Screening of several disinfectants to assess their efficacy in controlling mycelia growth, sporangia germination, and recovery of viable Phytophthora ramorum

Crop Protection 42 (2012) 186e192

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Screening of several disinfectants to assess their efficacy in controlling mycelia growth, sporangia germination, and recovery of viable Phytophthora ramorumq Delano James a, *, Aniko Varga a, Elisa Becker b, Grace Sumampong b, Karen Bailey c, Marianne Elliott d, Saad Masri a, Simon F. Shamoun b a

Sidney Laboratory, Canadian Food Inspection Agency, 8801 East Saanich Rd., Sidney BC, Canada, V8L 1H3 Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC, Canada, V8Z 1M5 Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada, S7N 0X2 d Puyallup Research and Extension Center, Washington State University, Puyallup, WA 98371, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2012 Received in revised form 11 July 2012 Accepted 15 July 2012

Phytophthora ramorum is the causal agent of sudden oak death, a very serious disease that causes dieback and death of several important forest species in the USA. It was recently associated with a destructive disease of larch in the United Kingdom. The pathogen has a wide host range including ornamentals such as Rhododendron and Viburnum, and has been recovered from nursery soil samples. Diligent use of effective disinfectants in nurseries can contribute to integrated management strategies for P. ramorum. Nine disinfectants, some at different concentrations and duration, were evaluated in vitro and in vivo for their efficacy in controlling the growth of P. ramorum mycelia and the germination of sporangia. ChemprocideÒ (1.35%), PerCeptÔ (diluted 1:16) and 15% bleach treatments were the most consistent in their effectiveness for inhibiting both mycelia growth and sporangia germination. ChemprocideÒ (1.35%) and 10% bleach treatments were effective also for preventing P. ramorum recovery from contaminated plastic plant saucers and metal surfaces. Isolates representing the three clonal lineages (EU1, NA1, and NA2) of P. ramorum were included in all studies. Interestingly, 10 min treatments of ethanol (70% or 95%) were effective in preventing colony forming unit (CFU) growth, but were not as effective for preventing mycelia growth. Ethanol treatments for 5 min were among the least effective, and in every case was not significantly different from the water-treated controls. Concentration and treatment time were critical factors influencing efficacy in these studies. Differences in CFU production were observed among the clonal lineages with a high of 18
1.19 (n ¼ 66) for EU1 isolates, and a low of 9.0
0.89 (n ¼ 74) for NA2 isolates. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.

Keywords: Phytophthora ramorum Disinfectants Sporangia germination Mycelia growth Colony forming unit (CFU) Oomycete recovery

1. Introduction Phytophthora ramorum (S. Werres, A.W.A.M. de Cock & W.A. Man in’t Veld) has been identified as the causal agent of sudden oak death (SOD). SOD is a serious and important disease that has killed thousands of trees in California, USA (Garbelotto et al., 2001; Rizzo et al., 2002; Dart et al., 2007) and has been described as reaching epidemic proportions (Rizzo et al., 2002). P. ramorum has a very wide host range that includes many ornamental and forest species of ecological, economical and cultural significance (Rizzo et al.,

q Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the Canadian Food Inspection Agency, Agriculture & Agri-Food Canada, or the Canadian Forest Service. * Corresponding author. Tel.: þ1 250 363 6650x235; fax: þ1 250 363 6661. E-mail address: [email protected] (D. James).

2005; Grünwald et al., 2008a). Recently the pathogen has been associated with a destructive disease of larch in the UK, identified as sudden larch death (Brasier and Webber, 2010). The pathogen has been found in nurseries infecting a range of ornamental plants including various cultivars of Rhododendron and Viburnum (Garbelotto et al., 2001; Werres et al., 2001; Werres and Kaminski, 2005). P. ramorum has been recovered from soil samples in ornamental nurseries (Dart et al., 2007). Nursery stock is moved between regions of a country, and between countries (Tjosvold et al., 2009). It is essential therefore that attempts at controlling the spread of P. ramorum include control of the pathogen in nurseries, since it is an important source of inoculum (Dart et al., 2007; Grünwald et al., 2008b). Three distinct clonal lineages (EU1, NA1 and NA2) have been identified (Ivors et al., 2006; Grünwald et al., 2008a, 2009; Elliott et al., 2009). They differ in their growth rate, aggressiveness, and distribution. The NA2 lineage is distinct from the EU1 and NA1

0261-2194/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2012.07.007

D. James et al. / Crop Protection 42 (2012) 186e192

lineages, and to date has been found only in North American nurseries. P. ramorum also produces two types of asexual propagules; sporangia and chlamydospores (Grünwald et al., 2008b). The sporangia can germinate directly, or under the right conditions release swimming zoospores. The sporangia and chlamydospores of Phytophthora species are associated with distribution and/or long term survival (Hwang and Ko, 1978; Linderman and Davis, 2006). Linderman and Davis (2006) found that sporangia and chlamydospores can survive (in media or soil) for 6 months and 12 months, respectively, with the potential for spread in potting media. Chemical treatments, including fungicides and disinfectants, are important components of integrated pest management practices aimed at controlling and limiting the spread of P. ramorum in nurseries (Garbelotto et al., 2001; Linderman and Davis, 2008; Tjosvold et al., 2008; Garbelotto et al., 2009). Disinfectants are used commonly to decontaminate surfaces and equipment for pathogen control (Tomasino, 2005; Cheah et al., 2009). The active ingredients of most disinfectants belong to a few classes of chemicals that include; hypochlorites, quaternary ammonium compounds, phenolics, iodines and alcohols (PHAC, 2004). The effectiveness of a disinfectant can be influenced by a number of factors including concentration, contact time, chemical/mode of action, presence of organic matter and the target organism (Best et al., 1990; Gehr et al., 2003). In this study, a range of disinfectants (including some that contain hypochlorite, quaternary ammonium compounds, or alcohol) were analysed for their efficacy in controlling germination and growth of P. ramorum. The term spore (Judelson and Blanco, 2005) or colony forming unit (CFU) is used to measure survival and/or dispersal structures. In vitro assay systems were used for the initial evaluation of the disinfectants. Despite quarantine restrictions preventing actual field testing, real-world simulations were conducted using in vivo experiments. These were designed to evaluate the recovery of P. ramorum from surfaces commonly encountered in nurseries, in order to provide recommendations to local nurseries who may unintentionally acquire the disease. 2. Materials and methods 2.1. P. ramorum isolates Isolates representing the three known clonal lineages of P. ramorum (Ivors et al., 2006; Elliott et al., 2009; Grünwald et al., 2009) were included in the study (Table 1). The isolates were maintained under quarantine in three laboratories: Canadian Food Inspection Agency (CFIA), Sidney Laboratory, Sidney BC; Canadian Forest Service, Pacific Forestry Centre, Victoria BC; and Agriculture & Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK. 2.2. Culture conditions for mycelia production All cultures were grown on 15% V8 media amended with 1% CaCO3 in Petri dishes. The cultures were maintained at 20  C in the dark. Seven mm (#4 cork borer) plugs of P. ramorum cultures were excised from the leading edge of 2 week-old cultures and used for evaluation of the various disinfectants for inhibiting the growth of P. ramorum. 2.3. Experimental design and treatments of mycelia plugs Three isolates of each of the three clonal lineages of P. ramorum were included and evaluated (Table 1, isolates used in this study are indicated). The complete experiment was done twice, Trial 1 and

187

Table 1 Isolates of Phytophthora ramorum that were used in this study and which represent the three clonal lineages.a Genotype Isolate

Original ID

EU1 NA1 NA1 NA1 NA2 NA2 NA2

MSOD03-0002b MSOD030002 PFC-5086c CSL 2268 CSL2266 PFC-5084b,c BBA9/95 PFC 5039b,c PR-04-002 b,c PR-04-001 PFC 5038 b,c PR-03-001 PFC 5046 b,c PR-04-007 PFC 5054 SOD05-11007b SOD05-11077 SOD05-16207b SOD05-16207 SOD05-17017b SOD05-17017

NA2 NA2

PFC-5063c PFC-5073c

WSDA 3765 RHCC 4

NA2

PFC-5074c

RHCC 23

EU1 EU1 EU1

Host

Origin

Rhododendron sp. cv. Holland Vulcan Rhododendron grandiflora UK R. catawbiense Germany Viburnum plicatum Lithocarpus densiflora L. densiflorus Pieris japonica Magnolia sp. Prunus lusitanica cv. Lolita Camellia sinensis var. Teabreeze Rhododendron, Colonel Coen cultivar Rhododendron, Colonel Coen cultivar

USA USA USA USA Canada Canada Canada USA USA USA

a See Ivors et al., 2006; Grünwald et al., 2008b; Elliott et al., 2009; Grünwald et al., 2009 for descriptions of the three lineages. b Isolates used in the mycelia plug experiments. c Isolates used in the sporangia treatments.

Trial 2. In each experiment, three plates per treatment were evaluated. Table 2 provides details including active agents of the various disinfectants. The concentrations used are for the most part that recommended by the manufacturer. Untreated controls were included with each treatment for direct comparison. The Hand Sanitizer treatment was not included in Trial 2, because no inhibition was observed in Trial 1. The inhibition of growth was studied as follows: All plugs were soaked in disinfectant solution for 10 min, except PerCeptÔ for 5 min, and the 5 min ethanol treatments. The plug, mycelia side down, was streaked across ½ of a Petri dish containing 15% V8 media amended with 1% CaCO3, to assess CFU growth. The plug was then placed on the other ½ of the Petri dish, mycelia side down, to assess radial mycelia growth. Controls were included for each treatment and consisted of plates that were streaked with untreated plugs (water only) across ½ of the plate, while the untreated plug was placed on the other ½ of the Petri dish, mycelia side down. The treated and untreated cultures were observed at regular intervals over a two-week period. A total of 117 (3  39) plates each were assessed for EU1, NA1, and EU1 isolates in Trial 1; and in Trial 2 a total of 114 (3  38) plates were assessed for each clonal lineage. It is assumed that the CFUs may have resulted from mycelia fragments, chlamydospores, sporangia, or any segment capable of initiating colony development. In preliminary studies 7 mm plugs of P. ramorum cultures were examined by light microscopy. Both sporangia and chlamydospores were observed, with the latter in much greater abundance (data not shown). 2.4. Data analysis of mycelia plug treatments Data were screened for normality using the ShapiroeWilk test, and for homogeneity of variance using the F-test for variances. Data from repeated trials were combined when the F-test was not significant. Statistical analysis was carried out using SPSS v. 10.0 (SPSS, Inc. Chicago, IL., USA). A chi-squared test was performed to examine differences between treatments for radial growth data followed by a modified Tukey multiple comparison test for proportions (Zar, 1999). Differences among treatments for CFU data were evaluated using one-way ANOVA on square root transformed

188

D. James et al. / Crop Protection 42 (2012) 186e192

Table 2 List of disinfectants. Disinfectants

Recommended concentration

Exposure time

Composition

Producer

Notes

Chemprocide

8.0 ml/L

10 min

Pace Chemicals, Burnaby

Fungicide, bactericide

Chemprocide

13.5 ml/L

10 min

Pace Chemicals, Burnaby

Fungicide, bactericide

Ethanol Ethanol Hyperox Hyperox Javex Liquid Bleach Javex Liquid Bleach SMTS Part A

75% 95% 1:256 dilution 1:128 dilution 5%

5 min, 10 min 5 min, 10 min 10 min 10 min 10 min

Didecyl dimethyl ammonium chloride 7.5%; isopropyl alcohol 10%; ethanol 1.5% Didecyl dimethyl ammonium chloride 7.5%; isopropyl alcohol 10%; ethanol 1.5% Ethanol and 25% water Ethanol Peracetic acid 5%, Hydrogen peroxide 10e30% Peracetic acid 5%, Hydrogen peroxide 10e30% Sodium Hypochlorite (3e7%)

15%

10 min

Sodium Hypochlorite (3e7%)

Neat (undiluted)

10 min

Quaternary ammonium formulation

PerCept

1:16 dilution

5 min

Hand Sanitizer Virkon

as dispensed 1% w/v solution

5 min 10 min

Virucidal Extra

1% solution

10 min

Hydrogen peroxide (5e10% weight); Phosphoric acid (1e5%); Arylsulfonated sodium salts (0.5e1.5%); 1-Hydroxyethylidene-1,1-diphosphonic acid (3e7%); Dodecyl-benzenesulfonic acid (1e5%) ethyl alcohol (60e62%) Potassium peroxomonosulfate (30e60% weight); Sulfamic acid (3e7%); Benzenesulfonic acid and alkyl derivatices, sodium salts (7e13%); Sodium toluenesulfonate (1e5%); Malic acid (7e13%); Dipotassium persulfate (1e3%) Potassium monopersulphate (23%), Sodium dichloro-s-triazinetrione (5%) (also contains Persulfate, Chlorinated isocyanurate, and Sulfamic acid, dye and fragrance)

data, followed by Dunnett’s T3 multiple comparison test. For all tests the probability level used was P  0.01. 2.5. P. ramorum sporangia isolation for microplate assays Isolates of each of the three clonal lineages were included in the microplate assays. This was based on the microbioassay described by Kuhajek et al. (2003), except that sporangia were used instead of zoospores. Preliminary growth experiments were conducted to identify the optimal concentrations of sporangia and the timing of growth in synthetic Roswell Park Memorial Institute (RPMI) media, using measurements of relative optical density at 650 nm. Cultures for sporangia isolation were initiated using an agar plug (seven mm, #4 cork borer) of P. ramorum grown on V8 media in 60 mm Petri dishes. The plugs were used to inoculate 10 ml aliquots of V8 broth, grown for 4e6 days in the dark at 20  C, and then washed twice with sterile distilled water. The media was replaced with sterile water and the cultures were incubated for a further 2e4 days before harvesting of the sporangia. Cultures were poured into 50 ml Falcon tubes, shaken to dislodge the sporangia, filtered through sterile cheesecloth into a sterile beaker, then if required, concentrated by centrifugation at 115  g for 20 min. The supernatant was removed without disturbing the sporangia and the remaining liquid, approximately 5 ml, was used to determine sporangia concentration using a haemocytometer. Sporangia concentration was adjusted to approximately 1.0  105 sporangia/ ml. For the timed exposures, 100 ml of a 2 concentrated solution of each disinfectant was mixed with an equal volume of sporangia, then diluted 10 min after with 10 ml of sterile water, a 50 fold dilution. Sporangia of each of nine P. ramorum isolates were exposed to each of five disinfectant treatments; ChemprocideÒ (13.5 ml/100 ml), HyperoxÒ (1:128), 1% VirkonÒ, 15% bleach and 5% bleach. Controls included a water treatment as

Vetoquinol N.-A, Inc Vetoquinol N.-A, Inc Clorox Company of Canada, Ltd. Clorox Company of Canada, Ltd. Mold Solutions LLC., FL

Disinfectant Disinfectant Germicide Germicide Disinfectant Disinfectant

Virox Technologies Inc.

Disinfectant, cleaner, mildewstat, fungicide, sanitizer, virucide, and deodorizer virucide, disinfectant

Vetoquinol N.-A, Inc

sanitizer disinfectant

Can-Vet Animal Health Supples Ltd

bactericide, virucide, fungicide

an untreated control, and uninoculated RPMI media as contamination controls. After treatment, 100 ml of sporangia at a concentration of 1.0  103 sporangia/ml was plated in each well, with100 ml RPMI media in each of 9 wells per treatment per isolate combination. The relative absorbance of the cultures at wavelength 650 nm was recorded using a microplate reader, with the initial reading taken at time zero, then every 24 h for 3 days. A total of four replicate experiments were performed. 2.6. Data analysis of sporangia isolation and in vitro treatments One-way ANOVA using SigmaStat was performed to identify any differences between replicates. Replicates 1, 3 and 4 were not significantly different and were combined and two separate oneway ANOVA were carried out to analyse genotype and treatment effects. The data were not normal so a KruskaleWallis ANOVA on Ranks was performed using the untransformed data. A multiple comparison test (Dunn’s Test) was performed where significant differences were identified. For all tests, the probability level used was P < 0.05. 2.7. In vivo disinfection of P. ramorum on plastic and metal surfaces Based on the results from in vitro testing, three disinfectants were evaluated for their ability to kill sporangia of P. ramorum on plastic and metal surfaces which are typically encountered as plant pots and trays or tools and bench tops, respectively, under greenhouse and nursery conditions. The disinfectants were 5% JavexÒ Liquid bleach, 10% JavexÒ Liquid bleach and 1.35% ChemprocideÒ. Disinfectants were compared to water controls. JavexÒ Liquid bleach was not tested at 15% because it was too corrosive for the metal surfaces. Three isolates of P. ramorum, each representing a different clonal lineage, were evaluated for their susceptibility to

D. James et al. / Crop Protection 42 (2012) 186e192

the disinfectants: PFC 5039 (EU1), PFC 5074 (NA2) and PFC 5054 (NA1). Plastic plant-pot saucers (8 cm diameter) and metal tins (8 cm diameter) were filled with 10 ml of inoculum suspension consisting of approximately 7000 sporangia ml1 plus chlaymdospores and mycelial fragments. The latter two propagule types were present consistently in the suspension, but not easily quantified because their numbers were too low to be counted on a haemocytometer grid. The inoculum suspension was left in the saucers and tins for 1 h and then the excess poured off immediately before treating with the disinfectant. It was important to treat with disinfectant before the surfaces dried because during protocol development preliminary testing showed that there was no recovery of P. ramorum from the dried surfaces based on a swab test (as described below). The disinfectants were applied at different rates and for varying times. The low application rate was delivered as a fine mist of approximately 3 ml by hand-pumping a spray bottle four times (equivalent to 0.06 ml cm2 over a 50.24 cm2 plastic or metal surface). A swab test was conducted at 1 and 30 min after disinfectant application. The high application rate delivered 25 ml to the saucers and tins (equivalent to 0.5 ml cm2 over a 50.24 cm2 plastic or metal surface) and was left to soak for 60 min before pouring off the excess and conducting a swab test. The swab test used sterile cotton tips moistened with sterile water that were rubbed across the treated surfaces then streaked in an S-pattern across 9 cm diameter Petri dishes containing 15% V-8 agar amended with 0.15% calcium carbonate, 0.01% streptomycin and 0.005% vancomycin. The culture plates were examined under the microscope to ensure sporangia were present after swabbing and then incubated at 19  C in the dark for 3 and 7 days. The study was set up as a complete random design with four replicates and tests conducted twice. The percentage recovery was determined based on the presence or absence of fungal growth after seven days on each of four replicate culture plates per trial. The count data are presented without further analysis due to minimal or zero variability observed. 3. Results 3.1. Effects of disinfectants on P. ramorum growth from agar plugs The radial growth of mycelia from agar plugs and the formation of CFUs streaked from the surface of plugs were observed on all untreated (water) control plates, for all replicates of the nine isolates in both trials. Fig. 1B shows a water control treatment with CFUs on the left, and a plug with mycelia growth on the right, while Fig. 1A shows a PerCeptÔ-treated plug with no growth of CFUs or mycelia, 2 weeks post-treatment. ChemprocideÒ (0.8% and 1.35%), HyperoxÒ 1:128 dilution, Part A, PerCeptÔ and 15% Bleach were the most effective treatments for controlling mycelia growth from plugs. They were not significantly different from each other at P < 0.01 (Table 3). ChemprocideÒ (1.35%) and PerCeptÔ treatments provided the most consistent control in inhibiting the growth of mycelia of all isolates of all clonal lineages, up to two weeks posttreatment (Table 3). Higher concentrations of ChemprocideÒ, HyperoxÒ and bleach displayed better control of all isolates of P. ramorum. ChemprocideÒ at 1.35% completely inhibited mycelia growth of all isolates representing the 3 clonal lineages of P. ramorum, but 0.8% ChemprocideÒ was significantly less effective in its control of mycelia growth of NA1 isolates. Two-fold dilution of HyperoxÒ (1:256) significantly reduced its efficacy for controlling mycelia growth across all clonal lineages (Table 3). Ethanol treatments (70% and 95%) for 5 min were the least effective chemical treatments and the mycelia growth observed was not significantly

189

Fig. 1. Culture plates of Phytophthora ramorum showing: A. PerCeptÔ treated plug at 2 weeks post-treatment. The agar plug of P. ramorum culture was treated for 10 min with PerCeptÔ, then the area on the left was smeared with the treated plug, mycelia side down, then the plug was placed on the right side of the plate. B. Control plate after 2 weeks. Mycelia plug on the right was treated with water (showing mycelia growth) and the region to the left streaked with the water-treated plug with the mycelia surface down (showing discreet colony forming units).

different from the untreated controls. Extending the treatment times to 10 min resulted in significant improvements, with 95% ethanol being more effective than 75% (Table 3). There was no statistically significant difference among the treatments for controlling the growth of CFUs except in the case of the 5 min treatments with ethanol (70% and 95%) (Table 4). Ethanol at 5 min provided no control of CFU growth, and the number of CFUs associated with the 5 min ethanol treatments was not significantly different from the untreated controls. Ethanol at 10 min (70% and 95%), PerCeptÔ and 15% bleach were the most consistent and completely inhibited growth of CFUs of all isolates (Table 4). Significant differences in the production of CFUs among clonal lineages were observed (Fig. 2). Isolates of EU1 were the most productive (18
1.19, n ¼ 66), and isolates of NA2 were the least productive (9.0
0.89, n ¼ 74). This pattern was observed in the untreated controls, and also for 5 min ethanol (70% and 95%) treated cultures (Table 4). In one case the level of inhibition was inversely related to the production of CFUs (0.8% ChemprocideÒ, Table 4). In another case the opposite trend was observed (VirkonÔ), but in general no relationship between CFU production and the efficacy of the disinfectants was observed.

D. James et al. / Crop Protection 42 (2012) 186e192

Table 3 Proportions of radial mycelia growth from plugs for each disinfectant treatment compared to untreated controls for Phytophthora ramorum isolates and for each P. ramorum lineage considered separately, two weeks post-treatment. Proportions are relative to the untreated controls assigned a value of 1.00, with n ¼ 18. Treatments with the same letter within a column are not significantly different (Pearson’s Chi-squared test, modified Tukey multiple comparison procedure for proportions). Treatment

All P. ramorum

NA1

NA2

EU1

Untreated ChemprocideÒ 0.8% ChemprocideÒ 1.35% HyperoxÒ 128 HyperoxÒ 256 VirkonÒ Virucidal ExtraÒ SMTSÔ Part A 70% Ethanol, 5 min 95% Ethanol, 5 min 70% Ethanol, 10 min 95% Ethanol, 10 min PerCeptÔ 5% Bleach 15% Bleach Y2 P-value <

1.00 c 0.00 a 0.00 a 0.02 a 0.54 b 0.48 b 0.11 a 0.07 a 1.00 c 0.91 c 0.48 b 0.07 a 0.00 a 0.09 a 0.04 a 232.30 0.01

1.00 c 0.17 b 0.00 a 0.00 a 0.72 c 0.17 b 0.00 a 0.00 a 1.00 c 0.83 c 0.56 c 0.11 b 0.00 a 0.17 b 0.00 a 97.70 0.01

1.00 d 0.00 a 0.00 a 0.06 ab 0.56 c 0.67 cd 0.006 b 0.00 b 1.00 d 0.89 d 0.33 bc 0.11 b 0.00 a 0.00 a 0.00 a 92.41 0.01

1.00 d 0.00 a 0.00 a 0.00 a 0.33 bc 0.50 c 0.17 b 0.00 a 1.00 d 1.00 d 0.56 c 0.00 a 0.00 a 0.11 a 0.00 a 101.30 0.01

c

20

Mean colony forming units

190

18 16 14 12

b a

10 8 6 4 2 0 NA1

NA2

EU1

P. ramorum Lineage Fig. 2. Variation among the three clonal lineages of Phytophthora ramorum in the number of colony forming units streaked from untreated (water) control plugs. Error bars indicate standard error of the mean. Data from trial 1 and trial 2 were combined. Lineages with different letters were significantly different at p ¼ 0.05 (ANOVA, Dunnett’s T3 test). See Ivors et al., 2006; Grünwald et al., 2008b, Elliott et al., 2009; Grünwald et al., 2009 for descriptions of the three clonal lineages of P. ramorum.

3.3. In vivo disinfection of P. ramorum on plastic and metal surfaces 3.2. Effects of disinfectant treatments on sporangia growth in microplate assays Ten minute exposure to ChemprocideÒ (1.35%), HyperoxÒ 1:128 dilution, or 15% bleach were the most effective treatments for inhibiting germination of the sporangia (99.6%e100%, Table 5). After three days of growth in liquid culture, there was no statistically significant difference among these treatments (P < 0.001, KruskaleWallis One Way ANOVA; P < 0.05, Dunn’s Method) (Table 5). VirkonÔ was not as effective, but still exhibited high levels of inhibition at 87.7% compared to the control treatment. All isolates of the three clonal lineages of P. ramorum displayed similar patterns of response to the various disinfectants (data not shown), and there was no significant effect of genotype on treatment inhibition (P ¼ 0.234, n ¼ 54). Table 4 Mean number of colony forming units (standard error of mean) for each disinfectant treatment for all Phytophthora ramorum isolates and for each P. ramorum lineage considered separately, one week post-treatment. Data for trial 2 are shown (n ¼ 9, per lineage). Treatments with the same letter within a column are not significantly different (one way ANOVA, Dunnett T3 multiple comparison test). Treatment

All P. ramorum NA1

Untreated Chemprocide 0.8% Chemprocide 1.3% Hyperox 128 Hyperox 256 Virkon Virucidal Extra SMTS Part A 70% Ethanol, 5 min 95% Ethanol, 5 min 70% Ethanol, 10 min 95% Ethanol, 10 min PerCept 5% Bleach 15% Bleach P-value <

17.85 (1.01) b 0.11 (0.06) a

18.06 (1.37) b 15.53 (1.96) d 0.11 (0.11) a 0.00 (0.00) a

NA2

20.56 (1.61) c 0.22 (0.15) a

EU1

0.07 (0.05) a

0.00 (0.00) a

0.00 (0.00) a

0.22 (0.15) a

0.04 (0.04) a 0.07 (0.05) a 0.56 (0.23) a 0.11 (0.06) a 0.07 (0.05) a 15.11 (2.00) b

0.00 (0.00) a 0.00 (0.00) a 0.56 (0.44) a 0.00 (0.00) a 0.00 (0.00) a 14.89 (4.34) b

0.11 (0.11) a 0.22 (0.15) ab 0.78 (0.55) abc 0.22 (0.15) ab 0.22 (0.15) ab 10.67 (2.16) cd

0.00 (0.00) a 0.00 (0.00) a 0.33 (0.17) ab 0.11 (0.11) ab 0.00 (0.00) a 19.78 (3.17) c

10.00 (1.42) b

9.78 (2.91) b

9.22 (2.52) bcd 11.00 (2.17) bc

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a

0.00 (0.00) a 0.04 (0.04) a 0.00 (0.00) a 0.001

0.00 (0.00) a 0.00 (0.00) a 0.00 (0.00) a 0.001

0.00 (0.00) a 0.11 (0.11) a 0.00 (0.00) a 0.001

0.00 (0.00) a 0.00 (0.00) a 0.00 (0.00) a 0.001

On both plastic and metal surfaces there was 100% recovery of P. ramorum from the treatment with water controls in all but one case, where there was only 25% recovery in Trial 2 for isolate PFC 5074 (data not shown). The three disinfectants were effective consistently against isolates PFC 5039 and PFC 5054, where there was 0% recovery regardless of the application rate and exposure time. For isolate PFC 5074, the disinfectant treatments were similarly effective in Trial 1 but in Trial 2 there was 25% recovery from the low rate of application with 1 min exposure for Chemprocide on metal and 5% JavexÒ bleach on plastic surfaces (data not shown). 4. Discussion Disinfectants are important tools used in controlling the spread of pathogens (Best et al., 1990; Gehr et al., 2003; Tomasino, 2005; Cheah et al., 2009). Several disinfectants including ChemprocideÒ (0.8%, 1.35%), HyperoxÒ, Ethanol (70% and 95%), PerCeptÔ and 15% bleach significantly inhibited P. ramorum mycelia growth and CFU growth in vitro. However, 1.35% ChemprocideÒ and PerCeptÔ were the most consistent and effective treatments for all three isolates of each of the three clonal lineages. A concentration effect was observed in controlling mycelia growth. Higher concentrations of ChemprocideÒ, HyperoxÒ and bleach were more effective than lower concentrations, especially in the case of mycelia growth inhibition by HyperoxÒ. A 2-fold dilution of HyperoxÒ resulted in significantly less inhibition of mycelia growth. In this study higher

Table 5 The effects on sporangia germination and growth of 10 min exposures to different disinfectant treatments, 3 days post-treatment, relative to controls (untreated). Disinfectant and concentration

Sample number

Mean % inhibitiona

Standard error of the mean

Chemprocide 1.35% Hyperox 1:128 Virkon 1% Javex liquid bleach 15% Control (no disinfectants)

27 27 27 27 27

100.2 a 99.6 ac 87.7 c 100.0 ac 0.06 b

0.11 0.15 3.80 0.12 0.02

a Mean % inhibitions among disinfectants were significantly different at P  0.001; H ¼ 95.410 with 5 degrees of freedom according to the KruskaleWallis One Way ANOVA on Ranks. Mean % inhibition followed by a similar letter indicates no significant difference according to the Dunn’s Method for multiple comparison test (P < 0.05).

D. James et al. / Crop Protection 42 (2012) 186e192

concentrations were always more effective for controlling mycelia growth, supporting earlier claims that concentration is an important factor affecting efficacy (Best et al., 1990; Gehr et al., 2003). Time of treatment also is critical since 5 min treatments with ethanol were no better than water in controlling CFU growth, but significant improvements were observed with 10 min treatments of both 70% and 95% ethanol. Increased treatment time with ethanol was more effective in controlling CFU growth, rather than mycelia growth. Chemoprocide and Javex Ò liquid bleach were effective disinfectants in preventing the recovery of P. ramorum from plastic and metal surfaces which are common in greenhouse and nursery situations. Some caution should be taken when using the low rates of application via a fine mist and short exposure times as one isolate was recovered at low levels from both plastic and metal surfaces. Delivering a large volume of disinfectant and or increasing the length of the exposure time should reduce the risk of potential cross contamination. The Javex Ò liquid bleach, even at the 5% and 10% concentrations, caused some corrosion of the metal surfaces after exposure for 30 min or longer, so Chemprocide may be the better choice for disinfecting metal equipment. In Trial 2 of the in vivo study, there was 25% recovery of P. ramorum for the water control treatment of isolate PFC 5074 (NA2 lineage). It is likely that the surface in this case may have been allowed to dry too much after decanting the inoculum suspension, and before the water treatment was applied, thus reducing the viability of the sporangia. The results from the in vitro studies using mycelia and sporangia and the in vivo study using sporangia with mycelia and chlamydospores were very comparable. Similar responses have been observed in other studies where the evaluation of disinfectants in one system was valid under other conditions (Best et al., 1990). This was supported further by Cheah et al. (2009) who found similar patterns of efficacy in both in vitro and field tests. This corroborates the fact that recommendations made from our in vitro and in vivo simulation studies should be valid under different field conditions. Differences in the production of CFUs were observed in this study, with EU1 isolates being the most productive isolates compared to isolates of NA1 or NA2. Whether this has anything to do with pathogenicity or efficiency of spread is yet to be determined. The conditions used, such as maintaining the cultures at 20  C, were optimal for chlamydospore production (Englander et al., 2006), as indicated in preliminary analysis of mycelia plugs (data not shown). The CFUs in this study, therefore, may have been predominantly fragments of mycelia and chlamydospores. Werres et al. (2001) found that older cultures of P. ramorum often required flooding to induce efficient sporangia formation. CFUs may be identified as ‘spores’ since they represent survival and dispersal structures (Judelson and Blanco, 2005). Most disinfectants were effective in inhibiting the germination of CFUs. There was no significant difference between treatments that showed inhibition of CFU growth. Where different concentrations of a chemical were evaluated, the lower concentrations were as effective as the higher concentrations with no significant difference in the levels of inhibition observed. In general the CFUs were more susceptible to the treatments used in this study, compared to mycelia growth, and therefore easier to control. The CFUs were likely on the surface of the mycelia plugs, possibly resulting in greater exposure to the disinfectants. PerCeptÔ and 15% bleach treatments showed complete inhibition of CFU growth. Sporangia represent another entity for Phytophthora survival and dispersal (Hwang and Ko, 1978; Linderman and Davis, 2006). They are generally of intermediate susceptibility to disinfectants compared to thick-walled chlamydospores, and the fragile short-

191

lived zoospores. Several treatments that effectively inhibited mycelia and CFU growth were selected and evaluated in vitro for sporangia growth inhibition. The microplate assays were designed to evaluate a timed exposure of sporangia to disinfectants. Ten minutes of exposure to ChemprocideÒ (1.35%), HyperoxÒ or 15% bleach were very effective in inhibiting sporangia germination. The treatments were effective on sporangia obtained from isolates of all three clonal lineages of P. ramorum, and no significant genotype-related differences were observed, indicating great potential for reliable control of sporangia germination. Two disinfectants (HyperoxÒ and PerCeptÔ) used in this study contained hydrogen peroxide. PerCeptÔ was very effective in inhibiting mycelia growth and CFU germination. HyperoxÒ was the only chemical with a combination of hydrogen peroxide (H2O2) (10e20%) and peracetic acid (CH3CO3H) (5%). Hydrogen peroxide is a commonly used decontaminant. Its activity is known to be affected by concentration, light, pH, temperature and other factors (Russell, 1990). Hydrogen peroxide may cause disruption of any coating material, which facilitates penetration of the disinfectant into the cortex and protoplast of bacterial spores (Russell, 1983). Peracetic acid may act by releasing active oxygen which disrupts sulfhydryl (SeH) and sulphur (SeS) bonds within enzymes in the cell membrane (Lefevre et al., 1992), or it may cause the release of hydroxyl radicals (Lubello et al., 2002). There is some suggestion that H2O2 and CH3CO3H may be synergistic (Gehr et al., 2003), although experiments by Lubello et al. (2002) suggest that it is the hydrogen peroxide which is responsible for the biocidal activity. PerCeptÔ contains H2O2 but is more complex in its formulation (Table 2). They were not significantly different in their efficacy for inhibiting mycelia growth and CFU germination, but PerCeptÔ was more reliable. Lubello et al. (2002) suggests that H2O2 may be the sole factor contributing to antimicrobial activity. The fact that the working concentration of PerCeptÔ contains 0.3% H2O2, compared to 0.08% for HyperoxÒ, using the minimum concentration of H2O2 for each disinfectant formulation, further supports the concentration effect. ChemprocideÒ and bleach were relatively effective also, with the higher concentrations being more effective. Chemprocide at 1.35% completely inhibited mycelia growth from cultures embedded in agar, but was less effective for inhibiting the growth of CFUs from exposed parts of the plugs of all isolates. ChemprocideÒ consists of a quaternary ammonium compound and alcohol, while sodium hypochlorite is the main ingredient of bleach. Sodium hypochlorite is a well known anti-microbial agent or disinfectant (DeQueiroz and Day, 2008), and it uses radicalmediated reactions to oxidize organic material that result in disruption of coating material (Russell, 1983). Some inconsistencies were observed between trials. This possibly reflects lower effectiveness, or variability of conditions and chemical composition between trials. Variability in factors such as temperature, relative humidity, and contact time can influence the effectiveness of disinfectants (Russell, 1999). It is clear however that treatment with the best performing disinfectants can contribute significantly to reducing the spread of P. ramorum, when compared to the untreated controls. ChemprocideÒ (1.35%) and 15% bleach were the most consistent in significantly inhibiting mycelia growth, CFU germination, and the germination of isolated sporangia. Both chemicals are likely effective also in limiting the movement of P. ramorum inocula, since they were effective in preventing P. ramorum recovery from plastic and metal surfaces, common surfaces in greenhouses and nurseries. Based on the results of these studies, these two disinfectants would be suitable for incorporation in any integrated management program for controlling the spread of P. ramorum.

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Acknowledgements We wish to thank and are very grateful to Dr. N.J. Grünwald (USDA, ARS,Corvallis), and S.C. Brière (CFIA, Ottawa) for generously providing the Phytophthora ramorum isolates used in this study. We wish to thank also the Canadian Food Inspection Agency (CFIA Research Partnership Strategy Program), the Canadian Forest Service (CFS), Agriculture & Agri-Food Canada (AAFC), and the Natural Sciences and Engineering Research Council Canada (NSERC) for their kind and generous financial support.

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