Arbuscular mycorrhizal fungus influence maize root growth and … · 2017. 12. 13. · 212 P. Priyadharsini & T. Muthukumar Anales de Biología 39, 2017 Introduction Phosphorus (P)

Anales de Biología 39: 211-222, 2017 ARTICLEDOI: http://dx.doi.org/10.6018/analesbio.39.22

Arbuscular mycorrhizal fungus influence maize root growthand architecture in rock phosphate amended tropical soil

Perumalsamy Priyadharsini & Thangavelu Muthukumar

Root and Soil Biology Laboratory, Department of Botany, Bharathiar University, Coimbatore - 641046, Tamil Nadu, India.

Resumen

CorrespondenceT. MuthukumarE-mail: [email protected]: 23 April 2017Accepted: 2 November 2017Published on-line: 13 December 2017

Influencia del hongo arbuscular micorrícico en el crecimiento yarquitectura radicular en suelo tropical modificado con fosforita

Hemos ensayado la influencia del homgo micorrícizo arbuscular(AM) Scutellospora calospora en la estructura, crecimiento, asimi-lación de nutrientes, actividad fosfatasa y dependencia micorrizalde raíces de maíz por adicción de 0-5% de fosforita (RP) en suelosdeficientes de fósforo (P). La adicción de RP aumentó significativa-mente la longitud total de la raíz, el número de raíces a diferentesniveles y el diámetro de los pelos radiculares de las plantas AM. Elhongo AM influyó positivamente el crecimiento del maíz y la asimi-lación de nutrientes. Las actividades fosfatasa ácida y alcalina fue-ron mayores en las plantas AM en suelos mejorados. Al aumentarlas concentraciones RP se redujeron no linealmente el porcentajede colonización del hongo AM. Entonces, la inoculación de hongosAM junto a la mejora de fósfoso proveniente de RP podría sustituirfertilizantes químicos y hacer disponible el P proveniente de RP.

Palabras clave: Hongo AM, Dependencia micorrizal, Nutrientes,Fósforo, Fosfatasa, Pelos radiculares.

Abstract

We evaluated the influence of arbuscular mycorrhizal (AM) fungusScutellospora calospora on root architecture, growth, nutrientuptake, root phosphatase activity and mycorrhizal dependency ofmaize in 0-5% rock phosphate (RP) amended phosphorus (P)deficient soil. RP amendment significantly increased total rootlength, number of roots in different orders, and root hair diameter ofAM plants. The AM fungus positively influenced maize growth andnutrient uptake. Acid and alkaline phosphatase activities werehigher for AM plants in RP amended soils. In contrast, increasingconcentrations of RP reduced the percentage of AM funguscolonization non-linearly. Thus, AM fungus inoculation along withRP amendment could substitute chemical fertilizers and makeavailable the P in RP to the plants.

Key words: AM fungi, Mycorrhizal dependency, Nutrients,Phosphorus, Phosphatase, Root hairs

212 P. Priyadharsini & T. Muthukumar Anales de Biología 39, 2017

Introduction

Phosphorus (P) is one of the most essential, butleast available macronutrient for plant growth inmany tropical soils. Phosphate, the available formof P though modulate the fundamental trait ofplants, it is neither soluble in the soil solution, norreadily transported by mass flow (Bagyaraj et al.2015). Many soils contain more organic forms ofphosphate (Po) compared to inorganic forms.Therefore, frequent application of inorganic P (Pi)in the form of synthetic fertilizers becomes oblig-atory in crop production systems. But, the use ofinorganic synthetic fertilizers on a regular basis isnot only expensive but also environmentally un-desirable (Arcand & Schneider 2006). Therefore,the current tendency is either to avoid or trimdown the use of synthetic fertilizers and to in-crease the use of natural materials like rock phos-phate (RP) in crop production (Arcand & Schnei-der 2006).

The microorganisms involved in solubilizingminerals and organic phosphates are rife in soil(Barea & Richardson 2015). In nutrient deficientsoils, these microorganisms utilize the energyderived from the breakdown of fresh carbon com-pounds to release Pi from organic sources (Arcand& Schneider 2006). Nevertheless, this effectmight be even more important in the presence ofthe obligate endosymbiotic arbuscular mycor-rhizal (AM) fungi which aid in the uptake of thereleased Pi from the soil. The AM fungi belongingto the phylum Glomeromycota are ubiquitous inmost plant communities and interconnect theintraradical with the extraradical environment(Smith & Read 2008). Although, the capacity ofAM fungi to solubilize P is controversial, itscapacity to enhance the uptake of P from the soilis well proven (Smith & Read 2008).

Plant species exhibit different strategies toacquire nutrients from nutrient-deficient soils.These include changes in root morphology, exuda-tion of enzymes and organic acids into the rhizo-sphere that dissolves and release nutrients fromorganic compounds (Arcand & Schneider 2006).Although roots play an important role in theacquisition of nutrients from the soil by plants, itsarchitecture is a highly plastic trait. Root archi-tecture differs with plant species and is stronglycontrolled by plant growth regulators, inherentgenetic factors, and soil conditions (Niu et al.

2012). The availability of P in the soil alsoinduces changes in root architecture like primaryroot length, branching, number and length of lat-eral roots and root hair development (Niu et al.2012). However, the AM fungal colonizationdepends on the anatomical features of the rootsystem, and also it could alter the host root mor-phology (Dreyer et al. 2014). Further, it is gener-ally believed that mycorrhizal plants have a coarseroot system with no or few short root hairs andexhibit increased growth response to AM fungalcolonization. Nevertheless, this view has beenrecently disputed by Maherali (2014). As AMfungi can induce changes both in plant hormoneand nutrient contents (Smith & Read 2008), itwould be interesting to assess the role of AMfungi in plant root architecture.

Like other soil fungi, AM fungi also exhibitphosphatase activity both in alkaline and acid pHranges. In addition, AM fungi can also enhanceplant’s uptake of Pi from P-deficient soils throughtheir extraradical hyphae that scavenge soil vol-umes beyond the reach of plant roots (Smith &Read 2008). In mycorrhizal plants, phosphataseenzyme appears during the initial stages of mycor-rhization and peaks just prior to or during mycor-rhizal establishment, thus playing a key role in theP assimilation (Khade et al. 2010). Both, intracel-lular and extracellular phosphatase produced byAM fungi has been shown to liberate Pi from Po(Khade et al. 2010). Acid phosphatase localized inmature arbuscules, intraradical hyphae and at thefungal entry points hydrolyse vacuolar polyphos-phate to Pi and release them into the plant-fungalinterface (Abdel-Fattah 2001). In contrast, alka-line phosphatases that are mainly present in vac-uoles in addition to arbuscules and intraradicalhyphae are also involved in the transfer of P fromthe fungus to the host (Kojima et al. 1998).Although many studies have examined the solubi-lization of RP by phosphate solubilizing micro-organisms and its influence on plant growth(Arcand & Schneider 2006 and references therein), studies on the effect of RP on AM formationand function are limited.

Maize (Zea mays L.), the major food sourcefor humans and livestock in many parts of theworld has a unique root system which is highlyefficient not only in anchoring the plant to the soilbut also in acquiring nutrients and water from thesoil (Hochholdinger & Tuberosa 2009). In addi-

Anales de Biología 39, 2017 AM fungus influences root growth 213

tion, the root system of maize also possesses se-veral morphological and metabolic traits that areessential for increased efficiency like adventitiousroots, long and dense root hairs, basal-root shal-lowness, root etiolation and cortical aerenchyma(Hochholdinger & Tuberosa 2009). Several stud-ies reported the mycorrhizal status and coloniza-tion patterns like Arum-, Paris- and intermediate-type in maize (Muthukumar & Prakash 2009;Muthukumar & Tamilselvi 2010; Chandra Gandhiet al. 2017). Further, maize genotypes exhibitvariation in their responsiveness to mycorrhizalcolonization (Kaeppler et al. 2000). Recently,Wang et al. (2017) in a long term experimentshowed that increasing P fertilization in spite ofreducing root colonization and community struc-ture of AM fungi can still contribute substantiallyto P nutrition of maize plants. Nevertheless, thisAM fungal benefit depends on the growth stage ofmaize plants and the soil layer in which roots arelocated. Though previous studies have examinedthe influence of RP on AM fungal colonization ofroots or on root architecture, there is no informa-tion on the simultaneous application of RP andAM fungi on root architecture. So, the mainobjective of the present study was to assess theinfluence of AM fungal inoculation and RPamendment on root growth and architecture, andits related changes in plant growth, nutrientuptake, root phosphatase enzyme activities andmycorrhizal dependency of maize in a P-deficienttropical soil.

Materials and methods

Experimental site and soil preparation

The experiment was conducted in the shade houseof the Botany Department, Bharathiar University(11º 01’ N, 96º 93’ E, altitude 410 MASL), Coim-batore, India. The physicochemical characteristicsof the soil as determined by standard methods(Jackson 1971) were as follows: pH 6.1, electricalconductivity 0.11 dSm-1, 45 mg/kg of total N, 8.5mg/kg of available P and 500 mg/kg of exchange-able K. The micronutrients iron (Fe), manganese(Mn), zinc (Zn) and copper (Cu) were 4.75, 2.64,1.06 and 1.50 mg/kg as determined by the DTPAmethod (Lindsay & Norvell 1978). The soil washeat sterilized at 121 ºC at 15 psi for 3 h thricewith 24 h intervals between subsequent steriliza-tion and shade dried for 15 days for refixation of

liberated nutrients.

Plant material and AM fungus inoculum

Maize (cv CO 6) seeds procured from Tamil NaduAgricultural University, Coimbatore, India wasused for this study. The AM fungus Scutellosporacalospora (Nicol. & Gerd.) Walker & Sandersused in this study originated from the semi-aridgrassland of Maruthamalai Hills, Coimbatore. Thefungus was maintained as a pure culture in sterilesoil with Plectranthus scutellarioides (L.) R.Br.,as the host plant. The fungal inoculum consistedof spores, hyphae, and mycorrhizal roots. Thenumber of AM fungal propagules per gram of theinoculum was assessed using the most probablenumber (MPN) technique (Feldmann & Idczak1992). Results were expressed as the number ofAM infective propagules per gram of soil.

Experimental design

A two-factorial experiment [concentrations of RP(containing 10.23% P2O5) and AM inoculation]was conducted in a completely randomized blockdesign with ten replicates. Inoculation of AM fun-gus was assigned to the main plot and RP concen-trations to the subplots. The RP treatment con-sisted of six different concentrations of RP (0%,1%, 2%, 3%, 4% and 5%, v/w) mixed thoroughlywith the sterilized soil. A 225 g of the soil mixwas filled in each of the 120 (6×2×10) plastic pots(9×7.5 cm, height ×width). Half of the total num-ber of pots in each treatment was inoculated withthree grams of the AM fungal inoculum consistingof 220 infective propagules per gram of inoculum.The AM fungal inoculum was layered 4 cm belowthe soil surface and each pot was seeded with twomaize seeds. Twenty milliliters of microbial washprepared from AM fungal inoculum as perMuthukumar et al. (2001) was added to pots notinoculated with the AM fungus to equalize thebackground microflora except the AM fungusacross treatments. The pots were rearranged everyweek to expose all the plants to uniform condi-tions. Plants were watered as necessary with tapwater and no nutrients were added. The destruc-tive harvest of the plants was carried out after 58days of growth.

Harvest and measurements

At harvest, half of the plants from each treatmentwere used for phosphatase assay and for AM colo-


nization determination. The roots of these plantswere washed thoroughly free of soil and a portionof the roots was removed and preserved in FAAsolution for the assessment of AM colonization.The remaining portion was used for assessing acidand alkaline phosphatase activity. The other halfof the replicates was used for determination ofgrowth, root characteristics and nutrient parame-ters. Shoots and roots were separated and dried at40 ºC for 72 hrs for determination of dry mass.Plant growth was assessed using standard parame-ters like shoot and total root length, and dryweights of shoot and root. In addition, other mor-phological parameters like root diameter, root hairnumber, length and diameter, and lateral rootnumbers in differed root orders were also mea-sured. Total root lengths of plants in differenttreatments were measured according to Newman(1966). Root diameter and root hair characteristicswere measured at ten 1-cm root bits floated in wa-ter using a calibrated ocular micrometer. The 1st

-and 2nd - order roots in each root system were de-termined manually after spreading the entire plantroot system in a 40×30 cm (l×b) plastic tray con-taining water. The roots were divided into two or-ders, primary roots directly arising from thecrown roots, 1st - order arising from the primaryroot and roots of 2nd - order arising from the 1st -order roots.

Phosphatase assay

One gram of fresh root tissue was homogenized in10 ml of ice-cold 50 mM of citrate buffer (pH-5.3) for acid phosphatase or 50 mM glycine-NaOH buffer (pH-10.4) for alkaline phosphatasein a pre-chilled pestle and mortar. The ho-mogenate was filtered and centrifuged at 10,000 gfor 10 min. The supernatant was used as the en-zyme source. Phosphatase activity was measuredat 405 nm in a spectrophotometer (UV-1601 visi-ble spectrophotometer) and expressed in terms ofm moles of p-nitrophenol released per gram offresh tissue per min at 37 ºC (Sadasivam & Man-ickam 1992).

Estimation of macronutrients in planttissues

In the shoot, N was determined by the micro-Kjel-dahl digestion method using concentrated H2SO4

digest and potassium sulphate - copper sulphate in5:1 ratio as a catalyst. After wet-ashing the plantsamples in a nitric-sulphuric-perchloric acid mix-

ture, the P was determined by the Vanado molyb-date method (Jackson 1971) using a spectropho-tometer at 470 nm and the K was estimated byflame photometry (Jackson 1971).

Preparation of roots and assessment of AMfungal colonization

Fixed roots were washed free of FAA, cut into 1-cm bits, cleared in 2.5 % KOH at 90 ºC, acidifiedwith 5N HCl and stained with trypan blue (0.05%in lactoglycerol) overnight. The stained roots wereexamined with a compound microscope (400 ×)for AM fungal structures and the percentage ofroot length colonization was estimated accordingto a magnified intersection method (McGonigle etal. 1990).

Mycorrhizal dependency

Mycorrhizal dependency was determined by usingthe biomass of AM and non-AM plants as perPlenchette et al. (1983).

Statistical analysis

Analysis of variance (ANOVA) was performed onall data to compare treatment effects and influenceof AM and RP application on maize plant growthand phosphatase activity. Means were separatedusing Duncan’s Multiple Range Test (DMRT).Pearson’s correlation and regression analysis wereused to assess the relationship between differentvariables. Percentage data on mycorrhizal colo-nization were arcsine square root transformedprior to analysis (SPSS, windows Version 9).

Results

Plant growth

Maize plants raised in various concentrations ofRP showed significant variations in growth pa-rameter both under mycorrhizal and non-mycor-rhizal conditions (Table 1). Non-mycorrhizalplants grown in soils amended with 2%RP werethe tallest and those grown in 1%RP amendedsoils were the shortest. A decline in plant heightwas observed in plants raised in 2% to 5%RPamended soils. In contrast, increasing concentra-tions of RP increased plant height under mycor-rhizal condition.

The RP amendment had no significant influ-ence on shoot and root dry weights of maizeplants (Table 1). AM fungal inoculation signifi


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cantly affected root dry weight, but not the shootdry weight in maize. Non-mycorrhizal plantsraised in 2% and 4%RP amended soils had themaximum and minimum shoot dry weightsrespectively. In contrast, the maximum shoot dryweight of mycorrhizal plants occurred in 5%RP,and minimum in 0% and 3%RP amended soilsrespectively. Like shoot dry weight, maximumand minimum root dry weights of non-mycor-rhizal maize plants occurred in 2%RP and otherconcentrations of RP except 3% at RP amend-ment. Among AM plants, maximum root dryweight was recorded in 5%RP and minimum rootdry weight was recorded in 4%RP respectively.

The interaction of different concentrations ofRP application and AM inoculation on R/S ratiosof maize plant was evident (Table 1). Neverthe-less, neither AM inoculation nor the various con-centrations of RP amendment had any influenceon maize R/S ratios. Generally, the R/S ratios ofAM plants were almost similar or lower comparedto their non-AM conspecifics (except at 1%RP).Lowest R/S was recorded for plants raised in the0%RP amendment under non-AM and in 4%RPamendment under AM conditions.

Influence of RP on root characters

Different concentrations of RP had no significantinfluence on total root length in maize. Neverthe-less, AM inoculation significantly enhanced thetotal root length of maize both under RP amendedand non-amended conditions (Table 1). Nonmyc-orrhizal plants raised in the highest concentrationof RP (5%) had the minimum root length, whilethose raised on 2%RP had the maximum rootlength. In contrast, AM maize plants raised in 5%and 1%RP amended soils had the maximum andminimum root lengths (Table 1).

Application of RP at different rates had no sig-nificant influence on lateral root numbers,whereas, AM fungus inoculation significantlyinfluenced lateral root production (Table 1). Gen-erally, AM-plants had significantly higher numberof lateral roots except for plants in 3%RP wherethe primary and 1st - order root numbers of AMplants was 24% and 42% lower than non-AMplants. The primary root production was 8% to198% higher for AM-plants compared to non-AMplants.

Mycorrhizal inoculation significantly influ-enced the 1st - and 2nd - order root numbers. The 1st

- order root numbers of AM plants were 4% to

110% higher compared to non-AM plants. The2nd- order root numbers of AM plants were 0.1%to 432% higher than non-AM plants (Table 1). Incontrast to root numbers, the primary root diame-ter was not influenced by RP concentrations, AMfungus inoculation and their interactions (Table1).

Root hair characteristics

Rock phosphate application had no significant ef-fect on root hair numbers (Table 1). Nevertheless,AM-plants had 18 to 84% more root hairs com-pared to non-AM plants (except in plants in2%RP). Similarly, root hairs of AM plants were 6to 81% thicker than non-AM plants (Table 1).Contrarily, neither AM inoculation nor RP appli-cation rates had any significant influence on roothair length (Table 1).

Plant tissue nutrients

Application of RP and AM inoculation signifi-cantly affected tissue N, P and K concentrations inmaize shoots (Fig. 1). There was a sequential in-crease in the shoot N, P and K contents of AM andnon-AM maize with increasing concentration ofRP (Fig. 2). The maximum and minimum shootnutrient concentrations in AM and non-AM maizeplants occurred in 5% and 0%RP applicationrates. Average N, P, and K concentrations were15%, 20%, and 22% higher of AM plants com-pared to non-AM plants.

Phosphatase enzyme activity

Both inoculation of AM fungus and RP applica-tion rates significantly influenced acid and alka-line phosphatase activities of maize roots. Never-theless, the interactions between these factorswere only significant for alkaline phosphatase ac-tivity (Table 2). Acid phosphatase activity of AMroots was only 0.2% to 6% higher than non-AMroots, whereas alkaline phosphate activity of AMroots was 1.4% to 26.9% higher than non-AMroots.

Extent of AM fungus colonization

No AM fungal structures were observed in theroots of uninoculated plants. The percentage of to-tal root length colonized by S. calospora(%RLTC) varied significantly (F5,89=18.83;P<0.001) with concentrations of RP and rangedfrom 22.20% (2%RP) to 46.78% (0%RP). The%RLTC decreased with increasing concentrations


Figura 1. Concentraciones de nitrógeno, fósforo, potasio entejidos de brote de plantas de maíz cultivadas a distintasconcentraciones de fosforita (RP) e inoculadas (+AM) o no (-AM) por el hongo micorrícico arbuscular Scutellosporacalospora. Estadísticos: Las diferencias entre el numerador y eldenominador se representa entre paréntesis. **, **, ns:significantivo a P<0,01, P<0,001 o no significativo,respectivamente. Las barras de error indican ± el error estándar.Las barras para +AM o -AM que muestran la misma(s) letra(s) noson significativamente diferentes (P>0,05) de acuerdo con elDMRT.

Figure 1. Shoot tissue nitrogen, phosphorus, potassiumconcentrations of maize plants raised on different concentrationsof rock phosphate (RP) and inoculated (+AM) or uninoculated (-AM) with the arbuscular mycorrhizal (AM) fungus Scutellosporacalospora. F-statistics: Numerator and denominator differencesare presented in parenthesis. **, ***, ns: significant at P<0.01,P<0.001 and non significant respectively. Error bars indicate ±standard errors. Bars for +AM or -AM bearing same letter(s) arenot significantly different (P>0.05) according to DMRT.

Treatmentsa

Enzyme activity (mM/g FW/min)

Acid phosphataseAlkaline

phosphatase-AM +AM -AM +AM

0%RP 0.498a 0.506ab 0.386a 0.413b1%RP 0.472ab 0.496bc 0.368a 0.373c2%RP 0.482a 0.483bc 0.378a 0.386bc3%RP 0.490a 0.501abc 0.376a 0.424ab4%RP 0.450b 0.477c 0.349a 0.408bc5%RP 0.498a 0.525a 0.357a 0.453aMean 0.482 0.498** 0.369 0.410***RP concentr. (A5,48) 7.16*** 2.72*AM (B1,48) 9.60** 33.51***A × B(5,48) <1 ns 4.10**

Means in a column followed by a same letter(s) are not significantly(P>0.05) different according to Duncan’s Multiple Range Test.*, **, ***, ns: significant at P<0.05, P<0.01, P<0.001 and nonsignificant respectively.

Tabla 2. Actividad fosfatasa ácida y alcalina en raíces de maíz endiferentes concentraciones de fosforita e inoculadas (+AM) o no(-AM) por hongo micorrícico arbuscular.

Table 2. Acid and alkaline phosphatase activity in maize plantroots raised on different concentrations of rock phosphate andinoculated (+AM) or uninoculated (-AM) with arbuscularmycorrhizal fungus.

Figura 2. Relación de las concentraciones de nitrógeno (a),fósforo (b) y potasio (c) con diferentes proporciones de adicciónde fosforita con micorrización (AM) no (NM). *, **, **:significativo P<0,05; P<0,01 y P<0,001 respectivamente.

Figure 2. Relationship of shoot tissue nitrogen (a), phosphorus(b) and potassium (c) concentrations to different rates of rockphosphate amendment under non mycorrhizal (NM) ormycorrhizal (AM) conditions in maize. *, **, *** significant atP<0.05; P<0.01 and P<0.001 respectively.


Figura 3. Colonización micorrícica arbuscular (AM) en raíces demaíz cultivadas en diferentes concentraciones (0-5%) de fosforita.RLH, RLHC, RLA/RLAC y RLTC indican el porcentaje delongitud de raíz conteniendo hifas, ovillos, arbúsculos/ovillosarbusculares y el total de colonización total AM. La barras quepresentan letra(s) similar(s) no son significativamente diferentes(P>0,05) de acuerdo con el DMRT.

Figure 3. Arbuscular mycorrhizal (AM) colonization in maizeroots raised in different concentrations (0-5%) of rock phosphate.RLH, RLHC, RLA/RLAC and RLTC indicate percentage rootlength containing hyphae, hyphal coils, arbuscules/arbusculatecoils and AM total colonization. Bars for an AMstructure/colonization bearing same low case letter(s) are notsignificantly (P>0.05) different according to DMRT.

Figura 4. Relación entre la proporción de longitud de raízconteniendo ovillos hifales (RLHC)/ longitud de raíz conteniendoarbúsculos/ovillos arbusculares (RLA/RLAC) y las diferentesconcentraciones de fosforita. ** significante a P<0,01.

Figure 4. Relationship between the ratio of percentage root lengthcontaining hyphal coils (RLHC) to root length containingarbuscules/arbusculate coils (RLA/RLAC) and differentconcentrations of rock phosphate. ** significant at P<0.01.

of RP, but the decrease was not linear (r=−0.469;P>0.05) (Fig. 3). The percentage of root lengthwith linear hyphae (%RLH) was significantly in-fluenced by RP application (F5,89=13.75; P<0.001)and ranged from 4% (4%RP) to 17% (0%RP) anddecreased non-linearly (P>0.05) with increasingconcentrations of RP. The percentage of rootlength with hyphal coils (%RLHC) ranged from7% (2%RP) to 17% (5%RP) and significant

Figura 5. Dependencia de micorrización del maíz cultivado adiferentes concentraciones de fosforita. Las barras con con lasmismas letras no son significativamente diferentes (P>0.05) deacuedo con el DMRT. Las barras de error indican ± 1 SD.

Figure 5. Mycorrhizal dependency of maize raised on differentconcentrations of rock phosphate. Bars bearing same low caseletter(s) are not significantly different (P>0.05) according toDMRT. Error bars indicate ± 1 SD.

ly varied (F5,89=6.99; P<0.001) with RP applica-tion rates. Likewise, percentage root length witharbuscules/arbusculate coils (%RLA/%RLAC)ranged from 8% (2%RP) to 18% (0%RP) and var-ied significantly (F5,89= 8.83; P<0.001) with RPapplication rates. The ratio of %RLHC to %RLA/%RLAC linearly increased with increasing con-centrations of RP (Fig. 4).

Mycorrhizal dependency

Mycorrhizal dependency was significantly influ-enced by RP application (F5,54=3.022; P<0.018)and ranged from 3.29% (3%RP) to 157.63%(5%RP) under different concentrations of RPamendment (Fig. 5).

Discussion

In this study, increasing concentrations of RP ap-plication reduced plant height, and this negativeeffect of RP on plant growth was ameliorated byAM fungus inoculation. Ramirez et al. (2009) alsoreported a reduction in maize growth in responseto RP application and a reversal of the effect byinoculation of the AM fungus Gigaspora mar-garita W.N. Becker & I.R. Hall. Stimulation inplant height in response to dual application of RPand AM fungi has also been reported in severalcereal crops (Costa et al. 2015; Wahid et al.2016). The increase in plant height in response toAM colonization can be attributed to the efficientuptake and utilization of P solubilized from RP bythe AM plants. This increased efficiency is due to


increased root absorbing area brought about bythe extraradical mycelial network of AM fungusin the soil (Maherali 2014).

Rock phosphate application had no significantinfluence on shoot and root dry weights. Never-theless, the shoot and root dry weights graduallyincreased with increasing concentrations of RP inAM plants. The higher biomass of AM plants canbe attributed to the increased activity of theextraradical mycelial network of AM fungi aroundthe roots as well as increased photosynthesis(Ayoob et al. 2011). Generally, the R/S ratios ofAM plants in the present study were similar orlower compared to their non-AM conspecifics(except at 1%RP). Khade et al. (2010) alsorecorded higher R/S ratios in mycorrhizal papayaat the end of period of four months of growth,which was attributed to the increased carbon allo-cation for root production in mycorrhizal plantsthan non-mycorrhizal plants. Usually, mycorrhizalplants exhibit lower R/S ratios than non-mycor-rhizal plants in response to greater increment inshoot mass relative to root mass due to an increasein efficiency of the roots by AM fungi (Maherali2014).

The increased total root length in response toAM inoculation in maize is similar to thosereported by Dickson et al. (1999), where Alliumcepa L. colonized by S. calospora and Glomus sp.‘City Beach’ (Glomus Tul. & C. Tul.) had longerroots than their non-AM counterparts. In thepresent study, inoculation of the AM fungus alongwith RP application affected the root proliferationin maize. Berta et al. (1990) also demonstrated anoverall increase in branching of the root system inAllium porrum L. when colonized by a Glomussp., in spite of the fact that individual roots wereshorter than those of non-AM plants. Similarly,Kaldorf & Müller (2000) also showed an increasein the percentage of fine roots in maize plants col-onized by Rhizophagus irregularis C. Walker &Schuessler. The increased branching of the rootsystem in AM plants has often been attributed tothe influence of tissue P content on root geometryand partitioning of assimilates to lateral root for-mation (Niu et al. 2012). Although not significant,the reduction in the number and length of roothairs observed in maize is expected as the elonga-tion and density of root hairs is dependent on Pavailability in the soils (Niu et al., 2012). How-ever, inoculation of S. calospora influenced allthese characteristics except root hair length. This

effect on root hair traits could be attributed to themycorrhizal mediated changes in plant hormoneslike auxins that are known to promote root hairformation (Kaldorf & Müller 2000).

There was a sequential increase in the shoot N,P and K concentrations in AM and non-AM maizeshoots with increasing concentration of RP appli-cation. This is in accordance with studies whereRP application has been shown to improve theconcentration of P and other nutrients in plant tis-sues (Barea & Richardson 2015). The increasednutrient content of AM plants as observed in thepresent and other studies may be due to the extraradical hyphal contribution and probably alsofrom the increased supply of nutrients at the rootsurface and mass flow resulting in an increasednutrient acquisition by roots (Frey & Schüepp1993). Although experimental evidence for thepossibility of active involvement of AM fungi inaccessing P from RP independently of othermicroorganisms is scarce, such a phenomenoncould not be ruled out.

Inoculation of S. calospora clearly led to a dis-tinctive increase in the activities of enzymesinvolved in P dynamics. The acid and alkalinephosphatase activities of AM maize roots werehigher when raised at higher concentrations of RPwhich is in agreement with the observations ofAbdel-Fattah (2001) where acid and alkalinephosphatase activities of soybean roots werefound to be significantly higher for AM than non-AM roots. Though Abdel-Fattah (2001) suggestedthat high phosphatase activities were related to thedegree of active mycorrhizal colonization, only%RLH in the present study was correlated to alka-line phosphatase activity (r=0.925; P<0.01; n=6).This is similar to the observations of Kojima et al.(1998) who showed that only the intraradicalmycelium of G. margarita colonizing Allium ceparesponds metabolically to plant P concentrationand external P availability. The observations ofthe present study are supported by another studywhere root colonization by R. irregularisincreased the activity of alkaline phosphataseenzyme in Trifolium alexandrinum L. (Raisei &Ghollarata 2006). Nevertheless, the activity ofalkaline phosphatase in maize roots was signifi-cantly higher only at higher concentration (3% to5%) of RP application. Acid phosphatase activitywas significantly higher in the AM than in non-AM roots. In contrast, either a lack of anyresponse or a significant reduction in acid phos-


phatase activity to AM fungi inoculation orincreasing levels of P amendment have beenreported in different plant species (Baligar et al.2005). However, the acid phosphatase activitywas 31% and 21% higher in non-AM and AMmaize roots than the respective alkaline phos-phatase activities. This is in accordance withKhade et al. (2010) who also found a consistentlyhigher acid root phosphatase activity than alkalineroot phosphatase activity in four papaya varieties.These increased acid phosphatases activities thanalkaline phosphatase activity could be due to thefact that the enzyme acid phosphatase is producedby both plants and microorganisms whereas alka-line phosphatases are exclusively produced bymicroorganisms (Khade et al. 2010). Further, thepresence of a significant positive correlationbetween acid and alkaline phosphatase activities(r=0.714; P<0.009; n=12) suggests the involve-ment of common factors in the induction of theseenzymes.

Even as the application of P to soils deficientin P is known to stimulate AM fungal formation(Sorenson et al. 2005), the %RLTC as well as%RLA/%RLAC, decreased with different concen-trations of RP application. Takács et al. (2006)also showed that arbuscules were more sensitiveto the supply of soil P than other AM structures.The negative effects of P application on AM fungiwithin roots often result from the reduction in themycorrhizal dependence in response to high P inplant tissues (Takács et al. 2006). In contrast tothe present study, the mycorrhizal formation wasfavored by high soil P in Allium porrum (Soren-son et al. 2005). An interesting observation madein the present study was that the proportion of%RLHC to %RLA/%RLAC increased linearlywith increasing concentrations of RP. A similarobservation was made by Cavagnaro et al. (2003)to increasing application of P in Asphodelus fistu-losus L. It has been suggested that the long life-span of the hyphal coils than arbuscules and theirlarge surface area can be the structural adaptationof the fungus to increasing concentrations of P inthe soil and plant tissues (Cavagnaro et al. 2003).In addition to the transfer of nutrients to theplants, hyphal coils could also store P and carbonwhich could benefit both the fungus and the plantsduring unfavourable periods (Cavagnaro et al.2003).

Mycorrhizal dependency defined as the degreeof change in plant growth associated with AM

colonization (Plenchette et al. 1983) was signifi-cantly affected by RP applications. The averagemycorrhizal dependency value (54.81%) for theweakly mycorrhizal-dependent maize in thepresent study falls well within the range (-30% to83%) reported for maize in other studies (Khalilet al. 1994; Kaeppler et al. 2000). The concentra-tions of P in the soil and the ability of the plantroots to acquire P from the soil play a very impor-tant role in determining the mycorrhizal depen-dency of a plant species. Application of differentconcentrations of RP increased the mycorrhizaldependency of maize plants nonlinearly as againstthe decreases reported in previous studies(Tawaraya 2003; Takács et al. 2006). This is inaccordance with an observation where differentconcentrations of soil P had little influence on themycorrhizal dependency of Hancornia speciosaGomes plantlets (Cardoso-Filho et al. 2008). Thelow mycorrhizal dependency values obtained formaize plants in certain concentrations of RP appli-cation suggests that AM association may not befully functional under these conditions as noted byBethlenfalvay et al. (1983). Mycorrhizal depen-dency has been shown to be influenced by hostand fungal species, the activity of soil micro-organisms, and soil type in addition to the concen-trations of nutrients in the soil (Cardoso-Filho etal. 2008; Oliveira-Júnior et al. 2017; Zangaro etal. 2007). Application of RP might have increasedthe soil microbial populations and the competitionfor P which might have elevated the mycorrhizaldependency of the maize plants in response to RPapplication (Nakhro & Dkhar 2010; Ndungu-Magiroi et al. 2015).

Conclusions

The AM fungal inoculation along with differentconcentrations of rock phosphate application hadvaried response in terms of root architecture, plantgrowth, nutrient content, enzyme (acid and alka-line phosphatase) activities and AM fungal colo-nization in maize plants. The performance ofmaize plants was better when RP application wascombined with AM inoculation. This suggests thatAM fungi could enhance the availability of P inRP to plants. Hence, AM fungal inoculation hasproven to be a better alternative to the chemicalfertilizers, especially P to increase and maintainthe productivity of crops. Long-term fertility trialswith efficient strains of mycorrhizal inoculums


and RP application are needed to test the perfor-mance of the maize under field conditions.

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Arbuscular mycorrhizal fungus influence maize root growth and … · 2017. 12. 13. · 212 P. Priyadharsini & T. Muthukumar Anales de Biología 39, 2017 Introduction Phosphorus (P)