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

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

Summary

The project is based on a farmer’s observation in 2002 that pinto beans intercropped with annual ryegrass did not exhibit iron-deficiency chlorosis and produced better than beans grown without the ryegrass intercrop. The purpose of this project is to determine if an annual ryegrass-pinto bean intercrop can mitigate iron deficiency in the high pH calcareous soils that are prevalent in the State of Wyoming. The study will also investigate recent observations that iron deficiency chlorosis can be overcome by more resistant cultivars of dry beans, close bean plant spacing, and low levels of nitrate nitrogen in these soils. In addition, the project will determine if interplantings of annual ryegrass can mitigate zinc deficiency in acidic soils in western Kenya.

Objectives/Performance Targets

• 1.0: To determine the effectiveness of intercropping annual ryegrass with pinto beans in mitigating iron deficiency in calcareous soils (DBO5). Nested within this objective: • A comparison with intercrops of wheat as a grass known to exude compounds capable of chelating iron • An assessment of the effect of selective herbicide in facilitating machine harvesting of dry beans • A comparison with the potential of close spacing and high plant density of pinto beans in mitigating iron deficiency • An assessment of the effect of nitrate-nitrogen on iron availability for uptake by dry beans Arising from the above objective: 1.1: To determine the effectiveness of annual ryegrass in mitigating iron deficiency in black beans and Navy beans as two dry bean cultivars known to be more susceptible to iron deficiency chlorosis compared to pinto beans (DBO6). 2.0 To determine the effect of intercropping annual ryegrass with dry beans on iron deficiency in dry beans in a controlled environment. Nested within this objective: 2.1 An assessment of two different temperature levels in influencing iron deficiency. 2.2 An assessment of temperature and different nitrate-nitrogen levels in influencing iron deficiency. 3.0 To determine the effectiveness of intercropping annual ryegrass with pinto beans in mitigating zinc deficiency in acid soils in comparison with intercrop of wheat, a grass known to exude compounds capable of chelating zinc, and millet, a hardy cereal food crop popular in western Kenya.

Accomplishments/Milestones

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) pinto beans intercropped with annual ryegrass; 2) pinto beans intercropped with annual ryegrass and sprayed with ‘Select’ herbicide; 3) pinto beans intercropped with wheat; 4) pinto beans intercropped with wheat and sprayed with ‘Select’ herbicide; 5) a monoculture of pinto beans at low density (163,090 plants/ha); 6) a monoculture of pinto beans at high density (326,179 plants/ha); 7) a monoculture of annual ryegrass; 8) a monoculture of wheat; 9) bare soil check. Annual ryegrass was planted at 22 kg/ha, spring wheat at 44 kg/ha and Pinto beans at 72 cm rows. Soil samples were collected from each plot at four different dates and analyzed for iron, zinc, nitrate-N, organic matter, electrical conductivity, pH, molybdenum, and phosphates. ‘Select’ herbicide was applied one month after planting. Tissue samples were collected at three different dates and tested for iron, zinc, nitrate-N, and molybdenum. Leaf cholophyll data was taken on the youngest fully expanded bean leaves using a SPAD-503Plus chlorophyll meter at three different dates. Three meters of row from each plot was harvested and dry bean yield was determined. Data was analysed using Split-plot-in-space and Split-plot-in-time general linear model procedures of the analysis of variance in SAS.

There were no differences between treatments for yield for the 2008 study. Hand-weeding of the study was ineffective in eliminating persistent weeds such as common lambsquarters, redroot pigweeds, black and hairy nightshades, and green foxtail. 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 the pinto bean plants during the first month after planting, the beans were generally able to overcome these symptoms as the season progressed. There was, however, 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 role of temperature in influencing micro- and macro-nutrient availability for uptake by dry beans will be tested in a growth chamber study at the UW Greenhouse set at two temperature regimes.

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 the plant leaves, depress chlorophyll synthesis and induce iron deficiency chlorosis (Christensen and Johnson, 2008). A growth chamber and greenhouse study will be established in 2010 to determine the influence of nitrate-nitrogen in micro- and macro-nutrient availability and uptake by plants.

All plots were planted on June 19 compared to June 11 in 2008. Planting date in 2009 was delayed due to unexpected prolonged 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.

‘Treflan’ herbicide was applied pre-emergence to the 2009 study. Control of weeds was quite effective as a result. High density pinto beans produced at least twice higher yields (p < 0.0001) than all the other treatments in 2009 (Table 4). Pinto beans intercropped with wheat produced the lowest yields (p < 0.0001) suggesting that wheat has a competitive advantage over dry beans. 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 annual ryegrass had less competitive effect on dry bean yields compared to wheat.

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. 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. A growth chamber study will attempt to establish the role of temperature in iron availability and acquisition by dry beans.

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 seemed to be more adversely affected by iron deficiency chlorosis that could potentially reduce their yields. This suggests that different dry bean cultivars have different tolerances to micronutrient deficiency.

A field experiment was established on the irrigated field at Sustainable Agriculture Research and Extension Center near Lingle, Wyoming, in summer 2009 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 4.267 meter plots for the other treatments in a 4 by 5 factorial randomized complete block design replicated four times (Figure 1). Twelve treatments planted in four blocks included three bean cultivars; ‘Buckskin’ pinto beans, ‘Schooner’ navy beans, and ‘T-39’ black beans each intercropped with common 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, annual ryegrass planted at 22 kg/ha, and spring wheat and oats at 44 kg/ha on June 19. Pioneer 38N88 corn was planted into 30-inch rows at a density of 79,073 seeds per hectare on June 19. 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. ‘Select’ herbicide was applied on all intercropped plots on July 30. Soil samples were collected from each plot at four different dates and analyzed for iron, zinc, nitrate-N, organic matter, electrical conductivity, pH, molybdenum, and phosphates. Tissue samples were collected at three different dates and tested for iron, zinc, nitrate-N, and molybdenum. Leaf cholophyll data was taken on the youngest fully expanded bean leaves using a SPAD-503Plus chlorophyll meter at two different dates. Three meters of row from each plot was harvested on September 25 and yield was determined in the field. Data was analysed using the Split-plot and Split-plot-in-time procedure of the analysis of variance in SAS

There was significant iron chlorosis on navy and black beans (p < 0.0001) compared to pinto beans (Figure 4 and 5). The results also found that bean monocultures were more chlorotic than grass-intercopped beans (p < 0.0001). Interestingly, however, the more chlorotic beans accumulated more iron and nitrate nitrogen (p = 0.003) in their tissues than less chlorotic beans. This can be explained by a phenomenon referred to as the “chlorosis paradox” described by Abadía (1992); Marschner (1995); and Morales et al. (1998). These discrepancies are attributed in part to the localization and binding state of iron in the leaves (Marschner, 1995). A proportion of iron may be precipitated in the apoplasm of leaves and not physiologically available (Mengel and Geurtzen, 1988; Marschner, 1995).

All three monocropped dry bean cultivars produced higher yields (p = 0.0018) than all the intercropped plots. “Select’ herbicide is applied to remove grass intercrops from dry beans one month after planting. One month after planting has been determined from previous studies as ample time needed for any benefits beans can obtain from the intercropped grasses (Omondi et al., 2010). Since pinto beans failed to germinate and had to be reseeded, application of ‘Select’ herbicide was delayed by 12 days. Given that it takes about two weeks for ‘Select’ herbicide to kill its target plant, competitive effects from the grass intercrops may have offset any benefits that may have accrued. ‘Select’ herbicide will be sprayed two weeks earlier when the study is repeated in summer 2010 to confirm or dispel these speculations.

There were no differences between treatments for soil iron and zinc concentration. Increase of soil iron concentration with time in all treatments was marginal attributed to heavy and prolonged precipitation in 2009 that may have reduced soil temperatures hence micronutrient availability. 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. There was, however, significantly higher soil nitrate nitrogen (p = 0.02) in pinto and navy bean monocrop plots compared to the other treatments.

A growth chamber study was established at the UW Plant Sciences greenhouse 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 on October 27, 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. Each growth chamber was designed by Percival Scientific Inc, Model 130BLLC8; SO#009991-001; Serial Number 9991.01-02.07. Treatments consisted of pinto beans intercropped with annual ryegrass and a monoculture of pinto beans. Intercropped pots were sprayed with ‘Select’ herbicide on November 10, 2009. 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 was determined on January 1, 2010.

Bean seedlings developed long internodes and bolted toward the fluorescence light tubes in the chamber suggesting that light intensity was insufficient for those plants. Both chambers were designed for smaller statured plants and organisms. Additional fluorescence tubes would be installed and day light period increased to 15 hours to minimize this effect in subsequent studies utilizing these chambers.

Results from this study are still being analyzed. Initial observations, however, indicated that plants in the warmer growth chamber were larger than plants in the cool growth chamber (Figure 4). Plants in the cool growth chamber were also more chlorotic than those in the warm growth chamber. Surprisingly, there were much denser root biomass and larger and more root nodules on plants from the cool growth chamber compared to those from the warm growth chamber. This may partly be due to less effective method used to separate the roots from the soil. The study will be repeated in summer 2010 to ascertain findings- and in cognizant of lessons learned from the first experiment.

A growth chamber study was established at the UW Plant Sciences greenhouse to test the potential of annual ryegrass to mitigate iron deficiency in black beans under two temperature and nitrate-nitrogen 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 on January 9, 2010. 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 15/11 hours. Each growth chamber was designed by Percival Scientific Inc, Model 130BLLC8; SO#009991-001; Serial Number 9991.01-02.07. Treatments consisted of pinto beans intercropped with annual ryegrass and a monoculture of pinto beans. Each of these treatments was split between two nitrate nitrogen rates; 28kg/ha and 150kg/ha incorporated in the soil as Calcium nitrate before planting, in accordance with Aktas and van Egmond (1979). Intercropped pots were sprayed with ‘Select’ herbicide on February 10, 2010. Leaf chlorophyll data will be 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 will be determined at the end of the study.

Adjusting the day-light period from 13 to 15 hours shortened bean seedling internodes and reduced the tendency of the plants to bolt toward the light source. Germination of plants at higher N level was much slower than at lower N level. Initial observations indicate that plants at lower N level are larger and better developed than those at higher N level (Figure 3). Plants in the cool growth chamber are generally more chlorotic than those in the warm growth chamber, with the chlorosis more pronounced on the monocropped beans at the higher N level (Figure 5-7). The study is ongoing.

A study was carried out in summer 2006 at the farm of Mr. Joseph Kamuto near Kitale town in western Kenya to determine the effect of annual ryegrass intercrop in mitigating zinc deficiency in pinto beans. The study consisted of 1.5 by 6 meter plots in a randomized complete block design with three replications. Seven treatments were planted in three blocks, including: 1) pinto beans intercropped with annual ryegrass; 2) pinto beans intercropped with wheat; 3) pinto beans intercropped with finger millet; 4) a monoculture of pinto beans 5) a monoculture finger millet; 6) a monoculture of annual ryegrass; 7) a monoculture of wheat. All land preparation, planting, weeding, and management was done manually by hand (Figure 9 and 10). Beans were hand-planted at 60 cm rows and 7.5 cm within the row (Figure 8); Finger millet and annual ryegrass were hand-broadcasted in 30 cm rows at 25 kg/ha and wheat hand-broadcasted in 30 cm rows at 45 kg/ha. Soil samples were collected from each plot at four different dates, air-dried and shipped to UW Soils lab to be analyzed for iron, zinc, nitrate-N, organic matter, electrical conductivity, pH, molybdenum, and phosphates. Tissue samples were collected at three different dates, air-dried, and shipped to UW Soils lab to be tested for iron, zinc, nitrate-N, and molybdenum. Hail storm at harvest time destroyed the crop before bean yield data could be collected. Data was analysed using the PROC MIX procedure of the analysis of variance in SAS.

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. There was also significantly higher (p <0.001) organic matter in these plots compared to the other treatments. 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 5, Figure 9). There are two rainy seasons in Trans Nzoia district where this study was carried out. The first season, commonly referred to as the long rains because that is when the highest precipitation is received, occurs between March and late May. The second season (short rains) occurs between July and September. Temperatures are generally cooler during those periods than the rest of the year. The higher concentration of zinc during the second sampling date on June 26 therefore suggests that temperature may have played a role in increasing zinc availability. There is need for further studies to ascertain these results. There were no differences between treatments for iron, pH, and nitrate nitrogen.

• Mean soil nitrate nitrogen concentration for all treatments between sampling dates
• Figure 6: Plants grown at high N level in the cool chamber (green tags) compared to the warm chamber (white tags).
• Figure 8: Plants grown at high N level in the cool chamber; left two pots contain bean monocrop while right two bean plants were intercropped with annual ryegrass.
• Figure 9: Manor House Agricultural Center (MHAC) students prepare the study site in Mucharage, Kitale, Kenya in 2006.
• Black and Navy bean monoculture were more chlorotic on July 9 than corresponding beans intercropped with grasses
• Mean soil iron concentration for all treatments between sampling dates
• Bean yield sampled from 3m rows in grams
• Mean monthly temperatures in 2008 and 2009 in degrees Fahrenheit (Weather
• Black and navy bean monocultures were more chlorotic than corresponding bean in intercrop with grasses
• Warm growth chamber bean plants (left) compared to cool growth chamber plants
• Figure 5A: Plants from cool chamber. Green tags were planted at high N level.
• Figure 5C: Plants from warm chamber. White tags were planted at high N level
• Figure 10: Boaz Oduor, a MHAC student precisely measures the distance between seeds in a row using a ‘measuring stick’ cut to size during establishment of the Kenya study in summer 2006.
• Plot plan for new 2009 study (DBO6)
• Figure 5B: Plants from warm chamber. White tags were planted at high N level
• Figure 7: Plants grown at low N level in the cool chamber (blue tags) compared to the warm chamber (red tags)

Impacts and Contributions/Outcomes

This study has not yet categorically established the role of grass intercrops in mitigating micronutrient deficiency. A new study established in summer 2009 and which will be repeated in 2010 that compares three dry bean cultivars with different tolerances to iron deficiency, both in monoculture and intercropped with grasses, hopes to shed more light in this area.

Results from the 2008-2009 study have also led to the initiation of a growth chamber study that undertakes to determine the effect of temperature and nitrate nitrogen under two different temperature regimes in influencing iron deficiency in dry beans.

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.

It will, however, not be possible to repeat the Kenya study, to establish the usefulness of the results obtained from the initial study, without significant financial infusion.

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 the 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 the 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 practices. The study has the potential to both save on the costs of farming as well as increase yields.

• Figure 11: Changes in zinc concentration with sampling time between pinto bean monoculture and pinto bean-annual ryegrass intercrop treatments.
• Table 5: Mean soil zinc concentration for all treatments between sampling dates

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