Enhancement of Rice Seed Germination and Vigour by Trichoderma spp

The present study was undertaken to examine the effectiveness of Trichoderma spp. to enhance rice germination and vigour. An in vitro experiment was carried out to assess the effect of seven isolates of Trichoderma spp. in enhancing rice germination and vigour. The results showed that all isolates of Trichoderma spp. significantly increased rice seedling growth, germination rate, vigour index and speed of germination with Trichoderma sp., SL2 showing the greatest increase in all the four parameters. Trichoderma sp., SL2 treated rice seeds attained values of 4.48 and 6.00 cm, 0.0084 and 0.0048 g and 1016.56 and 44.75 seeds/day for seedling shoot length seedling root length, shoot weight, root weight, vigour index and speed of germination respectively. We may conclude that Trichoderma spp. is able to enhance seed germination and vigour. The results of the study adds to the further understanding of the role of beneficial fungi in improving rice resistance to stress, yield and quality through seed invigoration.


INTRODUCTION
Rice (Oryza sativa L.) is one of the major staple food in the world, especially in Asia, the region that consumes the most rice (FAO, 2012).Rice farming nowadays is facing many environmental problems caused by input of chemical fertilizers and pesticides.The use of chemical fertilizers and pesticides has led to soil a decline in fertility, ecosystem damage, elimination of soil biota and emergence of resistant pathogens.Therefore the use of eco-friendly biofertilizers should be encouraged.Beneficial microorganisms have been reported to be involved in maintaining agricultural production, protecting the ecosystem and decreasing the use of chemical fertilizers (Adesemoye and Kloepper, 2009).
Seed priming is a pre-germination seed treatment whereby seeds are placed in partially hydrated condition (Bradford, 1986).Seed priming is considered as a method to enhance seedling quality and vigour (Basra et al., 2004).Seed priming is also involved in enhancing rice resistance to environmental stress (Goswami et al., 2013), weed suppressive ability and rice yield (Juraimi et al., 2012).Previous studies have shown that rice seeds respond to seed priming in the early part of the germination stage and revealed that it can increase seed germination, vigour index and germination speed (Anwar et al., 2013;Zarei and Sinaki, 2012).Priming rice seed with plant growth regulators is one of the beneficial treatments that can be used to invigorate rice seed and improve rice seed quality (Farooq et al., 2010).
Plant Growth Promoting Fungi (PGPF) is considered an alternative way to enhance rice growth and yield.The mechanisms by which PGPF act to enhance rice growth and yield includes production of phytohormones, phosphate solubilization, cellulose degradation and siderophore production.PGPF are generally abundant in the soil system and roots.One of the well-known fungi species listed in the PGPF group is the Trichoderma spp.(Doni et al., 2013).Currently, Trichoderma spp.are widely used in industrial processes and agriculture due to their ability to produce enzymes and secondary metabolites (Jiang et al., 2011;Mukherjee et al., 2008).In bio-fertilizer production, Trichoderma spp. is well recognized because of its ability to establish mycorrhizal-like association with plants, control of root and foliar pathogens, change the micro-floral composition in roots, enhance nutrient uptake, enhance root development, increase root hair formation, aid the plant in acquiring systemic resistance, degrade cellulose, solubilize phosphate and produce siderophores (Saravanakumar et al., 2013;Saba et al., 2012;Harman et al., 2004;Harman, 2000;Yadidia et al., 1999).
Germination stage is reported to be the most critical phase in the plant life cycle, because during this stage, plants have a high vulnerability to injury, disease and environmental stress (Rajjou at al., 2012).Research on the role of Trichoderma spp. in enhancing rice seed germination is very important to be undertaken due to the potential for Trichoderma spp. to act as plant growth regulators.Previous research studies on the effect of Trichoderma spp. on the improvement of seed germination have been conducted for soybean (Tančić et al., 2013), mustard (Lalitha et al., 2012), maize and beans (Okoth et al., 2011) and chili (Asaduzzaman et al., 2010).However, very little information is available with regards to enhancement of rice seed germination and vigour by Trichoderma spp. to improve rice seed germination and vigour.This research was carried out to examine the effect of Trichoderma spp. on rice (Oryza sativa L.) seed germination and vigour.

MATERIALS AND METHODS
Trichoderma spp.spore preparation: Isolates of Trichoderma spp.namely Trichoderma sp., SL1, Trichoderma sp., SL2, 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 isolates were grown separately in potato dextrose agar and incubated for seven days at 30°C.After incubation, spores of the Trichoderma spp.were immediately transferred to Erlenmeyer flasks containing sterilized water.
Rice seed preparation: Organic rice seeds of the variety MRQ74 were surface sterilized with 70% ethanol, followed by 5% sodium hypochlorite and washed by sterilized distilled water.One hundred rice seed grains were selected for each treatment then soaked in the respective Trichoderma spp. in a flask containing 10 7 spores/mL spore suspension for 30 min.Rice seeds soaked in sterilized distilled water served as control.The treated seeds were incubated for 5 days in sterilized petri dishes at 28±2°C fitted with filter paper and each petri dish was irrigated with 10 mL sterilized water.
Seedling length and weight: The root and shoot length were measured by selecting five seeds randomly for each treatment.Shoot length was measured from the base of the primary leaf to the base of the hypocotyl and root length was measured from the tip of the primary root to the base of hypocotyl, with all measurements expressed in centimeters.Shoot and root weight were examined using digital weight scales and expressed in grams.All measurements were according to the method used in Dash (2012).
Seed germination rate: Germination rate is the average of number of seeds that germinate over the 5and 10-day periods.).In this experiment data was calculated on day 5. Germination rate was computed using the formula proposed by IRRI (2011): Seed vigour index: Seed vigour index was examined after five days of incubation using the formula given by Abul-Baki and Anderson (1973) based on the product of germination (%) and seedling length (cm): S.V.I = Germination (%) ×Seedling Length Speed of germination: Numbers of seedlings emerging daily are counted from day 0 until day 5 of planting.Thereafter a Germination Index (G.I) is calculated by using the following formula as proposed by Gupta (1993): Statistical analyses: All the data were from triplicates and were represented as mean±standard deviation.Further, the variations in the results obtained with respect to different seedling length and seedling weight, as well as different seed germination rate, vigour index and speed of germination were statistically analyzed using one way Analysis of Variance (ANOVA) and tested for significance using Fisher's protected Least Significant Difference (LSD) at p≤0.05.

RESULTS AND DISCUSSION
Seedling length and weight: The length of shoot and root seedling by Trichoderma spp.were significantly greater than the control (untreated).However, it was not significant on the weight of shoot and root basis (Table 1 and 2).The result revealed that Trichoderma sp., SL2 has the highest ability to stimulate seedling root elongation compared to other strains (Fig. 1).The role of Trichoderma spp. in the production of auxins and gibberellins is the key factor to enhance rice seedling length (Chowdappa et al., 2013;Martínez-Medina et al., 2011).Gibberellins were first identified as metabolites of Gibberela fujikuroi which causes bakanae fungal disease (Bomke and Tudzynski, 2009;Phinney, 1983).Auxins produced by Trichoderma spp.are able to stimulate root initiation and cell elongation, while gibberellins are involved in cell division.Production of auxins and gibberellins by Trichoderma spp.enhances rice seedling performance during seed germination (Fig. 1).
Trichoderma spp.which has the ability to promote rice seedling length can be an alternative way to be    used in sustainable crop production.Among the seven isolates of Trichoderma spp.studied, Trichoderma sp., SL2 showed the highest consistency in the ability to enhance rice seedling weight (Fig. 2).The results demonstrate that the length of shoot and root was significantly higher for Trichoderma sp., SL2 than other strains and control (Fig. 3).It is envisioned that rice seedlings infected with Trichoderma sp., SL2 strain having better root growth and higher physiological activity would be transporting larger amounts of cytokinin from roots to shoot, resulting in a lower rate of leaf senescence in the later stages of development (San-oh et al., 2006).
Rice seed germination rate, vigour index and speed of germination: The results showed that the inoculation of the rice seeds with Trichoderma spp.significantly increased rice seed germination rate, vigour index and speed of germination compared to untreated control (Table 3 and Fig

CONCLUSION
Seed priming has become fashionable among agricultural practitioners in the effort to improve crop stand under normal and adverse conditions.From this study, the higher seed vigour and germination rates of rice seeds treated with Trichoderma spp. in comparison to untreated seeds underscores the potential of beneficial fungal species such Trichoderma spp. as seed priming material to improve rice seed germination.This augurs well with the search for effective biological agents to enhance rice seed quality.

Fig. 4 :Fig. 5 :
Fig. 4: Relationship between germination rate and vigour index with different treatments (Trichoderma strains) during seed germination . 4).Germination percentages were high in the rice seeds treated with Trichoderma spp. to the order of 96 to 99% compared to the untreated control seeds which registered 90% germination.The speed of germination was also high for the rice seeds treated with Trichoderma spp.compared to control.The range of germination speed in the rice seeds treated with Trichoderma spp.ranged from 36.2 to 44.75 seeds/day compared to the untreated control seeds which registered 35.86 seeds/day.The growth hormone which is produced by Trichoderma spp.enhances germination and speed.Trichoderma spp.also reduces the growth of pathogenic fungi by colonizing the rice seedlings as shown in Fig.5.These results are in support of the findings ofJayalakshmi et al. (2009) who reported the biological control potential of Trichoderma harzianum.Vigour index was highest in the treatment of Trichoderma sp., SL2 followed by Trichoderma sp., SL1 Trichoderma sp., SL6 Trichoderma sp., SL7 Trichoderma sp., SL5, Trichoderma sp., SL4, Trichoderma sp., SL3 and the lowest recorded in the untreated control (Fig.4).Zheng and Shetty (2000) reported that Trichoderma spp.induced phenolic compound production in the plant during seed germination and phenolic compounds produced by Trichoderma spp.led to enhancement of seed vigour.FurtherCai et al. (2013) reported that a secondary metabolite namely harzianolide produced by Trichoderma spp.can influence the early stage of plant development through the enhancement of root length.

Table 1 :
Comparison of length of shoot, length of root, weight of shoot and weight of root in different treatments

Table 2 :
Comparison of length of shoot, length of root, weight of shoot and weight of root in different treatments (Trichoderma strains) Means with different small letter was significantly different between treatments at p<0.05; Means with ''ns'' was not significantly different with all treatment at p<0.05

Table 3 :
Rice seed germination rate, vigour index and speed of germination by different treatments (Trichoderma strains) during seed germination