Experiment I: Evaluation of availability and uptake of P from rock phosphate by four agroforestry tree species in two acid soils of Kenya

The experiment was conducted in a glasshouse using an Acrisol from Western (Kakamega) and an Andosol from Central (Gituamba) Kenya. Soil samples were collected from the arable layer (0-20 cm) and the main properties of these soils are given in Table I. The trees species used in this study were Senna siamea, Leucaena leucocephala, Grevillea robusta and Eucalyptus grandis. The seeds of L. leucocephala, G. robusta, C. siamea and E. grandis obtained from the seed center of the Kenya Forestry Research Institute (KEFRI) were first pregerminated on agar-plates, then sown into sand trays in the glasshouse after two weeks. However L. leucocephala and C. siamea (legumes) seeds were first pretreated (to soften seed coat) by leaving them overnight in hot water before pregerminating.

The seedlings were pricked out into the pots 30 days after sowing into the sand trays.

Minjingu phosphate rock (12.9% P) was applied at three rates: 0 (PR₀), 52 (PR₁) and 77 (PR₂) kg P ha^-1 equivalent to 0, 25.8 and 38.7 mg P/ kg soil respectively. Two kilograms of air-dried soil were placed into plastic bags (gauge 300, 4" wide x 7" length), the required amount of Minjingu RP was added and mixed thoroughly with the soil. About 200 ml of water was added to bring the soil to field capacity and left for a week to allow for equilibration [16]. The seedlings were pricked out into the pots 30 days after sowing into the sand trays. A basal nutrient solution containing 200 mg N as NH₄NO₃, 400 mg K as K₂SO₄ ^.H₂O and 100 mg Mg as MgSO₄.7H₂O was also applied to each pot at planting. An amount of water not exceeding 250 ml was added to the pots on a weekly basis to maintain the soils at field water capacity. The pots were laid out in a completely randomized design (CRD) in the glasshouse.

In this experiment, height (ht.) and root collar diameter (rcd.) were recorded on a weekly basis up to 19 weeks after transplanting (19 WAT) using a meter rule and diameter tape respectively. The data at 19 weeks after planting (19 WAT) were subjected to an analysis of variance (ANOVA) and a Tukey's Test (P< 0.05) was used to compare the means using SAS software program.

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TABLE I. SOME PROPERTIES OF THE SOILS FROM THE ANDOSOL AND ACRISOL

1 Walkley-Black method, ²Kjeldahl method, ³Olsen’s Method, ⁴TSBF titrimetric method, ⁵ EDTA method,

6Ammonium acetate extraction method, ⁷ Hydrometer method

2.3. Experiment II: Response to rock phosphate fertilization and mycorrhizae inoculation by G. robusta and L. leucocephala in an Acrisol from Kenya

The MPR fertilizer rates were maintained as in Experiment I. Ten kilograms of soil from the Acrisol, Kakamega was weighed into plastic bags (gauge 300, 8"wide x 12" length). 2 g (RP₁) and 3 g (RP₂) of Minjingu RP was then added and mixed thoroughly. Two agroforestry tree species used in the study were L. leucocephala and Grevillea robusta and the procedures for raising and pricking out of the seedlings was as mentioned in experiment I. Mycorrhizae inoculant were prepared as described by [17]. The infected roots of Acacia spp. and freshly collected soils from Kakamega were placed on roots of host plant in this case, maize. A portion of the infected roots of maize and soil with endomycorrhizal associates were then used to inoculate Acacia tortilis seedlings. Two grams of the mixture soil and roots was spread halfway down the pots (except pots of treatment I) just before the pre-germinated seedlings were pricked out. The treatments used in this experiment were:

I. RP0 IV. RP0 + Mycorrhizae (M).

II. RP1 V. RP1 + M

III. RP2 VI. RP2 + M

Experiment II was an RCBD replicated four times. Some seedlings were grown and set aside for destructive sampling.

Monthly readings height (ht.) and root collar diameter (rcd.) were recorded for 12 months. Plants in experiment II were harvested at 12 months after transplanting (12 MAT). Parameters assayed were shoot and root dry matter for both tree species and nodule number and fresh weight for L.

leucocephala only. The data, as indicated above for experiment I were subjected to an analysis of variance (ANOVA) and a Tukey's Test (P< 0.05) was used to compare the means using SAS software program.

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202 2.4. Experiment III: Evaluation of P-uptake from Minjingu rock phosphate and TSP

by six agroforestry tree species growing in an Acrisol from W. Kenya, using the ³²P isotope dilution technique

The study was carried out in the glasshouse at the University of Nairobi. Surface samples (0-20 cm) from an Acrisol, Kakamega were collected randomly from an experimental farm. Samples were bulked and mixed thoroughly from which an amount was set aside for analysis and about 100 g was used for the P dynamic studies. The two phosphate sources were: Minjingu Rock Phosphate (MRP), 12.9%P and Triple Super Phosphate (TSP), 21.0%P

2.4.1. Choice of P fertilizer rates for pot experiments

The rate of 400 kg ha^-1 MRP was selected based on the results obtained in experiments I and II and an equivalent amount in % P content in the form of TSP. In isotope-aided experiments only one single fertilizer rate of application is normally utilized, namely that recommended for optimum yield [15]. The Andosols from Gituamba showed little or no response to added P fertilizer, therefore only the Acrisol from Kakamega was chosen for experiments III and IV. The methods used were adopted from [18]. The six trees species selected for this study included three legumes: Sesbania sesban, Leucaena leucocephala, Gliricidia sepium and three non-legumes: Grevillea robusta, Cassia siamea and Eucalyptus grandis. The seeds were obtained from the seed center of KEFRI. The seedlings were pricked out after 30 days into 5 kilogram air-dried soils, which were already mixed thoroughly with either Minjingu RP or TSP as per the treatments listed in section 4.8. L. leucocephala, S. sesban and G. sepium, were inoculated with appropriate Rhizobium strain(s). A basal nutrient solution containing 200 mg N as NH4NO3, 400 mg K as K2SO4, H2O and 100 mg Mg as MgS04.7H2O was also applied to each pot at planting. An amount of water not exceeding 250 ml was added on a weekly basis to maintain the soils at field water capacity. A set of six treatments listed below was used in this experiment. However treatments I, II and III received ³²P labeled KH2PO4 solution (10 ppm of P concentration) with an activity of 185 x 10 Bq ³²P /kg soil (50µCi ³²P/kg soil) applied as 20 ml aliquots per pot:

i: Soil + KH232PO4

ii: Soil + Minjingu rock phosphate [ MRP (25.8 mg P/kg soil) ]+ - KH₂ ³²PO₄ iii: Soil + Triple superphosphate [TSP (25.8mg P/kg soil)] + -KH2 32PO4

iv: Soil + Minjingu rock phosphate [MRP (25.8 mg P/kg soil)]

v: Soil + Triple superphosphate [TSP (25.8mg P/kg soil)].

vi: Soil alone (check treatment) 2.4.2. Experimental design and layout

The experiment was set-up as a Randomized Complete Design with 3 replications and three harvests for each tree species. Harvesting was done at 3 month intervals and the experiment lasted for 9 months. The ³²P-labeled solution was applied sequentially, immediately after pricking out of the seedlings into the pots (period 1), then was applied to 3 months-old seedlings for period 2 and this coincided with the first harvest. This was repeated two more times. The harvests were thus referred to as 3 MAT (months after transplanting), 6 MAT and 9 MAT respectively. Height and root collar diameter increase were measured in centimeters (by use of a meter rule) at monthly intervals for up to 9 months. The first destructive harvest was carried out at 3 MAT. The pots with unlabelled plants were harvested first. Roots and shoots were carefully separated. The roots (only from unlabelled plants) were washed out carefully on a 0.5 mm sieve by a water nozzle at low pressure and the nodules (for legume trees) carefully removed. Nodule count and characterization followed immediately.

The soils were carefully collected in a bucket per replicate and later transferred to polyethylene sheets for drying. This was later analyzed for micro and macronutrients. The shoots and roots from unlabelled plants were air-dried in an oven at 80°C till a constant weight was attained and shoot and root dry matter in grams (SDW and RDW respectively) were recorded. The shoot samples were then ground for determination of tissue nutrient P, C, N, Na, Ca, K and Mg contents.

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At each harvest the shoots were cut at 1cm from the soil surface chopped into small sections (approximately 1 cm), placed in porcelain crucibles and dried at 105°C in a muffle furnace for 24 hours to obtain dry weight. This was followed by subsampling into 2g portions which were then dry-ashed (in a muffle furnace at 250-300^oC for 2 hours) and dissolved into 20 ml HCl after cooling.

A 5 ml aliquot was then obtained for plant tissue P determination by colorimetric method. The rest of the solution was transferred to vials for measuring the ³²P activity of the plant samples in disintegration per-second (dps) in a liquid scintillation counter at National Agricultural Research Laboratories, Kabete, Nairobi. A series of calculations were made to evaluate the relative agronomic effectiveness (RAE) of Minjingu rock phosphate.

2.5. Experiment IV: A glasshouse evaluation of the response of L. leucocephala and G. sepium to P fertilization and VA-mycorrhizae inoculation

Seedlings of L. leucocephala and G. sepium were raised the same way as in the previous experiments, whereas VA-mycorrhizae inoculation was done as in experiment II and application of ³²P labeled solution as in experiment III. At six months after transplanting nodule number, nodule dry weight, biomass and P uptake were assessed.

3. RESULTS AND DISCUSSION

3.1. Experiment I: Growth response of four agroforestry species to Minjingu PR application