EFFECT OF ROOT ROT PATHOGENS AND FUNGICIDE SEED TREATMENT ON NODULATION IN FOOD GRAIN LEGUMES

EFFECT OF ROOT ROT PATHOGENS AND FUNGICIDE SEED TREATMENT ON NODULATION IN FOOD GRAIN LEGUMES J.W. Muthomi, P.E. Otieno, G.N. Chemining wa and J.H. Nderitu Department of Plant Science and Crop Protection, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Abstract Greenhouse experiments were conducted over 2 cropping cycles to investigate the effect of seed treatment and fungal root rot pathogens on nodulation and dry matter accumulation of selected food legumes. The legumes were common bean (Phaseolus vulgaris L.var GLP 2), green gram (Vigna radiata L., variety M66) and lablab (Lablab purpureus L.). Treatments included, inoculation of legumes with pathogen alone (Fusarium oxysporum f.sp. phaseoli or Macrophomina phaseolina or Sclerotinia sclerotiorum or Rhizoctonia solani), or with appropriate rhizobia alone or application of (copper oxychloride) or their combinations. Results of the study indicated that seed treatment reduced disease incidence on Sclerotinia and Rhizoctonia inoculated plants. However, treatment significantly (p=0.05) depressed nodulation of the legumes but its effect on nodulation was significantly suppressed when applied together with rhizobia on infected seeds. Fungicide application significantly reduced seedlings mortality (pre-emergence damping off) and number of nodules per plant but had no effect on dry matter accumulation. Combination of and rhizobia inoculation improved nodulation as well as reducing disease incidence. It is therefore concluded that this combination yields better results if the aim is to reduce root rot incidence while improving nodulation concurrently. Introduction Grain legumes are next to maize in order of importance of food crops in Kenya. They form a major source of proteins of high biological value, energy, minerals and vitamins for many people in tropical Africa where main diets consist mostly of starchy staples and minimal animal protein (Taylor et al., 2005). The protein content of edible species of legumes, with exception of soybeans and groundnuts, varies between 18 and 32% (Wiley, 1975). However, dry seed yields in most African countries have been declining due to diseases, low soil fertility, insect pests and low, erratic rainfall (Otsyula et al., 1998; Nderitu et al., 1997; Allen, 1990). Commercial fertilizers are expensive and out of reach for most small-scale farmers. Therefore, cheap sources of nitrogen like biological nitrogen fixation, combined with better pest and disease management options need to be optimised if yields are to be sustained and food security attained. All agriculturally important legume species have the ability to symbiose with Rhizobium and fix free atmospheric nitrogen. World wide, some 44 to 66 million tones of nitrogen are fixed annually, providing nearly half of all the nitrogen used in agriculture (Giller, 2001). The amount of nitrogen fixed by legumes varies with species and may supply a major part or the entire nitrogen needed by a crop (Chui et al., 2003). Reduced dependence on nitrogen fertilizers and attention to farming practices that favour the more economically viable and environmentally friendly nitrogen fixation through legume-rhizobium symbiosis has a special relevance to the developing countries (Giller 2000; Obara et al., 2000). Symbiotically-fixed nitrogen has been considered as a useful source of nitrogen to non-fixing plants in intercropping systems (Fujita et al., 1992; Sanginga et al., 1994; Hudgens, 1996; Carsky et al., 2000). Root rot diseases are a major limiting factor in legume production. The diseases depress seedling germination and cause post emergence damping off, resulting in poor crop stand and low yields. The disease causal agents are seed borne but most farmers often use seeds saved from previous harvest, a practice that negates the principle of sanitary practices (Buruchara, 1990). Effective control strategies against root rot fungal pathogens have not been fully developed. Sanitation and use of clean planting material is the primary way of preventing damping-off and other root diseases. Chemical seed treatment is a common practice before planting to prevent seed and seedling rots, damping off and other fungal diseases. However, problems arise when the chemical seed treatments are to be used in conjunction with rhizobia inoculants. In some cases the applied seed may fail to protect against the intended pathogen or suppresses the effectiveness of the rhizobia bacteria. Therefore, this study was carried out to investigate the interaction of seed treatment and rhizobia inoculation in the management of fungal root rots and their effect on nodulation and dry matter accumulation of selected food grain legumes. Materials and methods Pathogen isolation and inoculums production Root or hypocotyl portions of legume plants showing symptoms of root rot were collected from the University of Nairobi s Field Station, Kabete. Tissue portions were surface sterilised and aseptically plated on potato dextrose agar (PDA) medium at room temperature (25 o C) for 7 14 days. Pure cultures were obtained by hyphal tip transfer of each of the colonies onto fresh PDA media. Identification of the pathogens was done using cultural (colour of aerial mycelium, pigmentation) and morphological characteristics (septation of hyphae, conidia shape and size, septation of conidia). Inoculum was prepared by growing each pathogen separately on PDA for 7 14 days at room 1

temperature (25 o C). Whole cultures of each pathogen were macerated with sterile distilled water in a blender to make a slurry using sterile distilled water. Experimental design and treatments The experimental design was a Completely Randomised Design with 3 replicates. The grain legumes used were common bean (Phaseolus vulgaris L.var GLP 2), green gram (Vigna radiata L.) and lablab (Lablab purpureus L.). Treatments included, inoculation with pathogen (Fusarium oxysporum f.sp. phaseoli or Macrophomina phaseolina or Sclerotinia sclerotiorum or Rhizoctonia solani), or with appropriate rhizobia or application of (copper oxychloride) or their combinations. Prior to inoculation and treatment, the seeds were surface-sterilised in 10% sodium hypochlorite and rinsed twice in sterile distilled water. Common bean was inoculated with bean rhizobia while green gram and lablab were inoculated with cow pea cross-nodulating Rhizobia strain. Slightly wetted seeds were thoroughly mixed with the appropriate Rhizobia inoculant. Sucrose was added to the mixture to enable the Rhizobia stick to the seed surface and also to offer initial nutrients for the bacteria. Fungicide treatment was done by mixing seeds with the copper oxychloride powder. Pathogen slurry (15 ml) containing 5 x 10 5 propagules per mm for each of the pathogens was mixed into steam-sterilised soil in plastic pots and five appropriately treated seeds were then placed on the surface of the soil and covered with 1cm layer of soil. Watering was done regularly as required using distilled water. Each treatment was replicated four times and the experiment was arranged in a completely randomised design. Assessment of root rot, nodulation and dry matter accumulation Root rot was assessed on the basis of percent seedling emergence, seedling mortality and plant dry weight. Percent seedlings emergence was recorded after germination while percent seedling mortality was recorded on the second, fourth and sixth week after emergence. The experiment was terminated on the sixth week after emergence. The potting medium was carefully washed off the roots and the number of nodules counted for each plant. The plant were then separated into roots and shoots and dried in an oven at 110 o C for 48 hours for dry weight determination. Data was subjected to analysis of variance (ANOVA) using Genstat software and the treatment means separated using Tukey LSD test at 5% probability level. Results Pre- and post-emergence damping-off The interaction between the treatments and legume species on pre-emergence damping-off was not significant (P=0.05) (Table 1). However, significant differences were observed among the treatments and legume species. Rhizoctonia and Sclerotinia spp. significantly increased pre-emergence damping off relative to the control. However, Fusarium and Macrophomina spp. had no effect on pre-emergence damping-off. Fungicide application significantly reduced pre-emergence damping-off on Rhizoctonia and Sclerotinia spp. inoculated seeds compared to when the pathogens were applied alone and with rhizobia in the case of Sclerotinia. Green gram showed higher preemergence damping-off compared to common bean and lablab. Treatments, legume species and their interaction had a significant (P=0.05) effect on the percent post-emergence damping-off (Table 2). All the pathogens except Rhizoctonia had no effect on post emergence damping-off. There was a slight reduction in post-emergence dampingoff when rhizobium was inoculated together with Fusarium sp. on common bean and green gram. Fungicide application significantly reduced post-emergence damping-off on Rhizoctonia and Sclerotinia treated seeds relative to pathogen alone. However, seed treatment had no effect on Fusarium and Macrophomina treated seeds. Combined inoculation with, Rhizoctonia and rhizobia reduced post emergence damping-off in green grams relative to the pathogen alone. Fungicide and rhizobia in combination with Fusarium, Macrophomina and Sclerotinia spp had no effect on post emergence damping off. 2

Table 1 Mean percentage emergence of legumes treated with and inoculated with different root rot fungi and rhizobia inoculant Treatment Lablab Common bean Green gram Treatment Means Control 100a 100 a 100 a 100 a Rhizobia 100 a 100 a 100 a 100 a Rhizobia + 100 a 100 a 100 a 100 a Fungicide alone 100 a 100 a 100 a 100 a Fusarium alone 100 a 100 a 93.33 a 97.78 a Fusarium + rhizobia 100 a 100 a 93.33 a 97.78 a Fusarium + 100 a 100 a 100 a 100 a Fusarium + rhizobia + 100 a 100 a 100 a 100 a Macrophomina alone 100 a 100 a 100 a 100 a Macrophomina + rhizobia 100 a 100 a 93.33 a 97.78 a Macrophomina + 100 a 100 a 100 a 100 a Macrophomina + rhizobia + 100 a 100 a 100 a 100 a Rhizoctonia alone 86.67 b 80 b 66.67 c 77.78 a Rhizoctonia + rhizobia 86.67 b 86.67 b 66.67 c 80 b Rhizoctonia + 93.33 a 86.67 b 80 b 86.67 b Rhizoctonia + rhizobia + 93.33 a 86.67 b 73.33 c 84.44 b Sclerotinia alone 93.33 a 80 b 86.67 b 85.67 b Sclerotinia + rhizobia 93.33 a 80 b 86.67 b 86.67 b Sclerotinia + 93.33 a 93.33 a 93.33 a 93.33 a Sclerotinia + rhizobia + 93.33 a 93.33 a 93.33 a 93.33 a Legume Means 96.67 94.33 91.33 LSD (P 0.05) Legumes = 2.727; Treatments = 7.042; Treatments Legumes = ns; Means followed by different letters within columns are significantly different (P 0.05) Table 2 Mean percentage post-emergence damping off at the 6 th after emergence for 3 legume species treated with and inoculated with different root rot fungi and rhizobia inoculant Treatment Lablab Common bean Green gram Treatment Means Control 0.0 d 0.0 c 0.0 e 0.0 d Rhizobia 0.0 d 0.0 c 0.0 e 0.0 d Rhizobia + 0.0 d 0.0 c 0.0 e 0.0 d Fungicide alone 0.0 d 0.0 c 0.0 e 0.0 d Fusarium alone 6.7 c 13.3 b 8.3 e 9.4 c Fusarium + rhizobia 6.7 c 16.7 b 6.7 e 10.0 c Fusarium + 13.3 b 13.3 b 6.7 e 11.1 c Fusarium + rhizobia + 6.7 c 6.7 b 6.7 e 6.7 c Macrophomina alone 0.0 d 13.3 b 6.7 e 6.7 c Macrophomina + rhizobia 0.0 d 13.3 b 8.3 e 7.2 c Macrophomina + 6.7 c 13.3 b 8.3 e 9.4 c Macrophomina + rhizobia + 6.7 c 13.3 b 13.3 de 11.1 c Rhizoctonia alone 46.7 a 36.7 a 66.7 b 50.0 c Rhizoctonia + rhizobia 46.7 a 38.3 a 100.0 a 61.2 a Rhizoctonia + 15.0 b 15.0 b 45.1 c 25.0 c Rhizoctonia + rhizobia + 20.0 b 15.0 b 62.2 b 32.5 c Sclerotinia alone 13.3 b 30.0 a 25.0 d 22.8 c Sclerotinia + rhizobia 13.3 b 30.0 a 25.0 d 22.8 b Sclerotinia + 15.0 b 15.0 b 15.0 d 15.0 c Sclerotinia + rhizobia + 15.0 b 15.0 b 16.7 d 15.6 c Legume Means 11.6 14.9 21.1 LSD (P 0.05) Legumes = 4.88; Treatments =12.61; Treatments Legumes = 21.84 Means followed by different letters within columns are significantly different (P 0.05) Number of nodules per plant The interaction between the treatments and the legume species on number of nodules per plant was significant (P=0.05). In addition, significant differences were observed among the treatments and the legume species (Table 3). Rhizobia inoculation and its combination with and Fusarium spp. significantly increased the number of 3

nodules per plant in all the legume species except green gram, which showed no effect. However, combination of Fusarium and Rhizobia with and rhizobia had no significant effect on nodulation in dolichos. Inoculation of rhizobia, Macrophomina and Rhizoctonia spp. increased the number of nodules per plant in common bean. However, application of on Rhizobia inoculated seeds significantly reduced the number of nodules per plant in common bean but had no effect in lablab and green gram. Also, treatment significantly reduced the number of nodules per plant in Fusarium and Rhizobia inoculated common bean as compared to the pathogen alone. Table 3 Mean number of nodules per plant of legumes treated with and inoculated with different root rot fungi and rhizobia inoculant at 6 weeks after emergence Treatment Lablab Common Green gram Treatment Means bean Control 2.6 d 3.8 e 0.0 a 2.1 d Rhizobia 8.9 d 15.8 a 1.4 a 8.7 a Rhizobia + 7.7 d 12.4 b 0.0 a 6.7 b Fungicide alone 2.4 d 2.8 f 0.0 a 1.7 d Fusarium alone 3.0 c 4.3 e 0.0 a 2.4 d Fusarium + rhizobia 7.7 a 14.6 a 0.0 a 7.5 a Fusarium + 4.4 c 4.6 e 0.0 a 3.1 d Fusarium + rhizobia + 6.5 d 11.0 b 0.0 a 5.9 b Macrophomina alone 2.4 d 3.7 e 0.0 a 2.0 d Macrophomina + rhizobia 6.6 d 15.4 a 0.0 a 7.4 a Macrophomina + 2.5 d 3.9 e 0.0 a 2.1 d Macrophomina + rhizobia + 5.6 d 10.3 c 0.0 a 5.1 b Rhizoctonia alone 2.9 d 3.8 e 0.0 a 2.2 d Rhizoctonia + rhizobia 4.7 c 12.4 b 1.3 a 6.1 b Rhizoctonia + 2.9 d 7.7 d 0.0 a 3.9 b Rhizoctonia + rhizobia + 4.8 c 10.6 c 0.0 a 5.1 b Sclerotinia alone 2.1 d 5.2 e 0.0 a 2.8 d Sclerotinia + rhizobia 7.1 d 10.5 c 0.0 a 5.9 b Sclerotinia + 2.3 d 5.2 e 0.0 a 2.8 d Sclerotinia + rhizobia + 6.0 b 8.7 c 0.0 a 4.9 c Legume Means 5.2 9.6 0.1 LSD (P 0.05) Legumes = 0.7; Treatments = 1.8; Treatments Legumes = 3.1 Means followed by different letters within columns are significantly different (P 0.05) Dry matter accumulation The interaction between treatments and legume species on shoot and root dry matter was not significant (Table 4 and 5). However, significant (P=0.05) differences were observed among the treatments and legume species for both shoot and root dry matter. All the pathogens had no effect on shoot dry matter of the legume species. Rhizobia inoculation alone significantly increased shoot dry matter but not when applied in conjunction with the pathogens and. Fungicide alone had no effect on shoot dry matter but its combination with rhizobia improved this plant attribute on Rhizoctonia treated pants relative to the control. A combination of rhizobia and had no effect on shoot dry matter relative to the control. All the pathogens had no significant effect on root dry matter although Macrophomina, Sclerotinia and Rhizoctonia spp. showed reduced root dry matter. Fungicicide application and its combination with pathogens or with rhizobia had no effect on root dry matter. 4

Table 4 Mean shoot dry weight per plant (g) of three grain legume species treated with and inoculated with different root rot fungi and rhizobia inoculant at 6 weeks after emergence Treatment Lablab Common bean Green gram Treatment Means Control 1.10b 1.10 c 0.40 c 0.87 b Rhizobia 1.33 a 1.37 b 0.70 b 1.13 Rhizobia + 1.00 b 1.10 c 0.53 b 0.89 b Fungicide alone 1.00 b 1.07 c 0.40 c 0.82 b Fusarium alone 0.79 c 1.05 c 0.54 b 0.79 b Fusarium + rhizobia 1.06 b 1.18 c 0.42 c 0.89 b Fusarium + 1.00 b 1.20 c 0.45 c 0.89 b Fusarium + rhizobia + 0.90 b 1.00 d 0.45 c 0.78 b Macrophomina alone 0.78 c 1.22 c 0.51 b 0.84 b Macrophomina + rhizobia 1.10 b 1.35 b 0.48 b 1.00 b Macrophomina + 0.83 c 1.35 b 0.42 c 0.87 b Macrophomina + rhizobia + 0.92 b 1.30 b 0.52 b 0.91 b Rhizoctonia alone 1.08 b 0.76 d 0.53 b 0.79 b Rhizoctonia + rhizobia 0.82 c 1.50 b 0.60 b 0.96 b Rhizoctonia + 0.86 c 1.45 b 0.78 a 1.03 a Rhizoctonia + rhizobia + 1.03 b 1.74 a 0.97 a 1.25 a Sclerotinia alone 0.93 b 0.77 d 0.55 b 0.75 b Sclerotinia + rhizobia 1.15 a 1.15 c 0.51 b 0.94 b Sclerotinia + 0.88 b 1.27 b 0.30 c 0.82 b Sclerotinia + rhizobia + 0.84 c 1.39 b 0.46 c 0.90 b Legume Means 0.97 1.22 0.53 LSD (P 0.05): Legumes 0.0875; Treatments 0.2260; Treatments x legumes ns; Means followed by different letters within columns are significantly different (P 0.05) Table 5 Mean root dry weight per plant (mg) of three grain legume species treated with and inoculated with different root rot fungi and rhizobia inoculant at 6 weeks after emergence Treatment Lablab Common bean Green gram Treatment Means Control 343 c 514 bc 223 a 360 a Rhizobia 377 b 867 a 287 a 510 a Rhizobia + 322 c 803 a 243 a 456 a Fungicide alone 323 c 788 a 243 a 452 a Fusarium alone 258 c 827 a 44.0 c 376 a Fusarium + rhizobia 288 c 790 a 187 a 422 a Fusarium + 294 c 603 bc 235 a 378 a Fusarium + rhizobia + 307 c 678 b 233 a 406 a Macrophomina alone 220 c 478d 54.0 c 251 b Macrophomina + rhizobia 73.0 d 577bc 127 a 326 b Macrophomina + 307 c 347 d 123 a 259 b Macrophomina + rhizobia + 342 c 530 bc 170 a 347 a Rhizoctonia alone 333 c 596 bc 42.0 c 324 b Rhizoctonia + rhizobia 657a 557 bc 306 a 506 a Rhizoctonia + 292 c 487 d 45.0 c 274 b Rhizoctonia + rhizobia + 533 a 523 bc 142 a 400 a Sclerotinia alone 305 c 419 d 57.0 c 260 b Sclerotinia + rhizobia 323 c 470 d 119 b 304 b Sclerotinia + 268 c 437 d 138 a 281 b Sclerotinia + rhizobia + 312 c 580 bc 235 a 376 a Legume Means 334 594 163 LSD (P 0.05): Legumes 64.8; Treatment 167.0; Treatment x Legume NS; Means followed by different letters within columns are significantly different (P 0.05) Discussion Inoculation of the legumes with Rhizoctonia and Sclerotinia spp. significantly increased damping-off whereas Fusarium and Macrophomina spp. had no effect. This indicated that Rhizoctonia and Sclerotinia spp. were more pathogenic at the conditions provided during the experiment. Similar findings were reported by Wong et al. (2003). The insignificant effect of Fusarium and Macrophomina spp. may be due to the unfavourable conditions 5

characterised by moderate temperature and high moisture content provided during the experiment. Fusarium and Macrophomina spp. thrive well under moisture stress and high temperatures but disease development is reduced in flooded soils (Afouda, 1999; Allen et al., 1996; Ratnoo et al., 1997). Inoculation with rhizobia improved nodulation and shoot dry matter. However, application significantly reduced disease incidence but negatively impacted on nodulation. But when was used in combination with rhizobia, the disease incidence was significantly reduced and nodulation increased than when applied alone. This indicating that simultaneous use of rhizobia and s for root rot management and nodulation enhancement is recommendable for use by farmers. However, studies to determine s that are less toxic to rhizobia ought to be done. Inoculation with rhizobia improved nodulation and shoot dry matter. The negative effect of the on nodulation indicates that it had some bactericidal effects on the rhizobia. Heweidy et al. (2005) reported that copper oxychloride was the most inhibiting to Bradyrhizobial strains, even though it significantly decreased the infection percentage with Macrophomina phaseolina, Fusarium oxysporum and Sclerotium rolfisii compared to other tested s. The increased number of nodules when legume seeds were inoculated with fungal pathogen, rhizobia and treatment indicates that simultaneous application of seed treatment and rhizobia inoculation is beneficial with respect to root rot and biological nitrogen fixation management in grain legumes. The fact that combined inoculation with pathogen and Rhizobia generally resulted in significantly higher number of nodules compared to all the other treatment combinations suggests a synergistic effect between Rhizobia and fungal pathogen inoculation. 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