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Crop Protection 26 (2007) 782–787

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Effects of soil solarization and Trichoderma on strawberry production

M. Porras�, C. Barrau, F. Romero

Department of Plant Pathology, CIFA Las Torres-Tomejil, IFAPA, CICE Junta de Andalucıa, 41200-Alcala del Rıo, Sevilla, Spain

Received 6 July 2006; accepted 10 July 2006

Abstract

Soil solarization and Trichoderma, alone and combined, were tested in three consecutive annual production cycles in Huelva

(southwestern Spain) an environment representative of the coastal strawberry production area, to evaluate the effectiveness in enhancing

strawberry yield and the relationship between Trichoderma soil population, root colonization by Trichoderma, yield and root weight.

Solarization was conducted during the summer, using clear 50 mm low-density polyethylene mulch. Trichoderma spp. were applied via

drip irrigation and dip, adding to the soil 7-days before planting (108 conidia/m2), and strawberry roots were dipped in a suspension of

Trichoderma (106 conidia/ml) prior to planting. Mean soil temperatures in solarized plots averaged 46 1C at 5 cm depth, 43 1C at 10 cm,

and 38 1C at 20 cm. Solarization reduced Trichoderma soil populations. Nevertheless, Trichoderma soil populations increased over the

three consecutive years in solarized plots, and no differences were observed after three repeated treatments of the same site. Solarization

did not significantly reduce root colonization by Trichoderma. Combination of solarization with Trichoderma applications significantly

increased Trichoderma soil populations relative to the solarization alone treatment, and root colonization by Trichoderma compared to

the untreated control and the solarization alone treatment. Soil solarization, alone or combined with Trichoderma applications, increased

strawberry yield 77.6% and 78.2% in year 2, and 11.0% and 43.2% in year 3, respectively. Trichoderma spp. became established in soil,

and by the end of each season it could be recovered from soil samples and from root segments. Trichoderma applications increased

Trichoderma soil populations, root colonization, root weight and strawberry yield 84.9% in year 2 and 17.6% in year 3. Significant

positive correlations were observed between Trichoderma soil populations and strawberry yield, and between Trichoderma soil

populations and root colonization by Trichoderma.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Biological control; Soil solarization; Strawberry; Trichoderma harzianum; Trichoderma viride

1. Introduction

Huelva (southwestern Spain) is the most important areaof strawberry (Fragaria� ananassa Duch.) production inEurope (Lopez-Aranda, 1999). In 2002, 7078 ha wascultivated, yielding 261,236 metric tons of strawberry fruit,80% of which was exported (Anonymous, 2006).

Methyl bromide is being phased out globally because it isconsidered an ozone depleting compound (Anonymous,2000). Since 2005, the use of methyl bromide has beenbanned in European Union (Batchelor, 2002). Chemical,physical, and biological alternatives have been evaluated instrawberry fruit production (Rieger et al., 2001; Duniway,

e front matter r 2006 Elsevier Ltd. All rights reserved.

opro.2006.07.005

ing author. Tel.: +34955045613; fax: +34955045625.

ess: maria.porras.ext@juntadeandalucia.es (M. Porras).

2002; LaMondia et al., 2002; Martın and Bull, 2002; Porraset al., 2003; De los Santos et al., 2003).Solarization is a process that uses solar radiation to heat

soil under a transparent plastic film to temperatures thatare detrimental to soilborne pathogens. Increased soiltemperatures can decrease populations of plant pathogens(Katan, 1981; Pinkerton et al., 2002). Solarization canenhance the effectiveness of other pest managementapproaches (Katan, 1981; Ben-Yephet et al., 1987;Ramirez-Villapudua and Munnecke, 1988) and has theadditional advantages of being a nonchemical alternativemethod for pathogen control (Batchelor, 2002).

Trichoderma spp. are among the microorganisms thatsurvive the solar heating (Ben-Yephet et al., 1987; DeVay,1991; Sivan and Chet, 1993; Otieno et al., 2003). Soilfumigation affects the environment and also producesvariations in Trichoderma populations since these fungi can

ARTICLE IN PRESSM. Porras et al. / Crop Protection 26 (2007) 782–787 783

reproduce rapidly after methyl bromide treatments (Mun-necke et al., 1981). The potential of Trichoderma species asbiocontrol agents of plant diseases was first recognized inthe early 1930s (Weindling, 1932), and in subsequent years,control of many diseases has been added to the list(Lindsey and Baker, 1967; Howell, 1987; Smith et al., 1990;Wilcox et al., 1992; Washington et al., 1999; Harman,2000). This has culminated in the commercial productionof several Trichoderma species for the protection andgrowth enhancement of plants (Howell, 2003).

Cook and Baker (1983) defined biological control as ‘‘thereduction of inoculum density or disease-producing activ-ities of a pathogen or parasite in its active or dormant state,by one or more organisms, accomplished naturally orthrough manipulation of the environment, host, orantagonist, or by mass introduction of one or moreantagonists’’. Disease suppression by biocontrol agents isthe sustained manifestation of interactions between theplant, the pathogen, the biocontrol agent, the microbialcommunity on and around the plant and the physicalenvironment (Handelsman and Stabb, 1996). Species ofTrichoderma are primarily studied for their ability tocontrol plant disease through antagonism, rhizospherecompetence, enzyme production, induction of defenseresponse in plants, metabolism of germination stimulantsand beneficial growth of the host following root coloniza-tion (Weindling, 1932, 1934, 1937; Wright, 1956; Eladet al., 1981; Benhamou and Chet, 1993; Zimand et al.,1996; Bailey and Lumsden, 1998; Gams and Bissett, 1998;Washington et al., 1999; Howell, 2003). Determination ofthese effects depends on many interactions that take placein the soil between Trichoderma spp., other microorgan-isms, the plant root, and the soil environment (Bailey andLumsden, 1998).

The objective of our research was to evaluate the abilityof soil solarization, Trichoderma, and a combination of thetwo to enhance in-field production of strawberry.

2. Materials and methods

2.1. Field experiments

Experiments were conducted in an experimental straw-berry farm located in Moguer (Huelva, SW Spain; 61W,371N), for three consecutive growing seasons from Octoberto May (2000–2001, 2001–2002, and 2002–2003). The soil(90% sand, 2.8% slime, and 7.2% clay) had never beenpreviously fumigated, and strawberry had not been grownthere prior to the start of this work. A randomizedcomplete block design with four replications (blocks) wasused. Each plot was 12.5� 3.3m and had three raised beds.Treatments were soil solarization and applications ofTrichoderma spp., alone or combined (Solarization; Tri-

choderma; Solarization+Trichoderma), and the untreatedcontrol. Treatments were done in the same plots each year.Plots were solarized from 10 July to 12 September 2000,from 11 July to 18 September 2001, and from 11 July to 27

September 2002, using clear 50 mm low-density polyethy-lene mulch. Soil temperatures at 5, 10, and 20 cm weremeasured daily in single solarized and nonsolarized plots at8:00 a.m., 1:00 p.m., and 6:00 p.m., using soil thermo-meters, to estimate the thermal inactivation potential ofsolarization.A premix of Trichoderma harzianum and Trichoderma

viride (Tusals, 5� 108 conidia/g; Newbiotechic, S.A. Paseode Bollullos de la Mitacion, 6 Parque Industrial A-49,PIBO, 41110 Bollullos de la Mitacion, Seville, Spain) wasapplied via drip irrigation and dip. Drip irrigation(108 conidia/m2) was done once (7 days before planting),during 10min. Furthermore, the roots of strawberryplants were dipped, during 10min, in a suspension ofT. harzianum and T. viride (106 conidia/ml) immediatelyprior to planting.Each year in the last week of October, strawberries

cultivar Camarosa were planted. Plants were grown in anintensive annual system on drip-irrigated raised beds(35 cm high and 50 cm wide) with black plastic mulch.Plants were set 25 cm apart within each of two rows perraised bed, and the rows were spaced 25 cm apart. Bedswere covered with 70 cm-tall polyethylene perforated filmtunnels from late November to late March (Lopez-Arandaand Bartual, 1999).

2.2. Quantifying of Trichoderma spp. propagules

Soil samples were collected in July (prior to solarization),in October (10 days before planting), and weekly from thedate of planting to the end of the trials, each year. Samplesconsisted of three cores (4.5 cm in diameter by 20 cm deep)collected between two plants on the same row of the centralraised bed. Soil samples were held at 4 1C, from 1 to 7 days,and processed as follows.Ten-gram aliquots of the soil mixture were suspended in

100ml of sterile water agar (0.1%), shaken for 15min at150 rpm, and 100 ml aliquots from undiluted suspensionswere spread on Petri dishes with Trichoderma SelectiveMedium adapted by Askew and Laing (1993). Dry weightof soil samples was determined using 50 g subsamples.Ten replicate dishes were used for each plot. Plates were

incubated at 25 1C in dark for 7 days. Based onmorphological characteristics, Trichoderma spp. colonieswere identified and counted (Gams and Bissett, 1998) andresults were expressed as colony forming units per gram ofdry soil (CFU/g).Persistence of inoculum in soil over time can be

visualized as a survival curve (Benson, 1994). Therefore,results are presented as the area under the curve (AUC) soilpopulation of Trichoderma spp. (CFU/g) versus time(days).

2.3. Strawberry yield

All ripe fruit of 10 randomly selected plants per plot wereharvested once per week from February to May. All fruit

ARTICLE IN PRESS

Table 1

Soil temperatures in solarized and nonsolarized plots at the experimental

strawberry farm located in Moguer (Spain)a

Treatment Depth (cm) Hour Soil temperatureb (1C)

Solarized 5 8:00 a.m. 23.3

1:00 p.m. 46.4

6:00 p.m. 45.6

10 8:00 a.m. 25.2

1:00 p.m. 38.5

6:00 p.m. 43.3

20 8:00 a.m. 27.4

1:00 p.m. 31.2

6:00 p.m. 37.6

Non-solarized 5 8:00 a.m. 17.2

1:00 p.m. 31.4

6:00 p.m. 33.3

10 8:00 a.m. 20.1

1:00 p.m. 27.2

6:00 p.m. 32.4

20 8:00 a.m. 22.4

1:00 p.m. 23.8

6:00 p.m. 28.3

aTemperature were recorded at 8:00 a.m., 1:00 p.m., and 6:00 p.m.

Temperature data were collected from 10 July to 12 September 2000, from

11 July to 18 September 2001, and from 11 July to 27 September 2002.bMean temperature. The mean temperatures were averaged for the

years 2000, 2001 and 2002.

M. Porras et al. / Crop Protection 26 (2007) 782–787784

were counted and sorted based on marketability criteria.Fruit were categorized as unmarketable if they had Botrytisfruit rot, leather rot, anthracnosis, or the presence of otherdiseases. Only marketable fruit were weighed.

2.4. Root weight and colonization of the root by

Trichoderma

At the end of each annual production cycles, fresh rootweight of the 10 randomly selected plants per plot wereobtained.

Plant root systems were also examined for symptoms ofdisease prior to fungal isolation in Potato dextrose agarand V8 juice agar (data not shown).

In order to determine the colonization of the root byTrichoderma spp., 12 root segments were selected atrandom from a pooled sample of asymptomatic segmentsfrom the proximal, mid and distal regions of each plant(Dashwood et al., 1993). Roots were surface-disinfested for30 s in 1% sodium hypochlorite solution, washed indistilled water, and air-dried. Root segments were platedon Trichoderma Selective Medium (Askew and Laing,1993). Plates were incubated at 25 1C in dark for 7 days. Atotal of 480 segments per treatment was assessed at eachyear. The percent root colonization by Trichoderma spp.was calculated as the number of root segment withTrichoderma divided by the total number of root segmentfor each plant individually.

2.5. Statistical analysis

Analysis of variance (Statistix 8, Analytical Software forWindows) was performed for AUC. Mean separation wasconducted using the Tukey’s Studentized Range (HSD)comparison method at Po0.05.

Analysis of variance (ANOVA) was used to determinethe effects of treatments on the root colonization byTrichoderma spp. Arc sine transformation of percentagedata was used to stabilize variances. Tukey HSD compar-ison method was used to separate means after ANOVA atP ¼ 0.05.

Strawberry production and weights of roots data weresubjected to ANOVA and mean separation using theTukey HSD test (Po0.05).

In order to determine the effect of variation inTrichoderma soil population on variation in yield, rootcolonization by Trichoderma and root growth, Pearsoncorrelation values were calculated.

3. Results

3.1. Soil temperatures

Mean soil temperatures at 6:00 p.m. in solarized plotsaveraged 46 1C at 5 cm, 43 1C at 10 cm, and 38 1C at 20 cm,compared to 33, 32, and 28 1C, respectively, in nonsolar-ized plots (Table 1).

3.2. Trichoderma soil population and strawberry yield

Trichoderma applications increased Trichoderma soilpopulation 9.0% in year 1, 403.1% in year 2, and277.9% in year 3 relative to the untreated control; anddifferences were observed in years 2 and 3 (Table 2).

Trichoderma applications increased yield 6.5% in year 1,84.9% in year 2, and 17.6% in year 3 relative to theuntreated control; and differences were observed in years 2and 3 (Table 2).Pearson correlation values were calculated to describe

the relationship between Trichoderma populations andmarketable yield. In the first year there was no significantcorrelation. However, after the second treatment, signifi-cant positive correlations between Trichoderma popula-tions and yield in both years were consistently observed(Table 3).Solarization alone reduced Trichoderma soil population

70.1% in year 1, 79.0% in year 2, and 59.3% in year 3; anddifferences were observed in years 1 and 2. Nevertheless,the combination of solarization and Trichoderma increasedTrichoderma soil population 77.7% in year 1, 506.5% inyear 2, and 272.5% in year 3 relative to the solarizationalone; and differences were observed in years 2 and 3(Table 2).The combination of solarization and Trichoderma

increased significantly yield relative to the untreatedcontrol in years 2 and 3; and relative to the solarizationalone treatment in year 3 (Table 2). No differences wereobserved in yield between the Trichoderma alone treatment

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Table 3

Correlation coefficients between the Trichoderma soil populations

(AUTC), strawberry yield (Kg/ha), root colonization by Trichoderma

(%), and fresh root weight (g/plant)

YEAR TSP-Y TSP-RCT RCT-RW

2000–2001 �0.0792 0.4119* 0.7080**

2001–2002 0.5617* 0.7294** 0.6667**

2002–2003 0.5807* 0.7942*** 0.6144*

Symbols *, **, and *** indicate significant at Po0.05, 0.01, and 0.001,

respectively.

TSP ¼ Trichoderma soil populations (AUTC);Y ¼ strawberry yield (kg/

ha); RCT ¼ root colonization by Trichoderma (%); RW ¼ fresh root

weight (g/plant).

Table 2

Effects of Trichoderma and soil solarization on Trichoderma soil populations (AUTC), and strawberry yield (kg/ha)a

Year Treatment Trichoderma populations (AUTC) Yield (kg/ha)

2000–2001 Solarization 10,257 c 16,883

Trichoderma 37,370 a 18,113

Solarization+Trichoderma 18,231 bc 17,442

Control 34,284 ab 17,007

P 0.00151 0.94603

2001–2002 Solarization 118,309 c 26,085 ab

Trichoderma 2,272,000 a 27,150 a

Solarization+Trichoderma 599,276 b 26,157 ab

Control 563,668 b 14,682 c

P 0.00007 0.04516

2002–2003 Solarization 244,254 c 39,300 b

Trichoderma 1,668,000 a 41,645 ab

Solarization+Trichoderma 665,597 b 50,738 a

Control 600,320 bc 35,400 c

P 0.00107 0.01594

aData are the means of four replications (plots). Mean values within the same columns followed by the same letter are not significantly different

according to the Tukey HSD test (Po0.05). P ¼ significant probability values associated with the F tests.

M. Porras et al. / Crop Protection 26 (2007) 782–787 785

and the combination of solarization and Trichoderma

(Table 2).Trichoderma soil population and root colonization by

Trichoderma were highly correlated (Table 3).

3.3. Root weight and colonization of the root by

Trichoderma

Trichoderma applications increased root colonization byTrichoderma 360.6% in year 1, 50.4% in year 2, and200.4% in year 3 relative to the untreated control; anddifferences were observed each year (Table 4).

Trichoderma applications increased root weight 32.9% inyear 1, 44.8% in year 2, and 98.4% in year 3 relative to theuntreated control; and differences were observed in years 2and 3 (Table 4).

Pearson correlation values were calculated to describethe relationship between root colonization by Trichoderma

and root weight. Significant positive correlations wereobserved in years 1, 2, and 3 (Table 3).Soil solarization alone did not significantly reduce the

rate of colonization of the root by Trichoderma spp.relative to the untreated control. However, combination ofTrichoderma applications with soil solarization signifi-cantly increased the number of root segments colonizedby Trichoderma spp. relative to the untreated control andthe solarization alone treatment (Table 4).No differences were observed in root weight between the

solarization alone treatment and the untreated control.Nevertheless, the combination of solarization and Tricho-

derma increased root weight 15.5% in year 1, 32.7% in year2, and 44.6% in year 3 compared to the untreated control;and differences were observed in year 3 (Table 4).

4. Discussion

Potential use of biological control agents as replacementsor supplements for chemical fungicides have been ad-dressed in many reports (Weindling, 1932, 1934, 1937;Wright, 1956; Lindsey and Baker, 1967; Elad et al., 1981;Benhamou and Chet, 1993; Zimand et al., 1996; Bailey andLumsden, 1998; Gams and Bissett, 1998; Washington et al.,1999; Howell, 2003), but few have been tested under fieldconditions. In this study, the potential of Trichoderma spp.and soil solarization, alone or combined, for enhancing in-field production of strawberry was evaluated over a 3-yearperiod.The results indicate a significant correlation between

Trichoderma populations and yield after repeated Tricho-

derma applications in the same site. The closest relationshipbetween Trichoderma soil populations and marketable

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Table 4

Effects of Trichoderma and soil solarization on root colonization by Trichoderma (%), and fresh root weight (g/plant)a

Year Treatment Root colonization by Trichoderma (%) Roots (g/plant)

2000–2001 Solarization 0 b 50.60

Trichoderma 7.50 a 61.18

Solarization+Trichoderma 5.50 a 53.18

Control 2.08 b 46.03

P 0.00173 0.47383

2001–2002 Solarization 18.53 c 53.13 ab

Trichoderma 47.18 a 66.25 a

Solarization+Trichoderma 34.53 ab 60.70 ab

Control 31.38 bc 45.75 b

P 0.00139 0.04435

2002–2003 Solarization 26.45 b 70.40 cd

Trichoderma 72.93 a 106.63 a

Solarization+Trichoderma 70.43 a 77.70 bc

Control 36.40 b 53.75 d

P 0.00000 0.04977

aData are the means of four replications (plots). Mean values within the same columns followed by the same letter are not significantly different

according to the Tukey HSD test (Po0.05). P ¼ significant probability values associated with the F tests.

M. Porras et al. / Crop Protection 26 (2007) 782–787786

yield were obtained after 2 years of Trichoderma applica-tions, when Trichoderma populations were at their highest.

Although solarization reduced Trichoderma soil popula-tions, Trichoderma are among the microorganisms thatsurvive the solar heating (Ben-Yephet et al., 1987; DeVay,1991), or rapidly recolonize. According to this work,Trichoderma soil populations increased over the threeconsecutive years in solarized plots, and no differenceswere observed in Trichoderma soil populations between thesolarization alone treatment and the untreated controlafter three repeated soil solarization of the same site.Furthermore, root colonization by Trichoderma increaseover the three consecutive years in solarized plots, and nodifferences were observed between solarized and nonsolar-ized plots in all years.

No differences were observed in strawberry yieldbetween treatments in year 1, presumably due to the lowpropagule densities because the soil had never beenpreviously inoculated with Trichoderma, and strawberryhad not been grown there prior to the start of this work.Nevertheless, solarization and Trichoderma, alone orcombined, significantly increased strawberry yield relativeto the untreated control after repeated treatments of thesame site. Differences in marketable yield are more difficultto detect when disease pressure is low. During 2001–2002season, Colletotrichum spp. were isolated from most of theplants, causing anthracnosis crown rot (Porras et al., 2003).Consequently, differences in marketable yield betweentreated plots and untreated plots were the most in year 2.

Our study demonstrates the potential of Trichoderma

strains to establish in the soil and colonize the roots ofstrawberry plants, enhancing root weight and strawberryyield. The joint action of soil solarization and Trichoderma

applications increased root colonization by Trichoderma

relative to the untreated control and the solarization alonetreatment; and no differences were observed between theTrichoderma alone treatment and the combination withsolarization. Therefore, the combination of soil solariza-tion and Trichoderma applications could be considered ifthe soil are infested by soil pathogen populations, becauseit could be reduced the most (Porras, 2005).

Trichoderma spp. got to became established in the soil,and by the end of each season it could be recovered fromthe soil samples and from the root segments.Plant growth promotion by species of Trichoderma has

been reported before (Lindsey and Baker, 1967). Accordingto this, Trichoderma applications increased root coloniza-tion by Trichoderma and root weight relative to theuntreated control, and significant positive correlationswere observed between both. Furthermore, Trichoderma

soil population and root colonization by Trichoderma werehighly correlated.

Acknowledgments

This research was supported by a Research Grant fromInstituto Andaluz de Investigacion y Formacion Agraria,Pesquera, Alimentaria y de la Produccion Ecologica(IFAPA), Junta de Andalucia.

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