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Journal of Plant Physiology 171 (2014) 1378–1384

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

iochemistry

richoderma spp. alleviate phytotoxicity in lettuce plantsLactuca sativa L.) irrigated with arsenic-contaminated water

ntonio G. Caporalea,∗, Alessia Sommellaa, Matteo Loritoa,b, Nadia Lombardia,b,hah M.G.G. Azama, Massimo Pignaa, Michelina Ruoccob

Dipartimento di Agraria, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, Napoli, ItalyIstituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Portici, Napoli, Italy

r t i c l e i n f o

rticle history:eceived 12 March 2014eceived in revised form 14 May 2014ccepted 15 May 2014vailable online 26 June 2014

eywords:

a b s t r a c t

The influence of two strains of Trichoderma (T. harzianum strain T22 and T. atroviride strain P1) on thegrowth of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water, and their effect on theuptake and accumulation of the contaminant in the plant roots and leaves, were studied. Accumulation ofthis non-essential element occurred mainly into the root system and reduced both biomass developmentand net photosynthesis rate (while altering the plant P status). Plant growth-promoting fungi (PGPF) ofboth Trichoderma species alleviated, at least in part, the phytotoxicity of As, essentially by decreasing

richodermarsenicio-fertilizationhytotoxicityGPF

its accumulation in the tissues and enhancing plant growth, P status and net photosynthesis rate. Ourresults indicate that inoculation of lettuce with selected Trichoderma strains may be helpful, beside theclassical biocontrol application, in alleviating abiotic stresses such as that caused by irrigation with As-contaminated water, and in reducing the concentration of this metalloid in the edible part of the plant.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

Arsenic (As) is an ubiquitous metalloid considered one of theajor hazardous element for the environment and particularly in

armland ecosystems (Mandal and Suzuki, 2002; Su et al., 2010).ue to its high water solubility and mobility, both in ground-nd surface-waters, As contamination is widespread (Beni et al.,011; Smedley and Kinniburgh, 2002). The use of As-contaminatedaters for crop irrigation can have dramatic toxic effects for

oth the environment and the human health through contami-ated food consumption, which can cause carcinogenesis and otherevere chronic diseases (Bhattacharjee et al., 2013). Although As isxtremely toxic also for plant tissues, these can accumulate a smallmount of the inorganic forms, such as the arsenite (AsIII) through

quaporin channels and the arsenate (AsV) through the phos-hate transporter system (Zhao et al., 2009). Once absorbed, thesearmful As-derivatives may deleteriously affect photosynthesis,

Abbreviations: ANOVA, analysis of variance; As, arsenic; AsIII , arsenite; AsV, arse-ate; C, carbon; CEC, cation exchange capacity; N, nitrogen; P, phosphorous; PDB,otato dextrose broth; spp., species.∗ Corresponding author. Tel.: +39 0812539175.

E-mail address: ag.caporale@unina.it (A.G. Caporale).

ttp://dx.doi.org/10.1016/j.jplph.2014.05.011176-1617/© 2014 Elsevier GmbH. All rights reserved.

respiration, growth regulation and reproduction and, in the mostsevere conditions, lead to death (Stoeva et al., 2005; Tripathi et al.,2007). However, the level of As toxicity for the plant depends on thecombination of several factors, including the cultivar genetic back-ground, the agronomic management of the crop and the qualityof the rhizospheric microbiome (Tripathi et al., 2013). Trichodermaspecies (spp.) includes strains that are worldwide known as highlyrhizospere-competent fungi, thus able to colonize the entire rootsystem and actively persist on it for the entire lifespan of differentannual crops (Tucci et al., 2011).

Therefore, several species are extensively exploited in agricul-ture for plant growth promotion, biological control and, in general,as positive modifiers of crop metabolism (Harman, 2004; Hermosaet al., 2012). It has been well established that Trichoderma mayenhance the development of root system (Battaglia et al., 2013;Contreras-Cornejo et al., 2009; Harman, 2004) by promoting therelease of nutrients from the soil (Altomare et al., 1999; Harman,2004; Harman et al., 2004b; Vinale et al., 2013), while generally itseems to improves soil health (Shukla et al., 2008). Recent studies(Srivastava et al., 2011; Zeng et al., 2010) revealed that Trichoderma

spp. are able to tolerate moderate concentrations of As in vitro, aswell as alleviate As-mediated stress for instance on chickpea (Cicerarietinum L.) under greenhouse conditions (Tripathi et al., 2007).Nevertheless, further investigations are still necessary to better

A.G. Caporale et al. / Journal of Plant Ph

Table 1Physical and chemical properties of the soil used in the study.

Soil properties Amount

Sand (g kg−1) 467 ± 26Silt (g kg−1) 348 ± 19Clay (g kg−1) 185 ± 8pH (in H2O) 7.23 ± 0.20Organic carbon (g kg−1) 10.42 ± 0.31CEC (mequiv 100 g−1) 18.65 ± 0.77Total N (g kg−1) 0.97 ± 0.05C/N 10.74

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Available P (mg P2O5 kg−1) 11.09 ± 0.62Total As (mg kg−1) 5.83 ± 0.25

nderstand the role of Trichoderma spp. in ameliorating the tol-rance of the plants to certain abiotic stresses, as it might be thene caused by irrigation of water contaminated with As. LettuceLactuca sativa L.) is an important leafy vegetable that is consumedrimarily fresh or in salads, and it is considered to be a healthyood. The benefits of lettuce consumption are due to the presencef vitamin C, phenolic compounds and fibers (Gusman et al., 2013).herefore, an As accumulation in its edible portion (i.e., leaves)ould pose a risk for the consumer health (Tapio and Grosche, 2006).o this scope, in this paper we evaluated the ability of two strainsf Trichoderma, T22 and P1, applied as bio-fertilizers, in decreasinghe accumulation and toxicity of As in lettuce plants irrigated withontaminated water (5 or 10 mg As per liter).

aterials and methods

n vitro As removal by Trichoderma harzianum T22 andrichoderma atroviride P1

Trichoderma ability to grow in arsenic (As) contaminated mediaas tested by adding different concentrations of As (0; 3; 5; 10; 15;

0 mg As L−1) (NaAsIIIO2, Sigma–Aldrich, USA, was used as source)nto flasks containing 50 ml of potato dextrose broth (PDB). Afterterilization of the media, 50 �l of a 107 spore ml−1 of T22 or P1ere used for the inoculum. The cultures were let to grow for 7ays on the orbital shaker at 150 revolutions per minute (rpm)t 25 ◦C, with 3 replicates for each treatment. After this time theycelia were separated from the culture liquid through a vacuum

lter and collected separately. The lyophilized mycelia and the cul-ure filtrates were then used to detect the quantity of residual As.he ability of two fungal strains to volatilize and bioaccumulate Asas calculated, following the protocol by Srivastava et al. (2011),

y comparing the As content in the liquid medium before cultiva-ion and that in the fungal biomass plus the medium at the end ofultivation.

xperiment on lettuce plants

oil preparation and characterizationThe soil used in this experiment was collected from the sub-

urface layer (10–30 cm) of a grassland in the Naples area. Itsrincipal physical and chemical properties are reported in Table 1.ccording to the soil classification of Food and Agriculture Orga-ization (FAO) World Reference Base for Soil Resources, we canonsiderate this soil as a Calcari-Vitric Cambisol. The soil samplesor lettuce cultivation and chemical analysis, after air-drying, wereassed through a 2 mm sieve.

Soil fractions (sand, silt and clay) were separated using a

ipette and a sieving method, following a pretreatment with H2O2o oxidize organic matter, and dispersion was aided by sodiumexametaphosphate. Soil pH was measured by potentiometry inistilled water (1:2.5 soil/water ratio). The soil organic carbon (C)

ysiology 171 (2014) 1378–1384 1379

content was determined by wet digestion using the Walkley–Blackprocedure. For determination of cation exchange capacity (CEC),the soil was extracted with 1 M NH4OAc at pH 7.0. The total soilnitrogen (N) was determined using a NCS Elemental Analyzer(NA 1500 Series 2). Available phosphorous (P) concentration wasdetermined by the colorimetric method using 0.5 M NaHCO3 asthe extractant (Olsen procedure). Available As concentration wasextracted with 0.05 M (NH4)2SO4 for 4 h at 20 ◦C using the first stepsequential extraction procedure (SEP) (Wenzel et al., 2001). The Asbackground in the soil was determined by extracting the soil withconcentrated HNO3 and HF at a 5:1 ratio.

Experimental designLettuce seeds (Lactuca sativa L., cv. Marvel) were seeded into a

polystyrene alveolar seedbed consisting of 90 holes (25 mm diame-ter, 40 mm depth) containing soil. At the moment of transplantinginto pots containing 6 kg of soil (one plant per pot, 10 days afterplant emerging), the roots of 1/3 of the obtained seedlings weresprayed with a T22 spore suspension (107 spores ml−1), 1/3 weresprayed with a P1 spore suspension (107 spores ml−1) and 1/3were used as a control and sprayed with pure water (-T). Plantswere placed inside a glasshouse with controlled light regimen,temperature and humidity. All of them were initially irrigatedwith As-uncontaminated water (first 5 days after transplanting)and thereafter irrigated for 30 days with water containing sodiumarsenite (NaAsIIIO2, Sigma–Aldrich, USA) at two different concen-trations: 5 (As 5) and 10 (As 10) mg As L−l. The experiment includeda control set of plants irrigated with As-uncontaminated water(control) for 30 days. Constant volumes of irrigation waters werefrequently added to each pot, in order to maintain the soil moistureat 65% of the field capacity, avoiding any phenomenon of leach-ing. A basal fertilizer, consisting of 1.87 g N pot−1 (as NH4NO3 andKNO3), 0.95 g P pot−1 (as KH2PO4) and 2.31 g pot−1 K (as KNO3 andKH2PO4) was supplied to the lettuce plants as nutrient solution.The pots were organized in a completely randomized design with4 replicates for each treatment, and rearranged every 5 days. Thepopulations of both strains of Trichoderma in the rhizosphere of theinoculated plants were kept constant by water applications (10 and20 days after transplanting) of T22 and P1 spores suspension (107

spores ml−1). The watering of the lettuce plants was stopped 3 daysbefore harvesting.

Measurement of net photosynthesis rateThe measurement of net photosynthesis rate was made with

a portable leaf gas exchange analyzer (ADC Bioscientific Ltd.LCA4, UK). Measurements were taken on 2–3 leaves per plant(only mature, fully expanded, As toxicity symptom-free, well-exposed leaves were chosen), at three different days, from11:00 am to 1:00 pm, under conditions of fully saturating radia-tion (when photosynthetically active radiation (PAR) was in excessof 1500 mmol m−2 s−1).

Harvest and plant samples collectionThe lettuce plants were harvested when they reached the typi-

cal market size 30 days after transplanting. Roots and leaves weresampled separately at harvest time. Above-ground biomass wasremoved by cutting the base of the plant 1 cm above the soil sur-face. The fresh tissues of the lettuce plants were weighed, washedwith deionized water to remove soil residues and then dried in anoven for 2 days at 70 ◦C.

Portions of fresh roots were cut and weighed and used for the

determination of the rate of root colonization by the two Tricho-derma strains (T22 and P1). The remaining parts of the roots weredried and weighed and then ground using a PM 200 ball mill(Retsch).

1 ant Physiology 171 (2014) 1378–1384

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380 A.G. Caporale et al. / Journal of Pl

easurement of As and P concentration in plant tissues, PDBiquid medium and mycelium samples

Dried root, leaf and mycelium samples were digested in aicrowave (Milestone, Digestor/Dring Ethos 900); 0.5 g of dried

lant tissues or 0.1 g of mycelium samples were weighed intoolytetrafluoroethylene (PTFE) vessels and digested in 5 ml ofNO3 (65%), 0.5 ml of HF (50%) and 2 ml of H2O2. All solutionsbtained were passed through a 0.22 mm filter and diluted to0 ml with deionized water. Thereafter, the measurement of Asnd P concentrations, both in the filtrates and PDB liquid medium,as performed by a flow-injection hydride generation atomic

bsorption spectrometer (Perkin Elmer AAnalist 700 interfacedith the FIAS 100 hydride generator), and a inductively coupledlasma atomic emission spectroscopy (ICP-AES, Varian, Liberty50), respectively. The As and P detection limits of these meth-ds were 1.5 × 10−3 mg L−1 and 1.2 × 10−2 mg L−1, respectively. Allnalyses were carried out in triplicate. Each analytical batch con-ained at least one reagent blank and one internationally certifiedeference material (CRM) such as oriental tobacco leaves CTA-OTL-.

tatistical analysis

Mean values were compared by analysis of variance (ANOVA)nd the Tukey’s test at the significance level p < 0.05. Data on Asioaccumulation in mycelium samples and bio-volatilization, plantiomass, As concentration and content in plant tissues, and avail-ble As and P in the soils, were treated by two-way ANOVA withactors As and Trichoderma, and tested for normality using thehapiro–Wilk test.

esults and discussion

n vitro As removal by Trichoderma harzianum strain T22 andrichoderma atroviride strain P1

The results showed that both strains (T22 and P1) could toler-te increasing concentration of As into the growing media, withhe production of fungal biomass being in some cases promotedy the presence of As (Table 2). For instance, the obtained biomassf either T22 or P1 was more than doubled at 20 mg L−1 of As ofhat in the untreated control (Table 2). Some As-tolerant fungaltrains, indeed, may gain metabolic energy from As during the cul-ivating process and as a result their growth may be promoted to aertain extent (Zeng et al., 2010). The content of bio-accumulatednd bio-volatilized As was between 0.45–1.55% and 0.22–0.83% ofhe total applied toxicant, respectively (Table 2). Our results are inccordance with those reported for Trichoderma asperellum strainssolated from As contaminated soil (Srivastava et al., 2011; Su et al.,010, 2012; Zeng et al., 2010).

nfluence of the treatments on plant growth

The growth of lettuce plants (i.e., dry matter production ofoots and leaves) was severely impaired by the irrigation with thes-contaminated water (Table 3). The phytotoxic effect of As deter-ined a strong inhibition of root growth and, to a lesser extent,

eaf development. For instance, lettuce plants irrigated with waterontaining 10 mg L−1 of As produced less than a third of the rootiomass of the control (Table 3). Cozzolino et al. (2010) and Gusman

t al. (2013) also reported the sensitivity of this leaf vegetable to As,hich resulted in a stunted growth in contaminated soil or nutrient

olution. In the conditions used in our study, plants showed symp-oms of As toxicity, such as the appearance of red-brown necrotic Ta

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A.G. Caporale et al. / Journal of Plant Physiology 171 (2014) 1378–1384 1381

Table 3Effect of Trichoderma harzianum strain T22 and T. atroviride strain P1 on the biomass of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water.

Treatment Roots (g) Leaves (g) Total biomass (g)

Control -T 5.54 ± 0.42 b 11.38 ± 1.02 bc 16.92 ± 1.44 bcControl T22 7.67 ± 0.61 a (+38.4)a 14.36 ± 1.14 ab (+26.2)a 22.02 ± 1.75 a (+30.1)a

Control P1 6.68 ± 0.59 a (+20.6)a 13.55 ± 1.25 a (+19.1)a 20.23 ± 1.84 ab (+19.6)a

As 5 -T 3.04 ± 0.24 c 7.70 ± 0.72 de 10.74 ± 0.96 dAs 5 T22 5.14 ± 0.33 b (+68.9)a 9.81 ± 0.87 cd (+27.3)a 14.95 ± 1.20 c (+39.1)a

As 5 P1 4.83 ± 0.25 b (+58.8)a 9.92 ± 0.96 cd (+28.8)a 14.75 ± 1.21 c (+37.3)a

As 10 -T 1.43 ± 0.11 d 4.27 ± 0.37 f 5.70 ± 0.48 eAs 10 T22 2.45 ± 0.20 cd (+71.8)a 6.25 ± 0.58 ef (+46.1)a 8.69 ± 0.78 de (+52.5)a

As 10 P1 2.13 ± 0.16 cd (+49.3)a 5.51 ± 0.61 ef (+29.0)a 7.64 ± 0.77 de (+34.0)a

As level p < 0.0001 p < 0.0001 p < 0.0001Trichoderma treatment p < 0.0001 p < 0.0001 p < 0.0001As vs Trichoderma p < 0.05 p = 0.6953 p = 0.4658

Data are expressed as mean values ± SD (n = 4) and have been analyzed by two-way analysis of variance. Mean values followed by the same letter within columns are notsignificantly different by Tukey’s test at the 5% level. Total biomass is the sum of the root and leave biomass. Control, As 5 and As 10 refer to As concentrations of 0, 5 and1 fers toT

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always less than 50% of that found in the entire plant (Table 4), withthe only exception of As 10 -T treatment. The same phenomenonhas been reported for several crops, including rice (Liu et al., 2004;Xie and Huang, 1998), bean (Caporale et al., 2013; Cobb et al., 2000)

0

2

4

6

8

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(µm

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a a a

b

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Fig. 1. Effect of As concentration in the irrigation water on the net photosynthesisrate (�mol CO2 m−2 s−1) of lettuce plants (Lactuca sativa L.) inoculated with Tricho-derma harzianum T22 or Trichoderma atroviride P1. Circles, rhombi and trianglesrefer to As concentrations of 0, 5 and 10 mg L−1 in the irrigation water, respectively.

0 mg L−1, respectively; -T refers to plants non-inoculated with Trichoderma; T22 rerichoderma atroviride.

a Values in parenthesis are the percent increase vs. control non treated with Trich

pots on the leaves 12–15 days after the first irrigation with the As-ontaminated water, but none of them died from As intoxication.

On the contrary, a significant plant growth-promotion effectas obtained by inoculating lettuce with either strains of Tricho-

erma (T22 or P1) regardless of the level of As in the irrigation waterTable 3). Hence, it is evident that the two fungi used, and T22 inarticular, were able to mitigate, at least in part, the negative effectf As on plant growth. It is noteworthy that the higher As level inhe irrigation water, the greater the beneficial effect of the inocula-ion with T22 on plants growth. In fact, the percentage increase ofotal biomass was 30.1, 39.1 and 52.5 at 0, 5 and 10 mg L−1 of As inhe irrigation water, respectively (Table 3). The growth-promotingffect of Trichoderma spp. has been demonstrated on a wide vari-ty of plants in both laboratory-based and field trials (Adams et al.,007; Ousley et al., 1994), and it is attributed to a number of factors,

ncluding (i) pathogen biocontrol, (ii) increased soil nutrient solu-ilization (Altomare et al., 1999) and uptake by the plant (Harman,000), (iii) systemic improvement of carbohydrate metabolism andlant defense mechanisms, and (iv) stimulation by hormone-basedechanisms (Harman et al., 2004a, 2004b).

ffect on the net photosynthesis rate of lettuce

Increased As concentrations in the irrigation water cor-esponded to a stronger reduction of the net photosynthe-is rate (Fig. 1). The value in the control was, on aver-ge, 10.85 �mol CO2 m−2 s−1, while that of the plants irri-ated with As was 8.05 �mol CO2 m−2 s−1 for 5 mg L−1 and.51 �mol CO2 m−2 s−1 for 10 mg L−1. The reduction of photosyn-hetic rate is considered to be one of the most damaging effect ofhis metalloid (Gusman et al., 2013; Stoeva et al., 2005), whichan negatively affect one or both photosynthesis steps. As cannterfere in the electron transporter chain (ETR) limiting the pro-uction of nicotinamide adenine dinucleotide phosphate (NADPH)nd adenosine triphosphate (ATP) and/or promoting fluorescencemissions or energy loss as heat. As can also affect stomatalonductance, restricting CO2 concentration in the plant or inhib-ting ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO)ctivity (Gusman et al., 2013). Nevertheless, it is interesting toote that application of Trichoderma, and in particular strain T22,lightly enhanced, as compared to the un-inoculated control, thehotosynthesis rate of the plants stressed by As (Fig. 1). Accord-

ngly, the correlation between the stimulatory effect of Trichodermapp. on plant growth and an increased net photosynthesis rateas been reported in the literature (Harman et al., 2004a; Tripathit al., 2013). Shoresh and Harman (2008) also demonstrated that

plants inoculated with Trichoderma harzianum; P1 refers to plants inoculated with

a.

the increase of the photosynthesis rate in Trichoderma-inoculatedmaize seedlings was essentially due to the up-regulation of car-bohydrate metabolism and genes related to the photosyntheticprocess, which supports the enhanced growth response.

Arsenic accumulation in plant tissues

In comparison to untreated controls, significantly higher con-centrations of As were found in root and leaf tissues when theplants were irrigated with water containing increasing levels of As(Table 4). Most of the As taken up by plants was found in the rootsystem, probably due to the high sensitivity of the leaves to the Ascompounds. The As concentration in the lettuce roots was up to 4fold higher than that observed in the leaves (Table 4). The lowerconcentration of the metalloid in the leaves may indicate a limitedtranslocation ability of the plant at a systemic level (Smith et al.,2009). Although leaf biomass was much more than that of roots(Table 3), the amount of As accumulated in the lettuce leaves was

-T refers to plants non-inoculated with Trichoderma; T22 refers to plants inocu-lated with Trichoderma harzianum; P1 refers to plants inoculated with Trichodermaatroviride. Circles, rhombi and triangles represent mean values at least four differ-ent replicates ± SD (n = 4); those followed by the same letter are not significantlydifferent by Tukey’s test at the 5% level.

1382 A.G. Caporale et al. / Journal of Plant Physiology 171 (2014) 1378–1384

Table 4Arsenic concentration (mg kg−1) in roots and leaves and As content in leaves (percentage of total As content accumulated in the leaf biomass) of lettuce plants (Lactuca sativaL.) irrigated with As-contaminated waters, at harvest time.

As concentration in plant tissues As content in the leavesa (%)

Treatment Roots (mg kg−1) Leaves (mg kg−1)

Control -T 0.160 ± 0.008 e 0.060 ± 0.004 d 43.5 ± 0.7 bControl T22 0.110 ± 0.006 e 0.031 ± 0.003 d 34.5 ± 1.0 cControl P1 0.120 ± 0.010 e 0.036 ± 0.002 d 37.8 ± 0.6 cAs 5 -T 3.84 ± 0.21 c 1.24 ± 0.13 c 44.9 ± 1.6 bAs 5 T22 2.95 ± 0.10 d 0.92 ± 0.08 c 37.3 ± 1.8 cAs 5 P1 3.14 ± 0.25 cd 0.91 ± 0.07 c 37.1 ± 1.0 cAs 10 -T 7.30 ± 0.54 a 2.66 ± 0.25 a 52.2 ± 0.7 aAs 10 T22 6.13 ± 0.32 b 2.03 ± 0.22 b 45.8 ± 1.7 bAs 10 P1 6.25 ± 0.29 b 2.10 ± 0.18 b 46.4 ± 1.9 b

As level p < 0.0001 p < 0.0001 p < 0.0001Trichoderma treatment p < 0.0001 p < 0.0005 p < 0.0001As vs Trichoderma p < 0.01 p < 0.05 p = 0.1576

Data are expressed as mean values ± SD (n = 4) and have been analyzed by two-way analysis of variance. Mean values followed by the same letter within columns are notsignificantly different by Tukey’s test at the 5% level. Control, As 5 and As 10 refer to As concentrations of 0, 5 and 10 mg L−1, respectively; -T refers to plants non-inoculatedw efers

iding

i

aw

n(ap(tnmtaste

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rb1aatsT

((NH4)2SO4-extractable-As) was minimal (less than 4% increase)(Table 5). This finding is important because it suggests the useof Trichoderma for selectively enhancing the phytoavailability ofnutrients vs. that of toxicants, also in the case of chemical analogs

Fig. 2. Effect of As concentration in the irrigation water on the concentration of P(g kg−1) in the roots (A) and leaves (B) of lettuce plants (Lactuca sativa L.) inoculatedwith Trichoderma harzianum T22 or Trichoderma atroviride P1. As 0, As 5 and As 10

ith Trichoderma; T22 refers to plants inoculated with Trichoderma harzianum; P1 ra Percentage of total arsenic content found in the leaves (%) was calculated by div

n the entire plant (leaves + roots) × 100.

nd tomato (Burlo et al., 1999; Pigna et al., 2012), where As contentas roots > stems > leaves.

In all cases, plants inoculated with Trichoderma exhibited a sig-ificant lower concentration of As both in the roots and the leavesTable 4). These results may be related to the abiotic stress toler-nce induced in plant by Trichoderma, as supported by enhancedroduction of biomass and nutrient use efficiency and availabilityHarman et al., 2004b). The percentage of total As content found inhe leaves of the Trichoderma-inoculated plants resulted to be sig-ificantly lower in comparison to untreated control (Table 4). Thisay be due to the increased foliar biomass and/or to a reduced

ranslocation of the pollutant, as a consequence of the beneficialctivity of the fungus. In any case, the treatment with Trichodermapp. may help to reduce the concentration of inorganic toxicants inhe edible portion of crops in addition to alleviating the phytotoxicffect, with a positive outcome on yield and food quality.

ffect of Trichoderma on P status of As-treated plants

The inoculation of lettuce with either one of the Trichodermatrains raised significantly the P status, both in the roots (Fig. 2A)nd the leaves (Fig. 2B), regardless of the As treatment. A recentaper (Srivastava et al., 2012) also reports that P uptake by plantsan be augmented by inoculation with soil mineral-phosphate-olubilizing fungi. In our experiment, an increase of P concentrationp to 16% was found in lettuce roots treated with TrichodermaFig. 2A). Since As and P are transported into the plant by the sameystem (Gonzalez-Chavez et al., 2002), it is conceivable that theresence of Trichoderma on the roots promotes the uptake of P thus

imiting that of As. Therefore, plants highly susceptible to As, such asettuce, may be made more resistant by a treatment with beneficial

icrobes that enhance P availability and uptake.

ffect of Trichoderma on the availability of As and P in the soil

The presence of the Trichoderma strains (T22 and P1) in thehizospheric soil enhanced significantly the concentration of theiologically available P fraction (NaHCO3-extractable-P) (up to4.7%) (Table 5). Plant growth-promoting microbes, in fact, have thebility to convert nutritionally important elements from unavail-

ble to available forms (Khan et al., 2007; Srivastava et al., 2012),herefore increasing their uptake. This has been already demon-trated by de Silva et al. (2000) for the two Trichoderma species. virens and T. harzianum. However, it should be noted that the

to plants inoculated with Trichoderma atroviride.the amount of As accumulated in the leaf biomass by the total amount of As stored

addition of As has been reported to increase per se the P availablefraction because of the partial replacement of the nutrient by thecontaminant on the soil Al- and Fe-(hydr)oxides (Violante, 2013).

Unlike the significant raising of the available P fraction, theeffect of the two fungal strains (T22 and P1) on the As availability

refer to As concentrations of 0, 5 and 10 mg L−1 in the irrigation solution. Black,white and grid bars refer, respectively, to no Trichoderma, T. harzianum T22 and T.atroviride P1 treatment. Bars represent mean values at least four different repli-cates ± SD (n = 4); those followed by the same letter are not significantly differentby Tukey’s test at the 5% level.

A.G. Caporale et al. / Journal of Plant Ph

Table 5Concentrations of NaHCO3-extractable-P and (NH4)2SO4-extractable-As (biologi-cally available inorganic P and As) in the soils irrigated with As-contaminated waters,at plant harvest time.

Treatment P available (mg P kg−1) As available (�g As kg−1)

Control -T 11.98 ± 0.67 d 21.1 ± 0.9 cControl T22 13.42 ± 0.73 abcd (+12.0)a 22.3 ± 1.3 c (+5.7)a

Control P1 13.31 ± 0.54 abcd (+11.1)a 22.4 ± 1.5 c (+6.2)a

As 5 -T 12.42 ± 0.49 cd 321.8 ± 20.6 bAs 5 T22 14.20 ± 0.72 abc (+14.3)a 330.6 ± 18.6 b (+2.7)a

As 5 P1 13.97 ± 0.66 abc (+12.5)a 329.0 ± 22.3 b (+2.2)a

As 10 -T 12.86 ± 0.68 bcd 518.9 ± 37.5 aAs 10 T22 14.75 ± 0.46 a (+14.7)a 536.2 ± 39.4 a (+3.3)a

As 10 P1 14.53 ± 0.75 ab (+12.9)a 538.0 ± 35.8 a (+3.7)a

As level p < 0.01 p < 0.0001Trichodermatreatment

p < 0.0001 p < 0.6696

As vs Trichoderma p = 0.9780 p = 0.9726

Data are expressed as mean values ± SD (n = 4) and have been analyzed by two-wayanalysis of variance. Mean values followed by the same letter within columns arenot significantly different by Tukey’s test at the 5% level. Control, As 5 and As 10refer to As concentrations of 0, 5 and 10 mg L−1, respectively; -T refers to plantsnon-inoculated with Trichoderma; T22 refers to plants inoculated with Trichodermah

c

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C

ittttnsi

A

PvSLEPSGat

R

A

asperellum under laboratory conditions. Curr Microbiol 2010;61:261–6.

arzianum; P1 refers to plants inoculated with Trichoderma atroviride.a Values in parenthesis are the percent increase vs. control non treated with Tri-

hoderma.

uch as As and P. In particular, As-tolerant plant growth-promotingungi (PGPF), such as those used in this study, may be practicallyseful to support crop production in As-contaminated environ-ents. Recent studies (Srivastava et al., 2011, 2012; Turpeinen et al.,

004) demonstrated that some Trichoderma strains can maintainheir metabolic activities in contaminated soils, eventually adsorb-ng, volatizing and/or detoxifying harmful elements like As. Possible

echanisms include valence transformation, extra- and intracellu-ar precipitation, active uptake and methylation.

onclusions

Trichoderma spp. reduces phytotoxicity of As-containingrrigation water, possibly by increasing nutrient (i.e. P) uptake andhus enhancing plant growth. This work indicates that it is possibleo combine bio-fertilization and alleviation of As toxicity in impor-ant food crops by using selected strains of Trichoderma, includinghose already available on the market. However, further studies areecessary to fully understand the molecular mechanisms that allowome beneficial microbes to enhance the tolerance of the plants tonorganic toxicants.

cknowledgments

The research leading to these results has received funding from:RIN MIUR 2010–2011 n. 2010JBNL17 005, MAE – CISIA: Inno-azione e Sviluppo del Mezzogiorno – Conoscenze Integrate perostenibilità ed Innovazione del Made in Italy Agroalimentare –egge n. 191/2009; PRIN MIUR 2008 – prot. 2008SNPNC2, Theuropean Union Seventh Framework Programme (FP7/2007–2013)URE under the grant agreement n◦265865; PON MICROBIOTECH,IMBA Regione Campania, PSR 124 Regione Calabria MI.P.RE.VEGE.,HW – Ministero dello Sviluppo Economico Made in Italy. Authorscknowledge Drs. Cirillo and Pannino, who kindly collaborated inhe measurement of net photosynthesis rate.

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