**2.2 High Fe-EDDHA rates**

Research to reduce or alleviate IDC in soybean by applying various seed, soil, or foliar Fe chelates or fertilizers has been conducted for decades. Although the results have been mixed (Mortvedt, 1986), and are seldom directly comparable, positive responses to foliar (Randall, 1981), seed (Karkosh et al., 1988), and soil (Penas et al., 1990; Wiersma, 2005) application have been reported. Other researchers have observed only small, if any, response to similar treatments (Goos and Johnson, 2000; Goos and Johnson, 2001; Heitholt et al., 2003). Lack of consistent results may be related to differing levels of chlorosis severity among experiments; soil, environmental, or genetic differences; and/or the low rates of Fe often applied to ensure economic feasibility. Low rates of Fe probably do not satisfy the requirement of a continuous supply of Fe as plant development progresses (Goos and Johnson, 2001).

Responses to higher (beyond economic feasibility) rates of Fe-EDDHA appear variety specific and occur over an extended period, manifest at maturity (Fig. 2). As plant development progresses, there are earlier, limited responses to low rates of Fe-EDDHA, whereas higher rates appear to provide Fe continuously and to promote later, larger responses.

Importance of Seed [Fe] for

Variety Fe

**B.** 

probability.

Initial

conc.

Norpro 2.6 B 79.3

grown at six rates of Fe-EDDHA applied at planting.

Improved Agronomic Performance and Efficient Genotype Selection 27

(µg g-1) ---------------------- field visual chlorosis rating (1-5) -

LSD (0.05) 0.7 NS NS . . . CV 14.9 26 15.7 . . .

MN0302 88.6 2.1 B† 1.2 A 1.1 A 1.0 A 1.0 A 1.0 A 1.6 GC3104 73.1 3.4 A 1.9 A 1.2 A 1.0 A 1.0 A 1.0 A 2.5 Norpro 91.3 2.6 B 1.4 A 1.2 A 1.0 A 1.0 A 1.0 A 3.3 S2000 2020 57.6 3.8 A 1.5 A 1.1 A 1.0 A 1.0 A 1.0 A 3.7

 Published Initial chlorosis Fe-EDDHA (kg Fe ha-1) seed Variety rating 0.00 2.24 4.48 6.72 8.96 11.20 Fe conc.

(1-5) ---------------------- harvest seed [Fe] (µg g-1 seed) -----

MN0302 2.1 B 64.1 B 64.7 B 66.3 B 66.3 B 77.7 A 74.3 A 88.6 GC3104 3.4 A 42.4 C 49.5 C 47.8 C 51.3 C 53.2 B 52.7 B 73.1

S2000 2020 3.8 A 41.3 C 46.4 C 45.5 C 45.4 C 45.4 B 49.8 B 57.6

† Means followed by the same letter within a column are not statistically significant at the 5 % level of

Table 1. Field visual chlorosis rating recorded at V3 and seed [Fe] at harvest of four cultivars

resistant cultivars were consistently higher than that of susceptible cultivars whether chlorosis was nil or severe. This observation is similar to that of Beebe et al. (2000) and Blair et al. (2009) who, from work done with dry beans (*Phaseolus vulgaris* L.), concluded that seed micronutrient densities of Fe (and Zn) were consistent, reliable estimates of resistance to Fe deficiency. Genotypically superior and inferior cultivars could be identified consistently across years and locations (Bouis et al., 2003; Nestle et al., 2006; Ghandilyan et al., 2006). Other research (Wiersma, 2005) has shown that plotting relative grain yield vs seed [Fe] for several environments exhibits a narrow range of seed [Fe] associated with wide ranges in relative yield and that there are consistent seed [Fe] differences between resistant and susceptible cultivars regardless of relative yield. These conclusions have led to the concept that individual genotypes have a seed [Fe] "threshold" that is presumably, genetically

LSD (0.05) 0.7 8.3 6.0 5.8 6.3 12.2 9.7 CV 14.9 9.2 6.3 6.2 6.5 12.0 9.4

**A.** Published

seed Fe-EDDHA (kg Fe ha-1) chlorosis

0.00 2.24 4.48 6.72 8.96 11.20 rating



<sup>A</sup>77.1 A 78.6 A 76.4 A 76.9 A 80.0 A 91.3

Fig. 1. Visual chlorosis, grain yield, and harvest seed [Fe] of resistant and susceptible cultivars in response to seeding density and Fe-EDDHA rate.

However, it should be noted that the severity of Fe deficiency and the plant characters used to measure treatment response are both crucial when deciding the suitability of various treatments for improving Fe acquisition. For example, measures of field visual chlorosis at low rates of Fe-EDDHA discriminate nicely between resistant and susceptible cultivars, but at higher Fe rates, almost all cultivars have similar scores (Table 1A). In contrast, measures of harvest seed [Fe] provide nearly identical discrimination among cultivars at each rate of Fe-EDDHA, and are nearly the same as the [Fe] of the seed used for planting (Table 1B).

At lower rates of Fe-EDDHA, resistant cultivars often exceed susceptible cultivars in plant height, seed number, and grain yield, whereas at higher rates, susceptible cultivars approach values similar to resistant cultivars (Fig. 2). With only slight chlorosis (Fig. 3, Fisher, MN 2003) seed [Fe] changed very little in response to added Fe for either resistant or susceptible cultivars. With severe chlorosis (Fig. 3, Crookston, MN, 2003), resistant cultivars increased harvest seed [Fe] about 15% in each portion of the canopy, whereas susceptible cultivars changed harvest seed [Fe] little in any portion of the canopy. Harvest seed [Fe]s of

Fig. 1. Visual chlorosis, grain yield, and harvest seed [Fe] of resistant and susceptible

However, it should be noted that the severity of Fe deficiency and the plant characters used to measure treatment response are both crucial when deciding the suitability of various treatments for improving Fe acquisition. For example, measures of field visual chlorosis at low rates of Fe-EDDHA discriminate nicely between resistant and susceptible cultivars, but at higher Fe rates, almost all cultivars have similar scores (Table 1A). In contrast, measures of harvest seed [Fe] provide nearly identical discrimination among cultivars at each rate of Fe-EDDHA, and are nearly the same as the [Fe] of the seed used for planting (Table 1B). At lower rates of Fe-EDDHA, resistant cultivars often exceed susceptible cultivars in plant height, seed number, and grain yield, whereas at higher rates, susceptible cultivars approach values similar to resistant cultivars (Fig. 2). With only slight chlorosis (Fig. 3, Fisher, MN 2003) seed [Fe] changed very little in response to added Fe for either resistant or susceptible cultivars. With severe chlorosis (Fig. 3, Crookston, MN, 2003), resistant cultivars increased harvest seed [Fe] about 15% in each portion of the canopy, whereas susceptible cultivars changed harvest seed [Fe] little in any portion of the canopy. Harvest seed [Fe]s of

cultivars in response to seeding density and Fe-EDDHA rate.


† Means followed by the same letter within a column are not statistically significant at the 5 % level of probability.

Table 1. Field visual chlorosis rating recorded at V3 and seed [Fe] at harvest of four cultivars grown at six rates of Fe-EDDHA applied at planting.

resistant cultivars were consistently higher than that of susceptible cultivars whether chlorosis was nil or severe. This observation is similar to that of Beebe et al. (2000) and Blair et al. (2009) who, from work done with dry beans (*Phaseolus vulgaris* L.), concluded that seed micronutrient densities of Fe (and Zn) were consistent, reliable estimates of resistance to Fe deficiency. Genotypically superior and inferior cultivars could be identified consistently across years and locations (Bouis et al., 2003; Nestle et al., 2006; Ghandilyan et al., 2006). Other research (Wiersma, 2005) has shown that plotting relative grain yield vs seed [Fe] for several environments exhibits a narrow range of seed [Fe] associated with wide ranges in relative yield and that there are consistent seed [Fe] differences between resistant and susceptible cultivars regardless of relative yield. These conclusions have led to the concept that individual genotypes have a seed [Fe] "threshold" that is presumably, genetically

Importance of Seed [Fe] for

deficiency (Fisher, MN, 2003).

**2.3 Fe-EDDHA rates - canopy position** 

Improved Agronomic Performance and Efficient Genotype Selection 29

Fig. 3. Seed [Fe] at harvest for different canopy positions of resistant and susceptible

cultivars grown under mild to severe Fe deficiency (Crookston, MN, 2003) and nil to mild Fe

Ten consecutive plants within row two of each plot in the Fe-EDDHA trials mentioned above were harvested at R7-R8, the total number of main stem nodes was counted, averaged, and used to separate plants into the upper, middle, and lower thirds of the plant. Sections were combined and the number of seeds, total seed weight, and seed [Fe] of the three sections of the canopy were determined. Averaged across cultivars, seed [Fe] decreased from approx. 50 µg g-1 at the lower canopy position, to 45 µg g-1 in the middle

predetermined, yet seldom exceeded, and that seed [Fe] could supplement or replace VCS as a measure of resistance to IDC.

Fig. 2. Agronomic measures of resistance to Fe deficiency in soybean in response to increasing rates of applied Fe-EDDHA averaged over three environments (panels A, C, and E). SPAD measures were recorded at three stages of development in one environment (panels B, D, and F).

predetermined, yet seldom exceeded, and that seed [Fe] could supplement or replace VCS as

Fig. 2. Agronomic measures of resistance to Fe deficiency in soybean in response to

increasing rates of applied Fe-EDDHA averaged over three environments (panels A, C, and E). SPAD measures were recorded at three stages of development in one environment

a measure of resistance to IDC.

(panels B, D, and F).

Fig. 3. Seed [Fe] at harvest for different canopy positions of resistant and susceptible cultivars grown under mild to severe Fe deficiency (Crookston, MN, 2003) and nil to mild Fe deficiency (Fisher, MN, 2003).

### **2.3 Fe-EDDHA rates - canopy position**

Ten consecutive plants within row two of each plot in the Fe-EDDHA trials mentioned above were harvested at R7-R8, the total number of main stem nodes was counted, averaged, and used to separate plants into the upper, middle, and lower thirds of the plant. Sections were combined and the number of seeds, total seed weight, and seed [Fe] of the three sections of the canopy were determined. Averaged across cultivars, seed [Fe] decreased from approx. 50 µg g-1 at the lower canopy position, to 45 µg g-1 in the middle

Importance of Seed [Fe] for

with seed (µg Fe m-2).

cultivars.

**3.2 Variety x Fe-EDDHA rate** 

evaluations conducted on similar soils.

**3.1 Roundup ready vs conventional cultivars** 

Improved Agronomic Performance and Efficient Genotype Selection 31

grasses and legumes have been found to be reliable and consistent across both years and locations. Research conducted on dry bean (*Phaseolus vulgaris* L.), wheat (*Triticum aestivum* L.), and rice (*Oryza sativa* L.) cultivars demonstrated that genotypes with high micronutrient densities of Fe and Zn during one year at one location will also be among the highest at another location in another year (Gregorio, 2002; Shen et al., 2002; Bouis et al., 2003; Nestle et al., 2006; Blair et al., 2009; Blair et al., 2010). Perhaps, measures of resistance to Fe deficiency in soybean should involve integrated estimates of uptake, transport, and accumulation of Fe that are manifest at maturity, such as Fe content 1000-1 seeds, seed [Fe], and/or iron removal

As Roundup Ready™ (RR) cultivars were first being released there was local concern among growers and crop consultants that resistance to Fe deficiency may not have been incorporated during development of earlier releases. During 2002, ten RR™ and ten 'conventional' cultivars were grown at two rates of Fe-EDDHA (0 and 8.96 kg ha-1) at the University of Minnesota Northwest Research and Outreach Center (NWROC) on soils with a known history of mild to severe Fe deficiency. A relatively high rate of application of Fe-EDDHA increased relative chlorophyll readings at V3 about 13% (4.6 SPAD units) and increased grain yield nearly 18% (434 kg ha-1). Roundup Ready cultivars out-yielded conventional cultivars by approximately the same amount, 19% (453 kg ha-1). Nonetheless, seed [Fe] at harvest did not differ between Fe-EDDHA rates, nor between RR and conventional cultivars (Table 3). Seed [Fe] at harvest was moderately related to both published visual chlorosis score (r2=0.452) and Fe concentration of the seed used for planting (r2=0.458). Classifying cultivars on the basis of their published VCS and then their planting seed [Fe] resulted in the same cultivars being in each class and, consequently, having the same r2 values. This research involved a relatively small sample of cultivars grown under harsh conditions and may not have fairly represented the importance of Fe 1000-1 seeds, seed [Fe], and/or Fe removal. Similarly, it is important to remember that these results cannot be extended to all RR and conventional soybean

Four cultivars (two Fe deficiency resistant, two Fe deficiency susceptible) and six rates of Fe-EDDHA (0, 2.24, 4.48, 6.72, 8.96, and 11.2 kg ha-1) were evaluated at one location in 2002 and six cultivars (two Fe deficiency resistant, two moderately resistant, and two susceptible) were evaluated at two locations in 2003. Visual chlorosis scores recorded at V3 could distinguish resistant from susceptible cultivars only when no Fe-EDDHA was applied, whereas harvest seed [Fe] could discriminate among resistant and susceptible cultivars at all six rates of Fe-EDDHA and in exactly the same order at each level of added Fe chelate (Table 1). Although grain yield increased markedly with added Fe chelate (Fig. 1. C), seed [Fe] changed very little (approx. 11%) (Fig. 1. F). The rank order of cultivars for harvest seed [Fe] was also the same as that of the cultivars' initial or planting seed [Fe], providing some evidence that seed [Fe] reflects varietal differences in resistance to Fe deficiency. Similar results recorded for the two 2003 trials, where two additional cultivars were evaluated under different severities of Fe deficiency, extend the applicability of this concept to

one-third, to 40 µg g-1 in the top one-third (Fig. 3). This decrease occurred under both nil and severe Fe chlorosis and suggests that developmentally younger and older seeds respond similarly to increasing Fe-EDDHA rates. Increases in seed [Fe] occur primarily in resistant cultivars grown under harsh Fe deficiency. Susceptible cultivars show little response to added Fe-EDDHA whether Fe deficiency is nil or severe.

With limited Fe deficiency (Fisher, MN, 2003), both resistant and susceptible cultivars attain their genetically predetermined seed [Fe] (Fig. 3). Taken together, these results suggest that developmentally younger, intermediate, and older seed accumulate Fe at similar rates, but for different lengths of time and that cultivars and canopy positions have very similar regression slopes, but different intercepts or "thresholds" (Fig. 4).

### **2.4 Nitrogen rates**

The response of soybean cultivar resistance to IDC to differing N rates was evaluated in a field study. Six rates of fertilizer N (0, 34, 68, 102, 136, and 170 kg ha-1) were applied to six cultivars differing in resistance to IDC (2 Fe efficient, 2 moderately Fe efficient, and 2 Fe inefficient) over a three year period. Nodulation decreased linearly in response to added N for all cultivars, regardless of their Fe efficiency characterization or yearly growing conditions. In contrast, relative foliar chlorophyll concentrations (SPAD readings) differed markedly among cultivars, but showed little consequential response to increasing nitrogen rates (NR) (Fig. 5). Plant height, seed number, grain yield, and seed [Fe] decreased linearly in response to increasing NRs for Fe-inefficient cultivars, whereas these responses in Feefficient and moderately efficient cultivars changed little as NR increased (Figs. 5 and 6). Despite these differences, the ranking of cultivars based on seed [Fe] was only slightly affected by increasing NRs.
