Effect of drying on conidial viability of Penicillium

frequentans, a biological control agent against peachbrown rot disease caused by Monilinia spp.

BELEN GUIJARRO, INMACULADA LARENA,

PALOMA MELGAREJO, & ANTONIETA DE CAL

Department of Plant Protection, INIA, Madrid, Spain

(Received 31 January 2005; returned 28 March 2005; accepted 26 July 2005)

AbstractThe effects of drying methods (freeze-, spray-, and fluid bed-drying) on viability of Penicilliumfrequentans conidia were compared. Viability, estimated by germination of fluid bed- and freeze-dried conidia, was similar to that of fresh conidia. Skimmed milk alone, or in combination withother protectants, was added to conidia before freeze-drying. After the freeze-drying process, allprotectants used, except glycerol improved conidial viability. Freeze-dried P. frequentans conidiadid not maintain viability after 30 days of storage at room temperature, while conidia dried by fluidbed-drying showed 28% viability following 180 days after drying. This work also demonstrated arelationship between conidial viability after 1 year of storage at room temperature, moisturecontent after fluid bed-drying and initial weight of sample. Conidial moisture contents must bereduced to 5�/15% for optimal storage at room temperature. P. frequentans conidia dried by fluidbed-drying were as effective as fresh conidia in controlling brown rot of peaches.

Keywords: Biological control, brown rot disease, fluid bed-drying, Monilinia spp., Penicillium

frequentans

Introduction

Penicillium frequentans Westling, a component of the resident mycoflora of peach twigs

and flowers (Melgarejo et al. 1985), reduces peach twig blight caused by Monilinia

laxa (Aderh. et Ruhl.) Honey (Melgarejo et al. 1986; De Cal et al. 1990).

P. frequentans induced significant reductions in disease severity (from 38 to 80%)

compared to the fungicide captan (De Cal et al. 1990) when it was included in some

preparations. Recently, De Cal et al. (2002) have developed a mass production

method for P. frequentans conidia by solid-fermentation.

Biological control agents must be formulated as products capable of storage,

distribution and application in the agricultural market to be of practical use. The

major drawback in commercialising biocontrol products is the development of stable

formulated products that retain similar efficacy to that of the agent’s fresh cells

Correspondence: P. Melgarejo, Department of Plant Protection, INIA, Crtra. De la Coruna Km. 7, 28040

Madrid, Spain. Tel: 34 91 3476846. Fax: 34 91 3572293. E-mail address: melgar@inia.es

First published online 24 January 2006

ISSN 0958-3157 print/ISSN 1360-0478 online # 2006 Taylor & Francis

DOI: 10.1080/09583150500335897

Biocontrol Science and Technology, 2006; 16(3/4): 257�/269

(Janisiewicz et al. 1997). An adequate shelf life for a biological product in the market

requires stability for at least 1 year (Baker & Henis 1990; Rodham et al. 1999). The

product must preferably be stored at room or warm temperatures (Baker & Henis

1990). Some methods of preserving cultures are based mainly on the reduction of the

metabolic rate of the organisms by removing the available water (Foster 1962).

Product dehydration preserves the inoculum for a long time with high viability. In

addition, dry products are good formulations of microbial agents since they can be

handled using the normal channels of distribution and storage (Burges 1998). Dried

and stable products, with a high population of viable and uninjured conidia of

P. frequentans, are needed as biopesticides. Drying can be accomplished by the use of

different methods including freeze-drying, drying on silica gel, and spray- and fluid

bed-drying (Mujumdar 1987; Kudra & Mujumdar 2001).

The objective of our study was to characterise and to compare the effects of freeze-,

spray- and fluid bed-drying on the survival of P. frequentans conidia to obtain a good

and stable, dried biocontrol product. In addition, the relationship between conidial

viability and moisture content after drying was determined.

Materials and methods

Cultures

The isolate of P. frequentans (ATCC 66108) was stored at 48C on potato dextrose agar

(PDA) slants and grown in darkness at 20�/25 8C for 7 days on PDA in Petri dishes to

produce conidia for inoculating the fermentation substrate. Conidia of P. frequentans

were produced in a solid fermentation system as previously described (De Cal et al.

2002). The fungus was grown on a mixture of peat (Gebr. Brill substrate GmbH &

Co. KG, Germany):vermiculite (Termita, Asfaltex, S.A., Barcelona, Spain):lentil

meal (1:1:0.5, w/w/w). Fifty grams of the substrate described above (40% moisture

content, v/w) were placed in a plastic bag (600 cm3) especially designed for solid

fermentation (Valmic†), sealed, and sterilised by autoclaving at 117.67 kPa and

1238C for 1 h on three consecutive days. The substrate was then inoculated with a

conidial suspension of P. frequentans produced on PDA plates to achieve a final

concentration of 105 conidia g�1 dry substrate. Bags were sealed again and incubated

in darkness at 20�/258C for 5 days. To separate the conidia from the substrate, the

mixture in the fermentation bags was resuspended in sterile distilled water, shaken in a

rotary shaker at 200 rpm for 10 min (Lab-Line Instruments, Inc., model 3527,

Melrose Park, IL, USA), and filtered through glass wool. Afterwards, conidia were

concentrated by centrifugation at 14 040�/g for 10 min. These fresh conidia were

used for testing the effect of temperature on viability and in drying experiments.

Effect of temperature on conidial viability

Two replicated experiments were carried out to study the effect of temperature on

conidial viability of P. frequentans . Water suspension (5 mL) of fresh conidia (108

conidia mL�1) was dispensed in pre-heated glass tubes (6�/1 cm). Tubes were

maintained for 0.5, 1, 1.5 and 2 min in a hot water bath (wet heat) or in an incubator

(dry heat) at temperatures ranging from 20 to 1008C. Four tubes were prepared for

each time, temperature and method combination. Conidial viability was estimated by

measuring germination according to the bioassay described by De Cal et al. (1988)

258 B. Guijarro et al.

before and after heat treatment. Briefly, sterile glass slides were placed in glass 15-mm

diameter Petri dishes lined with moist paper. On each slide, a 15-mL droplet of diluted

conidial suspension of P. frequentans (106 conidia mL�1) was mixed with a 30-mL

droplet of sterile Czapek broth. Slides were incubated for 16 h at 20�/258C in

darkness, after which germination of 50 conidia was assessed in each replicate. Three

replicate drops were made for each sample, and the whole experiment was repeated

twice. A spore was considered to have germinated when a germ-tube was longer than

the length of the spore.

Effect of drying method on conidial viability

Freeze-drying. Two replicated experiments were carried out to determine the effect of

freeze-drying on viability of P. frequentans conidia. Eppendorf tubes (1.5 mL)

containing 0.5-mL suspensions of fresh conidia (106 conidia mL�1) were placed at

�/20 8C for 24 h. Several protectants were added to these suspensions: reconstituted

non-fat skimmed milk (NFSM) (Sveltesse, Nestle, Vevey, Switzerland) (10%, w/v);

NFSM (10%, w/v)�/Tween 20 (10%, v/v); NFSM (10%, w/v)�/peptone (10%,

w/v); NFSM (10%, w/v)�/sucrose (10%, w/v); NFSM (10%, w/v)�/glucose (10%, w/

v); NFSM (10%, w/v)�/glycerol (10%, v/v); NFSM (10%, w/v)�/starch (10%, w/

v);NFSM (10%, w/v)�/peptone (5%, w/v)�/starch (5%, w/v). Three 1.5-mL tubes

were used/protectant. After overnight storage in a freezer, samples were connected to a

freeze drier (Mod. CRYODOS �/808C, 230 V, 50 Hz with vacuum bomb 2G6, Telstar

S.A., Spain) operating at �/808C for 24 h. After freeze-drying, samples were stored at

room temperature. Viability of conidia was estimated just after being frozen, after

freeze-drying, and after 30, 60, 90, and 120 days of storage. Sterile distilled water

(5 mL) was added to the dried conidia, shaken in a laboratory mixer (MS 2

Minishaker Ika-Works, Inc., USA) operated at full speed for 1 min and incubated at

room temperature for 1 h. Conidial moisture content after freeze-drying was

determined with a humidity analyser (BOECKEL, GmbH �/Co, Hamburg,

Germany). Before the bioassay, conidial samples were resuspended in sterile distilled

water to give a concentration of 1�/106 conidia mL�1.

Spray-drying. The effect of spray-drying on viability of P. frequentans conidia was

determined. Fresh conidia, obtained as above, were resuspended in sterile distilled

water (50 mL) to obtain a concentration of 9.6�/108 mL�1 with or without

protectants (used also as carriers in this process), and then spray-dried in a

laboratory-scale spray-dryer (SD-05, Lab Plant, UK). Reconstituted non-fat skimmed

milk (NFSM) and MgSO4 were used as protectants and carriers, both at 10% (w/v).

Feed suspensions were delivered by a peristaltic pump at 200 mL min�1. Moisture in

the spray droplets produced by a jet nozzle (0.2 mm in diameter) was evaporated into

the drying chamber (0.2 m in diameter and 0.5 m long). The inlet temperature was

fixed at 1508C. The powder passed through a single cyclone separator and was

collected in a collector bottle. Recovered powder was measured by the difference

between the initial and the final weight of the collector bottle. Conidial viability was

determined by measuring germination utilising the bioassay described above. Before

the bioassay, conidia were resuspended in sterile distilled water to obtain concentra-

tions of 1�/106 conidia mL�1, shaken in a laboratory mixer (MS 2 Minishaker Ika-

Works, Inc., USA) operated at full speed for 1 min and incubated at room

temperature for 1 h.

Drying of Penicillium frequentans conidia 259

Fluid bed-drying. Two replicate experiments were carried out to determine the effect of

fluid bed-drying on viability of P. frequentans conidia. Fresh conidia, obtained as

described above, were resuspended in sterile distilled water and filtered through 1-mm

filter paper in a Buchner funnel. Two grams of this conidial paste was placed in four

tubes of a fluid bed dryer 350s (Burkard Manufacturing Co Ltd., Hertfordshire, UK)

for 20 min at the highest air-flow rate and a temperature range from 30 to 408C.

Samples were disaggregated during drying. After fluid bed-drying, samples were

stored at room temperature for 180 days. Conidial viability was estimated by

measuring their germination after fluid bed-drying, and at 30, 60, 90, 120, 150,

and 180 days of storage performing the bioassay described above. Before the bioassay,

conidia were resuspended in sterile distilled water to give a concentration of 106

conidia mL�1, shaken in a laboratory mixer at full speed for 1 min and incubated at

room temperature for 1 h. Conidial moisture content after fluid bed-drying was

determined with a humidity analyser as described before.

Effect of conidial moisture content after drying on conidial viability

Two replicated experiments were carried out to determine the effect of conidial

moisture content after fluid bed-drying on viability of P. frequentans conidia stored at

room temperature. Fresh conidia were resuspended in sterile distilled water and

filtered through 1-mm filter paper in a Buchner funnel. Samples of 5, 25, 50 and 100 g

of this conidial paste were placed in a fluid bed dryer at 408C, as described above, for

10, 20, 40, 60, 80, 100, 120, 160, 180, and 200 min. Three replicates were made for

each sample size and drying time combination. After fluid bed-drying, conidial

moisture content of each sample was measured using a humidity analyser. Each

sample of dried conidia was stored at room temperature. Viability of conidia was

estimated by measuring their germination just after fluid bed-drying, and at 30, 90,

180, and 360 days of storage at room temperature using the bioassay described above.

Before the bioassay, conidia were resuspended as described above.

Biocontrol efficacy of dry conidia

Two replicate experiments were conducted to determine the biocontrol efficacy of

fresh and dried conidia obtained by fluid bed-drying. Viability of these conidia was

tested as described above. Healthy peaches were surface sterilised by dipping in 1%

NaOCl for 5 min, followed by 70% ethanol for 1 min, and in sterile distilled water for

1 min as recommended by Sauer and Burroughs (1986). The surface of the peaches

was then dried by sterile air in a flow-cabinet for 2 h. Three artificial wounds of 1 mm

were made at separated locations on each fruit. Each peach was sprayed with 10 mL of

conidial suspension (106 conidia mL�1) of fresh or dried P. frequentans (69 and 73%

viability, respectively). Surfaces of peaches were dried again for 2 h by sterile air and

then 10 mL of conidial suspension of M. laxa (104 conidia mL�1, 86% viability) was

sprayed onto each peach. Fruits were incubated for 4�/7 days at 20�/258C under 99�/

100% RH in the darkness. Control treatments were: (i) fruits not inoculated with

M. laxa and treated with fresh or dried P. frequentans ; (ii) fruits inoculated

with M. laxa and not treated with P. frequentans ; (iii) fruits not inoculated with either

M. laxa or P. frequentans. Six peaches were used for each treatment. At the end of the

assay, disease incidence as percentage of rotten wounds caused by M. laxa in peaches

was recorded.

260 B. Guijarro et al.

Statistical analyses

Data were analysed by analysis of variance (ANOVA). Prior to analysis, data were

subjected to arc sin (arcsin), square root (sqrt), arc sin�/square root (arcsinsqrt), or

log10 transformation to improve homogeneity of variances. When the F-test was

significant at P�/0.05, means were compared by the Student�/Newman�/Keul’s

multiple range test (Snedecor & Cochram 1980). Since replicated experiments

yielded similar results, data from each assay was pooled and analysed. Conidial

viability for 0, 30, 90, 180, and 360 days of storage gave a viability progress curve, and

the area under this conidial viability progress curve for a year (AUCVPC) was

calculated as described for the area under the disease progress curve (AUDPC)

(Campbell & Madden 1990) using the formula:

AUCVPC�X n�1

i�1[(ti�1�ti)(vi�vi�1)=2]; (1)

where t is days of storage after drying, v is the percentage of conidial viability at each

estimation date and n is the number of estimations. The AUCVPC was calculated for

each time of drying within each sample conidial weight using an Excel spreadsheet.

The AUCVPC expressed the dynamic of conidial viability for 1 year of storage as a

single value and is useful to compare different viability dynamics. Multiple regression

analysis was carried out with (i) log10 conidial moisture content after drying (mc) on

time of drying (dt) and sample conidial weight (sd) conidial, and (ii) log10 AUCVPC

on sample conidial weight (sw) and conidial moisture content after drying (mc) on

time of drying (dt).

Results

Effect of temperature on conidial viability

Using wet heat (Figure 1A), viability of conidia maintained at 408C for 0.5�/2 min and

at 508C for 0.5 min was similar to those maintained at 208C. A reduction in conidial

viability was observed when temperature or time was increased (wet heat) (Figure

1A). However, when dry heat was used, viability of conidia maintained at 40�/1008Cfor 0.5 min was similar to those maintained at 208C. A reduction in conidia viability

was observed by exposure at 808C for 2 min, or 90 and 1008C for 1 min (Figure 1B).

Effect of drying method on conidial viability

Freeze-drying. Conidial viability was 85% before freeze-drying. Storage at �/208Covernight did not result in any reduction in conidial viability with or without

protectants (Figure 2). A significant reduction (78%) in conidial viability was

observed after freeze-drying without protectants or with NFSM�/glycerol. All of the

protectants used in this study maintained conidial viability of P. frequentans after

freeze-drying, except NFSM�/glycerol (0% viability after freeze-drying). Viability of

freeze-dried samples stored at room temperature was drastically reduced after 30 days

of storage at room temperature following freeze-drying. Conidial moisture content

after freeze-drying was in a range between 5.3 and 8.4%.

Spray-drying. All conidial suspensions of P. frequentans with or without protectants

(carriers) gave problems during the drying process, such as plugging in the jet nozzle

or sticking in the cyclone. Conidial viability was 95% before spray-drying and

Drying of Penicillium frequentans conidia 261

decreased to B/27% after spray-drying, especially with conidial suspensions contain-

ing protectants (carriers), where viability was B/1% (Table I). The outlet temperatures

were �/848C in all cases. The yield of the powered conidia recovered was B/44% in all

cases.

Fluid bed-drying. Conidial viability was 92% before the fluid bed-drying process.

Conidia of P. frequentans dried by fluid bed-drying maintained 100% viability after

7 days (Figure 3). No significant differences between temperatures used were shown.

Conidial moisture content after fluid bed-drying was 13%. Viability of fluid-bed-dried

samples stored at room temperature was reduced during storage; conidial viability

declined 24% after 15�/60 days of storage, 48% after 90 days, and 65% after 120�/180

days. Conidial viability after 180 days was 28%.

Effect of conidial moisture content after fluid bed-drying on conidial viability

Conidial viability after a year of storage is shown in Figure 4 (as AUCVPC) together

with conidial moisture content in relation to different drying times and different

sample weights. The highest AUCVPC after 1 year of storage was recorded for conidia

containing an initial moisture content �/5% and B/17, 12, 13, or 9% for 5-, 25-, 50-,

20 40 50 60 70 80 90 1000

20

40

60

80

100

120

% V

iabi

lity

% V

iabi

lity

(a)

**

***

* * ****

20 40 50 60 70 80 90 100Temperature °C

0

20

40

60

80

100

120(b)

*

* *

*

*

Figure 1. Percentage conidial viability of P. frequentans after 0.5 min ( ), 1 min ( ), 1.5 min ( ), and 2

min ( ) in wet heat (a) and in dry heat (b) at different temperatures. Columns with the same symbol

between each temperature are not significantly different according to the Student�/Newman�/Keul’s range

test. Data are the mean of eight replicates, with three drops/replicate.

262 B. Guijarro et al.

and 100-g samples, respectively (Figure 4). When sample weight increased, it was

necessary to dry samples for a longer time to obtain better conidial viability for a year

(Figure 4). Five grams of conidial paste was needed from 40 to 80 min of drying time

treatment to obtain a high AUCVPC, while more weighted samples needed �/80 and

B/200 min (Figure 4). The moisture content of freshly prepared conidial paste was up

to 65%. Moisture was eliminated from conidia quicker in smaller samples; B/10% was

obtained only after 40 min of drying for 5-g samples, while 80, 100 or 180 min of

drying was required for 25-, 50- or 100-g samples, respectively (Figure 4).

The area under the conidial viability progress curve (AUCVPC) was negatively

correlated to conidial moisture content after fluid bed-drying (mc) and to time of

drying (dt), and positively correlated to sample weight (sw) (R2�/41.67) (Figure 5A),

giving the following equation:

log AUCVPC�4:10-0:02mc-0:001dt�0:02sw: (2)

There was a statistically significant relationship between the variables at the 99%

confidence level (Figure 5A). Conidial moisture content after fluid bed-drying (mc)

0 Afterfreezing

0 30 60 90 120Days after drying

0

20

40

60

80

100

120

% V

iabi

lity

aa

bb

c

c

Figure 2. Percentage conidial viability of P. frequentans before and after freeze-drying and after storage at

room temperature. Conidia of P. frequentans were resuspended in water without protectants (m), with 10%

reconstituted non-fat skimmed milk alone (NFSM) ("), or with 10% NFSM plus other protectors such as

Tween 20 ('), peptone ( ), sucrose (X), glucose ( ), glycerol ( ), starch ( ) and peptone�/starch ( ).

Within each storage time, points with the same letter are not significantly according to the Student�/

Newman�/Keul’s range test. Data are the mean of six replicates/protector with three drops/replicate.

Table I. Recovered powder and viability of P. frequentans conidia after spray-drying using non-fat skimmed

milk (NFSM) (10%) and MgSO4 (10%) as carriers.a

Carriers % Viability Recovered powder (mg) Recovered powder (conidia mg �1)

None 26.79/4.05b 70 3.12�/106

NFSM 09/0 2940 7.2�/106

MgSO4 0.79/0.7 1500 10.5�/106

a50 mL of a conidial suspension of P. frequentans (9.6�/108 conidia mL �1) with or without carriers was

spray-dried in a laboratory-scale spray-dryer. bData are the mean of three replicates9/standard error of the

mean.

Drying of Penicillium frequentans conidia 263

was negatively correlated to time of drying (dt) and positively correlated to sample

weight (sw) (R2�/77.88) (Figure 5B), giving the equation:

log mc� 1:28-0:004dt�0:004sw (3)

There was a statistically significant relationship between the variables at the 99%

confidence level (Figure 5B).

Biocontrol efficacy of dried conidia

Peaches inoculated with M. laxa showed brown rot symptoms in the inoculated

wounds after 4�/7 days of incubation. Peaches uninoculated with M. laxa did not

show any symptoms. Fresh or dried P. frequentans conidial suspensions applied to

peaches before M. laxa inoculation significantly reduced (P�/0.05) the number of

rotten wounds (by 54 and 64%, respectively, Table II). No significant differences

between peaches treated with fresh or dried cells were found.

Discussion

P. frequentans conidia with a viability of 100% could be obtained after drying by fluid

bed- or freeze-drying, but protective additives were required to obtain this viability for

freeze-dried conidia. Similar results were obtained with other biocontrol fungi such as

E. nigrum (Larena et al. 2003a), or P. oxalicum (Larena et al. 2003b). Substances such

as polymers, sugars, albumin, milk, honey, polyols and amino acids have been tested

for their protective effect during freeze-drying (Font de Valdez et al. 1983;

Champagne et al. 1991). The protection obtained by a given additive during freeze-

drying depends on the species studied (Font de Valdez et al. 1983). Factors including

culture age, growth conditions, processing and dehydration conditions influence the

ability of microorganisms to survive freeze-drying (Champagne et al. 1991). Materials

0 20 40 60 80 100 120 140 160 180Days after drying

0

20

40

60

80

100

%V

iabi

lity

aa

bbc

bcd

cd

deef

f

Figure 3. Percentage conidial viability of P. frequentans after drying at 408C in a fluid bed-dryer. Means with

the same letter are not significantly different according to Student�/Newman�/Keul’s range test. Data are the

mean of eight replicates with three drops/replicate.

264 B. Guijarro et al.

are firstly frozen and dried in the lyophilisation process and, therefore, death of P.

frequentans conidia could occur at any of these steps. In the present study, death of

conidia occurred in the drying step (Figure 2). Conidia freeze-dried with the skimmed

milk mixture and other protectants (Tween 20, peptone, sucrose, glucose, starch and

peptone�/starch) had the same viability as conidia freeze-dried with skimmed milk

alone. Proteins present in milk provide a protective coat for the cells and seem to

restore injured cells during dehydration, avoiding osmotic shock, disruption and death

of cells (Champagne et al. 1991). Glycerol had a negative effect in the lyophilisation of

P. frequentans conidia. Glycerol is an effective cryoprotectant widely used in frozen

concentrates (Baumann & Reinbold 1964), but it causes problems with the

dehydration step. The internal water potential of fungal cells (Brown 1978) can be

reduced. Cells could be more tolerant to osmotic changes and, therefore, the presence

of glycerol would make the freeze-drying process. Pascual (1998) reported that an

enhanced accumulation of glycerol as a compatible solute in spores of E. nigrum did

not result in a higher survival rate after freeze-drying. Moreover, glycerol provided no

protection for cells of lactic acid bacteria (Font de Valdez et al. 1983), Candida sake

10 20 40 60 80 100 120 160 180 200Drying time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

% M

oisture content

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

% M

oisture content

0

5

10

15

20

AU

VC

PC

(x1

000)

10 20 40 60 80 100 120 160 180 200Drying time (min)

0

5

10

15

20

AU

VC

PC

(x1

000)

5 g 25 g

50 g 100 g

Figure 4. Area under the conidial viability progress curve for a year (AUCVPC) ( ) and conidial moisture

content ( ) after drying 5, 25, 50 or 100 g of P. frequentans conidia by fluid bed-drying during different

drying times.

Drying of Penicillium frequentans conidia 265

(Abadias et al. 2000), E. nigrum (Larena et al. 2003a), Penicillium oxalicum (Larena et

al. 2003b) and Pantoea agglomerans (Costa et al. 2000) in freeze-drying, and in

dehydration of Agrobacterium radiobacter and Rhizobium japonicum in polysaccharide

gels (Mugnier & Jung 1985).

Freeze-dried P. frequentans conidia did not maintain viability for a long time. After

30 days storage at room temperature, all the conidia lost their viability. However,

Figure 5. Relationships between area under the conidial viability progress curve (AUVCPC) of P.

frequentans with conidial moisture content after fluid bed-drying (mc), time of drying (dt), and sample

weight (sw) (A); and between P. frequentans conidial moisture content after fluid bed-drying (mc ) with time

of drying (dt), and sample weight (sw) (B). AUCVPC and mc were Log10 transformed. There was a

statistically significant (P�/0.05) relationship between the variables at 99% confidence level.

Table II. Effect of P. frequentans (Pf) on disease incidence caused by M. laxa (Ml) on peaches 4�/7 days

after inoculation.

Treatment Disease incidence (% rotten wounds)

Pf dried conidia treated�/Ml inoculated 229/9a b

Pf fresh conidia treated�/Ml inoculated 289/9 b

Pf untreated �/ Ml inoculated 619/11 a

Pf dried conidia treated�/Ml uninoculated 0

Pf fresh conidia treated�/Ml uninoculated 0

Pf untreated �/ Ml uninoculated 0

aData are the mean of 18 wounds9/standard error of the mean. Means followed by different letters differ

significantly at P�/ 0.05 by the Student�/Newman�/Keul’s range test.

266 B. Guijarro et al.

conidia of other fungi, such as P. oxalicum or E. nigrum, freeze-dried with skimmed

milk maintained 50 or 100% of their initial viability, respectively, after 30 days storage

at room temperature (Larena et al. 2003a,b). Microorganisms dried by freeze-drying

do not become totally inert and respiration should not be completely shut down

(Burges & Jones 1998). The observed loss in viability of dried P. frequentans conidia

after storage at room temperature could be due to temperature and/or the presence of

air in contact with the spores. Lyophilised microorganisms must be protected from

moisture and kept at low temperature to remain alive and active (Foster 1962). The

relationship between mortality and storage temperature is well known (Lievense &

Van’t Riet 1994). Many researchers have reported the effect of temperature and

package atmosphere on stability of storage-dried microorganisms (Champagne et al.

1991). Oxygen is thought to interact with the membranous system, causing damage to

the initiation of DNA synthesis (Israeli et al. 1975). However, commercial preparation

of biocontrol agents must be stable during storage at room temperature (Abadias et al.

2000). Beker and Rapoport (1987) suggested the addition of antioxidants, such as

thiourea, sorbitane esther, and some other additives to Saccharomyces cerevisiae cells

prior to their drying in order to increase the stability of dried cells during storage,

especially at room temperature.

Viability of P. frequentans conidia after spray-drying was B/27%. Death of conidia of

P. frequentans during spray-drying could be due to the high temperatures recorded.

High temperatures are devastating to microorganisms and non-thermostable enzymes

(Hutter et al. 1995). Viability of conidia of P. frequentans was reduced after a few

minutes at temperatures �/40 or �/708C in a water bath or an incubator, respectively.

Similar results were obtained with other fungi such as P. oxalicum and E. nigrum ;

conidial viabilities were reduced to 20 and 10%, respectively, after spray-drying

(Larena et al. 2003a,b). Nevertheless, spray-drying would be an economic alternative

for preserving conidia if a spray-drying apparatus with a high volume spray dryer were

used to reduce the inlet air temperature (Hutter et al. 1995). Low air temperature is

desirable for drying when working with heat-labile microorganisms (Hutter et al.

1995).

Viability of fluid bed-dried samples (2-g samples with 13% moisture content after

fluid bed-drying) after storage for 30 days at room temperature was �/75%. This

viability was significantly higher than those of freeze-dried conidia stored for the same

time. In addition, samples dried using freeze-drying did not survive after storage at

room temperature if they had moisture contents from 5.3 to 8.4%. In the case of fluid

bed-drying, samples of conidia with moisture contents of 13% had viabilities �/60%

after 30 days storage. This work demonstrated that the moisture content of conidia

after fluid bed-drying was critical in terms of viability with storage time. The conidial

moisture content of P. frequentans conidia needs to be reduced to 5�/15% for optimal

room temperature storage for 1 year. These values were slightly higher than those

obtained for Metarhizium flavoviride conidia, which were maintained between 4 and

5% (Moore et al. 1996). However, longevity of P. oxalicum conidia declined when

moisture content was below about 5%. As sterile water was directly added to dried

conidia, these spores may have suffered rehydration damage (Moore et al. 1997). This

damage could be avoided by exposing dried spores to a humid atmosphere for 30 min

before adding water. Nevertheless, P. frequentans conidia dried by fluid-bed-drying

maintained good viability after 30 days of storage, and moisture content should be

B/15%. Similar results were previously obtained with other biocontrol agents, such as

Drying of Penicillium frequentans conidia 267

P. oxalicum , and E. nigrum . E. nigrum conidia dried by fluid bed-drying maintained

100% viability for 90 days (Larena et al. 2003a). Similarly, P. oxalicum spores

maintained 40% viability (Larena et al. 2003b). The benefit of drying the conidia is

the reduction of the metabolic activity that minimises the loss of storage reserves and

the production of toxic metabolites (Baker & Henis 1990). Survival of bacteria such as

Rhizobium , Agrobacterium and Arthrobacter spp. was also related to water activity

(Mugnier & Jung 1985).

Fluid bed-drying operates at lower temperatures than spray-drying and consumes

less time and energy than the freeze-drying technique. Freeze-drying is an expensive

dehydration process because of the low drying rates, high capital and energy costs due

to refrigeration and vacuum units, directly dependent on the drying duration

(Lombrana & Villaran 1997). Using fluid bed-drying, we obtained the best conidial

survival without protectants and also the best survival after storage. P. frequentans

conidia dried by fluid bed-drying were as efficient as fresh conidia in controlling

brown rot of peaches. P. frequentans is a promising biological control agent to both

brown rot and peach twig blight (Melgarejo et al. 1986; De Cal et al. 1990, 2002).

Research is in progress to obtain an improved shelf life of P. frequentans conidia dried

by fluid bed-drying using different storage conditions and some protectant additives,

which will enhance and stabilise the performance of P. frequentans in a formulated

product.

Acknowledgements

This research was supported by Project AGL2002-04396-CO2-O1 (Plan Nacional de

I�/D�/I, Ministerio de Educacion y Ciencia, Spain). We thank the Postharvest

Laboratory of IRTA (Lerida, Spain) for kindly providing their laboratory-scale spray

dryer. B. Guijarro received a fellowship from the Ministerio de Educacion y Ciencia

(Spain).

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