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Increased shelf life of a bioherbicidethrough combining modifiedatmosphere packaging and lowtemperaturesMiron P. Teshler a , Gavin J. Ash b , Yevgen Zolotarov a & Alan K.Watson aa Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, Québec, Canadab EH Graham Centre for Innovative Agriculture, School ofAgricultural and Veterinary Sciences, Charles Sturt University,Wagga Wagga, NSW, Australia
Version of record first published: 13 Apr 2007
To cite this article: Miron P. Teshler, Gavin J. Ash, Yevgen Zolotarov & Alan K. Watson (2007):Increased shelf life of a bioherbicide through combining modified atmosphere packaging and lowtemperatures, Biocontrol Science and Technology, 17:4, 387-400
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Increased shelf life of a bioherbicide throughcombining modified atmosphere packaging and lowtemperatures
MIRON P. TESHLER1, GAVIN J. ASH2, YEVGEN ZOLOTAROV1, &
ALAN K. WATSON1
1Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada, and2EH Graham Centre for Innovative Agriculture, School of Agricultural and Veterinary Sciences,
Charles Sturt University, Wagga Wagga, NSW, Australia
(Received 29 September 2006; returned 16 October 2006; accepted 13 December 2006)
AbstractThe effects of air temperatures (4, 14 and 248C) and modified atmosphere packaging (MAP)(0% CO2/100% N2; 20% CO2/80% N2 or 40% CO2/60% N2) on vigour of a Sclerotinia minorbarley formulation during 6 months storage were evaluated. The study was performed using amultilevel factorial experimental design and response surface methodology (RSM) and aimed todetermine the optimum combination of the above factors that resulted in retention of S. minorvigour during storage. Temperature and storage duration are the main factors that affectS. minor vigour. CO2 concentration had no effect on S. minor vigour during storage. However,oxygen displacement from storage containers by CO2 and N2 resulted in significant decrease ofvigour reduction of S. minor as compared to ambient air control. An acceptable level of S. minorvigour reduction (ALVR) during storage was developed and determined to be ALVR�31.7914.8% (mean995% CI). Contour plot analysis indicated that the S. minor barley formulation at0.4 water activity could be stored for 6, 12 or 26 weeks without exceeding the upper ALVRthreshold (ca. 46%) at air temperatures not higher than 20, 17 or 118C, respectively.
Keywords: Sclerotinia minor, barley formulation, storage, modified atmosphere packaging
(MAP), storage temperature, acceptable level of vigour reduction (ALVR)
Introduction
With the enactment of laws in Canada, USA and Europe restricting or banning the
use of chemical herbicides, a need for alternative means of weed control has arisen.
Biological control agents, such as fungi or bacteria, often present viable alternatives to
chemical herbicides. However, to be able to compete with chemicals, biological
control agents have to be economically efficient. One way to improve economical
efficiency of bioherbicides is to increase their shelf life.
Our research was focused on increasing the shelf life of a fungal plant pathogen
Sclerotinia minor (Stewart-Wade et al. 2002; Abu-Dieyeh & Watson 2006) comprised
Correspondence: Miron P. Teshler, Department of Plant Science, McGill University, 21,111 Lakeshore
Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9. Tel: �1 514 398 7851, ext. 8746. Fax: �1 514
398 7897. E-mail: [email protected]
ISSN 0958-3157 print/ISSN 1360-0478 online # 2007 Taylor & Francis
DOI: 10.1080/09583150701213695
Biocontrol Science and Technology, 2007; 17(4): 387�400
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of a unique formulation system of mycelia grown on barley (Hordeum vulgare L.) grits,
which serve a double purpose of being a nutrient source for the biocontrol agent and a
delivery propagule at the same time. The combination of solid state fermentation with
a granular formulation resulted in a superior product at a projected lower cost than
alternative formulations (Stewart-Wade et al. 2002). One drawback of this formula-
tion is that the fungal mycelia it contains as the active ingredient are in a metabolically
active state that tends to be less tolerant to environmental stress, has a shorter shelf
life, and requires specific packaging to enable gas and moisture exchange compared
with quiescent resting propagules (e.g. spores or sclerotia) (Paau 1988).
From first view, it would be logical to use sclerotia in formulation of sclerotia-
producing fungus as S. minor . There are indeed several attempts to use microsclerotia-
based formulation for the biological control of weeds. For example, microsclerotial
formulations of Colletotrichum truncatum incorporated into soil to control hemp
sesbania seedlings (Sesbania exaltata) (Jackson et al. 1996b) or Mycoleptodiscus
terrestris applied against hydrilla (Hydrilla verticillata) in aquatic environments
(Shearer & Jackson 2006). The above mentioned fungi produce microsclerotia that
germinate both hyphally and sporogenically significantly enhancing the infection
process. There are, however, several reasons why the S. minor mycelia formulation
was chosen for our biocontrol project. First, sclerotia of S. minor infect via eruptive or
mycelial germination followed by invasion of plant parts in contact with infested soil
(Hollowell et al. 2003) and, therefore, contact with the target plant is limited and
depends mainly on number of sclerotia introduced. Second, for post-emergence
application, mycelium on the surface of barley grits is superior to sclerotia since it
causes rapid disease initiation, whereas sclerotia require additional time to germinate.
Third, sclerotia survival in field conditions is an important safety issue since S. minor ,
has a wide host range (Melzer et al. 1997) and, therefore, sclerotia distribution and
persistence are undesirable. At the same time, mycelia persistence and dissemination
in field conditions are limited.
Many excellent methodologies are used in food technology to improve product
shelf-life, such as modified atmosphere packaging (MAP), storage at lower water
activity and temperature. These technologies could be adapted to increase the
biological control agent survival in storage. MAP, for example, has been used in the
food industry for a relatively long time (Smith 1987) and was proven to have a positive
effect on shelf life of various products by inhibiting the growth of microorganisms
thriving on foodstuffs (Wolfe 1980; Magan & Lacey 1984; Church 1994). MAP
reduces the rate of respiration thus decreasing microbial metabolism and substrate
uptake (Zagory & Kader 1988), which results in prolonged nutrient availability and
decreased release of toxic metabolites. To the best of our knowledge, no research has
yet been reported on the effects of MAP on storage life of S. minor or other biological
control agents.
Response surface methodology (RSM) has been successfully used for a wide range
of optimization studies (Zaika et al. 1994; Devlieghere et al. 1998; Tellez-Luis et al.
2003; Chen et al. 2004) as a tool to solve multivariate problems, determine
interrelationships among test variables and represent the combined effect of all test
variables as a response surface.
This investigation was undertaken to develop and optimize a process of storage of
S. minor barley grit formulation and to evaluate the effect of temperature and MAP on
S. minor survival in storage.
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Materials and methods
Fungal formulation
Sclerotinia minor isolate IMI 344141 from the Weed Science Laboratory (McGill
University) fungal collection was grown on Modified Richard’s solution (MRS)
(Briere et al. 2000) in flask cultures on a rotary shaker at 200 rpm, 208C in the dark
for 6 days. Mycelia from the flask cultures was homogenized and used to inoculate
autoclaved ground barley seeds (3 mL of MRS-mycelium slurry per 100 g of ground
barley) inside 44�20.5 cm transparent autoclavable plastic bags equipped with 0.02-
mm filter discs (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Inoculated
barley grits were incubated for 6 days at 208C in the dark inside a Conviron-E15
growth chamber (Controlled Environments Ltd., Winnipeg, MB, Canada). At day 6,
S. minor-colonized barley grits (pH 5.24) were removed from the bags and dried on
metal mesh trays (44.5�22.5�7 cm) inside a Conviron-E15 growth chamber for
20 h at 208C in the dark. Dry material was passed through 2-mm sieve and collected
on 1.4-mm sieve (Canadian standard sieve series, W.S. Tyler Ltd., St. Catherines,
ON, Canada) to obtain the formulation with particle size 1.4�2 mm.
Preparation of Sclerotinia minor formulation for storage
Adjusting the water activity. During drying, the water activity of S. minor barley
formulation was measured using a HygroLab-3 water activity system (Rotronic AG,
Bassersdorf, Switzerland) with the accuracy of 90.003 at 23928C. After drying for
20 h at 208C, the S. minor barley formulation had ca. 0.6 water activity. The target
water activity of 0.4 and 11% moisture content was attained within 5 h of additional
drying at 208C and using the CD-35 low temperature dehumidifier (Ebac Industrial
Products Inc, VA, USA) placed inside the Conviron growth chamber. Dried barley
grits were transferred to a 50�33�28-cm plastic box, equipped with rubber gloves
permanently fixed and projected inside the box, for packaging the S. minor barley grit
formulation. This box allowed free hand manipulation during packaging and minimal
moisture exchange between the S. minor barley samples and the surrounding air. A
high gas and moisture barrier 9�23 cm top-lock PAKVF4 foil bags (Impak Co., Los
Angeles, CA, USA) with a water vapour transmission rate (WVTR)�0.005 g/
100 inch2 per 24 h and oxygen transmission rate (OTR)�0.001 cc/m2 per 24 h at
238C were used. A TISH-300 impulse sealer (TEW Electric Heating Equipment Co.
Ltd., Taipei, Taiwan) was used for sealing the foil bags. A 5-g sample of colonized S.
minor barley grit formulation was placed into each foil bag.
Experimental design
Storage conditions. To study the effect of storage treatments on inoculum vigour, a two-
factor experimental design of CO2 content of 0, 20 and 40% (balanced by nitrogen)
and storage temperatures of 4, 14 and 248C consisting of nine treatments was used.
The experimental design with responses is presented in Table I. Sclerotinia minor
barley formulations stored in packages with ambient air (ca. 78% N2, 21% O2 and 1%
other gases) at 4, 14 and 248C served as controls. The Multivac A300 packaging
system (Pro Air Conveying, St. Paul, MN, USA) was used to establish atmospheres by
flushing storage containers of S. minor formulation using different CO2/N2 mixtures.
Storage of Sclerotinia minor: Role of MAP and temperature 389
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Mean residual O2 content after flushing the foil bags was 0.26%. Conviron E-15
growth chambers with accuracy of 918C were used for storage of S. minor in foil
pouches at 14 and 248C. A low temperature 2020 series incubator (Sheldon
Manufacturing Inc., Cornelius, OR, USA) with accuracy of 90.58C was used for
S. minor storage at 48C.
Once per month, the gas composition (CO2, N2 and O2 content) inside the storage
containers (experimental and control treatments) was evaluated using a Servomex-
1400 food package analyzer (Servomex Company Inc., Sugar Land, TX, USA). Gas
composition was tested individually in three foil bags (three replications). To measure
the water activity of the S. minor barley-based formulations the HygroLab 3 indicator
was used. Water activity was evaluated once a month for each experimental and
control treatment in the three replications.
Inoculum vigour. For each experimental and control treatment, the vigour of the
S. minor formulation was evaluated at 2-week intervals and gas composition and water
activity every 4 weeks of storage. The experiment lasted 6 months. After opening for
samplings, the foil bags with S. minor formulation were resealed using the plastic
zipper and stored for an additional 4 weeks in the refrigerator at 48C. Two additional
estimations of fungal vigour were performed on the second and fourth week of
additional storage. On each sampling day, two foil bags of a S. minor grit formulation
were removed from the storage location and thoroughly mixed together. Ten S. minor
colonized barley grits were randomly taken from the mixture and placed individually
in the middle of a potato dextrose agar (PDA) plate (9 cm diameter) to evaluate the
radial colony growth. Colony diameter of the S. minor grit samples on PDA plates
were measured using a dial caliper (Manostat, Switzerland) after 48 h of incubation at
20918C in the dark. The vigour of a freshly prepared S. minor formulation (control
treatment) at 0.4 water activity was 69.891.4 mm in diameter within 48 h.
Table I. Effect of modified atmosphere packaging and air temperature on survival of barley-based
formulation of Sclerotinia minor in storage: experimental design and responses.
Factor Response
Storage conditions Mean vigour reduction (%)
Treatment
X1, atmosphere
(percent CO2 in N2, v/v)
X2,
temperature (8C) 6 weeks 12 weeks 26 weeks
1 0 4 19.0 37.3 44.4
2 20 4 28.4 32.7 44.2
3 40 4 34.8 37.8 38.9
4 0 14 36.1 44.1 66.5
5 20 14 42.3 49.8 63.5
6 40 14 36.2 47.9 54.7
7 0 24 67.2 92.2 100.0
8 20 24 77.6 97.2 100.0
9 40 24 71.4 96.8 100.0
10 Ambient 4 39.9 38.2 57.1
11 Ambient 14 44.6 62.8 78.0
12 Ambient 24 88.5 100.0 100.0
Treatments 10, 11, 12�control treatments, Sclerotinia minor stored at ambient conditions (ca. 78% N2,
21% O2, 1% other gases).
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Since previously conducted experiments (Table II) demonstrated that vigour of
S. minor (radial growth on PDA within 48 h) has a strong significant correlation with
S. minor virulence (lesion diameter on detached dandelion leaves) measured within
48 h (R�0.818, PB0.001) and also significantly correlated (R�0.774, PB0.001)
with fungal field efficacy (disease severity on dandelion plants observed 7 days after
treatment) only S. minor vigour on PDA media was therefore monitored during the
storage experiment.
Acceptable level of vigour reduction
A quality indicator, the acceptable level of vigour reduction (ALVR), was developed to
estimate the loss of vigour during storage that does not result in significant loss of
efficacy in controlling dandelion plants in the field. To develop the ALVR, five field
trials were conducted at the Macdonald Campus of McGill University, Ste-Anne-de-
Bellevue, QC, Canada in 2004 and 2005 (Table II). For each field trial, batches of S.
minor barley formulation with vigour ranging from low (10�30 mm diameter/48 h) to
high (45�65 mm diameter/48 h) were applied at 60 g/m2 on dandelion-infested
turfgrass. Plots were 50�50 cm2 and each treatment (S. minor batch) was replicated
four times. Weather conditions (e.g. mean, maximum and minimum air temperatures
and total precipitation within 7 days after S. minor application) were recorded.
Disease severity on dandelion plants was evaluated 7 days after treatment using the
Horsfall-Barratt scale (Cambell & Madden 1990). The batch stored at 48C with the
highest vigour was designated as the control. Multiple comparison tests were used to
Table II. Calculation of ALVR and correlation between disease severity, virulence and vigour.
Field trial
July 9,
2004
August 13,
2004
August 25,
2004
September
22, 2005
October 3,
2005
Number of S. minor batches tested
Parameter 12 14 17 7 9
A. Mean vigour (mm/48 h)
Control batch (vc) 57.7 57.6 55.7 50.1 61.0
Marginal batch (vm) 45.7 33.4 43.8 26.8 44.2
B. Mean disease severity (%) 7 DAT
Control batch 93.4 99.4 100 88.6 98.4
Marginal batch 78.6 75.1 94.4 83.3 98.0
C. ALVR (%) 20.7 41.9 21.5 46.6 27.6
Mean ALVR (995%CI) 31.7914.8
D. Pearson correlation (P values presented in brackets)
Vigour and disease severity 0.841 0.766 0.809 0.776 0.872
(B0.001) (0.001) (B0.001) (B0.05) (0.001)
Vigour and virulence 0.870 0.855 0.606 0.850
(B0.001) (B0.001) (B0.05) (0.01) n/a
DAT, days after treatment; Control batch, S. minor batch with the highest vigour stored at 48C prior the
field testing; Marginal batch, S. minor batch with the lowest vigour, which is not significantly different from
the control batch in disease severity; Virulence, S. minor rate of growth on detached dandelion leaves
expressed as a lesion diameter (mm/48 h).
Storage of Sclerotinia minor: Role of MAP and temperature 391
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compare disease severity caused by different batches. The batch stored at 48c with the
lowest vigour (marginal batch, vm) that did not differ statistically in disease severity
from the control batch (vc) was chosen. Difference in vigour (Dv) between batches vm
and vc was expressed as percent of vc vigour (i.e. Dv�vc � vm
vc
�100%): The mean Dv
from the five experiments was defined as the ALVR.
Data analysis
Results were analyzed using the response surface experimental design procedure of
StatGraphics Plus v. 4 (Manugistics Inc, Rockville, MD, USA). Linear regression
analysis, slope and intercept comparison of regression lines (Zar 1996) was performed
using the GraphPad version 4.03 (GraphPad Software, San Diego, CA, USA).
Correlation analysis was conducted using SigmaStat 2.03 (SPSS, Chicago, USA) and
presented as a Pearson correlation coefficient (R) and level of significance (P).
Sclerotinia minor vigour (V) data were presented as a percentage of vigour reduction
in comparison with vigour of a freshly prepared S. minor formulation (i.e.
/V �vcontrol � vstored
vcontrol
�100%; where, vcontrol is the vigour of fresh material, vstored is
the vigour of material stored in ambient air or MAP).
After bags were opened, they were stored for an additional 2 and 4 weeks at 48C.
Vigour of S. minor from these bags was measured and compared to the vigour
measured after the initial opening of the bags to assess the possibility of additional
storage in normal atmosphere. This comparison was done using the one-way ANOVA
combined with Tukey multiple comparison procedure at the 5% level of significance
using SigmaStat v.2.03.
Since S. minor vigour reduction data ranged from 10 to 100% and can be expected
to have a non-normal frequency distribution (Gomez & Gomez 1984), these data were
arcsine transformed prior to statistical analysis.
Results
ALVR
The estimated ALVR (mean995% CI) based on S. minor vigour tested within 48 h
was 31.7914.8% (mean995% CI) (Table II).
Analysis of contour lines
After statistical analysis of results (Table I), the second-order polynomial equations
were generated (Table III) and contour lines were plotted for 6, 12 and 26 weeks of
storage (Figure 1). The analysis of variance for the response variables (Table III),
indicates that the models developed for the S. minor vigour reduction within 6, 12 and
26 weeks of storage are adequate, possessing no significant lack of fit, and explain
55.4, 72.8 and 78.2% of the variability of the responses, respectively. Based on
ANOVA, the temperature and quadrate of temperature have a significant effect on
fungal vigour in storage (Table III). The relationship between factors and responses
can best be understood by examining the series of contour plots. Analysis of contour
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plots indicates that S. minor in the barley formulation could be stored for up to 6
weeks at up to 208C (Figure 1A), for 12 weeks at up to 178C (Figure 1B) or for 26
weeks at up to 118C (Figure 1C) without exceeding the ALVR (upper threshold); and
CO2 concentration had no effect on S. minor vigour (the contour lines of vigour
reduction are predominantly parallel to x-coordinate representing the CO2 concen-
tration) (Figure 1).
Modified air versus ambient air storage
Effect of modified air conditions (0% CO2/100% N2, 20% CO2/80% N2, 40% CO2/
60% N2) on S. minor vigour during storage in comparison with the vigour of S. minor
in control treatments stored at 4, 14 and 248C and ambient air is presented in
Figure 2. Statistical analysis of regression lines is presented in Table IV. All linear
regression models representing different S. minor treatments were significant at 95%
probability level except the model for treatment 3 that was significant at the lower
probability level of 90% (Table IV).
At 48C, most data points that represent vigour reduction did not exceed the upper
ALVR threshold (Figure 2A�C). At 148C, the upper ALVR threshold of S. minor was
observed after ca. 60 days (ambient air storage) and after ca. 100 days (modified air
storage). After 180 days in storage, S. minor vigour reduction was ca. 80% (ambient
conditions) and ca. 60% when S. minor stored in modified air conditions (Figure 2D�F). At 248C, the vigour reduction of all S. minor treatments exceeded the upper ALVR
threshold (ca. 46%) within 2�3 weeks of storage (Figure 2G�I). Fungal death was
Table III. Selected statistics for the quadratic regression models: Sclerotinia minor vigour reduction in
storage.
Model
Quadratic model Regression terms Significance (P�jt j) r2 Lack of fit (P�F )
Intercept�39.71 0.0001
X1�1.97 0.1267
X2�15.23 0.0001
Model 1 (6 weeks) X1�X1��2.73 0.2212 0.554 0.624
X2�X1��2.11 0.1823
X2� X2�8.19 0.0003
Intercept�44.30 0.0001
X1�1.17 0.3797
X2�23.03 0.0001
Model 2 (12 weeks) X1�X1�0.03 0.9881 0.728 0.806
X2�X1�1.08 0.5072
X2�X2�15.30 0.0001
Intercept�53.27 0.0001
X1��1.15 0.2885
X2�24.23 0.0001
Model 3 (26 weeks) X1�X1��2.12 0.2851 0.782 0.847
X2�X1�0.871 0.5107
X2�X2�13.17 0.0001
P, probability; r2, coefficient of determination; t , Student value; F, Fisher criterion; X1, percent CO2 in N2;
X2, temperature (8C).
Storage of Sclerotinia minor: Role of MAP and temperature 393
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observed within ca. 70 days (all treatments stored in ambient conditions) and ca. 100
days for treatments stored in modified air (Figure 2G�I).
Comparison of linear regressions representing S. minor vigour reduction in different
modified air treatments with vigour reduction of S. minor stored in ambient air
(comparison with control treatments) is presented in Table V. Sclerotinia minor was
12
23
35
46
58
4
8
12
16
20
24
4
8
12
16
20
24
4
8
12
16
20
24A
B
C
Tem
pera
ture
(°C
)
35
46
58
69
81
CO2 Concentration (%)0 10 20 30 40
35
46
58
69
81
A LV R
A L V R
A L V R
Figure 1. Contours of constant vigour reduction (%) of Sclerotinia minor as a function of CO2 concentration
and air temperature: (A) 6 weeks in storage; (B) 12 weeks in storage; (C) 26 weeks in storage.
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significantly more viable when stored in packages with modified air in comparison
with storage in ambient air. Non-significant difference among equation slopes
(treatments compared with respective controls) indicated that regression lines
are parallel; and significant difference of intercepts indicated that the regression
lines are not identical (i.e. significantly different) (Table V). In one instance, the
regression slopes were significantly different as well (treatment 6 compared with
treatment 11).
Comparison of different modified air conditions
There was no effect of different MAP conditions on S. minor vigour. Slopes and
intercepts of regression lines (group comparison) were not significantly different
(Table V) indicating that the regression lines are identical.
Sclerotinia minor vigour after opening the storage containers
Storage containers with S. minor that were stored for 2, 4, 6, 8, 10, 12 and 16 weeks
and opened for vigour sampling were re-sealed to evaluate the effect of additional
storage at 48C. No significant difference in S. minor vigour (P B0.05) was observed
for S. minor formulations for 4 weeks of additional storage at 48C after re-opening
storage containers.
A
0102030405060708090
100
0102030405060708090
100
0102030405060708090
100
D
Vig
or
Red
uct
ion
(%
)
G
200180160140120100806040200 200180160140120100806040200 200180160140120100806040200
C
F
I
B
Storage Period (day)
E
H
Figure 2. Effect of storage time on Sclerotinia minor vigour reduction: (A�C) 48C and CO2�0, 20 and
40%, respectively; (D�F) 148C and CO2�0, 20 and 40%, respectively; (G�I) 248C and CO2�0, 20 and
40%, respectively. (-m-) Various MAP treatments (1�9); (-k-) ambient air control treatments (10, 11, 12);
(dotted line) upper ALVR level. Statistical significance is based on arcsine-transformed data; non-
transformed data were plotted.
Storage of Sclerotinia minor: Role of MAP and temperature 395
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Discussion
Significant information has been gathered about factors responsible for growth and
survival of various fungi and bacteria. These factors include fermentation process,
formulation, compatible solutes and nutrients, storage temperature, water activity,
packaging system and gas composition (Brown & Hellebust 1978; Kemp et al. 1981;
Beuchat 1983; Sperber 1983; Mugnier & Jung 1985; Hallsworth & Magan 1994;
Fravel et al. 1995; Connick et al. 1996; Jackson et al. 1996a; Quimby et al. 1999;
Shabana et al. 2003).
In this study effect of air temperatures and gas composition on S. minor in a
granular barley-based formulation was evaluated. Prior to storage, S. minor formula-
tion was air-dried to 0.4 water activity (aw) since in our preliminary storage trials
(unpublished), samples of S. minor barley grit formulation stored at 0.4 water
activities had superior shelf life to samples with 0.5 or higher water activities. The
water activity value indicates how much free water is available to microorganisms, as
opposed to water that is strongly bound to formulation components, and is more
meaningful in the study of dried microorganisms than water content (Troller 1980). In
general, as aw is reduced, the rate of microbial growth is delayed, and, usually, causes
some reduction in metabolites secreted by the cell (Troller 1980). Reduced aw can
also lead to increased resistance of a fungus to high temperatures (heat-tolerance), and
often favours long-term vigour (Connick et al. 1996). For example, survival of
Table IV. Selected statistics for the linear regression models: Sclerotinia minor vigour reduction in storage.
Treatment (linear regression model) Regression terms Significance* P �jt j r2
1 (0% CO2�48C) Intercept�26.71 0.0001 0.590
Slope�0.071 0.0013
2 (20% CO2�48C) Intercept�29.09 0.0001 0.613
Slope�0.063 0.0009
3 (40% CO2�48C) Intercept�30.58 0.0001 0.237
Slope�0.038 0.0777
4 (0% CO2�148C) Intercept�29.79 0.0001 0.742
Slope�0.136 0.0001
5 (20% CO2�148C) Intercept�28.76 0.0001 0.707
Slope�0.137 0.0002
6 (40% CO2�148C) Intercept�32.37 0.0001 0.634
Slope�0.105 0.0007
7 (0% CO2�248C) Intercept�36.69 0.0001 0.868
Slope�0.371 0.0001
8 (20% CO2�248C ) Intercept�41.48 0.0001 0.867
Slope�0.339 0.0001
9 (40% CO2�248C) Intercept�39.63 0.0001 0.868
Slope�0.349 0.0001
10 (ambient�48C) Intercept�31.19 0.0001 0.345
Slope�0.059 0.0272
11 (ambient�148C) Intercept�32.42 0.0001 0.845
Slope�0.183 0.0001
12 (ambient�248C) Intercept�47.01 0.0001 0.769
Slope�0.307 0.0001
r2, coefficient of determination; P, probability; t , Student value. Treatments 10, 11, 12�controls. *The
significance of the regression model is the same as the significance of the ‘slope’ term of a regression
equation.
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Table V. Comparison of linear regression models.
Slope Intercept
Comparison Comparison
Value (9SE) With control Group Value (9SE) With control Group
1 0.07190.017 F1,22�0.303, P�0.588 26.7191.62 F1,23�13.244, P�0.0014
2 0.06390.015 F1,22�0.061, P�0.807 F2,35�0.581, P�0.565 29.0991.37 F1,23�6.263, P�0.020 F2,37�1.352, P�0.271
3 0.04690.019 F1,21�0.213, P�0.649 30.4491.76 F1,22�4.644, P�0.042
10 0.05890.017 32.5291.67
4 0.13690.023 F1,24�2.138, P�0.157 29.7992.18 F1,25�12.51, P�0.002
5 0.13790.025 F1,24�1.858, P�0.186 F2,36�0.585, P�0.562 28.7692.39 F1,25�15.27, P�0.0006 F2,38�0.233, P�0.793
Tre
atm
ent
6 0.10590.023 F1,24�5.925, P�0.023 32.3792.16 ***
11 0.18390.023 32.2492.13
7 0.52290.045 F1,16�1.532, P�0.234 29.7492.98 F1,17�11.15, P�0.0039
8 0.53890.040 F1,15�1.280, P�0.276 F2,25�0.031, P�0.970 32.7892.30 F1,16�4.687, P�0.046 F2,27�1.930, P�0.165
9 0.53190.054 F1,15�1.193, P�0.292 31.5993.09 F1,16�6.381, P�0.022
12 0.64990.100 33.1194.83
Treatments: 1, 2, 3�S. minor stored at 0% CO2 and 4, 14, 248C, respectively; 4, 5, 6�20% CO2 and 4, 14, 248C, respectively; 7, 8, 9�40% CO2 and 4, 14, 248C,
respectively; 10, 11, 12�control treatments, at ambient air (ca. 78% N2, 21% O2, 1% other gases) and 4, 14, 248C, respectively.
Stora
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Role
ofM
AP
and
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bacteria such as Salmonella sp. is doubled or quadrupled at the temperature studied as
the aw is decreased from 0.71 to 0.34 (Sperber 1983). Heat resistance of ascospores of
Byssochlamys nivea and chlamydospores of Humicola fuscoatra increased significantly
when the relative humidity (and presumably the aw in test cells) decreased from 100 to
60 or 30%, respectively. Further reduction in relative humidity to 0% caused an
increase in sensitivity to heat and cell death, indicating that protective effect of free
water is maximum at a critical moisture level, which may be lower than required for
growth but not approaching zero (Beuchat 1983). Water activity usually works in
combination with temperature to slow down the metabolism of microbial organisms
(Mugnier & Jung 1985; Eyles et al. 1993). A range of air temperatures selected in this
study simulates refrigerated storage (48C); cool-room storage (148C) and room
temperature storage (248C).
Modified atmosphere packaging is another important component against microbial
food spoilage. Different combinations of oxygen, carbon dioxide and nitrogen are
usually used in MAP (Church 1994). For example, it was found that the atmosphere
containing concentrations of CO2 as low as 20% (balanced by nitrogen) inhibited the
growth of Pseudomonas sp. on the surface of buffered Brain Heart Infusion agar plates
at 5 and 158C (Eyles et al. 1993). Carbon dioxide is used in MAP mainly due to its
bacteriostatic and fungistatic properties (Sivertsvik et al. 2002). CO2 inhibits the
growth of microorganisms by dissolving in the water-phase of a stored product, which
forms bicarbonate ions that lower the pH (Devlieghere et al. 1998). However, the
results of our experiments show no effect of CO2 on S. minor vigour in storage. Vigour
reduction, for example, in treatments where bags were flushed with 100% N2 did not
differ significantly from the treatments where either 20 or 40% CO2 was used. This
can be explained by the fact that barley-grit formulation of S. minor has low moisture
content (11%), and, therefore low amounts of CO2 were dissolved in the water-phase
of the formulation. On the other hand, a decrease in vigour reduction was observed in
all MAP treatments compared with ambient air controls. This is primarily caused by
the decrease of oxygen concentration in the storage containers, which was displaced
during gas flushing. Aerobes require oxygen for cellular respiration, where it is
employed as an electron acceptor in oxidative phosphorylation, therefore decreased
oxygen content inhibits the growth of aerobic microorganism like S. minor . Oxygen
was not completely removed from storage containers during flushing, residual
amounts of approximately 0.26% were left in the bags and that amount did not
change significantly during storage. Therefore, the main finding of the present study is
that S. minor in a barley-based formulation appears to be tolerant and even benefits
from the low O2 content (near anaerobic conditions) in storage containers. Further
studies on the effects of different oxygen concentrations on S. minor vigour in storage
should be conducted. In addition, our studies have demonstrated that the application
of MAP technology is not needed for S. minor , and O2 displacement could be
provided by less expensive methods (e.g. vacuum packaging or O2 scavengers).
An integral part of any storage system is proper packaging of a product to control
gas exchange, movement of moisture, and to prevent contamination (Powell 1992).
The aluminium foil pouches used in this study provided excellent stability of key
storage parameters (insignificant moisture and gases fluctuation for up to 6 months).
However, a drawback of using the aluminium foil pouches is their relatively high cost.
More research needs to be done to select less expensive storage containers such as, for
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example, high or low density polyethylene bags to provide a sufficient moisture and
gas control during long-term storage of S. minor .
In this study, the ALVR parameter was developed based on the evaluation of the
field performance of S. minor batches with different vigour levels. It is necessary to
take into consideration, however, that ALVR is only valid for the specific weather
conditions present when experiments were conducted. For example, based on five
field trials, the mean average, minimum and maximum air temperatures were ca. 21,
16, and 258C, respectively; and mean total precipitation was 16 mm for the first 7 days
after S. minor application. According to previous observations (unpublished data),
these temperature and moisture conditions are near the optimum for fungal growth
and pathogenicity. Trials with less favorable weather conditions for S. minor need to
be carried out to extend the applicability of ALVR.
Acknowledgements
Authors grateful for the financial support from the Natural Sciences and Engineering
Research Council of Canada Discovery and I to I programs and the valuable
contributions from colleagues: Simona Teshler and Boris Touvykine (McGill
University, Canada) for S. minor vigour testing and data preparation for statistical
analysis; Professor E. Mencher (Tiraspol University, Moldova) and Dr V. Catana
(Oklahoma State University, USA) for useful suggestions on RSM applications;
Bernard Cayouette for help with MAP set up, gas composition and water activity
monitoring throughout the experiment; Allison Pollock for reviewing the manuscript
and Professor J. P. Smith (Food Science Department, McGill University) for sharing
his extensive knowledge of various methods used in food technology; the two
anonymous reviewers for their valuable suggestions to improve the manuscript.
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