For Peer Review

Evaluating the survival and environmental fate of the biocontrol agent

Trichoderma atroviride SC1 in vineyards in northern Italy

Journal: Applied Microbiology

Manuscript ID: JAM-2008-0650.R2

Journal Name: 1 Journal of Applied Microbiology - JAM

Manuscript Type: JAM - Full Length Paper

Date Submitted by the Author:

16-Oct-2008

Complete List of Authors: Longa, Claudia; Fondazione Edmund Mach, SafeCrop Centre Savazzini, Federica; Fondazione Edmund Mach, SafeCrop Centre Tosi, Solveig; Università degli Studi di Pavia, Ecologia del Territorio e degli Ambienti Terrestri Elad, Yigal; The Volcani Center, Department of Plant Pathology and Weed Research Pertot, Ilaria; Fondazione Edmund Mach, SafeCrop Centre

Key Words: environmental fate, fungal ecology, population dynamics, risk assessment, biocontrol agent

For Peer Review

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Evaluating the survival and environmental fate of the biocontrol agent Trichoderma 1

atroviride SC1 in vineyards in northern Italy 2

C.M.O. Longa1, F. Savazzini

1, S. Tosi

2, Y. Elad

3 and I. Pertot

1 3

1 Safecrop Centre, FEM, Via Mach 1, San Michele all’Adige, Trento 38010, Italy 4

2 Università degli Studi di Pavia, S. Epifanio 14, Pavia 27100, Italy 5

3 Department of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 6

50250, Israel 7

8

Running headline: Survival of Trichoderma atroviride SC1 9

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Correspondence: C.M.O. Longa, SafeCrop Centre, Fondazione Edmund Mach, Via E. Mach, 11

1 38010 S. Michele all'Adige (TN) – Italy; E-mail address: claudia.longa@iasma.it, Tel.: + 39 12

0461 615 505, Fax: + 39 0461 615 500 13

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ABSTRACT 27

28

Aims: To study the survival in the soil and the dispersion in the environment of Trichoderma 29

atroviride SC1 after soil applications in a vineyard. 30

Methods and Results: T. atroviride SC1 was introduced into soil in two consecutive years. 31

The levels of T. atroviride populations at different spatial and temporal points following 32

inoculation were assessed by counting the colony-forming units and by a specific quantitative 33

real-time PCR. A high concentration of T. atroviride SC1 was still observed at the 18th

week 34

after inoculation. The vertical migration of the fungus to a soil depth of 0.4 m was already 35

noticeable during the first week after inoculation. The fungus spread up to 4 m (horizontally) 36

from the point of inoculation and its concentration decreased with increasing distance 37

(horizontal and vertical). It was able to colonize the rhizosphere and was also found on 38

grapevine leaves. One year after soil inoculation, T. atroviride SC1 could still be recovered in 39

the treated areas. 40

Conclusions: T. atroviride SC1 survived and dispersed becoming an integrant part of the 41

local microbial community under the tested conditions. 42

Significance and impact of the study: The persistence and rapid spread of T. atroviride SC1 43

represent good qualities for its future use as biocontrol agent against soilborne pathogens. 44

45

Key Words: environmental fate, fungal ecology, population dynamics, risk assessment, 46

biocontrol agent 47

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Introduction 48

49

Species belonging to the anamorphic genus Trichoderma have been extensively mentioned in 50

scientific literature as biocontrol agents (BCAs) active against numerous plant pathogens and 51

several commercial biocontrol products based on specific strains of Trichoderma species have 52

been developed for use in greenhouses and on open field crops (Elad 2000; Harman 2000; 53

Paulitz and Bélanger 2001; Howell 2003). The high ecological adaptability of members of the 54

genus Trichoderma makes these species good candidates for use in biocontrol applications in 55

a variety of habitats (Hjeljord and Tronsmo 1998). 56

Strains of Trichoderma atroviride P. Karst. have been used against a wide range of 57

economically important airborne and soilborne plant pathogens (Dodd et al. 2003). The 58

antagonistic activity of T. atroviride against fungal hosts is attributed to a synergistic effect of 59

successful nutrient competition, the production of cell wall-degrading enzymes and antibiosis 60

(Lu et al. 2004; Brunner et al. 2005). 61

T. atroviride strain SC1, originally isolated in northern Italy from decaying hazelnut wood, 62

can effectively control several plant pathogens (patent pending; Pertot et al., 2008). This 63

fungal strain is particularly active against Armillaria grapevine root rot, which is an emerging 64

disease in traditional grape-growing areas that is caused by Armillaria mellea (Vahl) P. 65

Kumm (Pertot et al. 2008). T. atroviride SC1 can survive under and adapt to a wide range of 66

environmental conditions (soils and phyllosphere environments). It grows at temperatures 67

between 10 and 30°C, with optimal growth at 25°C. This fungus tolerates a wide range of pH 68

levels, but its growth is reduced on alkaline media (pH ≥ 8), and can utilize a large number of 69

carbon and nitrogen sources (Longa et al. 2008). 70

Under experimental conditions, T. atroviride has been shown to reduce the amount of A. 71

mellea inoculum in soil and prevent infections of strawberry plants if high concentrations of 72

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conidia are applied directly into the soil (patent pending; Pertot et al., 2008). In soil, A. mellea 73

inoculum can survive for several months protected by root debris, even in the absence of a 74

living host plant (Pertot et al. 2008). Therefore, the successful biocontrol of A. mellea by T. 75

atroviride SC1 depends on its ability to survive and remain active for a long time. The 76

survival and spread of a microorganism after it has been introduced into the soil depends on 77

its interactions with the abiotic and biotic components of the soil (Bae and Knudsen 2005) 78

and the environmental parameters of the local ecosystem (Kredics et al. 2003). 79

In addition to effective biocontrol activity and environmental adaptability, a prerequisite for 80

the use of a BCA is that any non-target effects on the environment and/or non-target 81

organisms should be at least tolerable, if not negligible (Winding et al. 2004). European 82

regulations (Anon 1991) concerning the introduction of plant protection products into the 83

market require a rigorous risk assessment for the registration of microorganisms as 84

fungicides. The collection of basic information on colonization ability and persistence, spread 85

and possible dispersal routes of the microorganism under conditions typical of the 86

environment in which it will be used are milestones in the development of a BCA. 87

Most studies of Trichoderma population dynamics in soil have been conducted under 88

microcosm conditions (Weaver et al. 2005; Cordier et al. 2007; Savazzini et al., 2008). There 89

is some information available about the survival and spread of Trichoderma species once they 90

have been applied in the field (Elad et al. 1981a; 1981b), but no study has addressed the 91

spatial dispersal of Trichoderma spp. over time, following soil applications. In spite of the 92

effective biocontrol that T. atroviride SC1 provides and its inherent adaptability, the fate of 93

this fungus in the natural environment after its introduction is still unknown. 94

T. atroviride SC1 grown on growth media could be mixed into surface soil in established 95

vineyards to prevent A. mellea infections. In new vineyards, the T. atroviride SC1 could be 96

placed directly into the holes into which young vines are transplanted. In the grapevine trellis 97

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systems used around the world, the planting distance between rows is generally no more than 98

4 m. Thus, the aims of this study were to monitor the survival and horizontal and vertical 99

migration of T. atroviride SC1 in soil and its dispersal among aboveground parts of the crop 100

(grapevine leaves) under natural field conditions in northern Italy. 101

102

Materials and Methods 103

104

Site description 105

106

Field experiments were carried out in two vineyards located in the Adige Valley (Trentino, 107

northern Italy). The first experiment was established in a sandy clay soil with a pH of 7.78 108

and conducted from May 15th

through September 17th

, 2006. The second experiment was 109

established in a sandy loam soil with a pH of 7.64 and conducted from August 1st through 110

December 4th

, 2007. Meteorological data (rain fall and soil temperature at a depth of 0.1 m) 111

were recorded by an automated meteorological station (http://meteo.iasma.it/meteo/). 112

A preliminary screening of indigenous populations of Trichoderma species, for each 113

vineyard, was carried out at the two sites by counting colony-forming unit (CFU) in 30 soil 114

samples of 10 g each. Soils were plated on a semi-selective medium composed of potato 115

dextrose agar (PDA; Oxoid, Cambridge, UK) supplemented with 100 µg g-1

rose bengal 116

(Sigma-Aldrich, St. Louis, MO, USA), 100 µg g-1

streptomycin (Sigma-Aldrich) and 50 µg g-1

117

chloramphenicol (Sigma-Aldrich). This semi-selective medium was used to culture samples 118

for CFU counts in all experiments. Trichoderma species were identified on the basis of their 119

morphology, using the techniques described by Rifai (1969). 120

121

Microorganism, inoculum and sampling 122

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123

T. atroviride SC1 (CBS 122089) was maintained on PDA in slants at 4°C and, for long-term 124

storage, in Microbank tubes (Pro-Lab Diagnostics, Cheshire, UK) at -80°C. The inoculum 125

was prepared by inoculating sterilized boiled rice (Gams et al. 1998) with T. atroviride SC1 126

and incubating the cultures at 25°C for 21 days. The conidia that were grown on the rice to be 127

used as inoculum always had an average viability close to 100%. In order to test the effect of 128

the media on native Trichoderma spp., equal quantities of sterile autoclaved rice and soil were 129

mixed, in a manner similar to that used in the experiments with T. atroviride SC1 that 130

involved five replicates. Five untreated replicates served as an untreated control. Individual 131

Trichoderma spp. were counted and identified as previously described. In all experiments, the 132

concentration of viable T. atroviride SC1 was estimated by collecting 1-g samples of soil 133

from each plot, placing each sample in 10 ml of sterile water, making 10-fold serial dilutions 134

and plating 1 ml of each dilution onto semi-selective media. Fungal colonies were counted 135

after seven days of incubation and the Trichoderma population were presented in terms of log 136

CFU g -1

dry soil. The soil dry weight was estimated after the incubation of the sample (or 137

sub-sample) at 60°C for 48 h. T. atroviride SC1 colonies were distinguished from other 138

Trichoderma species by their characteristic aerial mycelia (white at first, then rapidly turning 139

yellowish green to olive green). For the unequivocal identification of our isolate, the identity 140

of almost 10% of the colonies from each plate that had been morphologically identified as T. 141

atroviride was confirmed by PCR analysis using primers and a Taq-Man probe set based on a 142

base mutation of the endochitinase gene (Ech42) that is specific for T. atroviride SC1 143

(Savazzini et al., 2008). 144

145

Survival and vertical dispersion in soil 146

147

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The aim of these experiments was to determine whether T. atroviride SC1 can survive and 148

migrate vertically in the soil. Experiments consisted of six plots of 0.6 × 0.6 m each, which 149

were located between grapevine plants in the row in the vineyards. The row of grapevines was 150

mechanically kept free of any other vegetation (common agricultural practice in the area). 151

Three plots were inoculated with T. atroviride SC1 using 500 g of boiled-rice inoculum . The 152

inoculum was mixed into the soil surface layer (approximately 30 mm deep). The initial 153

concentration of the fungal inoculum in this layer was estimated to be 1.2 × 108 CFU g

-1 dry 154

soil. Three additional uninoculated plots were used as non-treated control. In each plot, the 155

soil was sampled by excavating the external part of one side of the plot and exposing the soil 156

profile. Sampling was done by collecting three transverse core samples of soil (50 ml, 30 mm 157

in diameter × 70 mm height) at different depths (on the surface and at 0.1, 0.2, 0.3 and 0.4 m) 158

and time points (at inoculation time and 1, 5, 9 and 18 weeks after T. atroviride SC1 159

inoculation). An additional soil sampling was made one year after the inoculation of the first 160

experiment. 161

A sub-sample of soil (1 g) was removed from each sample and placed in 10 ml 0.01% Tween 162

80 (Acros Organics, Geel, Belgium), shaken for 4 min using a vortex (Heidolph Instruments, 163

Schwabach, Germany) and left to stand for 1 min. A dilution series in sterile distilled water 164

were completed and the diluted suspensions were plated on Petri dishes containing the semi-165

selective media. These Petri dishes were then incubated at 25°C and final colony counts were 166

made on plates with 30-300 colonies per plate after five days of incubation. There were three 167

replicates for each soil sample. The identities of T. atroviride SC1 colonies were confirmed as 168

described above. 169

In 2006, real-time PCR was used to confirm the identity of the T. atroviride colonies counted 170

in the experiment and to determine the numbers of T. atroviride SC1 genome copies (CN) in 171

each of the samples. Since a good correlation between CFU and CN was observed in 2006, 172

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indicating the identity of T. atroviride SC1 CFU, the real-time PCR procedure was not 173

repeated in 2007. In 2007, the CFU counting was coupled with qualitative PCR to confirm the 174

identity of a representative sample of colonies. The CFU method was also preferred because it 175

quantifies the living propagules while the real-time PCR may indicate only the presence of the 176

specific DNA as describe in Savazzini et al., 2008. For the real-time PCR analysis, two 177

independent sub-samples were collected for each combination of depth and time and the DNA 178

extraction and real-time PCR analysis of each sample were performed as described by 179

Savazzini et al. (2008). 180

181

Spatial dispersal and environmental fate 182

183

The aim of this experiment was to verify whether the T. atroviride SC1 applied to the soil at 184

planting would spread to nearby plants in the same row and/or the soil between the rows. The 185

dispersion of T. atroviride SC1 conidia was examined in 2006. Holes measuring 0.3 × 0.3 × 186

0.3 m were dug in the vineyard row between grapevine plants. Ten holes were filled up with a 187

mixture of the dug soil and T. atroviride inoculum (400 g hole-1

). The initial fungal inoculum 188

concentration was 1.6 × 106 CFU g

-1dry soil. Ten additional holes were filled back in with the 189

non-treated dug soil. A one-year-old grapevine plant (Pinot gris on Kober 5BB) was planted 190

in each hole. 191

Two sets of soil samples were collected. The first sampling was carried out nine weeks after 192

inoculation in both the treated and non-treated holes (0 m) and at horizontal distances of 0.5 193

and 2.0 m from the hole. At each of these distances, soil samples were collected at three soil 194

depths (0, 0.1 and 0.3 m). A second sampling was performed 18 weeks after inoculation and, 195

at that time, soil samples were collected only on the surface (first 30 mm of soil) of the holes 196

and at horizontal distances of 2.0 and 4.0 m from the inoculation sites. Samples were 197

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collected and the number of CFU in each sample was determined as previously described. 198

Dispersion was evaluated in terms of T. atroviride SC1 concentration (CFU g-1

dry soil) and 199

frequency (percentage of soil samples with at least one CFU). 200

The migration of T. atroviride SC1 from the soil to the leaves of the grapevines was evaluated 201

10 weeks after soil inoculation. Three apical (younger, 160-180 cm above the soil surface) 202

leaves and three bottom (older, 70-90 cm above the soil surface) leaves were removed from 203

each plant (each plant had an average of 15 leaves) growing in the treated and non-treated 204

holes. Each freshly harvested leaf was transferred to a Falcon tube containing 30 ml of sterile 205

distilled water plus 0.01% Tween 80. These tubes were shaken for 3 minutes and 1 ml of each 206

of the resulting suspensions was then transferred to a Petri dish containing the semi-selective 207

medium. CFU were counted following seven days of incubation at 25°C. Leaf area was 208

calculated using an image processing and analysis program, Image Tool version 2.0 209

(UTHSCSA, San Antonio, TX, USA). The results were expressed as CFU mm leaf -2

. 210

At the end of the experiment, plants were removed from the soil. Soil that did not tightly 211

adhere to roots was carefully removed with light shaking and the roots were then shaken 212

vigorously in a plastic bag to dislodge the rhizosphere soil (Meriles et al. 2006). Sampling of 213

the rhizosphere soil and CFU counting for these samples (sub-samples of 1 g; three replicates) 214

was done as previously described. 215

To evaluate the influence of T. atroviride SC1 on plant growth, measurements of total length, 216

stem length and the numbers of leaves and shoots were taken for each plant in the treated and 217

non-treated areas in the ninth and 18th

weeks after soil inoculation. The root dry weight was 218

also determined for each plant at the end of the experiment (18th

week after inoculation). 219

220

Statistical analysis 221

222

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A constant value of “1” was attribute to “0” values to allow the inclusion of those data points 223

in the analysis and counts were log10 transformed to satisfy the assumption of homogeneity of 224

variance. The significance of any differences (α = 0.05) between means was tested after 225

ANOVA, using Tukey’s test. The Pearson product-moment correlation coefficient was used 226

to compare the mean concentrations of conidia at all soil depths and time points in the two 227

years, and to compare results obtained using the dilution plates and with those obtained using 228

real-time PCR methods in 2006. Statistical analyses were conducted using Statistica version 229

7.0 (StatSoft, Inc., Tulsa, OK, USA). 230

231

Results 232

233

Survival and vertical dispersion in soil 234

235

The soil temperature varied between 15.3 and 25.0°C in 2006 and between 4.5 and 22.5°C in 236

2007 (Fig 1). The amount of rain fall and its distribution were similar in the two years, with 237

averages of 18.8 mm week-1

and 21.7 mm week-1

in 2006 and 2007, respectively. 238

Preliminary analyses of soil samples from the two vineyards before T. atroviride SC1 239

introduction showed low, but detectable levels of indigenous Trichoderma species in 12 and 240

15% of the samples in 2006 and in 2007, respectively. These strains were identified as T. 241

virens, T. longibrachiatum and T. rossicum, and the concentration of all naturally occurring 242

Trichoderma spp. varied from 1 × 101 to 3.9 × 10

2 CFU g

-1 dry soil. All colonies of the 243

identified Trichoderma species were morphologically different from those of T. atroviride; 244

therefore, CFU counts of our fungus were possible. Moreover, this identification was 245

confirmed by molecular identification of a representative sample of the T. atroviride SC1-like 246

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colonies in each Petri dish. The addition of sterile rice medium to the soil did not result in a 247

significant change in the native Trichoderma spp. population. 248

The observations concerning the population dynamics and vertical dispersion of T. atroviride 249

SC1 were similar in the two years of the study (Fig 1) and there was a high Pearson’s 250

correlation coefficient (r = 0.94) between the data sets from the two years. T. atroviride SC1 251

was able to survive at a high concentration (1.15 × 107 and 5.1 × 10

6 CFU g

-1 dry soil 252

respectively in 2006 and 2007) in the first layer of soil (soil surface) through the last 253

assessment (18 weeks after inoculation). Following application, T. atroviride SC1 migrated 254

vertically in the soil very quickly, reaching a depth of 0.4 m at one week after treatment. A 255

gradient in population densities was observed in both years, with greater numbers of T. 256

atroviride SC1 CFU at the surface and fewer CFU in the deeper soil layers. At the last 257

assessment and at soil depths of 0.1, 0.2, 0.3 and 0.4 m an average of 1.2 ×104, 1.1 ×10

4, 1.7 258

×101 and 1.4 × 10

3 CFU g

-1 dry soil were detected in 2006. 2.1 × 10

5, 5.4 × 10

3, 3.6 × 10

2 and 259

6.5 × 103 CFU g

-1 dry soil

were detected in 2007. Differences in the population dynamics 260

observed in the two years were only observed for the CFU concentrations at the soil surface at 261

five and nine weeks after treatment; these values were significantly different (Tukey’s test; α 262

=0.05). In particular, 9 weeks after treatment in 2006, the T. atroviride SC1 CFU count on the 263

soil surface was even greater than the initial inoculum concentration (4.9 × 109 CFU g

-1 dry 264

soil), following an initial decrease observed during the 5th

week. In 2007, was observed a 265

slight and continuous decrease in CFU counts until the 18th

week. T. atroviride SC1 was still 266

present in the treated soil one year after the 2006 treatment, with populations ranging from 1× 267

102 to 1 × 10

3 CFU g

-1 dry soil, at a concentration similar to that of the populations of 268

indigenous Trichoderma species in the non-treated plots. Similar numbers of T. atroviride 269

SC1 (1.3 × 102 to 2.1 × 10

3 CFU g

-1 dry soil) were found in the soil surface two years after 270

2006 treatment . One year after 2007 treatment, the population of T. atroviride SC1 observed 271

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was 5.2 × 103, 2.2 × 10

3 and 1.8 × 10

2 CFU g

-1 dry soil at the soil surface, 0.1 and 0.3 m of 272

soil depth, respectively. 273

The results of the real-time PCR SC1 survival assays in 2006 confirmed the absence of the 274

SC1 strain before its introduction and the persistence of the strain in the soil after inoculum. 275

The linear relationship between the results obtained using the two quantification methods (Fig 276

2) presents a high Person’s coefficient correlation (r = 0.82). 277

278

Spatial dispersal and environmental fate 279

280

The numbers of indigenous Trichoderma species in the non-treated plots varied from 0 to 103 281

CFU g-1

dry soil and no significant differences were observed among populations at the soil 282

surface and those at depths of 0.1 and 0.3 m (Tukey’s test; α = 0.05). 283

At 9 weeks after inoculation, T. atroviride SC1 was found on the soil surface at horizontal 284

distances of 0.5 and 2.0 m from the treated holes and at depths of 0.1 m and 0.3 m (Table 1). 285

The CFU concentration of the introduced fungus was significantly higher (Tukey’s test; α = 286

0.05) than that of the indigenous Trichoderma species isolated from the soil surface in the 287

non-treated area and at horizontal distances of 0.5 and 2.0 m from the treated holes, and at a 288

depth of 10 cm at a distance of 0.5 m from the treated holes. The concentrations of SC1 289

conidia at soil depths of 0.1 and 0.3 m, at distances of 0.5 and 2.0 m away from the treated 290

holes, were not significantly higher (Tukey’s test; α = 0.05) than that of the indigenous 291

Trichoderma in the non-treated area. The frequency of T. atroviride SC1 in the samples was 292

high (100%) at 0.5 m from the treated holes at all soil depths and decreased to 90, 70 and 30% 293

at a distance of 2.0 m away from the treated holes at soil depths of 0.0, 0.1 and 0.3 m, 294

respectively. 295

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Eighteen weeks after inoculation, it was still possible to recover T. atroviride SC1 from the 296

soil surface at horizontal distances of both 2 and 4 m away from the treated holes. These CFU 297

concentrations were not significantly different (Tukey’s test; α = 0.05) from the concentration 298

of conidia of the indigenous Trichoderma species in the non-treated area. Even if low 299

concentrations of the introduced fungus were detected after 18 weeks, the frequency of T. 300

atroviride SC1 remained high at this time point (80 and 70% at 2.0 and 4.0 m from the treated 301

holes, respectively). 302

T. atroviride SC1 was found on leaves of vines planted in the treated soil. The population of 303

T. atroviride SC1 on the leaf surface of plants growing in treated soil was significantly higher 304

(Tukey’s test; α = 0.05) than the number of indigenous Trichoderma spp. isolated from plants 305

growing in the non-treated areas (Fig 3). There was a significant difference (Tukey’s test; α = 306

0.05) in the population of T. atroviride SC1 from the apical and bottom leaves of plants in 307

treated areas (Fig 3). 308

Eighteen weeks after planting, the concentration of T. atroviride SC1 in the grapevine 309

rhizospheres in the treated holes was 1.2 × 106 CFU g

-1 dry soil. 310

There were no significant differences between the number of leaves, number of shoots, dry 311

root weights, stem lengths and total lengths of the grapevine plants planted in the non-treated 312

soil and those planted in the soil treated with T. atroviride SC1 (Tukey’s test, α = 0.05; data 313

not shown). 314

315

Discussion 316

317

T. atroviride SC1 grown on boiled rice and applied to the soil surface persisted in the soil for 318

a long time after its release. In fact even two years after its introduction, the fungus was still 319

present at a concentration of 103 CFU g

-1dry soil in the soil surface. This fact suggests that 320

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this fungus is able to maintain a stable population under the tested conditions. This finding is 321

in accordance with Leandro et al. (2007), who obtained similar results in their study of a 322

strain of T. hamatum, which maintained a population level close to 103 CFU g

-1 soil in field 323

plots to which compost was added throughout the eight months of that trial. The isolation of 324

T. atroviride SC1 one (and two years for 2006 treatment) after inoculation indicates that this 325

fungus can tolerate low winter temperatures and humidity fluctuations in the soil. 326

In 2006, the population decreased after five weeks and then increased in the ninth week. In 327

contrast to this, an opposite trend was observed in 2007. This difference in population 328

dynamics may be related to differences in soil temperatures (Fig 1). The relatively high 329

temperature (closer to the optimal temperature of 25°C) in the ninth week after inoculation in 330

2006 (Fig 1) could have favored the proliferation of the fungus (Longa et al., 2008). 331

The long and stable persistence of T. atroviride SC1 under field conditions may be related to 332

the fact that the fungus was introduced on rice media that supplied an adequate and accessible 333

food base for fungal growth and sporulation. The use of this type of media may have 334

stimulated fungal proliferation. Several studies have demonstrated that when BCAs are 335

introduced together with a nutrient source, there may be an increase of their proliferation in 336

the soil (Beagle-Ristaino and Papavizas 1985; Sivan and Chet 1989). The long persistence of 337

T. atroviride SC1 may be regarded as a trait that will make this fungus a good candidate for 338

use against A. mellea. In fact, A. mellea can survive for a long time inside root debris. 339

Therefore, it is important that the activity of a biocontrol agent directed against this pathogen, 340

which is commonly related to its survival in soil, be adequately persistent (Pertot et al., 2008). 341

Fungal migration patterns permit the establishment of populations of this fungus least at 342

depths of up to 0.4 m, following inoculation of the soil surface. This finding agrees with 343

observations of indigenous populations of Trichoderma spp. that are present at higher 344

concentrations in the upper soil horizons and whose concentrations decrease at greater depths 345

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(Sariah et al. 2005). The applied strain of the fungus was present in all soil samples; however, 346

lower CFU values were observed for samples taken from deeper soils. At depths of 0.3 and 347

0.4 m, the low density of conidia and their irregular distribution in the soil were reflected in 348

higher standard deviation values. In fact, the coefficients of variation associated with deeper 349

soil layers were greater than those associated with the upper layers. This observation may be 350

explained by the high spatial variability associated with undisturbed soil (Angle et al. 1995). 351

Mixing a large number of conidia into the surface soil layer allowed the uniform distribution 352

of those conidia, which greatly reduced the spatial variability effect in this soil layer. 353

Some isolates of Trichoderma spp. have the ability to colonize and grow in association with 354

plant roots, a property that was termed ‘rhizosphere competence’ (Ahmad and Baker 1988a). 355

This has also been demonstrated by T. atroviride SC1. The concentration (1 × 106 CFU g

-1 356

dry soil) in the grapevine rhizosphere 18 weeks after soil inoculation was even higher than 357

that reported by McLean et al. (2005) for T. atroviride C52 on onion roots (105 CFU g

-1 soil) 358

at 16 weeks after inoculation. This result is also encouraging as concerns the fungus’s use as a 359

biocontrol agent against A. mellea. In the vineyard, A. mellea is spread mainly by 360

rhizomorphs; when a rhizomorph reaches a new root, a new infection may occur. 361

The presence of T. atroviride SC1 in the rhizosphere could potentially provide protection 362

against the establishment of A. mellea on the roots. Root colonization by Trichoderma spp. 363

can also have significant effects on plant growth and productivity. Trichoderma species have 364

long been recognized for their ability to increase plant growth and development. Some studies 365

have shown that Trichoderma spp. can stimulate the growth of a number of vegetable and 366

bedding plant crops (Chang et al. 1986; Ahmad and Baker 1988b; Lynch et al. 1991). 367

However, under our experimental conditions, the strain did not improve plant growth. 368

T. atroviride SC1 was found on grapevine leaves, away from the treated soil. The migration 369

of the biocontrol agent to the leaves was probably passive. It occurred by the impaction of soil 370

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particles onto the leaf surface, gravity settling or sedimentation, or rain splash dispersal to the 371

leaf surface, which is strongly influenced by the physical environment, including the amount 372

and intensity of rainfall (Kinkel 1997). During the sampling process, the presence of soil 373

particles was observed on the abaxial surface of bottom leaves; this soil was probably 374

transported to that site along with rain splash. This is probably how the conidia from the soil 375

reached the aerial parts of the plants. The results also indicate that their proximity to the 376

inoculated soil gave the lower leaves greater exposure to the inoculum than the upper leaves. 377

As expected for a species belonging to the Trichoderma genus, T. atroviride SC1 is not 378

phytotoxic to or a pathogen of grapevine. Indeed, we did not notice any disease symptoms on 379

any grapevine after soil treatment, even if the fungus had been detected on its leaves. 380

T. atroviride SC1 can move beyond the treated soil area. It was found at the soil surface 2.0 381

and 4.0 m horizontally away from the inoculated site at 9 and 18 weeks after inoculation, 382

respectively. Our conclusion is that if T. atroviride SC1 is applied during the planting of a 383

new vineyard, the fungus may migrate from the treated row to an untreated row. 384

Concentrations of T. atroviride SC1 conidia decreased with increasing distance from the 385

inoculated plot. At a horizontal distance of 2.0 m from the treated plots, the concentration of 386

T. atroviride SC1 conidia decreased over time (between the ninth and18th

week after 387

inoculation). Based on the fact that low concentrations of this strain were recovered from the 388

soil surface at 18 weeks after inoculation and the fact that this concentration is similar to that 389

of indigenous Trichoderma spp. present in the area, it can be inferred that T. atroviride SC1 390

did disperse, but that this dispersion was limited and the fungus did not proliferate 391

significantly in the vineyard soil under the examined environmental conditions. Moreover, 392

one year after inoculation, T. atroviride SC1 was found in the inoculated area at the same 393

abundance level (concentration and frequency) as indigenous Trichoderma species. Based on 394

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these results, we can conclude that this biocontrol strain could become an integrant part of the 395

local microbial community following its release into the soil. 396

According to Brimner and Bolland (2003), the most important negative effect of BCAs 397

applied against fungal pathogens is a reduction in the biodiversity and/or abundance of non-398

target indigenous soil microorganisms. This could be an expected impact of a microorganism 399

that establishes itself in the soil and persists for long periods of time. For this reason, the 400

effect of applications of T. atroviride SC1on indigenous soil microbial communities should be 401

investigated in a future work. 402

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Acknowledgements 403

404

The authors thank Susanna Micheli for her technical assistance. This research was supported 405

by SafeCrop Centre, funded by Fondo per la Ricerca, Autonomous Province of Trento. 406

407

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Winding, A., Binnerup, S.J. and Pritchard, H. (2004) Non-target effects of bacterial biological 499

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Fig 1. Survival and vertical dispersion of Trichoderma atroviride SC1 in the vineyard soil at 501

different depths (surface , 0.1 m , 0.2 m , 0.3 m and 0.4 m soil depth) in two consecutive 502

years (white circles: 2006; black circles: 2007). Average soil temperature at a depth of 0.1 m 503

is indicated in the graph in the upper part of the figure (broken line: 2006; solid line: 2007). 504

Data points represent the averages of three replicates transformed by log (x + 1). Error bars 505

represent the standard deviations of the means. Different letters for time × depth points 506

indicate values that are significantly different (α = 0.05) according to Tukey’s test. 507

508

Fig 2. Correlation between Trichoderma atroviride SC1 population figures determined using 509

dilution plates and real-time PCR. The strain was introduced (108 CFU g

-1 dry soil) at the 510

surface of the soil of a vineyard in northern Italy in 2006. Soil samples were collected at 511

different times (at the time of inoculation and 1, 5, 9 and 18 weeks after inoculation) and at 512

different soil depths (surface, 0.1, 0.2, 0.3 and 0.4 m). Results are shown as log10 colony-513

forming units (CFU) and number of copies of haploid genomes (CN) per gram of dry soil. 514

Data are means of three replicates (CFU) and two replicates (CN) transformed by log (x+1). 515

516

Fig 3. Presence of Trichoderma atroviride SC1 (CFU) in the grapevine phylloplane. Data are 517

means of 30 replicates transformed by log (x+1). Values followed by the same letter are not 518

significantly different (α = 0.05) according to Tukey’s significant difference test. Error bars 519

indicate ± standard deviation of the means. The CFU values for the non-treated leaves (apical 520

and bottom) represent indigenous Trichoderma species. 521

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Table 1 Spatial changes in Trichoderma atroviride SC1 populations 9 weeks and 18 weeks 522

after soil inoculation at different horizontal distances and depths from the inoculation point in 523

2006. 524

525

Trichoderma atroviride SC1 CFU g-1

dry soil ± SD and frequency of detection

0 weeks 9 weeks 18 weeks

Soil depth (m) Horizontal

distance (m) 0 0 0.1 0.3 0

0 5.85 ± 0.29

100%

6.52 ± 0.36 a

100%

5.92 ± 0.27 a

100%

5.57 ± 0.60

a

100%

6.53 ± 0.58 a

100%

0.5 0 3.52 ± 0.96

b

100%

2.66 ± 1.26 b

90%

2.77 ± 0.34 b

100%

-

2.0 0 3.05

± 1.14

b

90%

1.69 ± 1.19 bc

70%

0.61 ± 0.99 c

30%

2.86 ± 1.53 b

80%

4.0 0

- - - 2.48 ± 1.76

b

70%

0 (Untreated) 0

0 0 0

0

0 (Untreated)* 1.38±1.15

60%

0.77 ± 0.99

c

40%

0.94 ± 1.25 c

40%

1.83 ± 0.99 d

40%

2.23 ± 1.56 b

70%

T. atroviride was mixed with the soil in the holes. 526

The untreated plot was prepared just like the treated plot, except for the fact that no fungus 527

was applied. 528

Data are means of 10 replicates (holes) transformed by log (x + 1) and represent 529

concentrations of CFU. 530

Frequency of detection is the percentage of samples that had at least one T. atroviride SC1 531

CFU and is described below each population level. 532

Values followed by the same letter in each column are not significantly different (α = 0.05) 533

according to Tukey’s test. 534

* Indigenous Trichoderma spp. population in untreated plots. 535

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