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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: miron.teshler@mcgill.ca

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.

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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.

References

Abu-Dieyeh MH, Watson AK. 2006. Effect of turfgrass mowing height on biocontrol of Taraxacum officinale

with Sclerotinia minor . Biocontrol Science and Technology 16:509�524.

Beuchat LR. 1983. Influence of water activity on growth, metabolic activities and survival of yeasts and

molds. Journal of Food Protection 46:135�141.

Briere SC, Watson AK, Hallett SG. 2000. Oxalic acid production and mycelial biomass yield of Sclerotinia

minor for the formulation enhancement of a granular turf bioherbicide. Biocontrol Science and

Technology 10:281�289.

Brown LM, Hellebust JA. 1978. Sorbitol and proline as intracellular osmotic solutes in the green algae,

Stichococcus bacillaris . Canadian Journal of Botany 56:676�679.

Campbell CL, Madden LV. 1990. Introduction to plant disease epidemiology. New York: John Wiley &

Sons. p 532.

Chen MJ, Chen KN, Lin CW. 2004. Optimization of the viability of probiotics in a fermented milk drink by

the response surface method. Asian-Australasian Journal of Animal Sciences 17:705�711.

Church N. 1994. Developments in modified-atmosphere packaging and related technologies. Trends in

Food Science and Technology 5:345�352.

Connick WJ Jr, Daigle DJ, Boyette CD, Williams KS, Vinyard BT, Quimby PC Jr. 1996. Water activity and

other factors that affect the vigor of Colletotrichum truncatum conidia in wheat flour-kaolin granules

(‘Pesta’). Biocontrol Science and Technology 6:277�284.

Devlieghere F, Debevere J, Van Impe J. 1998. Concentration of carbon dioxide in the water-phase as a

parameter to model the effect of a modified atmosphere on microorganisms. International Journal of

Food Microbiology 43:105�113.

Eyles MJ, Moir CJ, Davey JA. 1993. The effects of modified atmospheres on the growth of psychrotrophic

pseudomonas on a surface in a model system. International Journal of Food Microbiology 20:97�107.

Storage of Sclerotinia minor: Role of MAP and temperature 399

Dow

nloa

ded

by [

McG

ill U

nive

rsity

Lib

rary

] at

08:

19 1

0 A

ugus

t 201

2

Fravel DR, Lewis JA, Chittams JL. 1995. Alginate prill formulations of Talaromyces flavus with organic

carriers for biocontrol of Verticillium dahliae . Phytopathology 85:165�168.

Gomez KA, Gomez AA. 1984. Statistical procedures for agricultural research. 2nd ed. New York: John

Wiley & Sons. p 680.

Hollowell JE, Shew BB, Cubeta MA. 2003. Weed species as hosts of Sclerotinia minor in peanut fields. Plant

Disease 87:197�199.

Hallsworth JE, Magan N. 1994. Effect of carbohydrate type and concentration on polyhydroxy alcohol and

trehalose content of conidia of three entomopathogenic fungi. Microbiology 140:2705�2713.

Jackson MA, Schisler DA, Slininger PJ, Boyette CD, Silman RW, Bothast RJ. 1996a. Fermentation

strategies for improving the fitness of a bioherbicide. Weed Technology 10:645�650.

Jackson MA, Shasha BS, Schisler DA. 1996b. Formulation of Colletotrichum truncatum microsclerotia for

improved biocontrol of weed hemp sesbania (Sesbania exaltata ). Biological Control 7:107�113.

Kemp JD, Langlois BE, Fox JD. 1981. Effects of vacuum packaging and potassium sorbate on yield, yeast

and mold growth, and quality of dry-cured hams. Journal of Food Science 46:1015�1018.

Magan N, Lacey J. 1984. Effects of gas composition and water activity on growth of field and storage fungi

and their interactions. Transactions of the British Mycological Society 82:305�314.

Melzer MS, Smith EA, Boland GJ. 1997. Index of plant hosts of Sclerotinia minor . Canadian Journal of Plant

Pathology 19:272�280.

Mugnier J, Jung G. 1985. Survival of bacteria and fungi in relation to water activity and the solvent

properties of water in biopolymer gels. Applied and Environmental Microbiology 50:108�114.

Paau AS. 1988. Formulations useful in applying beneficial microorganisms to seeds. Trends in

Biotechnology 6:276�279.

Powell KA. 1992. Biological product fermentation, formulation and marketing. In: Tjamos EC, Papavizas

GC, Cook RJ, editors. Biological control of plant diseases (progress and challenges for the future). New

York: Plenum Press. pp 381�387.

Quimby PC Jr, Zidack NK, Boyette CD, GreyWE. 1999. A simple method for stabilizing and granulating

fungi. Biocontrol Science and Technology 9:5�8.

Shabana YM, Muller-Stover D, Sauerborn J. 2003. Granular Pesta formulation of Fusarium oxysporum f. sp.

orthoceras for biological control of sunflower broomrape: efficacy and shelf-life. Biological Control

26:189�201.

Shearer JF, Jackson MA. 2006. Liquid culturing of microsclerotia of Mycoleptodiscus terrestris , a potential

biological control agent for the management of hydrilla. Biological Control 38:298�306.

Sivertsvik M, Jeksrud WK, Rosnes JT. 2002. A review of modified atmosphere packaging of fish and fishery

products � significance of microbial growth, activities and safety. International Journal of Food Science

and Technology 37:107�127.

Smith J. 1987. Modified atmosphere packaging � packaging for the future. The Macdonald Journal 48:12�14.

Sperber WH. 1983. Influence of water activity on foodborne bacteria � a review. Journal of Food Protection

46:142�150.

Stewart-Wade SM, Green S, Boland GJ, Teshler MP, Teshler IB, Watson AK, Sampson MG, Patterson K,

DiTommaso A, Dupont S. 2002. Taraxacum officinale (Weber), dandelion (Asteraceae). In: Mason PG,

Huber JT, editors. Biological control programmes in Canada 1981�2000. Wallingford, Oxon: CABI

Publishing. pp 427�430.

Tellez-Luis SJ, Moldes AB, Alonso JL, Vazquez M. 2003. Optimization of lactic acid production by

Lactobacillus delbrueckii through response surface methodology. Journal of Food Science 68:1454�1458.

Troller JA. 1980. Influence of water activity on microorganisms in foods. Food Technology 34:76�83.

Wolfe SK. 1980. Use of CO- and CO2-enriched atmospheres for meats, fish and produce. Food Technology

:55�58 34:63.

Zagory D, Kader AA. 1988. Modified atmosphere packaging of fresh produce. Food Technology 42:70�77.

Zaika LL, Moulden E, Weimer L, Phillips JG, Buchanan RL. 1994. Model for the combined effects of

temperature, initial pH, sodium chloride and sodium nitrate concentrations on anaerobic growth of

Shigella flexneri . International Journal of Food Microbiology 23:345�458.

Zar JH. 1996. Biostatistical analysis. 3rd ed. Upper Saddle River, NJ: Prentice Hall. p 718.

400 M. P. Teshler et al.

Dow

nloa

ded

by [

McG

ill U

nive

rsity

Lib

rary

] at

08:

19 1

0 A

ugus

t 201

2