Potential of Managing Iron and Zinc Deficiency in Dry Beans with Interplantings of Annual Ryegrass and Increased Bean Density

Final Report for GW08-016

Project Type: Graduate Student
Funds awarded in 2008: $18,928.00
Projected End Date: 12/31/2009
Grant Recipient: University of Wyoming
Region: Western
State: Wyoming
Graduate Student:
Major Professor:
Andrew Kniss
University of Wyoming
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Project Information

Summary:

Based on a farmer’s observation in 2002 that pinto beans intercropped with annual ryegrass did not exhibit iron-deficiency chlorosis and outperformed bean monocultures, the objective of this project was to determine if interplantings of annual ryegrass can mitigate iron deficiency in dry beans in high pH calcareous soils in Wyoming and zinc deficiency in acidic soils in western Kenya. The project also investigated recent observations that iron deficiency chlorosis can be overcome by more resistant cultivars of dry beans, temperature, close bean plant spacing and low levels of nitrate nitrogen in these soils.

Introduction

Iron Deficiency in Calcareous Soil

Iron deficiency chlorosis in dry edible beans is common in the high pH, calcareous soil prevalent in the western Great Plains (Stevens and Belden, 2005). In general, this condition is not due to absolute lack of soil iron, as total iron concentration in the soil is usually adequate (Aktas and Egmond, 1979). The deficiency is normally due to the conversion of the more bio-available ferrous (Fe2+) iron into less soluble ferric (Fe3+) oxides, hydroxides, oxihydroxides and ferrihydrites (Cornell and Schwertmann, 2003).

Higher plants have developed two different strategies that help them to increase iron availability in soils (Alcaniz et al., 2005). Strategy I is developed by dicot and non-graminaceous monocot species in response to iron deficiency and involves acidification of the rhizosphere root zone by the plants through proton (H+) extrusion, increasing Fe3+ – chelates’ solubility and the concomitant reduction by a ferric reductase to ferrous iron which can then be up-taken by plants (Robinson et al., 1999). In Strategy II plants developed by graminaceous (Poaceae family) species such as wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oriza sativa) and maize (Zea mays), iron-induced morphological and physiological changes adopted by Strategy 1 plants are absent. Instead methionine derivatives belonging to the mugineic acid family (phytosiderophores) are synthesized by the plant roots which are secreted in the rhizosphere root zone, where they chelate ferric iron and make it more bioavailable (Marschner and Romheld, 1994; Marschner, 1995; Vansuyt et al., 2000). Strategy I and II plants can also be referred to as iron-efficient plants. In high pH calcareous soils with high bicarbonate content, the activity of Strategy I can be neutralized causing iron deficiency chlorosis in the plants (Alcaniz et al., 2005). Studies by Venkat Raju and Marschner (1972) showed that reducing substances released by iron-efficient Strategy I plants under iron deficiency declined when the pH of the medium was sustained at high level during the growth of sunflower in nutrient solution.

Many microorganisms can also produce Fe3+ – chelating compounds known as hydroximate siderophores (HS) with high affinity for Fe3+and capable of transporting iron (Sylvia et al., 2005). Studies by Powell et al. (1980; 1983) found that HS occur in the soil at levels that can be sufficiently high enough to promote absorption of iron by plants. Uptake of iron by iron-efficient plants such as oats can therefore be due to the release of phytosiderophores by their roots or their ability to obtain iron chelated by microbial HS. Annual ryegrass has not yet been established in literature as an iron-efficient graminaceous plant capable of producing iron-chelating phytosiderophores. The role of microorganisms in alleviating iron deficiency in dry edible beans has also not yet been conclusively reported in literature.

Studies by Aktas and Egmond (1979), Mengel (1994), and Mengel et al. (1994) have shown that high nitrate nitrogen levels in the soil can also induce iron chlorosis. Besides the proton extrusion strategy developed by plants in response to iron stress, nitrogen nutritional status considerably influences proton or hydroxyl (OH-) ion excretion from plant roots (Aktas and Egmond, 1979). Plant species growing in complete nutrient solution with nitrate-nitrogen exude OH- or HCO- into the nutrient medium as long as there is enough nitrate in the medium (Kashirad and Marschner, 1974; Aktas and Egmond, 1979). Iron efficient plants secrete H+ ions into the rhizosphere root zone when iron stress develops, regardless of the nitrate status of the soil, and continue to excrete protons even after soil nitrate supply is depleted (Aktas and Egmond, 1979). Iron inefficient plants on the other hand continue to secrete OH- and HCO-ions into the rhizosphere root zone when nitrate is sufficiently available even when iron stress develops (Aktas and Egmond, 1979; Romheld et al., 1984). Excretion of protons by these plants only begins after nitrate supply in the soil has been depleted.

Practiced worldwide for many generations, mixed cropping – especially of legumes and grasses – can enhance on-farm biodiversity, promote biological nitrogen fixation, increase dry matter production and grain yield and enhance resource use efficiency (Agboola and Fayemi, 1972; Willey, 1979; Searle et al., 1981; Aggarwal et al., 1992; Fujita et al., 1992; Okereke and Anyama, 1992; Shaxton and Tauer, 1992; Kim and Rees, 1994; Sylvia et al., 2005). Different plant cultivars and species exhibit different susceptibility to iron chlorosis (Aktas and Van Egmond). Plant cultivars that are tolerant to iron deficiency selectively intercropped with susceptible plant cultivars can alleviate iron chlorosis in the latter. In comparative studies between iron-efficient sunflower plant species and iron-inefficient corn species, Venkat Raju and Marschner (1972) and Kashirad and Marschner (1974) showed that under iron deficiency conditions, sunflower plants lowered the pH of the nutrient solution resulting in increased uptake of inorganic Fe3+ evidenced by re-greening of the sunflower plants. In contrast, corn plants were not able to lower the pH of the nutrient solution as a result of which they were unable to utilize Fe3+ as their source of iron. When the two plant species were intercropped in the nutrient solution under iron-stress, the iron efficient sunflower lowered the pH of the nutrient solution, enabling corn plants to also re-green with Fe3+ as the source of iron. The role of annual ryegrass in a similar cropping system to mitigate iron deficiency in dry edible beans has not yet been reported in literature.

The purpose of this study was to investigate the role of interplantings of annual ryegrass, temperature, soil microorganisms, and nitrate-nitrogen in mitigating iron deficiency in susceptible dry beans in calcareous soil.

Zinc Deficiency in Acidic Soil

One of the most widespread nutritional constraints in crop plants (Sillanpaa;, 1982), zinc is an essential constituent of many vital enzymatic systems in plants (CPHA, 2002; Ludwick et al., 2002). Zinc is usually taken up by plants as a divalent cation (Zn2+), which can function as a metal component of enzymes or as a “functional, structural, or regulatory cofactor of a large number of enzymes” (Kickens, 1995). Among other important enzymatic activities, zinc plays a key role in the synthesis of indoelacetic acid, which is an important plant growth regulator (CPHA, 2002). Zinc deficiency in dry beans results in interveinal chlorosis starting at the terminal growth areas, decrease in stem length, rosetting of terminal leaves, reduced fruit bud formation and dieback of twigs after the first year (Ludwick et al., 2002). Low availability of zinc in soils can reduce crop yields by up to 50% (Cakmak, 2002).

The concentration of zinc in the soil between 0.5 and 2.0 mg kg-1, depending on the methods of extraction, are considered deficient for plant development (Jacobsen et al., 2003; Singh et al., 2005). While the total concentration of zinc in the soil is mainly due to parent rock materials, its application in fertilizers, pesticides and waste materials can also determine its content (Halina, 2003). Zinc deficiency commonly occurs on neutral and calcareous soils, intensively cropped soils, paddy soils, poorly drained soils, sodic and saline soils, peat soils, soils with high available phosphorus and silicon, sandy soils and highly weathered acid and coarse-textured soils (Singh et al., 2005). Factors such as topsoil drying, disease interactions and high cost of fertilizer also contribute to zinc deficiency (Sillanpaa;, 1982). Zinc deficiency is common in the old highly weathered acidic soils of western Kenya, where dry beans are a major source of protein for most low-income communities in Kenya (Sombroek et al., 1992; Itulya and Aguyoh, 1998).

Although zinc is thought to be adsorbed on cation exchange sites of clay minerals under acidic conditions, this adsorption is reduced by other cations competing for the exchange sites resulting in more bioavailability of zinc under low pH conditions (Kabata-Pendias, 2001). However, bioavailability of zinc in acid light soils is reduced through immobilization in soils rich in Ca and P, in well-aerated soils with S compounds and in soils containing substantial amounts of certain Ca-saturated minerals such as allophone, imogolite, and montmorillonite, as well as hydrous oxides (Kabata-Pendias, 2001).

Although zinc is thought to be adsorbed on cation exchange sites of clay minerals under acidic conditions, this adsorption is reduced by other cations competing for the exchange sites resulting in more bioavailability of zinc under low pH conditions (Kabata-Pendias, 2001). However, bioavailability of zinc in acid light soils is reduced through immobilization in soils rich in Ca and P, in well aerated soils with S compounds and in soils containing substantial amounts of certain Ca-saturated minerals such as allophone, imogolite, and montmorillonite, as well as hydrous oxides (Kabata-Pendias, 2001).

In Kenya, dry beans are commonly interplanted in rows between rows of maize to increase productivity per unit of land, given that land sizes among most farmers are quite limited, averaging 1.5 hectares per family of six (Woomer and Mukhwana, 2004). Interplanting dry beans with maize not only provides farmers with two staple food crops within the same growing season, but some of the nitrogen fixed by dry beans through its association with nitrogen fixing bacteria can be transferred to the maize growing in close proximity, thereby increasing the cropping system’s yield and efficiency of nitrogen use (Fujita et al. 1992). The role of annual ryegrass in such a cropping system to mitigate zinc deficiency in legumes has not yet been reported in literature.

The purpose of this study was also to investigate the role of annual ryegrass intercropped with dry beans in mitigating zinc deficiency in the acidic soils of western Kenya.

Project Objectives:

1. To determine the effectiveness of intercropping annual ryegrass with pinto beans in mitigating iron deficiency in calcareous soils. This was compared to interplantings of wheat as a grass known to exude compounds capable of chelating iron. Within this objective included an investigation into the following:

1.1 The role of temperature and high bean plant density/close spacing

1.2 The role of bean cultivar susceptibility/tolerance and microorganisms

1.3 The role of nitrate-nitrogen on iron deficiency

2. To determine the effectiveness of intercropping annual ryegrass with pinto beans in the field in mitigating zinc deficiency in more acid soils. This was compared to interplantings of wheat as a grass known to exude compounds capable of chelating zinc and interplantings of millet, a hardy cereal food crop popular in western Kenya.

Cooperators

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  • Andrew Kniss

Research

Materials and methods:
1.1 Effect of Interplantings of Annual Ryegrass, High Bean Plant Density, and Temperature on Iron Deficiency in Pinto Beans

Field Study

A field experiment was established on the irrigated field at Sustainable Agriculture Research and Extension Center near Lingle, Wyoming in summer 2008 and repeated in summer 2009. The study consisted of 3 by 9 meter plots in a randomized complete block design with four replications. Nine treatments were planted in four blocks including: 1) ‘Buckskin’ pinto beans intercropped with ‘Gulf” annual ryegrass; 2) ‘Buckskin’ pinto beans intercropped with ‘Gulf” annual ryegrass and sprayed with ‘Select’ herbicide; 3) ‘Buckskin’ pinto beans intercropped with spring wheat; 4) ‘Buckskin’ pinto beans intercropped with spring wheat and sprayed with ‘Select’ herbicide; 5) a monoculture of ‘Buckskin’ pinto beans at low density; 6) a monoculture of ‘Buckskin’ pinto beans at high density; 7) a monoculture of ‘Gulf” annual ryegrass; 8) a monoculture of spring wheat; 9) bare soil check.

All planting was done on June 11 for the 2008 study and July 19 for 2009 study. Planting date in 2009 was delayed due to unexpected heavy rains during the normal planting period. Due to a mechanical failure on the planter used for this study, pinto beans failed to germinate and had to be reseeded on June 30. Annual ryegrass was seeded at the rate of 22 kh ha-1 (Hannaway et al., 1999) and wheat seeded at 44 kg ha-1 (Kirkland et al., 2000) using a Tye double disc drill on 8 inches (20.3 cm) spacing. Dry beans were then drilled into the plots at 163,000 plants ha-1 for the normal monoculture treatment and 326,000 plants ha-1 for the double density monoculture treatment using a John Deere Maxemerge 7300, 4-row, 30-inch (76.2 cm) vacuum planter. All irrigation was done using parallel overhead irrigation equipment. Hand weeding was done in mid July and early August for the 2008 study. However, ‘Treflan’ (trifluralin) herbicide was applied pre-emergence to the 2009 study. Prevalent weeds were Common lambsquarters (Chenopodium album), Redroot pigweed (Amaranthus retroflexus), Hairy nightshade (Solanum saracchoides), and Green foxtail (Setaria viridis). ‘Select’(clethodim) grass herbicide was applied one month after planting to remove grasses from treatments 2 and 4 once benefits from the grasses was determined to have occurred.

Soil samples were collected from each plot at planting, mid-season, and at completion of the experiment for soil analysis. Each soil sample consisted of five soil cores randomly collected from each plot at 15.24 cm depth using a 2.54 cm soil probe. Cores from each plot were thoroughly mixed together into a composite sample, dried at 60 degrees centigrade for 72 hours, and then shipped to Ward Laboratories, Inc. Kearney, Nebraska for analysis. Soil samples were analyzed for iron, zinc, nitrate-N, organic matter, electrical conductivity, pH, manganese, molybdenum, and phosphate. The pH of the samples was determined using the saturation paste method (Gavlak et al., 2003) while soil organic matter was determined using the ‘loss on ignition’ method based on Storer (1984). Soil iron and zinc were extracted from soil samples using diethylenetriaminepentaacetic acid (DTPA) micronutrient extraction method developed by Lindsay and Norvell (1978). Inductively coupled plasma atomic emission spectrometry with a detection limit of 0.1 ppm (Gavlak et al., 2003) was used to determine the analytical concentration of the micronutrients.

Bean leaf samples were collected at plant establishment, mid-season, and maturity, dried at 60 degrees centigrade for 72 hours, and then shipped to Ward Laboratories, Inc. Kearney, Nebraska for analysis. The first tissue samples were collected after the second set of trifoliate leaves developed on 50% of the beans. Two to three of the youngest fully expanded leaves were collected from an average of 30 consecutive plants from the two middle rows of each plot (Hamrick, 2003). These samples were analyzed for Fe, Zn, Mo, Mn, P, S, and nitrate-nitrogen using the dry ash method and atomic absorption spectrometry with a detection limit of 0.1 ppm (Gavlak et al., 2003).

Two to three of the youngest fully expanded leaves from an average of 3 consecutive plants from the two middle rows of each plot were measured for chlorophyll content using a SPAD-503Plus chlorophyll meter at two different dates. Three meters of row from each plot was harvested in September and yield was determined in the field. Data was analysed using the Split-plot-in-space and Split-plot-in-time procedure of the analysis of variance in SAS (version 9.1 SAS Institute, 2008). Treatment differences for soil and tissue nutrient concentration as well as chlorophyll content were determined using Fisher’s protected LSD (alpha= 0.05).

Growth Chamber Study

A growth chamber study was established at the UW Plant Sciences greenhouse in 2009 and repeated in 2010 to test the potential of annual ryegrass to mitigate iron deficiency in black beans under two temperature regimes. Ten centimeter pots were filled with homogenized soil obtained from the irrigated fields at Sustainable Agriculture Research and Extension Center near Lingle. Two sets of two treatments replicated three times in a complete randomized design were planted in these pots in two growth chambers (Percival Scientific Inc, Model 130BLLC8; SO#009991-001; Serial Number 9991.01-02.07). In 2009, one growth chamber was set at day/night temperatures of 25/20oC; relative humidity of 50%; and Light/Dark period of 13/11 hours. The cooler growth chamber was set at day/night temperatures of 21/11oC; relative humidity of 50%; and Light/Dark period of 13/11 hours. The light/dark period was adjusted to 15/13 in 2010 to increase bean plant exposure to light after plants developed long internodes and bolted toward fluorescent light tubes in the previous year. Treatments consisted of black beans intercropped with annual ryegrass and a monoculture of black beans.

Planting was done on October 27 for the 2009 study and May 23 for the 2010 study. Intercropped pots were sprayed with ‘Select’ herbicide three weeks after planting. Leaf chlorophyll data was taken on the youngest fully expanded bean leaves using a SPAD-503Plus chlorophyll meter at three different dates. Bean shoots and roots biomass as well as root nodule counts were determined two months after planting.

Data was analysed using the PROC MIX procedure of the analysis of variance in SAS version 9.2 (SAS Institute, 2008). Treatment differences for bean shoots and roots biomass as well as root nodule counts were determined using Fisher’s protected LSD (alpha= 0.05).

1.2 Effect of Interplantings of Annual Ryegrass and Soil Microorganisms on Iron Deficiency in Susceptible Dry Edible Bean Cultivars

Field Study

Although pinto beans in 2008 and 2009 study did not exhibit sustained iron deficiency chlorosis symptoms, soy beans and black beans planted on adjacent plots by another researcher exhibited sustained iron deficiency chlorosis symptoms over the growing period. This suggested that different dry bean cultivars have different tolerances to micronutrient deficiency which led to the introduction of two additional bean cultivars in a separate study in 2009.

A field experiment was established on the irrigated field at Sustainable Agriculture Research and Extension Center near Lingle, Wyoming in summer 2009 and repeated in 2010 to assess the effectiveness of annual ryegrass in mitigating iron deficiency in more susceptible dry bean cultivars. The study consisted of 3 by 3 meter plots for treatments with corn and 3 by 4 meter plots for the other treatments in a 4 by 5 factorial randomized complete block design replicated four times (Figure 2). Twelve treatments planted in four blocks included three bean cultivars: ‘Buckskin’ pinto beans, ‘Schooner’ navy beans, and ‘T-39’ black beans each intercropped with ‘Gulf’ annual ryegrass, spring wheat, ‘Russell’ oats, and ‘Pioneer 38N85’ field corn as well as a bare soil check. Beans were planted at 163,090 plants ha-1 and Pioneer 38N88 corn was planted into 30-inch rows at a density of 79,073 seeds per hectare. Beans and corn were planted using a John Deere Maxemerge 7300, 4-row, 30-inch (76.2 cm) vacuum planter. Annual ryegrass was seeded at the rate of 22 kg ha-1 (Hannaway et al., 1999) and wheat seeded at 44 kg ha-1 (Kirkland et al., 2000) using a Tye double disc drill on 8 inches (20.3 cm) spacing. All planting was done on June 19 in 2009 and June 1 in 2010. Due to a mechanical failure on the planter used for this study in 2009, pinto beans failed to germinate and had to be reseeded on June 30. Although application of ‘Treflan’ (trifluralin) herbicide pre-emergence was effective in season-long weed control in 2009, hand –weeding had to be carried out mid-season in 2010 as weed control by ‘Treflan’ did not last throughout the season. Prevalent weeds were Common lambsquarters, Redroot pigweed, Hairy nightshade, and Green foxtail. ‘Select’ herbicide was applied on all intercropped plots one month after planting.

Soil samples were collected from each plot at four different dates for soil analysis using soil sampling and analysis methods described in section 1.1 above. Soil samples for phopholipid fatty acid analysis were collected from plots containing the following treatments:
1. ‘T-39’ black beans intercropped with ‘Gulf’ annual ryegrass
2. ‘T-39’ black beans intercropped with spring wheat
3. ‘T-39’ black beans intercropped with ‘Russel’ oats
4. Monoculture of ‘T-39’ black beans.
These samples were cooled in a cool box filled with ice and transported to the UW greenhouse where it was frozen prior to analysis. Phospholipids were extracted from soil in chloroform phase by sonicating and shaking in a buffered solution for two hours. Phospholipids were separated from soil material and other lipids through a series of centrifugations, chloroform rinses, and chromatography in a solid phase extraction column. Phospholipids isolated from these steps were methylated under mild alkali methanolysis and purified with chromatography in an amino pyralid column. All fatty acid methyl esters were analyzed for relative abundance on a gas chromatograph and final abundance was calculated based on chromatography response of an internal standard added to the chromatography solvent.

Bean leaf samples were collected mid-season and at maturity, dried at 60 degrees centigrade for 72 hours, and then shipped to Ward Laboratories, Inc. Kearney, Nebraska for analysis. Bean sampling and analysis methods used were similar to those described in section 1.1 above.

Two to three of the youngest fully expanded leaves from an average of 3 consecutive plants from the two middle rows of each plot were measured for chlorophyll content using a SPAD-503Plus chlorophyll meter at two different dates. Three meters of row from each plot was harvested in September and yield was determined in the field. Data was analysed using the Split-plot-in-space and Split-plot-in-time procedure of the analysis of variance in SAS. Treatment differences for soil and tissue nutrient concentration as well as chlorophyll content were determined using Fisher’s protected LSD (alpha= 0.05).

Greenhouse Study

A greenhouse study was established at the UW Plant Sciences greenhouse in 2010 to test the potential of annual ryegrass and soil microorganisms to mitigate iron deficiency in black beans (Figure 3). Soil obtained from the irrigated fields at Sustainable Agriculture Research and Extension Center near Lingle [Haverson and McCook loams (42% loams, 37% silt, 21% clay, 1.4% organic matter, pH 7.9)] were homogenized and split into two sets. One set was sterilized by steam heating under pressure of 6.8 Kg cm-1 at a temperature of 121oC for 4 hours. Each of the two sets of soil was used to fill ten-centimeter pots into which two duplicate studies were established; one using sterilized soil and the other using unsterilized soil. Each study consisted of three treatments replicated three times in a complete randomized design as follows:
1. ‘T-39’ black beans intercropped with ‘Gulf’ annual ryegrass
2. ‘T-39’ black beans intercropped with spring wheat
3. Monoculture of ‘T-39’ black beans

Beans were planted on 7.6 cm centers (40 plants m-2), which was equivalent to recommended field spacing in accordance with Blackshaw et al. (2000). Annual ryegrass was broadcast seeded at the equivalent rate of 22 kg ha-1 (Hannaway et al., 1999) and spring wheat seeded at the rate of 44 kg ha-1. Planting was done on July 29 for the 2009 study and May 23 for the 2010 study. All plants were watered with 0.01M potassium hydroxide solution to maintain soil pH above 8. Intercropped pots were sprayed with ‘Select’ herbicide three weeks after planting. Leaf chlorophyll data was taken on the youngest fully expanded bean leaves using a SPAD-503Plus chlorophyll meter at three different dates. Bean-shoot and root-biomass as well as root-nodule counts were determined two months after planting.

Data was analysed using the split-plot-in-space and split-plot-in-time procedure of the analysis of variance in SAS. Treatment differences for chlorophyll content, bean shoots and root-biomass as well as root-nodule counts were determined using Fisher’s protected LSD (alpha= 0.05).

1.3 Effect of Interplantings of Annual Ryegrass and Nitrate Nitrogen on Iron Deficiency in Black Beans

A growth chamber study was established at the UW Plant Sciences greenhouse in 2010 to examine the effect of nitrate nitrogen and interplantings of annual ryegrass on iron assimilation by dry edible black beans under two temperature regimes. The study consisted of a 2 x 2 factorial design replicated three times in a complete randomized design in each of the two growth chambers (Percival Scientific Inc, Model 130BLLC8; SO#009991-001; Serial Number 9991.01-02.07). One growth chamber was set at day/night temperatures of 25/20oC, relative humidity of 50% and Light/Dark period of 15/13 hours. The low temperature growth chamber was set at day/night temperatures of 21/11oC, relative humidity of 50% and Light/Dark period of 15/13 hours. Ten-centimeter pots were filled with homogenized soil obtained from the irrigated fields at the Sustainable Agriculture Research and Extension Center near Lingle WY. Two nitrogen levels were used in this study: 3.2 grams and 16 grams of calcium nitrate each in 6 kg soil (equivalent to 34 and 150 kg N ha-1) in accordance with Aktas and Edmund (1979). The pH of the soil was raised to about 8.5 by incorporating 12 grams of potassium hydroxide in 6 kg of soil. Potassium hydroxide and calcium nitrate were thoroughly mixed into the soil prior to filling the pots and planting. Treatments consisted of black beans intercropped with annual ryegrass and a monoculture of black beans.

Beans were planted on 7.6 cm centers (40 plants m-2), which was equivalent to recommended field spacing in accordance with Blackshaw et al. (2000). Annual ryegrass was broadcast seeded at the equivalent rate of 22 kg ha-1 (Hannaway et al., 1999) and spring wheat seeded at the rate of 44 kg ha-1. Planting was done on January 9, 2010. All plants were watered with 0.01M potassium hydroxide solution to maintain soil pH above 8. Intercropped pots were sprayed with ‘Select’ herbicide three weeks after planting. Leaf chlorophyll data was taken on the youngest fully expanded bean leaves using a SPAD-503Plus chlorophyll meter at three different dates. Bean shoots and roots biomass, as well as root nodule, counts were determined two months after planting.

Data was analysed using the complete Split-plot-in-space and Split-plot-in-time procedure of the analysis of variance in SAS. Treatment differences for chlorophyll content, bean shoots and roots biomass, as well as root nodule, counts were determined using Fisher’s protected LSD (alpha= 0.05).

2. Effect of Interplantings of Annual Ryegrass on Zinc Deficiency in Pinto Beans in Western Kenya

A field study was established in the summer of 2006 at the farm of Mr. Joseph Kamuto in Sikhendu near Kitale town in Trans Nzoia district, western Kenya (0o54?N, 34o55?W; elevation, 1,738 m above sea level). Soils at the site were very old, highly-weathered and leached ferralsols and acrisols with poor fertility (Sombroek et al., 1982). Mean annual rainfall in the area ranges from 1000–1200 mm (Medvecky et al., 2007). Precipitation is unimodal with an eight–nine month rainy season (March–November) and three–five month dry season (November–March) (Medvecky et al., 2007). Temperature ranges from an average minimum of 11.2 degree centigrade to a maximum of 25 degrees centigrade (Medvecky et al., 2007). The study consisted of 1.5 x 6 meter plots in a randomized complete block design with three replications. Seven treatments were planted in three blocks, including:

1. Intercropped – ‘Nodak’ pinto beans with ‘Gulf’ annual ryegrass (PR)

2. Incorporated – ‘Nodak’ pinto beans with finger millet (PM)

3. Incorporated – ‘Nodak’ pinto beans with spring wheat (PW)

4. Monoculture – ‘Gulf’ annual ryegrass only (R)

5. Monoculture – spring wheat only (W)

6. Monoculture – finger millet only (M)

7. Monoculture – ‘Nodak’ pinto beans only, as the control treatment (P)

All land preparation, planting, weeding and management was done manually by hand (Figure 11). Land preparation and planting were done on May 19. Beans were hand-planted at 60 cm rows and 7.5 cm within the row (Figure 12); finger millet and annual ryegrass were hand-broadcasted in 30 cm rows at 25 kg ha-1, and wheat was hand-broadcasted in 30 cm rows at 45 kg ha-1. Plots were hand-weeded three times; on June 24, July 15 and August 12. Prevalent weeds were Broadleaf woodsorrel (Oxalis latifolia), Quack grass (Agropyron repens), Black jack (Bidens pilosa) and Mexican Marigold (Tagetus minuta). Mosquito nets were used to cover all plots on May 27 to control bird damage of the beans (Figure 13 and 14).

Soil samples were collected from each plot on April 26, June 26 and August 19. Each soil sample consisted of five soil cores randomly collected from each plot to 15.24 cm depth using a 2.54 cm soil probe. Cores from each ploy were thoroughly mixed together into a composite sample, air dried and then shipped to UW soils lab for analysis. At the UW soils lab, soil samples were ground and analyzed using the same methods described in section 1.1.

Bean leaf samples were randomly collected from each plot on June 6 and August 19, air-dried and shipped to UW Soils lab for tissue analysis. Tissue sampling and analysis were also done using methods described in section 1.1 of this report.

Data was analysed using the PROC MIX procedure of the analysis of variance in SAS version 9.2 (SAS Institute, 2008). Treatment differences for soil and tissue iron and zinc concentration were determined using Fisher’s protected LSD (alpha= 0.05). Soil and tissue nutrients were compared with soil organic matter using the Pearson Correlation method of SAS version 9.2 (SAS Institute, 2008). Means of soil zinc concentration was regressed against soil organic matter using a simple linear regression analysis in SAS (PROC REG) version 9.2 (SAS Institute, 2008).

Research results and discussion:
1.1 Effect of Interplantings of Annual Ryegrass, High Bean Plant Density, and Temperature on Iron Deficiency in Pinto Beans

2008 Field Study Results and Discussion

There were no differences between treatments for yield for the 2008 study. Hand-weeding of the study was ineffective in eliminating persistent weeds. This may have affected the yields obtained for dry beans.

There were no differences for soil and tissue iron and zinc between treatments. Though iron chlorosis symptoms were observed in pinto bean plants during the first month after planting, the beans were generally able to overcome these symptoms as the season progressed.

Although pinto beans in 2008 study did not exhibit sustained iron deficiency chlorosis symptoms, soy beans and black beans planted on adjacent plots by another researcher were observed to be more adversely affected by iron deficiency chlorosis that could potentially reduce their yields. This suggested that different dry bean cultivars have different tolerances to micronutrient deficiency. A study planned for 2010 would attempt to determine the potential role of annual ryegrass to mitigate iron deficiency in more susceptible dry bean cultivars.

There was a significant increase in mean iron concentration (p<0.0001) with respect to sampling time. Iron concentration was significantly higher in all plots during the last two sampling dates in August compared to the earlier sampling dates in June and July (Table 1). Since there was no interaction between treatments and sampling dates, a seasonal variable may have been responsible for the increased iron concentration. Gradual increase in mean temperature between May and August (Table 2) may have been responsible for these results. The mechanism influenced by temperature that increased iron availability over time was not determined by this study. We hypothesize that increasing temperature as the season progressed may have increased microbial activity and root interactions which may have processed more soil iron through means such as siderophore exudation and chelation.

There was a significant decrease in mean nitrate nitrogen (p<0.0001) with time (Table 3). Nitrate loses through the season can be expected from utilization by plants, volatilization and leaching though irrigation and precipitation. Reduction in nitrate nitrogen in the soil may partly explain the observed recovery of some cultivars of dry beans from the initial iron deficiency symptoms exhibited early in the season (late spring to early summer). High levels of nitrate nitrogen in soils that already have high levels of salt and free calcium carbonate are thought to interfere with iron metabolism in plant leaves, depress chlorophyll synthesis and induce iron deficiency chlorosis (Christensen and Johnson, 2008).

2009 Field Study Results and Discussion

There were no differences for soil and tissue iron and zinc concentration between treatments. Just as in the 2008 study, iron chlorosis symptoms observed in the pinto bean plants during the first month after planting disappeared as the season progressed. Unlike 2008, however, increase of soil iron concentration with time in all treatments was only marginal in 2009 (p=0.07). Unexpected heavy and prolonged precipitation in 2009 may have contributed to a reduction in environmental and soil temperature (Table 2), resulting in lower soil iron concentration compared to the previous year. Different sampling dates, due to delay in planting and failure of pinto beans to germinate, may also have failed to capture the time period when iron concentration was expected to rise.

Pre-emergent application of ‘Treflan’ herbicide resulted in effective season-long control of the prevalent weeds observed in the previous study. High density pinto beans produced at least twice higher yields (p<0.0001) than all the other treatments (Table 4). The effect of high density beans on soil and tissue iron availability, however, could not be ascertained from this study. Pinto beans intercropped with wheat produced the lowest yields (p<0.0001), suggesting that wheat out-competed dry beans for nutrients and light and may therefore not be a suitable crop to interplant dry beans with. There were no differences in yield between the lower density beans, beans intercropped with annual ryegrass and beans intercropped with annual ryegrass sprayed later with select herbicide, suggesting that the slower-growing annual ryegrass exerted less competition for nutrients and light on dry beans compared to wheat. Annual ryegrass may therefore be a better companion for dry beans when the two crops are intercropped. The specific role played by annual ryegrass in increasing iron availability was not established by this study. Subsequent studies utilizing less tolerant dry bean market classes intercropped with annual ryegrass will attempt to address this need.

Growth Chamber Study Results and Discussion

In 2009 study, bean seedlings developed long internodes and bolted toward fluorescent light tubes in the chamber, suggesting that light intensity was insufficient for those plants. Both chambers were designed for smaller statured plants and organisms. Adjusting the day-light period from 13 to 15 hours in subsequent study shortened bean seedling internodes and reduced the tendency of the plants to bolt toward the light source. Bean plants in the warmer growth chamber were larger than plants in the cool growth chamber (Figure 1).

There were no differences between treatments within each growth chamber for above and below ground biomass, as well as root nodule counts. However, temperature had a significant effect on the shoot biomass (Table 5). Bean plants in the high temperature growth chamber had significantly higher shoot biomass (p = 0.0004) than bean plants in the low temperature growth chamber. The youngest fully expanded leaves of the plants in the high temperature chamber had higher chlorophyll content (p<0.0001) than plants in the low temperature chamber. These results suggest that temperature increases plant nutrient availability and uptake and are consistent with our field study results. Just as in the field study, however, this study did not determine the actual mechanism influenced by high temperature that increased iron availability and prevented development of chlorosis symptoms.

Intercropped bean plants had significantly higher chlorophyll content than monoculture bean plants in the high temperature chamber (p<0.0001). There was no significant difference between treatments for chlorophyll content in the low temperature chamber. These results are, however, not consistent with our 2008-2009 field study results, partly because we used a bean cultivar (‘T-39’ Black bean) thought to be more susceptible to iron-deficiency in our growth chamber study, compared to the more tolerant pinto beans used for the field study.

Results from our field and growth chamber studies suggest that seasonal temperature changes can influence soil iron availability for plant uptake. Further studies are needed, however, to determine the specific mechanisms in the soil that change iron availability when temperatures change. Results from these studies also suggest that annual ryegrass has the potential to mitigate iron deficiency chlorosis in more susceptible dry bean cultivars. Additional studies to investigate this potential for three dry bean cultivars with varying susceptibility to iron deficiency were carried out in 2009 and repeated in 2010. Our results did not ascertain the potential of high dry bean plant density to alleviate iron deficiency.

1.2 Results: Effect of Interplantings of Annual Ryegrass and Soil Microorganisms on Iron Deficiency in Susceptible Dry Edible Bean Cultivars

2009 Field Study: Soil and Tissue Nutrient analysis

The study found significant differences between treatments for chlorophyll content. Navy and black beans had significantly less chlorophyll content (and therefore more chlorotic) compared to pinto beans (p < 0.0001). Grass intercropped bean plants had significantly higher chlorophyll content (p < 0.0001) than bean monocultures (Figure 4; Table 6).

There were significant differences between treatments for bean tissue iron concentration (p<0.0001). While there were no differences for tissue iron between Navy bean monoculture and navy-ryegrass intercropped treatments, these two treatments had the highest mean iron concentration in their tissues compared to all the other treatments (Table 6). Similarly, although there were no differences for tissue iron concentration between black bean monocultures and black beans interplanted with corn and oats, these three treatments had significantly higher mean iron concentration in bean tissues than black beans interplanted with wheat. Paradoxically, bean plants in treatments with less chlorophyll content accumulated more iron and nitrate-nitrogen (p = 0.003) in their tissues than less chlorotic beans (Table 6). All intercropped treatments had less mean nitrate-nitrogen in their tissues (p<0.0001) than all bean monoculture treatments (Table 6).

The study found significant differences between treatments for bean tissue manganese concentration (p=0.0006). Whereas there were no differences for tissue manganese between navy bean and black beans monocultures, these two treatments had the highest mean manganese concentration in their tissues compared to all the other treatments (Table 1). Apart from bean-corn intercrop, which had higher manganese concentration than bean-grass intercropped treatments with oats and wheat, all the other intercropped treatments were not significantly different for tissue manganese concentration (Table 1). There were no differences between treatments for soil manganese concentration.

There was a significant decline in tissue iron concentration (p=0.0026) and, at the same time, a significant increase in tissue zinc concentration (p = 0.0031) between the first and second sampling date for all treatments, though there were no differences between treatments for bean tissue zinc concentration (Figure 5 and 6). These results were reversed in soil zinc and soil iron concentration, (Figure 7) whereby there was a significant increase in soil iron for all treatments between the first and second sampling dates (p=0.02) and a significant decline in soil zinc concentration for all treatments between the first and second sampling date (p=0.042), though there were no differences between treatments for soil iron and soil zinc concentration.

All three monocropped dry bean cultivars produced higher yields (p = 0.0009) than all the intercropped beans.

Phopholipid Fatty Acid Analysis

The results of the Gas Chromatographic PLFA assay of soil samples obtained from four representative treatment plots found no significant differences between treatments. However, there was a general trend showing higher total bacterial, fungal and protozoan abundance in intercropped treatments (Figure 8).

Greenhouse Study Results

The study carried out between July 29 and September 5 found no differences between treatments for chlorophyll content. Bean plants grown in unsterilized soil, however, exhibited a significant increase in chlorophyll content (p=0.024) between the first and second sampling date. The reverse was the case in bean plants grown in sterilized soil, whereby there was a marginal decrease in chlorophyll content (p=0.06) between the first and second sampling date (Figure 9 and 10; Table 7).

When the study was repeated between September 4 and October 18, treatment had highly significant effects (p=0.009) on chlorophyll content for bean plants in sterilized soil (Figure 11; Table 8). Whereas there was no difference in chlorophyll content in bean plants between bean-wheat and bean-annual ryegrass intercropped treatments, both treatments had significantly higher chlorophyll content than bean monoculture treatment. There were also statistical differences between sampling dates (p=0.002), whereby the highest chlorophyll content was recorded on the first sampling date, September 26, and the lowest chlorophyll content recorded on the last sampling date, October 18 (Table 7).

Whereas there were no statistical differences between sampling dates for beans planted in unsterilized soil during this study period, bean plants in intercropped treatments had significantly higher chlorophyll content (p=0.026) than bean plants in monoculture treatment in the September 4-October 18 study (Table 8).

The study found significant differences in root nodule counts between the two soil media during the first study period (July 29-September 5). Bean root nodule counts were significantly higher (p=0.004) in unsterilized soil compared with bean plants in sterilized soil, with the highest counts in bean-annual ryegrass and bean-wheat intercropped treatments (Table 9). There were also significant differences in total above- and below-ground bean dry matter biomass weights between treatments across the two soil media (p=0.04). Bean monoculture and bean intercropped with wheat in the sterile soil media had the highest dry matter biomass compared to the other treatments (Table 9).

1.2 Discussion

Field Study

Although black and navy bean monocropped treatments in the field study exhibited significantly more chlorosis than the intercropped treatments, the bean monoculture treatments had more tissue iron and zinc concentration, and ultimately higher yields than the intercropped plots. Bean plants with higher iron concentration in their tissues exhibiting less chlorophyll content may be explained by a phenomenon referred to as the “chlorosis paradox” described by Abadía (1992); Marschner (1995); and Morales et al. (1998). These inconsistencies may be attributed to the localization and binding state of iron in leaves (Marschner, 1995), whereby some of the iron may precipitate in the apoplasm of leaves and become less available physiologically (Mengel and Geurtzen, 1988; Marschner, 1995). These results are consistent with similar findings by Omondi et al. (2010), who observed that bean plants exhibiting iron deficiency chlorosis symptoms had more iron concentration in their tissues than beans that were less chlorotic.

Minimum soil Zn concentration before Zn chlorosis symptoms can occur is 0.5 ppm (Jacobsen et al., 2003). On the other hand, the deficiency range of soil iron concentration is 2.5 – 5 ppm (Jacobsen et al., 2003). Soil zinc concentration from the field study (2ppm) was far above the threshold found by Jacobsen et al. (2003) to cause zinc chlorosis. Average soil iron concentration on the other hand was 5 ppm which was within the range of 2.5 – 5ppm found by Jacobsen at al. (2003) as capable of causing iron chlorosis. These results therefore suggest that the chlorosis symptoms observed were caused by soil iron deficiency rather than zinc deficiency.

While soil iron concentrations from this study were low according to Jacobsen et al., (2003), they were generally above the critical concentration level of 5 ppm before the performance of dry beans can be seriously impacted by iron deficiency. This suggests that the performance of dry beans was influenced by competition from grass intercrops more than micronutrient deficiency. ‘Select’ herbicide is applied to remove grass intercrops from dry beans one month after planting. This allows for adequate root interaction between the two species to enable any benefits expected from the grass to accrue. However, given that it takes about two weeks for ‘Select’ herbicide to kill its target plant, competitive effects from the grass intercrops can offset any benefits that may have been obtained from intercropping. There is therefore a need to carry out further studies to determine the optimum time of grass removal that can mitigate yield losses due to grass competition.

An interesting result of this study was whereby tissue iron concentration declined at the same time as tissue zinc concentration increased between sampling dates. Curiously, the situation was reversed with regard to the soil micronutrients, whereby soil zinc concentration declined in apparent opposition to soil iron concentration, which increased as the season progressed. These results may be explained by studies by Ambler et al. (1970) which found that soil zinc interfered with the translocation of iron in soy beans by inhibiting the capacity of the root conversion of ferric to ferrous iron or by accentuating other reactions detrimental to iron transport. Both nutrient supply and nutrient balance are important considerations in plant nutrition, as the concentration of one nutrient in the soil will often affect the uptake or transport of another nutrient within the plant (Biertman and Rosen, 2005). Nutrient interactions not only involve the relationship between nutrient supply in the soil and plant growth, but also nutrient concentrations in plant tissue and plant growth (Biertman and Rosen, 2005). According to Biertman and Rosen (2005), “the precise nature of nutrient interactions depends on the nutrients involved and can vary for different plant species. Furthermore, the actual mechanism for the interaction may not be completely understood.” Concentration of soil zinc in our study was relatively low according to Jacobsen et al., (2003) and may, therefore, have not substantially affected performance of beans in accordance with Ambler et al. (1970). Higher zinc concentration may benefit from a polycultural cropping design such as this experiment, which would enable more utilization of the micronutrient and alleviate its potential interference with iron uptake.

Our study found significantly higher bean tissue manganese concentration in black and navy bean monocultures compared to the intercropped treatments. Mean manganese concentration was 117 ppm and 123 ppm in black beans and navy beans respectively. Studies by Fageria (2001) to determine adequate and toxic levels of copper and manganese in upland rice, common beans, corn and soybeans found that manganese concentration of 128 ppm caused toxicity symptoms in common beans. Mean tissue manganese concentration for black and navy beans in our study were close to the threshold established by Fageria (2001) as capable of influencing manganese toxicity and/or inducing iron deficiency chlorosis. Sideris and Young (1949) suggested that heavy metals such as manganese with similar ionic radii, valency and electronic configuration as iron can compete with iron for reaction sites that lead to chlorophyll formation. Similar results were reported by Epstein (1972), Marschner (1986) and Mengel and Kirkby (1987). Manganese, rather than iron, can react with porphyrin compounds, thereby inactivating them for subsequent conversion to chlorophyll (Sideris and Young, 1949). Studies by Twyman (1951) to determine the relationship between iron and manganese in the tissue and growth media on iron deficiency chlorosis found that high manganese concentration under adequate iron bioavailability caused chlorosis in various plants grown in nutrient medium, the intensity of which was correlated with manganese concentration in the medium. This chlorosis was attributed to competition between iron and manganese for active iron acceptors, antagonism between iron and manganese, whereby manganese slowed the entry of iron and encouraged formation on inactive iron receptors and a direct toxic action of manganese on processes involved in iron metabolism (Twyman, 1951). Although there were no differences between treatments for soil manganese concentration in our study, mean soil manganese concentration was 6.39 ppm for navy beans and 7.19 ppm for black beans. Fageria (2001) determined toxic concentration of soil manganese to be 6 ppm for dry beans. Results from our study, therefore, suggest that high levels of manganese were present in the soil, and that grass intercrops prevented excessive uptake and accumulation of the micronutrient in the bean tissues, thereby alleviating induction of iron deficiency chlorosis.

Findings by this study that all intercropped treatments had less mean nitrate-nitrogen in their tissues than all the bean monoculture treatments suggest that high nitrate-nitrogen in the monoculture treatments may have played a role in causing iron deficiency chlorosis in those treatments. These results are consistent with findings by Christensen and Johnson (2008) that high levels of nitrate-nitrogen can interfere with iron metabolism in the plant leaves, depress chlorophyll synthesis and induce iron deficiency chlorosis. Kosegarten et al. (1999) hypothesized that nitrate nutrition might induce iron deficiency chlorosis by inactivation of iron in the leaf apoplast. Intercropping dry beans with annual ryegrass as a means to reduce nitrate-nitrogen in the soil and bean tissues may therefore provide a potential solution to iron deficiency chlorosis, induced by high nitrate-nitrogen, in dry beans.

Annual ryegrass performance in the field study was comparable to those of oats and corn with regard to mitigating iron deficiency chlorosis. Navy and black beans intercropped with these three grasses were less chlorotic and had higher tissue iron concentration compared to treatments with spring wheat. Oats and corn have been found to have the ability to mitigate Fe deficiency chlorosis through root exudates of phytosiderophores which can chelate iron and make it more available (Romheld and Marschner, 1990; Singh et al., 2005). These results suggest that annual ryegrass is a potentially suitable grass to include in a dry bean intercropping management program designed to combat iron deficiency.

Greenhouse Study

Results from the greenhouse study found a decrease in chlorophyll content for bean plants growing in sterilized soil and a corresponding increase in chlorophyll content for beans grown in unsterilized soil. Increased chlorophyll content in bean plants grown in unsterilized soil suggest that, with time, greenhouse conditions (adequate temperature, light, moisture and air) may have helped to increase microbial activity in the soil, some of which may have produced siderophores that chelated iron and made it more available for plant uptake. Increased root nodule counts in this medium confirms that microbial action was at play, including biological dinitrogen fixation by root-nodule-forming bacteria of the genus rhizobia that exist in symbiotic association with leguminous plants. Considered as one of the most limiting nutrients to plants in terrestrial ecosystems (Sylvia et al., 2005), nitrogen is used by plants to synthesize amino acids, the primary components of proteins, and is also required by plants for other vital components such as chlorophyll and enzymes (Ludwick et al., 2002). Biological dinitrogen fixation is catalyzed by the nitrogenase enzyme system which consists of two metalloproteins, the iron (Fe-) protein and the molybdenum-iron (MoFe-) protein (Kim and Rees, 1994). The legume–rhizobia symbiosis is particularly sensitive to iron deficiency, given its critical role as a vital structural and functional component of the nitrogenase enzyme (Tang et al., 1991).

Significantly lower root nodule counts on bean plants grown in autoclaved soil is attributed to the high temperatures involved in autoclaving soil, which may have destroyed microorganisms in the soil, including rhizobia. High initial chlorophyll content in these bean plants may therefore be a result of resident mineral nitrogen in the soil and an increase in available micronutrients such as iron due to the high autoclaving temperatures. In a study by Abou-Shanab et al. (2003), autoclaving increased extractable Fe, Mn and Co but had no significant effect on other elements examined. Reduction of chlorophyll content in bean plants in autoclaved soil in our study, as the season progressed, may have been due to depletion of nitrogen, iron and other nutrients in the soil as the plant developed. However, interveinal chlorosis that developed in these plants could also have been caused by manganese toxicity. Studies by Boyd (1971) found that interveinal leaf chlorosis occurring in Argentine peanuts growing in autoclaved soil was the result of manganese toxicity. The toxic level of manganese was attributed to direct release of manganese complexed with organic fraction of the soil and the killing of microorganisms that normally transform available manganese into higher, less available and therefore less toxic oxides (Boyd, 1971). As has already been described elsewhere in this paper, high manganese concentration can induce iron deficiency chlorosis by competing with iron for reaction sites that lead to chlorophyll formation (Hewitt, 1948; Sideris and Young, 1949; Twyman, 1951; Epstein, 1972; Marschner, 1986; and Mengel and Kirkby, 1987). Our greenhouse study finding that bean plants intercropped with annual ryegrass and wheat growing in autoclaved soil had higher chlorophyll content than monoculture bean plants in the same medium are therefore consistent with our field studies that grass intercrops prevented excessive uptake and accumulation of manganese in bean tissues, thereby alleviating induction of iron deficiency chlorosis.

PLFA Analysis

Phospholipid fatty acid analysis (PLFA) provides a means to study in-situ microbial communities without the usual problems associated with their isolation or removal of cells from the environment (Petersen and Klug, 1994). This method examines the entire microbial community, thus providing a quantitative description of the structure and function of the micro-flora within a particular environment (Sinsabaough et al., 1999). PLFA characterizes the composition of microbial biomass by identifying extracted cellular phospholipid ester-linked fatty acids (Zelles et al., 1992). Microbial communities under different agricultural management profiles can also be distinguished using a simplified extraction of cellular fatty acid methyl esters (Cavigelli et al., 1995). The assay is based on the fact that all organisms except archaea have membranes made of phospholipids. Many different fatty acids exist in microbial lipids and can be used to identify microorganisms (Sylvia et al., 2005). Phospholipid fatty acids are an integral part of cell membrane and are rapidly metabolized when the cell dies in the soil and can therefore provide an accurate measurement of living organisms (Sylvia et al., 2005). PLFA assay involves extraction of lipids from the soil into a mixture of water, chloroform and methanol, followed by separation of different classes of lipids (Sylvia et al 2005). The PLFA fraction is then analyzed after hydrolysis and methylation under alkaline conditions. The PFLAs are extracted with organic solvents and analyzed by Gas Chromatography. Typically 20 to 50 fatty acids can be detected and differentiated (Sylvia et al 2005). Statistical analysis of fatty acid composition can enable identification of specific fatty acids that distinguish between microbial communities in different soils or in specific soils under different management conditions (Sylvia et al 2005). PFLA assays can therefore tell us which kinds of microorganisms are present and their relative amounts and diversity in the soil.

Although the Gas Chromatographic PLFA assay analysis of soil samples found no significant differences between treatments, there was a general trend showing higher total bacterial, fungal and protozoan abundance in intercropped treatments, which suggests that iron-chelating microorganisms may have interacted with grass root exudates or organic matter in these treatments to increase iron availability resulting in higher chlorophyll content. The exact nature that such interactions may occur was not established by this study and should be a subject for further investigation.

While results from our study showed that annual ryegrass can mitigate iron deficiency chlorosis in dry beans, grass intercropping did not result in higher dry bean grain or biomass yields in both field and greenhouse studies. Additional studies are required to determine the appropriate annual ryegrass and dry bean densities, as well as the optimum time of removal of annual ryegrass from the intercropped treatment that can not only alleviate iron deficiency chlorosis symptoms but also increase dry bean yields.

1.3 Results: Effect of Interplantings of Annual Ryegrass and Nitrate Nitrogen on Iron Deficiency in Black Beans

The study found significant differences between treatments for chlorophyll content. Bean-annual ryegrass intercropped treatments had significantly higher chlorophyll content (p=0.004) than bean monoculture, regardless of the nitrate-nitrogen treatment or growth chamber temperature (Table 10). Treatments with low soil nitrate-nitrogen had significantly higher (p=036) chlorophyll content compared with treatments with high soil nitrate-nitrogen (Table 2). Bean monoculture in high nitrate-nitrogen treatment in the low temperature chamber had the lowest chlorophyll content.

Treatments with low soil nitrate-nitrogen had significantly higher (p=024) root nodule counts compared with treatments with high soil nitrate-nitrogen, regardless of growth chamber temperature (Table 10). Treatments in the high temperature growth chamber in low nitrate-nitrogen treated soil media had significantly higher (p=0.018) root nodule counts than all the other treatments (Table 11). In this case, however, bean monoculture in high nitrate-nitrogen treatment in the high temperature chamber had the lowest root nodule count.

There were no differences between treatments for root fresh weight or dry weight biomass. However, temperature had a marginal effect (p=0.085) on above ground fresh weight biomass, with mean fresh weight of 10.6g per bean plant in the high temperature growth chamber and 6.0g per bean plant in the low temperature growth chamber. Temperature also had a significant interaction with nitrate-nitrogen treatment for above ground fresh biomass (p=0.018). Mean fresh weight of bean plants in low nitrate-nitrogen treatment was significantly higher (p=0.007) than mean fresh weight of bean plants in high nitrate-nitrogen treatment (Table 10) even though there were no differences in fresh weight between monoculture and intercropped bean plants in either low or high nitrate-nitrogen treatment. The study did not find any differences between above ground bean dry biomass as well as total above and below ground bean dry biomass. However, total above and below ground bean dry biomass showed a similar trend to the above ground fresh biomass weights.

1.3 Discussion

Findings by this study that bean plants growing in annual ryegrass-intercropped treatments had higher chlorophyll content than monoculture bean plants are in agreement with field and greenhouse studies by Omondi et.al. (Unpublished) as well as field studies by the same author (Omondi et al., 2010), which showed that interplantings of annual ryegrass alleviated chlorosis symptoms in dry edible beans. These studies suggest that annual ryegrass may be an iron efficient plant that can utilize Strategy II mechanism, whereby root exudates containing phytosiderophores can be secreted into the rhizosphere root zone, thereby chelating iron and making it more bio-available for uptake by both the grass and dry beans growing in close proximity (Marschner and Romheld, 1994; Marschner, 1995; Vansuyt et al., 2000). However, there is still a need to conduct HPLC analyses to confirm the ability of annual ryegrass to excrete phytosiderophores in nutrient medium.

Although high temperature improved the performance of bean plants in all treatments, the study found that high nitrate-nitrogen reduced chlorophyll content, root nodule counts and above ground fresh biomass of bean plants regardless of the temperature treatment. These results not only suggest that high nitrogen induces iron deficiency, but also that black beans are iron inefficient plants which can secrete OH- and HCO- ions into the rhizosphere root zone in the presence of high nitrate-nitrogen (Aktas and Egmond, 1979; Romheld et al., 1984). These results are in agreement with Smolders et al. (1997), who found that increased nitrate assimilation by sharpflower rush (Juncus acuti?orus) led to increased apoplastic pH and to simultaneous immobilisation of iron and/or lower Fe3+ reduction. These results are also in conformity with studies by Aktas and Egmond (1979), which showed that high nitrate-nitrogen decreased dry matter production, caused ionic imbalances and reduced chlorophyll content of iron inefficient soybean cultivar ‘T-203’.

2. Results: Effect of Interplantings of Annual Ryegrass on Zinc Deficiency in Pinto Beans in Western Kenya

The study found significant differences in soil zinc concentration between treatments. There was significantly higher (p = 0.042) zinc concentration in pinto beans-annual ryegrass intercropped plots compared to pinto beans monoculture (Table 12). There was also significantly higher (p<0.001) organic matter in beans-ryegrass intercropped plots compared to the other treatments (Table 12). A linear regression analysis of the means of soil zinc concentration against soil organic matter content found a significant correlation (p = 0.49; r2 = 0.33) between soil zinc concentration and soil organic matter (Figure 17).

Sampling date had a significant effect on zinc availability on all plots (p = 0.05). There was a significant increase in zinc concentration between the first and second sampling date, with the highest increase in the bean-annual ryegrass plots (Table 13, Figure 16). Peak soil zinc concentration was observed on June 26 (Figure 16).

There were no differences between treatments for soil iron, soil pH and soil nitrate-nitrogen. There were also no differences between treatments for tissue iron, tissue zinc and tissue nitrate-nitrogen.

2. Discussion

Findings by this study that there was significantly higher soil zinc concentration in the bean-annual ryegrass intercropped treatment compared with the monoculture treatment suggest that intercropping annual ryegrass with beans has the potential to mitigate zinc deficiency in the highly weathered acidic soils in western Kenya. Corresponding high organic matter content in the bean-annual ryegrass intercropped treatment compared to the other treatments (Table 12) suggests that organic matter contributed to the increase in soil zinc concentration. A linear regression analysis of the means of soil zinc concentration against soil organic matter content found that organic matter content explained 33% of the variability observed in soil zinc concentration between pinto-beans/annual ryegrass intercropped plots and pinto beans monoculture (Figure 17). However, the relatively low r2 value of this analysis discounts the possibility that soil organic matter by itself contributed to the increased soil zinc content in the intercropped plots.

Higher soil organic matter in the bean-annual ryegrass intercropped plots in this study suggests that the extensive rooting system of annual ryegrass may have interacted with the rooting system of pinto beans resulting in increased below ground biomass. Soil organic matter increases the buffering capacity of soil and plays an important role in nutrient availability and soil aggregate stability (Hussain et al., 1999). Soil organic matter can undergo partial decomposition and give rise to organic acids and other breakdown products such as carbon dioxide and water, which combine to form soluble micronutrient-organic complexes (Chen and Hadar, 1991). Soil organic matter provides many beneficial biological, chemical and physical properties to the soil. With regard to biological properties, soil organic matter provides slow-release carbon and energy source which can support a large, diverse and metabolically active microbial community (Sylvia et al., 2005). Soil organic matter is also a source of certain compounds that can promote plant growth (Sylvia et al. 2005). Chemically, soil organic matter increases the cation exchange capacity, buffering capacity, provides a slow-release supply of organically bound nutrients such as nitrogen, phosphorus, and sulfur, and enhances chelation thus increases bioavailability of micronutrients to plants (Hussain et al., 1999, Sylvia et al. 2005%2

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

Publications emerging from this project include the following:

• Omondi, E. C. (Unpublished). The potential of managing iron and zinc deficiency in dry beans with interplantings of annual ryegrass. Dissertation to be submitted as partial fulfillment for a Doctorate of Science in Agronomy at the University of Wyoming in spring 2011.

• Omondi, E. C., J. Kamuto, A. Kniss, and R. Smith. (Unpublished). The Potential of Managing Zinc Deficiency in Dry Beans with Interplantings of Annual Ryegrass in Western Kenya. To be submitted to the African Journal of Plant Science.

• Omondi, E. C., M. Ridenour, C. Ridenour, and R. Smith. 2010. The Effect of Intercropping Annual Ryegrass with Pinto Beans in Mitigating Iron Deficiency in Calcareous Soils. Journal of Sustainable Agriculture 34: 3, 244 — 257.

• Omondi, E. C. 2008. The Effect of Intercropping Annual Ryegrass with Pinto Beans in Mitigating Iron Deficiency in Calcareous Soils. Abstract in 89th Annual Meeting of the Pacific Division of the American Association for the Advancement of Science. Western Society of Crop Science, Hawaii, U.S.A. (Figure 23).

• Omondi, E. C. 2007. The Effect of Intercropping Annual Ryegrass with Pinto Beans in Mitigating Iron Deficiency in Calcareous Soils. Thesis submitted as partial fulfillment for a Master of Science in Agronomy at the University of Wyoming in December 2007.

• Omondi, E. C. 2007. The potential of managing iron and zinc deficiency in dry beans with interplantings of annual ryegrass. Abstract In ASA-CSSA-SSSA International Annual Meetings, New Orleans, U.S.A (Figure 22).

In addition to the above publications, I have made a presentation of this project at the UW SAREC annual field day near Lingle twice every year for the last four years. Over 100 farmers and researchers from the state participate in these field days each year (Figure 18-21).

Project Outcomes

Project outcomes:

Our study results show that mechanisms leading to iron deficiency chlorosis are complex and varied. Iron deficiency is a global problem in crop production and can be caused by any factor that interferes with its absorption and translocation or impairs its utilization in metabolic processes (Brown, 1961; Welch et al., 1991). Several chemical and biological mechanisms are involved through various antagonistic and complementary interactions. Information published in existing literature is obscure on exactly how these interactions occur and may therefore be an area of further scientific investigation. However, our study results have consistently shown that, regardless of the known and hypothesized mechanisms involved in inducing deficiency, annual ryegass intercropped with dry beans has the potential to alleviate iron deficiency chlorosis in dry beans in calcareous, alkaline soil and zinc deficiency chlorosis in dry beans in acidic soil.

Conventional control of iron deficiency in dry beans is achieved by multiple foliar applications of 1% iron sulfate solution applied at 20-30 gallons per acre, or similar applications of more expensive iron chelates at approximately half the rate of iron sulfate (Stevens and Belden, 2005). A non-chemical cultural practice, such as this project hopes to recommend, would be a welcome alternative for organic and natural bean producers and would also provide a more sustainable and potentially more affordable solution for conventional bean growers. It also provides an opportunity for more conventional farmers to adopt sustainable and/or organic farming.

Conventional control of zinc deficiency in dry beans is achieved by multiple foliar applications of 0.5-1.5% zinc EDTA applied at 20-30 gallons per acre, or similar applications of more expensive zinc chelates at approximately half the rate of zinc sulfate (Stevens and Belden, 2005). Recommendations from this project will be especially useful to Kenyan farmers, as it not only provides them with an alternative to expensive chemicals, but it fits in with their small scale hand-labor farming routine. The study has the potential to both save on the costs of farming, as well as increase yields.

This study will provide useful information to growers on dry bean cultivar tolerances and susceptibility to micronutrient deficiency and also determine the effectiveness of grass intercrops in mitigating such deficiencies. Determining the role of nitrate-nitrogen and temperature in influencing micronutrient deficiency will also be very useful in helping growers to accurately determine the causes of observed deficiency symptoms in their crops and to save on expenses by taking appropriate remedial actions, for example, timing of planting and optimum use of nitrogen fertilizers.

Economic Analysis

This study did not have an economic analysis component built into it. In addition, our results did not establish the grain yield advantage of using annual ryegrass as an intercrop to control iron and zinc deficiency in dry beans. Further studies to determine optimum plant densities and/or time of removal of annual ryegrass to eliminate yield losses due to competition are recommended. However, use of annual ryegrass as a cultural means to alleviate micronutrient deficiency has the distinct potential to reduce farming costs while at the same time minimizing negative environmental impacts due to use of chemical alternatives. Examples of how this can be accomplished are enumerated below:

*According to Stevens and Belden (2005), multiple foliar applications of 1% iron sulfate solution applied at 187–280 L ha-1 (20-30 gallons per acre) are required to control iron chlorosis, whereby application frequency depends on severity of deficiency. One application would cost about $62 ha-1 ($25 per acre) (Wortmann et al., 2008). According to Hearne Seeds and Seedland, it would cost on average $74 ha-1 ($30 per acre) to drill-seed annual ryegrass at the 22 kg ha-1 rate used in this study. An additional $24 ha-1 ($10 per acre) would be needed if annual ryegrass is removed after one month using ‘Select’ herbicide (Bernards et al., 2009), adding up to $98 ha-1 ($40 per acre) as the total cost of using annual ryegrass to control iron deficiency in dry beans. A farmer forced to apply two to three foliar application of iron sulfate to control severe iron deficiency would therefore save $25-$88 ha-1 ($10-$35 per acre) by using annual ryegrass instead.

*Annual ryegrass is a suitable crop to use as a cover crop due to its ability to improve soil tilth through its extensive root system and can therefore provide excellent soil erosion control (Bowman et al., 1998). Annual ryegrass’ ability to suppress other plant species attributed to good ground cover and production of allelochemicals (McKell and Cameron, 1969; Weston, 1996; Bauder, 2000; Liebman and Davis, 2000) provides it with a natural ability to outcompete weeds, reducing the need to cultivate or hand-weed. Season-long growing of annual ryegrass intercropped with dry beans, if optimum plant density can be achieved to minimize grass competition effects, can therefore potentially save on weeding and erosion control costs.

*Extensive rooting system of annual ryegrass can increase organic matter in the soil. Studies by Omondi et al.,(2010) found that annual ryegrass significantly increased soil organic matter when intercropped with pinto beans compared to pinto beans monoculture. According to Oregon Ryegrass Growers Seed Commission, 34–100 kg ha-1 of nitrogen can be recycled back into the soil as nitrogen credit for the following season’s plants through growing annual ryegrass as a cover crop, which is then incorporated into the soil before the next season planting. Increased soil organic matter, as well as nitrogen credits from annual ryegrass acting as N-trap crop, could tremendously save on nitrogen fertilizer costs.

Farmer Adoption

This project is still in its infant stage. Some aspects of it are still ongoing; for example, soil and tissue analysis from the 2010 study have not yet been completed. One greenhouse and one growth chamber study associated with this project are also still ongoing. Efforts to disseminate findings from the study and determine its acceptability and adoption will therefore be pursued after final completion of the study.

Even though some results from this study have been made available to producers at the annual UW SAREC Field Days, the impact of this information and extent to which it has influenced adoption by farmers has not yet been studied and established. Plans are still underway for Manor House Agricultural Centre’s (MHAC’s) Training and Extension program to coordinate the dissemination of research findings to farmers countrywide in Kenya after my graduation and return to the country. Published literature from this study will also be used by MHAC in teaching; both in its two-year certificate course program and its farmer workshops.

Recommendations:

Areas needing additional study

1. There is need to determine potential synergism that may exist between increased organic matter and root exudates produced by annual ryegrass in a grass-bean intercrop, as well as enhanced activities of microorganisms to produce microbial siderophores – all of which result in increased availability of iron, zinc and other micronutrients in acidic and alkaline soils.

2. Peak soil zinc concentration in the Kenyan study was observed mid-season, corresponding with the time of most rapid plant development, suggesting that microbial activity in the rhizosphere root zone was also at its highest, thereby increasing zinc availability at this time. Quantification of microbial biomass and diversity between treatments and between sampling time points need to be undertaken to try to establish this possibility.

3. Given that a hail storm during harvest time destroyed the Kenyan crop before bean yield data could be collected, the potential of intercropping beans and annual ryegrass to increase zinc availability and save on zinc-based fertilizer costs could not be determined. Further studies in this regard are therefore required.

4. While results from all our studies showed that annual ryegrass can mitigate iron deficiency chlorosis in dry beans, grass intercropping did not result in higher dry bean grain or biomass yields in both field and greenhouse studies. Additional studies are required to determine the appropriate annual ryegrass and dry bean densities, as well as the optimum time of removal of annual ryegrass from the intercropped treatment that can not only alleviate iron deficiency chlorosis symptoms but also increase dry bean yields.

5. Our greenhouse study to determine the effect of annual ryegrass and microorganisms on iron deficiency using autoclaved and unsterilized soil could not establish whether the sustained interveinal chlorosis of dry beans in autoclaved soil was due to manganese toxicity (Boyd, 1971) or depletion of micronutrients by bean plants. The study did not also categorically establish if the significantly higher chlorophyll content of bean plants intercropped with annual ryegrass in sterilized soil was due to production of phytosiderophores by ryegrass roots and could only speculate on the possibility. In addition, the study did not ascertain whether the higher chlorophyll content of bean plants growing in unsterilized soil compared to those in sterilized (autoclaved) soil was due to microorganisms or root exudates. There is therefore a need to carry out soil and tissue analyses from these studies to provide some answers to these questions. There is also a need to conduct HPLC analyses to confirm the ability of annual ryegrass to excrete phytosiderophores in nutrient medium, thereby establish it as an iron- and zinc-efficient plant.

6. Although Gas Chromatographic PLFA assay analysis of soil samples found no significant differences between treatments, there was a general trend showing higher total bacterial, fungal and protozoan abundance in intercropped treatments, which suggests that iron-chelating microorganisms may have interacted with grass root exudates or organic matter in these treatments to increase iron availability, resulting in higher chlorophyll content in these treatments. The exact nature that such interactions may occur was not established by this study and should be a subject for further investigation. These results were obtained from samples collected from only one sampling time point. There is need to compare microbial biomass and abundance between treatments and between several sampling time points to better ascertain the role of microorganisms in our study observations and findings.

Acknowledgements

I want to express my sincere gratitude to Western SARE for their generous grant of $18,928, without which this study would not have been possible. Due to the great interest and additional scientific questions that the proposed project generated, the grant enabled our study to cover much more than was originally envisaged with supplemental funding from UW Plant Sciences department. For example, one additional field study was established in 2009 and repeated in 2010 to examine the effects of interplantings of annual ryegrass on iron deficiency in more susceptible dry bean cultivars (Blacks and Navies) apart from pinto beans. With timely permission from Western SARE, we were able to carry out various greenhouse and growth chamber studies that went a long way to complement our field results. I hope that I can still count on Western SARE for additional support to carry out follow-up studies off-shooting from this one, some of which are listed above.

I also want to sincerely thank Mike and Cindy Ridenour for identifying the original research question, securing Western SARE funds, kindly providing their farm for my MS thesis study and their continued support to date.

My heartfelt appreciation also goes to my former Advisor, Dr. Rik Smith, who played a key role in development of our successful proposal to Western SARE and continue to provide personal and professional friendship to me as I work through my dissertation research. Dr. Smith reamins a member of my PhD Program Committee.

Finally, my utmost appreciation to my current Advisor, Dr. Andrew Kniss, who took over from Dr. Smith and ensured a smooth transition. I am eternally grateful to Dr. Kniss for his continuing support, both in terms of advice and financial, that has enabled me to keep working on my PhD studies.

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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.