Physiological and growth response of rice plants (Oryza sativa L.) to Trichoderma spp. inoculants
© Doni et al.; licensee Springer. 2014
Received: 13 March 2014
Accepted: 4 May 2014
Published: 29 May 2014
Trichoderma spp., a known beneficial fungus is reported to have several mechanisms to enhance plant growth. In this study, the effectiveness of seven isolates of Trichoderma spp. to promote growth and increase physiological performance in rice was evaluated experimentally using completely randomized design under greenhouse condition. This study indicated that all the Trichoderma spp. isolates tested were able to increase several rice physiological processes which include net photosynthetic rate, stomatal conductance, transpiration, internal CO2 concentration and water use efficiency. These Trichoderma spp. isolates were also able to enhance rice growth components including plant height, leaf number, tiller number, root length and root fresh weight. Among the Trichoderma spp. isolates, Trichoderma sp. SL2 inoculated rice plants exhibited greater net photosynthetic rate (8.66 μmolCO2 m−2 s−1), internal CO2 concentration (336.97 ppm), water use efficiency (1.15 μmoCO2/mmoH2O), plant height (70.47 cm), tiller number (12), root length (22.5 cm) and root fresh weight (15.21 g) compared to the plants treated with other Trichoderma isolates tested. We conclude that beneficial fungi can be used as a potential growth promoting agent in rice cultivation.
Rice growth is influenced by a combination of genotype, environment and management factors. Balancing and improving soil fertility is one of the main factors in enhancing rice growth and yield. The intensive cultivation of rice that depends on chemical fertilizers and pesticides have led to the decrease in soil fertility and deteriorating soil health. The excessive use of chemical fertilizers in the current decades has led to soil toxicity through the presence of toxic heavy metals and adversely affecting the health of rice plants (Habibah et al. ). To overcome the decrease in soil fertility and deterioration in soil health caused by the use of chemical fertilizers, it is necessary to look for alternative ways to improve soil fertility and stimulate the growth of rice plants.
Microbes have been reported to be a key factor in maintaining soil quality and increasing rice yield and growth. The use of microbes to enhance rice growth while making the plant resistant to pathogens has been reported as an eco-friendly way to maintain the ecosystem. For decades, the application of microbes in a sustainable agroecological manner has increased rapidly due to their ability to act as plant growth promoters (Sakthivel and Gnanamanickam ; Carreres et al., ; Malik et al., ; Anhar et al., ; Pedraza et al., ; Amprayn et al., ; Anizan et al., ).
Trichoderma spp. recently was suggested as a Plant Growth Promoting Fungi (PGPF) due to their ability to produce siderophores, phosphate-solubilizing enzymes, and phytohormones (Doni et al., ). Trichoderma species play important roles in decomposition, mycoparasitism, and even in cellulose degradation (Jiang et al., ; Druzhinina et al., ; Samuels, ). Trichoderma spp. was reported to be able to increase growth in plants such as strawberries, tomatoes, soya beans, apples, cotton and gray mangroves (Saravanakumar et al.,; Raman, ; John et al., ; Shanmugaiah et al., ; Morsy et al. ; Porras et al., ). However, very little research has been conducted on the potential of Trichoderma spp. for improving rice growth.
Recently, we reported the successful isolation of several Trichoderma spp. and which have been proven to possess positive plant growth promotion potential in enhancing rice seed germination and vigour (Doni et al., ). This research was conducted to examine the effect of Trichoderma spp. on rice growth and physiological response.
Materials and methods
This experiment was conducted at the Fermentation Technology Laboratory, Genetics Molecule Laboratory and Greenhouse, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia. A completely randomized design (with nine treatments and three replications) was used for this experiment. Rice plants were placed in soil with different Trichoderma spp. inoculants and soil with NPK fertilizer 100 kg ha−1. The NPK fertilizer used is in accordance to the rate employed in Micheal et al., () for rice variety MRQ74. Sterilized soil without any application was used for control.
Trichoderma spp. inoculants preparation
Local isolates of Trichoderma spp. which was isolated from System of Rice Intensification Paddy Field, Sik, Kedah, Malaysia namely Trichoderma sp. SL1, Trichoderma sp. SL2 (Public Accession number: UPMC 1021), Trichoderma sp. SL3, Trichoderma sp. SL4, Trichoderma sp. SL5, Trichoderma sp. SL6 and Trichoderma sp. SL7 were obtained from the Fermentation Technology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia. Each isolate was grown separately in potato dextrose broth and incubated for seven days in a thermo shaker at a speed of 200 rpm and 30°C. After incubation, Trichoderma spp. inoculants were filtered using filter paper and stored in sterile polyethylene plastic bags.
Soil preparation and Trichoderma spp. inoculation
Autoclaved homogenous sandy clay loam soils were used for this experiment; the dosage of Trichoderma spp. inoculants was set at 5 g per 1 kg soil. The inoculated soil was immediately placed in 15 × 15 cm plastic containers.
Rice plant preparation
Rice seeds of the variety MRQ74 were surface sterilized with 70% ethanol, followed by 5% sodium hypochlorite and washed by sterilized distilled water. The rice seeds were grown in autoclaved sandy clay loam soil under green house condition with 30 ± 4°C temperature, 320 ± 3 μmol light intensity, 80 ± 3% humidity and 11 h 11 m 17 s ± 9 s photoperiod, and placed in a seedling tray. Seven day-old rice seedlings of MRQ74 were transplanted singly in 15 × 15 cm plastic containers containing different Trichoderma spp. treatments, NPK treatment and control. Water was maintained at 2 cm level from the soil surface and actively aerated by physically disturbing and breaking-up the soil surface once every ten days.
Measurement of rice growth and physiological components
Rice physiological and growth components were measured 30 days after transplanting. Measurements for the physiological characteristics were made on flag leaves. Measurements of net photosynthesis (μmol CO2 m−2 s−1), leaf stomatal conductance (mol H2O m−2 s−1), and transpiration rate (mmol H2O m−2 s−1) were monitored using a LICOR 6400 portable photosynthesis system (Lincoln, Nebraska, USA) and infrared gas analyser (IRGA). To assess the trade-off between CO2 uptake and water loss, instantaneous water-use efficiency (WUE) was calculated as the ratio between photosynthetic rate and transpiration rate (μmol CO2/μmol H2O). These measurements were taken on a clear sunny day between 09:45 am until 11:30 am under a saturated light condition (solar radiation > 1200 μmol.m−2.s−1). Plant height (cm) measured from ground level to the tip of the longest leaf, tiller number and leaf number were counted for each treatment and control. For rice root length (cm) and root fresh weight (g) measurements, the rice plants were separated carefully from the soil. Root length was measured from the base of the stem to the longest root using a ruler and root fresh weight was measured using digital scales. Rice root dry weight (g) measurement was done after rice roots were dried in the oven at a temperature of 65°C for seven days.
All data were statistically analyzed using one-way analysis of variance (ANOVA). The significance of the effect of the treatment was determined using F-test and to determine the significance of the difference between the means of the treatments, least significant difference (LSD) was calculated at 5% probability level. Regression relationship was determined using the data analysis SPSS software version 20.
Rice plant growth performance
Comparison of plant height, leaf number, tiller number, root length, root dry weight and root wet weight in different treatments
Plant height (cm)
Root length (cm)
Root dry weight (g)
Root fresh weight (g)
Trichoderma sp. SL1
Trichoderma sp. SL2
Trichoderma sp. SL3
Trichoderma sp. SL4
Trichoderma sp. SL5
Trichoderma sp. SL6
Trichoderma sp. SL7
Rice plant physiological characteristic
Comparison of net photosynthetic rate, stomatal conductance, transpiration and internal CO 2 concentration in different treatments
Net photosynthetic rate (μmolCO2m−2 s−1)
Stomatal conductance (mmolH2O m−2 s−1)
Internal CO2concentration (ppm)
Trichoderma sp. SL1
Trichoderma sp. SL2
Trichoderma sp. SL3
Trichoderma sp. SL4
Trichoderma sp. SL5
Trichoderma sp. SL6
Trichoderma sp. SL7
Rice plant growth performance
The fresh weight of rice root plants treated with Trichoderma spp. was significantly greater than the NPK treated plants and control. However, the weight of dry rice root plants was not significant among treatments. Rice plants inoculated with Trichoderma sp. SL2 have the highest increase in root fresh weight. The capacity of Trichoderma spp. to produce growth hormones such as auxins and gibberelins were reported as the main factor that contributes to the ability of Trichoderma spp. to support root growth and increase water absorption from soil (Arora et al., ; Contreras-Cornejo et al., ; Martínez-Medina et al., ). The present findings are also in agreement with previous research by Viterbo et al. () that revealed the role of Trichoderma asperellum in promoting canola seedling root elongation via ACC deaminase (ACCD) activity.
Our study also reports a lack of response of the rice variety MRQ74 to NPK fertilizer. Recently, research done by Khairiah et al. () revealed that the heavy metal content of the rice variety MRQ 74 plants in chemical fertilizers-used rice field area was high due to excessive usage of NPK fertilizers. Previous research by Yap et al. () also suggested that the use of NPK fertilizers in rice cultivation significantly increase heavy metal content in rice plants. Further, Yadav () stated that heavy metals may cause oxidative stress inside the plants leading to cellular damage. Our research is in agreement with previous researches which revealed the lack response of this variety to NPK fertilizer.
Rice plants physiological characteristics
Rice growth performance is subjected to environmental factors which affect the physiological processes inside rice plant cells. Improving rice physiological characteristics is considered to be desirable due to its agronomic importance towards the achievement of high rice yield (Makino, ; Li et al., ). From Table 2, net photosynthetic rates, stomatal conductance, and internal CO2 concentration were significantly different between treatments. High photosynthetic rates were observed for Trichoderma sp. SL1 and Trichoderma sp. SL2. It is known that large amounts of sucrose exudates provided by rice roots to Trichoderma sp. SL1 and Trichoderma sp. SL2 can facilitate good root colonization by Trichoderma spp. as well as promote good coordination of defense mechanism of rice plants (Vargas et al., ). The results imply that the root physiological activity of Trichoderma sp. SL2 is the highest among the Trichoderma spp. studied. The results also revealed that Trichoderma sp. SL2 has the lowest internal CO2 concentration and the highest photosynthetic rate. This indicates that the activity of carboxylation by CO2 fixation for glucose production as carbohydrate metabolism in the rice plants was very active. The results agree with the findings of Thakur et al. ().
Research by Thakur et al. () found that high photosynthetic rates coupled with low transpiration rates indicate high water use efficiency. This report supports our experiment on Trichoderma sp. SL2 which showed high values for these parameters. The activity of Trichoderma spp. that contributes to the enhancement of root growth and distribution was also considered as a key factor to the prolonged photosynthetic activity and the delayed senescence in rice plants (Mishra and Salokhe, ). Further, Trichoderma spp. was recently reported as having the potential to degrade cellulose (Jiang et al., ). Cellulose degradation may release a large amount of N in rice plant rhizosphere. High N concentration uptake has positive correlation with photosynthetic rate.
In Figure 2, water use efficiency of Trichoderma sp. SL2 was the highest among the Trichoderma spp. isolates registering at 1.15 μmolmmol−1. Stomatal conductance was significantly correlated with photosynthesis. Stomatal conductance plays an importance role in generating photosynthesis in rice plants because H2O and CO2 which are involved in photosynthetic process must pass through the stomata before they enter mesophyll cells and chloroplast stroma (Fu et al., ). Further, Harman et al. () stated that mechanisms employed by Trichoderma spp. in enhancing nutrient availability by solubilization and chelation of minerals can increase plant metabolism leading to the enhancement of plant physiological activity.
The present study concludes that Trichoderma spp. have the potential to enhance rice physiological processes and growth. In this respect, the present experiment proved that Trichoderma sp. SL2 was the best strain compared to six others strains.
We wish to thank Captain Zakaria Kamantasha and Tuan Hj. Marzuki Md. Zin from SRI Lovely, Sik, Kedah, for providing rice seeds for this research. This study was financially funded by Universiti Kebangsaan Malaysia under grant ETP-2013-070, Komuniti-2012-001, Komuniti-2012-007 and DLP-2013-023.
- Amprayn K-O, Michael TR, Kecskés M, Pereg P, Nguyen HT, Kennedy IR: Plant growth promoting characteristics of soil yeast ( Candida tropicalis HY) and its effectiveness for promoting rice growth. App Soil Ecol 2012, 61: 295–299. 10.1016/j.apsoil.2011.11.009View ArticleGoogle Scholar
- Anhar A, Doni F, Advinda L: Respons pertumbuhan tanaman padi ( Oryza sativa L.) terhadap introduksi Pseudomonad flouresen . Eksakta 2011, 1(1):1–11.Google Scholar
- Anizan I, Ahmad A, Rosenani AB, Habibah J: SRI rice crop establishment. Trans Malaysian Soc Plant Physiol 2012, 20: 11–15.Google Scholar
- Arora DK, Elander RP, Mukherji KG: Fungal Biotechnology. In Handbook of Applied Mycology. Marker Dekker, New York; 1992:4p.Google Scholar
- Cai F, Yu G, Wang P, Wei Z, Fu L, Shen Q, Chen W: Harzianolide, a novel plant growth regulator and systemic resistance elicitor from Trichoderma harzianum . Plant Physiol Biochem 2013, 73: 106–113. 10.1016/j.plaphy.2013.08.011View ArticlePubMedGoogle Scholar
- Carreres RGT, Sendra J, Ballesteros R, Fernández Valiente E, Quesada A, Nieva M, Leganés F: Effect of nitrogen rates on rice growth and biological nitrogen fixation. J Agric Sci 1996, 127: 295–302. 10.1017/S002185960007845XView ArticleGoogle Scholar
- Chowdappa P, Kumar SPM, Lakshmi MJ, Upreti KK: Growth stimulation and induction of systemic resistance in tomato against early and late blight by Bacillus subtilis OTPB1 or Trichoderma harzianum OTPB3. Biol Control 2013, 65: 109–117. 10.1016/j.biocontrol.2012.11.009View ArticleGoogle Scholar
- Contreras-Cornejo HA, Macías-Rodríguez L, Cortés-Penagos C, López-Bucio J: Trichoderma virens , a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in arabidopsis. Plant Physiol 2009, 149: 1579–1592. 10.1104/pp.108.130369PubMed CentralView ArticlePubMedGoogle Scholar
- Doni F, Al-Shorgani NKN, Tibin EMM, Abuelhassan NN, Anizan I, Che Radziah CMZ, Wan Mohtar WY: Microbial involvement in growth of paddy. Curr Res J Biol Sci 2013, 5(6):285–290.Google Scholar
- Doni F, Anizan I, Che Radziah CMZ, Salman AH, Rodzihan MH, Wan Mohtar WY (2014) Enhancement of rice seed germination and vigour by Trichoderma spp. Res J App Sci Eng Technol 7(21): (in press)Google Scholar
- Druzhinina IS, Kopchinsky AG, Kubicek CP: The first 100 Trichoderma species characterized by molecular data. Mycoscience 2006, 47: 55–64. 10.1007/S10267-006-0279-7View ArticleGoogle Scholar
- Fu Y, Zheng-wei L, Zhi-chun W, Yuan C: Relationship between diurnal changes of net photosynthetic rate and influencing factors in rice under saline sodic stress. Rice Sci 2008, 15(2):119–124. 10.1016/S1672-6308(08)60024-4View ArticleGoogle Scholar
- Habibah J, Lee PT, Khairiah J, Ahmad MR, Fouzi BA, Ismail BS: Speciation of heavy metals in paddy soils from selected areas in Kedah and Penang, Malaysia. African J Biotechnol 2011, 10(62):13505–13513.Google Scholar
- Harman GE (2006) Overview of mechanisms and uses of Trichoderma spp. Phytopathol. doi:10.1094/PHYTO-96–0190Google Scholar
- Harman GE, Petzoldt R, Comis A, Chen J: Interactions between Trichoderma harzianum strain T22 and maize inbred line Mo17 and effect of this interaction on diseases caused by Pythium ultmum and Colletotricum graminicola . Phytopathol 2004, 94(2):147–153. 10.1094/PHYTO.2004.94.2.147View ArticleGoogle Scholar
- Jiang X, Geng A, He N, Li Q: New Isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production. J Biosci Bioeng 2011, 111: 121–127. 10.1016/j.jbiosc.2010.09.004View ArticlePubMedGoogle Scholar
- John RP, Tyagi RD, Prévost D, Brar SK, Pouleur S, Surampalli RY: Mycoparasitic Trichoderma viride as a biocontrol agent against Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes and as a growth promoter of soybean. Crop Prot 2010, 29: 1452–1459. 10.1016/j.cropro.2010.08.004View ArticleGoogle Scholar
- Khairiah J, Azie RR, Habibah J, Zulkifle I, Ismail BS: Heavy metal content of paddy plants in Langkawi, Kedah, Malaysia. Aust J Basic Appl Sci 2013, 7(2):123–127.Google Scholar
- Li X, Bu N, Li Y, Ma L, Xin S, Zhang L: Growth, photosynthesis and antioxidant responses of endophyte infected and non-infected rice under lead stress conditions. J Hazard Mater 2012, 213: 55–61. 10.1016/j.jhazmat.2012.01.052View ArticlePubMedGoogle Scholar
- Lorito M, Woo SL, Harman GE, Monte E: Translational research on Trichoderma : from ‘omics to the field. Annu Rev Phytopathol 2010, 48: 395–517. 10.1146/annurev-phyto-073009-114314View ArticlePubMedGoogle Scholar
- Makino A: Photosynthetic, grain yield, and nitrogen utilization in rice and wheat. Plant Physiol 2011, 155: 125–129. 10.1104/pp.110.165076PubMed CentralView ArticlePubMedGoogle Scholar
- Malik KA, Rakhshanda B, Mehnaz B, Rasul G, Mirza MS, Ali S: Association of nitrogen-fixing, Plant-Growth-Promoting Rhizobacteria (PGPR) with Kallar grass and rice. Plant Soil 1997, 194: 37–44. 10.1023/A:1004295714181View ArticleGoogle Scholar
- Martínez-Medina A, Roldán A, Albacete A, Pascual JA: The Interaction with Arbuscular Mycorrhizal Fungi or Trichoderma harzianum alters the shoot hormonal profile in melon plants. Phytochemistry 2011, 72: 223–229. 10.1016/j.phytochem.2010.11.008View ArticlePubMedGoogle Scholar
- Micheal JSAS, Juraimi AS, Selamat A, Man A, Anwar MP, Uddin MK: Critical period of weed control in aerobic rice system. AJCS 2013, 7(5):665–673.Google Scholar
- Mishra A, Salokhe VM: Rice growth and physiological responses to SRI water management and implications for crop productivity. Paddy Water Environ 2011, 9: 41–52. 10.1007/s10333-010-0240-4View ArticleGoogle Scholar
- Morsy EM, Abdel-Kawi KA, Khalil MNA: Efficiency of Trichoderma viride and Bacillus subtilis as biocontrol agents gainst Fusarium solani on tomato plants. Egypt J Phytopathol 2009, 37(1):47–57.Google Scholar
- Nawrocka J, Malolepsza U: Diversity in plant systemic resistance induced by Trichoderma. Biol Control 2013, 67: 149–156. 10.1016/j.biocontrol.2013.07.005View ArticleGoogle Scholar
- Neumann B, Laing M: Trichoderma : An Ally in the Quest for Soil System Sustainability. In Biological Approaches to Sustainable Soil System. Edited by: Uphoff N, Fernandes E, Herren H, Husson O, Laing M, Palm C, Pretty J, Sanchez P, Sanginga N, Thies J. Taylor & Francis, Boca Raton, FL; 2006:491–500. 10.1201/9781420017113.ch34View ArticleGoogle Scholar
- Pedraza RO, Bellone CH, de Bellone SC, Sorte PMFB, dos Santos Teixeira KR: Azospirillum Inoculation and Nitrogen Fertilization Effect on Grain Yield and on the Diversity of Endophytic Bacteria in the Phyllosphere of Rice Rainfed Crop. European J Soil Biol 2009, 45: 36–43. 10.1016/j.ejsobi.2008.09.007View ArticleGoogle Scholar
- Porras M, Barrau C, Romero F: Effects of soil solarization and Trichoderma on strawberry production. Crop Prot 2007, 26: 782–787. 10.1016/j.cropro.2006.07.005View ArticleGoogle Scholar
- Raman J: Response of Azotobacter, Pseudomonas and Trichoderma on Growth of Apple Seedling. International Conference on Biological and Life Sciences IPCBEE, IACSIT Press, Singapore; 2012.Google Scholar
- Saba HDV, Manisha M, Prashant KS, Farham H, Tauseff A (2012) Trichoderma – a promising plant growth stimulator and Biocontrol agent. Mycosphere. doi:10.5943/mycosphere/3/4/14Google Scholar
- Sakthivel N, Gnanamanickam SS: Evaluation of Pseudomonas fluorescens for suppression of sheath rot disease and for enhancement of grain yields in rice ( Oryza sativa L.). Appl Environ Microbiol 1987, 53: 2056–2059.PubMed CentralPubMedGoogle Scholar
- Samuels GJ: Trichoderma : a review of biological systemics of the genus. Mycol Res 1996, 100(8):923–935. 10.1016/S0953-7562(96)80043-8View ArticleGoogle Scholar
- Saravanakumar K, Shanmuga Arasu V, Kathiresan K: Effect of Trichoderma on soil phosphate solubilization and growth improvement of Avicennia marina . Aquat Bot 2013, 104: 101–105. 10.1016/j.aquabot.2012.09.001View ArticleGoogle Scholar
- Shanmugaiah V, Balasubramanian N, Gomathinayagam S, Manoharan PT, Rajendran A: Effect of single application of Trichoderma viride and Pseudomonas fluorescens on growth cotton plants. African J Agri Res 2009, 4(11):1220–1225.Google Scholar
- Shukla N, Awasthi RP, Rawat L, Kumar J: Biochemical and physiological responses of rice ( Oryza sativa L.) as influenced by Trichoderma harzianum under drought stress. Plant Physiol Biochem 2012, 54: 78–88. 10.1016/j.plaphy.2012.02.001View ArticlePubMedGoogle Scholar
- Tchameni SN, Ngonkeu MEL, Begoude BAD, Wakam Nana L, Fokom R, Owona AD, Mbarga JB, Tchana T, Tondje PR, Etoa FX, Kuaté J: Effect of Trichoderma asperellum and Arbuscular Mycorrhizal Fungi on Cacao growth and resistance against black pod disease. Crop Prot 2011, 30: 1321–1327. 10.1016/j.cropro.2011.05.003View ArticleGoogle Scholar
- Thakur M, Sohal BS (2013) Role of elicitors in inducing resistance in plants against pathogen infection: a review. ISRN Biochem :1–10. doi:10.1155/2013/762412Google Scholar
- Thakur AK, Uphoff N, Antony E: An assessment of physiological effects of system of rice intensification (sri) practices compared with recommended rice cultivation practices in india. Cambridge J 2010, 46(1):77–98.Google Scholar
- Vargas WC, Mandawe JC, Kenerley CM: Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant Physiol 2009, 151: 792–808. 10.1104/pp.109.141291PubMed CentralView ArticlePubMedGoogle Scholar
- Viterbo A, Landau U, Kim S, Chernin L, Chet I: Characterization of AAC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203. FEMS Microbiol Lett 2010, 305: 42–48. 10.1111/j.1574-6968.2010.01910.xView ArticlePubMedGoogle Scholar
- Yadav SK: Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S Afr J Bot 2010, 76: 167–179. 10.1016/j.sajb.2009.10.007View ArticleGoogle Scholar
- Yap DW, Adezrian J, Khairiah J, Ismail BS, Ahmad-Mahir R: The uptake of heavy metals by paddy plants ( Oryza sativa ) in Kota Marudu, Sabah, Malaysia. Am Eurasian J Agric Environ Sci 2009, 6(1):16–19.Google Scholar
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