Plant Science 167 (2004) 977–986

In vitro characterization of manganese toxicity in relation tophosphorus nutrition in potato (Solanum tuberosum L.)

D. Sarkara,∗, S.K. Pandeya, K.C. Sudb, A. Chanemougasoundharama

a Division of Crop Improvement, Central Potato Research Institute, Shimla 171 001, Himachal Pradesh, Indiab Division of Crop Production, Central Potato Research Institute, Shimla 171 001, Himachal Pradesh, India

Received 15 January 2004; received in revised form 10 May 2004; accepted 11 May 2004

Available online 9 June 2004

Abstract

This study investigated the effect of P (0.0, 2.5 and 6.25 mM) on potato response to excess Mn (0.1, 1.0, 2.5 and 5.0 mM) at two pH (4.0and 5.8) conditions in vitro using KH232PO4 and54MnSO4·H2O as dual isotopic tracers. Toxicity due to excess Mn supply resulted in stemstreak necrosis with significant inhibition in microplant rooting and growth. Addition of P had a significant beneficial effect in controllingthe severity and progression of Mn toxicity symptoms. This beneficial effect of P in relation to Mn supply was, however, pH-dependent andgenotype-specific with its effect more prominent at pH 4.0 than 5.8.54Mn uptake in microplants was much higher at pH 5.8 than 4.0 dueto interference by H+ ions under low pH. P enhanced the uptake of54Mn under an excess supply of Mn up to 2.5 mM, but this increasedconcentration was not accompanied with any concomitant increase in severity of toxicity symptoms, suggesting that P exerted its synergisticeffect by inactivating Mn within the plant. Mn at a very high level (5.0 mM) depressed the32P concentration in microplants and tissue contentof total inorganic P, and was, therefore, irreversibly toxic even in the presence of high levels of external P supply. Restricted P accumulationinside the plant could be the reason as to why, in general, P was unable to check the progression of toxicity symptoms under excess Mn supplybeyond a critical level.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Manganese toxicity; Microculture; Phosphorous nutrition; Potato; Stem streak necrosis; Tissue culture

1. Introduction

Manganese (Mn) is an essential trace element for plantsystems. It is involved in photosynthesis, respiration andactivation of several enzymes including superoxide dismu-tase, NADPH-specific decarboxylating malate dehydroge-nase and nitrate reductase[1]. However, Mn is toxic whenin excess[2], and consequently it represents an importantfactor in environmental contamination with various phyto-toxic effects[3,4]. Mn toxicity is often more common thanMn deficiency in acid soils for a number of plant species[5,6]. A typical symptom of Mn toxicity in vascular plantsis intervenial chlorosis resembling Fe deficiency[7,8]. Thisis because Mn is known to play an important role in the

∗ Corresponding author. Tel.:+91 177 2625692x506;fax: +91 177 2624460.

E-mail addresses: d.sarkar@excite.com, debabratas@yahoo.com(D. Sarkar).

biosynthesis of chlorophyll and several enzymes along theisoprenoid biosynthetic pathway[9,10]. Thus excess Mninhibits chlorophyll synthesis, and induces a decline inphotosynthetic rate[10].

Potato (Solanum tuberosum L.) plants are very sensitiveto excess manganese[11], and Mn toxicity in potatoeshas been reported on many acid soils[12–14]. It has beenshown that stem streak necrosis in potatoes may be causedby Mn toxicity [13–16]. This disorder is characterizedby an early necrotic flecking on stems and petioles thatsoon develop into long, narrow streaks; in severe casesirregular small brown necrotic areas appear between themidrib on the leaves followed by leaf abscission. Com-plete stem browning and leaf abscission ultimately resultin premature death of the plant. Despite these studies in-dicating that stem streak necrosis of potato is associatedwith excess of soluble Mn occurring at low pH levels,they are inconclusive because stem streaking has also beenshown to be a symptom common to several disorders

0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.plantsci.2004.05.022

978 D. Sarkar et al. / Plant Science 167 (2004) 977–986

of the potato plant caused by fungal and viral infections[16–18].

The incidence of Mn toxicity shows a strong dependenceon environment, plant species, genotype and accompanyingfertilizer nutrients[1,5]. In potato, Mn toxicity has beenshown to be influenced by amino acids[19], various anions[20], soil pH [14,21], temperature[22] and aluminum[11].However, of different factors influencing Mn uptake and tox-icity in potato, the role of phosphorus (P) is still unresolvedand contentious. Enhanced uptake of Mn under high soil Phas been demonstrated by several workers, especially whenMn is deficient[23,24]. Although the potential beneficial ef-fect of P on the incidence of Mn toxicity has been suggested[5,25], there are no reports, as to the best of our knowledge,confirming such effect in potato. Employing microcultureexperiments Marsh et al.[26] have shown that Mn toxicityin potato plants have little dependence on phosphate levels.However, in these microculture experiments, Mn× P inter-action was studied under low levels of external supplies ofMn (0.132–2.0 mM), and a general Mn toxicity rating, in-stead of stem streak necrosis per se, was assessed to examinethe interaction effects. In addition, these experiments useda single cultivar ofS. tuberosum L. In this context, it needsto be emphasized that phosphate uptake is also known to beinfluenced by pH[27]. However, there are no data availableto evaluate Mn uptake and toxicity in relation to inorganicphosphorous nutrition under varying pH conditions.

In a preliminary study, we examined the effects of awide range of Mn (0.0–12.0 mM) concentrations on potatomicroplants at standard P level (1.25 mM as in MS medium[28]) in a microculture system in vitro, and assessed theprofile and progression of Mn toxicity symptoms, especiallywith reference to stem streak necrosis. We observed thatthe symptoms corresponded to stem streak necrosis, andincreased with Mn concentrations in the medium. Based onthis preliminary study we conducted the present investiga-tion to study Mn uptake and toxicity in relation to inorganicP nutrition under varying pH regimes. The objectives of thisinvestigation were (1) to determine the effects of excess Mnon development and progression of toxicity symptoms withreference to stem streak necrosis, (2) to assess Mn uptakein relation to P nutrition under varying pH conditions, (3)to study P uptake and accumulation in relation to varyingMn levels under different pH conditions, and (4) to relateMn and P uptake/concentration in microplants to toxicitysymptoms.

2. Materials and methods

2.1. Plant material

Two tetraploid (2n= 4x = 48) potato (S. tuberosum L.)genotypes, viz., Kufri Ashoka (S. tuberosum subsp.tubero-sum; early maturing) and Kufri Sindhuri (S. tuberosumsubsp. andigena × S. tuberosum subsp. tuberosum hy-

brid; late maturing) were used in the study. In vitro cul-tures of these genotypes were established from indexedtuber-sprouts, and they were made virus-free by stan-dard meristem culture procedure[29]. Virus-freedom of invitro stock cultures was checked by immunosorbent elec-tron microscopy as described by Garg and Khurana[30].Disease-free stock cultures were maintained and multipliedthrough shoot cuttings on semisolid (7.0 g l−1 Nobel agar;HiMedia, Mumbai) MS[28] medium supplemented with8.39�M d-calcium pantothenate and 30 g l−1 sucrose. Theculture and incubation conditions in vitro were as describedearlier[31].

2.2. 54Mn and 32P labelling (treatments)

The micronutrient Mn was labelled with54Mn (144.0�Ci ml−1 as chloride in dilute HCl solution; 99% radio-chemical purity) obtained from the Board of Radiation andIsotope Technology (BRIT), Bhaba Atomic Research Cen-tre (BARC), Trombay, Mumbai (India). Working solution oflabelled MnSO4 having an activity of 1.44�Ci ml−1 54Mnwas prepared by adding 1.0 ml of54Mn of 144.0�Ci ml−1

to 99 ml of 200 mM MnSO4·H2O. This 54Mn-labelledMnSO4 stock was used to obtain four concentrations of Mn(0.1, 1.0, 2.5 and 5.0 mM) as used in the present experi-ment. The macronutrient P was labelled with carrier-free32P (4.0 mCi ml−1 as orthophosphoric acid in dilute HCl)obtained from the same source as above. Working solutionof labelled KH2PO4 having an activity of 20.0�Ci ml−1

32P was prepared by adding 0.5 ml of carrier-free32P to99.5 ml of 220.59 mM KH2PO4. This32P-labelled KH2PO4stock solution was used to obtain three different concentra-tions of inorganic P (0.0, 2.5 and 6.25 mM) in the presentexperiment. The macronutrients of MS medium[28] weremodified to obtain these three different concentrations ofinorganic P while maintaining the ionic balance of othermacronutrients constant. The macronutrient composition ofdifferent P-media is shown inTable 1.

Table 1Composition of different macronutrients adjusted to obtain media withthree levels of inorganic P in the manganese experiment in potato

Macronutrienta P (mM)

0.0 2.5 6.25

CaCl2·2H2O 3.000 2.375 0.500Ca(NO3)2·4H2O – 0.625 2.500KCl 1.250 – –KH2PO4

b – 2.500 6.250KNO3 18.790 17.540 13.790MgSO4·7H2O 1.500 1.500 1.500NH4NO3 20.620 20.620 20.620

a Concentration is in mM; a constant ionic balance of Ca2+ (2.99 mM),K+ (20.04 mM), Mg2+ (1.50 mM), N (60.03 mM), NH4+ (20.60) andNO3

− (39.41 mM) was maintained as in MS medium[28].b KH2PO4 was labelled with carrier-free32P having an activity of

20.0�Ci ml−1.

D. Sarkar et al. / Plant Science 167 (2004) 977–986 979

2.3. In vitro microculture experiment

Single node cuttings (SNCs) dissected from 21-day-oldaseptically grown microplants were cultured on MS mediumsupplemented with 30 g l−1 sucrose and different concen-trations of54Mn (0.1, 1.0, 2.5 and 5.0 mM) plus32P (0.0,2.5 and 6.25 mM). SNCs were essentially derived from mid-dle nodes of the microplants for maintaining explant homo-geneity. The pH of the medium was adjusted to either 4.0or 5.8 with 0.1 N HCl/NaOH before autoclaving at 121◦Cfor 20 min, and solidified with 2.0 g l−1 gelrite (Duchefa,Haarlem). A single SNC was cultured per tube (25 mm×150 mm) containing 10 ml of medium, and the culture tubeswere closed with Steristoppers (TMHeinz Herenz, Hamburg).The cultures were maintained at 24◦C under a 16-h photope-riod (approximately 60�mol m−2 s−1 light intensity) pro-vided by cool-white fluorescent lamps. The experiment wasconducted in a factorial (2× 2× 3× 4) completely random-ized design with two genotypes, two pH conditions, threelevels of P and four levels of Mn. Each treatment comprisedfive replicate culture tubes.

2.4. Morphological data observations

After 5 weeks’ incubation, observations were recorded onmicroshoot length (mm), number of green leaves/microplant,number of necrotic leaves/microplant, degree of stem necro-sis, degree of rooting and microplant fresh mass (mg).Degree of stem necrosis was scored on a 0–5 scale ofincreasing severity of necrotic streaking on stems, 5 repre-senting complete necrotic flecking of stems leading to tipnecrosis and wilting of the microplant. Leaf necrosis index(LNI) was measured using the formula as described earlier[32]: LNI = √

(x + 0.5)/√

(y + 0.5), wherex = numberof necrotic leaves/microplant andy = number of greenleaves/microplant. Degree of rooting was scored on a 0–5scale of increasing intensity of rooting (0= no rooting and5 = profuse rooting).

2.5. 54Mn and 32P uptake

At harvest, the microplants were cut 1.0 cm above thelevel of the culture medium to prevent potential contam-ination of samples with54Mn and 32P. The microshoots(plus leaves) were weighed, dried at 70◦C for 48 h, digestedwith 3.0 ml tri-acid mixtures of nine parts HNO3: four partsHClO4:1 part H2SO4, and finally made up to 10.0 ml withdouble distilled water. For32P estimation, 3.0 ml scintil-lation cocktail (on a dioxane–naphthalene–butyl PBD ba-sis) was added to 1.0 ml digested sample, and the activity(�-radiation) was counted using a Tri-Carb® Liquid Scintil-lation Analyzer Model 1600 TR (Packard, Connecticut) witha counting efficiency of 66%. The activity (�-radiation) of54Mn was measured using a Berthold LB 951G AutomaticGamma Sample Changer (Laboratorium Prof. Dr. Berthold,Wildbad). The results were reported as nanomole (54Mn)

or�M (32P)/microplant on a fresh weight basis followingthe standard method[32].

2.6. P estimation

Total inorganic P was estimated following the methodas described by Jackson[33]. To 1.0 ml of digested tis-sue sample, 2.0 ml of 5.0% ammonium molybdate wasadded followed by 20.0 ml of distilled water and 0.5 ml of1,2,4-aminonaphtholsulfonic acid solution. After 30 min,the colour (blue) intensity was measured at 660-mu us-ing a Beckman DU®-64 Spectrophotometer (BeckmanInstruments Inc., Fullerton). The results were expressedas�M/microplant on a fresh weight basis.

2.7. Statistical analyses

Before statistical analyses, data on degree of stemnecrosis and degree of rooting were transformed intosquare roots (

√x + 0.5); fresh mass into logarithmic scale

(log10 x); 32P concentration (�M/microplant) and P con-tent (�M/microplant) into square roots (

√x + 0.5); and

54Mn concentration (nanomole/microplant) into logarith-mic scale (log10 x). The growth data on microshoot lengthand leaf necrosis index did not require any data transfor-mation. Statistical analyses of the data were performedusing MSTAT-C (Michigan State University, Michigan,USA). Four-way analyses of variance were carried out toexamine the main and interaction (two-way, three-way andfour-way) effects of genotype, pH, P and Mn on the growthand mineral uptake/accumulation parameters describedabove. Mean separations were carried out by least signif-icant difference (LSD) test, and the term significant hasbeen used to indicate differences for whichP ≤ 0.05. Therelationship between various characters was determined bynon-parametric method of Spearman’s coefficients of rankorder correlation using the standard procedures[34].

3. Results

3.1. Morphological effects

In the absence of P, as expected, a general decline inmicroplant growth occurred at both the pH conditions, andexcept for fresh mass, there was no significant differencein microplant growth response between the two pH levels(Table 2). Even in the absence of P, there was a signifi-cant improvement in fresh mass up to 2.5 mM Mn supply,when the microplants were grown at pH 5.8 as comparedto pH 4.0. At pH 5.8, under control Mn supply (0.1 mM),microshoot length improved significantly in the presenceof 2.5 mM P, whereas at pH 4.0, its significant improve-ment was recorded only at higher level of P (6.25 mM).In contrast, significant improvement in fresh mass, undercontrol Mn supply (0.1 mM), was obtained with 2.5 mM P

980 D. Sarkar et al. / Plant Science 167 (2004) 977–986

Table 2Effects of Mn on potato microplant growth at various concentrations of inorganic P (in mM) under two pH conditions in vitro

Mn (mM) pH 4.0 pH 5.8

0.0 2.5 6.25 0.0 2.5 6.25

Microshoot length (mm)a

0.1 41.5 42.3 63.5 56.4 101.3 87.21.0 38.6 74.3 61.0 50.9 116.1 100.02.5 35.1 53.8 85.1 61.8 102.0 78.25.0 24.5 37.5 34.3 42.0 45.4 46.3

Microplant fresh mass (mg)b

0.1 34.0 (1.44) 121.7 (2.03) 73.6 (1.83) 72.1 (1.81) 152.0 (2.17) 125.3 (2.02)1.0 30.2 (1.45) 112.9 (2.02) 81.1 (1.73) 51.6 (1.67) 142.5 (2.14) 126.3 (2.05)2.5 16.5 (1.14) 59.2 (1.61) 109.1 (1.96) 44.1 (1.58) 100.7 (1.99) 77.4 (1.79)5.0 29.0 (1.44) 36.0 (1.54) 23.2 (1.32) 27.3 (1.37) 38.0 (1.48) 38.5 (1.55)

a LSD0.05 = 19.4.b Values in parenthesis are log10 x transformed values; LSD0.05 = 0.20.

at both the pH conditions. Microshoot length, under boththe P levels (2.5 and 6.25 mM), was significantly inhibitedat pH 4.0 with 0.1–1.0 mM Mn. However, no significantdifference in fresh mass was observed between pH 4.0 and5.8 at low to intermediate supply of Mn (0.1–1.0 mM) incombination with P. In general, there was a gradual declinein microplant growth with increasing concentrations of Mnsupply under both the pH conditions. At pH 4.0, a signif-icant reduction in microplant growth occurred when themedium was supplemented with Mn and P each at 2.5 mM,and this Mn-induced detrimental effect was found to be sig-nificantly improved with increasing P supply (6.25 mM). Incontrast, at pH 5.8, the detrimental effect of Mn in relationto P nutrition was not consistent over various microplantgrowth parameters. At pH 5.8, no significant decline inmicroshoot length and fresh mass occurred under 2.5 mMMn supply in combination with 2.5 mM P, and increasingP supply (6.25 mM) was rather accompanied with signif-icant reduction in both the parameters. Irrespective of pHand P levels, an inhibition of microplant growth occurredwhen the medium was supplemented with 5.0 mM Mn(Table 2).

3.2. Root growth response

Rooting was significantly inhibited in the absence of Psupply irrespective of Mn concentrations (Fig. 1). A sig-nificant improvement in rooting occurred under control Mnsupply (0.1 mM), when the medium was supplemented with2.5 mM P, and further increase in P supply (6.25 mM) didnot result in any concomitant improvement in rooting. Atboth the P levels, a gradual decline in rooting occurred withincreasing concentrations of Mn. At 2.5 mM P, a progressivedecline in root growth occurred with≥2.5 mM Mn supply.In contrast, under 6.25 mM P supply, Mn had an adverseeffect on rooting at 1.0 mM, and further decline in rootingoccurred with >1.0 mM Mn supply. Irrespective of P levels,Mn at 5.0 mM significantly inhibited rooting.

3.3. Mn toxicity effects

Irrespective of P levels, there was no significant changein leaf necrosis index under control Mn supply (0.1 mM) atboth the pH regimes (Fig. 2). In the absence of P, at pH 4.0,there was a significant increase in leaf necrosis at 2.5 mMMn supply in both the genotypes, whereas at pH 5.8 signif-icant increase in leaf necrosis occurred only at higher Mnsupply (5.0 mM) in cv. Kufri Ashoka, but not in cv. KufriSindhuri. In cv. Kufri Sindhuri, at pH 4.0, incorporation ofP in the medium significantly decreased the leaf necrosis at2.5–5.0 mM Mn supply; but, at pH 5.8, P decreased the leafnecrosis only under 1.0 mM Mn supply, and rather a signif-icant increase in leaf necrosis occurred at 5.0 mM Mn sup-ply in combination with 2.5 mM P. In cv. Kufri Ashoka, leafnecrosis was significantly increased under 2.5–5.0 mM Mnsupply at both the P levels (2.5 and 6.25 mM) irrespective ofpH conditions. However, the beneficial effect of P nutrition

Fig. 1. Interaction between Mn and P on degree of rooting in potatomicroplants. Degree of rooting was scored on a 0–5 scale of increasingintensity of rooting with 0 representing complete inhibition of rooting.Data were analyzed in square root (

√x + 0.5) transformed scale and pre-

sented in original scale. Means with common letters are not significantlydifferent atP ≤ 0.05, according to LSD test.

D. Sarkar et al. / Plant Science 167 (2004) 977–986 981

Fig. 2. Leaf necrosis index (LNI) as affected by Mn nutrition at various concentrations of P under two different pH levels in potato microplants in vitro.LNI = √

(x + 0.5)/√

(y + 0.5), wherex = number of necrotic leaves/microplant andy = number of green leaves/microplant.

on checking leaf necrosis under high Mn supply (5.0 mM)was only evident at pH 5.8.

Early necrotic flecking on petioles was followed by yel-low green chlorosis in the leaves in areas between the veins,and as the symptoms progressed the chlorosis became moresevere and finally the leaves became necrotic and dried up(Fig. 3A). Stem streak necrosis appeared as dark brown pit-ted areas on the outside of the stem near the base, whichprogressively became long and narrow, extending into thecentre of stem (Fig. 3B). Early necrotic flecking on stem alsoappeared adjacent to affected petioles that subsequently de-veloped into long, narrow streaks (Fig. 3A). Stem necrosisdid not appear under control Mn supply (0.1 mM) irrespec-

Table 3Effects of different combinations of Mn and P on degree of stem necrosisa under varying pH conditions in two potato genotypes in vitro

Mn (mM) P (mM)

0.0b 2.5 6.25

(a) cv. Kufri Ashoka0.1 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71)1.0 2.2 (1.64) 0.8 (1.04) 0.2 (0.81) 0.2 (0.81) 0.6 (0.99) 1.4 (1.37)2.5 2.4 (1.68) 2.2 (1.59) 3.6 (2.00) 2.6 (1.76) 1.6 (1.43) 3.6 (2.01)5.0 2.6 (1.75) 4.4 (2.19) 4.4 (2.19) 3.4 (1.96) 3.4 (1.94) 2.8 (1.77)

(b) cv. Kufri Sindhuri0.1 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71) 0.0 (0.71)1.0 0.8 (1.09) 2.4 (1.62) 0.0 (0.71) 0.0 (0.71) 0.6 (0.99) 0.4 (0.91)2.5 2.8 (1.78) 3.8 (2.03) 0.6 (0.99) 1.8 (1.43) 1.8 (1.50) 1.8 (1.46)5.0 4.2 (2.15) 3.4 (1.92) 1.2 (1.14) 3.8 (2.03) 1.4 (1.37) 2.0 (1.52)

a Degree of stem necrosis was scored on a 0–5 scale of increasing severity of necrotic streaking on stems with 5 representing complete necroticflecking of stems.

b Values in light and bold faces represent data under pH 4.0 and 5.8, respectively; data in parenthesis are square root (√

x + 0.5) transformed values;LSD0.05 = 0.41.

tive of pH and P levels in both the genotypes (Table 3). Incv. Kufri Ashoka, at pH 4.0, a significant increase in stemnecrosis occurred under 1.0 mM Mn supply in the absenceof P, but incorporation of P (2.5–6.25 mM) in the mediumsignificantly decreased it. However, at pH 5.8, in cv. KufriAshoka, a significant increase in stem necrosis was apparentat 2.5 mM Mn supply in the absence of P, and addition ofP did not result in any decline in stem necrosis; rather therewas a significant increase in stem necrosis with 6.25 mM P.In cv. Kufri Sindhuri, at pH 4.0, stem necrosis significantlyincreased under 2.5–5.0 mM Mn supply when the mediumwas not supplemented with P. However, at both the pH lev-els in cv. Kufri Sindhuri, there was a significant reduction

982 D. Sarkar et al. / Plant Science 167 (2004) 977–986

Fig. 3. Development and progression of toxicity symptoms in potato cv. Kufri Ashoka in vitro under excess supply of Mn. (A) Microplants growing inMS medium supplemented with 1.0 mM Mn at pH 4.0 after 7 (left), 14 (middle) and 21 days (right); early necrotic flecking on the petiole (arrow) beingfollowed by progression of necrosis in the lamina and adjacent stem region (circle). (B) Long necrotic streaks on stems of microplants after 35 days’culture in MS medium supplemented with 1.0 mM Mn at pH 4.0 (left and middle); microplants under same culture conditions showing no appearanceof toxicity symptoms (right) in the presence of 6.25 mM P.

in stem necrosis under 2.5–5.0 mM Mn supply, when themicroplants were grown in the presence of P.

3.4. 54Mn and 32P accumulation

At both the pH conditions, a significant gradual increasein 54Mn level in microplants occurred with increasing Mnsupply up to 2.5 mM, and beyond that no further significantincrement was recorded (Fig. 4). Although, under controlMn supply (0.1 mM), there was no difference in54Mnconcentration between the pH levels, there was always asignificant increase in54Mn concentration in microplantswith increasing Mn supply at pH 5.8 as compared to pH 4.0.When P was limiting in the medium, there was a gradualincrease in54Mn uptake with increasing concentrations ofMn supply, reaching maximum at 5.0 mM. In the presenceof 2.5 mM P,54Mn concentration in microplants increasedsignificantly up to 2.5 mM of Mn as compared to thatwhen P was limiting. However, increasing P levels beyond2.5 mM did not further improve the uptake efficiency of54Mn, and rather, there was a significant decline in uptakeunder 5.0 mM Mn supply when the medium was supple-mented with 6.25 mM P. Mn had a significant effect on32Pconcentration in microplants. When the microplants weregrown in medium containing 2.5 mM P, a significant decline

in 32P level in microplants occurred at 2.5 and 5.0 mM Mnsupply in cvs Kufri Sindhuri and Kufri Ashoka, respectively(Fig. 5). However, at 6.25 mM P, a significant increase in32Pconcentration was observed under 1.0–2.5 mM Mn supplyas compared to control (0.1 mM Mn) in cv. Kufri Ashoka,but not in cv. Kufri Sindhuri. In general, Mn at 5.0 mMsignificantly retarded32P uptake at both the P levels.

Highly significant (P ≤ 0.01) positive correlations couldbe established between54Mn concentration in microplantsand the toxicity symptoms (Table 4). However, no signifi-cant relationship was apparent between54Mn concentrationand microplant growth parameters. This is not unexpectedconsidering significant (P ≤ 0.01) positive correlations be-tween32P concentration and microplant growth parameters.32P concentration in microplants, however, showed signifi-cant negative correlations with toxicity symptoms like leafand stem necrosis.

3.5. P accumulation

In the absence of P, Mn did not have any effect on inor-ganic P content in microplants (Table 5). In cv. Kufri Ashoka,Mn at 5.0 mM significantly depressed the P accumulation atboth the levels of external P supply. However, in cv. KufriSindhuri, supplying Mn even at intermediate concentrations

D. Sarkar et al. / Plant Science 167 (2004) 977–986 983

Fig. 4. Interactions of Mn× pH (top) and Mn× P (bottom) on con-centration of54Mn in potato microplants.54Mn concentration data wereanalyzed in log10 x transformed scale and presented in original scale.Means with common letters are not significantly different atP ≤ 0.05,according to LSD test.

(1.0–2.5 mM) significantly retarded the P accumulation inmicroplants. Increasing the external P supply from 2.5 to6.25 mM resulted in a significant rise in tissue P contentat 1.0 mM Mn level in cv. Kufri Ashoka, but at 2.5 mMMn level in cv. Kufri Sindhuri. The pH condition alone orin combination with other factors did not have any signif-icant effect on the rate of P accumulation in microplants.There was significant (P ≤ 0.01) positive correlations be-tween tissue P content and microplant growth parameters(Table 4). However, tissue P content showed significant neg-ative correlations with toxicity symptoms like leaf and stemnecrosis.

Table 4Spearman rank order correlationsa between microplant growth/toxicity parameters and mineral concentration/accumulation in potato microplants in vitro

Mineral concentration/accumulation Microplant growth/toxicity symptoms

Microshoot length Microplantfresh mass

Degree of rooting Leaf necrosis index Degree ofstem necrosis

54Mn, nanomole/microplant 0.232 nsb 0.087 ns −0.182 ns 0.505∗∗ 0.635∗∗32P, �M/microplant 0.623∗∗ 0.759∗∗ 0.659∗∗ −0.407∗∗ −0.281∗P, �M/microplant 0.570∗∗ 0.760∗∗ 0.676∗∗ −0.434∗∗ −0.328∗

a rs = 1- (6∑

d2i )/(n − 1)n(n + 1), wheredi is the difference for theith pair, andn is the number ofd’s; n = 48.

b ns, non-significant.∗ Significant atP = 0.05.∗∗ Significant atP = 0.01.

Fig. 5. Concentration of32P in microplants at different levels of externalMn in relation to P supplies in potato cvs Kufri Ashoka (top) and KufriSindhuri (bottom).32P concentration data were analyzed in square root(√

x + 0.5) transformed scale and presented in original scale. Means withcommon letters are not significantly different atP ≤ 0.05, according toLSD test.

4. Discussion

The present study clearly documented that Mn toxicityresulted in stem streak necrosis in potato. Stem streak necro-sis in potato has also been reported to be caused by certainstrains of virus Y[35], and fungiVerticillium albo-atrum[17,36], Phytophthora infestans andRhizoctonia [37]. Evenit has been speculated to be caused by a non-systemictospovirus like the tomato spotted wilt virus[18], but theauthors did not provide conclusive evidences by serological

984 D. Sarkar et al. / Plant Science 167 (2004) 977–986

Table 5Effects of Mn on accumulation of total inorganic P (in mM) (�M/microplant) in two potato genotypes in vitro

Mn (mM) cv. Kufri Ashoka cv. Kufri Sindhuri

0.0 2.5 6.25 0.0 2.5 6.25

0.1 1.63 (1.45)a 5.99 (2.47) 4.73 (2.23) 1.33 (1.35) 5.99 (2.48) 5.54 (2.42)1.0 1.45 (1.39) 4.99 (2.31) 7.01 (2.69) 1.49 (1.40) 4.53 (2.23) 3.33 (1.91)2.5 1.33 (1.34) 4.88 (2.29) 6.16 (2.53) 1.43 (1.39) 3.02 (1.86) 5.22 (2.32)5.0 1.32 (1.35) 2.93 (1.84) 2.99 (1.84) 1.38 (1.37) 2.30 (1.67) 2.77 (1.79)

a Values in parenthesis are square root (√

x + 0.5) transformed values; LSD0.05 = 0.33.

or electron microscopic examination of diseased tissue. Thepresent study has for the first time provided an unambigu-ous evidence for the involvement of excess Mn in causingstem streak necrosis in potato by employing a uniquemicroculture-based experimental condition in vitro, wherethe experimental materials were completely free from dis-eases, especially the viruses. The previous studies on Mntoxicity in potato were either based on qualitative assess-ment of toxicity symptoms[11,12,14,15]or quantitativestem streak index[13,16] or general toxicity rating[22,26].By comparison, we quantitatively assessed the Mn toxicityin relation to stem streak necrosis by studying the devel-opment and progression of toxicity symptoms on both theleaves and stems of potato microplants. In contrast to otherstudies[22,26], we investigated the effect of Mn even in thecomplete absence of external supply of P. This consequentlyallowed us to analyze Mn× P interaction more effectively,and to examine the development and progression of Mn tox-icity symptoms under P-deficient conditions also. This studyshowed that excess Mn supply accounted for significantincreases in leaf and stem necrosis. Irrespective of P levels,stem streak necrosis symptoms did not develop at controlMn (0.1 mM) level, thus eliminating the possible role ofP in toxicity development under limiting or excess supply.Addition of P significantly checked the severity of both leafand stem necrosis, but this beneficial effect of P was de-pendent on pH conditions, and was genotype-specific. Thebeneficial effect of P in controlling the progression of leafand stem necrosis was most conspicuous at pH 4.0 than 5.8.The progression of Mn toxicity symptoms was also closelyrelated to maturity levels of the genotypes. This differentialsusceptibility of genotypes to stem necrosis in relation toexcess Mn supply under P-limiting conditions could notfind support from Mn accumulation rate in microplants,which will be discussed in following sections. Late matur-ing genotype Kufri Sindhuri produces lanky shoots withsmall green leaves under standard culture conditions at pH5.8 as compared to that of early maturing genotype KufriAshoka. This microplant type can be assumed to be moresusceptible to excess Mn supply. It is well recorded thateven varieties within a species have different degrees oftolerance to Mn[4,8].

The study also showed that increasing the Mn concen-tration to 10 times that in the standard MS medium[28]had no significant effect on potato microplant growth. This

may perhaps be because Mn toxicity symptoms occur wellbefore any substantial reduction in vegetative yield occursin potato [5,11,15]. That increased stem elongation is anearly sign of Mn toxicity[11], however, could not be con-firmed in this study since there was always a significantreduction in microshoot length with increasing concentra-tions of Mn supply under both the pH conditions. This isnot quite unexpected because Lee[11] had used a very lowlevel of Mn (0.2 mM) as compared to Mn treatments usedin this experiment. Perhaps the most conspicuous effect ofMn on microplant growth was exhibited by root growthresponse under excess Mn supply. In none of the previousstudies on Mn toxicity in potato[12,13,15,16,22,26], therewas any attempt to study root growth response under excessMn supply. Increasing Mn concentrations were associatedwith significant decline in root growth, and there was asignificant Mn× P interaction on this detrimental effect.

The pH condition was an important factor in controllingthe uptake and accumulation of Mn in potato microplants.In a similar way to other divalent cation species, Mn2+ par-ticipates in cation competition[27]. At the lower pH therewas a greater concentration of H+ ions around the root, andthese can effectively compete with other metal ions includ-ing Mn2+. This results in a strong inhibitory effect on theuptake of Mn[2]. Interference of Mn uptake by H+ ions atlow pH has also been reported inPhragmites australis [38].Mn precipitation on root surfaces has been shown to be re-lated to pH of culture solutions inTriticum aestivum [10].The concentration of54Mn in microplants was significantlyaffected by P nutrition. Enhanced uptake of Mn at high Plevels as also reported by Marsh et al.[26] is perhaps due togreater growth and transpiration under adequate P; the moremature tissue would have larger vacuoles, and a better abilityto accumulate Mn[26]. Increased Mn accumulation at highP levels has been shown to lead to increased Mn toxicity inpotato by Marsh et al.[26], and the authors did not find anybeneficial effect of P in reducing the appearance of Mn tox-icity symptoms. In contrast, despite enhanced accumulationof Mn at high P levels, P did show a beneficial effect on con-trolling the severity of Mn toxicity symptoms in the currentstudy. It is plausible that addition of P can reduce Mn toxic-ity by rendering it inactive within the plant[1,5]. However,whether this inactivation is mediated by detoxification of Mnby precipitation within the plant[39,40] or more vacuolaraccumulation of Mn[41,42] or its enhanced compartmen-

D. Sarkar et al. / Plant Science 167 (2004) 977–986 985

talization as a protective mechanism for segregating excessMn from the key metabolic centre[43] is not clear. Furtherresearch is required to understand the exact mechanism ofthis P-mediated inactivation of Mn at the cellular level. Thedifferential effect of P nutrition on microplant Mn concentra-tion as reported in this study can also be attributed to varyingCa:P ratio in the nutrient medium. Le Mare[44] has shownthat when the Ca:P mole ratio was 1:1 and 1:2, Mn concen-tration was greater in cotton plants grown with lower concen-tration of P in nutrient solution than in plants without addedP or more P. In the present experiment, Ca:P mole ratio (ata fixed concentration of 3.0 mM Ca) approximated 1:1 and1:2, and consequently, it might have influenced the Mn accu-mulation in microplants with increasing levels of P nutrition.

The present study further showed that Mn had a significanteffect on P uptake and accumulation in potato microplants.About two to three times reduction in32P concentration inmicroplants occurred at a very high level of Mn (5.0 mM)supply; there was also a significant reduction of tissue levelsof inorganic P at this high level of external Mn supply. Itmay be that higher cation uptake under a very high levelof external Mn supply somehow depresses the uptake andaccumulation of H2PO4

− anions inside the plant. Restricted32P uptake and tissue P content under very excessive Mnsupply might perhaps be the reasons as to why external Psupply did not have any beneficial effect in controlling theseverity of Mn toxicity symptoms at 5.0 mM Mn.

Acknowledgements

We thank Dr. S.M. Paul Khurana, Director, Central PotatoResearch Institute, Shimla for providing the facilities andMr. S.K. Dey for photographic assistance. Comments andsuggestions on the manuscript from two anonymous review-ers are gratefully acknowledged.

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