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: [email protected], 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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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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