Abstract: Background and Objective: There has been intriguing nutrient soil conservation status associated with mycorrhizal fungus associations with plants. The influence of arbuscular mycorrhiza fungus (AMF) Glomus mosseae on disease incidence and severity, nematode population and residual nutrient on soybean was investigated under both screenhouse and field conditions. This was with the view to providing information on the bio protective potential of arbuscular mycorrhiza fungi. Materials and Methods: Inoculation of G. mosseae, S. rolfsii and M. incognita singly and simultaneously near root zones of soybean seedlings was carried out 2 weeks after planting. Symptomological characteristics were taken two weeks after inoculation till maturity. Soils samples were analyzed for nutrient and nematode population before planting and at harvest. Results: Soybean plants inoculated with either Sclerotium rolfsii or Meloidogyne incognita (pathogens) alone expressed higher disease incidence than mycorrhizal plants. The combined application of G. mosseae, S. rolfsii and M. incognita caused a significant reduction in nematode population while plants inoculated with M. incognita alone had the highest population in both sites of studies. Residual phosphorus and nitrogen were obtained in soil treated with mycorrhiza. Conclusion: This study shows that mycorrhizal inoculation could be used as a bio-protective agent to suppress the incidence and severity of S. rolfsii and M. incognita infection in soybean.
INTRODUCTION
Mycorrhizas are widespread symbiotic associations between soil fungi and plants which are present in 92% of all land plant families1. The benefits of AMF infection in plants include efficient nutrient uptake2 especially in infertile soils3 improved plant growth4,5 and disease protection against soil-borne pathogens6. Arbuscular mycorrhizal (AM) fungi has been found to have dramatic but stimulatory effects on phosphorus nutrition, water transport, N2 fixation7, growth and seed production of soybeans8. An additional benefit of mycorrhizal symbiosis often reported for soybean and other crops is the suppression of sedentary nematode parasites typically accompanied by improved host tolerance to the nematode9. The ubiquitous nature of arbuscular-mycorrhizal fungi (AMF) has been confirmed by available information and the importance of its inoculation has been demonstrated overwhelmingly in recent decades10-12.
Thus, the exploration of biological control approaches with AMF is therefore necessary to protect man and his environment from the hazardous effects associated with the use of synthetic pesticides in crop production. In addition, the approach will constitute valuable tool in sustainable agriculture. Against this background, this study was conducted to evaluate the effects of G. mosseae on disease incidence and severity of S. rolfsii and M. incognita in soybean.
MATERIALS AND METHODS
Propagation of Glomus mosseae (mycorrhiza) inoculum: Mycorrhizal inoculum was multiplied on Zea mays using the bucket method. After 3 months of growth, the upper part of the plant was removed and the colonized root were chopped and mixed with dry soil. The mean fungal spore density observed from assessment of soil sample was 800 spore/200 g.
Sclerotium rolfsii inoculum: Five-day old pure cultures of the S. rolfsii were comminuted in a warring blender after removing the aerial mycelium. About 1000 mL of sterile distilled water was added to the culture and 16 mL of the suspension served as inoculum for S. rolfsii.
Meloidogyne incognita cultures: Chopped root pieces of Celosia infected with M. incognita were shaken vigorously with 0.5% sodium hypochlorite solution to extract eggs of the nematode13.
Screen house experiment: Seeds of soybeans were sown in 15 cm diameter pots filled with steam sterilized topsoil and the pots, were arranged in a Completely Randomized Block Design. Seedlings were thinned to 4/pot, 6 days after emergence. Each pot was inoculated with G. mosseae, S. rolfsii and M. incognita singly and simultaneously near the root zone of soybean seedlings 2 weeks after planting. One thousand eggs of M. incognita were inoculated close to the roots of plants in the screenhouse. About 30 g of the soil /root fragment mixture served as fresh mycorrhizal inoculum.
Disease incidence was determined by counting diseased leaves expressing it as a percentage of the total number of plants observed. The scoring was based on easily distinguishable symptomatological characteristics using a modified 0-5 scale as described by Tarr14:
Where:| 0 | = | All leaves without symptoms |
| 1 | = | 1- 25% total number with symptoms |
| 2 | = | 26-50% total leaf number with symptoms |
| 3 | = | 51-75% total leaf number with symptoms |
Field experiment: Field trials were conducted between 2009 and 2010 at the Teaching and Research Farm of Obafemi Awolowo University Ile-Ife, located at 7° 28 N, 4°33 E at 22 m above the sea level in the tropical rainforest area of Nigeria. A plot of 10.2×27.5 m was cleared, ploughed and harrowed. The experiment was laid out in a Randomized Complete Block Design. The field was divided into four blocks of 1.8×1.5 m with an alley of 0.5 m between plots and 1 m between blocks. Seeds were drilled at the spacing of 0.36 cm and 5 m with 5 rows in each plot. The three middle rows were inoculated and sampled while outer row serves as guard rows. The time, method of inoculation and data collected were similar to that of screen house with slight modification in nematode treated plots. While eggs of M. incognita were used in the screenhouse, equal quantity of chopped galled Celosia infected roots infected with M. incognita was used for the field inoculation.
Statistical analysis: All data were transformed using natural logarithm (x = loge y+1) and subjected to analysis of variance. Treatment means were separated using Duncan’s Multiple Range Tests (DMRT) at 5% level of probability.
RESULTS
The incidence and severity of disease was higher in plants inoculated with single pathogens alone in both the screen house and under field conditions (Table 1 and 2, transformed values on Table 3). In the first screen house experiment at 4 weeks after inoculation (WAI) mild to moderate chlorotic symptoms were expressed on all soybean plants except in un -inoculated soybean plants. At 6 WAI more severe basal stem lesions were observed in plants treated with single pathogen alone than in those simultaneously inoculated with the pathogens, in the second trial, at 4 WAI, chlorotic symptoms were moderate on TGX1903-7F and TGX1449+Glomus moseae+Sclerotium rolfsii. Basal stem lesion was moderate on TGX 1449+Sclerotium rolfsii but only mild on TGX1903-7F+Sclerotium rolfsii. The uninoculated control treatment only showed mild symptoms of chlorosis. At 6 WAI soya bean plants treated with S. rolfsii alone showed moderate symptoms of basal stem lesion. Chlorotic symptoms were observed on plants as early as 6 WAI in both field experiments except uninoculated control plants which showed no symptoms of chlorosis. At 6 WAI, moderate dieback was observed in addition to severe necrotic lesions in plants inoculated with pathogens alone whereas plants treated with Glomus either singly or simultaneously showed no symptoms of dieback. Similar trend was observed in the 2 soybean cultivars and for nematode population. The results presented in Table 4 and 5 show that varieties treated with the combination of M. incognita and G. mosseae had the highest percentage reduction in population of M. incognita (93.33%) and (90.90%) under screen house conditions in the first and second trials, respectively. The results in Table 5 show that in the first field experiment, TGX 1449 inoculated with M. incognita alone had significantly higher percentage increase in nematode population (181.76%). Results show that the effects of mycorrhiza on residual soil nutrient (NPK).
| Table 1: | Disease incidence of basal stem rots of soybean at 6 weeks after inoculation under screen house conditions |
0: No symptoms, 1: Mild symptoms, 2: Moderate symptoms, 3: Severe symptoms, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita | |
| Table 2: | Disease incidence of basal stem rots of soybean on the field at 6 weeks after inoculation on the field |
0: No symptoms, 1: Mild symptoms, 2: Moderate symptoms, 3: Severe symptoms, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita | |
The soil residual N was increased in both field plantings. The highest value was observed in TGX1903-7F+Meloidogyne incognita while the least value was in TGX1449+Glomus mosseae+Sclerotium rolfsii+Meloidogyne incognita. Soil N level in the screen house trials increased only in TGX1903-7F+Srand TGX1449 (control treatments). The residual soil P decreased in all field plantings. TGX1449+Glomus mosseae+Sclerotium rolfsii+Meloidogyne incognita gave highest soil P level while the least value was obtained in TGX1449+Meloidogyne incognita. P level only increased in the screen house in the following treatments TGX1903-7F+Sclerotium rolfsii, TGX1903-7F+Glomus mosseae+Sclerotium rolfsii+Meloidogyne incognita while it decreased in all other treatments. Residual nutrient in screen house and field experiment were presented in Table 6 and 7, respectively.
| Table 3: | Effects of Glomus mosseae on soybean cultivars inoculated with Meloidogyne incognita and Sclerotium rolfsii |
Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita, Gm: Glomus mosseae | |
Residual K level varied with the various the treatments on the field while it was generally reduced in all the screen house treatments except in TGX1903-7F+Glomus mosseae which remained unchanged from the initial level.
DISCUSSION
Disease development and symbiotic establishment of the mycorrhizal fungus is a function of host-pathogen-interaction and time. The influence of mycorrhizal fungus on disease incidence and severity had been attributed to the fact that mycorrhiza usually affect the physiology of the host plant positively by creating or inducing a form of mechanical barrier against the invading pathogen2. Findings from both screen house and field experiments in the current study showed that soybean plants inoculated with pathogens alone expressed higher disease incidence and severity while mycorrhizal plants were more resistant to the test pathogens. Similarly, disease severity of pepper caused Fusarium oxysporum2, soybean infected with S. rolfsii was observed on inoculation with AM fungi15. Nematode population suppression by G. mosseae was also observed in this study. In earlier studies, the parasitization of plants by nematode was found to be influenced by the establishment of AM fungi by Todd et al.9, Carling et al.16, Siddiqui and Mahmood17 and Pinochet et al.18.
| Table 4: | Effects of Glomus mosseae, Sclerotium rolfsii on soil population of Meloidogyne incognita (number/200 mL soil) infested soybean plants under screen house conditions |
Each value is a mean of 4 replicates, values with the same letter in the same column are not significantly different (p = 0.05) by DMRT, negative values indicate decrease in nematode population, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita | |
| Table 5: | Effects of Glomus mosseae, Sclerotium rolfsii on soil population (number/200 mL soil) of Meloidogyne incognita infested soybean plots under field conditions |
Each value is a mean of 4 replicates, values with the same letter in the same column are not significantly different (p = 0.05) by DMRT, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita | |
| Table 6: | Effects of Glomus mosseae, Sclerotium rolfsii and Meloidogyne incognita on residual soil nutrient under screen house conditions |
Values with the same letters in the same column coded final are not significantly different (p = 0.05) by DMRT, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita, initial N: 0.39, initial. P: 26.22, initial K: 0.60 | |
| Table 7: | Effects of Glomus mosseae, Sclerotium rolfsii and Meloidogyne incognita on residual soil nutrient under field conditions |
Values with the same letters in the same column coded final are not significantly different (p = 0.05) by DMRT, Gm: Glomus mosseae, Sr: Sclerotium rolfsii, Mi: Meloidogyne incognita, initial N: 0.39, initial. P: 26.22, initial K: 0.60 | |
The population suppression of and (or) improved host tolerance to the root knot nematode (M. incognita) by AMF may vary with fungal species and phosphorus nutrition9. The increased residual N and P associated with Glomus mosseae in this study supports the findings of Chen et al.19. It has also been reported that Glomus mosseae mycorrhizal plants improves phosphorus uptake of the plant9,19. Leaf chlorosis was a prominent above ground symptom observed in this study especially in plants inoculated with M. incognita alone. In vitro bioactivity of S. rolfsii against M. incognita was reported by Adekunle and Akinsanmi20 hence this compatible combination can be responsible for lower pathogenic infection. This suggests that root colonization of G. mosseae has a negative effect on the development of nematodes. According to Diedhiou et al.21, the pre-inoculation of tomato plants with Glomus coronatum or Fo62 (non-pathogenic Fusarium strain) stimulated plant growth and reduced M. incognita infestation. Similarly Dos Anjos et al.6 reported the protection of Scutellospora heterograma (mycorrhizal fungus) of passion fruit against M. incognita. The suggested mechanisms of plant protection against soil borne pathogens includes lignifications of cell wall, production of other polysaccharides to prevent penetration of plants (Sharma and Johri7 and Alban et al.11). An evidence of localized biochemical defense of Glomus fasciculatum colonization against root knot nematode on tomato plant had also been reported by Nagesh and Reddy22. The main effects of AM fungi on nematodes seem to be in competition for space nutrient and as well as prevention of juvenile penetration. Since the protective ability of AM fungi against soil-borne diseases is as a result of complex interacting factors at a specific time and situation which include, effect of nematode, fungi, plant species, environmental conditions, time of mycorrhization, period of exposure, while generalization of each unique interaction seems difficult, the research for compatible combinations will be important in agriculture and ecology because of the benefits they provide to the majority of plant cultivars and the conservation of the environment by acting as bio-protectors, bioremediation, bio- fertilizers and bio-control agents.
SIGNIFICANCE STATEMENT
This study discovered that inoculation of soybeans with Glomus mosseae (arbuscular mycorrhiza) reduced disease incidence caused by Sclerotium rolfsii, suppressed populations of Meloidogyne incognita (nematode) and increased the residual soil nitrogen and phosphorus. Thus, a new theory on the interactive effect of Glomus mosseae on the test pathogens of soybean and residual soil nutrient may be arrived at.
ACKNOWLEDGMENT
The assistance offered by Mr. Osude the Laboratory Technologist of the Pathology Unit, Department of Crop Production and Protection, Obafemi Awolowo University, Ile-Ife, Nigeria is acknowledged.