Influence of Intercropping Systems, Mycorrhizal Inoculation and Fertilizer Levels on Root Colonization, Root Attributes and Yield of Hybrid Maize

A Field experiment was conducted at

Phosphorus is known to stimulate early and extensive development of root systems, which enables rapid maize growth and to mature early (Sankaran et al., 2005).Enhancement of P uptake by mycorrhizal hyphae can also be indirectly attributed to the faster uptake rate of P by the hyphae and the disturbance of the solution solid P equilibrium, which will increase the sorption of absorbed phosphate into soil solution.
The initial slow growth of the maize crop roots introduction of legume as intercrop in the inter row space and such a system will also enhance the productivity.Most of research work and approaches to develop production technologies for maize in the *Corresponding author email: ananthu12@gmail.compast, but now there is need to work with cropping systems that farmers can practice to exploit location specific agro-climatic conditions for enhanced production.Considering the limited net sown area, it will be necessary to raise the cropping intensity so as to grow more crops on the same piece of land.The initial slow growth of the maize crop roots offers a choice for introduction of legume as intercrop in the inter row space and such a system will also enhance the productivity.Hence, an attempt was made to study the yield of hybrid maize as influenced by different intercropping systems, mycorrhizal inoculation and fertilizer levels during winter seasons.

Materials and Methods
Field experiments were conducted at Tamil Nadu Agricultural University Farm, Coimbatore during winter 2011-12 to study the influence of maize under different intercropping systems, mycorrhizal inoculation and fertilizer levels on root growth and grain yield of hybrid maize under irrigated condition.The experiment was laid out in a split-split design with three replications.Three intercropping systems viz., sole maize, maize+cowpea and maize + green gram were the treatments under main plot.Two mycorrhizal treatments viz., no mycorrhizal inoculation (control) (M-) and inoculation of mycorrhiza (M+) were included under sub plot.Three fertilizer levels viz., 75% RDF (F1), 100% RDF (F2), and 125 % RDF (F3) were studied under sub-sub plot.The soil of the experimental field was sandy clay loam in texture belonging to Typic Ustropept.The nutrient status of soil was low in available nitrogen (234 kg ha-1), medium in available phosphorus (14.6 kg ha-1) and high in available potassium (612.0 kg ha-1).Maize hybrid, NK 6240, a high yielding single cross hybrid released by syngenta private ltd, India was chosen for the study.
Seeds of maize hybrids were sown on the flat beds by adopting a spacing 60 x 25 cm along with vermiculite based mycorrhizal inoculum at a depth of 5 cm below the seeds.The mycorrhizal inoculum (Glomus intraradices TNAU-03-08) used in this study was purchased from the Department of Agricultural Microbiology, Tamil Nadu Agricultural University.This strain was cultured in maize plants and propagules comprised of infected root bits and spores were blended in sterile vermiculite.The inoculum with the spore density of 10 spores g-1 was applied as a thin layer beneath the seeds one week after sowing @ 100 kg ha-1.Seeds were dibbled at the rate of one seed hill-1.
As an intercrop, cowpea CO (CP) 7 and greengram (CO 6) were raised as per the treatments with spacing of 30 x 10 cm and a seed rate of 10 kg ha-1.One row of intercrop was sown in between the rows of main cop as additive series.Adjacent to the treatment plots, sole cowpea and greengram were also raised in dummy plots with same management practices to calculate the yield advantages.
Well-decomposed farmyard manure at the rate of 12.5 t ha-1 was applied uniformly over the field before last ploughing.ZnSO4 @ 37.5 kg ha-1 was applied uniformly as basal to all the plots.The recommended fertilizer dose followed for maize was 150:75:75 kg NPK ha-1.As per the treatment schedule, nitrogen as urea was applied in three splits viz., 25: 50: 25 per cent as basal and on 25 and 45 DAS, respectively.The entire dose of phosphorus as single super phosphate was applied basally.The potassium as muriate of potash was applied in two equal split doses viz., basal and at 45 DAS.
The root and shoot samples were collected at 45 and 60 DAS.The root architecture of mycorrhizal and non mycorrhizal plants was observed in terms of root length, root volume, and root dry mass.

Plant mycorrhizal colonization
The root colonization of maize plants was favorably influenced by intercropping systems, mycorrhizal inoculation and fertilizer levels.Mycorrhizal colonization was assessed in AM treated and untreated plants at 45 and 60 DAS (Fig 1).The beneficial effects of intercropping are, at least in pat, due to the contribution of AM fungal hyphae.Intercropping with legume improved mycorrhizal fungal colonization of roots of both maize and intercrops.
The results in this study showed that intercropping maize with cowpea and greengram could promote colonization on the roots of both maize and legumes by Glomus intraradices.In general, many factors such as root density, root exudates and nutrient status of the rhizosphere, contribute to regulate colonization by AMF (Smith and Read, 1997).Similar finding was also reported by Harinikumar et al. (1990) in maize and soybean intercropping system.
The intercropping system did not significantly increase root density in this study, however, the diffusion of root exudates to the adjacent maize roots was relatively free in this treatments.Since root exudates govern signalling between AMF and their host plants (Hause and Fester 2005), the additional exudates from intercropped roots would stimulate the establishment of AMF symbiosis.Moreover, the small difference in colonization between maize and legume roots in this study suggests that traversed AM hyphae could possibly multiply the infection rates in the intercropping system.
In this study, the percentage of colonization was found to be higher (30-60%) in AM treated plants than untreated plants (20-30%) at 60 DAS when compared to 45 DAS.The plants in the non mycorrhizal treatment were also colonized by AM fungi, which indicated that indigenous AM fungi occurred in the soils, a few of AM fungal species belonging to the genera Glomus and Acaulospora were found but at a low spore density and with a limited species.AM fungi can colonize the roots of maize, mycorrhizal colonization rates were lower relative to the mycorrhizal inoculation.The reasons may be the propagules in the soils were far lower than those in mycorrhizal inoculation, so the overall effect was probably lower in non mycorrhizal soil under field conditions (Wang et al., 2006).
A significant increase in the proportion of mycorrhizal colonization in arbuscular mycorrhiza inoculated roots (M+) from non-inoculated (M-) maize plants at all the fertilizer levels were noticed.The higher proportion of mycorrhizal colonization in the M+ maize plants was consistent across mycorrhizal structures such as arbuscules, vesicles and external mycelium, and across time of sample collection (45 and 60 DAS).
The development of mycorrhizal structures such as arbuscules and vesicles had significant changes at varying fertilizer levels, but the growth of external mycelium was decreased at higher levels of fertilizer application in AM+ plants.Monocots like grasses with rapidly developing roots are the ideal stock plants for AM, but any host plant which is readily colonized by AM and which can be easily grown in the greenhouse can also serve as the stock plant (Ferguson and Woodhead, 1982).The reduction of mycorrhizal infection in the presence of added phosphorus is owing to a self regulatory mechanism of plant discarding the mycorrhizal fungus when its phosphorus requirement is more than that satisfied (Hayman, 1982) .Similar results were reported by Mehraban et al. (2009) and Albert et al. (2009) in sorghum.
The fertilizer level significantly increased root colonization in this study.Inoculation of Glomus intraradices increased the percentage of colonization at both the stages even at lower levels of fertilizer application i.e. 75% RDF.In contrast, higher level 125%RDF caused a slight inhibitory effect on colonization.

Root morphology
Root morphological features such as length, volume, root dry mass and rootshoot ratio were significantly increased by intercropping systems, mycorrhizal inoculation and fertilizer levels at 45 and 60 DAS (Table 1).respectively) was associated with 125% RDF followed by 100% RDF but both were comparable.The interaction between fertilizer levels and mycorrhizal inoculation was significant.Increased root length was observed under 100 % RDF along with mycorrhizal inoculation (23.75 and 29.73 cm at 45 and 60 DAS, respectively) and was comparable with 125% RDF with mycorrhizal inoculation and (M+ F3) fertilizer level 75 % RDF without mycorrhizal inoculation (M-F1) recorded the least root length (Table 2).

Root volume (cc plant -1)
In general, intercropping systems, fertilizer levels and mycorrhiza influenced root volume significantly.Among the intercropping system, higher root volume (88.4 and 147.2 cm at 45 and 60 DAS, respectively) was associated with sole maize followed by maize intercrop with cowpea.Among mycorrhizal treatments, mycorrhizal inoculation recorded higher root volume (92.2 and 150.9 cm at 45 and 60 DAS, respectively), than the non mycorrhizal inoculation.Regarding the fertilizer treatments, the higher root volume (86.0 and 141.7 cm at 45 and 60 DAS, respectively) was associated with 125% RDF followed by 100% RDF and both were comparable among themselves.
The interaction between fertilizer levels and mycorrhizal inoculation was significant at 60 DAS only.Increased root volume was observed under 100 % RDF along with mycorrhizal inoculation (154.2 cm at 60 DAS) and was comparable with 125% RDF with mycorrhizal inoculation (M+F3) and 75 % RDF without mycorrhizal inoculation (M-F1) recorded the least root volume (Table 3).root dry mass (531 and 1100 kg at 45 and 60 DAS, was associated with sole maize followed by maize respectively) was associated with 125% RDF cowpea intercropping and both were comparable followed by 100% RDF but it was comparable with with each other at 45 DAS.Inoculation of mycorrhiza 100% RDF.The fertilizer level 75 % recorded the had substantial effect on root dry mass.Mycorrhizal least root length at all the stages.The interaction between fertilizer levels and mycorrhizal inoculation was significant.Increased root dry mass was observed under 100 % RDF along with mycorrhizal inoculation (585 and 1265 at 45 and 60 DAS, respectively) and was comparable with 125% RDF with mycorrhizal inoculation (F3M+).The treatment combination 75 % RDF without mycorrhizal inoculation (F1M-) recorded the least root dry mass (Table 3).

Root-shoot ratio
The rootshoot ratio declined from 45 DAS to 60 DAS.Regarding the intercropping systems, higher rootshoot ratio (0.140 and 0.158 at 45 and 60 DAS, respectively) was associated with sole maize followed by maize cowpea intercropping and both were comparable with each other at all the stages (Table 1) .Inoculation of mycorrhiza had substantial effect on rootshoot ratio.Mycorrhizal inoculated plants recorded higher rootshoot ratio (0.140 and 0.161 at 45 and 60 DAS, respectively), than the non mycorrhizal plants.Regarding the fertilizer treatments, the higher rootshoot ratio of 0.139 at 45 DAS was associated with 100% RDF followed by 125% RDF and both were comparable.At 60 DAS, the higher rootshoot ratio of 0.156 was recorded with 125% RDF.
The root length is an important morphological parameter involved in improving water and nutritional status of the plant.Knowledge about the root system of a crop is a pre-requisite for understanding many problems related to crop production.Root characters such as length and distribution and especially root volume help in divulging the pattern of water use and nutrient uptake by the crop.In general, the root characters such as root length, volume and its dry weight showed a gradual increase from 45 to 60 DAS.
Among the intercropping systems, sole maize recorded higher root length (6.3 and 4.1 %), root volume (12.4 and 9.2%) and root dry mass (6.8 and 11.6%), in comparison with maize + greengram intercropping at both 45 and 60 DAS, respectively.This might be due to better utilization of spacing, nutrients, light and moisture.
In the case of fertilizer levels, 125% RDF increased the root length (10.1 and 7.9 %), root volume (8.0 and 4.3 %) and root dry mass (6.1 and 9.9 %) in comparison with 75% RDF level of fertilizers at both 45 and 60 DAS.With regard to the N and P application, the root characters such as total root length and root volume increased with increasing fertilizer levels in the present investigation.This is in corroboration with the findings of Dhurandher and Tripathi (1999).Deeper root system might play an important role not only for continuous water uptake, but also for nutrient uptake under suboptimal water availability.
With regard to mycorrhiza, the measurements of root architecture variables viz., root length, root volume, root dry mass and root/shoot ratio in the mycorrhizal plants were significantly higher than nonmycorrhizal (M-) plants.The increase in root architecture measurements in mycorrhizal inoculated (M+) plants was found in both the root samples collected at 45 and 60 DAS.The extensive root growth of AM plants might be attributed to the improved P nutrition of host plants.The enhanced supply of P by mycorrhizal symbiosis has been unequivocally demonstrated (Hetrick et al., 1996;Smith and Read, 1997;Subramanian et al., 2006).At early stage of crop growth, AM fungal inoculated plants showed an increase in root mass while the shoot masses were similar.This may be attributed to the utilization of carbon for establishment of functional symbiosis as reported by Jakobsen and Rosendahl (1990).
In the case of fertilizer levels, 125% RDF increased the oot length (10.1and 7.9%), root volume (8.0 and 4.3%) and root dry mass (6.1 and 9.9%) in comparision with 75% RDF level of fertilizers at both 45 and 60 DAS.With regard to the N and P application, the root characters such as total root length and root volume increased with increasing fertilizer levels in the present investigation.This is in corroboration with the findings of Dhurandher and Tripathi (1999) and Ananthi et al. (2011a).Deeper root system might play an important role not only for continuous water uptake, but also for nutrient uptake under suboptimal water availability.

Grain and stover yield (Table 5)
The maize grain yield was significantly influenced by intercropping systems, mycorrhizal inoculation and fertilizer levels.

Effect of intercropping systems
Among the intercropping systems, sole maize recorded the highest grain yield of 8531 kg ha-1 but was comparable with maize intercropped with cowpea.The yield reduction due to intercropping cowpea was negligible (0.89 per cent) comparing the sole maize yield.Higher yield under sole maize was due to zero competition for sunlight, space, water and nutrients compared to intercropping which might have curtailed efficient utilization of natural resources and restrict growth of maize from initial stages to harvest resulting in yield competition in intercrop (Yilmaz et al., 2008).Similar findings were also obtained by Haque et al. (2008) and Choudhary et al. (2012).

Effect of mycorrhizal inoculation
Mycorrhizal inoculation recorded higher grain yield (8892 kg ha-1) than no inoculation (7981 kg ha-1) and reduction in yield being 10.25 per cent.Mycorrhiza had positive influence on grain yield of maize crop.The improved nutritional status of AM fungus -inoculated plants resulted in higher grain yield in comparison to uninoculated treatments.This yield gain in mycorrhizal treatments was mainly caused by the intense flow of minerals and metabolites from the leaf to the developing kernel.The increased yields of AM fungus inoculated treatments thus suggest that significant amounts of P and N were translocated from the source to the sink to support kernel development and grain yield (Subramanian and Charest, 1997).A higher yield of maize due to mycorrhizal inoculation has been reported previously by Subramanian et al. (2008) and Ananthi et al. (2011b).

Effect of fertilizer levels
Comparing the yield of maize grain obtained under different fertilizer levels, 125 % RDF recorded the highest grain yield of 8572 kg ha-1 and was comparable with 100% RDF.Fertilizer level of 75% RDF recorded lower grain yield and was 4.7 per cent and 4.5 per cent lesser than 125% and 100% RDF.This increase in yield was probably due to effective utilization of applied nutrients, increased sink capacity and nutrient uptake by crop.The yield potential of maize is mainly governed by the growth and yield components.The positive and significant improvement in yield attributes and nutrient uptake would have resulted in enhanced grain yield.The present findings are in line with the findings of Maddonni et al. (2006).The positive responses of hybrid maize upto 250 kg N ha-1 as reported by Srikanth et al. (2009) lend support to the present findings.
Since N is the major structural constitute of cells, as N level increased, the rate of vegetative and reproductive growth also increased in plants due to increase in assimilating surface of plants as well as total photosynthesis.In physiological terms, the grain yield of maize is largely governed by source (photosynthesis) and sink (grain) relationship which is directly related to N.These resulted in more grain yield when N was higher.

Interaction effect
The interaction between mycorrhizal inoculation and fertilizer levels on maize grain yield was significant.The highest grain yield (9157 kg ha-1) was recorded under the treatment combination mycorrhizal inoculation with 100% RDF (M+F2) followed by 125% RDF and was comparable with each other (Table 6).
The interaction between intercropping systems, mycorrhizal inoculation and fertilizer levels on maize grain yield was significant.The treatment combination sole maize with 125% RDF along with mycorrhizal inoculation (I1F3M + ) recorded significantly higher yield (8561 kg ha-1) (Fig 2).In sole maize, there was no competition for various resources except intra-species competition and the immediate supply of nutrients due to the addition of inorganic fertilizers and the significant amounts of P and N were translocated from the source to the sink in mycorrhizal inoculated maize plants, might be the reason for increase in growth and yield parameters which would have increased the yields in the treatments mentioned.Even though maize intercropped with cowpea and 100% RDF along with mycorrhizal inoculation (I2F2M + ) recorded lesser yield than the I1F3M + , the yield reduction was not significant.The reduction of maize yield was probably due to intercrop competition between maize and cowpea.However, additional yield from cowpea not only compensated the deficit, but also gave extra income.This finding is in conformity with that of Quayyum and Maniruzzaman (1995), Uddin et al. (2003) and Pandey et al. (2003).Under maize + cowpea intercropping systems, 100% RDF with mycorrhizal inoculation produced higher yield.This might be due to mycorrhizal fungi increased the root efficiency to absorb nutrient and in nutrient depleted soil, mycorrhizal fungi develop strand in the soil and absorb phosphorus through the root hairs, thereby increase nodulation and nitrogen, this character of the fungi enhanced plant growth and yield Muok, et al. (2009).Mycorrhizal inoculum significantly increased the growth and yield of maize and cowpea resulting from the interaction between mycorrhizal fungi and rhizobia.Stover yield did exhibit similar trend as that of grain yield.Regarding intercropping systems, sole maize recorded the highest stover yield (12560 kg ha-1) followed by maize intercropped with cowpea which was comparable with sole maize.This might be due to higher plant height, LAI and DMP in sole maize and also there is no inter plant competition.Mycorrhizal inoculated plants recorded significantly higher (12849 kg ha-1) stover yield, which might be due to increase in plant height, leaf area index and total biomass as evidenced in the present investigation.Similar results of increase in stover yield due to mycorrhizal inoculation were also reported earlier by Lauzon and Miller (1997) in maize.
Increasing the fertilizer levels increased the stover yield significantly. Fertilizer level of 125% RDF recorded higher stover yield (12494 kg ha-1) followed by100% RDF.The positive and significant improvement in LAI and DMP at different stages and higher nutrient uptake due to higher dose of fertilizer would have resulted in enhanced stover yield.These results are in conformity with the findings of Srikanth et al. (2009).
The interaction between mycorrhizal inoculation and fertilizer levels was significant.The treatment combination mycorrhizal inoculation with 100% RDF (M+F2) recorded higher stover yield (13155 kg ha-1) followed by mycorrhizal inoculation with 125% RDF (M+F3) and both were comparable with each other (Table 6).

Conclusion
Finally, it can be concluded that the root parameters viz., root length, root volume, root dry mass and root-shoot ratio and root colonization were higher under sole maize, mycorrhizal inoculation and fertilizer level of 125% RDF at both 45 and 60 DAS.Root colonization was higher under maize/cowpea intercropping among intercropping systems, mycorrhizal inoculation among the mycorrhiza treatments and 75% RDF among the fertilizer levels.Among the intercropping systems, sole maize recorded higher grain and stover yield.With respect to mycorrhiza, mycorrhiza inoculated treatments and among fertilizer levels 125% RDF recorded higher grain and stover yield.

Fig. 1 .
Fig.1.Effect of intercropping systems, mycorrhizal inoculation and fertilizer levels on mycorrhizal colonization of maize hybrid at 45 and 60 DAS during winter 2011-12

Fig 2 .
Fig 2. Interaction effect of intercropping, mycorrhiza and fertilizer levels on grain yield (kg ha-1) of maize hybrid during winter 2011-12