A comparative study of the tolerance of salt marsh plants to manganese

Summary Salicornia europaea, Puccinellia maritima, Triglochin maritima, Aster tripolium, Plantago maritima, Armeria maritima, Juncus gerardii andFestuca rubra, collected as seed from a salt marsh at Portaferry, County Down, were grown on saline (340 mM NaCl) and non saline nutrient solutions at five concentrations of manganese sulphate (0.025–10.0 mM). After an eight week growing period, shoot and root yields and the concentrations of sodium, potassium, calcium and manganese in the shoots were determined.Except forS. europaea the saline treatments had a strongly limiting effect on plant growth. Each of the species investigated showed a degree of tolerance to high concentrations of manganese which was similar to that of calcifuge species and plants characteristic of waterlogged sand dune slack communities, but which was very much greater than that ofArrhenatherum elatius a species usually excluded from acidic soils. There was little evidence to support the hypothesis that tolerance of high manganese concentrations was correlated with the position of the experimental plants in the salt marsh ecotone or that the manganese nutrition of halophytic and glycophytic marsh species differs. Whilst manganese uptake increased proportionally with solution manganese concentration, there were few other major effects of manganese on the balance of shoot cation concentrations in the plants investigated. Both antagonistic and synergistic effects of sodium on manganese uptake were recorded for different species.     

Population differentiation within Festuca rubra L. with regard to soil salinity and soil water

Summary Seed and transplanted adult plants from populations of Festuca rubra, collected from inland, salt-marsh and sand-dune sites were grown on culture solution with added sodium chloride. The growth of the populations of the three habitats was reduced differentially by salt. The salt marsh ecotype Festuca rubra ssp. litoralis was only slightly affected and the inland ecotype F. rubra ssp. rubra was severely retarded at 60 mM NaCl. The dune ecotype F. rubra ssp. arenaria had an intermediate tolerance. The tolerant ecotypes accumulated less sodium chloride as compared to the sensitive ecotype, suggesting that salt tolerance is caused in part by salt exclusion.In addition, the dune ecotype F.r. arenaria appeared to be more drought tolerant than the salt marsh ecotype. Abscission of salt-saturated leaves does not function as an adaptation to salinity in Festuca rubra.All three ecotypes accumulated proline with increased salinity. The response was most pronounced in the drought tolerant F.r. arenaria, indicating that proline accumulation is a response to osmotic stress rather than to ion-specific effects of salinity. The observed differences in salt tolerance may be explained by differential sensitivity to toxic effects of sodium chloride.The occurrence on a beach plain of closely adjacent populations of F.r. arenaria and F.r. litoralis, differing markedly in salt tolerance, is briefly discussed.     

锰胁迫下盐肤木锰富集及生理响应特征

为揭示盐肤木(Rhus chinensis)锰积累特征与耐受机制,该研究通过盆栽试验方法,分析0(CK)、1、5、10和20 mmol·L^-1Mn²⁺胁迫对半年生盐肤木幼苗生长、生理生化特征及其锰富集特征的影响。结果表明：(1)盐肤木在Mn²⁺浓度为0～10 mmol·L^-1条件下生长发育状况良好,且在5 mmol·L^-1Mn²⁺处理下叶片舒展,叶片颜色较深,生长最佳,而在20 mmol·L^-1Mn²⁺条件下部分叶片出现褐色斑点、萎蔫卷边的现象;随着Mn²⁺浓度的升高,盐肤木幼苗的生物量呈先升高后下降的趋势,并在5 mmol·L^-1Mn²⁺胁迫时最高。(2)随着Mn²⁺浓度的升高,盐肤木叶片中光合色素含量呈先升后降的趋势,且在Mn²⁺浓度为5 mmol·L^-1时达到峰值。(3)随着M...     

Intracellular sequestration of manganese and phosphorus in a metal-resistant fungus <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis> from deep-sea sediment

A heavy metal resistant fungus was isolated from the sediment of Pacific Ocean, and identified to be Cladosporium cladosporioides. It grew normally in a medium containing 60 mM Mn²⁺ and could endure 1,200 mM as the highest concentration tested. Quantification analysis confirmed a high accumulation of Mn which was 58 mg/g in dried biomass. Under transmission electron microscope, many intracellular crystals were observed in the cytoplasm of the hypha cells grown in a Mn-rich medium, and varied from a few nanometers to 200 nm in length. Energy dispersive X-ray (EDX) analysis showed that the crystals were composed of manganese and phosphorus in atomic ratio of 1.6:1 (Mn/P). Further, factors which might influence the resistance of this fungus were investigated. As a result, its high resistance to Mn²⁺ was found dependent on the presence of Mg²⁺, and could be further enhanced by phosphate. However, the effect of phosphate was not observed without the presence of Mg²⁺. In addition, the resistance was also influenced by pH of the medium, which was lost above pH 8. This is the first report on a fungus which showed a hyper resistance to manganese by forming a large quantity of intracellular Mn/P crystals.     

Development and effectiveness of a soluble manganese silicate compound in controlling manganese deficiency in plants

Summary A study was made of soluble manganese silicates prepared by adding a solution of manganese sulfate to different concentrations of sodium silicate in alkaline medium. The solubility of manganese increased with increasing amounts of sodium silicate and reached its maximum at a molar Na₂SiO₃/MnSO₄ ratio between 10 and 15. At molar Na₂SiO₃/MnSO₄ ratios of 5 and more a red-brown coloured manganese silicate is formed, which is stable in the pH range of 4 to 12. Gel chromatographic analysis indicated that this compound contains 1 molecule Mn per 15 molecules SiO₂. At a molar Na₂SiO₃/MnSO₄ ratio of 15 the yield of soluble manganese could be raised to 98.5% of the theoretical amount by preventing the rapid oxidation of the Mn²⁺-ions in the alkaline solution. A pot experiment with oats was conducted to evaluate the effectiveness of the red-brown manganese silicate in controlling manganese deficiency. The effect of the Mn-carrier on Mn-content and yield of grain and on the colour scores was compared with that of Mn-EDTA, Mn-DTPA and MnSO₄·4H₂O. The manganese silicate was found to be a more effective manganese source than Mn-EDTA, Mn-DTPA or MnSO₄·4H₂O.     

九州虫草菌丝体对Mn的耐性及富集

重金属耐性真菌的研究是生物修复的重要研究内容。本文研究了九州虫草（Cordyceps kyusyuensis）对于Mn的耐性及富集。在液体培养基中添加不同浓度（0—60 g/L）的Mn离子,测定其菌丝生物量、菌丝Mn含量、菌丝抗氧化酶活性和过氧化水平以及菌体细胞离子交换量、Mn在细胞中的分布的变化情况。实验结果表明九州虫草菌丝生物量与Mn浓度呈显著负相关,Mn浓度60 g/L为九州虫草菌丝生长极限浓度。菌丝中Mn含量随培养基中Mn浓度的增大而显著升高,10 g/L Mn时,菌丝细胞中Mn积累量达到细胞干重的1.0013%。九州虫草菌丝中过氧化产物丙二醛（MDA）、可溶性蛋白（SP）含量、可溶性糖浓度与培养基中Mn浓度呈负相关,实验组与对照组差异显著。抗氧化酶（过氧化氢酶（CAT）、过氧化物酶（POD）、超氧化物歧化酶（SOD））活性随着培养基中Mn浓度增大而显著升高,但变化趋势不同。九州虫草菌丝细胞不可溶性组分中Mn的量（91.51%—98.6%）显著高于可溶部分（1.40%—8.49%）。九州虫草菌丝细胞壁离子交换量（CEC）随着培养基中Mn浓度的升高变化不明显。说明在九州虫草菌丝对Mn的富集过程中,其细胞壁、细胞膜和细胞器对于Mn结合发挥了主要作用,细胞质中可溶性成分对Mn的结合发挥次要作用。在Mn的胁迫下,增强抗氧化酶系统的协同作用以清除大量自由基是细胞对锰耐性的重要机制。     

Chronic exposure to soil salinity in terrestrial species: Does plasticity and underlying physiology differ among specialized ground-dwelling spiders?

In salt marshes, the alternation of low and high tides entails rapid shifts of submersion and aerial exposure for terrestrial communities. In these intertidal environments, terrestrial species have to deal with an osmotic loss in body water content and an increase in sodium chloride concentration when salt load increases. In salt marshes, spiders represent an abundant arthropod group, whose physiological ecology in response to variations of soil salinity must be further investigated. In this study, we compared the effect of salinity on the survival and physiology of three species of Lycosidae; two salt marsh species (Arctosa fulvolineata and Pardosa purbeckensis) and one forest species (P. saltans). Spiders were individually exposed at three salinity conditions (0‰, 35‰ and 70‰) and survival, changes in body water content, hemolymph ions (Na⁺, Ca²⁺, Mg²⁺, K⁺; ICP-MS technique) and metabolites (mainly amino acids, polyols, sugars; LC and GC techniques) were assessed. The survival of the forest species P. saltans was very quickly hampered at moderate and high salinities. In this spider, variations of hemolymph ions and metabolites revealed a quick loss of physiological homeostasis and a rapid salt-induced dehydration of the specimens. Conversely, high survival durations were measured in the two salt-marsh spiders, and more particularly in A. fulvolineata. In both P. purbeckensis and A. fulvolineata, the proportion of Na⁺, Ca²⁺, Mg²⁺, K⁺ remained constant at the three experimental conditions. Accumulation of hemolymph Na⁺ and amino acids (mainly glutamine and proline) demonstrated stronger osmoregulatory capacities in these salt-marsh resident spiders. To conclude, even if phylogenetically close (belonging to the same, monophyletic, family), we found different physiological capacities to cope with salt load among the three tested spider species. Nevertheless, physiological responses to salinity were highly consistent with the realized ecological niches of the spiders.     

锰胁迫对盐肤木种子萌发、幼苗生长及理化特性的影响

为揭示植物适应锰胁迫的生理机制,通过在不同Mn²⁺浓度(0、1、5、10、15、20 mmol/L)下开展盐肤木(Rhus chinensis)种子萌发以及幼苗生长实验,检测锰胁迫处理7、15、30 d后幼苗生理生化特性的变化。结果表明:(1)随着Mn²⁺浓度的升高,盐肤木种子发芽率变化不显著,在80.0%-81.6%之间,发芽势、发芽指数和活力指数则呈先升后降的趋势;其幼苗生物量也呈现先升后降的趋势;(2)随着Mn²⁺浓度的升高与胁迫时间的延长,盐肤木幼苗叶绿素a、叶绿素b含量均呈现先增加后降低的趋势,类胡萝卜素含量呈现下降的趋势;(3)胁迫7 d时,随着Mn²⁺浓度的升高,盐肤木幼苗超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性均显著上升;胁迫15、30 d时,高Mn²⁺浓度(15-20 mmol/L)下POD、CAT活性则均降低;(4)胁迫7 d时,随着Mn²⁺浓度的升高,可溶性糖、可溶性蛋白、游离脯氨酸含量升高;胁迫15、30 d时,在Mn²⁺浓度为20 mmol/L时可溶性蛋白与游离脯氨酸含量显著降低;(5)随着Mn²⁺浓度的升高与胁迫时间的延长,丙二醛(MDA)含量均升高。研究说明盐肤木具有较强的耐受锰胁迫能力,它可通过增强抗氧化酶活性、积累渗透调节物质含量来应对锰胁迫。     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export. Received: 9 January 1998 / Accepted: 4 May 1998     

Germination and early establishment of lower salt-marsh species in grazed and mown salt marsh

Abstract. Lower salt-marsh species occur more in the grazed than in the mown sites of a salt marsh in Schiermonnikoog in the Netherlands. This was demonstrated by a sowing experiment which determined which characteristic of the stand structure, height of the canopy or percentage of bare soil, is responsible for this difference. The total number of seedlings which emerged was negatively related to the height of the canopy and positively to the percentage of bare soil. The survival of seedlings of Suaeda maritima and Plantago maritima could be explained by the height of the canopy and for the latter species also by the percentage of bare soil. The survival of Aster tripolium and Plantago maritima was higher in grazed than in mown sites. Since the amount of bare soil was higher than seemed necessary for germination and survival, it was concluded that the taller canopy was responsible for the absence of lower salt-marsh species in the mown sites.     

Nucleolytic activities and appearance of a new DNase in relation to nickel and manganese accumulation inAlyssum murale

Serpentine and non-serpentine plants of Alyssum murale, a nickel (Ni) accumulator plant, from North Greece, were studied in order to examine: (1) The ability of natural plants to accumulate metals; (2) the ability of their seedlings to tolerate increasing concentrations of Ni²⁺ or Mn²⁺ (0, 0.16, 0.32, 0.5 and 1 mmol/L), when grown in nutrient solution; (3) the activities and electrophoretic patterns of root and shoot DNases and RNases under the above conditions. Measurements of metal concentrations in serpentine and non-serpentine natural plants and the respective soils revealed: (1) Very low calcium (Ca)/magnesium (Mg) (0.16) ratio and high concentration of Ni in serpentine soil; (2) very high Ca/Mg (17) ratio and high concentration of manganese (Mn) in non-serpentine soil; (3) the ability of serpentine natural plants to accumulate Ni and the inability of plants of both serpentine and non-serpentine populations to accumulate Mn. A. murale plants grown in nutrient solution with increasing Ni²⁺ or Mn²⁺ concentrations showed a negative correlation between the Ni²⁺ or Mn²⁺ concentrations in the nutrient solution, and the chlorophyll concentration, shoot and especially root length. The accumulation of Ni²⁺ or Mn²⁺ in the plant showed a positive correlation with increasing Ni²⁺ or Mn²⁺ concentrations in the nutrient solution. Application of 0.5 mmol/L Ni²⁺ or Mn²⁺ resulted in the inhibition of DNase activities and the appearance of a new DNase form, in both root and shoot detected by electrophoresis in active ssDNA polyacrylamide gel. The new gel-extracted DNase showed nicking action against plasmid DNA and has been characterised as an endo-DNase. In contrast, electrophoretic patterns and RNase activities were unaffected. According to our studies on growth, both serpentine and non-serpentine plants of A. murale have a constitutive ability to tolerate and accumulate Ni²⁺ or Mn²⁺; they have similar DNase and RNase electrophoretic patterns and show a new DNase form under Ni²⁺ or Mn²⁺ stress. This is the first report on the response of nucleolytic enzymes under metallic elements hyperaccumulation.     

Manganese‐oxidizing bacteria mediate the degradation of 17α‐ethinylestradiol

Manganese (II) and manganese‐oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17α‐ethinylestradiol (EE2) at trace concentrations. Mn²⁺‐derived higher oxidation states of Mn (Mn³⁺, Mn⁴⁺) by Mn²⁺‐oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn²⁺‐dependent degradation of EE2 was found to be a slow process, which requires multi‐fold excess of Mn²⁺ and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2‐derived degradation products were shown to no longer exhibit undesirable estrogenic activity.     

Conversion of adamsite (phenarsarzin chloride) by fungal manganese peroxidase

Fungal manganese peroxidase was found to convert the persistent chemical warfare agent adamsite (phenarsarzin chloride) in a cell-free reaction mixture containing sodium malonate, Mn²⁺ ions, and reduced glutathione. The organo-arsenical compound disappeared completely within 48 h accompanied by the formation of a more polar metabolite with a clearly modified UV spectrum. Thus, As(III) in the adamsite molecule was oxidized by manganese peroxidase to As(V) which added dioxygen and released chloride.     

Accumulation and precipitation of Mn2+ by the cells of Oscillatoria terebriformis

The cyanobacterium Oscillatoria terebriformis was shown to exhibit resistance to high manganese concentrations, remaining viable at 2.5 mM MnCl₂ in the medium. Cyanobacterial cells were capable of considerable manganese consumption from the medium. The dynamics of Mn sorption by the cells were the same in all experimental variants, independent of the manganese concentration. Manganese concentration in the biomass peaked after 2–3 days and depended on Mn²⁺ concentration in the medium and on the amount of biomass introduced. In the case of O. terebriformis, manganese removed from the medium may be subdivided into Mn absorbed by the cell, Mn bound to the cell wall, Mn absorbed by the glycocalix, and chemically precipitated Mn. Of the total 21.25 ± 1.0 mg of consumed manganese, biological absorption and chemical precipitation were responsible for 11.78 ± 0.98 and 9.2 ± 0.8 mg, respectively. In the presence of cyanobacteria, Mn removal from the medium was 2.28 times higher than in the control. This process depended considerably on Mn sorption by exopolysaccharides. At 1.3 mM Mn²⁺, a lamellar mat was formed with interlayers of manganese carbonate.     

锰对外生菌根真菌生长、养分吸收、有机酸分泌和菌丝体中锰分布的影响

外生菌根真菌对于酸性和锰污染土壤的植树造林和生态恢复有重要作用。采用液体培养方法,以大白菇Rd Fr(Russula delica Fr.)、彩色豆马勃Pt 715(Pisolithus tinctorius 715)、土生空团菌Cg Fr(Cenococcum geophilum Fr.)和厚环粘盖牛肝菌Sg Kl S(Suillus grevillei(Kl.)Sing)为供试对象,研究了Mn2+对外生菌根真菌生长、养分吸收、有机酸和氢离子分泌的影响,以及锰在菌丝细胞内外的分布情况。结果表明:在0—800 mg Mn2+/L的培养液中,Mn2+对Rd Fr生长无显著影响;低浓度的Mn2+刺激Sg Kl S生长,中、高浓度无抑制作用;但大幅度降低Pt 715和Cg Fr的生物量,说明Rd Fr和Sg Kl S抗(耐)锰的能力较强。在Mn2+胁迫下,供试菌株的氮、钾含量和吸收量显著降低;含磷量和吸收量,以及草酸和柠檬酸的分泌速率因菌株不同而表现出多样性,说明在减轻Mn2+毒的过程中,磷酸盐(或聚磷酸盐)对Mn2+固定作用和有机酸的络合作用因菌株不同而异。但是,Mn2+显著降低Rd Fr和Sg Kl S的氢离子分泌速率,菌丝和原生质中的含Mn量显著低于敏感性菌株,说明降低Mn2+的活性和减少吸收可能是外生菌根真菌抗(耐)Mn2+的重要机制。此外,菌丝吸收的Mn2+绝大部份存在于质外体,少量进入细胞,前者是后者的5.23—9.21倍,说明原生质膜是外生菌根真菌防御Mn2+进入细胞的重要屏障。     

Population-specific Traits and their Implication for the Evolution of a Drought-adapted Ecotype in Armeria maritima

Traits contributing to drought resistance of Armeria maritima were investigated by comparing six populations of this species from sandy grasslands, heavy metal mines and salt marsh sites. The sandy soil ecotype that is influenced by periodic drought was found to allocate constitutively a higher proportion of biomass to the root system, especially to the fine roots, than the other two ecotypes. The selective advantage of this lower shoot-root ratio is presumably the delayed onset of the critical water saturation deficit because of the ability to take up additional water from deeper soil layers. Under water stress, all populations of A. maritima showed a decrease in the shoot-root ratio. Additionally, under moderate long-term water stress a decrease in (= more negative) osmotic potential was found in leaves of plants from all populations. Lowest osmotic potentials were shown by the salt marsh ecotype and the highest by one of the heavy metal populations. Osmotic adjustment was achieved passively by a reduction in osmotic volume. Accumulation of osmotically active compounds was seen only under severe water stress, when the considerable betaine contents found in all populations of A. maritima even under control conditions were further increased by severe drought. The evolution of traits related to drought adaptation in the sandy soil ecotype of A. maritima is discussed.     

Sporulation ofBacillus subtilis as a possible measure of available manganese in calcareous soils

Summary Sporulation ofBacillus subtilis ATCC 9799 in nutrient broth was correlated with Mn⁺⁺ concentrations at 10^–7 to 10^–6 M. This specific response was employed for the bioassay of plant available soil manganese. A correlation between sporulation ofB. subtilis in growth medium supplemented with soil samples sterilized by gamma rays or by ethylene oxide, and growth response ofAvena sativa to added manganese was observed in 5 of 6 loessial and rendzina highly calcareous soil types. No correlation between chemical tests and response to manganese fertilization of this soil could be observed.The possible use of this method for the bioassay of plant-available soil manganese is discussed.     

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn²⁺ in the basal medium to 32% with 16 µM Mn²⁺ supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn²⁺ concentration. By varying Mn²⁺ and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn²⁺ concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn²⁺ on high mannose glycan content depends on glucose availability. At each Mn²⁺ supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non‐limiting concentration. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:460–467, 2015     

Variation in the natural abundance of 15N in the halophyte,Salicornia virginica,associated with groundwater subsidies of nitrogen in a southern California salt-marsh

To provide insight into the importance of the salt-marsh ecotone as a sink for inorganic nitrogen in perched groundwater, measurements were made of the natural abundance of ¹⁵N in dissolved NO₃-N and NH₄-N and in the salt-marsh halophyte, Salicornia virginica, along an environmental gradient from agricultural land into a salt-marsh. The increase in the natural abundance of ¹⁵N (expressed by convention as ¹⁵N) of NO₃-N, accompanied by the decrease in NO₃-N (and total dissolved inorganic N, DIN) concentration along the gradient, suggested that the salt-marsh ecotone is a site of transformation, most likely through denitrification, of inorganic nitrogen in groundwater. ¹⁵N enrichment in S. virginica (and the parasitic herb, Cuscuta salina), along the tidal marsh boundary, relative to high and middle marsh locations, indicated the retention of groundwater nitrogen as vegetative biomass. The correlation between ¹⁵N _Salicornia and ¹⁵N_NH4 suggested a preference for NH₄-N over NO₃-N during uptake by this plant. Groundwater inputs enhanced the standing crop, above-ground productivity, and nitrogen content of S. virginica but the ralative effects of pore water salinity and DIN concentration on these parameters were not determined. ¹⁵N enrichment of marsh plants by groundwater DIN inputs could prove useful in tracing the fate of these inputs in the marsh food web.     

Mycorrhizal and nonmycorrhizal host growth in response to changes in pH and P concentration in a manganiferous oxisol

Glomus aggregatum and Leucaena leucocephala were allowed to interact in a manganese-rich oxisol at pH 4.3–6.0 and at soil P concentrations considered optimal for mycorrhizal host growth and sufficient for nonmycorrhizal host growth. At 0.02 mg P l^-1, vesicular-arbuscular mycorrhizal fungal (VAMF) colonization of roots increased as soil pH increased from 4.3 to 5.0. However, VAMF colonization of roots did not respond to further increases in pH. At pH 6.0, growth of mycorrhizal Leucaena observed at 0.02 mg P was comparable with that observed at 0.8 mg P l^-1. Increasing P concentration from 0.02 to 0.8 mg P 1^-1 increased target soil pH from 4.3 to 4.7 and reduced the concentration of available soil Mn from 15.1 to 1.9 mg 1^-1. Thus, the normal plant growth observed at the higher P concentration at pH<5 was mainly due to the alleviation of Mn toxicity as a result of its precipitation by excess P. VAMF colonization levels observed at pH 5.0–6.0 were similar, but maximal plant growth occurred at pH 6.0, suggesting that the optimal pH for mycorrhizal formation was substantially lower than for VAMF effectiveness. The poor growth of Leucaena at the lower P concentration in the unlimed soil was largely due to high concentrations of Mn²⁺ and H⁺ ions.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3910