Directory UMM :Data Elmu:jurnal:E:Environmental and Experimental Botany:Vol44.Issue3.Nov2000:
Crop Protection 18 (1999) 365}371
Reduced dosages of methyl bromide applied under gas-impermeable
plastic "lms for controlling soilborne pathogens of vegetable crops
Andrea Minuto, Giovanna Gilardi, M.L. Gullino*, Angelo Garibaldi
Dipartimento di Valorizzazione e Protezione delle Risorse agro-forestali, Patologia Vegetale, Via Leonardo Da Vinci, 44, 10095 Grugliasco TO, Italy
Received 30 October 1998; received in revised form 26 February 1999; accepted 26 March 1999
Abstract
Four experimental trials were carried out at Albenga (Northern Italy) to evaluate the e!ectiveness of reduced dosages of methyl
bromide (MB) applied under gas-impermeable "lms in controlling soilborne pathogens of vegetable crops. Forty and thirty g/m2 of
MB applied under di!erent types of gas-impermeable "lms controlled Rhizoctonia solani on bean, lettuce and basil, Fusarium
oxysporum f. sp. basilici on basil and Sclerotinia sclerotiorum on lettuce. Such treatments greatly decreased survival of arti"cially
introduced pathogen inoculum in the soil, with Fusarium oxysporum f. sp. basilici being the most di$cult pathogen to control. Gas
impermeable "lms permitted increased retention of MB, thus resulting in higher C]¹ (concentration ] time) product values and
reduced emissions into the atmosphere, as indirectly shown by measurements carried out by using a portable Fumiscope gas
detector. ( 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Soil fumigation with methyl bromide (MB), applied
at 60 g/m2, under low density polyethylene (LPDE)
plastic "lm, is a common practice for the control of a
wide spectrum of soilborne pathogens in many crops
(Garibaldi and Gullino, 1995) and, particularly, vegetable
crops (Katan, 1984). However, during recent years, concern regarding the potential of MB to interfere with the
ozone layer led to its inclusion on the list of ozonedepleting substances regulated by the Montreal Protocol
(Bell et al., 1996; Ristaino and Thomas, 1998). The strict
rules that limit its usage, prior to its phasing-out, stimulated the development of methods to reduce dosages and,
consequently, emissions of MB into the atmosphere
(Gamliel et al., 1997a).
The study in Northern Italy evaluates the possibility of
adopting gas-impermeable "lms with reduced dosages of
MB for the control of important pathogens of vegetable
crops.
2.1. Layout of trials
* Corresponding author. Tel.: 39-011-6708539; fax: 39-011-6708541.
E-mail address: [email protected] (M.L. Gullino)
Three of the trials at Albenga (Northern Italy) at
the Centro di Sperimentazione e Assistenza Agricola
(CeRSAA) of the Chamber of Commerce of Savona in
1996 (Table 1) assessed the e$cacy against soilborne
diseases as well as MB concentration under the tarp. In
the fourth trial only MB concentration under the plastic
"lm was measured. All trials were carried out in
a sand : silt : loam (75 : 20 : 5) soil, pH 8, 2.5% organic
material. Soil moisture ranged from 6 to 14% during the
di!erent stages of the trials. In trials 1 and 2, the e!ect of
the fumigation treatment on pathogen survival was also
evaluated. Climatic conditions during the fumigation and
the period immediately after fumigation were recorded.
2.2. Soil infestation with pathogens
To achieve a uniform soil infestation, and higher disease incidence, the inoculum of the following pathogens
was incorporated into the soil by rototilling at a depth of
5}10 cm prior to fumigation at the dosages reported in
Table 1: Rhizoctonia solani, as infected wheat kernels in
trial 1; R. solani, as infected kernels, Fusarium oxysporum
0261-2194/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 1 - 2 1 9 4 ( 9 9 ) 0 0 0 3 6 - 8
366
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Table 1
Outline of the four experimental trials
Trial number and code
Location, plots surface and
number of replicates
Arti"cial infestation on
Soil infestation with
Crop grown
1. MU 1
Ce.R.S.A.A.,
Albenga,
plastic tunnel,
60 m2,
3 replicates
15/03/96
Basil (cv Genovese
gigante)
2. SC 1
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2,
3 replicates
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2
3 replicates
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2
3 replicates
05/04/96
Rhizoctonia solani, 30 g/m2
of infected kernels Fusarium
oxysporum f. sp. basilici
1]105 CFU/ml of soil of
chlamydospores on talc
Basil plants infected with
Colletotrichum gloeosporioides
R. solani, 30 g/m2 of infected
kernels
30/07/96
R. solani, 30 g/m2 infected
kernels Sclerotinia sclerotiorum, 30 g/m2 infected
kernels
Bean (cv Bobis,
Canellino) Lettuce
(Gheisa, Foglia di
quercia)
*!
*
*
3. SC 3
4. CO 3
Bean (cv Bobis,
Canellino, Anellino)
Lettuce (cv Lido)
!No arti"cial infestation.
f. sp. basilici (FOB) as chlamydospores suspended in talc
(Locke and Coulhoun, 1974) and Colletotrichum gloeosporioides as heavily infected basil plant pieces in trial 2;
R. solani and Sclerotinia sclerotiorum as infected kernels
in trial 3. In addition, in order to evaluate the direct e!ect
of fumigation on the survival of the pathogens in trials
1 and 2, the inoculum of the pathogens was buried in
the soil at 10 and 20 cm depths, in the central part of the
plots. In trial 1 R. solani and F. o. basilici were used, while
R. solani, F. o. basilici and Pythium ultimum were used
in trial 2. R. solani and P. ultimum were introduced as
infected kernels; F. basilici as talc infested with
chlamydospores. In the case of F. o. basilici infested talc,
a mixture (1 : 1.5 talc : perlite v/v) was used; perlite was
mixed to talc in order to improve gas permeability of the
last material. Seven grams inoculum of each pathogen
was put separately into small gas permeable tissue bags
(5 cm diam) and buried into the soil 12}18 h before the
fumigation treatment. Two bags per pathogen were introduced into each plot, one at 10 and one at 20 cm
depth. The bags were taken out 7 days after fumigation
when the soil was uncovered.
(VIFs): Bromotech (LMG Smith Brothers. Bristol UK,
30 lm) coded as LMG, Barromid (Plastopil, Kibbutz
Hazorea, Israel, 30 lm) coded as PP. Moreover, two
coloured impermeable "lms, provided by Societa'
Italiana Sterilizzazioni (Vittoria, Italy), and coded as SIS
white and SIS grey were tested in trial 4.
Tarps were manually removed from soil seven days
after the fumigation treatment, in accordance with Italian
law.
2.3. Plastic mulch
Seven days after fumigation, when the soil was unmulched, the bags containing the inocula were taken out of
the soil and immediately trransferred to the laboratory
in a portable refrigerator (8}103C), and processed. In
the case of F. o. basilici, talc was suspended at di!erent
Soil mulching was carried out by using standard,
transparent, LPDE (Ei!el, Fontanellato, Italy, 40 lm
thick) and two virtually impermeable, transparent "lms
2.4. Fumigation treatments
Methyl bromide (Metabrom, 98% a.i., Bromine Compounds, Israel) was applied by licensed fumigators
through drip lines placed under the "lm, using the hot
gas method, at the dosages reported in Tables 2}9. In
trial 1, one treatment consisted in the addition of 5 T/ha of
cow manure to the soil before fumigation (on March 11,
1996) in order to evaluate a possible negative interaction
between fertilization with manure and MB fumigation.
2.5. Pathogen inoculum survival
A. Minuto et al. / Crop Protection 18 (1999) 365}371
dilutions in sterile water and plated on Komada medium
(Komada, 1975). After 7 days of incubation at 243C, the
number of colony forming units (CFU) was counted. In
the case of R. solani and P. ultimum, 100 infected kernels/
bag were directly plated on Petri plates (10 kernels/plate)
on a Rhizoctonia semi-selective (Migheli et al., 1990) and
on a oomycete selective medium (Masago et al., 1977),
respectively. Inoculated plates were incubated for 5}7
days at 243C. The number of kernels from which mycelium grew was counted, and the data expressed as
percent of plated kernels.
soil was unmulched on March 27, 1996, then rototilled
and irrigated on April 2, 1996. Two subsequent crops
of basil (cv Genovese gigante) were grown. The "rst
sowing took place on April 4, 1996; the second one
on May 20, 1996. In both the cases, 3 g of seed/m2 were
used.
In experiment 2, carried out in the open "eld, fumigation was done on April 17, 1996, soil was unmulched on
April 26, 1996, then rototilled and irrigated on May 15,
1996. Bean (cv Bobis, Canellino and Anellino) and lettuce
(cv Lido) were grown. Bean was seeded at 200 seeds/plot
on May 24, 1996; lettuce was transplanted (160 plants/
plot) on May 28, 1996.
In experiment 3, carried out in the open "eld, soil
fumigation was carried out on August 6, 1996, soil was
unmulched on August 13, 1996, then rototilled and irrigated on August 20, 1996. One crop of bean (cv Bobis
and Canellino) and two crops of di!erent cultivars of
lettuce (cv Gheisa and Foglia di quercia) were grown.
Bean was seeded at 200 seeds/plot on September 2, 1996;
lettuce cv Gheisa was transplanted (100 plants/plot),
while cv Foglia di quercia was sown at 300 seeds/m2 on
September 2, 1996.
In trial 4 no crop was grown.
All crops were sprinkle irrigated and grown according
to the cultural practices adopted by commercial growers
in the region.
2.6. Crops grown
In experiment 1, carried out under plastic tunnel,
soil fumigation was carried out on March 20, 1996,
Table 2
E!ect of fumigation with methyl bromide under di!erent plastic mulch
on the survival of soil buried pathogens in trial 1
Treatment, CFU/g of F. basilici at
a depth of
with MB,
g/m2 and
plastic cover!
10 cm
*
60/LPDE
30/LPDE
30/LMG
30/PP
60/LPDE
Man#
3.1]107
2.1]101
1.7]102
8.2]101
1.7]102
4.6]101
Percentage of kernels
infected with R. solani at
a depth of
20 cm
b"
a
a
a
a
a
8.7]107
1.5]102
7.2]101
5.2]101
6.7]101
1.4]102
b
a
a
a
a
a
10 cm
20 cm
98.0
2.0
1.0
1.0
0.3
0.0
100.0
1.0
0.0
0.0
0.3
0.0
b
a
a
a
a
a
367
b
a
a
a
a
a
2.7. Disease evaluation
Disease development was evaluated at regular intervals by counting the number of plants with symptoms
(incidence) or by using a subjective disease index scale for
the di!erent pathogens ranging from 0 (healthy plant) to
5 (dead plant) (disease severity).
All data collected were statistically analyzed, according to Duncans's Multiple Range Test.
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
#Plots were treated with manure (5 T/ha).
Table 3
E!ect of fumigation with methyl bromide under di!erent plastic mulch on the survival of soil buried pathogens in trial 2
Treatment, with
MB, g/m2 and
plastic cover!
*
60/LPDE
40/LPDE
20/LPDE
40/LMG
20/LMG
40/PP
20/PP
CFU/g of F. basilici at a depth of
Percentage of kernels infected with
R. solani at a depth of
Percentage of kernels infected
with R. solani at a depth of
10 cm
10 cm
20 cm
10 cm
20 cm
95.0
0.0
0.0
0.3
0.0
0.5
0.0
0.0
94.8
0.0
0.0
0.0
0.0
0.0
0.0
0.3
97.0
0.5
0.0
1.8
0.0
1.0
0.0
0.3
94.3
0.0
0.0
0.5
0.0
0.5
0.0
1.0
9.0]107
3.6]102
2.4]103
5.1]104
7.9]102
1.0]104
4.3]102
1.8]103
20 cm
b"
a
a
a
a
a
a
a
6.0]107
6.5]102
1.7]103
1.7]104
4.3]102
7.2]103
3.3]102
2.4]102
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gas-impermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
b
a
a
a
a
a
a
a
368
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Table 4
E!ect of fumigation with methyl bromide (MB) under di!erent mulch "lms on the incidence of Rhizoctonia solani, Fusarium oxysporum f. sp. basilici and
Colletotrichum gloeosporioides infections and on yield of basil (cv Genovese gigante) (trial 1, "rst crop)
Treatment with MB,
g/m2 and plastic cover!
*
60/LPDE
30/LPDE
30/LMG
30/PP
60/LPDE Man#
Percentage of infected plants 34 days after sowing
Yield (g/m2)
R. solani
C. gloeosporioides
F. basilici
Total
28.9
4.3
1.9
3.2
2.1
0.3
5.8
0.1
1.6
0.2
0.2
0.4
16.0
0.3
1.1
0.5
1.9
0.6
50.7
4.7
4.6
4.0
4.3
1.4
b"
a
a
a
a
a
b
a
a
a
a
a
b
a
a
a
a
a
b
a
a
a
a
a
2172
3404
3030
3193
2785
2906
b
a
a
a
ab
ab
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gas-impermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
#Plots were treated with manure (5 T/ha).
Table 5
E!ect of fumigation with methyl bromide (MB) under di!erent mulch
"lms against Rhizoctonia solani infections on three cultivars of bean.
Data expressed as percent of infected plants (IP) and disease index (DI,
0}5) (trial 2, "rst crop)
Percentage of infected plants (IP) and disease index (0}5)
Treatment
58 days after sowing
with MB,
g/m2 and
plastic cover!
cv Bobis
*
60/LPDE
40/LPDE
20/LPDE
40/LMG
20/LMG
40/PP
20/PP
%
80
12
54
38
8
36
9
34
IP
d"
a
c
b
a
b
a
b
DI
2.0
0.1
0.9
0.6
0.1
0.6
0.1
0.5
cv Anellino
d
a
c
bc
a
bc
a
ab
%
78
9
43
45
8
30
11
28
IP
d
a
c
c
a
c
ab
bc
DI
1.9
0.1
0.8
0.9
0.1
0.5
0.1
0.3
cv Canellino
c
a
b
b
a
ab
a
a
%
86
11
41
46
16
23
14
36
IP
d
a
a
c
ab
abc
ab
abc
DI
2.2
0.1
0.7
0.9
0.2
0.3
0.4
0.6
b
a
a
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
2.8. MB measurements
MB concentrations in the air trapped under the tarp
were measured at constant intervals after fumigation
using a FUMISCOPE D (Key Chemical & Equipment
Co., Clearwater, FL, USA), a portable instrument commonly used for measuring the concentration of MB in
fumigation chambers. The instrument is based on the
principle of thermal conductivity (Chakrabarti, 1996)
and can detect concentration of the gas in the atmosphere up to 1999 g/m3.
Measurements were taken according to a technique developed by Laboratoire National d'Etude des
Table 6
E!ect of fumigation with methyl bromide (MB) under di!erent plastic
mulch "lms on the incidence of Sclerotinia sclerotiorum infections on
lettuce (cv Gheisa) (trial 3, "rst crop)
Treatment with MB, g/m2 and
plastic cover!
Percentage of infected plants 78
days after transplanting
*
60/LPDE
40/LPDE
30/LPDE
40/LMG
30/LMG
14.1
3.9
1.6
2.3
3.9
3.1
b"
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
Table 7
E!ect of fumigation with methyl bromide (MB) under di!erent plastic
mulch "lms against Rhizoctonia solani on bean (cv Bobis and Canellino), expressed as percent of emerged plants (% E) and Disease Index
(DI, 0}5), 53 days after sowing (trial 3, "rst crop)
Treatment
with MB,
g/m2 and
plastic cover!
cv Bobis
%E
*
60/LPDE
40/LPDE
30/LPDE
40/LMG
30/LMG
19.8
48.0
39.4
45.5
50.0
41.9
cv Canellino
DI
b"
ab
ab
ab
a
ab
1.3
0.8
0.7
0.6
0.7
0.8
%E
b
a
a
a
a
a
50.0
71.3
58.0
60.0
67.8
60.3
DI
a
a
a
a
a
a
1.6
0.9
0.6
0.8
0.6
1.0
b
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
A. Minuto et al. / Crop Protection 18 (1999) 365}371
369
Table 8
Methyl bromide (MB) measured under the tarp at di!erent intervals
after fumigation (trial 3)
Table 9
Methyl bromide (MB) measured under the tarp at di!erent intervals
after fumigation (trial 3)
Treatment (g/m3) of MB at minutes from the start of fumigation
with MB,
g/m2 and
plastic
cover!
Treatment (g/h/m3) at minutes from the start of fumigation
with MB,
g/m2 and
plastic
cover!
6
60/LPDE
40/LPDE
40/LMG
30/LMG
919
1035
1568
720
60
bc"
b
a
c
363
395
645
303
120
bc
b
a
c
332
266
348
284
180
a
b
a
b
259
252
251
279
600
a
a
a
a
185
197
181
214
1200
ab
ab
b
a
156
174
157
171
a
a
a
a
6
60/LPDE
40/LPDE
40/LMG
30/LMG
122
115
151
73
b"
b
a
c
60
120
654 b
608 b
1004 a
474 b
997
926
1480
763
180
b
b
a
b
1288
1180
1781
1035
600
b
b
a
b
2706
2707
3322
2658
1200
b
b
a
b
4381
4540
4987
4537
b
ab
a
ab
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
Techniques de Fumigation et de Protection des DeH nreH es
StockeH es (L.N.D.S) du Ministère de l'Agriculture at
Bordeaux (France) (P. Ducom and M. Guinet. pers.
comm.). The concentration of MB present in the air
trapped under the plastic "lm was measured at a distance
of at least 1.5 m inward from the plot border, and of
4}5 m from the fumigant injection points. Measures
started 6 min after beginning of fumigation. Data are
expressed as g/m3 and as C]¹ (concentration]time)
products. This last value is the product of multiplying
MB concentration (C) by the duration of the exposure
time (¹).
3.2. Ewect of xlm type and MB dosages on disease control
3. Results
3.1. Ewect of xlm type and MB dosages on pathogen
survival
All treatments signi"cantly reduced (98}100% reduction) survival of the pathogens arti"cially introduced
into the soil in both trials 1 and 2 (Tables 2 and 3).
Also, the lowest tested dosage of MB (20 g/m2) under
gas-impermeable plastic "lm strongly reduced the survival of the pathogens, both at 10 and 20 cm depths.
However, only R. solani and P. ultimum were completely
inactivated by some treatments. In the case of FOB,
the tested treatments strongly reduced but did not
completely kill the inoculum (Tables 2 and 3). Considering the e!ect on FOB, the e$cacy of the treatments
was higher in trial 1 than in trial 2. No signi"cant di!erences on the e!ect on the pathogen's inocula were observed among treatments, indicating that all tested
dosages of MB, applied under di!erent "lms, gave similar
results.
In the trial carried out against R. solani, FOB and
C. gloeosporioides, all the tested treatments signi"cantly
reduced the incidence of all three pathogens (1}28% reduction) on the "rst crop of basil (Table 4). No signi"cant
di!erences among treatments were observed concerning
the level of disease control and basil yield (Table 4). The
addition of manure before treatment with MB, applied at
60 g/m2 under standard LPDE "lm, did not a!ect the
e$cacy of the treatments (Table 4).
In a second trial, carried out against R. solani on bean,
60 g/m2 of MB applied under standard LPDE or 40 g/m2
under LMG "lm provided the best disease control on the
three tested cultivars of bean (Table 5). The results obtained by applying 40 g/m2 under PP were only slightly
inferior. Forty grams of MB under standard LPDE or
20 g/m2 under gas impermeable "lm did not provide
a satisfactory disease control (Table 5). As in trial 2, R.
solani symptom incidence was signi"cantly reduced by all
treatments in the case of lettuce (cv Lido), in comparison
with the control, but yield was not a!ected by any of the
treatments (data not shown).
In trial 3, S. sclerotiorum symptom incidence was signi"cantly reduced by all tested treatments on lettuce (cv
Gheisa) (Table 6) as well as on the second crop (cv Foglie
di quercia) (data not shown). On cv Bobis, only 40 g/m2
of MB, applied under LMG, signi"cantly reduced the
incidence of R. solani, while on cv Canellino, no signi"cative di!erences were observed among treatments (Table 7).
3.3. Permeability of xlms
In trial 3, the plots treated with 40 g/m2 of MB and
covered with LMG "lm retained the highest concentrations of MB in the atmosphere trapped under the "lm, as
370
A. Minuto et al. / Crop Protection 18 (1999) 365}371
compared with those covered by LMG and treated with
30 g/m2 of MB and those covered with standard LPDE,
fumigated with 60 and 40 g/m2 (Table 8). The same trend
is shown when C]¹ products are considered: the highest concentrations of MB were observed when the
fumigant was applied at 40 g/m2, under gas-impermeable
"lm, in comparison with those achieved under LPDE
with an initial dosage of 60 g/m2 of MB (Table 9). This
indirectly shows that emissions of MB into the atmosphere are signi"cantly reduced by the use of gas-impermeable "lms, in comparison with the standard dosage of
MB under LPDE.
In trial 4, LMG "lm applied on plots fumigated with
40 g/m2 of MB con"rmed its capability of retaining MB
under the tarp (data not shown).
In both trials the di!erences of the MB concentrations
measured under the di!erent plastic "lms signi"cantly
decreased over time. The di!erences between some C]¹
products remained statistically signi"cant. Particularly,
such value remained signi"cantly higher in the case of
the treatments carried out under LMG at 40 g/m2. Less
promising results were obtained with the two experimental "lms coded as SIS (data not shown).
4. Discussion
The results presented, obtained in a variety of experiments, with di!erent crops and pathogens, show the
possibility of reducing the dosages of MB for soil disinfestation from 60 g/m2 to 40 and 30 g/m2, with still very
e!ective control of important soilborne pathogens, when
gas-impermeable "lms are used for mulching. The results
obtained in terms of disease control were generally similar to those achieved with 60 g/m2 of MB under LPDE.
The dosage of 20 g/m2, although still e!ective in some
cases, did not always ensure satisfactory disease control.
For this reason, this dosage cannot be advised under
practical conditions, at least under those circumstances
where fungal pathogens are the major problem.
The results obtained by evaluating the direct e!ect of
MB applied at di!erent dosages under di!erent "lms,
con"rm the relatively high sensitivity of P. ultimum and
R. solani to the fumigant in comparison to F. oxysporum,
as already shown (Munnecke et al., 1978; Ebben et al.,
1983). The Fusarium population was, however, strongly
reduced. It is possible that the level of inoculum of F.
basilici surviving the treatment is not su$cient to cause
disease: actually, it has been shown that 3]102 CFU/ml
of soil are necessary in order to cause the appearance
of symptoms on the cv `Genovese gigantea of basil
(A. Garibaldi and G. Minuto, unpublished). In similar
experiments carried out in Israel by infesting soil with
basil stems infected with F. basilici, the pathogen was
completely killed by MB treatment at full dosage under
LPDE or at reduced dosage under gas-impermeable "lm
(Gamliel et al., 1998b). The lower mortality observed in
our experiments can be explained by the higher resistance to the fumigation treatment of chlamydospores of
the pathogen in comparison to spores and mycelium
present on infected stems.
The results obtained in the tests on pathogen survival
con"rm the results obtained in terms of disease control:
20 g/m2 of the fumigant, applied under any of the tested
gas-impermeable "lms did not give complete control of
R. solani and P. ultimum.
The di!erent levels of control of R. solani achieved with
reduced dosages of MB under gas-impermeable "lm on
bean, lettuce and basil could be, at least partially, explained with a di!erent virulence of the pathogen on
the tested crops, bean being a very susceptible crop to
R. solani infections.
A long-term e!ect of MB at a reduced dosage under
gas-impermeable "lm was observed in trial 3 on lettuce.
This long-term management of soilborne pathogens on
more than one crop allows a greater interval between
fumigation treatments, with positive implications from
an economic point of view. Moreover, a long-term e!ect,
coupled with the reduction in dosage by using gas-impermeable "lm should reduce the emissions of MB into the
atmosphere, thus further adding to the environmental
and economic bene"ts.
Among the tested gas-impermeable "lms, both LMG
and PP provided consistent results both in terms of
disease control and retention of the fumigant under the
tarp, as indirectly measured with our system. In other
studies, both "lms were very e!ective in retaining higher
concentration of MB in the soil and reducing MB emissions into the atmosphere under laboratory and "eld
conditions (Gamliel et al., 1998a,b).
In order to implement the use of gas-impermeable
"lms on a large scale, particularly in the open "eld, these
"lms should have su$cient mechanical properties, so as
to maintain their impermeability in the "eld (Gamliel
et al., 1997a). Resistance to mechanical stretching, temperature #uctuation, tearing and holes are among the
essential requirements for commercial application. From
a practical point of view, currently available "lms still
present some technical problems because of their tendency to tear. Due to their high rigidity, small lides can
become long cuts, which can nullify the positive e!ect of
their impermeability to the fumigant. Moreover, glueing
VIFs is more complicated than glueing standard LPDE.
The measurements of MB concentration under the
plastic "lms provided useful information concerning the
behaviour of the gas. Although the method used in this
work gave an indirect evaluation of the concentration of
the fumigant in the soil, it clearly indicated that a higher
concentration of the fumigant is trapped under gas-impermeable "lms giving higher C]¹ values and providing good disease control while reducing the emissions
of MB into the atmosphere (Gamliel et al., 1997a).
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Reduced dosages of MB applied under gas-impermeable "lm also were demonstrated in other pathogen-crop
systems (Cebolla et al., 1996; Gamliel et al., 1997b; Gullino et al., 1996; Minuto et al., 1998a,b). The availability
of better quality gas-impermeable "lms will allow expansion of the application technique in an increasing number
of situations. At this stage, VIFs are more expensive than
LPDE, but allowing MB dosage reduction may compensate for the additional cost. Hopefully, expanding their
usage will help reducing their production costs.
The usage of VIFs with reduced dosages of MB will
help agriculture, in the short term, to conform to Montreal Protocol requirements, while reliable, long term
solutions are developed.The application of reduced dosages of MB under gas-impermeable plastics, if combined
with other methods of soilborne disease management,
may enable even further reductions of MB emissions into
the atmosphere.
Acknowledgements
Work carried out with grants from Ministry of Environment } S.I.A.R., Italy and Regione Liguria. The
authors thank Prof. Jaacov Katan and Prof. Alberto
Matta for critically reviewing the manuscript and the
anonymous reviewers for their valuable comments.
The "rst author carried the experimental "eld work,
collected and elaborated the data and participated into
the planning of the experiments during the preparation of
his Ph.D thesis, the second author cooperated into part
of the "eld work and carried the laboratory experiments
during the preparation of her Master thesis, the third
author planned the work and wrote the manuscript. The
fourth author participated into the planning of the experiments and reviewed the manuscript.
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Locke, T., Coulhoun, J., 1974. Contribution to a method of testing oil
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Minuto, A., Gullino, M.L., Baruzzi, G., Faedi, W., Lucchi, P., 1998a.
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Reduced dosages of methyl bromide applied under gas-impermeable
plastic "lms for controlling soilborne pathogens of vegetable crops
Andrea Minuto, Giovanna Gilardi, M.L. Gullino*, Angelo Garibaldi
Dipartimento di Valorizzazione e Protezione delle Risorse agro-forestali, Patologia Vegetale, Via Leonardo Da Vinci, 44, 10095 Grugliasco TO, Italy
Received 30 October 1998; received in revised form 26 February 1999; accepted 26 March 1999
Abstract
Four experimental trials were carried out at Albenga (Northern Italy) to evaluate the e!ectiveness of reduced dosages of methyl
bromide (MB) applied under gas-impermeable "lms in controlling soilborne pathogens of vegetable crops. Forty and thirty g/m2 of
MB applied under di!erent types of gas-impermeable "lms controlled Rhizoctonia solani on bean, lettuce and basil, Fusarium
oxysporum f. sp. basilici on basil and Sclerotinia sclerotiorum on lettuce. Such treatments greatly decreased survival of arti"cially
introduced pathogen inoculum in the soil, with Fusarium oxysporum f. sp. basilici being the most di$cult pathogen to control. Gas
impermeable "lms permitted increased retention of MB, thus resulting in higher C]¹ (concentration ] time) product values and
reduced emissions into the atmosphere, as indirectly shown by measurements carried out by using a portable Fumiscope gas
detector. ( 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Soil fumigation with methyl bromide (MB), applied
at 60 g/m2, under low density polyethylene (LPDE)
plastic "lm, is a common practice for the control of a
wide spectrum of soilborne pathogens in many crops
(Garibaldi and Gullino, 1995) and, particularly, vegetable
crops (Katan, 1984). However, during recent years, concern regarding the potential of MB to interfere with the
ozone layer led to its inclusion on the list of ozonedepleting substances regulated by the Montreal Protocol
(Bell et al., 1996; Ristaino and Thomas, 1998). The strict
rules that limit its usage, prior to its phasing-out, stimulated the development of methods to reduce dosages and,
consequently, emissions of MB into the atmosphere
(Gamliel et al., 1997a).
The study in Northern Italy evaluates the possibility of
adopting gas-impermeable "lms with reduced dosages of
MB for the control of important pathogens of vegetable
crops.
2.1. Layout of trials
* Corresponding author. Tel.: 39-011-6708539; fax: 39-011-6708541.
E-mail address: [email protected] (M.L. Gullino)
Three of the trials at Albenga (Northern Italy) at
the Centro di Sperimentazione e Assistenza Agricola
(CeRSAA) of the Chamber of Commerce of Savona in
1996 (Table 1) assessed the e$cacy against soilborne
diseases as well as MB concentration under the tarp. In
the fourth trial only MB concentration under the plastic
"lm was measured. All trials were carried out in
a sand : silt : loam (75 : 20 : 5) soil, pH 8, 2.5% organic
material. Soil moisture ranged from 6 to 14% during the
di!erent stages of the trials. In trials 1 and 2, the e!ect of
the fumigation treatment on pathogen survival was also
evaluated. Climatic conditions during the fumigation and
the period immediately after fumigation were recorded.
2.2. Soil infestation with pathogens
To achieve a uniform soil infestation, and higher disease incidence, the inoculum of the following pathogens
was incorporated into the soil by rototilling at a depth of
5}10 cm prior to fumigation at the dosages reported in
Table 1: Rhizoctonia solani, as infected wheat kernels in
trial 1; R. solani, as infected kernels, Fusarium oxysporum
0261-2194/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 1 - 2 1 9 4 ( 9 9 ) 0 0 0 3 6 - 8
366
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Table 1
Outline of the four experimental trials
Trial number and code
Location, plots surface and
number of replicates
Arti"cial infestation on
Soil infestation with
Crop grown
1. MU 1
Ce.R.S.A.A.,
Albenga,
plastic tunnel,
60 m2,
3 replicates
15/03/96
Basil (cv Genovese
gigante)
2. SC 1
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2,
3 replicates
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2
3 replicates
Ce.R.S.A.A.,
Albenga,
open "eld,
50 m2
3 replicates
05/04/96
Rhizoctonia solani, 30 g/m2
of infected kernels Fusarium
oxysporum f. sp. basilici
1]105 CFU/ml of soil of
chlamydospores on talc
Basil plants infected with
Colletotrichum gloeosporioides
R. solani, 30 g/m2 of infected
kernels
30/07/96
R. solani, 30 g/m2 infected
kernels Sclerotinia sclerotiorum, 30 g/m2 infected
kernels
Bean (cv Bobis,
Canellino) Lettuce
(Gheisa, Foglia di
quercia)
*!
*
*
3. SC 3
4. CO 3
Bean (cv Bobis,
Canellino, Anellino)
Lettuce (cv Lido)
!No arti"cial infestation.
f. sp. basilici (FOB) as chlamydospores suspended in talc
(Locke and Coulhoun, 1974) and Colletotrichum gloeosporioides as heavily infected basil plant pieces in trial 2;
R. solani and Sclerotinia sclerotiorum as infected kernels
in trial 3. In addition, in order to evaluate the direct e!ect
of fumigation on the survival of the pathogens in trials
1 and 2, the inoculum of the pathogens was buried in
the soil at 10 and 20 cm depths, in the central part of the
plots. In trial 1 R. solani and F. o. basilici were used, while
R. solani, F. o. basilici and Pythium ultimum were used
in trial 2. R. solani and P. ultimum were introduced as
infected kernels; F. basilici as talc infested with
chlamydospores. In the case of F. o. basilici infested talc,
a mixture (1 : 1.5 talc : perlite v/v) was used; perlite was
mixed to talc in order to improve gas permeability of the
last material. Seven grams inoculum of each pathogen
was put separately into small gas permeable tissue bags
(5 cm diam) and buried into the soil 12}18 h before the
fumigation treatment. Two bags per pathogen were introduced into each plot, one at 10 and one at 20 cm
depth. The bags were taken out 7 days after fumigation
when the soil was uncovered.
(VIFs): Bromotech (LMG Smith Brothers. Bristol UK,
30 lm) coded as LMG, Barromid (Plastopil, Kibbutz
Hazorea, Israel, 30 lm) coded as PP. Moreover, two
coloured impermeable "lms, provided by Societa'
Italiana Sterilizzazioni (Vittoria, Italy), and coded as SIS
white and SIS grey were tested in trial 4.
Tarps were manually removed from soil seven days
after the fumigation treatment, in accordance with Italian
law.
2.3. Plastic mulch
Seven days after fumigation, when the soil was unmulched, the bags containing the inocula were taken out of
the soil and immediately trransferred to the laboratory
in a portable refrigerator (8}103C), and processed. In
the case of F. o. basilici, talc was suspended at di!erent
Soil mulching was carried out by using standard,
transparent, LPDE (Ei!el, Fontanellato, Italy, 40 lm
thick) and two virtually impermeable, transparent "lms
2.4. Fumigation treatments
Methyl bromide (Metabrom, 98% a.i., Bromine Compounds, Israel) was applied by licensed fumigators
through drip lines placed under the "lm, using the hot
gas method, at the dosages reported in Tables 2}9. In
trial 1, one treatment consisted in the addition of 5 T/ha of
cow manure to the soil before fumigation (on March 11,
1996) in order to evaluate a possible negative interaction
between fertilization with manure and MB fumigation.
2.5. Pathogen inoculum survival
A. Minuto et al. / Crop Protection 18 (1999) 365}371
dilutions in sterile water and plated on Komada medium
(Komada, 1975). After 7 days of incubation at 243C, the
number of colony forming units (CFU) was counted. In
the case of R. solani and P. ultimum, 100 infected kernels/
bag were directly plated on Petri plates (10 kernels/plate)
on a Rhizoctonia semi-selective (Migheli et al., 1990) and
on a oomycete selective medium (Masago et al., 1977),
respectively. Inoculated plates were incubated for 5}7
days at 243C. The number of kernels from which mycelium grew was counted, and the data expressed as
percent of plated kernels.
soil was unmulched on March 27, 1996, then rototilled
and irrigated on April 2, 1996. Two subsequent crops
of basil (cv Genovese gigante) were grown. The "rst
sowing took place on April 4, 1996; the second one
on May 20, 1996. In both the cases, 3 g of seed/m2 were
used.
In experiment 2, carried out in the open "eld, fumigation was done on April 17, 1996, soil was unmulched on
April 26, 1996, then rototilled and irrigated on May 15,
1996. Bean (cv Bobis, Canellino and Anellino) and lettuce
(cv Lido) were grown. Bean was seeded at 200 seeds/plot
on May 24, 1996; lettuce was transplanted (160 plants/
plot) on May 28, 1996.
In experiment 3, carried out in the open "eld, soil
fumigation was carried out on August 6, 1996, soil was
unmulched on August 13, 1996, then rototilled and irrigated on August 20, 1996. One crop of bean (cv Bobis
and Canellino) and two crops of di!erent cultivars of
lettuce (cv Gheisa and Foglia di quercia) were grown.
Bean was seeded at 200 seeds/plot on September 2, 1996;
lettuce cv Gheisa was transplanted (100 plants/plot),
while cv Foglia di quercia was sown at 300 seeds/m2 on
September 2, 1996.
In trial 4 no crop was grown.
All crops were sprinkle irrigated and grown according
to the cultural practices adopted by commercial growers
in the region.
2.6. Crops grown
In experiment 1, carried out under plastic tunnel,
soil fumigation was carried out on March 20, 1996,
Table 2
E!ect of fumigation with methyl bromide under di!erent plastic mulch
on the survival of soil buried pathogens in trial 1
Treatment, CFU/g of F. basilici at
a depth of
with MB,
g/m2 and
plastic cover!
10 cm
*
60/LPDE
30/LPDE
30/LMG
30/PP
60/LPDE
Man#
3.1]107
2.1]101
1.7]102
8.2]101
1.7]102
4.6]101
Percentage of kernels
infected with R. solani at
a depth of
20 cm
b"
a
a
a
a
a
8.7]107
1.5]102
7.2]101
5.2]101
6.7]101
1.4]102
b
a
a
a
a
a
10 cm
20 cm
98.0
2.0
1.0
1.0
0.3
0.0
100.0
1.0
0.0
0.0
0.3
0.0
b
a
a
a
a
a
367
b
a
a
a
a
a
2.7. Disease evaluation
Disease development was evaluated at regular intervals by counting the number of plants with symptoms
(incidence) or by using a subjective disease index scale for
the di!erent pathogens ranging from 0 (healthy plant) to
5 (dead plant) (disease severity).
All data collected were statistically analyzed, according to Duncans's Multiple Range Test.
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
#Plots were treated with manure (5 T/ha).
Table 3
E!ect of fumigation with methyl bromide under di!erent plastic mulch on the survival of soil buried pathogens in trial 2
Treatment, with
MB, g/m2 and
plastic cover!
*
60/LPDE
40/LPDE
20/LPDE
40/LMG
20/LMG
40/PP
20/PP
CFU/g of F. basilici at a depth of
Percentage of kernels infected with
R. solani at a depth of
Percentage of kernels infected
with R. solani at a depth of
10 cm
10 cm
20 cm
10 cm
20 cm
95.0
0.0
0.0
0.3
0.0
0.5
0.0
0.0
94.8
0.0
0.0
0.0
0.0
0.0
0.0
0.3
97.0
0.5
0.0
1.8
0.0
1.0
0.0
0.3
94.3
0.0
0.0
0.5
0.0
0.5
0.0
1.0
9.0]107
3.6]102
2.4]103
5.1]104
7.9]102
1.0]104
4.3]102
1.8]103
20 cm
b"
a
a
a
a
a
a
a
6.0]107
6.5]102
1.7]103
1.7]104
4.3]102
7.2]103
3.3]102
2.4]102
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gas-impermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
b
a
a
a
a
a
a
a
368
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Table 4
E!ect of fumigation with methyl bromide (MB) under di!erent mulch "lms on the incidence of Rhizoctonia solani, Fusarium oxysporum f. sp. basilici and
Colletotrichum gloeosporioides infections and on yield of basil (cv Genovese gigante) (trial 1, "rst crop)
Treatment with MB,
g/m2 and plastic cover!
*
60/LPDE
30/LPDE
30/LMG
30/PP
60/LPDE Man#
Percentage of infected plants 34 days after sowing
Yield (g/m2)
R. solani
C. gloeosporioides
F. basilici
Total
28.9
4.3
1.9
3.2
2.1
0.3
5.8
0.1
1.6
0.2
0.2
0.4
16.0
0.3
1.1
0.5
1.9
0.6
50.7
4.7
4.6
4.0
4.3
1.4
b"
a
a
a
a
a
b
a
a
a
a
a
b
a
a
a
a
a
b
a
a
a
a
a
2172
3404
3030
3193
2785
2906
b
a
a
a
ab
ab
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gas-impermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
#Plots were treated with manure (5 T/ha).
Table 5
E!ect of fumigation with methyl bromide (MB) under di!erent mulch
"lms against Rhizoctonia solani infections on three cultivars of bean.
Data expressed as percent of infected plants (IP) and disease index (DI,
0}5) (trial 2, "rst crop)
Percentage of infected plants (IP) and disease index (0}5)
Treatment
58 days after sowing
with MB,
g/m2 and
plastic cover!
cv Bobis
*
60/LPDE
40/LPDE
20/LPDE
40/LMG
20/LMG
40/PP
20/PP
%
80
12
54
38
8
36
9
34
IP
d"
a
c
b
a
b
a
b
DI
2.0
0.1
0.9
0.6
0.1
0.6
0.1
0.5
cv Anellino
d
a
c
bc
a
bc
a
ab
%
78
9
43
45
8
30
11
28
IP
d
a
c
c
a
c
ab
bc
DI
1.9
0.1
0.8
0.9
0.1
0.5
0.1
0.3
cv Canellino
c
a
b
b
a
ab
a
a
%
86
11
41
46
16
23
14
36
IP
d
a
a
c
ab
abc
ab
abc
DI
2.2
0.1
0.7
0.9
0.2
0.3
0.4
0.6
b
a
a
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
2.8. MB measurements
MB concentrations in the air trapped under the tarp
were measured at constant intervals after fumigation
using a FUMISCOPE D (Key Chemical & Equipment
Co., Clearwater, FL, USA), a portable instrument commonly used for measuring the concentration of MB in
fumigation chambers. The instrument is based on the
principle of thermal conductivity (Chakrabarti, 1996)
and can detect concentration of the gas in the atmosphere up to 1999 g/m3.
Measurements were taken according to a technique developed by Laboratoire National d'Etude des
Table 6
E!ect of fumigation with methyl bromide (MB) under di!erent plastic
mulch "lms on the incidence of Sclerotinia sclerotiorum infections on
lettuce (cv Gheisa) (trial 3, "rst crop)
Treatment with MB, g/m2 and
plastic cover!
Percentage of infected plants 78
days after transplanting
*
60/LPDE
40/LPDE
30/LPDE
40/LMG
30/LMG
14.1
3.9
1.6
2.3
3.9
3.1
b"
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
Table 7
E!ect of fumigation with methyl bromide (MB) under di!erent plastic
mulch "lms against Rhizoctonia solani on bean (cv Bobis and Canellino), expressed as percent of emerged plants (% E) and Disease Index
(DI, 0}5), 53 days after sowing (trial 3, "rst crop)
Treatment
with MB,
g/m2 and
plastic cover!
cv Bobis
%E
*
60/LPDE
40/LPDE
30/LPDE
40/LMG
30/LMG
19.8
48.0
39.4
45.5
50.0
41.9
cv Canellino
DI
b"
ab
ab
ab
a
ab
1.3
0.8
0.7
0.6
0.7
0.8
%E
b
a
a
a
a
a
50.0
71.3
58.0
60.0
67.8
60.3
DI
a
a
a
a
a
a
1.6
0.9
0.6
0.8
0.6
1.0
b
a
a
a
a
a
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
A. Minuto et al. / Crop Protection 18 (1999) 365}371
369
Table 8
Methyl bromide (MB) measured under the tarp at di!erent intervals
after fumigation (trial 3)
Table 9
Methyl bromide (MB) measured under the tarp at di!erent intervals
after fumigation (trial 3)
Treatment (g/m3) of MB at minutes from the start of fumigation
with MB,
g/m2 and
plastic
cover!
Treatment (g/h/m3) at minutes from the start of fumigation
with MB,
g/m2 and
plastic
cover!
6
60/LPDE
40/LPDE
40/LMG
30/LMG
919
1035
1568
720
60
bc"
b
a
c
363
395
645
303
120
bc
b
a
c
332
266
348
284
180
a
b
a
b
259
252
251
279
600
a
a
a
a
185
197
181
214
1200
ab
ab
b
a
156
174
157
171
a
a
a
a
6
60/LPDE
40/LPDE
40/LMG
30/LMG
122
115
151
73
b"
b
a
c
60
120
654 b
608 b
1004 a
474 b
997
926
1480
763
180
b
b
a
b
1288
1180
1781
1035
600
b
b
a
b
2706
2707
3322
2658
1200
b
b
a
b
4381
4540
4987
4537
b
ab
a
ab
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
!LPDE: low density polyethylene; LMG: LMG Smith Brothers gasimpermeable "lm; PP: Plastopil gas-impermeable "lm.
"Means of the same column, followed by the same letter, do not
signi"cantly di!er following Duncan's Multiple Range Test (P"0.05).
Techniques de Fumigation et de Protection des DeH nreH es
StockeH es (L.N.D.S) du Ministère de l'Agriculture at
Bordeaux (France) (P. Ducom and M. Guinet. pers.
comm.). The concentration of MB present in the air
trapped under the plastic "lm was measured at a distance
of at least 1.5 m inward from the plot border, and of
4}5 m from the fumigant injection points. Measures
started 6 min after beginning of fumigation. Data are
expressed as g/m3 and as C]¹ (concentration]time)
products. This last value is the product of multiplying
MB concentration (C) by the duration of the exposure
time (¹).
3.2. Ewect of xlm type and MB dosages on disease control
3. Results
3.1. Ewect of xlm type and MB dosages on pathogen
survival
All treatments signi"cantly reduced (98}100% reduction) survival of the pathogens arti"cially introduced
into the soil in both trials 1 and 2 (Tables 2 and 3).
Also, the lowest tested dosage of MB (20 g/m2) under
gas-impermeable plastic "lm strongly reduced the survival of the pathogens, both at 10 and 20 cm depths.
However, only R. solani and P. ultimum were completely
inactivated by some treatments. In the case of FOB,
the tested treatments strongly reduced but did not
completely kill the inoculum (Tables 2 and 3). Considering the e!ect on FOB, the e$cacy of the treatments
was higher in trial 1 than in trial 2. No signi"cant di!erences on the e!ect on the pathogen's inocula were observed among treatments, indicating that all tested
dosages of MB, applied under di!erent "lms, gave similar
results.
In the trial carried out against R. solani, FOB and
C. gloeosporioides, all the tested treatments signi"cantly
reduced the incidence of all three pathogens (1}28% reduction) on the "rst crop of basil (Table 4). No signi"cant
di!erences among treatments were observed concerning
the level of disease control and basil yield (Table 4). The
addition of manure before treatment with MB, applied at
60 g/m2 under standard LPDE "lm, did not a!ect the
e$cacy of the treatments (Table 4).
In a second trial, carried out against R. solani on bean,
60 g/m2 of MB applied under standard LPDE or 40 g/m2
under LMG "lm provided the best disease control on the
three tested cultivars of bean (Table 5). The results obtained by applying 40 g/m2 under PP were only slightly
inferior. Forty grams of MB under standard LPDE or
20 g/m2 under gas impermeable "lm did not provide
a satisfactory disease control (Table 5). As in trial 2, R.
solani symptom incidence was signi"cantly reduced by all
treatments in the case of lettuce (cv Lido), in comparison
with the control, but yield was not a!ected by any of the
treatments (data not shown).
In trial 3, S. sclerotiorum symptom incidence was signi"cantly reduced by all tested treatments on lettuce (cv
Gheisa) (Table 6) as well as on the second crop (cv Foglie
di quercia) (data not shown). On cv Bobis, only 40 g/m2
of MB, applied under LMG, signi"cantly reduced the
incidence of R. solani, while on cv Canellino, no signi"cative di!erences were observed among treatments (Table 7).
3.3. Permeability of xlms
In trial 3, the plots treated with 40 g/m2 of MB and
covered with LMG "lm retained the highest concentrations of MB in the atmosphere trapped under the "lm, as
370
A. Minuto et al. / Crop Protection 18 (1999) 365}371
compared with those covered by LMG and treated with
30 g/m2 of MB and those covered with standard LPDE,
fumigated with 60 and 40 g/m2 (Table 8). The same trend
is shown when C]¹ products are considered: the highest concentrations of MB were observed when the
fumigant was applied at 40 g/m2, under gas-impermeable
"lm, in comparison with those achieved under LPDE
with an initial dosage of 60 g/m2 of MB (Table 9). This
indirectly shows that emissions of MB into the atmosphere are signi"cantly reduced by the use of gas-impermeable "lms, in comparison with the standard dosage of
MB under LPDE.
In trial 4, LMG "lm applied on plots fumigated with
40 g/m2 of MB con"rmed its capability of retaining MB
under the tarp (data not shown).
In both trials the di!erences of the MB concentrations
measured under the di!erent plastic "lms signi"cantly
decreased over time. The di!erences between some C]¹
products remained statistically signi"cant. Particularly,
such value remained signi"cantly higher in the case of
the treatments carried out under LMG at 40 g/m2. Less
promising results were obtained with the two experimental "lms coded as SIS (data not shown).
4. Discussion
The results presented, obtained in a variety of experiments, with di!erent crops and pathogens, show the
possibility of reducing the dosages of MB for soil disinfestation from 60 g/m2 to 40 and 30 g/m2, with still very
e!ective control of important soilborne pathogens, when
gas-impermeable "lms are used for mulching. The results
obtained in terms of disease control were generally similar to those achieved with 60 g/m2 of MB under LPDE.
The dosage of 20 g/m2, although still e!ective in some
cases, did not always ensure satisfactory disease control.
For this reason, this dosage cannot be advised under
practical conditions, at least under those circumstances
where fungal pathogens are the major problem.
The results obtained by evaluating the direct e!ect of
MB applied at di!erent dosages under di!erent "lms,
con"rm the relatively high sensitivity of P. ultimum and
R. solani to the fumigant in comparison to F. oxysporum,
as already shown (Munnecke et al., 1978; Ebben et al.,
1983). The Fusarium population was, however, strongly
reduced. It is possible that the level of inoculum of F.
basilici surviving the treatment is not su$cient to cause
disease: actually, it has been shown that 3]102 CFU/ml
of soil are necessary in order to cause the appearance
of symptoms on the cv `Genovese gigantea of basil
(A. Garibaldi and G. Minuto, unpublished). In similar
experiments carried out in Israel by infesting soil with
basil stems infected with F. basilici, the pathogen was
completely killed by MB treatment at full dosage under
LPDE or at reduced dosage under gas-impermeable "lm
(Gamliel et al., 1998b). The lower mortality observed in
our experiments can be explained by the higher resistance to the fumigation treatment of chlamydospores of
the pathogen in comparison to spores and mycelium
present on infected stems.
The results obtained in the tests on pathogen survival
con"rm the results obtained in terms of disease control:
20 g/m2 of the fumigant, applied under any of the tested
gas-impermeable "lms did not give complete control of
R. solani and P. ultimum.
The di!erent levels of control of R. solani achieved with
reduced dosages of MB under gas-impermeable "lm on
bean, lettuce and basil could be, at least partially, explained with a di!erent virulence of the pathogen on
the tested crops, bean being a very susceptible crop to
R. solani infections.
A long-term e!ect of MB at a reduced dosage under
gas-impermeable "lm was observed in trial 3 on lettuce.
This long-term management of soilborne pathogens on
more than one crop allows a greater interval between
fumigation treatments, with positive implications from
an economic point of view. Moreover, a long-term e!ect,
coupled with the reduction in dosage by using gas-impermeable "lm should reduce the emissions of MB into the
atmosphere, thus further adding to the environmental
and economic bene"ts.
Among the tested gas-impermeable "lms, both LMG
and PP provided consistent results both in terms of
disease control and retention of the fumigant under the
tarp, as indirectly measured with our system. In other
studies, both "lms were very e!ective in retaining higher
concentration of MB in the soil and reducing MB emissions into the atmosphere under laboratory and "eld
conditions (Gamliel et al., 1998a,b).
In order to implement the use of gas-impermeable
"lms on a large scale, particularly in the open "eld, these
"lms should have su$cient mechanical properties, so as
to maintain their impermeability in the "eld (Gamliel
et al., 1997a). Resistance to mechanical stretching, temperature #uctuation, tearing and holes are among the
essential requirements for commercial application. From
a practical point of view, currently available "lms still
present some technical problems because of their tendency to tear. Due to their high rigidity, small lides can
become long cuts, which can nullify the positive e!ect of
their impermeability to the fumigant. Moreover, glueing
VIFs is more complicated than glueing standard LPDE.
The measurements of MB concentration under the
plastic "lms provided useful information concerning the
behaviour of the gas. Although the method used in this
work gave an indirect evaluation of the concentration of
the fumigant in the soil, it clearly indicated that a higher
concentration of the fumigant is trapped under gas-impermeable "lms giving higher C]¹ values and providing good disease control while reducing the emissions
of MB into the atmosphere (Gamliel et al., 1997a).
A. Minuto et al. / Crop Protection 18 (1999) 365}371
Reduced dosages of MB applied under gas-impermeable "lm also were demonstrated in other pathogen-crop
systems (Cebolla et al., 1996; Gamliel et al., 1997b; Gullino et al., 1996; Minuto et al., 1998a,b). The availability
of better quality gas-impermeable "lms will allow expansion of the application technique in an increasing number
of situations. At this stage, VIFs are more expensive than
LPDE, but allowing MB dosage reduction may compensate for the additional cost. Hopefully, expanding their
usage will help reducing their production costs.
The usage of VIFs with reduced dosages of MB will
help agriculture, in the short term, to conform to Montreal Protocol requirements, while reliable, long term
solutions are developed.The application of reduced dosages of MB under gas-impermeable plastics, if combined
with other methods of soilborne disease management,
may enable even further reductions of MB emissions into
the atmosphere.
Acknowledgements
Work carried out with grants from Ministry of Environment } S.I.A.R., Italy and Regione Liguria. The
authors thank Prof. Jaacov Katan and Prof. Alberto
Matta for critically reviewing the manuscript and the
anonymous reviewers for their valuable comments.
The "rst author carried the experimental "eld work,
collected and elaborated the data and participated into
the planning of the experiments during the preparation of
his Ph.D thesis, the second author cooperated into part
of the "eld work and carried the laboratory experiments
during the preparation of her Master thesis, the third
author planned the work and wrote the manuscript. The
fourth author participated into the planning of the experiments and reviewed the manuscript.
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