyll contents of leaf tissues were expressed as µg mg−1 FW. Proline content Proline content was measured according to the proce- dure of Bates et al. (1973). Leaf samples (0.5 g) were ho- mogenized with 5 mL sulphosalicylic acid (3% w/v) and the homogenate was filtered on Whatman No. 1 filter paper. Fig. 1. Trichoderma harzianum was cultured in potatoes dextrose agar containing 100 mM NaCl. YASMEEN R., SHAHEED SIDDIQUI Z. 156 ACTA BOT. CROAT. 76 (2), 2017 Then, 2 mL of extract in test tube was taken and 2 mL of glacial acetic acid and 2 mL of acid ninhydrin where added. The mixture was heated in a boiling water bath at 100 °C for an hour. A brick red color developed. After cooling of the reaction mixture, 4 mL toulene was added and mixed vigorously for 15 to 20 seconds. Chromophore containing toluene was separated from the aqueous phase. Then the mixture was allowed to reach room temperature. The absor- bance was recorded at 520 nm against a toluene blank. Pro- line content in sample was estimated by referring to a stan- dard curve made from known concentrations of proline by taking following formula: µmol proline g–1FW=(µg proline/mL×mL toulene)/ /115.5 µg/µmol)/(g sample)/5 H2O2 production Hydrogen peroxide content was measured according to the procedure of Velikova et al. (2000). Freshly harvested leaf samples (100 mg) were homogenized with 3 mL of 0.1% (w/v) trichloroacetic acid in an ice bath and the ho- mogenate was centrifuged at 12,000 g for 15 min. Later, 0.5 mL of 10 mM phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide (KI) were added to 0.5 mL of the superna- tant. The absorbance of the supernatant was read at 390 nm. The amount of H2O2 was calculated using a standard curve and expressed as µmol g–1 FW. Enzyme assays Leaf samples (500 mg) were crushed and homogenized in 10 mL protein extraction buffer containing Tris-HCl pH 6.8, 50 mg polyvinylpyrrolidone and 0.05 mM ethylenediamine- tetraacetic acid (EDTA). Whole contents were centrifuged at 12,000 rpm for 10min in a Smart R-17, Hanil centrifuge. The supernatant was collected and used to determine the activities of catalase and superoxide dismutase. Total pro- tein was estimated by the method of Bradford (1976). Catalase (CAT; EC 1.11.1.6) activity was estimated by method of Patterson et al. (1984). The decomposition of H2O2 was measured at 240 nm taking Δε as 43.6 mM cm–1. The reaction mixture (3.0 mL) consisted of 10.5 mM H2O2 in 0.05 M potassium phosphate buffer (pH 7.0) and the re- action was initiated after the addition of 0.1 mL enzyme ex- tract at 25 °C. The decrease in absorbance at 240 nm was used to calculate the activity. One unit of CAT activity is defined as the amount of enzyme that catalyzes the conver- sion of 1 mM of H2O2 min–1 at 25 °C. Superoxide dismutase (SOD; EC 1.15.1.1) activity was recorded according to the method of Beyer and Fridovich (1987). The reaction mixture consisted of 27.0 mL of 0.05 M potassium phosphate buffer (pH 7.8), 1.5 mL of L-methi- onine (300 mg per 2.7 mL), 1.0 mL of nitrobluetetrazolium salt (14.4 mg per 10 mL), and 0.75 mL of Triton X-100. Ali- quots (1.0 mL) of this mixture were delivered into small glass tubes, followed by the addition of 20 mL enzyme ex- tract and 10 mL of riboflavin (4.4 mg per 100 mL). The cocktail was mixed and then illuminated for 15 minutes in an aluminum foil-lined box, containing 25 W fluorescent tubes. In a control tube the sample was substituted for by 20 mL of buffer and the absorbance was measured at 560 nm. The reaction was stopped by switching off the light and placing the tubes in the dark. Increase in absorbance due to the formation of formazan was measured at 560 nm. Under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples were calculated by determining the percentage inhi- bition per minute. One unit of SOD is the amount of enzyme that causes a 50% inhibition of the rate for reduction of ni- trobluetetrazolium salt under the conditions of the assay. Statistical analysis Statistical analysis was carried out using the personal computer software packages SPSS version 20. All data were subjected to SPSS and two-way ANOVA was performed. 1 1 2 3 Fig.1. Trichoderma harzianum was cultured in Potatoes dextrose agar containing 100 mM 4 NaCl. 5 0 50 100 150 0 50 100 150 R o o t (c m ) 5 10 15 20 Without Trichoderma With Trichoderma NaCl mM S h o o t (c m ) 0 10 20 30 40 R o o t (c m ) 2 4 6 8 10 12 14 16 Without Trichoderma With Trichoderma NaCl mM S h o o t (c m ) 0 5 10 15 20 25 a A a B C D a B C D a b C D Maize Rice b B c C d D A b c d A b c d A B c d * * * * * * * * * * * * * * * ns Fig.2. Effect of T. harzianum seed treatments on seedling growth of maize (Zea mays L.) var. 7 NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment . Vertical lines on bars 8 graph stand for means standard error (±). Same alphabets on the bar graphs showed non-9 significant difference within each treatment (*) stand for significant and (ns) for non-10 significant difference among the treatments (with and without Th). 11 Fig. 2. Effect of Trichoderma harzianum seed treatment on seedling growth of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without T. harzianum. PHYSIOLOGICAL RESPONSES OF CROPS AGAINST ENDOPHYTES ACTA BOT. CROAT. 76 (2), 2017 157 Results The results showed that shoot and root length signifi- cantly declined with the increase in salinity concentration in the soil (Fig. 2). However, seed treated with Trichoderma harzianum (Th) has shown substantial increase in plant growth. Shoot and root length increased significantly in Th- treated maize and rice plants subjected to 50, 100 and 150 mM NaCl treatments as compared to those plants that were not treated with Trichoderma (Fig. 2). Relative water content (RWC) of maize and rice signifi- cantly decreased at all salinity concentrations (50, 100, 150 mM) (Fig. 3). However, in Th-treated plants, the adverse effects of salinity were alleviated, showing a substantial in- crease in the RWC of both crop plants over Th-untreated plants. 2 12 0 50 100 150 0 50 100 150 NaCl mM R e la ti v e w a te r c o n te n t (% ) 0 20 40 60 80 100 Without Trichoderma With Trichoderma NaCl mM R e la ti v e w a te r c o n te n t (% ) 0 20 40 60 80 100Without Trichoderma With Trichoderma a b c d a A b B c C d D Maize Rice A B C A* * * ** ns ns ns Fig.3. Effect of T. harzianum seed treatments on relative water content and biomass 14 accumulation of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in 15 saline environment. Vertical lines on bars graph stand for means standard error (±).Same 16 alphabets on the bar graphs showed non-significant difference within each treatment (*) stand 17 for significant and (ns) for non- significant difference among the treatments (with and without 18 Th). 19 20 21 22 23 Fig. 3. Effect of Trichoderma harzianum seed treatment on relative water content and biomass accumulation of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treat- ments with and without T. harzianum. 3 00 50 100 150 50 100 150 C h l b ( g m g -1 F W ) 1 2 3 4 5 6 7 T o ta l c h l ( g m g -1 F W ) 2 4 6 8 10 12 14 NaCl mM C a ro te n o id s ( g m g -1 F W ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 C h l a ( g m g -1 F W ) 2 4 6 8Without Trichoderma With Trichoderma C h l b ( g m g -1 F W ) 1 2 3 4 5 6 7 T o ta l c h l ( g m g -1 F W ) 2 4 6 8 10 12 NaCl mM C a ro te n o id s ( g m g -1 F W ) 0.0 0.2 0.4 0.6 0.8 1.0 a A b B c C d D a A b B c d C D a A b c d B C D a A b B C D A B C D A B C D A B C D c d a b c d a b d c a b c d C h l a ( g m g -1 F W ) 2 4 6 8 Without Trichoderma With Trichoderma a A b B c C d D Maize Rice * * * * * * * * * * * * * * * * * * * * * * * * ns ns ns * ns ns ns ns Fig.4. Effect of T. harzianum seed treatments on photosynthetic pigments of maize (Zea mays 25 L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment . Vertical lines 26 on bars graph stand for means standard error (±).Same alphabets on the bar graphs showed 27 non-significant difference within each treatment (*) stand for significant and (ns) for non- 28 significant difference among the treatments (with and without Th ) 29 30 Fig. 4. Effect of Trichoderma harzianum seed treatments on photosynthetic pigments of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non-signifi- cant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without T. harzianum. YASMEEN R., SHAHEED SIDDIQUI Z. 158 ACTA BOT. CROAT. 76 (2), 2017 Salinity caused a considerable decrease in pigment con- tent but Th treatment mitigated the salinity affect and im- proved pigment concentration during all NaCl treatments (Fig. 4). The values of photosynthetic attributes like dark-adapt- ed quantum yield (Fv/Fm ratio), performance index (PIabs), photochemical quenching (qP) and stomatal conductance (gs) were reduced in all salt treatments but Th-treated plants showed higher values than Th-untreated plants (Fig. 5). In the present study, free proline and H2O2 contents of both crop plants were measured at different NaCl concen- trations (50, 100, and 150 mM) with and without the Tri- choderma inoculum (Fig. 6). Results revealed that proline and H2O2 contents were significantly influenced by the presence of Trichoderma in a saline environment. However, in comparison between the crop plants, free proline content was significantly higher in maize than in rice. Seeds that were treated with Trichoderma showed maximum accumu- lation of proline content in all saline treatments (0, 50, 100, 150 mM) as compared to those treatments without Tricho- derma. However, maximum proline content was recorded at 150 mM NaCl (Fig. 6). H2O2 production was elevated at all salinity levels in plants that were not treated with Tri- choderma (Fig. 6). However, H2O2 production significantly lowered with the increasing concentration of NaCl in Th- treated plants. Activity of antioxidant enzymes like SOD and CAT were measured at different NaCl concentrations with or without the Trichoderma inoculum (Fig. 7). Observations revealed that the SOD and CAT activities of both maize and rice plants were substantially increased with increased NaCl concentration. However, in a comparison between the crop plants, a maize plant showed greater antioxidant activ- ity than rice (Fig. 7). It was observed that the presence of Trichoderma in a saline environment additionally increased the activity of antioxidant enzymes as compared to plants in saline medium without Trichoderma. Discussion Salinity is a major abiotic factor that restricts plant growth and productivity, which not only causes osmotic stress but also alters physiological and biochemical mecha- nism in plants. Crops such as rice and maize (Poaceae) are sensitive or moderately sensitive to salinity. They are un- able to tolerate a higher amount of salt in soil. Results showed that the application of Trichoderma to the crop plants enhances tolerance to a high concentration of NaCl. Trichoderma is an endophytic symbiont, as its inoculation 4 31 32 0 50 100 150 0 50 100 150 P e rf o rm a n c e i n d e x (P I a b s ) 0.5 1.0 1.5 2.0 2.5 3.0 P e rf o rm a n c e i n d e x (P I a b s ) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 D a rk a d a p te d q u a n tu m y ie ld (F v /F m R a ti o ) 0.5 1.0 1.5 2.0Without Trichoderma With Trichoderma P h o to c h e m ic a l q u e n c h in g (q p ) 1 2 3 4 5 P h o to c h e m ic a l q u e n c h in g (q p ) 0.5 1.0 1.5 2.0 2.5 NaCl mM S to m a ta l c o n d u c ta n c e (m m o l m -2 s -1 ) 0 100 200 300 400 NaCl mM S to m a ta l c o n d u c ta n c e (m m o l m -2 s -1 ) 0 100 200 300 400 a b c d A B B C a A b c d B C D a A b c d B C D a A b c d B C D a b c d A B C D a A b B c d C D a A B C D b c d D a rk a d a p te d q u a n tu m y ie ld (F v /F m R a ti o ) 0.5 1.0 1.5 2.0 2.5 3.0 Without Trichoderma With Trichoderma a A b B c C d D Maize Rice * * * * * * * * * * * * ns * * * * * * * * ns ns ns ns ns ns ns ns ns ns ns Fig.5. Effect of T. harzianum seed treatments on photosynthetic attributes of maize (Zea mays 34 L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment . Vertical lines 35 on bars graph stand for means standard error (±).Same alphabets on the bar graphs showed 36 non-significant difference within each treatment (*) stand for significant and (ns) for non- 37 significant difference among the treatments (with and without Th ) 38 39 Fig. 5. Effect of Trichoderma harzianum seed treatments on photosynthetic attributes of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non-signifi- cant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without T. harzianum. PHYSIOLOGICAL RESPONSES OF CROPS AGAINST ENDOPHYTES ACTA BOT. CROAT. 76 (2), 2017 159 has antagonistic properties and therefore enhances the sys- temic tolerance to salt stress in plants (Harman et al. 2004). It was observed that salinity caused a substantial reduc- tion in growth and biomass of those plants without a Th treatment. The application of Th mitigates salt-related con- sequences in plants, which results in considerable increases in growth and biomass production. It was reported that the application of Th in a saline habitat improved biomass and growth parameters (Moud and Maghsoudi 2008, Rasool et al. 2013, Ahmad et al. 2014). The increase in growth rela- tive water content and biomass production with a Th appli- cation may be due to its ability to produce phytohormones like gibberellins and cytokine, which may not only promote the plant growth but also increase some degree of tolerance in a saline environment (Harman 2000, Benitez et al. 2004, Iqbal and Ashraf 2013, Ahmad et al. 2015). Relative water content decreased in maize and rice crops under NaCl stress but increased due to the application of Th. The maintenance of a substantial amount of relative water content in leaves is a main strategy for maintaining optimal growth of plants under salinity (Siddiqui et al. 2014). It was reported that T. harzianum provides better ability to regulate additional intracellular water relations due to biomass accumulation resulting from the uptake of more water under salt stress (Rawat et al. 2011, Hashem et al. 2014). 6 C A T ( U n it m g -1 o f p ro te in ) 20 40 60 80 100 120 140 160 180 Without Trichoderma With Trichoderma C A T ( U n it m g -1 o f p ro te in ) 20 40 60 80 100 120 140 160 Without Trichoderma With Trichoderma NaCl mM S O D ( U n it m g -1 o f p ro te in ) 0 20 40 60 80 100 120 140 160 NaCl mM S O D ( U n it m g -1 o f p ro te in ) 0 20 40 60 80 100 120 140 160 0 50 100 150 0 50 100 150 a A b B c C d D a A b B c C d D a A b B c C d D a A b B c C d D Maize Rice * * * * * * * * * * * * * * * * 49 Fig.7. Effect of T. harzianum seed treatments on catalase (CAT) and superoxide dismutase 50 (SOD) antioxidant enzyme activity of maize (Zea mays L.) var. NT6621 and rice (Oryza 51 sativa L.) var. Kernel in saline environment. Vertical lines on bars graph stand for means 52 standard error (±).Same alphabets on the bar graphs showed non-significant difference within 53 each treatment (*) stand for significant and (ns) for non- significant difference among the 54 treatments (with and without Th 55 56 57 PROVIDE PLEASE ALL FIGURES AS TIFF OR PDF WITH RESOLUTION OF 300 DPI 58 Fig. 6. Effect of Trichoderma harzianum seed treatments on proline and hydrogen peroxide content of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non- significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without T. harzianum. Fig. 7. Effect of Trichoderma harzianum seed treatments on catalase (CAT) and superoxide dismutase (SOD) antioxidant enzyme activity of maize (Zea mays L.) var. NT6621 and rice (Oryza sativa L.) var. Kernel in saline environment. Results are expressed as means±standard errors. Same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant differ- ence among the treatments with and without T. harzianum. YASMEEN R., SHAHEED SIDDIQUI Z. 160 ACTA BOT. CROAT. 76 (2), 2017 Photosynthetic attributes like Fv/Fm ratio, PIabs, qP and gs of Th-treated plants were increased in all NaCl treatments compared to Th-untreated plants. It has been demonstrated that content of plant photosynthetic pigments like chl a, chl b, total chlorophyll and carotenoids generally reduces under NaCl (Parida and Das 2005, Sairam et al. 2002). The result of the decrease in carotenoid contents under salt stress might be due to decrease in β-carotene and zeaxanthin for- mation (Sultana et al. 1999). Salt stress directly affects the chloroplast function, degrading enzymes which results in substantial reduction in photosynthesis of plants (Siddiqui et al. 2014). However, in the present study, the application of Trichoderma in a saline environment produced a consid- erable increase in all the tested photosynthetic attributes. It was reported that Th stimulates the synthesis of chlorophyll enzymes and phytohormones under different biotic stress in plants (Rawat et al. 2011, Zhang et al. 2013, Hashem et al. 2014). There was a maximum amount of photosynthetic pigments present in Th-treated plants in maize and rice crops as compared to untreated plants under NaCl condi- tion. Further, these results are in accordance with the results of Mishra and Salokhe (2011) who reported that inocula- tion of seed by Trichoderma enhanced pigment system PSII performance and produced a higher rate of transpiration in plants under salt stress conditions. In plants subjected to sa- linity stress, photosynthetic rate and stomatal conductance might be disturbed due to the higher amount of Na+ ions ac- cumulation which disturb the electron transport chain dur- ing photosynthesis (Kanwal et al. 2011). From the present investigation, it was clearly observed that the dark adapted quantum yield (Fv/Fm ratio), performance index (PIabs), pho- tochemical quenching (qP) and stomatal conductance (gs) were decreased with the increase in salinity concentration but increased by Th application. This could be due to an in- crease in chlorophyll concentration by the application of Trichoderma in saline environment (Sheng et al. 2008). Results showed a substantial decrease of proline and an increase of H2O2 contents in untreated plants subjected to salt stress, but increased proline with decreased H2O2 pro- duction in Th-treated plants was observed in both crops. It was suggested that proline accumulation provided protec- tion to the cell through balancing osmolyte concentration under salt stress conditions in tolerant plants (Greenway and Munns 1980). In an abiotic stress like salinity, elevated H2O2 production damages protein and lipid molecules (Sid- diqui et al. 2014). It is presumed that the application of Trichoderma lowers H2O2 production in salt stress due to proline accumulation. Moreover, it was reported that Trich- oderma enhances antioxidant enzymes such as glutathione S-transferases (GSTs) and peroxidase (POD) activities and lowers ROS production (Hajiboland et al. 2010, Wu et al. 2010, Alqarawi et al. 2014). It was reported that maximum quantum yield and pho- tosynthetic performance control the production of ROS (Siddiqui and Khan 2013, Siddiqui et al. 2014). Photosyn- thetic performance and quantum yield are inter-related and are often decreased in abiotic stress and under elevated ROS level. Fluctuating response with respect to quantum yield and performance index in stress environment are di- versified and specific for some plant species (Behera et al. 2002). Substantial photosynthetic performance index and quantum yield in maize treated with Trichoderma indicate that maize could develop better symbiotic relationship with Th in saline environment compared to rice. In our study, it was detected that the presence of Tri- choderma in a saline environment increased the activity of antioxidant enzymes as compared to those saline media that did not have Trichoderma. Earlier, it was reported that the activities of antioxidant enzymes like CAT, SOD, POD and ascorbate peroxidase (APX) were increased in a saline en- vironment (Siddiqui 2013). It is presumed that the presence of Trichoderma in a saline environment diminished H2O2 production due to elevated antioxidant enzyme activities as well as proline production. It was reported that antioxidant enzymes, production of osmolytes and polyols like proline, sorbitol etc. are important physiological strategy for coping with the consequences of abiotic stress and maintaining ion homeostasis (Sairam et al. 2002, Siddiqui and Khan 2011, Rasool et al. 2013). Further it was observed that a Tricho- derma inoculation enhanced antioxidant enzyme activities and decreased salt stress in plants (Hajiboland et al. 2010, Hashem et al. 2014, Ahmad et al. 2015). It can be concluded that application of Trichoderma harzianum enhances salt tolerance of maize and rice through higher antioxidant activities and high proline con- tent. Treatment with Trichoderma harzianum not only en- hanced some physiological parameters but also lowered the H2O2 concentration reducing the damaging effect of ROS within plants. 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