Germination of Fungal Conidia After Exposure to Low Concentration

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Postharvest Biology and Technology 83 (2013) 22–26

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Germination of fungal conidia after exposure to low concentration

ozone atmospheres

a b c,∗

Zilﬁna Rubio Ames , Erica Feliziani , Joseph L. Smilanick

Kearney Agricultural Center, University of California, 9240 South Riverbend Avenue, Parlier, CA 93648, USA

Department of Environmental and Crop Science, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona, Italy

USDA-ARS San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Avenue, Parlier, CA 93648, USA

a r t i c l e i n f o a b s t r a c t

Article history: The germinability of conidia of Alternaria alternata, Aspergillus ﬂavus, Aspergillus niger, Penicillium dig-

Received 13 July 2012

itatum, Penicillium expansum, or Penicillium italicum was determined periodically during exposure for

Accepted 11 March 2013 ◦

approximately 100 d to a humid atmosphere of air alone or with 150 nL/L ozone at 2 C. Conidia were

exposed on glass coverslips that were removed from chambers at intervals of one week and the ger-

Keywords:

mination of 100 conidia of each species was assessed after incubation for 24 h on potato dextrose agar.

Ozone

The period in d when 50% or 95% (ET50 and ET95, respectively) could not germinate and 95% conﬁdence

Alternaria alternata

intervals for these estimates were made using Finney’s probit analysis. ET50 and ET95 estimates were

Aspergillus ﬂavus

approximately one month and two to three months, respectively. Some natural mortality of the conidia

Aspergillus niger

occurred during these periods, so the entire decline in germinability was not solely due to ozone. The

Penicillium digitatum

Penicillium expansum age of the culture from which conidia were collected inﬂuenced their susceptibility to ozone. Conidia

Penicillium italicum were harvested from 7, 14, 21, and 28 d old potato dextrose agar cultures of P. digitatum and exposed to

◦

13,000 nL/L ozone at 2 C. After 48 h of exposure to ozone, none of the conidia from the seven-day old

culture germinated, while 30–35% of conidia from 14, 21, or 28 d in age cultures germinated.

Published by Elsevier B.V.

1. Introduction decay lesions caused by Penicillium spp. (Metzger et al., 2007; Palou

et al., 2003). Nothing about the mortality of the conidia of the citrus

Postharvest decay pathogens of fresh fruit and vegetables are pathogens was reported.

primarily ﬁlamentous fungi that reproduce by the production of In vitro studies revealed that ozone treatment suppressed

air borne conidia. The constant use of ozone in low concentrations the germination of conidia, with the degree of inhibitory action

during storage has been evaluated and showed some promise to dependent on the concentration and duration of exposure to ozone

extend storage life of several products by the control of these fungi (Tzortzakis et al., 2007). However, reports deﬁning ozone doses that

(Karaca and Velioglu, 2007; Palou et al., 2006; Smilanick et al., 2012; kill conidia of postharvest fungal pathogens are few, their ﬁnd-

Tiwari and Muthukumarappan, 2012). Sanitation of fruit surfaces ings vary widely, and few were conducted at cold temperatures

with ozone in air has been reported, but doses of ozone that kill and under humid conditions (Palou et al., 2006). Relative humidity

postharvest decay fungi in a few hours are very high (Ozkan et al., has a large effect on the sensitivity of conidia to ozone gas. Ozkan

2011), and to use them requires very ozone-tolerant commodi- et al. (2011) reported the concentration of ozone that killed 99%

ties because of the high risk of phytotoxicity. Ozone is harmful to of the conidia of Penicillium digitatum in 1 h decreased markedly,

␮

humans if even brief exposure occurs to these high concentrations from 11,410 to 817 L/L (parts per million), when the humidity

(Rice, 2012), so the use of constant, low concentrations of 100 to was increased from 35 to 95%. Tzortzakis et al. (2007) reported

◦

300 nL/L (parts per billion) is used to reduce this hazard to workers exposure to a constant concentration of 200 nL/L ozone at 13 C and

and the size of the ozone generation equipment needed. Storage 95% relative humidity reduced viability of conidia of Colletotrichum

under ozone gas at a concentration of 300 nL/L reduced or delayed coccodes by more than 90% after eight hours, while this concentra-

the natural decay of a variety of citrus fruit, destroyed ethylene in tion had no effect on germinability of conidia of Alternaria alternata

the storage room air, and retarded the production of conidia from after up to 72 h, the longest exposure evaluated. This concentration

of ozone reduced conidial production of A. alternata by about 50%

after 10–14 d, while that of C. coccodes was unchanged. Antony-

∗ Babu and Singleton (2009) compared the inﬂuence of a sustained

Corresponding author. Tel.: +1 559 596 2810; fax: +1 559 596 2777.

ozone atmosphere (200 nL/L) on the growth, sporulation, and via-

E-mail addresses: [email protected], [email protected]

(J.L. Smilanick). bility of conidia of Aspergillus nidulans and Aspergillus orchraceus at

0925-5214/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.postharvbio.2013.03.008

Z.R. Ames et al. / Postharvest Biology and Technology 83 (2013) 22–26 23

◦

18 C. A. nidulans cultured in ozone did not produce conidia, while Fremont, CA) and into a nurse tank (42.6 L), then into a distribution

A. orcharceus did, although at a much lower rate than in air. This manifold with needle valves and ﬂow meters that independently

concentration caused a reduction in conidial germination of both controlled the rate of ﬂow into a stainless steel chamber (116.8 L).

species by about 15% after 8 d. The gas stream was humidiﬁed by passage through water before it

Our goal was to determine the inﬂuence of ozone gas at a entered the chamber, and the high humidity (approximately 95%

constant concentration of 150 nL/L on the germinability of coni- RH) inside the chamber was conﬁrmed with an electronic relative

dia of A. alternata, Aspergillus ﬂavus, Aspergillus niger, P. digitatum, humidity sensor (Acurite model 359). Ozone concentration was

◦

Penicillium expansum, or Penicillium italicum at 2 C under humid conﬁrmed and monitored every 30 min with a UV ozone moni-

conditions. In a second study, we evaluated the effect of colony age tor (Model 465, Advanced Pollution Instrumentation Systems, San

on the sensitivity of conidia of P. digitatum to ozone at a higher Diego, CA).

concentration (13,000 nL/L).

2.3. Germination of conidia

2. Materials and methods

Following exposure to air or ozone, the cover glasses were

␮

2.1. Culture and preparation of conidia placed in a previously sterilized glass test tube. A volume of 100 L

of Triton X-100 was added and the tube was vortexed for 10 s.

␮

P. digitatum and P. italicum were isolated from infected lemons, Aliquots of 10 L of the conidial suspension were plated on PDA,

Penicillium expansum from an infected apple, and A. ﬂavus, A. niger, the conidial suspension dried on the PDA surface, the dishes were

◦

and A. alternata from contaminated almonds. All isolates originated closed and incubated for 18 to 24 h at 25 C, then 100 conidia were

from colonies initiated by a single-conidium and they were stored examined with a light microscope and the number of germinated

on silica gel. All were cultured on potato dextrose agar (Difco Lab- conidia were recorded.

oratories, Detroit, MI) for 2 weeks in a cycle consisting of 12 h

◦

in light (cool white ﬂuorescent) and 12 h in darkness at 20 C.

2.4. Statistical analysis

In experiments where the inﬂuence of colony age on the sensi-

tivity of conidia of P. digitatum to ozone gas was examined, the

The time when 50% and 95% mortality was estimated with 95%

fungus was cultured for 7, 14, 21, or 28 d before conidia were har-

conﬁdence intervals was determined using Finney’s probit analysis

vested. Conidia were rubbed from the media surface with a glass

(Finney, 1971; SPSS ver. 20, IBM Corporation, Armonk, NY) applied

rod after adding 2 mL of Triton X-100 (0.1% w/v). Conidia were ﬁl-

to the number of conidia that could germinate, the total number

tered through at two layers of cheese cloth to remove mycelial

examined, and the time in hours or d of exposure to air or ozone.

fragments. A volume of approximately 0.1 mL of dense (approx-

−7 Germination data from two experiments were used.

imately 10 conidia/mL) suspensions of conidia were dispersed

onto one side of a cover glass (18 mm × 18 mm, Corning Inc., Corn-

ing, NY, USA) with a compressed air sprayer (model DB-32, Paasche 3. Results

Airbrush Co., Harwood Heights, IL) operating at 2.8 × 10 Pa to dis-

tribute the conidia evenly over the entire surface. Conidia were 3.1. Conidial germination after exposure to air or ozone

mostly deposited in a single layer and clumping of conidia was

minimal. Two identical 150 mm × 15 mm Petri dishes (Fisher Sci- The mortality of conidia in the low ozone concentration atmo-

entiﬁc, Pittsburgh, PA) with twenty cover glasses per fungus were sphere was slow and began after two to three weeks of ozone

prepared. The plates, without lids, were inverted and placed in exposure (Fig. 1). None of the conidia exposed to 150 nL/L ozone

◦ ◦

the ozone or air (control) atmospheres at 2 C and high humidity at 2 C for 100 d could germinate. The rank of ozone tolerance,

(approximately 95% RH) that was measured with an electronic rel- as determined the ET50 values, with the most tolerant stated ﬁrst,

ative humidity sensor (Acurite model 359, Chaney Instrument Co., were A. ﬂavus > P. italicum > A. niger > P. expansum > P. digitatum > A.

Lake Geneva, WI). All experiments were repeated twice. alternata (Table 1). The ranks of ozone tolerance, as determined

the ET95 values, with the most tolerant stated ﬁrst, were A. alter-

nata > A. ﬂavus > P. italicum > A. niger > P. expansum > P. digitatum.

2.2. Ozone system

Some reduction in germination also occurred in air, but a large per-

centage of the conidia exposed to air could germinate at the end of

Two ozone systems were used. For the long-term exposure of

the experiment. After exposure to air for 100 d, conidia of P. digita-

the conidia to a relatively low concentration of ozone (150 nL/L

tum, A. alternata, and A. niger declined to about 50% of their initial

or 150 parts per billion), a commercial corona discharge ozone

germination percentages, while germination of the conidia of P.

generator system (model 4900, Purfresh, Inc., Fremont, CA) was

italicum, P. expansum, and A. ﬂavus was about 80% of their initial

installed on an environmental chamber. The generator concen-

percentages (Fig. 1).

trated oxygen from air, dried the air, passed it through an air-cooled

corona plates, and passed the ozone gas through Teﬂon lines to the

ozonated chamber. The dry oxygen produced by the system that 3.2. Inﬂuence of culture age on conidial sensitivity to ozone

was passed into the corona plates reduced the production of inter-

fering nitrogen radicals and nitric acid (Tapp and Rice, 2012). Ozone The age of the culture inﬂuenced the sensitivity of conidia to

concentration was monitored with an electrochemical detector ozone; conidia from colonies cultured for 7-day old cultures were

that controlled the ozone generator output. Ozone concentrations markedly more sensitive to ozone than those from older cultures

◦

were conﬁrmed and monitored weekly with a UV ozone moni- (Fig. 2). After 24 h of exposure to ozone at 13,000 nL/L at 2 C ger-

tor (Model 465, Advanced Pollution Instrumentation Systems, San mination of conidia from the 7-day old culture declined to 20%,

Diego, CA). For the short term exposure of conidia to a relatively while those from the 14-, 21-, and 28-day old cultures germinated

high concentration of ozone (13,000 nL/L or 13 parts per million), at 40, 70, and 60%, respectively. After 48 h of exposure to ozone,

air was dried by passage through a 1.4-m long by 6 cm wide column none of the conidia from the 7-day old culture could germinate,

ﬁlled with anhydrous calcium sulfate (W.A. Hammond Drierite Co., while 30–35% of the conidia from cultures 14, 21, or 28 d in age

Xenia, OH), passed through a corona discharge generator (Purfresh, germinated. None could germinate after 72 h exposure to ozone.

24 Z.R. Ames et al. / Postharvest Biology and Technology 83 (2013) 22–26

Fig. 1. Germination of conidia of Alternaria alternata, Aspergillus ﬂavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, or Penicillium italicum after exposure in

◦

d to atmospheres of air () or 150 nL/L ozone () at 2 C.

Table 1

Germinability of conidia of Alternaria alternata, Aspergillus ﬂavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, or Penicillium italicum after exposure in days

◦

to an atmosphere of 150 nL/L ozone at 2 C.

Fungus Days in ozone at 150 nL/L

ET50 (±95% CI) ET95 (±95% CI)

Alternaria alternata 18.7 (14.0, 22.3) 107.3 (99.3, 117.3)

Aspergillus ﬂavus 43.9 (41.5, 46.3) 86.8 (82.4, 92.0)

Aspergillus niger 36.2 (29.4, 42.9) 74.5 (64.3, 91.1)

Penicillium digitatum 21.7 (21.1, 30.7) 54.0 (46.2, 67.2)

Penicillium expansum 23.0 (18.4, 27.3) 69.5 (62.4, 79.1)

Penicillium italicum 42.9 (38.8, 47.0) 79.4 (72.7, 88.3)

Values indicate the period in d when 50% (ET50) or 95% (ET95) of the conidia could not germinate, followed by the 95% conﬁdence interval (CI). Values were estimated

using Finney’s probit analysis. Conidia, exposed to ozone on cover glasses, were removed from the ozone chamber at intervals of one week and the germination of 100 conidia

was assessed after incubation for 24 h on potato dextrose agar.

The germination of conidia after exposure to air for these periods Low concentrations of ozone gas have been applied to oxidize

was unchanged. ethylene gas to retard the accelerated ripening it causes, to reduce

worker exposure to ozone and the risk of phytotoxicity or other

harm to treated products, to minimize corrosiveness to rooms and

4. Discussion refrigeration coils compared to that caused by very high concentra-

tions of ozone or sulfur dioxide, and to facilitate the use of smaller

The germinability of conidia of the fungi exposed to constant ozone generators to reduce capital and operating costs (Palou et al.,

◦

low concentrations of ozone at 2 C with high humidity declined 2006). It has been amply demonstrated that ozone at low concen-

over a period of weeks; complete mortality did not occur until trations will alter, and usually decrease, the conidial production of

after two months or more. These results predict the rate of mor- many fungi (Antony-Babu and Singleton, 2009; Palou et al., 2002;

tality that could be expected to occur when ozone is used at low Tzortzakis et al., 2008). This is one of few studies that examined the

concentrations during the storage of horticultural products. rates of conidial mortality of common postharvest decay pathogens

Z.R. Ames et al. / Postharvest Biology and Technology 83 (2013) 22–26 25

of culture age on the sensitivity of A. niger and A. ﬂavus to ozone gas.

Cultures of A. ﬂavus became increasingly tolerant to ozone from

age 3 d to 9 d, while those of A. niger were unchanged and equally

sensitive at these ages.

5. Conclusions

Conidia of A. alternata, A. ﬂavus, A. niger, P. digitatum, P. expansum,

or P. italicum were exposed to ozone gas at 150 nL/L for 100 d at

◦

2 C. In the ozone atmosphere, about 50% of the conidia did not

germinate after 3–4 weeks, while 95% did not germinate after 2–3

months. In air, some mortality also occurred during these periods,

but 50% or more was alive after 3 months. It is probable that the

mode of action of low concentrations of ozone applied to control

fungi on stored products is not mortality of their conidia. Conidia

from older colonies of P. digitatum were more tolerant to ozone

than those from younger colonies.

Fig. 2. Inﬂuence of the age of the culture of Penicillium digitatum on the susceptibility

◦

of conidia to inhibition by 13,000 nL/L of ozone gas at 2 C.

Acknowledgements

We thank Kent Fjeld for the design, fabrication, and opera-

under conditions similar to those of commercial storage. Because tion of the high concentration ozone system, Dennis Margosan

mortality of conidia occurs relatively slowly, it is likely mortal- for help with cultures and germination assays, ﬁnancial support

ity of conidia alone is not the principle mechanism of ozone to of California Citrus Research Board and California Table Grape

control diseases caused by these fungi. In studies that reported Commission, Ozone International for loan of equipment and its

beneﬁts in storage life resulting from the use of ozone gas, sec- maintenance, Luciana Cerioni for her technical help, and Mary Lu

ondary effects (Palou et al., 2003; Metzger et al., 2007), such as Arpaia for administrative support.

retarded senescence of fruit or vegetables in storage caused by

the control of ethylene, a reduction in air-borne inoculum within

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