Background

Cacao (Theobroma cacao L.) belongs to the family of Sterculiaceae and the genus Theobroma. Recently, with the application of molecular marker, cacao was reclassified to belong to the family Malvaceae (Alvenson et al., 1999). Its natural habitat is the lower storey of the evergreen rainforest. There are over twenty species in the genus but Theobroma cacao is the only one cultivated widely. Since its discovery in the 18th century at the Amazon basin, its cultivation has spread to other tropical areas of South and Central America, and indeed West Africa, which became the major producer from the mid-1960 s (Opeke, 1987). Cocoa is one of the most important perennial type cultivation in the world, with an estimate world production of 3.6 Mt (FAO, 2012). Cocoa is a major cash crop in many tropical countries and it is produced within 10°N and 10°S of the equator where the climate is suitable for its growth. West Africa has been the center of cocoa cultivation for many decades, as two-thirds of the world’s cocoa is produced in West Africa (Duguma et al., 2001). Globally, the six main world cocoa producers are Ivory Coast, Ghana, Indonesia, Nigeria, Brazil, and Cameroon in descending order.

Cacao plant is highly sensitive to changes in climate from hours of sunshine to rainfall and application of water, soil condition and particularly to temperature due to effects on evapotranspiration (Almeida et al., 2002; Anim-Kwapong and Frimpong, 2005; Agele et al., 2016a). Water account for large proportion of living weight of plants, for example in some herbaceous species, water forms 70-90% of the fresh organic weight in many woody species, is essential for many physiological functions and it is most critical at certain periods in the life of cacao. Legavre et al. (2006) reported that varietal improvement for tolerance to abiotic and biotic stress factors has been identified as a priority of research programs of cocoa producing countries. The Nigerian National Cocoa Breeding Programme through selection resulted in “Established Ability Elites” which had better yields and adapted to marginal and drought prone areas (CRIN, 2011). CRIN has developed sets of improved cocoa varieties. Improvement implies development of varieties that are high yielding, pest and disease tolerant, tolerant to environmental stress. Cocoa varieties were registered by the National Centre for Genetic Resources and Biotechnology (NACGRAB) and presented at CRIN headquarters to the Nigerian public and farmers on September 27, 2011. These varieties were CRIN TC-1 (Kasimawo), CRIN TC-2 (Olowo), CRIN TC-3 (Peta-1), CRIN TC-4 (Eskes), CRIN TC-5 (Esan), CRINTC-6 (Iremiren), CRIN TC-7 (Semobita), CRIN TC-8 (Efeh.) (CRIN, 2011).

Most plants form symbiotic relationship with a group of fungi called mycorrhizal, which function as a bridge for the flow of energy and matter between plants and soils (Cardon and Whitbeck, 2007). Mycorrhizal fungi have a long history in having symbiosis relation with most plants families and they exist in most ecosystems (Saleh, 1998). The symbiotic association involves most plant species and certain fungal species which has great relevance to soil ecosystem functions, especially nutrient dynamics, microbial processes, plant ecology and agriculture. The mycorrhizal performs beneficial functions for crops, like other microorganisms such as; phosphate-solubilizing bacteria (Panhwar et al., 2009) and N2-fixing bacteria (Naher et al., 2009). The fungus colonizes the host plant's roots inside the cortical tissues, the association may be either intracellular like Arbuscular Mycorrhizal Fungi (AMF), or extracellularly as in Ectomycorrhizal fungi. The root infection by the mycorrhizal increases active absorptive surface area and stimulate nutrient and water uptake even in water stress condition. Increasing attention is paid to the potential role of mycorrhizae fungi in agriculture due to their ability to increase water and nutrient uptake by agricultural plants (Sardi, 1992). Improvement in plant performance may be due to the increase in the absorption of the roots as a result of the wide extension of fungus mycelium in the soil around the root system that allows the agricultural plant to have access to higher volume of soil (Hayman, 1983). Mycorrhizal-plant root association may be either intracellular such as AMF or extracellular by action of ectomycorrhizal fungi. The root infection by the mycorrhizal increases active absorptive surface area and stimulate nutrient and water up take even in water stressed condition (Agele et al., 2016b; Agele et al., 2017). Presently, there is paucity of information on the effect of cocoa variety and mycorrhizal inoculation on water use, growth and development of cocoa seedlings, Hence, this study evaluate the effect of variety and mycorrhizal inoculation on water use, growth and development of cocoa seedlings as affected by wet-dry cycle.

Cocoa seeds are sown in pots in the nursery to raise seedlings. In the nursery and on the field, cocoa seedlings are subjected to variable soil moisture status (wet-dry cycles) during its growth. Inoculation of crops with AMF is a common horticultural practice and variable responses of species to AMF inoculation had been reported. There is paucity of information on the effects of wet-dry cycles and arbuscular mycorrhizal inoculation on water use, growth and development of seedlings of cocoa varieties. The establishment and rehabilitation of cocoa farms, aimed at replacing ageing and non-productive cocoa stocks in the field may be limited by inadequacy of healthy cocoa seedlings (Agele et al., 2016a). Efforts to increase cocoa seedlings by smallholder farmers and seed production unit for planting and or replanting through the raising of cocoa seedlings in the nursery is associated with poor growth as a result of inadequate water application rate (Agele et al., 2016b). Therefore it is necessary to investigate suitable water application rate and the imports of other nursery management practices required to raise vigorous hybrid cocoa seedlings in the nursery to replace the old and nonproductive stock in the field for optimum and quality production of cocoa in Nigeria. The objectives of this study are to evaluate the effects of watering regime and mycorrhizal inoculation on the growth, development and seedling survival among cocoa hybrids, water use leaf chlorophyll and water soluble carbohydrate content and to relate measured agronomic variables with soil moisture deficit stress tolerance in cocoa seedlings. Findings from this study is expected to enhance understanding of the responses of newly released cocoa varieties and hybrids to soil moisture deficit stress the information will be relevant to the development of management practices for the production of cocoa seedlings. The results will contribute to the development of improved nursery practices for the production of vigorous cocoa seedlings.

1 Materials and Methods

1.1 Site of experiment and conditions

The experiment was conducted in the Screen House of the Department of Crop, Soil and Pest Management of the Federal University of Technology, Akure (Lat. 7.16°N and Long. 5.12°S) in the rain forest zone of Nigeria.

1.2 Planting materials

Seedlings of elite, high yielding and early maturing lines of cocoa (Theobroma cacao L.) were obtained from the Cocoa Research Institute of Nigeria (CRIN) Ibadan, Nigeria. The hybrids are: CRIN TC1, CRIN TC2, CRIN TC3, CRIN TC4 and CRIN TC5 and the varieties are T65/7 x N38, T65/7 x T57/22, T82/27 x T12/11, PA150 x T60/887 and T82/27 x T16/17 were obtained from the Cocoa Research Institute of Nigeria (CRIN) Ibadan, Nigeria. The seeds were planted into polybags perforated at the bottom to allow drainage and filled with 5 kg topsoil obtained from 10 years fallow vegetation. Arbuscular Mycorrhizal Fungus (AMF) was obtained from the International Institute of Tropical Agriculture IITA, Ibadan, Oyo State, Nigeria. 

1.3 Experimental design and treatments

The treatments imposed on seedlings of cocoa varieties/hybrids and watered at two sets of watering regimes with or without mycorrhizal inoculation. In Experiment One, watering regimes consisted of well watered conditions at 100% field capacity (FC) moisture content and at 60 and 40% FC which are equivalent to 1.5, 0.9 and 0.6 litres of water/pot. These were separately applied twice per week. For experiment two, there were three watering regimes which are 4- day, 8- day and 14- day watering intervals. The seedlings which were grown in perforated pots were arranged in rows of 3 consisting of 10 cocoa seedlings per row according to the watering regimes, with or without AMF inoculation. Each experiment was 5 x 3 x 2 factorial combinations arranged using Completely Randomized Design (CRD) with 3 replications.

1.4 Data collection

Measurements of agronomic variables commenced 2 weeks after transplanting (WAT). The variables included seedling water use using weighing method (kg) to determine the water use by the seedlings, plant height (cm), stem girth (stem diameter) (cm), number of leaves and branches, leaf drop, crude leaf area (product of length and width in cm²), weight of leaves per plant (g), root weight (g), tap root length (cm), sum total of root length (cm).

These parameters were determined in the following ways:

1.4.1 Seedling water use

Seedling water use was determined by weighing method sing a weighing balance, the weights of potted plants were measured before watering and a day after watering and the change in weight between measurement periods were recorded.

Plant height (cm): Plant height was done using a measuring tape calibrated in centimeters, measurements were taken form the base of the cocoa to the apical shoot level and recorded.

Stem girth (cm): The collar diameter of the seedling was assessed using digital Venier caliper and converted into girth calculating their circumference and recorded.

Number of leaves: Visual counting of the leaves on each plant in the pot was done and recorded.

Number of branches: Visual counting of the branches on each plant in the pot was done and recorded.

Plant Leaf Area (cm): The area of individual leaves and total leaf area/plant were measured using a Leaf Area meter (Delta, UK).

Leaf Fresh Weight (g): All the leaves on each plant were harvested and weighed using a weighing balance and were recorded.

Root Fresh Weight (g): The root of each plant was harvested and weighed using a weighing balance and was recorded.

Tap Root Length (cm): The tap root length of each plant was measured with a measuring tape and recorded.

Total Root Length (cm): This was carried out with a measuring tape and recorded.

1.4.2 Extraction and determination of leaf chlorophyll

Chlorophyll extraction and its determination were done at the laboratory of the Department of Crop, Soil and Pest Management, Federal University of Technology, Akure. The 2 uppermost leaves of legumes species from each treatment were harvested. One gramme of the fresh plant samples were cut into pieces and smashed in a mortar. The samples were put in a test tube and its chlorophyll content was repeatedly extracted with successive volume of 100 ml acetone/water (80:20 v/v) until no traces of green colour were noticed (residue became white). While adding the solvent (acetone), the test tubes containing the samples were kept boiling in hot water bath. The total volume of the extract was also recorded at end of the extraction. Three millimeter (3 ml) of the extract was taken and the absorbance was determined with a spectrophotometer (Spectronic 20) at two wave length of 663 nm and 645 nm that corresponds to maximum absorption of chlorophyll “a” and “b” respectively. The total chlorophyll content was calculated as follows:

Total chlorophyll content (mg/100 g tissue) = (20.2A645 + 8.02A663) (V/10 w)

Where, A645 = absorbance at 645 nm wavelength; A663 = absorbance at 663 nm wavelength; V is the final volume (cm³) of chlorophyll extract in 80% acetone and W is fresh weight (g) of tissue extracted.

1.4.3 Determination of leaf water soluble carbohydrate

About 2 ml of extracts were pipetted into a test tube. 10 ml of anthrone reagent was rapidly added and mixed by shaking and placed in a boiling water bath. The absorbance of the extract was determined on a spectrophotometer device (using a 10 mm diameter cuvette). About 0.5 g of plant samples were ground and transferred into 250 ml test tube and 220 ml of water was added. The bottles was capped and shaken on a shaker for about an hour and filtered. The first few ml was ejected and the filtrate was retained for the determination of soluble carbohydrate using Antrone reagents. 770 ml of concentrated H₂SO₄ was added to 330 ml of distilled water, in addition to 1 g of thiourea, 1 g of antrone, stired until dissolved and was stored in a refrigerator. Glucose stock solution, 1.0 g of anhydrous D (+) glucose in water and diluted to one litre prepared immediately before use. From the glucose working standard solutions, 10 ml of stock to 100 ml was diluted to produce100 ppm. From these, 0, 5, 10, 20, 40, 80 ml was pipetted and made up to 100 ml and these produced 0, 5, 10, 20, 40, 80 ppm. Samples of 2 ml of each glucose working standard solutions were pipetted into the glass test tube and rapidly, 10 ml of anthrone reagent was added and mixed by shaking. The test tube was loosely covered with a glass bulb stopper and placed immediately in boiling water for 20 minutes. The absorbance was measured using spectrophotometer device in a 10 mm optical cell at 620 mm. The graph of absorbance was plotted against glucose concentration in ppm and prepare standard graph with each batch of extracts examined. The glucose standard becomes 0, 0.8, 1.7, 3.3, 6.7, 13.3 ppm respectively.

1.5 Statistical analysis

The data collected were subjected to Analysis of Variance (ANOVA) to test the significance of the treatment means using the Statistical Analysis System (SAS) software package version 9.2 (2007). The means were separated using the Duncan’s Multiple Range Test (DMRT) (Duncan, 1955).

2 Results

2.1 Experiment one

2.1.1 Effects of variety, watering regime and mycorrhizal inoculation on growth and water use of cocoa seedlings

There were consistence pattern in the amount of water use among cocoa varieties (Table 1). Varieties T65/7 x T57/22 appeared to have consumed the highest volume of water while PA150 x T60/887 was the least. Consistently, variety T82/27 x T12/11 produced tallest plants while variety PA150 x T60/887 that was among the tallest in the earlier months declined in height development. Although, there seems to be no consistent trend in leaf production, variety T82/27 x T12/11 significantly had the highest number of leaves while T65/7 x N38 had the least number of leaves. Cocoa variety T65/7 x N38 had the least number of branches per plant. The effects of variety on plant biomass components (leaf and root weight and total root length) taken at 11 months after transplanting (MAT) are shown in Table 2. Among the varieties, leaf weight was highest in T82/27 x T12/11 and least in T65/7 x N38 and treatment differences between the two varieties were significant. The reverse was the case for root weight where the highest value occurred in T65/7 x N38 and the least value was in T82/27 x T12/11, which was significantly lower than values obtained in each of the other four cocoa varieties. T65/7 x N38 variety also had the longest total root length while T38/27 x T16/17 had the least and treatment differences between the two were significant. However, water use was highest in variety PA150 x T60/887 and least in T65/7 x T57/22. The effects of watering intervals were significant on water use of cocoa seedlings across most of the sampling dates (Table 2). Seedlings that were watered at 4- day intervals consistently used more water than those watered at 8- day intervals while seedlings watered at 14- day intervals used least amount of water. Table 2 shows the effects of watering regimes on plant height of cocoa seedlings. Seedlings that were watered at 4- day intervals were consistently taller than those watered at 8- and 14- day intervals across the different periods of observation. Significant interactions (P ≤ 0.05) were obtained between watering regimes and varieties for plant height, stem girth, number of leaves and branches per plant. At 6 MAT, seedlings watered at 4- day intervals were the tallest for all the varieties, apart from T82/27 x T12/11 and PA150 x T60/887 where those watered at 8- day intervals were tallest (Table 2). Effects of cocoa varieties and watering regimes on leaf development showed that watering at 8- day intervals led to enhanced leaf development than watering at 4- day intervals for varieties T65/7 x N38 (Table 8).

The effect of mycorrhizal inoculation was significant on plant water use measured at 8 WAT (Table 3). Mycorrhizal inoculation enhanced water use for varieties T65/7 x T57/22, T82/27 x T12/11 and PA150 x T60/887 than the non-inoculated. The effect of mycorrhizal inoculation was not significant on plant height, stem girth, number of leaves and branches as shown in Table 3. Plant height and stem girth were generally higher throughout in inoculated cocoa seedlings. Significant interactions (P ≤ 0.05) were obtained between mycorrhizal inoculation and watering regime for plant height, stem girth, number of leaves and branches per plant (Table 4). At 6 WAT, both inoculated and non-inoculated seedlings subjected to 4- and 8- day watering intervals were consistently taller than the inoculated. At 6 MAT, the varieties T65/7 x N38 and T82/27 x T12/11, that were inoculated were taller than those not inoculated. Interaction effects of mycorrhizal inoculation and watering regime on number of leaves showed that, more leaves developed for the inoculated seedlings than the non-inoculated seedlings except for variety PA150 x T60/887 which have the same leaves development (Table 4). Significant interactions (P ≤ 0.05) were obtained between mycorrhizal inoculations and watering regime for plant height, stem girth, number of leaves and branches per plant (Table 5). At 6 WAT, results showed that both inoculated and non-inoculated seedlings subjected to 4- and 8- day watering intervals were consistently taller than the inoculated and non-inoculated seedlings subjected to 14- day watering intervals. Interaction effects for stem girth and number of leaves followed similar trends as observed for plant height.

2.1.2 Effects of mycorrhizal inoculation, variety and watering regime on growth characters and water use of cocoa

The interaction effect of mycorrhizal inoculation, variety and watering regime for plant height of cocoa seedlings at 6 WAT were virtually not significant. However, seedlings of variety T65/7 x T57/22 that were not inoculated and were watered at 4- day intervals were the tallest while those watered at 14- day intervals were the shortest (Table 6a and Table 6b). All varieties both inoculated and non-inoculated watered at 4- and 8- day intervals were taller than those watered at 14- day intervals. Treatment differences for plant height were significant between 4- and 14- day watering regimes of T65/7 x T57/22 inoculated seedlings and, also, the non-inoculated seedling of T82/27 x T12/11. The interaction effect on number of leaves also showed consistent higher values for both inoculated and non-inoculated at 4- day and 8- day intervals respectively Mycorrhizal inoculation, watering interval and variety were profound for T82/27 x T16/17 on leaf formation for the inoculated and at 4- day watering intervals. Significant differences were also obtained for water use among the varieties tested. Leaf weight, tap root length and total root length showed no significant interactions (P ≤ 0.05) for effects of variety, mycorrhizal inoculation and watering regime (Table 7a and Table 7b). The tap root length was also least in T82/27 x T12/11 and highest in T65/7 x T58/22 and treatment differences were significant (Table 6a and Table 6b). However, variety T82/27 x T12/11 non-inoculated at 14- day watering intervals had the highest leaf weight while variety T65/7 x T57/22 non-inoculated at 4- day watering intervals had the highest value for both tap root length and highest total root length. Significant interactions occurred for root weight of the tested varieties, and T65/7 x T57/22 non-inoculated at 4- day watering intervals had the highest root weight while T82/27 x T12/11 inoculated at 14- day watering interval had the least value for root weight (Table 7a and Table 7b). Significant differences were also obtained in the level of water used among the varieties tested. Cocoa varieties T65/7 x T57/22 watered at 14- day interval and non-inoculated and T82/27 x T16/17 8- day inoculated watering intervals had the highest values for water use respectively (Table 8). However, varieties T65/7 x T57/22 that were watered at 8- day intervals but inoculated and T82/27 x T12/11 and watered at 14- days intervals but inoculated had the least values for water use among the varieties (Table 9).

2.2 Experiment two

2.2.1 Effect of variety and watering regime on the growth and water use of cocoa

The effect of cocoa variety and watering regime was significant (P < 0.05) on water use, plant height, stem girth and number of leaves per plant (Table 8). Seedlings grown at 100% FC (1.5 litres of water per pot) were tallest for all varieties in addition to larger stem diameter (girth). Among the varieties, TC4 and TC5 showed the least response, compared with varieties TC1, TC2, and TC3 (Table 10) varietal effects was significant on leaf development and water use, the varieties TC2, TC4 and TC5 100% FC had similar water use pattern. However TC1 consumed the highest volume of water (1.5 litres per day). There were significant differences (P<0.05) in the growth responses of cocoa varieties evaluated. The result showed that variety TC1 was tallest while TC4 had tallest plants at 14 WAT. Although, there seems to be no consistence trend in leaf production, variety TC3 significantly had the highest number of leaves across the period of measurement while TC4 had the least.

2.2.2 Effects of variety, watering regime, AMF inoculation on concentrations of chlorophyll and water soluble carbohydrate, plant biomass and water use of cocoa

Significant interactions (P < 0.05) were obtained for the effects of watering regime and variety on plant height, stem girth and numbers of leaves. Seedlings watered at 100% FC were the tallest for all the varieties except for varieties TC3 and TC4 that produce tallest plant at 60% FC. Effect of cocoa varieties and watering regimes on leaf development showed that watering at 100% FC enhanced leaf development than watering at 60% FC except for variety TC3 (Table 8). Watering at 40% produced least leaf development. Across the varieties inoculated and non-inoculated both watered at 100% FC and 60% FC were taller than at 40% FC. Treatment differences for plant height were significant between 100% FC and 40% FC watering for TC1 that was AMF inoculated, and similar trend obtains for variety TC3. Significant differences were also obtained for water use among the varieties tested. Varieties TC2 and TC5 at 40% FC and non-inoculated had the highest values for water use while varieties TC2 at 60% FC inoculated and TC3 at 40% FC watering regime and inoculated had the least values for water use. The effects of mycorrhizal inoculation and watering regime was not significant for tap root length (Table 9). However, variety TC3 non-inoculated at 40% FC watering had the highest leaf area (Figure 1) while variety TC5 inoculated at 100% FC watering regime had the highest value for both root length and highest shoot length. Significant interactions occurred for root weight of the tested varieties, and TC2 non-inoculated at 100% FC watering had the highest root weight while TC1 had the least at 100% FC inoculated. Table 10 shows the effects of AMF inoculation and watering regime on growth and water use of seedlings of cocoa. The seedlings that were grown at 100% FC were consistently taller than those watered with 60 and 40% FC across the period of observation, likewise, had lager stem girth and more leaves. The results of the effects of AMF inoculation and watering regime on growth characters of cocoa varieties are presented in Table 11. Cocoa seedlings grown at well watered condition (100% FC) were consistently taller and had lager stem girth than those watered with 60 and 40% FC, values were not different between the inoculated and non-inoculated treatments. However, differences were obtained for number of leaves produced per plant for the variously watered plants under the inoculated and non-inoculated treatments. There were no significant differences in total chlorophyll and water soluble carbohydrate of the leaves of cocoa variety TC2, TC3, but for TC1, TC4 and TC5 (Table 12). There were no significant different in the soluble carbohydrate content among the varieties except TC1. There were significant interactions for shoot weight and root weight among the varieties. The effect of watering regime and AMF were significant on water use of cocoa seedlings (Table 13). Seedlings that were watered at 100% FC watering regime consistently used more water than those watered at 60% FC while seedlings watered at 40% FC watering regime used least. The varieties TC2, and TC5 that were inoculated used more water than the non-inoculated. For varieties TC1 TC3 and TC4 water use in both the inoculated and non-inoculated seedlings were similar but differ significantly from TC1 TC3 and TC4 this imply that TC2 and TC5 cocoa varieties had inherent capacity for high water use. The effect of mycorrhizal inoculation for plant height and number of leaves was not significant (P > 0.05). While plant height and stem girth were generally higher in values for the inoculated cocoa seedlings, the trend was different for number of leaves per plant.

3 Discussion

3.1 Effects of watering regimes on growth and development of cocoa seedlings

The results of this study showed that the measured growth variables of cocoa seedlings responded to watering regimes and AMF mycorrhizal inoculation. The enhancement of plant height, stem girth, number of leaves and branches by more frequent watering may be attributed to higher moisture contents in the crop root zone. The measured growth variables of cocoa seedlings were statistically superior under 4- and 8- days watering regimes compared with 14- days watering interval. This implies that the cocoa seedlings require consistently moist root zone environment and favourable microclimate (Henson and Harun, 2007; Haeberle et al., 2015). Khalil and El-Noemani (2012) and Bahreininejad et al. (2013) and Haeberle et al. (2015) stated that, water stress reduces plant growth through inhibition of various physiological and biochemical processes, such as photosynthesis, respiration, translocation, ion uptake and nutrient metabolism. The cocoa seedlings that were well watered were taller than those watered poorly. This result further confirms how essential water is to growth and development of cocoa seedlings, a crop that is known to be very sensitive to a soil water deficit (Henson et al., 2005; Majumdar, 2010; Agele et al., 2016). Adequacy of soil moisture promotes leaf development as was obtained for seedlings that were well watered, possibly via enhance evapotranspiration. Adequate soil and plant water status are critical to the survival of cocoa seedlings during establishment especially in the premise of the unfavorable weather conditions of the dry season in Nigeria. The results of this study confirmed that cocoa seedlings cannot withstand soil moisture deficit stress as was obtained for seedlings that were watered with 40% FC and 12- days watering intervals). In plants, moisture deficit stress reduces leaf area, branching and biomass development. These responses mean that plants experiencing water stress will end up smaller and poor in vigour (Jones and Tardieu, 1997). In this respect, the cocoa varieties evaluated showed significant differences and values of measured parameters were statistically superior at well watered situation (4- days watering intervals and 100% FC) compared with the 40% FC (12- days watering intervals). Under serious water stress, the cocoa seedlings stopped growing, leaves turned yellow, wilted and later recovered or died in the 40% FC (12- days watering regimes). The terminal drought (dry spell) situation of the dry season is characterized by high intensity of soil and air moisture deficits and temperatures (Agele, 2003; Agele et al., 2016). These conditions have implications for survival and establishment of seedlings both in the nursery and on the field. Plants exposed to soil water stress elicit a number of physiological responses in an effort to conserve water, these include closing of stomatal and arresting cellular growth (Kulac et al., 2012; Haeberle et al., 2015). If water stress is not alleviated, plants will close stomatal and shut down photosynthesis, carbon assimilation, and normal metabolism (Haeberle et al., 2015). 

3.2 Effects of variety on growth and development of cocoa seedlings

There were significant differences on the effects of varieties on growth characteristics, which include plant height, stem girth, number of leaves and branches, root and shoot biomass and water use efficiency. This is in agreement with the finding that every growth characteristics in crop seedlings are affected by drought stress (Dias et al., 2007; Silva et al., 2004; Agele et al., 2015). In this respect, all the varieties tested showed differences and were statistically superior under the 4- days watering and 100% FC regimes compared with the 40% Fc and 12- days watering regimes. Under these soil moisture deficit stress conditions, the seedlings stopped growing, leaves turned yellow, wilted and later recovered or died. This observation supported the findings of Agele et al. (2015) and Agele et al. (2017) reported that growth parameters were reduced under drought stressed condition in Shea butter seedlings and rice varieties and legume species. Our findings were also substantiated by the reports of DaMatta (2004), Carr (2001) and Burkhardt et al. (2006) on the drought stress responses of coffee varieties.

3.3 Effects of mycorrhizal inoculation on growth and development of cocoa seedlings

Cocoa seedlings inoculated with mycorrhizal fungi and subjected to frequent watering were more vigorous. Nevertheless, there were no significant differences among most of the parameters measured 4- and 8- days and 14- day watering for inoculated seedlings. Across the varieties, cocoa seedling varieties that were subjected to well watered and mycorrhizal inoculation have significantly (P < 0.05) higher vigor than low watering regime. These findings support the observations of Levy and Kirkum (1983), Read and Boyd (1986) and Shinkafi (2000) who reported that mycorrhizal inoculation increased soil water extraction and root activities of tree seedlings. Agele et al. (2016) reported that mycorrhizal inoculation and mild water stress enhanced Shea butter seedling growth than the non-inoculated. In conformity with the findings of the present study, Osunubi (1992) reported that mycorrhizal inoculation enhanced leaf number, collar diameter and shoot biomass of Acacia species. Inoculation of seedlings with AMF promoted the growth of cocoa seedlings in the nursery. Mycorrhizae fungi form symbiotic (mutualistic) association with plant roots. Govindarajulu et al. (2005) reported that extra-radical hyphae take up inorganic nitrogen which is transported to intra-radical hyphae in the form of amino acids. In Shea butter, seedlings responses were greater for the inoculated plants (Agele et al., 2016). The enhancement of growth characters under these treatment combinations may be due to the activity of the mycorrhizal fungi which enabled the cocoa seedling to explore a greater volume of soil for increased nutrient absorption. Ibiremo et al. (2011) found that mycorrhizal inoculation increased the stem diameter of cashew. It is probable that mycorrhizal root colonization improved water and nutrient uptake of cocoa seedlings and consequently increased its growth.

4 Conclusions

The interaction among variety, watering regime, AMF Inoculation was profound on concentration of chlorophyll and water soluble carbohydrate, plant biomass and water use of cacao seedling. Mycorrhizal inoculation enhanced seedling growth while more frequent watering at 4- and 8- day intervals (100 and 60% FC) enhanced plant height compared with 14- day watering intervals (40% FC). Across varieties, both inoculated and non-inoculated seedlings that were subjected to 4- and 8- day watering regimes had enhanced growth and development. Myccorhizal inoculation combined with 4- and 8- day intervals enhanced height of plants and leaf development compared with 14- day watering intervals. While variety T82/27 x T12/11 showed greater response to 4- and 8- day watering and AMF inoculation, cocoa hybrid TC3 showed greater response to 100 and 60% FC watering and AMF inoculation. Based on the measured growth parameters, mycorrhizal inoculation and 8- day watering enhanced plant vigour over the 14- day watering. Therefore, for enhanced growth and development of cocoa seedlings in the nursery, it is recommended that variety T82/27 x T12/11 subjected to 4- day watering interval (100% FC) hybrid TC3 at 100 and 60% FC watering combined with AMF inoculation.

Acknowledgments

The authors acknowledge with gratitude, Cocoa research Institute of Nigeria (CRIN) for the supply of cacao elite lines and hybrids used for the experiments and laboratory and field staff of CRIN and FUT, Akure, Nigeria for technical support rendered in sampling, data collection and measurements.

References

Agele S.O., 2003, Sunflower responses to weather variations in rainy and dry cropping seasons in a tropical rainforest zone, International Journal of Biotronics, 32: 17-33 (Japan, 100% contribution)

Agele S.O., Iremiren G.O., and Ojeniyi S.O., 2011, Evapotranspiration, wáter use efficiency and yield of rainfed and irrigated tomato in the dry season in a humid rainforest zone of Nigeria, International Journal of Biology & Agricultural Sciences, 13: 469-476

Agele S.O., Osaigbovo A.U., Ogedegbe S.A., and Nwawe A.K., 2015, Effects of watering regime, organic manuring and mycorrhizal inoculation on the growth and development of Shea butter (Vitellaria paradoxa C.F.Gaertn) seedlings, International Journal of Agricultural Policy and Research, 4(3): 35-45

Agele S.O., Iremiren G.O., Aiyelari O.P., and Famuwagun I.B., 2016a, Mainstreaming adaptation, resilience and disaster risk reduction into extension of frontiers of cacao to marginal soils and climate of the humid tropics in the wake of climate and weather variabilities, 11^th Annual International Symposium on Environment, Athens, Greece, Athens Institute for Education and Research

Agele S., Famuwagun B., and Ogunleye A., 2016b, Effects of shade on microclimate, canopy characteristics and light integrals in dry season field-grown cocoa (Theobroma cacao L.) seedlings, Journal of Horticulture, 11(1): 47-56

Agele S.O., Ajayi A.J., and Olawanle F.M., 2017, Effects of watering regime and rhizobium inoculation on growth, functional and yield traits of four legume species, Int. J., Plant & Soil Sci., 17(4): 1-15

https://doi.org/10.9734/IJPSS/2017/32891

Almeida A-A.F., Brito R.C.T., Agular M.A.G., and Valle P.R., 2002, Water relations aspects of Theobroma cacao L. clones, Agrotropical, 14: 35-44

Alvenson W.S., Whitlock B.A., Feller R., Bayer C., and Baum D.A., 1999, Phylogeny of the Core Malvales: Evidence from NDHF sequence Data, American Journal of Botany, Vol. 86: 1474-1486

https://doi.org/10.2307/2656928

Anim-Kwapong G.J., and Frimpong E.B., 2005, Vulnerability of Agriculture to Climate-change impact of climate on Cocoa production, Cocoa Research Institute, New Tafo Akim, Ghana

Bahreininejad B., Razmjoo J., and Mirza M., 2013, Influence of water stress on morpho-physiological and phytochemical traits in Thymus daenensis. International Journal of Plant Production, 7(1): 151-166

Burkhardt J., Beining A., Kufa T., and Goldbach H.E., 2006, Different drought adaptation strategies of Coffea arabica populations along a rainfall gradient in Ethiopia

Cardon Z.G., and Whitbeck J.L., 2007, Elsevier Academic Press, The Rhizosphere, pp: 235

Carr M.K.V., 2001, The water relations and irrigation requirements of coffee, Experimental Agriculture, 37: 1-36

https://doi.org/10.1017/S0014479701001090

Cocoa Research Institute of Nigeria (CRIN), 2002, Information Booklet. CRIN News, Ibadan, Nigeria, A Quarterly Magazine, 3(4): 1-2

DaMatta F.M., 2004, Exploring drought tolerance in coffee: A physiological approach with some insights for plant breeding, Brazil Journal of Plant Physiology, 16: 1-6

https://doi.org/10.1590/S1677-04202004000100001

Famuwagun I.B., and Agele S.O., 2010, Effects of sowing methods and plant population densities on root development of cacao (Theobroma cacao L.) seedlings in the nursery, International Journal of Agricultural Research, 5: 445-452

https://doi.org/10.3923/ijar.2010.445.452

Food and Agriculture Organization (FAO), 2012, FAOCLIM: Environment and natural resources service, Working Papers No. 5. FAO, Rome

Govindarajulu M., Pfeffer P.E., Jin H., Abubaker J., Douds D.D., Allen J.W., Bucking H., Lammers P.J., and Shacker-Hill Y., 2005, Nitrogen transfer in the arbuscular mycorrhizal symbiosis, Nature, 435: 819-823

https://doi.org/10.1038/nature03610

PMid:15944705

Haeberle K.H., Agele S.O., Matyssek R., and Hennlich M., 2016, Aspects of Water Relations and Gas Exchange of Katsura and Tilia Seedlings Subjected to Wet-Dry Cycles: Indication of Strategies for Whole Plant Drought Tolerance, International Journal of Plant & Soil Science, 10(2), 1-13

https://doi.org/10.9734/IJPSS/2016/17154

Hayman D.S., 1983, The physiology of VA-endo Mycorrhizal symbiosis, Canadian Journal of Botany, 61: 944-963

https://doi.org/10.1139/b83-105

Henson I.E., Harun M.H., and Chang K.C., and Mohammed A.T., 2007, Predicting soil water status, evapotranspiration growth and yield of young oil palm in a seasonally dry region of Malaysia Journal of Oil Palm Research, 19: 398-415

Henson I.E., Noor M.R.M., Harun M.H., Yahya Z., and Mustakim S.N.A., 2005, Stress development and its detection in young oil palms in north Kedah, Malaysia Journal of Oil Palm Research, 17: 11-26

Ibiremo O.S., Daniel M.A., Oloyede A.A., and Iremiren G.O., 2011, Growth of coffee seedlings as influenced by Arbuscular mycorrhizal Inoculation and phosphate fertilizer in two soils in Nigeria: International Research Journal of Plant science (ISSN: 2141-5447), 2(6): 160-165

Khalil S.E., and El-Noemani A.A., 2012, Effect of irrigation intervals and exogenous proline application in improving tolerance of garden cress plant (Lepidium sativum L.) to water stress, Journal of Applied Sciences Research, 8(1): 157-167

Kulac S., Nzokou P., Guney D., Cregg B.M., and Turna I., 2012, Growth and physiological response of Fraser Fir [Abies fraseri (Pursh) Poir.] seedlings to water stress: Seasonal and diurnal variations in photosynthetic pigments and carbohydrate concentration, HortScience, 47(10): 1512-1519

Legavre T., Gramacho K., Duchamp M., Sounigo O., Debert P., Fouet O., Sabau X., Argout X., Wincker P., Da Silva C., and Lanaud C., 2006, Identification of Theobroma cacao genes differentially expressed during Phytophthora infection. Paper presented at the 15 International Cocoa Research Conference, Costa Rica

Levy Y., and Krikum J., 1983, Effect of irrigation, water and salinity and root-stock on the vertical distribution of Vesicular Arbuscular Mycorrhiza on citrus roots, New phytopathology L., 15: 397-403

Majumder A.L., Sengupta S., and Goswami L., 2010, Osmolyte Regulation in Abiotic Stress, pp: 349-370

Naher U.A., Radziah O., Shamsuddin Z.H., Halimi M.S., and MohdRazi I., 2009, Growth Enhancement and Root Colonization of Rice Seedlings by Rhizobium and Corynebacteriumspp, International Journal of Agricultural Biology, 11: 586-590

Navarro Garcia A., Del PilarBanon Arias S., Morte A., and Snachez-Blanco M.J., 2011, Effects of nursery preconditioning through mycorrhizal inoculation and drought in Arbutus unedo L. Mycorrhiza, 21: 53-64

https://doi.org/10.1007/s00572-010-0310-x

PMid:20405149

Opeke L.K., 1987, Tropical Tree Crops. John Willey and sons, Chichester, pp: 67-213

Osonubi O., and Mulongony K., 1992, Interaction between drought stress and vesicular Arbuscular mycorrhizal on growth of Faidherbia albida (Acacia albida) and Acacia nilotica in sterile and non-sterile soils, Biology and Fertility of Soils, 3: 159-165

https://doi.org/10.1007/BF00346056

Panhwar Q.A., Radziah O., Sariah M., and Mohd Razi I., 2009, Solubilization of phosphate forms by phosphate solubilizing bacteria isolated from aerobic rice, International Journal of Agricultural Biology, 11: 667-673

Read D.J., and Boyd R.C., 1986, Water relations of Mycorrhizal fungi and their host plant in Ayres.P.C Boddy L (eds), Water, Fungi and Plants, Cambridge University press, Cambridge U.K.

Sardi P., Saracchi M., Quaroni S., Petrolini B., Borgonovi G., and Meril S., 1992, Isolation of endophytic Streptomyces strains from surface sterilized roots, Applied Environmental Microbiology, 58: 2691-2693

Shinkafi M.A., 2000, Effects of macronutrients deficiency and mycorrhizal inoculation of Faidherbla albida (Del) A. chev. in a semiarid environment, Ph.D. Thesis, Usmanu Danfodiyo University Sokoto, pp: 148