Removal of Lithium Citrate from H3A for Determination of Plant Available P

The soil extractant, H3A, has undergone several iterations to extract calcium (Ca), iron (Fe), aluminum (Al), potassium (K), phosphorus (P), ammonium (NH4-N) and nitrate (NO3-N) under ambient soil conditions. Few soil extractants currently used by commercial and university soil testing laboratories can perform multi-nutrient extraction without overor under-estimating at least one nutrient. Soil pH and plant root exudates have a strong influence on nutrient availability and H3A was developed to mimic soil conditions. Lithium citrate was previously used in the H3A formulation, but resulted in a cloudy supernatant in some samples, complicating laboratory analyses. In this study, we removed lithium citrate and compared the nutrients extracted from the modified (H3A-4) to the established (H3A-3) solutions. We found that the new extractant, H3A-4, produced a clear supernatant even in soils with low pH and high iron and aluminum concentrations. H3A-4 accurately predicts plant available nutrients and is a viable choice for commercial and laboratory settings due to its ease of use.


Introduction
Few currently available soil extractants are capable of multi-nutrient extraction without sacrificing accuracy for one compound or another [1]. Plant-available soil P has been difficult to accurately assess across the naturally occurring pH range of soils (calcareous to acid) with a single extractant because soil pH and P solubility are highly interrelated [2] [3] and soil pH has a strong influence on soil-solution chemistry [4]. Thus multiple extractants have been developed to Mehlich 3 [5] was developed for use in neutral to acid soils, while the Olsen extractant was developed primarily to extract P from calcareous soils [6]. Their development demonstrates early awareness of the difficulty of accurately extracting plant available P across such a wide range in natural conditions. Despite its development for neutral to acid soils, Mehlich 3 is commonly used without regard to soil pH. In addition, Mehlich 3 cannot be used for NH 4 -N and NO 2 -N/NO 3 -N analysis.
The need for rapid analysis in commercial and university soil-testing labs contributed to use of soil extractants outside of the soil conditions for which they were developed. Using strong acid extractants such as Mehlich 3 (pH ~3) or alkaline extractants such as Olsen (pH ~8.5) without regard to soil pH can result in over-estimation of plant-available P. As Menon et al. (1988) stated "a major disadvantage in using extracting solutions; however, is that they may mobilize not only the phosphate available for plant use but also some otherwise stable and non-mobile soil components." For instance, Mehlich 3 releases significantly more P than other extractants [7] [8] [9] by dissolving relatively insoluble fractions of Ca-, Al-and Fe-associated P [10]. Menon et al. (1988) suggest that deionized water and the anion exchange resin method (FeAlO exchange) do not experience the limitations common with other extracting solutions. While FeAlO exchange is an accurate method for the measurement of plant available P [3] [7], it is slow and cumbersome. In addition, there are well known analytical issues with water extraction, such as cloudy extract resulting in instrument interference. Therefore, few, if any, commercial labs offer FeAlO and water extractions for large scale routine soil analysis. H3A-1 [11] and its subsequent modifications, H3A-2 and H3A-3 [12], were developed to mimic the plant root environment by utilizing organic acid plant exudates [13] [14] to extract nutrients at ambient soil pH, with the additional benefit of simultaneous N, P and K extraction. Many organic acids exuded by plant roots have been identified. Three exuded by plants species such as corn, wheat, and sorghum are malic, citric, and oxalic acid [15]. These organic acids are used in H3A and have a low buffering capacity, which allows the soil pH to dominate the pH of the extractant solution in soil [11]. The effort to mimic the soil-root environment is important since plants utilize root exudates to overcome P, Fe, Zn, and Mn deficiencies [13] [16] [17]. Ion toxicity and pathogen attack can also stimulate an exudate response from plants [18] [19] [20]. The mechanism for increased P availability from plant-root exudates is mediated by a decrease in soil pH at the plant root-soil interface, which induces ligand exchange, dissolution, and binding to exchange sites by organic acids exudates that release ligand-bound P to the soil solution [21]. As the pH of the soil increases, the acids struggle to extract P as the effects of soil calcium take effect, much like in the Texas Houston Black (fine, smectitic, thermic UdicHaplusterts) soils (pH generally see a crop response to P fertilizer additions. H3A has been able to accurately reflect limited availability of P in these soils as the extracting power of H3A decreases in proportion to increases in soil pH [22]. H3A begins to extract less soil P around pH 7.7 where free CaO 3 is abundant [12]. The original H3A formulation and processing methods were altered to address the occurrence (roughly 5%) of soil extracts that did not have a visually strips are considered to best represent plant available P in soils [3] [7], we compared the relationships between H3A-2 and H3A-3 extractable P with FeAlO P.H3A-3 extractable P was strongly correlated with P from FeAlO strip extraction results (r 2 > 0.96, P < 0.001) [22].
As we continually endeavor to mimic nature in the lab, it is logical to remove Li citrate since it is not a naturally occurring organic compound. In the past, we made the mistake of synthetically forcing a natural process to conform to a lab method rather than allowing the natural process to drive lab methodology. Lithium citrate buffered organic acids in H3A to stabilize extractant pH and extractable P across a range of soils; however, this process does not occur in the field. Plants control their response to P deficiency by changing the organic acid strength or type of acid they exude into the soil [23]. The objective of this study was to compare extractable P from H3A-3 to extractable P from H3A-3 without Li-citrate (H3A-4). We believe that the removal of Li citrateis a necessary change in inching us ever closer to extracting solutions that mimicplant root and soil interactions.

Methods
Extractable P from a total of106 soils were analyzed with H3A-3 and H3A-

Results and Discussion
Soil pH values range from 4.2 to 8.5. Averaged across all 106 samples from ICP analysis, H3A-3 extractable P ranges from 1.0 mg P kg −1 soil to 215 mg P kg −1 soil with a mean of 36.3 mg P kg −1 soil, while H3A-4 extractable P ranges from 1.0 mg P kg −1 soil to 227 mg P kg −1 soil with a mean of 34.6 mg P kg −1 soil.
H3A-4 extracted 95.4% of that extracted with H3A-3 from 106 samples (3672 mg P kg −1 soil compared to 3849 mg P kg −1 soil). H3A-4 extractable P was highly correlated with H3A-3 extractable P (r 2 = 0.98, p < 0.001, Figure 1). The roughly one to one slope is a strong indicator that H3A-4 is an accurate soil extractant for P when compared to H3A-3 (slope = 1.08). These data indicate that H3A-4 is extracting P within roughly 5% of H3A-3.  Figure 2). Note the slope is similar to that from   H3A-3 and H3A-4 (r 2 = 0.99 p < 0.001). Figure 5 illustrates the same data with the highest NH 4 -N value removed, demonstrating that the correlation holds well at lower levels of extractable NH 4 -N (r 2 = 0.99 p < 0.001). These data also indicate a slight increase in extractable NH 4 -N with H3A-4 compared to H3A-3.        The most notable difference between H3A-3 and H3A-4 was the increase in percent P saturation (%P sat = (extractable P/(Al + Fe)) * 100) with H3A-4 ( Figure 11). The rise in %P sat is due to the approximately two-fold decrease in Al and Fe extracted with H3A-4 and relatively no change to extractable P.
Phosphorus saturation percentages over 15 commonly indicate that P fertilizer has been applied or excessive P is available. Averaged across all samples, %P sat   Therefore, the new ratio would indicate adequate P at 30% P sat and excessive P above 30%. Percent P saturation is an excellent indicator of excessive P when we receive samples from fields to which no P fertilizer has been recently applied.
Phosphate recommendations are known to be inherently unreliable over a wide range of soils [24] [25] [26]; therefore our approach to soil testing differs from traditional soil tests for plant available P. Laboratories using Bray, Mehlich and Olsen find a critical value for extractable P and rate soils as having a low, medium or high P response probability. P recommendations are therefore based on P response curves to added P fertilizer from field trials of a few sample soils.
This approach does not account for the plants ability to naturally extract P through root exudates under varying climatic conditions as well as variations in management such as no-till versus conventional-till. Additionally, fertilizer response curves are known to be highly variable year to year [27], decreasing the likelihood that P recommendations will be on target with crop needs. The best soil tests are those that are insensitive to soil type [28] and thereby viable over a wide range of soils. Menon [29] stated at the time, only the water and anion exchange resin methods can be considered insensitive to the soil types.
Fertilizer recommendations from soil-testing with H3A-4 are dynamic because we account for the inherent soil chemistry and natural plant biochemistry, as has been shown by Somenahally [8] and Haney [11]. Somenahally [8] [30] found that within-field soil variability is greater than variability in weather when water availability is not a limiting factor so we must attempt to account for variations in soil in our fertilizer recommendations versus regional blanket approaches.

Conclusions
H3A-4 was developed to overcome the inherent over-or under-estimations of plant available nutrients when soil extractants are too harsh (i.e. very high or low pH) or too weak (water). Fertilizer recommendations based on response curves are outdated and ignore the inherently variable soil and climatic factors. While it is not ideal to forgo field studies for every nutrient extractant, there is a shortage of time, funding, and labor to conduct the far-reaching research that would be needed to test over the vast gamut of soils and climatic conditions. Coupled with the fact that farmers need updated nutrient management information now, and Open Journal of Soil Science not in another 20 to 40 years, it is critically important to advance our soil-testing techniques so we can give producers the best data available without extensive multi-year greenhouse or research plot experiments. A move toward on-farm research could promote advances in soil analyses, ultimately benefiting producers as they endeavor to yield high quality feed and fiber efficiently and economically.
We found it remarkable that extractable P values can be altered so easily by using a natural process as the foundation of development. The variations in the H3A extractant over time have been somewhat subtle, yet have effectively improved extractable P capitalizing on a biomimicry approach. The first iteration (H3A-1) was a little more aggressive in extracting P than was H3A-2. Transition soil extractant demonstrates that our endeavor to mimic the natural processes in the lab were successful. The results will undoubtedly improve our P fertilizer recommendations for producers.