EFFECTS OF AQUEOUS PLANT EXTRACTS AND INORGANIC FERTILIZER ON THE GERMINATION , GROWTH AND DEVELOPMENT OF MAIZE ( ZEA MAYS L . )

Screen house experiments were carried out to examine the effects of aqueous leaf extracts of Tithonia diversifolia (Ti) and Vernonia amygdalina (Ve), as well as NPK fertilizer (15-15-15) on the germination, growth and development of maize. Fresh leaves were collected, washed with tap water, chopped and pounded, soaked in distilled water and filtered. The two filtrates were used to prepare extracts at 50 and 100% w/v. Four maize seeds were placed in Petri dishes laid out in a completely randomized design with five replicates. In every Petri dish 10 ml of extract per treatment was added. A control experiment with distilled water was also set up. Also, in a completely randomized design with four replicates, 4-week-old potted maize plants were treated with 500 ml of each extract as well as with 1.52 g of NPK fertilizer. The results showed that the germination percentage of the seeds followed the order Ti50 Control Ve50. The seeds treated with aqueous extracts of T. diversifolia and V. amygdalina at 100% w/v produced lower but equal germination percentage. The seedling radicle growth was significantly inhibited by the aqueous extracts of Ti100, Ve50 and Ve100 (p≤0.05). The inhibition was dose-dependent and more pronounced in seeds treated with extracts of V. amygdalina. The aqueous extract of T. diversifolia (50% w/v) and control influenced radicle growth substantially. All the extracts inhibited the plumule development compared to the control. On the other hand, growth, development and yield were not significantly affected by the plant extracts.


Introduction
Tithonia diversifolia (Hemsl.)A.Gray, also known as the Mexican sunflower, is a common woody herb or succulent shrub belonging to the family Asteraceae (Orwa et al., 2009).This plant is widely distributed in humid and sub-humid tropical regions of the world including Asia, Central Africa and South America (Sonke, 1997).It is encountered in the South West of Nigeria where it occurs abundantly along roadsides, farmlands, lawns and river courses (Lordbanjou, 1991;Chukwuka, 2003;Chukwuka et al., 2007).Outside the tropics, T. diversifolia is also seen in almost pure stands, suggesting its invasive ability (Tongma et al., 1997).Ayeni et al. (1997) reported that the Mexican sunflower due to its great invasiveness has the ability to compete successfully with agricultural crops.Several bioactive compounds, mainly secondary metabolites, have been isolated from different parts of T. diversifolia (Schuster et al., 1992;Chon et al., 2000;Taiwo and Makinde, 2005;Bouberte et al., 2006;Otusanya and Ilori, 2012) and some of these are known plant growth inhibitors (Stiles et al., 1994).
In spite of its phytotoxic attributes, T. diversifolia presents several other uses including medicinal (Goffin et al., 2002;Bouberte et al., 2006;Sánchez-Mendoza et al., 2011) as well as agricultural ones.In effect, the green manure of T. diversifolia has been confirmed to be a good source of essential plant nutrients (Jama et al., 2000;Olabode et al., 2007;Partey, 2010).It can thus be averred that this plant possesses a lot of potentials in terms of both growth-inhibiting as well as growth-stimulating properties vis-à-vis several other plants, especially crops.
Vernonia amygdalina Del. otherwise known as 'bitter leaf' is a shrub belonging also to the family Asteraceae.The genus Vernonia is one of the most important in this family.It contributes about 1,000 species to the tropical regions of Asia, Africa and the Americas (Dematteis, 1998).Bitter leaf is indigenous to tropical Africa and is found wild or cultivated all over Sub-Saharan Africa (Bosch et al., 2005).Correct identification at sub-generic levels has proved difficult (Yeap et al., 2010).This may be due in part to the high number of species present in this genus as well as to the multitude of morphologically similar features among them.Thus chemotaxonomy and cytotaxonomy have been the commonly used taxonomic approaches taken to settle disputes in this genus (Mabry et al., 1975;Ayodele, 1999).The phytochemical composition of this plant has been the subject of many investigations and the results have been closely related to those obtained from studies of T. diversifolia.In effect, a wide range of phytochemicals that have been shown to mediate allelopathy in T. diversifolia have been also isolated from V. amygdalina and these include anthraquinones, flavonoids, alkaloids, saponins, phenolic acids, coumarins and terpenoids (Cimanga et al., 2004;Audu et al., 2012;Muraina et al., 2010).
Some of the uses of the plant that pervade the literature include the consumption of its leaves either as a vegetable or the use of aqueous extracts as tonics for the treatment of a variety of ailments ranging from nausea, diabetes, gastrointestinal tract problems, sexually transmitted diseases and others (Akah and Ekekwe, 1995;Igile et al., 1995;Awe et al., 1999;Cimanga et al., 2004;Abosi and Raseroka, 2003;Alawa et al., 2003).
Unlike T. diversifolia, there is lack of information on the effect of crude water extract of V. amygdalina as tested on any crop plant even though its chemical composition is similar to that of T. diversifolia.Thus, experiments were carried out to investigate the susceptibility of maize to crude water extracts (CWEs) prepared from leaves of both plants from germination to maturity.Similarly, the effect of an inorganic fertilizer on this crop was comparatively established.

Germination experiment
The seeds of maize downy mildew resistant yellow variety (DMRY) locally known as 'Auba Super' were collected from the International Institute of Tropical Agriculture, Ibadan; while fresh and healthy leaves of T. diversifolia and V. amygdalina were harvested on the University of Ibadan campus.The rest of the seeds were carefully kept for further use.One hundred grams of each of the plant materials were washed thoroughly with tap water, cut into small pieces and finely ground using a ceramic pestle and mortar.The ground plant materials were allowed to stand in two separate beakers for 24 hours with 100 ml of distilled water added to each beaker.The solutions were separately sieved and filtered using Whatman filter paper number 1.The two filtrates obtained served as 100% w/v aqueous extract of T. diversifolia (Ti 100 ) and 100% of aqueous extract of V. amygdalina (Ve 100 ).Solutions of lower concentration were prepared from each of the stock solution by adding an equal volume of distilled water to 50 ml of the stock solution.A total of four solutions namely aqueous extract of T. diversifolia and V. amygdalina at 100 and 50%, (Ti 100 and Ti 50 ) and (Ve 100 and Ve 50 ) respectively were used for seed bioassay.
Seeds were washed in tap water and sterilized in a 3.5% w/v solution of sodium hypochlorite for 5 min.They were then rinsed in distilled water four times.Four uniform seeds were sown in each of the disposable 9 mm diameter Petri dishes lined with one layer of Whatman paper number 1.In each dish, 10 ml of each aqueous extract was used to wet the seeds while an equal volume of distilled water was used in the control.The treatments were replicated five times in a completely randomized design.The seeds were put in a drawer and allowed to germinate at room temperature for 3 days.After incubation, the radicle and the plumule of each seed were measured with a metre rule and the germination percentage was determined using the formula below: G = × 100 where: N g = final number of emerged seeds, N t = total number of seeds sown.Seeds were considered as germinated when the radicle extended through the seed coat (emergence of 1 mm-long radicle).

Growth and development experiment
The remaining maize seeds from the germination experiment were used with 1 kg of inorganic fertilizer (NPK 15-15-15) while fresh and healthy leaves of T. diversifolia and V. amygdalina were harvested on the University of Ibadan campus to study the growth and development of maize plants.This experiment was carried out in the Screen House, Department of Botany, University of Ibadan.The extraction procedure was similar to that of the germination experiment but in this case, 6 kg of fresh and healthy leaves were used.The treatments were replicated four times in a completely randomized design and consisted of 500 ml of each of the extracts (Ti 100 , Ti 50 , Ve 100 and Ve 50 ) and 1.52 g of inorganic fertilizer (NPK 15-15-15), while 500 ml of tap water was used for the control.Two applications were made, namely at 4 weeks after sowing (WAS) and 7 WAS.
Twenty four big experimental polythene bags which were filled with 20 kg of top soil collected from the Department of Botany Research Farm were used for the study.Four seeds were sown in each of the twenty four bags.The bags were watered at alternate days, at 4 weeks after sowing (WAS), seedlings were thinned down to one plant per bag and initial growth parameters (stem girth, leaf length and breadth and plant height) were measured and recorded using the metre rule; this was done subsequently on a weekly basis.At the end of the experiment (harvest), the dry weight of maize cobs was measured using a digital electronic weighing balance and the leaf area was calculated thus: Individual leaf area = 0.75 × leaf length × maximal leaf width (Montgomery, 1911).
Data obtained from experiments were subjected to one way analysis of variance using statistical package for social sciences (SPSS) version 20.Treatment means were separated and compared using the Duncan's multiple range test (DMRT) at p≤0.05.

Results and Discussion
Analysis of the data revealed that different plant extracts significantly affected the germination percentage of maize in diverse ways (Figure 1).The extract of V. amygdalina and T. diversifolia at 100% (Ti 100 and Ve 100 ) inhibited the germination of maize to some extent while at 50% (Ve 50 and Ti 50 ) percentage germination was observed in the following order Ti 50 >Control >Ve 50 .It was observed that the degree of inhibition increased with the concentration of the solutions.Several workers reported similar findings in their studies.For example, Tongma et al. (1997) and Al-Watban and Salama (2012) reported that aqueous extracts obtained from different plant parts in the family Asteraceae affect germination percentage negatively as the concentration increases.However, few workers as Oyerinde et al. (2009) presented contradictory reports.They reported that the fresh aqueous shoot extracts of T. diversifolia do not significantly inhibit the germination of maize.Also, Musyimi et al. (2012) reported that higher concentration of shoot extracts of T. diversifolia stimulates the germination of the seeds of Cleome gynandra.Table 1 shows that plumule and radicle lengths were significantly inhibited (p≤0.05) as a result of the application of the extracts.The seedlings in the control had the highest mean radicle length (4.26±0.80cm).However, this was not significantly different from results obtained from seedlings irrigated with the aqueous extracts of T. diversifolia at 50% w/v (Ti 50 ) but was significantly different from the radical lengths obtained from other treatments (Ti 100 , Ve 100 and Ve 50 ).Germination % The lowest radicle mean length (1.29±0.26cm) was recorded in seedlings irrigated with aqueous extracts of V. amygdalina at 100% w/v (Ve 100 ).The treatment effects that produced the lowest plumule lengths were the extracts of T. diversifolia at 100% w/v and those of V. amygdalina at 100 and 50% (Ti 100 , Ve 100 and Ve 50 ).These significantly inhibited plumule development (p≤0.05)compared to Ti 50 and control.It can be concluded that all aqueous extracts at 100% w/v had inhibitory effects on plumule and radicle development of maize seedlings.
Plants, especially crops, respond in diverse ways to aqueous plant extracts depending on type of extracts (dry or fresh), concentration of extracts and extraction media, part from which the extract was derived from as reported by Taiwo and Makinde ( 2005 2012).Thus, it can be averred from Table 1 that V. amygdalina aqueous leaf extracts had inhibitory effects on plumule development and radicle elongation in comparison with extracts of T. diversifolia.In addition, the physiognomy of seedlings treated with the aqueous extracts at higher concentrations was affected.The seedlings irrigated with aqueous extracts of both plants at 100% had stunted and thin radicles as seen in Figure 2.There were remarkable differences in the radicle length of seedlings obtained from the control compared to seedlings obtained from other groups.The order is as follows: Control > Ti 50 > Ti 100 > Ve 50 > Ve 100 .
Table 2 shows the effects of the aqueous extracts of T. diversifolia and V. amygdalina and inorganic fertilizer on the height of maize.There were no significant effects of the treatments on the height of maize seedlings.These findings are at variance with Oyerinde et al. (2009) who reported that fresh shoot aqueous extracts of T. diversifolia stimulate the growth of older maize plants by significantly enhancing fresh weight and dry weight of cobs, leaf area and ratio and chlorophyll content.Similarly, Ilori et al. (2007) reported the inhibitory effects of aqueous extracts of T. diversifolia on rice.Other works by Taiwo and Makinde (2005) showed that shoot and root aqueous extracts of T. diversifolia enhance the growth of cowpea.Ti 50 = 50% aqueous extract of T. diversifolia, Ti 100 = 100% aqueous extract of T. diversifolia, Ve 50 = 50% aqueous extract of V. amygdalina and Ve 100 = 100% aqueous extract of V. amygdalina.WAS = Weeks after sowing.Mean of treatments ± SEM (n=4).Using Duncan's multiple range test, values within each column with the same letters are not significantly different at p≤0.05.
Table 3 shows the effects of aqueous extracts of T. diversifolia and V. amygdalina and inorganic fertilizer on the girth of maize.There were no significant treatment effects at 4 WAS and 9 WAS.However at 5, 6, 7 and 8 WAS, the inorganic fertilizer (NPK) increased the maize girth significantly (p≤0.05)compared to all other treatments.The highest stem girths were recorded in plants supplied with NPK throughout the study.Abu-Romman (2011) reported similar findings on the potential of Achillea biebersteinii in his study.
Table 4 shows the effects of the aqueous extracts of T. diversifolia and V. amygdalina and inorganic fertilizer on the leaf area of maize.The study showed that there were significant effects of treatments at 5, 6, 7 and 8 WAS (p≤0.05).This result is in agreement with Alabi et al. (2005), who reported that aqueous extracts of many plants including V. amygdalina enhance the leaf area index (LAI) of cowpea under field conditions.Table 5 shows the yield responses of maize to different treatments.The highest cob yield (30.30±1.14g) was observed in seedlings treated with 50% w/v aqueous extracts of T. diversifolia (Ti 50 ) which was significantly different from the yield obtained from plants that were irrigated with aqueous extracts of T. diversifolia (Ti 100 ) at 100% w/v (28.64±0.30g) as well as V. amygdalina at 50 and 100% w/v (Ve 50 and Ve 100 ) with yields of 26.83±0.92g and 27.76±2.37g respectively.Previous works by Jama et al. (2000); Nziguheba et al. (2002) and Olabode et al. (2007) showed that T. diversifolia is a rich source of essential nutrients and when used as organic fertilizer gives better maize yields than conventional fertilizers.Contrarily, the results of this study show that the maize yield was not significantly affected when aqueous extracts of T. diversifolia were supplied to the soil.This difference may be due to many factors including experimental conditions such as the quantity of material applied to the soil since better yields are obtained depending on the supply of organic manure, the study location (field or greenhouse study), the soil characteristics and the climate.The effects of the aqueous extracts of V. amygdalina on the yield of the maize crop were also not significant.This is not in agreement with the report of Alabi et al. (2005) who reported that fresh and dry leaf aqueous extracts of V. amygdalina improve the yield of cowpea plants in terms of pod and grain weight.

Conclusion
The aqueous extracts of T. diversifolia and V. amygdalina inhibited germination percentage, plumule and radical growth of maize seedlings.The germination percentage and the seedling growth parameters were also inhibited in seeds treated with aqueous extracts of V. amygdalina suggesting that V. amygdalina has an inhibitory effect on maize seedlings.Similarly, no significant treatment effects were observed on the height of the maize seedlings studied.However, the effects of the aqueous extracts of T. diversifolia, V. amygdalina and NPK fertilizers on the maize leaf area indicated increment from 4-6 WAS while they remained in stationary phase from 7-9 WAS, except for the control experiment.Also, T. diversifolia and V. amygdalina significantly influenced the yield of maize seedlings compared to NPK and control experiments.

Figure 1 .
Figure 1.Effects of aqueous extracts of T. diversifolia (Ti) and V. amygdalina (Ve) on the germination percentage of maize.

Figure 2 .
Figure 2. Effects of aqueous extracts of T. diversifolia (Ti) and V .amygdalina(Ve) on maize seed germination and seedling growth: (a) seed germination experiment and (b) seedling growth and development.

Table 1 .
Effects of extracts of T. diversifolia, V. amygdalina on plumule and radicle length of maize seedlings (cm).

Table 2 .
Effects of extracts of T. diversifolia, V. amygdalina and NPK fertilizer on maize plant height (cm).

Table 4 .
Effects of extracts of T. diversifolia, V. amygdalina and NPK fertilizer on maize leaf area (cm 2 ).

Table 5 .
Effects of extracts of T. diversifolia, V. amygdalina and NPK fertilizer on maize yield (g) per treatment.Ti 50 = 50% aqueous extract of T. diversifolia, Ti 100 = 100% aqueous extract of T. diversifolia, Ve 50 = 50% aqueous extract of V. amygdalina and Ve 100 = 100% aqueous extract of V. amygdalina.Mean of treatments ± SEM (n=4).Using Duncan's multiple range test, values within each column with the same letters are not significantly different at p≤0.05.