Glyphosate-tolerant alfalfa is compositionally equivalent to conventional alfalfa (Medicago sativa L.)

Glyphosate-tolerant alfalfa (GTA) was developed to withstand over-the-top applications of glyphosate, the active ingredient in Roundup agricultural herbicides. As a part of the safety assessment, GTA (designated J101 x J163) was grown under controlled field conditions at geographically diverse locations within the United States during the 2001 and 2003 field seasons along with control and other conventional alfalfa varieties for compositional assessment. Field trials were conducted using a randomized complete block design with four replication blocks at each site. Alfalfa forage was harvested at the late bud to early bloom stage from each plot at five field sites in 2001 (establishment year) and from four field sites in 2003 (third year of stand). The concentration of proximate constituents, fibers, amino acids, coumestrol, and minerals in the forage was measured. The results showed that the forage from GTA J101 x J163 is compositionally equivalent to forage from the control and conventional alfalfa varieties.     

Soil proprieties and alfalfa (Medicago sativa L.) responses to sustainable treated urban wastewater reuse

ABSTRACT

The present work aims to perform an experimental reuse study of raw and sustainable treated urban wastewaters in the irrigation of alfalfa (Medicago sativa L.) under semi-controlled conditions. The obtained results highlight a significant effect (p < 0.05) of water quality on soil monitored parameters. Compared to control soils irrigated with groundwater (GW), those irrigated with treated urban wastewaters from the modified activated sludge process (PTW) and raw urban wastewater (RW) showed significant increase in their organic matter (OM) and nutrients content. Indeed, in comparison to GW organic matter (OM) of RW and PTW was 5,37 and 3,84 times higher; Kjeldahl nitrogen (TKN) was 3,50 and 2,81 times higher and total phosphorus (TP) was 4,00 and 3,25 times higher, respectively. The results showed also a significant effect of water quality on yield, macro and microelements concentrations of alfalfa plant. Hence, the highest yield, macro and microelements uptake of the plant were obtained in response to irrigation with PTW and RW compared to control soil GW. Concerning trace metallic elements concentrations, pots irrigated with RW and PTW showed relatively higher concentrations than those of GW pots.     

Amino acid sequence of proteins induced in Fe-deficient stressed alfalfa (Medicago sativa L.) roots

Alfalfa (Medicago sativa L.) grows well in soils with a moderately high pH and dissolves insoluble iron in the rhizosphere. We have investigated active uptake mechanisms under Fe-deficient nutrient conditions and the effects of Fe-deficiency on plants. Previously, we observed that Fe-deficient alfalfa roots exuded many compounds (Masaoka et al. 1993) such as fiavonoids. We also identified a new compound “alfafuran” which is a phenol compound and is different from organic acids or phytosiderophore-type amino acid derivatives exuded by Fe-deficient plant roots. This compound is also very effective in dissolving ferric phosphate (Noguchi et al. 1994), suggesting that alfalfa may have developed several strategies against Fe-deficient stress including the exudation of organic compounds like alfafuran which accelerate the Fe³⁺-reducing activity on the root cell membrane to dissolve insoluble iron compounds. Suzuki et al. (1995, 1997) observed that in barley several peptide spots obtained by electrophoresis were induced under Fe-deficient stress when mugineic acid-family phytosiderophores were secreted from the roots. They suggested that these peptides control the mugineic acid synthesis and secretion. We examined the peptides induced in Fe-deficient alfalfa roots.     

Inoculation treatments affect the migration and colonisation of rhizobia in alfalfa (Medicago sativa L.) plants

Purpose: The purposes of this study were to characterise the migration and the colonisation dynamics of two different fluorescent-tagged rhizobia in various alfalfa tissues (especially in seeds); and also to develop efficient inoculation treatments to promote colonisation ability of target rhizobia in elite seed varieties.

Materials and methods: Four treatments (root drench, root damaging and drench, root drench with matrine, and flower spray) were applied to inoculate alfalfa with two fluorescent-tagged rhizobia, Ensifer meliloti LZgn5f (gn5f) and Ensifer meliloti 12531f (12531f), at three different growth stages; bud, flower and pod stages. The migration and colonisation dynamics of the two fluorescent tagged rhizobia strains were monitored using UV lamp detection and a stereo fluorescence microscopy.

Results: The results showed that both rhizobia strains mainly colonised the roots and could migrate to aerial tissues. In aerial tissues, when alfalfa plants were inoculated during the bud stage, both rhizobia strains mainly colonised the leaves and stems; during the flower stage, a spray inoculation treatment resulted in more 12531f colonising reproductive tissues, while during the pod stage, more rhizobial strains gn5f colonised seeds using the root drench with matrine treatment.

Conclusions: These results indicate that endophytic rhizobia are natural inhabitants of internal regions of roots, stems, leaves and that the endophytes may arise from reproductive tissues, such as seeds. Understanding the population dynamics of endophytic rhizobia in alfalfa would considerably improve the survival of target rhizobia during seed transfer. Combining target endogenous rhizobial species with good alfalfa seed varieties may lead to the development of a novel breeding method.     

Weed flora survey in alfalfa (Medicago sativa L.) fields of Shabestar (northwest of Iran)

Alfalfa is one of the most widely grown forage crops in Iran, especially in Shabestar county. Weeds play a significant role in reducing the feeding value of alfalfa. Weed survey was done in order to determine environmental factors on weed species distribution in alfalfa fields at Shabestar, Iran (2010–2011). The family dominance index (FDI) and relative dominance (RD) were used to find dominant families and species for infestation of Shabestar's alfalfa fields. Data were analyzed by principal component analysis (PCA) and canonical correspondence analysis (CCA). A total of 130 weed species belonging to 28 families were observed. The most dominant families, based on the FDI, were Asteraceae, Poaceae, and Brassicaceae with 66.3, 61.8, and 41.1 FDI, respectively. Alopecurus myosuroides and Convolvulus arvensis were the most dominant species according to the RD index, measuring 26.4 and 21.8, respectively. PCA showed that some weed species only infested specific areas, but a few species with broader environmental tolerances were found near origin. Most variation in species distribution was explained by altitude, which was followed by soil potassium and salinity content of the soil. Our results suggest that the effect of altitude was more important than other environmental factors.     

Effect of salt stress on Growth and Ion accumulation of alfalfa (Medicago sativa L.) cultivars

Alfalfa (Medicago sativa L.) yield and nutrient contents may be affected under salinity condition. Thus, this experiment was conducted to determine the effect of three salinity levels (60, 120, and 180 mM NaCl) on shoot and root dry weights, and mineral contents of three alfalfa cultivars. With the increasing salinity levels sodium (Na) and magnesium (Mg) contents increased; but potassium (K), nitrogen (N), phosphorous (P), calcium (Ca), zinc (Zn), and copper (Cu) contents and root and leaf weights decreased; however, changes in these traits depended on cultivar and salinity level. However, Rehnani, a tolerant cultivar, had the lowest Na and Mg contents and the highest K, N, P, Ca, Zn, and Cu contents and dry weights under all of the salinity levels. Moreover, leaf dry weight and leaf P content had the highest correlation with salt tolerance suggesting that these traits may be used as a marker for selecting salts that are tolerant among genotypes in alfalfa.     

Migration,colonization and seedling growth of rhizobia with matrine treatment in alfalfa (Medicago sativa L.)

Purpose: The purposes of this study were to determine the effect of matrine on the migration and the colonization dynamics of the two fluorescent-tagged rhizobia in Gannong No.5 alfalfa (Medicago sativa L. Gannong No. 5) tissues, and also to determine the effect of the combination treatments on alfalfa seedlings’ growth.

Materials and methods: 0, 100, 200, 300 and 400 mg L^?1 matrine levels were added into two cyan fluorescent protein (CFP)-tagged rhizobia; Ensifer meliloti LZgn5f (gn5f) and Ensifer meliloti 12531f (12531f), respectively; and drenched the alfalfa root with the inoculants. Then the migration and colonization of the two rhizobia in alfalfa on D7, D14, D21 and D28, and subsequently seedling growth were investigated.

Results: The results showed that the optimum matrine level enhanced the colonization of both fluorescent-tagged rhizobia in alfalfa roots and the highest colonization densities of log 6.31 cfu g^?1 and log 5.87 cfu g^?1 were achieved by adding 300 mg L^?1 matrine into 12531f and adding 100 mg L^?1 matrine into gn5f, respectively. They could migrate to the aerial tissues and most colonize stems through the application of adding 300 mg L^?1 matrine into 12531f and 100 mg L^?1 matrine into gn5f, respectively. No fluorescent-tagged rhizobia were detected in the control treatment. Alfalfa seedling growth parameters like leaf chlorophyll content, seedling growth rate, root length, seedling biomass and total N percentage also increased the most when 300 mg L^?1 matrine was added into 12531f and 100 mg L^?1 matrine added into gn5f treatments.

Conclusions: Our results suggest that 300 mg L^?1 matrine added into 12531f and 100 mg L^?1 matrine added into gn5f might be exploited to promote the colonization of rhizobia in alfalfa tissue and positively impact growth and yield, indicating possible benefits for plant cultivation.     

Response of alfalfa (Medicago sativa L.) to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti

The objectives of this work were to phenotypically and genetically characterize alfalfa rhizosphere bacteria and to evaluate the effect of single or mixed inoculation upon nodulation and biological nitrogen fixation. Thirty-two strains showed tricalcium phosphate solubilization ability, and two of them caused bigger or equal solubilization halos than the control strain P. putida SP22. The comparison of the 16S ribosomal DNA sequences indicated that these strains are phylogenetically related to Bacillus spp. and Pseudomonas spp. A beneficial effect of both isolates on alfalfa growth was observed in coinoculation assays. Pseudomonas sp. FM7d caused a significant increase in root and shoot dry weight, length, and surface area of roots, number, and symbiotic properties of alfalfa plants. The plants coinoculated with Sinorhizobium meliloti B399 and the Bacillus sp. M7c showed significant increases in the measured parameters. Our results indicating that strains Pseudomonas sp. FM7d and Bacillus sp. M7c can be considered for the formulation of new inoculants.     

Assessment of summer drought tolerance variability in Mediterranean alfalfa (Medicago sativa L.) cultivars under Moroccan fields conditions

This study was undertaken to assess levels of tolerance to summer drought in Medicago sativa genotypes and to comment on their abilities to adapt to Moroccan conditions. Sixteen alfalfa cultivars originating from the Mediterranean basin were tested at an experimental station in Morocco, located in the semi-arid bioclimatic area. Trials were conducted over three years and consisted of one continuously irrigated treatment and another irrigated treatment in which summer watering was withheld for 9 weeks. Results showed that summer water stress significantly reduced aboveground biomass in all of the cultivars tested. This reduction ranged between 23.9 and 42.6% compared with fully irrigated treatment. At the end of stress period, mean summer leaf senescence rate was 83.3%, with a significant difference between cultivars. Ground cover, estimated at the end of summer in the third year, decreased dramatically and ranged between 12 and 30.7%, depending on the cultivar. High intrinsic water-use efficiency was associated with a decrease in stomatal conductance rather than an increase in photosynthesis.     

Ridge-furrow planting of alfalfa (Medicago sativa L.) for improved rainwater harvest in rainfed semiarid areas in Northwest China

In rainfed semiarid areas of northwestern China, alfalfa (Medicago sativa L.) is usually cultivated in flat fields with spaced rows. The yield of the crop is normally low because of insufficient moisture in the soil. The purpose of this study was to examine the effect of soil ridging and plastic film, covering the ridges, on rainwater harvest and alfalfa yield and to identify the optimal design of the ridge-furrow system. In this system the ridges served as the runoff area and the furrows as the infiltration area, where alfalfa was grown. Three ridge width (RW) measured at the bottom of the ridge, namely 30, 45 and 60 cm, were tested under the condition of the ridge-furrow systems with or without plastic film covering the ridge. These treatments were compared with the flat soil (FS) planting system, having neither ridge nor plastic cover. No matter what RW (or in the case of FS spacing between planting belts), the width of furrow (planting belt) was the same as 0.6 m. Soil moisture was measured at 7–10 day intervals, in 10 cm increments, to soil depth of 2 m. Rainwater harvests were measured from two additional plots without planting, representing the covered ridge (CR) and uncovered ridge (UR) systems, respectively. The results showed that in terms of average alfalfa yield on a total area basis, ridge type and ridge width both had significant effects, with CR being better than UR, and UR in turn being better than FS. The 2-year average annual yield of alfalfa was 5114 kg DM ha⁻¹ for CR, 2531 kg DM ha⁻¹ for UR, and 1927 kg DM ha⁻¹ for FS. For the CR treatments, the ridge width (RW) of 0.3 m resulted in higher yield (5528 kg DM ha⁻¹) than RW of 45 cm (5095 kg DM ha⁻¹) and 60 cm (4719 kg DM ha⁻¹). For the UR treatments, yields with 30 cm RW (3088 kg DM ha⁻¹) were higher than with 45 cm RW (2434 kg DM ha⁻¹) and 60 cm RW (2071 kg DM ha⁻¹). The results also showed that the CR planting system had additional advantage of collecting more rainwater to improve plant effective soil moisture. In conclusion, the CR with a RW of 0.3 m and furrow width of 0.6 m is recommended for improved rainwater utilization and improved alfalfa yield in the semiarid areas where alfalfa production is largely rainfed.     

Circulating fluidized bed combustion product addition to acid soil: alfalfa (Medicago sativa L.) composition and environmental quality

To reduce S emissions, petroleum coke with a high concentration of S was combusted with limestone in a circulating fluidized bed (CFB) boiler. The combustion process creates a bed product that has potential for agricultural uses. This CFB product is often alkaline and enriched in S and other essential plant nutrients, but also contains high concentrations of Ni and V. Agricultural land application of CFB product is encouraged, but little information is available related to plant responses and environmental impacts. CFB product and agricultural lime (ag-lime) were applied at rates of 0, 0.5, 1.0, and 2.0 times the soil's lime requirement (LR) to an acidic soil (Wooster silt loam). The 2.0x LR application rate of CFB product was equivalent to 67.2 Mg ha(-1). Alfalfa yield was increased 4.6 times by CFB product and 3.8 times by ag-lime compared to untreated control. Application of CFB product increased the concentration of V in soil and alfalfa tissue, but not in soil water, and increased the concentration of Ni in soil and soil water, but not in alfalfa tissue. However, these concentrations did not reach levels that might cause environmental problems.     

南平市紫花苜蓿种植气候区划

在南平市1951-2000年的气象资料和紫花苜蓿试验成果的基础上,结合本市地形、地貌特征,通过GIS的空间分析和多层复合过程,融合牧草生长信息和农业气象专家的思想,进行南平市紫花苜蓿适宜种植区的划分和评价,为南平市紫花苜蓿种植调整提供科学依据。     

Phytoestrogenic compounds in alfalfa sprout (Medicago sativa) beyond coumestrol

Coumestrol has long been known as the phytoestrogenic compound in alfalfa. However, it has been demonstrated that the ethyl acetate extract of alfalfa sprout (AEA) attenuated the disease severity and increased survival and life span of autoimmune-prone MRL-lpr/lpr mice. Coumestrol, on the contrary, decreased the survival. This study thus aimed to isolate and identify phytoestrogenic compounds other than coumestrol in AEA. AEA was fractionated and separated by successive silica gel chromatography and preparative HPLC. The activity of collected fractions was tracked by a transactivation assay for ERα and ERβ, respectively. In addition to coumestrol, liquiritigenin, isoliquiritigenin, loliolide, and (4S,6S)- and (4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one were isolated and chemically identified. Except for loliolide, these compounds showed higher transactivation via ERβ than via ERα. The maximal activation via ERα of coumestrol reached 80% that of 1 nM 17β-estradiol (E(2)), whereas the activations of the remaining five compounds as well as AEA ranged from 8 to 49%. In addition, isoliquiritigenin, loliolide, and (4S,6S)- and (4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one, but not coumestrol, preferentially inhibited 1 nM E(2) induced ERα activation, compared to that ERβ activation. The selectivity of these phytoestrogens might account for the difference between the effects of AEA and coumestrol in autoimmune-prone MRL-lpr/lpr mice observed previously.     

Alfalfa (Medicago sativa L.) flavonoids. 1. Apigenin and luteolin glycosides from aerial parts

Nine flavones and adenosine have been identified in aerial parts of alfalfa, and their structures were established by spectral (FABMS and NMR) techniques. Five of the identified compounds, including apigenin 7-O-[beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranosyl]-4'-O-beta-D-glucuronopyranoside, apigenin 7-O-[2-O-feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranosyl]-4'-O-beta-D-glucuronopyranoside, apigenin 7-O-[2-O-feruloyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside], apigenin 7-O-[2-O-p-coumaroyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside], and luteolin 7-O-[2-O-feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranosyl]-4'-O-beta-D-glucuronopyranoside, have not been reported before in the plant kingdom. Additionally, five known compounds, including apigenin 7-O-beta-D-glucuronopyranoside, apigenin 4'-O-beta-D-glucuronopyranoside, apigenin 7-O-[beta-D- glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside], luteolin 7-O-beta-D-glucuronopyranoside, and adenosine, were identified.     

The effect of selenium biofortification in alfalfa (Medicago sativa)

Abstract

Biofortification of agricultural productions is important to enhance the quality of the products. The aims of this research were to investigate the possibility of selenium biofortification of alfalfa in the calcareous and non-calcareous soils by applying fertilizers differently (control, soil application, inoculant of seleno-bacteria, foliar application, combined soil, and foliar applications of selenate). Morpho-physiological properties and nutritional responses, such as shoot dry weight, phosphorus, potassium, selenium, zinc, and iron concentration in the plant’s tissue were measured. In all parameters, the use of selenium sources in the calcareous soils had a significant effect compared with the control treatment. Among the methods and sources of selenium, foliar application of sodium selenate gave the best response. By the usage of this treatment, Se concentration was increased up to 112.5% and 182%, respectively, compared with the control treatment in non-calcareous and calcareous soil. According to the results, inoculant of selenium bacteria and sodium selenate are recommended as a natural way to selenium biofortification in alfalfa and to improve the quality of the produced forage. In addition, attention to the biological potential of the soil and the use of native soil bacteria is also recommended.     

Alfalfa (Medicago sativa L.) flavonoids. 2. Tricin and chrysoeriol glycosides from aerial parts.

Ten flavone glycosides have been isolated and identified in aerial parts of alfalfa. These included six tricin, one 3'-O-methyltricetin, and three chrysoeriol glycosides. Most of these compounds were acylated with ferulic, coumaric, or sinapic acids, and acylation occurred on the terminal glucuronic acid. Eight of these compounds, including 7-O-beta-D-glucuronopyranosyl-3'-O-methyltricetin, 7-O-beta-D-glucuronopyranosyl-4'-O-beta-D-glucuronopyranosidechrysoeriol, 7-O-[2'-O-feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]chrysoeriol, 7-O-[2'-O-feruloyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]chrysoeriol, 7-O-[2'-O-sinapoyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, 7-O-[2'-O- feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, 7-O-[2'-O-p-coumaroyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, and 7-O-[2'-O-feruloyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, have not been reported previously in the plant kingdom. Two previously identified alfalfa flavones, 7-O-beta-D-glucuronopyranosidetricin and 7-O-[beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, were also isolated.     

Soil pH,rhizobia, and vesicular-arbuscular mycorrhizae inoculation effects on growth and heavy metal uptake of alfalfa (Medicago sativa L.)

Summary The interaction between soil pH and inoculation with rhizobia and vesicular-arbuscular mycorrhizae (VAM) was studied in an industrially polluted soil contaminated with high levels of Zn and Cd. A silt loam soil (pH 6.7) was amended with Ca(OH)₂ or elemental S to adjust the soil pH to 4.3, 5.3, 6.0, and 7.2. Alfalfa (Medicago sativa L.) was planted in each treated soil an subsequently inoculated with Rhizobium meliloti and/or a mixed VAM spore population. Alfalfa growing in soils at a pH of 4.3 and 5.3 failed to survive as a result of soil acidity and heavy metal toxicity. At the three higher pH values, growth and foliar N and P were significantly increased by inoculation with rhizobia or VAM. The greatest increase was observed when both VAM and rhizobia were inoculated together into the soil. With a soil pH of 6.0 and 6.7, the available heavy metal concentration in the soil was high and the VAM significantly decreased heavy metal uptake from these soils. The foliar concentration of Zn was reduced from 455 to 306 g g^–1 by inoculation with VAM (pH 6.0). At the highest soil pH (7.2), however, available heavy metal concentrations were generally lower and NAM significantly increased the heavy metal uptake. The influence of VAM on heavy metal uptake thus appears to be partly a function of the available heavy metal content in the soil.     

外源lea3基因转化紫花苜蓿的研究

将来源于大麦的lea3基因通过基因枪法导入紫花苜蓿栽培品种“中苜一号”的胚性愈伤组织中,经过膦丝菌素类除草剂(PPT)的4次筛选获得抗性愈伤组织,诱导分化后共获得21株抗性再生植株。经叶片涂抹除草剂试验和lea3基因的PCR分子检测证明,lea3基因已整合到紫花苜蓿的基因组中。与对照植株相比,获得的转基因植株具有显著增强的耐盐能力,表明遗传转化lea3基因可用于苜蓿抗旱耐盐新品种的选育。     

Presence of phylogeographic structure among wild diploid alfalfa accessions (Medicago sativa L. subsp. microcarpa Urb.) with evidence of the center of origin

The genetic structure of wild germplasm on a macrogeographic scale has implications for collection and conservation of wild genetic resources of economically important plants. Cultivated alfalfa is derived from a taxonomic group called the Medicago sativa–falcata complex, which includes a number of morphologically and genetically differentiated diploid and tetraploid subspecies representing the primary gene pool for alfalfa improvement. Although the origin of the taxa included in M. sativa–falcata complex has been described broadly, the center of diversity for each taxon is not clear. In this study, 60 individual genotypes from 16 accessions of the diploid taxon M. sativa subsp. microcarpa Urb. [M. sativa subsp. caerulea (Less. ex Ledeb.) Schmalh.] were obtained from the two proposed centers of diversity, Caucasia and Central Asia, and were genotyped with 89 simple sequence repeat markers. Phylogenetic reconstructions together with population genetics statistics (heterozygosity, allelic diversity, and F_ST) were used to describe the pattern of present genetic diversity, to deduce biogeographic history, and to infer the center of diversity. Our results revealed that the Central Asian accessions were distinct from the Caucasian accessions, and that overall accessions, genetic distance was positively correlated with geographical distance, indicating a spatial genetic structure. The accessions from the Caucasian region had higher mean F_ST values, higher allele diversity and higher heterozygosity, suggesting that the Caucasian region is the likely center of diversity for M. sativa subsp. microcarpa [M. sativa subsp. caerulea] and the accessions recolonized Central Asia from Caucasia after the glacial retreat.     

Micronutrient topdressing of alfalfa (medicago sativa L.) on a udollic albaqualf

Abstract

An established stand of alfalfa (Medicago sativa L.) was topdressed with two rates each of borax, zinc sulfate, and copper sulfate at two levels of applied P and K. Yields and plant composition were determined at five harvests over two seasons. Treatments were not effective in increasing yields but topdressing with B and Zn increased the concentration of these elements in the plant tissue. The Cu concentration in alfalfa tissue was not affected by topdressing with copper sulfate. Phosphorus application rates of about four times removal rates were needed to maintain the Bray 2 soil test level of the soil.