NigerJ.mycol Vol.9, 10-23

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1 CONTROL OF ROT DISEASE OF OKRA THROUGH FOLIAR APPLICATION WITH TRICHODERMA HARZIANUM *Udoh, M. E. and Olufolaji, D. B. Department of Crop, Soil and Pest Management, The Federal University of Technolgy, Akure. Nigeria *correspondence author; com NigerJ.mycol Vol.9, ABSTRACT Macrophomina phaseolina is one of the major pathogens of okra which causes considerable loss by reducing germinability, seedling establishment, and also causing black rot disease, wilting and damping-off with resultant damages on seeds, stem, flowers and pods. Studies were carried out to investigate the efficacy of Trichoderma harzianum as foliar treatment in the control of okra disease caused by M. phaseolina. Trichoderma harzianum, an antagonistic fungus to the pathogen used in this study, was isolated from decayed wood obtained from Teaching and Research Farm of The Federal University of Technology Akure, Ondo State, Nigeria and M. phaseolina was isolated from infected okra seeds variety NHAE 47-3, obtained from National Institute of Horticulture Research (NIHORT) in Ibadan, Oyo State, Nigeria. Treatments were; foliar application of spores of T. harzianum at 24, 48 and 72 hours before and after introduction of M. phaseolina for preventive and curative methods of control respectively. Mancozeb was also applied as the standard check. Uninfected seedlings and infected seedlings with no control measure applied, served as positive and negative controls. It was observed from this study, that the antagonist (T. Harzianum) inhibited the growth of the pathogen in vitro. In the in vivo studies, it was observed that the percentage seed germination and seedling establishment with T. harzianum 72 hours before inoculating M. phaseolina had significant (p< 0. 05) and highest germination of %. Disease incidence was significantly (p< 0. 05) higher in seeds treated with pathogen alone having %. Furthermore, disease severity was higher in control with pathogen alone with %. Also, okra seeds treated with T. harzianum 72 hours before infection with M. phaseolina, had significantly highest number of pods, yielding These suggest that the use of T. harzianum would be more beneficial ecofriendly, in okra cultivation and may be used as an alternative to synthetic fungicides to enhance plant growth and reduce disease incidence. This will subsequently result in high okra yield in Nigeria. Keywords: Trichoderma harzianum, Macrophomina phaseolina, rot disease, antagonistic, disease incidence, disease severity Nigerian Journal of Mycology Vol. 9 (2017) 10

2 Udoh & Olufolaji INTRODUCTION Okra, Abelmoschus esculentus (L. ) Moench belongs to the family Malvaceae, (Sarkar, 2010). It is an important vegetable crop grown mainly in the tropical or sub-tropical regions. It is regarded as an important vegetable throughout Nigeria. Okra seeds contain a considerable amount of good quality oil and protein which can be used as a substitute for coffee (Valeriana, 2010). Okra is mainly propagated through seeds and has a duration of days (Anab, 1976). The ripe seeds contain Thiamine (0. 07 mg), approximately 20% edible oil, and is a good source of vitamins A, B, C and minerals, (Nicotinic acid mg) and Iodine (Amade, 2010). Flowering is continuous, but highly dependent upon biotic and abiotic stress. Flower bud initiation, flowering, anthesis and stigma receptivity are influenced by genotype and climatic factors like temperature and humidity (Venkatramini, 1992). Okra is susceptible to a wide range of fungi which can cause damages in the field on germination, flowering and fruit production, however fruit storage is also affected (White, 2015). Macrophomina phaseolina is one of the major fungal pathogens that affect okra seeds leading to rot disease. Control of okra rot, could be carried out through the use of chemical fungicides like Mancozeb or Dithane M45 but the use of chemical fungicides has been discouraged by International Environmental Protection Agency (EPA) (Olufolaji, ) due to the harzards posed on the environment and human beings who consume the farm produce. Hence, the aim of this study is to source for an alternative to the chemical fungicides in controlling the rot disease of okra. MATERIALS AND METHODS Experimental site: This study was carried out in the Pathology Laboratory and Screen House of Crop, Soil and Pest management Department, Federal University of Technology Akure, Ondo State, Nigeria. ( N, E). Isolation and identification of Macrophomina phaseolina from okra Macrophomina phaseolina used in this study was isolated from infected okra seeds obtained from the National Institute of Horticultural Research (NIHORT) Ibadan, Nigeria using potato dextrose agar (PDA). Pure cultures obtained were identified at the Advanced Plant Pathology Laboratory of the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. Nigerian Journal of Mycology Vol. 9 (2017) 11

3 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum Isolation and identification of Trichoderma harzianum from decayed wood Trichoderma harzianum used in this study was isolated from decayed wood obtained from the Teaching and Research Farm of the Federal University of Technology Akure, Nigeria. Conidia and sclerotia from the isolates were used for the identification, which was further confirmed at the Advanced Plant Pathology Laboratory, IITA Ibadan Nigeria. Preparation of spore suspension of M. phaseolina and T. harzianum Spores from 7 day old pure cultures of M. phaseolina and T. harzianum was harvested using a sterile scalpel to dislodge the spores into 10ml of sterile distilled water in the Petri dish from the surface of PDA. The suspension obtained was poured into a 50ml beaker and the concentration of the spores of M. phaseolina and T. harzianum was standardized to 10 4 spores/ml and 10 8 spores/ml, respectively, with the aid of a haemocytometer. Standard chemical check Mancozeb suspension was used as a check in this study. The recommended rate of 0.25g was dissolved in 10ml of sterile distilled water 5ml portion of the suspension was sprayed on each plant. Sterilization of Soil Samples Sandy loam soil was sterilized using steam heat method to destroy the propagules of pathogens and other seeds. Five kilograms (5. 0kg) of sterilized soil was weighed into each plastic pot of 7L capacity. A total of twenty four plastic pots were used and each was perforated at the base to allow for free drainage and soil aeration. There were a total of eight treatments and each treatment was replicated thrice. Treatments application legend: M= Macrophomina phaseolina; T= Trichoderma harzianum; T24M, T48M and T72M; Preventive treatment (T24M -- Trichoderma harzianum spore suspension 24hours before introduction of M. Phaseolina) M24T, M48T and M72T; Curative treatment (M24T -- Macrophomina phaseolina spore suspension 24hours before introduction of T. Harzianum Mancozeb24 /M; Mancozeb (0. 25mg/ml) at 24hours before introduction of M. phaseolina (preventive) M72/Mancozeb; M. phaseolina spore Nigerian Journal of Mycology Vol. 9 (2017) 12

4 Udoh & Olufolaji suspension (at 72hours) before introduction of Mancozeb ( mg/ml) (curative) T t = Sterile distilled water (control) Experimental layout and data collection The experiment was laid out in a Completely Randomized Design (CRD) with three replications and data collected were subjected to Analysis of Variance (ANOVA) with the means separation using Turkey Test at 5% level of significance. Statistical Analysis was done with the aid of SPSS version Computer Software Statistical Package. Data were collected on seed germination, seedling establishment, effect of mancozeb on disease incidence and severity, number of flowers and pods of okra plant in the screen house. Disease incidence was determined using the expression: Disease Incidence = Also disease severity was assessed by visual observation and scoring was done using 0-4 scale on the basis of percentage of foliage yellowing or necrosis, according to the modified scale of Richard and Philip (2011) 0=0%, 1=1-33%, 2=34-66%, 3=67-100% and 4= dead plant. RESULTS Effect of treatment on okra plant height There was no significant difference in the heights of plants treated at weeks 3, 5, 6, and 7. But weeks 4 and 8 were significantly different from each other. The greatest height was recorded in plants treated through preventive method with mancozeb before the introduction of M. phaseolina for 24 hours having cm. This was followed by T 72 M having cm, while the shortest plant was recorded in curative method having cm in M 72 T. It was observed that there were significant (p 0. 05) differences among all treatments. (Table 1). Effect of each treatment on okra plant leaves Plants receiving preventive treatment had the highest number of leaves. Mancozeb before M24 had leaves per plant at 7 weeks after planting indicating the highest number, T48M had at 5 weeks after planting, the least number of leaves was recorded in M72 before Mancozeb having at 6 weeks after planting (Table 2). Effect of treatment on disease incidence on okra Disease incidence on okra plants at weeks 3, 4, 5, 6, 7 and 8 was significantly different (Table 3). Control Nigerian Journal of Mycology Vol. 9 (2017) 13

5 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum plant with the pathogen alone had the highest incidence of % at 4 weeks after planting. It was observed in curative control method that, all had disease incidence from week 3 through 8. Week 3 had the highest values of , and % at 24, 48 and 72 hours after foliar application respectively. Effect of treatment on disease severity on okra It was observed that all treatments were significantly (p 0. 05) different from one another. However, control okra plants that received the pathogen alone had the highest disease severity of % at 4 weeks after planting. M. phaseolina applied 72 before mancozeb at 3 weeks had %, while M 72 T had % at 3 weeks after planting. (Table 4). Furthermore, the seedlings that were treated for preventive did not show any rot symptoms from week 6 through 8 due to the establishment of T. harzianum on the okra plant. (Table 4 ) Effect of treatments on flowering in okra plants Table 5 showed that there were no significant differences on flowering in okra. All treated plants produce fruits equally, but those of curative suffered fruit and flower abortion indicating presence and impact of M. Phaseolina. Effect of treatments on weight of okra pods Findings from this study revealed that weight of okra pods varied from one treatment to the other. T72M at 9 weeks after planting had g. It was also observed that at 8 weeks after planting, control without pathogen had g while control with pathogen had 5. 27g. In the preventive treatments, T24M, T48M and T72M had , and g respectively at 7 weeks after planting. Furthermore, Mancozeb at 24 hours before M. phaseolina had g, while M. phaseolina 72 before Mancozeb had 2. 11g at 7 weeks after Planting (Table 6). Effect of treatments on number of okra pods Table 7, shows that the number of okra pods per plant varied depending on the treatment combinations. It was observed that seedling treated through curative methods had 0. 45, and for M24T, M48T and M72T respectively, while preventive had 4. 17, and for T72M, T48M and T24M respectively. Also M72 before Mancozeb had 0. 30, Mancozeb before M24 had all at 7 weeks after planting. E Nigerian Journal of Mycology Vol. 9 (2017) 14

6 Udoh & Olufolaji Means not followed by the same letter are significantly different at p 0.5 Turkey HSD abc NOTE: M = M. phaseolina 24,48 and 72 hours before and after T=T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 15

7 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M= M. phaseolina 24,48 and 72 hours before and after T=T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 16

8 Udoh & Olufolaji Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M= M. phaseolina 24,48 and 72 hours before and after T =T. harzianum Nigerian Journal of Mycology Vol. 9 (2017) 17

9 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum Table 4: Effect of treatment on disease severity in okra plant Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M = M. phaseolina 24,48 and 72 hours before and after T = T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 18

10 Udoh & Olufolaji Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M = M. phaseolina 24,48 and 72 hours before and after T=T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 19

11 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M= M. phaseolina 24,48 and 72 hours before and after T= T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 20

12 Udoh & Olufolaji Means that do not share a letter are significantly different at p 0.5 Turkey HSD abc NOTE: M= M. phaseolina 24,48 and 72 hours before and after T=T. harzianum. Nigerian Journal of Mycology Vol. 9 (2017) 21

13 Control of Rot Disease of Okra through Foliar Application with Trichoderma Harzianum DISCUSSION In this study, it was observed that T. harzianum enhanced okra growth and development in terms of its assessed parameters. These findings correlated with the work of Olufolaji et al. (2016) that the most effective concentration of T. harzianum in controlling pathogen is 10 8 spores/ml. It was also observed that T. harzianum and Mancozeb used as preventive method, were able to control the effect of the pathogen on okra plant through prevention of disease symptoms on growth parameters of the plants, such as number of leaves per plant and height of the plants. Furthermore, the bio-control agent used as preventive was better than the curative in that it exhibited appreciable effect in promoting reproduction of okra which brought about good yield characters. These are in consonance with the report of Elad et al., (2015), that treatment of okra plants with T. harzianum (10 8 spores/ml) for 24, 48 and 72 hours before introduction of M. phaseolina(10 4 spores/ml) had the highest number and weight of pods and no incidence of fruit and pods abortions. REFERENCES Arora, M. Z., Pawar, M. V, and Sakhare, B. A. (2012). Effect of seed treatment with Trichoderma harzianum on the prevalence of seed borne pathogens of okra. MS Thesis, Department of Plant Pathology, Bangladesh Agricultural University (BAU), Mymensingh. 59 pp. Chakraborty, A. P., Chakraboty, B. N and Chakrab, U. (2015). Bacillus megaterium from tea rhizosphere promotes growth and induces systemic resistance in tea against Sclerotium rolfsii. Indian Phytopath. 68 (3): Elad, S. B. (2015). Common Laboratory Seed Health Testing Methods for Detecting fungi. Published by ISTA, Bassersdorf, Switzerland. Pp Elad, D. C. (2016). Effect of seed borne fungi on germination of some vegetable seeds. MS Thesis, Department of Plant Pathology. Bangladesh Agricultural University, Mymensingh. 78 pp. Eni, O. B. (2015). The effect of Trichoderma harzianum pre treatment on germination, fungal growth and mitotic index of Abelmoschus esculentus L. (Okra). B. Sc. Dissertation, Babcock University, Nigeria. 37 pp. Howell, B. K., Tandon, R. N. and Verma, A. E. (2013). Storage fungi of lady s finger seed and their significance. J. Phytol. Res., 13 (1): Olufolaji, D. B., Ajayi, M. A., Ariyo, C. T. and Rowland, M. (2014). Nigerian Journal of Mycology Vol. 9 (2017) 22

14 Udoh & Olufolaji Biological control of Colletotrichum falcatum, the red rot fungus of sugar cane with Trichoderma asperellum in: Yang-Rui li, Nasr, M. I., Solomon, S. and Rao, G. P. (ed). Proceeding of international conference of professional in sugar and integrated technologies. International Association of Professional in sugar and integrated technologies, 174 East Daxue Road, Nanning, Guangxi , P. R China, pp, Olufolaji, D. B. (2016). Bio-control management of fungal plant diseases for sustainable agriculture in the tropics. In: Perspectives of plant pathology in genomic era. Ed. P. Chowdappa, Pratibha Sharma, Dinesh Singh, A. K. Misra. Pbl. Indian Phytopathological Society, Div. Plant Pathology, Indian Agric. Res Institute, Pusa, New Delhi India. Pp Olufolaji, D. B., Ajayi M. A. and Adetuyi R. O. (2016). Antifungal attributes of Trichoderma harzianum in the control of charcoal rot of cowpea caused by Macrophomina phaseolina. Indian Phytopath, 69 (4s): Richard, L. F. and Phillip, D. D. (2011). Southern blight (Sclerotium rolfsii Sacc. ) of cowpea: Genetic characterization of two sorus of resistance. Inter. J. Agronomy, Article ID pp6 Richardson, M. J. (2015). An annotated list of seed borne diseases. 4th edition, The International Seed Testing Association, Switzerland. pp Sharma, D. K. Ravishankar, M. D., Shailaja, T. V. and Kumar, H. S. (2013). Effects of microflora associated with okra (Abelmoschus esculentus L. ) Moench seeds and their phytopathological effects. An online Intl.. 2 (2): Available at cibtech. org/cjm. htm Sarka, A. K., Jain V. K., Rajni, J. A. and Nandini, S. H (2014). Studies on seed borne nature of Macrophomina phaseolina in okra. Annual Plant Prot. Sci., 9 (1): White, D. R. (2015). Study on health status of okra seeds collected from different locations of Nigeria. Department of Botany, University of Calabar, Cross River State, Nigeria. Pp Nigerian Journal of Mycology Vol. 9 (2017) 23