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Infection and decontamination of citrus canker-inoculated leaf surfaces
Crop Protection 30 (2011) 259e264
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Infection and decontamination of citrus canker-inoculated leaf surfaces C.H. Bock a, *, P.E. Parker b, A.Z. Cook b, J.H. Graham a, T.R. Gottwald c a
University of Florida-IFAS-CREC, 700 Experiment Station Rd., Lake Alfred, FL 33850, USA USDA-APHIS-PPQ, Moore Air Base, Edinburg, TX 78539, USA c USDA-ARS-USHRL, 2001 S. Rock Rd., Ft. Pierce, FL 34945, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2010 Received in revised form 29 September 2010 Accepted 8 October 2010
Citrus canker (Xanthomonas citri subsp. citri, Xcc) is now considered endemic in Florida and continues to spread. Various surfaces, including plant material, personnel and equipment can become contaminated. Decontamination is practiced in both disease-endemic and disease-free areas to reduce the risk of bacterial spread by man or machinery. We used grapefruit leaf surfaces to explore the efficacy of a commonly used personnel sprayer system applying a quaternary amine decontaminant. In three experiments, plants in flush (leaves 3/4 expanded) were inoculated (w105 CFU ml1). Immediately after inoculation, plants were passed through the sprayers 0, 1, 2, 3, or 6 times. Leaves were sampled at 0.5, 10 and 20 min after decontamination and tested for viable Xcc by dilution plating. There was a large and rapid decline in the quantity of live bacteria with one pass through the decontamination sprayer (>80% decrease in CFU ml1), and multiple sprays (2e6) resulted in up to 100% mortality of surface Xcc. Presumably more thorough coverage with multiple sprays killed remnant bacteria, although the first spray invariably caused the highest proportion of population mortality. The effect of the decontaminant spray was immediate (within 0.5 min only 3e11% of surface bacteria survived, and by 20 min <1e3% survived). Based on these results, use of a personnel sprayer with a quaternary amine compound is highly effective for reducing surface inoculum. A single spray kills a high proportion of the population, but multiple sprays increase mortality of Xcc. All the Xcc-inoculated plants subsequently developed symptoms of citrus canker. No significant difference in incidence or severity of grapefruit leaf infection was detected among decontamination treatments or compared to the untreated control. This finding indicates that infection occurred at, or very soon after, inoculation, and that Xcc was in protected sites inside the leaf before exposure to the decontaminant spray. Published by Elsevier Ltd.
Keywords: Epidemiology Infection Citrus canker Inoculum Disease spread Quaternary ammonium Amines Personnel sprayers
1. Introduction Citrus canker (Xanthomonas citri subsp. citri, Xcc, (ex Hasse) Gabriel et al.) is endemic in Florida and is dispersed in rain splash often associated with wind (Gottwald and Graham, 1992; Pruvost et al., 2002; Bock et al., 2005 and Bock et al., 2010a; Gottwald and Irey, 2007). The pathogen can also be spread by people directly, and potentially on various surfaces including plant material, clothing, and various equipment or implements (Graham et al., 1989, 2000; Gottwald et al., 1992). Contamination of these surfaces might occur by brushing against wet canker-infected leaves after heavy dew or rain, and during orchard management such as pruning, or fruit harvest.
* Corresponding author. Present address: USDA-ARS-SEFTNRL, 21 Dunbar Road, Byron, GA 31008, USA. Tel.: þ1 478 956 6421; fax: þ1 478 956 2929. E-mail address: [email protected] (C.H. Bock). 0261-2194/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.cropro.2010.10.001
Once dispersed and on a susceptible leaf surface, various factors are thought to play a role in infection including direct access of the pathogen into stomata through the force of wind-blown rain splash (Gottwald and Graham, 1992; Graham et al., 1992), injury (Christiano et al., 2007; Bock et al., 2010b), and temperature and leaf wetness duration (Dalla Pria et al., 2006; Christiano et al., 2009). Infection with Xcc is thought to occur when bacteria gain access to the leaf through stomata, and infecting host cells proximal to the endophytic population (Koizumi, 1976a, Koizumi, 1976b, Koizumi, 1977; Gottwald and Graham, 1992; Graham et al., 1992). Thus access to the stomata might occur immediately upon arrival of the bacteria on the leaf surface when they are driven into the stomata by rain splash (Gottwald and Graham, 1992) or over a period of several hours (Dalla Pria et al., 2006; Christiano et al., 2009), perhaps from a population of Xcc existing in the phyllosphere (Rigano et al., 2007). Bacteria are common inhabitants of leaf surfaces and often form epiphytic biofilms (Beattie and Lindow, 1995; Lindow and Brandl, 2003; Monier and Lindow, 2005).
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Epiphytic biofilms of bacteria that are also endophytes are thought to be involved in survival, and possibly infection (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009). Surface decontamination removes a proportion of bacteria associated with the phyllosphere e those on the surfaces that are exposed. Bacteria in protected sites such as the sub-stomatal cavity or in the mesophyll are not exposed to the disinfecting agent (Wilson et al., 1999; Niemira, 2007; Pruvost et al., 2009), or where bacteria have had time to form a biofilm (Beattie and Lindow, 1995; Rigano et al., 2007). Surface sterilization with various agents has been used to study development of epi- and endophytic populations of plant pathogenic and non-pathogenic bacteria (Wilson et al., 1999; Pruvost et al., 2009), as well as to study human pathogens (USDA-FDA, 2001; Solomon et al., 2002; Niemira, 2007), and surface decontamination procedures have been shown to reduce bacteria populations on citrus (Brown and Schubert, 1987; Gottwald et al., 2009). A decontaminant treatment that kills surface bacteria of Xcc could provide insight into the earliest timing of infection in relation to arrival of inoculum on the leaf surface, while providing data to demonstrate the efficacy of surface spray application. Xcc has been shown to be ephemeral on surfaces, surviving up to 72 h (Graham et al., 1989, 2000). Nevertheless, to minimize the risk of spreading the pathogen via contaminated personnel or equipment, individuals and their implements are commonly surfacedisinfected before leaving a grove or facility where the presence of canker is known or suspected. Quaternary amines are recommended for this purpose (Schubert and Sun, 2003; Roberts et al., 2004). On commercial groves and in government and state facilities these disinfectants are applied with hand-held sprayers to hands and feet, and also via walk-through sprayers. Walk-through sprayers consist of spray nozzles on two vertical tubes that produce a mist of disinfectant that coats the individual and any items (for example, carts, notebooks, bags) taken through the sprayer system. The assumption is that spray coverage is sufficient to cause high mortality, or complete kill of Xcc. Vehicles and machinery are decontaminated with similar, but often more potent disinfectants (Roberts et al., 2004) using a hoop, or arch structure with sprayer nozzles positioned to produce a cloud of disinfectant through which the equipment is passed. Quaternary amines are very effective at killing microbial contaminants and many kinds of bacteria (McDonnell and Russell, 1999; Gamage, 2003), but they rapidly decompose as they come in contact with material in the environment. While the efficacy of some personnel decontaminants like quaternary amines, and various application systems have been investigated in the medical, veterinary and food sciences (Jeffrey, 1997; McDonnell and Russell, 1999; Amass et al., 2000; Sutton et al., 2002; Gamage, 2003; Goeres et al., 2004; Sawant et al., 2008), they do not appear to have received much attention in relation to decontamination or management of plant pathogens (Letal, 1977; Teviotdale, 1991; Chase, 1992; Draper et al., 2003; Rugh and Rosenberger, 2005). In situations where containment or eradication is important, it would be useful to have data to support the efficacy of application methods and decontaminants used to reduce the risk of pathogen spread. It is of particular importance to establish the effectiveness of various delivery systems for disinfesting surfaces of complex topography, such as citrus leaves. To investigate the efficacy of a personnel sprayer delivery system and a common quaternary disinfectant, we used healthy, injury-free, susceptible grapefruit leaf surfaces inoculated with Xcc and immediately exposed them to a personnel decontaminant, and measured surface populations of Xcc after treatment, and also assessed subsequent disease development on these plants. Citrus
leaves were chosen over other surfaces as there exists a substantial literature on surface decontamination of leaf surfaces for comparative purposes (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Lindow, 2009; Niemira, 2007; Rigano et al., 2007; Pruvost et al., 2009; ). The objectives were to ascertain the efficacy of the personnel sprayer delivery system for killing surface bacteria of Xcc, and to gain some insight into whether bacteria gained access to protected sites at the time of inoculation. 2. Materials and methods 2.1. Experiment plan and procedure Three replicate experiments examined surface decontamination (experiments I, II and III). In all three experiments, inoculum of Xcc was grown on a semi-selective media composed of Nutrient Agar (NA) amended with kasugamycin (16 mg L1), cephalexin (35 mg L1) and chlorothalanil (12 mg L1 tetrachloroisophthalonitrile). Colonies were maintained in an incubator at 27 C for five days. Inoculum (105 bacteria ml1) was prepared in sterile distilled water (Table 1). In all experiments, Duncan grapefruit plants (Citrus x paradisi Macfad.) in flush (leaves 3/4 expanded) were sprayed to run-off with inoculum of Xcc prepared as above. Immediately after inoculation (30 s) the plants were exposed to a decontaminant disinfectant spray (Canker GuardÒ, Flo-Tec, Largo, FL; active ingredients are a proprietary blend of two quaternary amines, alkyl-dimethylbenzyl-ammonium chloride and alkyl dimethyl ethylbenzyl ammonium chloride). The decontamination spray was applied by passing the plants at a slow walk (approx 0.7 m s1) through a standard personnel decontamination spray system (Fig. 1) ensuring good droplet coverage of the plants with each pass. Three sprayers (Conejet X1, Spraying Systems Co., Wheaton, IL) arranged vertically on either side provided the spray (at heights of 60, 100 and 140 cm, with 76 cm horizontal distance between pairs of nozzles). Three replicate plants were passed through the decontaminant sprayer 1, 2, 3 or 6 times at a height of 120 cm. An additional set of plants was not passed through the sprayer, but were inoculated. These plants served as the positive control. Subsequent to passing the plants through the sprayer, a single leaf of approximately the same size was sampled from a plant receiving each spray treatment at 0 (no decontaminant spray), 0.5 min (immediately after the decontaminant spray), 10 and 20 min after the decontamination process. Each leaf was placed in a 50 ml tube containing 40 ml sterile distilled water and vortexed for 5 s (Xcc bacteria are naturally dispersed in rainwater, which has few dissolved impurities and therefore a low osmotic potential akin to distilled water). Bacteria recovered in the leaf wash were dilution plated on the semi-selective medium as described above. After incubation for 5 days at 27 C, the colony forming units (CFU) were counted and bacteria ml1 calculated. In two experiments (II and III) the plants were transferred to a greenhouse immediately after exposure to the decontaminant spray (greenhouse venting set to 27 C, heating set to 20 C). Inoculum and disinfectant were
Table 1 Dates and concentration of Xcc inoculum in sterile distilled water used for each experiment. Plants were sprayed to run-off with inoculum using a hand-held sprayer. Inoculum concentration was confirmed by dilution plating on KCB nutrient agar. Expt. no.
Date
Inoculum conc. (Xcc bacteria ml1)
I II III
19 Dec 2007 22 May 2008 23 Aug 2008
1.7 105 1.5 105 8.6 105
C.H. Bock et al. / Crop Protection 30 (2011) 259e264
261
Table 2 Characteristics of the exponential relationships between CFU ml-1 of Xanthomonas citri subsp. citri surviving on a leaf surface of Duncan grapefruit after different numbers of passesa through a personnel decontamination sprayer. Parametersb (y ¼ ae–bx)
Expt. no.
I II III
a
b
100.0 100.0 99.9
2.14 1.67 2.02
F-value (Pr>F)
R2c
7602 (<0.0001) 4609 (<0.0001) 886 (<0.0001)
>0.99 >0.99 >0.99
a
Control treatment received no disinfectant sprays. Parameters shown are for normalized bacteria counts, where for each sample the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment (Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment (Fig. 3B). c R2 ¼ coefficient of determination. b
Fig. 1. A personnel sprayer system used to decontaminate individuals, clothing and sundry equipment exposed to citrus canker showing the nozzles placements. Plants were carried through at waist height.
allowed to dry on the leaf surfaces in the greenhouse; it took approx. 1.5e2.0 h for the visible moisture to evaporate. After 28 days, symptoms of citrus canker were assessed visually. Disease incidence (% leaves infected) and disease severity on each leaf (% leaf area infected) on each plant was calculated.
2.2. Data analysis The relationship between number of CFU ml1 surviving and numbers of decontamination sprays (averages of spray number across sample times), and between CFU ml1 surviving with time after exposure to the decontamination treatment (averages of sample time across number of decontamination sprays) were analyzed using regression. An exponential function (y ¼ e(abx)) was
30000 25000 20000
There was a large decline in the CFU of Xcc with one pass through the decontaminant spray for all times of exposure (Fig. 2A). Decontaminant sprays (from one to six) resulted in up to 100% mortality of the recoverable, culturable population of Xcc associated with the surface of these citrus leaves. Different numbers of bacteria were isolated from leaves in the three experiments, but the relationship between the normalized canker populations and passes through the decontaminant spray was very similar for all three experiments, and was well described by an exponential function (Fig. 2B, Table 2). Thus rate of bacteria mortality declined with increasing numbers of sprays. After a single spray only 12e19% of the original surface population survived, after two sprays the remaining population was 0.02e6% that before the decontaminant
B Experiment I Experiment II Experiment III
15000 10000 5000 0
3. Results
0 1 2 3 6 Number of decontaminant sprays
Percent of maximum CFU
CFU (bacteria of Xcc ml–1)
A
fit to the normalized quantity of bacteria. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. The disease incidence and severity on plants inoculated with Xcc but receiving different numbers of decontamination sprays was compared using general linear modeling and a post hoc means separation using Tukey’s HSD test (P ¼ 0.05). The standard error of the mean for each treatment was calculated as the sample estimate of the population standard deviation divided by the square root of the sample size. All analyses were performed in SAS (SAS Systems, Cary, NC).
100
Experiment I Experiment II Experiment III Exp.fit, expt. I Exp.fit, expt. II Exp. fit, expt. III
80 60 40 20 0
0
1 2 3 4 5 6 7 Number of decontaminant sprays
Fig. 2. A. The quantity of bacteria of Xanthomonas citri subsp. citri on leaf surfaces of Duncan grapefruit with no exposure, one, two, three and six passes through a personnel decontaminant sprayer (results for three experiments shown). B. The exponential relationship between percent bacteria survival and number of passes through the personnel sprayers for each experiment. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, where Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment.
C.H. Bock et al. / Crop Protection 30 (2011) 259e264
spray was applied, and after six sprays, it was either zero or less than 1% of the control. The longer the period between disinfection and sampling, the lower the cfu of surviving bacteria (Fig. 3A). In the three experiments, within 0.5 min of the decontaminant spray only 3e11% of the population of bacteria of Xcc survived, and by 20 min only 0.5e3% remained. The rapid decline in the proportion of Xcc surviving was described by an exponential model in experiment II and III (Fig. 3B, Table 3). In experiment I, several missing values for time 0.5 min precluded fitting a model to these data, although the existing data support a similar relationship. The quantity of Xcc isolated from leaf surfaces of untreated plants at spray application, 10 and 20 min after application of the decontaminant showed no decline in the surface population for experiment I, II or III (Fig. 4). There was no significant effect of the decontamination spray on the development of symptoms of canker on the treated plants compared to the untreated control (Fig. 5AeD). Application of a decontaminant disinfectant did not appear to significantly reduce incidence or severity of infection in experiment II (F ¼ 0.70, P ¼ 0.62 and F ¼ 0.69, P ¼ 0.67, respectively) or III (F ¼ 0.35, P ¼ 0.84 and F ¼ 0.11, P ¼ 0.98, respectively). 4. Discussion Use of the personnel decontamination system on leaf surfaces resulted in high mortality of Xcc, despite the leaf surfaces being spray-inoculated to run-off with bacterial inoculum. This appears to be the first report of the efficacy of this type of delivery system for a surface decontaminant aimed at providing a thorough spray coverage to achieve a high level of kill for a plant pathogen on a surface. Although personnel decontamination sprayers are not widely used in plant pathogen decontamination situations, the citrus canker epidemic is one case where they have been recommended (Schubert and Sun, 2003; Roberts et al., 2004). We found >80% of the bacteria isolated and cultured from the heavily contaminated leaf surfaces were killed with a single pass through the personnel sprayer. With two or more passes through the sprayer, up to 100% mortality of surface bacteria was achieved. High mortality of surface bacteria on leaves and other plant surfaces has been demonstrated before using various other decontaminants and delivery systems (Brown and Schubert, 1987; Wilson et al., 1999; Solomon et al., 2002; Niemira, 2007; Gottwald et al., 2009; Pruvost et al., 2009). Presumably the sprayers ensured that the surfaces were well covered by the three-dimensional spray cloud of
CFU (bacteria of Xcc ml–1)
A
Expt no.b
II III
Parametersc (y ¼ e(a-bx)) a
b
100.0 100.0
4.42 6.71
F-value (Pr>F)
Rd,c
507 (0.002) 226 (0.0044)
>0.99 >0.99
a Data for time 0 (control data) received no disinfectant sprays and is included in the analysis. b A regression solution was not fit to data from experiment 1 due to missing data. c Parameters shown are for the normalized bacteria counts, where each sample is a percentage of the maximum (Q) collected in that experiment (Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. d R2 ¼ coefficient of determination.
disinfectant that contacted upper and lower surfaces of leaves regardless of orientation. Quaternary amines are known to be highly effective disinfectants against many bacteria (McDonnell and Russell, 1999), and several different types are available for decontamination of personnel or implements exposed to canker bacteria (Roberts et al., 2004). Although the first spray invariably killed the most surface bacteria, the observation that multiple sprays killed remnant, surviving bacteria is significant. The relationship between surviving Xcc population and passes through the decontaminant sprayer was described by an exponential model; thus there was significant efficacy of additional sprays for killing surface bacteria. A spray “tunnel” with two or more spray systems would provide greater mortality of surface bacteria. By six passes the number of surviving bacteria isolated was in some cases zero. In addition, the greater the period after application of the disinfectant contact prior to sampling, the lower the bacterial survival, although the greatest proportion of the bacterial population was killed within 0.5 min (>88%), compared to the control. Having established the efficacy of quaternary amines against Xcc for decontamination of citrus leaf surfaces, this evaluation should be extended to include other materials including those with porous or absorbent surfaces such as clothing or boots, and hard surfaces including plastic or metal. An unexpected observation from this study was the incidence of canker that developed in plants after spray treatments was not diminished despite the high efficacy for killing surface bacteriawithin
B
35000 30000
Experiment I
25000
Experiment III
Experiment II
20000 15000 10000 5000 0
Table 3 Characteristics of the exponential relationships between actual CFU ml-1 of Xanthomonas citri subsp. citri surviving on leaf surfaces of Duncan grapefruit and time since inoculation after passinga through the decontaminant spray.
0.5 10 20 0 Time since decontaminant spray (min)
Percent of maximum CFU
262
100 80 60
Experiment I Experiment II Experiment III Exp. fit expt. I Exp. fit exp. III
40 20 0
0 5 10 15 20 Time since decontaminant spray (min)
Fig. 3. A. The quantity of bacteria of Xanthomonas citri subsp. citri on leaf surfaces of Duncan grapefruit at different times after inoculation (data from no exposure, one, two, three and six passes through a personnel decontaminant sprayer (results for three experiments shown). B. The exponential relationship between percent bacteria survival and time after inoculation for each experiment. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, where Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. A regression solution was not fit to data from experiment 1 due to missing data.
5
Expt I
Expt II
Expt III
4 3 2 1 0
10 20 0 Time since decontaminant spray
Fig. 4. The quantity of bacteria of Xanthomonas citri subsp. citri re-isolated from leaf surfaces of control plants that did not receive a quaternary amine disinfectant spray at time 0 (time of decontaminant spray), 10 and 20 min after application of the decontaminant to treated plants. Results for the three different experiments, I, II and III are shown.
a short period. Some surface decontamination procedures have previously been shown to kill virtually all surface bacteria (Wilson et al., 1999; Solomon et al., 2002; Niemira, 2007; Pruvost et al., 2009). The time span of our study would be insufficient for bacteria to aggregate or form biofilms and perhaps be protected against a decontaminant as described for Xcc and other epiphytic bacteria (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009;). Such biofilms formed by bacteria are thought to be a survival strategy that several plant pathogenic bacteria have evolved to deal with stressful environments (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009). If Xcc had no time to form a biofilm between inoculation and exposure to the decontaminant (a period of 30 s), then infection that led to disease likely took place immediately upon exposure of the leaf to inoculum. This can be deduced because mortality of the surface inoculum by the disinfectant did not appear to significantly reduce
Percent leaves infected
A 100
B
80 60 40 20 0
a
a
a
a
a
0
1
2
3
6
24 20 16 12 8 4 0
Number of decontaminant sprays
D
6 5 4 3 2 1 0
a 0
a 1
a 2
a 3
a 6
Number of decontaminant sprays
a
a
a
a
a
0
1
2
3
6
Number of decontaminant sprays Severity per leaf (% area infected)
Severity per leaf (% area infected)
C
263
incidence or severity of infection in either experiment compared to the control. Quaternary amines are directly disruptive to the cell membrane integrity so it is unlikely the bacteria were rendered “viable but non-culturable” (VBNC), as might occur in response to some pesticides or chemicals (Wilson and Lindow,1992; Oliver, 2005; Campo et al.,2009). Furthermore, evenin theevent that a VBNC stateis induced, it appears that onlya small proportionof a population has the opportunity to enter this state yet remain pathogenic (Campo et al., 2009). As the amount of disease among treatments was not different, these data suggest the bacteria responsible for infection were immediately protected from the decontaminant, i.e., in the substomatal cavity or mesophyll. These data provide evidence that bacteria of Xcc in splash or spray dispersed droplets apparently gain immediate access to the substomatal chamber (Gottwald and Graham, 1992; Graham et al., 1992; Pruvost et al., 2002; Bock et al., 2010b). In the studies of Gottwald and Graham (1992) and Graham et al. (1992) pressure was applied to ensure the inoculum gained access to the leaf through the stomata. Although, prolonged leaf wetness may contribute to greater infection of undamaged leaves (Dalla Pria et al., 2006; Christiano et al., 2009), and biofilms might play a role in this infection (Jacques et al., 2005; Rigano et al., 2007; Gudesblat et al., 2009), our results suggest the importance of immediate access of the Xcc bacterium to the inside of the leaf, even with relatively gentle inoculation using a pump-action sprayer. The spray inoculum from the pump-action sprayer may have sufficient force for the droplets to penetrate the stomata. Hence, the relative role of immediate, direct penetration of the Xcc bacterium through sprayed inoculum or splash, pressurized inoculation and the role of epiphytic Xcc deserves a rigorous comparative investigation. Finally, we saw no evidence of phytotoxicity due to the quaternary amines when applied using a gentle decontaminant spray system. However, applying the disinfectant directly with a hand-held sprayer to run-off at the recommended concentration caused phytotoxicity to grapefruit leaves (C. H. Bock, unpublished
Percent leaves infected
Log CFU (bacteria of Xcc ml–1)
C.H. Bock et al. / Crop Protection 30 (2011) 259e264
0.6 0.5 0.4 0.3 0.2 0.1 0.0
a
a
a
a
a
0
1
2
3
6
Number of decontaminant sprays
Fig. 5. . Incidence (percent leaves infected A,B) and severity (percent area infected per leaf, C,D) on leaves of grapefruit plants spray-inoculated with bacteria of Xanthomonas citri subsp. citri and immediately passed through a personnel decontamination sprayer 1, 2, 3 or 6 times. Data shown are for the two experiments, II (A,C) and III (B,D). Treatments with the same letter within each experiment are not significantly different from each other based on Tukey’s HSD test. The standard error of the mean for each treatment is indicated, and was calculated as the sample estimate of the population standard deviation divided by the square root of the sample size.
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observations). In conclusion, these personnel decontaminant systems using quaternary ammonium compounds are an effective way of decontaminating at least some surfaces of Xcc. Other common materials should be tested using this system to confirm its efficacy in different situations. Acknowledgements This research was supported by the Florida Citrus Production and Research Advisory Council. Any mention of a trademark or proprietary product does not constitute an endorsement or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. References Amass, S.F., Vyerberg, B.D., Ragland, D., Dowell, C.A., Anderson, C.D., Stover, J.H., Beaudry, D.J., 2000. Evaluating the efficacy of boot baths in biosecurity protocols. Swine Health Prod. 8, 169e173. Beattie, G.A., Lindow, S.E., 1995. The secret life of foliar bacterial pathogens on leaves. Annu. Rev. Phytopathol 33, 45e172. Bock, C.H., Graham, J., Gottwald, T.R., Cook, A.Z., Parker, P.E., 2010a. Wind speed effects on the quantity of Xanthomonas citri sub sp. citri dispersed downwind from canopies of grapefruit trees infected with citrus canker. Plant Dis. 94, 725e736. Bock, C.H., Graham, J.E., Gottwald, T.R., Cook, A.Z., Parker, P.E., 2010b. Wind speed and wind associated injury affect severity of citrus canker on Swingle citrumelo. Euro. J. Plant Pathol. 128, 21e38. Bock, C.H., Parker, P.E., Gottwald, T.R., 2005. The effect of simulated wind-driven rain on duration and distance of dispersal of Xanthomonas axonopodis pv citri from canker infected citrus trees. Plant Dis. 89, 71e80. Brown, G.E., Schubert, T.S., 1987. Use of Xanthamonas campestris pv. vesicatoria to evaluate surface disinfectants for canker quarantine treatment of citrus fruit. Plant Dis. 4, 319e323. Campo del, R., Russi, P., Mara, P., Mara, H., Peyrou, M., de Leon, I.P., Gaggero, C., 2009. Xanthomonas axonopodis pv. citri enters the VBNC state after copper treatment and retains its virulence. FEMS Microbiol. Lett. 298, 143e148. Chase, A.R., 1992. Bacterial Disease Control on Ornamentals Using Aliette, Kocide, Greenshield and ASC-66825. Research Report RH-92e10. Central Florida Research and Education Center, Apopka, University of Florida, Fl. Christiano, C., Dalla Pria, M., Jesus, W.C., Amorim, L., Bergamin-Filho, A., 2009. Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness. Euro. J. Plant Pathol. 124, 1e7. Christiano, C., Dalla Pria, M., Jesus, W.C., Parra, J.R.P., Amorim, L., Bergamin-Filho, A., 2007. Effect of leaf-miner damage, mechanical damage and inoculum concentration on severity of symptoms of Asiatic citrus canker in Tahiti lime. Crop Prot. 26, 59e65. Dalla Pria, M., Christiano, R.C.S., Furtado, E.L., Amorim, L., Bergamin Filho, A., 2006. Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker. Plant Pathol. 55, 657e663. Draper, M.A., Burrows, R., Mills, J., 2003. Fireblight of Apples, Pears and Other Species. FS916. South Dakota State University, Cooperative Extension Service, South Dakota, 4 pp. Gamage, B., 2003. A Guide to Selection and Use of Disinfectants. British Columbia Centre for Disease Control, British Columbia, Canada, 18 pp. Goeres, D.M., Palys, T., Sandel, B.B., Geiger, J., 2004. Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model. Water Res. 38, 3103e3109. Gottwald, T.R., Graham, J.H., 1992. A device for precise and nondisruptive stomatal inoculation of leaf tissue with bacterial pathogens. Phytopathology 82, 930e935. Gottwald, T.R., Graham, J.H., Bock, C.H., Bonn, G., Civerolo, E.L., Irey, M., Leite, R.P., et al., 2009. The epidemiological significance of post-packinghouse survival of Xanthomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Prot. 28, 508e524. Gottwald, T.R., Graham, J.H., Egel, D.S., 1992. Analysis of foci of Asiatic citrus canker in a Florida citrus orchard. Plant Dis. 76, 389e396. Gottwald, T.R., Irey, M., 2007. Post-hurricane analysis of citrus canker II: predictive model estimation of disease spread and area potentially impacted by various eradication protocols following catastrophic weather events. Online. Plant Health Prog.. doi:10.1094/PHP-2007-0405-01-RS. Graham, J.H., Gottwald, T.R., Civerolo, E.L., McGuire, R.G., 1989. Population dynamics and survival of Xanthomonas campestris in soil in citrus nurseries in Maryland and Argentina. Plant Dis. 73, 423e427. Graham, J.H., Gottwald, T.R., Riley, T.D., Achor, D., 1992. Penetration through leaf stomata and strains of Xanthomonas campestris in citrus cultivars varying in susceptibility to bacterial diseases. Phytopathology 82, 1319e1325. Graham, J.H., Gottwald, T.R., Riley, T.D., Cubero, J., Drouillard, D.L., 2000. Survival of Xanthomonas campestris pv. citri on various surfaces and chemical control of
Asiatic citrus canker. Proceedings of the International Citrus Canker Research Workshop, June 20e22, 2000, Ft. Pierce, Fl, p. 7. Gudesblat, G.E., Torres, P.S., Vojnov, A.A., 2009. Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor. Plant Physiol. 149, 1017e1027. Jacques, M.A., Josi, K., Darrasse, A., Samson, R., 2005. Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans. Appl. Environ. Microbiol. 71, 2008e2015. Jeffrey, J.S., 1997. Sanitation-disinfection Basics. Poultry Fact Sheet No. 27. Cooperative Extension, Veterinary Medicine Teaching and Research Center, University of California, CA, p. 5. Koizumi, M., 1976a. Behavior of Xanthomonas citri (Hasse) Dowson in the infection process I. Multiplication of the bacteria and histological changes following needle prick inoculation. Ann. Phytopath. Soc. Japan. 42, 407e416. Koizumi, M., 1976b. Behavior of Xanthomonas citri (Hasse) Dowson in the infection process II. Multiplication of the bacteria and histological changes of the host plant following rubber block press or infiltration inoculation. Ann. Phytopath. Soc. Japan. 42, 407e416. Koizumi, M., 1977. Behavior of Xanthomonas citri (Hasse) Dowson in the infection process II. Multiplication of the bacteria and histological changes of the host plant following rubber block press or infiltration inoculation. Ann. Phytopath. Soc. Japan. 42, 407e416. Letal, J.R., 1977. Efficacy of disinfestants against potato ring rot and blackleg bacteria. Am. Potato J. 54, 405e409. Lindow, S.E., 2009. Plant factors and other bacterial residents modulate iron levels on leaves thereby influencing quorum sensing controlled epiphytic fitness and virulence in Pseudomonas syringae. Phytopathology 99, S154. Lindow, S.E., Brandl, M.T., 2003. Microbiology of the phyllosphere. App. Env. Microbiol. 69, 1875e1883. McDonnell, D., Russell, D., 1999. Antiseptics and disinfectants: activity, action, and resistance. Clin. Microbiol. Rev. 12, 147e179. Monier, J.-M., Lindow, S.E., 2003. Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces. Proc. Nat. Acad. Sci. U.S.A. 100, 15977e15982. Monier, J.-M., Lindow, S.E., 2005. Spatial organization of dual-species bacterial aggregates on leaf surfaces. App. Env. Microbiol. 71, 5484e5493. Niemira, B.A., 2007. Relative efficacy of sodium hypochlorite wash versus irradiation to inactivate Escherichia coli O157:H7 internalized in leaves of Romaine lettuce and baby spinach. J. Food Prot. 70, 2526e2532. Oliver, J.D., 2005. The viable but nonculturable state in bacteria. J. Microbiol. 43, 93e100. Pruvost, O., Boher, B., Brocherieux, C., Nicole, M., Chiroleu, F., 2002. Survival of Xanthomonas axonopodis pv citri in leaf lesions under tropical environmental conditions and simulated splash dispersal of inoculum. Phytopathology 92, 336e346. Pruvost, O., Savelon, C., Boyer, C., Chiroleu, F., Gagnevin, L., Jacques, M.-A., 2009. Populations of Xanthomonas citri pv. mangiferaeindicae from asymptomatic mango leaves are primarily endophytic. Microb. Ecol. 58, 170e178. Rigano, L.A., Siciliano, F., Enrique, R., Sendin, L., Filippone, P., Torres, P.S., Questa, J., Dow, J.M., Catagnaro, A.P., Vojnov, A.A., Marano, M.R., 2007. Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri. Mol. Plant Microbe Interact. 20, 1222e1230. Roberts, P.D., Chamberlain, H.L., Chung, K.R., Schubert, T.S., Graham, J.H., Timmer, L.W., 2004. 2005 Florida Citrus Pest Management Guide: Citrus Canker. University of Florida IFAS Extension PP-182. Rugh, A.L., Rosenberger, D.A., 2005. Quaternary Ammonium Sanitizer Eliminates Airborne Spores of Penicillium Expansum. Northeastern Divisional Meeting October 5e7, 2005-Geneva. American Phytopathological Society, New York. http://www.apsnet.org/members/divisions/ne/meetings/Pages/ 2005MeetingAbstracts.aspx. Sawant, A.A., Hegde, N.V., Donaldson, S.C., Buck, K.B., Jayarao, B.M., 2008. Bactericidal efficacy of quaternary ammonium compounds against species of bacteria isolated from feces of dairy cattle. J. Anim. Sci. 81, 132 (S524). Schubert, T.S., Sun, X., 2003. Bacterial Citrus Canker. In: Plant Pathology Circular No, vol. 377. Fl. Dept. of Agriculture and Cons. Svcs. Division of Plant Industry, Gainesville, Fl, 6 pp. Solomon, E.B., Yaron, S., Matthews, K.R., 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. App. Env. Microbiol. 68, 397e400. Sutton, S.V.W., Proud, D.W., Rachui, S., Brannan, D.K., 2002. Validation of microbial recovery from disinfectants. PDA J. Pharm. Sci. Tech. 56, 255e266. Teviotdale, B.L., 1991. Further tests on effectiveness of disinfectants for preventing transmission off fireblight. Plant Prot. Q. 1, 5e6. Kearney Plant Protection Group, Cooperative Extension, University of California. U.S. Food and Drug Administration, 2001. Analysis and evaluation of preventative control measures for the control and reduction/elimination of microbial hazards on fresh-cut produce, chap V. Methods to reduce/eliminate pathogens from fresh-cut produce Available at. http://www.cfsan.fda.gov/wcomm/ift3e5.html. Wilson, M., Lindow, S.E., 1992. Relationship of total viable and culturable cells in epiphytic populations of Pseuedomonas syringae. App. Env. Microbiol. 58, 3908e3913. Wilson, M., Hirano, S.S., Lindow, S.E., 1999. Location and survival of leaf-associated bacteria in relation to pathogenicity and potential for growth within the leaf. App. Env. Microbiol. 65, 1435e1443.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Infection and decontamination of citrus canker-inoculated leaf surfaces C.H. Bock a, *, P.E. Parker b, A.Z. Cook b, J.H. Graham a, T.R. Gottwald c a
University of Florida-IFAS-CREC, 700 Experiment Station Rd., Lake Alfred, FL 33850, USA USDA-APHIS-PPQ, Moore Air Base, Edinburg, TX 78539, USA c USDA-ARS-USHRL, 2001 S. Rock Rd., Ft. Pierce, FL 34945, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2010 Received in revised form 29 September 2010 Accepted 8 October 2010
Citrus canker (Xanthomonas citri subsp. citri, Xcc) is now considered endemic in Florida and continues to spread. Various surfaces, including plant material, personnel and equipment can become contaminated. Decontamination is practiced in both disease-endemic and disease-free areas to reduce the risk of bacterial spread by man or machinery. We used grapefruit leaf surfaces to explore the efficacy of a commonly used personnel sprayer system applying a quaternary amine decontaminant. In three experiments, plants in flush (leaves 3/4 expanded) were inoculated (w105 CFU ml1). Immediately after inoculation, plants were passed through the sprayers 0, 1, 2, 3, or 6 times. Leaves were sampled at 0.5, 10 and 20 min after decontamination and tested for viable Xcc by dilution plating. There was a large and rapid decline in the quantity of live bacteria with one pass through the decontamination sprayer (>80% decrease in CFU ml1), and multiple sprays (2e6) resulted in up to 100% mortality of surface Xcc. Presumably more thorough coverage with multiple sprays killed remnant bacteria, although the first spray invariably caused the highest proportion of population mortality. The effect of the decontaminant spray was immediate (within 0.5 min only 3e11% of surface bacteria survived, and by 20 min <1e3% survived). Based on these results, use of a personnel sprayer with a quaternary amine compound is highly effective for reducing surface inoculum. A single spray kills a high proportion of the population, but multiple sprays increase mortality of Xcc. All the Xcc-inoculated plants subsequently developed symptoms of citrus canker. No significant difference in incidence or severity of grapefruit leaf infection was detected among decontamination treatments or compared to the untreated control. This finding indicates that infection occurred at, or very soon after, inoculation, and that Xcc was in protected sites inside the leaf before exposure to the decontaminant spray. Published by Elsevier Ltd.
Keywords: Epidemiology Infection Citrus canker Inoculum Disease spread Quaternary ammonium Amines Personnel sprayers
1. Introduction Citrus canker (Xanthomonas citri subsp. citri, Xcc, (ex Hasse) Gabriel et al.) is endemic in Florida and is dispersed in rain splash often associated with wind (Gottwald and Graham, 1992; Pruvost et al., 2002; Bock et al., 2005 and Bock et al., 2010a; Gottwald and Irey, 2007). The pathogen can also be spread by people directly, and potentially on various surfaces including plant material, clothing, and various equipment or implements (Graham et al., 1989, 2000; Gottwald et al., 1992). Contamination of these surfaces might occur by brushing against wet canker-infected leaves after heavy dew or rain, and during orchard management such as pruning, or fruit harvest.
* Corresponding author. Present address: USDA-ARS-SEFTNRL, 21 Dunbar Road, Byron, GA 31008, USA. Tel.: þ1 478 956 6421; fax: þ1 478 956 2929. E-mail address: [email protected] (C.H. Bock). 0261-2194/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.cropro.2010.10.001
Once dispersed and on a susceptible leaf surface, various factors are thought to play a role in infection including direct access of the pathogen into stomata through the force of wind-blown rain splash (Gottwald and Graham, 1992; Graham et al., 1992), injury (Christiano et al., 2007; Bock et al., 2010b), and temperature and leaf wetness duration (Dalla Pria et al., 2006; Christiano et al., 2009). Infection with Xcc is thought to occur when bacteria gain access to the leaf through stomata, and infecting host cells proximal to the endophytic population (Koizumi, 1976a, Koizumi, 1976b, Koizumi, 1977; Gottwald and Graham, 1992; Graham et al., 1992). Thus access to the stomata might occur immediately upon arrival of the bacteria on the leaf surface when they are driven into the stomata by rain splash (Gottwald and Graham, 1992) or over a period of several hours (Dalla Pria et al., 2006; Christiano et al., 2009), perhaps from a population of Xcc existing in the phyllosphere (Rigano et al., 2007). Bacteria are common inhabitants of leaf surfaces and often form epiphytic biofilms (Beattie and Lindow, 1995; Lindow and Brandl, 2003; Monier and Lindow, 2005).
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Epiphytic biofilms of bacteria that are also endophytes are thought to be involved in survival, and possibly infection (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009). Surface decontamination removes a proportion of bacteria associated with the phyllosphere e those on the surfaces that are exposed. Bacteria in protected sites such as the sub-stomatal cavity or in the mesophyll are not exposed to the disinfecting agent (Wilson et al., 1999; Niemira, 2007; Pruvost et al., 2009), or where bacteria have had time to form a biofilm (Beattie and Lindow, 1995; Rigano et al., 2007). Surface sterilization with various agents has been used to study development of epi- and endophytic populations of plant pathogenic and non-pathogenic bacteria (Wilson et al., 1999; Pruvost et al., 2009), as well as to study human pathogens (USDA-FDA, 2001; Solomon et al., 2002; Niemira, 2007), and surface decontamination procedures have been shown to reduce bacteria populations on citrus (Brown and Schubert, 1987; Gottwald et al., 2009). A decontaminant treatment that kills surface bacteria of Xcc could provide insight into the earliest timing of infection in relation to arrival of inoculum on the leaf surface, while providing data to demonstrate the efficacy of surface spray application. Xcc has been shown to be ephemeral on surfaces, surviving up to 72 h (Graham et al., 1989, 2000). Nevertheless, to minimize the risk of spreading the pathogen via contaminated personnel or equipment, individuals and their implements are commonly surfacedisinfected before leaving a grove or facility where the presence of canker is known or suspected. Quaternary amines are recommended for this purpose (Schubert and Sun, 2003; Roberts et al., 2004). On commercial groves and in government and state facilities these disinfectants are applied with hand-held sprayers to hands and feet, and also via walk-through sprayers. Walk-through sprayers consist of spray nozzles on two vertical tubes that produce a mist of disinfectant that coats the individual and any items (for example, carts, notebooks, bags) taken through the sprayer system. The assumption is that spray coverage is sufficient to cause high mortality, or complete kill of Xcc. Vehicles and machinery are decontaminated with similar, but often more potent disinfectants (Roberts et al., 2004) using a hoop, or arch structure with sprayer nozzles positioned to produce a cloud of disinfectant through which the equipment is passed. Quaternary amines are very effective at killing microbial contaminants and many kinds of bacteria (McDonnell and Russell, 1999; Gamage, 2003), but they rapidly decompose as they come in contact with material in the environment. While the efficacy of some personnel decontaminants like quaternary amines, and various application systems have been investigated in the medical, veterinary and food sciences (Jeffrey, 1997; McDonnell and Russell, 1999; Amass et al., 2000; Sutton et al., 2002; Gamage, 2003; Goeres et al., 2004; Sawant et al., 2008), they do not appear to have received much attention in relation to decontamination or management of plant pathogens (Letal, 1977; Teviotdale, 1991; Chase, 1992; Draper et al., 2003; Rugh and Rosenberger, 2005). In situations where containment or eradication is important, it would be useful to have data to support the efficacy of application methods and decontaminants used to reduce the risk of pathogen spread. It is of particular importance to establish the effectiveness of various delivery systems for disinfesting surfaces of complex topography, such as citrus leaves. To investigate the efficacy of a personnel sprayer delivery system and a common quaternary disinfectant, we used healthy, injury-free, susceptible grapefruit leaf surfaces inoculated with Xcc and immediately exposed them to a personnel decontaminant, and measured surface populations of Xcc after treatment, and also assessed subsequent disease development on these plants. Citrus
leaves were chosen over other surfaces as there exists a substantial literature on surface decontamination of leaf surfaces for comparative purposes (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Lindow, 2009; Niemira, 2007; Rigano et al., 2007; Pruvost et al., 2009; ). The objectives were to ascertain the efficacy of the personnel sprayer delivery system for killing surface bacteria of Xcc, and to gain some insight into whether bacteria gained access to protected sites at the time of inoculation. 2. Materials and methods 2.1. Experiment plan and procedure Three replicate experiments examined surface decontamination (experiments I, II and III). In all three experiments, inoculum of Xcc was grown on a semi-selective media composed of Nutrient Agar (NA) amended with kasugamycin (16 mg L1), cephalexin (35 mg L1) and chlorothalanil (12 mg L1 tetrachloroisophthalonitrile). Colonies were maintained in an incubator at 27 C for five days. Inoculum (105 bacteria ml1) was prepared in sterile distilled water (Table 1). In all experiments, Duncan grapefruit plants (Citrus x paradisi Macfad.) in flush (leaves 3/4 expanded) were sprayed to run-off with inoculum of Xcc prepared as above. Immediately after inoculation (30 s) the plants were exposed to a decontaminant disinfectant spray (Canker GuardÒ, Flo-Tec, Largo, FL; active ingredients are a proprietary blend of two quaternary amines, alkyl-dimethylbenzyl-ammonium chloride and alkyl dimethyl ethylbenzyl ammonium chloride). The decontamination spray was applied by passing the plants at a slow walk (approx 0.7 m s1) through a standard personnel decontamination spray system (Fig. 1) ensuring good droplet coverage of the plants with each pass. Three sprayers (Conejet X1, Spraying Systems Co., Wheaton, IL) arranged vertically on either side provided the spray (at heights of 60, 100 and 140 cm, with 76 cm horizontal distance between pairs of nozzles). Three replicate plants were passed through the decontaminant sprayer 1, 2, 3 or 6 times at a height of 120 cm. An additional set of plants was not passed through the sprayer, but were inoculated. These plants served as the positive control. Subsequent to passing the plants through the sprayer, a single leaf of approximately the same size was sampled from a plant receiving each spray treatment at 0 (no decontaminant spray), 0.5 min (immediately after the decontaminant spray), 10 and 20 min after the decontamination process. Each leaf was placed in a 50 ml tube containing 40 ml sterile distilled water and vortexed for 5 s (Xcc bacteria are naturally dispersed in rainwater, which has few dissolved impurities and therefore a low osmotic potential akin to distilled water). Bacteria recovered in the leaf wash were dilution plated on the semi-selective medium as described above. After incubation for 5 days at 27 C, the colony forming units (CFU) were counted and bacteria ml1 calculated. In two experiments (II and III) the plants were transferred to a greenhouse immediately after exposure to the decontaminant spray (greenhouse venting set to 27 C, heating set to 20 C). Inoculum and disinfectant were
Table 1 Dates and concentration of Xcc inoculum in sterile distilled water used for each experiment. Plants were sprayed to run-off with inoculum using a hand-held sprayer. Inoculum concentration was confirmed by dilution plating on KCB nutrient agar. Expt. no.
Date
Inoculum conc. (Xcc bacteria ml1)
I II III
19 Dec 2007 22 May 2008 23 Aug 2008
1.7 105 1.5 105 8.6 105
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261
Table 2 Characteristics of the exponential relationships between CFU ml-1 of Xanthomonas citri subsp. citri surviving on a leaf surface of Duncan grapefruit after different numbers of passesa through a personnel decontamination sprayer. Parametersb (y ¼ ae–bx)
Expt. no.
I II III
a
b
100.0 100.0 99.9
2.14 1.67 2.02
F-value (Pr>F)
R2c
7602 (<0.0001) 4609 (<0.0001) 886 (<0.0001)
>0.99 >0.99 >0.99
a
Control treatment received no disinfectant sprays. Parameters shown are for normalized bacteria counts, where for each sample the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment (Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment (Fig. 3B). c R2 ¼ coefficient of determination. b
Fig. 1. A personnel sprayer system used to decontaminate individuals, clothing and sundry equipment exposed to citrus canker showing the nozzles placements. Plants were carried through at waist height.
allowed to dry on the leaf surfaces in the greenhouse; it took approx. 1.5e2.0 h for the visible moisture to evaporate. After 28 days, symptoms of citrus canker were assessed visually. Disease incidence (% leaves infected) and disease severity on each leaf (% leaf area infected) on each plant was calculated.
2.2. Data analysis The relationship between number of CFU ml1 surviving and numbers of decontamination sprays (averages of spray number across sample times), and between CFU ml1 surviving with time after exposure to the decontamination treatment (averages of sample time across number of decontamination sprays) were analyzed using regression. An exponential function (y ¼ e(abx)) was
30000 25000 20000
There was a large decline in the CFU of Xcc with one pass through the decontaminant spray for all times of exposure (Fig. 2A). Decontaminant sprays (from one to six) resulted in up to 100% mortality of the recoverable, culturable population of Xcc associated with the surface of these citrus leaves. Different numbers of bacteria were isolated from leaves in the three experiments, but the relationship between the normalized canker populations and passes through the decontaminant spray was very similar for all three experiments, and was well described by an exponential function (Fig. 2B, Table 2). Thus rate of bacteria mortality declined with increasing numbers of sprays. After a single spray only 12e19% of the original surface population survived, after two sprays the remaining population was 0.02e6% that before the decontaminant
B Experiment I Experiment II Experiment III
15000 10000 5000 0
3. Results
0 1 2 3 6 Number of decontaminant sprays
Percent of maximum CFU
CFU (bacteria of Xcc ml–1)
A
fit to the normalized quantity of bacteria. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. The disease incidence and severity on plants inoculated with Xcc but receiving different numbers of decontamination sprays was compared using general linear modeling and a post hoc means separation using Tukey’s HSD test (P ¼ 0.05). The standard error of the mean for each treatment was calculated as the sample estimate of the population standard deviation divided by the square root of the sample size. All analyses were performed in SAS (SAS Systems, Cary, NC).
100
Experiment I Experiment II Experiment III Exp.fit, expt. I Exp.fit, expt. II Exp. fit, expt. III
80 60 40 20 0
0
1 2 3 4 5 6 7 Number of decontaminant sprays
Fig. 2. A. The quantity of bacteria of Xanthomonas citri subsp. citri on leaf surfaces of Duncan grapefruit with no exposure, one, two, three and six passes through a personnel decontaminant sprayer (results for three experiments shown). B. The exponential relationship between percent bacteria survival and number of passes through the personnel sprayers for each experiment. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, where Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment.
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spray was applied, and after six sprays, it was either zero or less than 1% of the control. The longer the period between disinfection and sampling, the lower the cfu of surviving bacteria (Fig. 3A). In the three experiments, within 0.5 min of the decontaminant spray only 3e11% of the population of bacteria of Xcc survived, and by 20 min only 0.5e3% remained. The rapid decline in the proportion of Xcc surviving was described by an exponential model in experiment II and III (Fig. 3B, Table 3). In experiment I, several missing values for time 0.5 min precluded fitting a model to these data, although the existing data support a similar relationship. The quantity of Xcc isolated from leaf surfaces of untreated plants at spray application, 10 and 20 min after application of the decontaminant showed no decline in the surface population for experiment I, II or III (Fig. 4). There was no significant effect of the decontamination spray on the development of symptoms of canker on the treated plants compared to the untreated control (Fig. 5AeD). Application of a decontaminant disinfectant did not appear to significantly reduce incidence or severity of infection in experiment II (F ¼ 0.70, P ¼ 0.62 and F ¼ 0.69, P ¼ 0.67, respectively) or III (F ¼ 0.35, P ¼ 0.84 and F ¼ 0.11, P ¼ 0.98, respectively). 4. Discussion Use of the personnel decontamination system on leaf surfaces resulted in high mortality of Xcc, despite the leaf surfaces being spray-inoculated to run-off with bacterial inoculum. This appears to be the first report of the efficacy of this type of delivery system for a surface decontaminant aimed at providing a thorough spray coverage to achieve a high level of kill for a plant pathogen on a surface. Although personnel decontamination sprayers are not widely used in plant pathogen decontamination situations, the citrus canker epidemic is one case where they have been recommended (Schubert and Sun, 2003; Roberts et al., 2004). We found >80% of the bacteria isolated and cultured from the heavily contaminated leaf surfaces were killed with a single pass through the personnel sprayer. With two or more passes through the sprayer, up to 100% mortality of surface bacteria was achieved. High mortality of surface bacteria on leaves and other plant surfaces has been demonstrated before using various other decontaminants and delivery systems (Brown and Schubert, 1987; Wilson et al., 1999; Solomon et al., 2002; Niemira, 2007; Gottwald et al., 2009; Pruvost et al., 2009). Presumably the sprayers ensured that the surfaces were well covered by the three-dimensional spray cloud of
CFU (bacteria of Xcc ml–1)
A
Expt no.b
II III
Parametersc (y ¼ e(a-bx)) a
b
100.0 100.0
4.42 6.71
F-value (Pr>F)
Rd,c
507 (0.002) 226 (0.0044)
>0.99 >0.99
a Data for time 0 (control data) received no disinfectant sprays and is included in the analysis. b A regression solution was not fit to data from experiment 1 due to missing data. c Parameters shown are for the normalized bacteria counts, where each sample is a percentage of the maximum (Q) collected in that experiment (Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. d R2 ¼ coefficient of determination.
disinfectant that contacted upper and lower surfaces of leaves regardless of orientation. Quaternary amines are known to be highly effective disinfectants against many bacteria (McDonnell and Russell, 1999), and several different types are available for decontamination of personnel or implements exposed to canker bacteria (Roberts et al., 2004). Although the first spray invariably killed the most surface bacteria, the observation that multiple sprays killed remnant, surviving bacteria is significant. The relationship between surviving Xcc population and passes through the decontaminant sprayer was described by an exponential model; thus there was significant efficacy of additional sprays for killing surface bacteria. A spray “tunnel” with two or more spray systems would provide greater mortality of surface bacteria. By six passes the number of surviving bacteria isolated was in some cases zero. In addition, the greater the period after application of the disinfectant contact prior to sampling, the lower the bacterial survival, although the greatest proportion of the bacterial population was killed within 0.5 min (>88%), compared to the control. Having established the efficacy of quaternary amines against Xcc for decontamination of citrus leaf surfaces, this evaluation should be extended to include other materials including those with porous or absorbent surfaces such as clothing or boots, and hard surfaces including plastic or metal. An unexpected observation from this study was the incidence of canker that developed in plants after spray treatments was not diminished despite the high efficacy for killing surface bacteriawithin
B
35000 30000
Experiment I
25000
Experiment III
Experiment II
20000 15000 10000 5000 0
Table 3 Characteristics of the exponential relationships between actual CFU ml-1 of Xanthomonas citri subsp. citri surviving on leaf surfaces of Duncan grapefruit and time since inoculation after passinga through the decontaminant spray.
0.5 10 20 0 Time since decontaminant spray (min)
Percent of maximum CFU
262
100 80 60
Experiment I Experiment II Experiment III Exp. fit expt. I Exp. fit exp. III
40 20 0
0 5 10 15 20 Time since decontaminant spray (min)
Fig. 3. A. The quantity of bacteria of Xanthomonas citri subsp. citri on leaf surfaces of Duncan grapefruit at different times after inoculation (data from no exposure, one, two, three and six passes through a personnel decontaminant sprayer (results for three experiments shown). B. The exponential relationship between percent bacteria survival and time after inoculation for each experiment. For each sample, the bacteria counts were normalized as a percentage of the maximum (Q) collected in that experiment, where Q ¼ n/nmax 100, where n is the quantity cultured in each sample in the experiment, and nmax is the maximum quantity sampled in that experiment. A regression solution was not fit to data from experiment 1 due to missing data.
5
Expt I
Expt II
Expt III
4 3 2 1 0
10 20 0 Time since decontaminant spray
Fig. 4. The quantity of bacteria of Xanthomonas citri subsp. citri re-isolated from leaf surfaces of control plants that did not receive a quaternary amine disinfectant spray at time 0 (time of decontaminant spray), 10 and 20 min after application of the decontaminant to treated plants. Results for the three different experiments, I, II and III are shown.
a short period. Some surface decontamination procedures have previously been shown to kill virtually all surface bacteria (Wilson et al., 1999; Solomon et al., 2002; Niemira, 2007; Pruvost et al., 2009). The time span of our study would be insufficient for bacteria to aggregate or form biofilms and perhaps be protected against a decontaminant as described for Xcc and other epiphytic bacteria (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009;). Such biofilms formed by bacteria are thought to be a survival strategy that several plant pathogenic bacteria have evolved to deal with stressful environments (Monier and Lindow, 2003, 2005; Jacques et al., 2005; Rigano et al., 2007; Lindow, 2009). If Xcc had no time to form a biofilm between inoculation and exposure to the decontaminant (a period of 30 s), then infection that led to disease likely took place immediately upon exposure of the leaf to inoculum. This can be deduced because mortality of the surface inoculum by the disinfectant did not appear to significantly reduce
Percent leaves infected
A 100
B
80 60 40 20 0
a
a
a
a
a
0
1
2
3
6
24 20 16 12 8 4 0
Number of decontaminant sprays
D
6 5 4 3 2 1 0
a 0
a 1
a 2
a 3
a 6
Number of decontaminant sprays
a
a
a
a
a
0
1
2
3
6
Number of decontaminant sprays Severity per leaf (% area infected)
Severity per leaf (% area infected)
C
263
incidence or severity of infection in either experiment compared to the control. Quaternary amines are directly disruptive to the cell membrane integrity so it is unlikely the bacteria were rendered “viable but non-culturable” (VBNC), as might occur in response to some pesticides or chemicals (Wilson and Lindow,1992; Oliver, 2005; Campo et al.,2009). Furthermore, evenin theevent that a VBNC stateis induced, it appears that onlya small proportionof a population has the opportunity to enter this state yet remain pathogenic (Campo et al., 2009). As the amount of disease among treatments was not different, these data suggest the bacteria responsible for infection were immediately protected from the decontaminant, i.e., in the substomatal cavity or mesophyll. These data provide evidence that bacteria of Xcc in splash or spray dispersed droplets apparently gain immediate access to the substomatal chamber (Gottwald and Graham, 1992; Graham et al., 1992; Pruvost et al., 2002; Bock et al., 2010b). In the studies of Gottwald and Graham (1992) and Graham et al. (1992) pressure was applied to ensure the inoculum gained access to the leaf through the stomata. Although, prolonged leaf wetness may contribute to greater infection of undamaged leaves (Dalla Pria et al., 2006; Christiano et al., 2009), and biofilms might play a role in this infection (Jacques et al., 2005; Rigano et al., 2007; Gudesblat et al., 2009), our results suggest the importance of immediate access of the Xcc bacterium to the inside of the leaf, even with relatively gentle inoculation using a pump-action sprayer. The spray inoculum from the pump-action sprayer may have sufficient force for the droplets to penetrate the stomata. Hence, the relative role of immediate, direct penetration of the Xcc bacterium through sprayed inoculum or splash, pressurized inoculation and the role of epiphytic Xcc deserves a rigorous comparative investigation. Finally, we saw no evidence of phytotoxicity due to the quaternary amines when applied using a gentle decontaminant spray system. However, applying the disinfectant directly with a hand-held sprayer to run-off at the recommended concentration caused phytotoxicity to grapefruit leaves (C. H. Bock, unpublished
Percent leaves infected
Log CFU (bacteria of Xcc ml–1)
C.H. Bock et al. / Crop Protection 30 (2011) 259e264
0.6 0.5 0.4 0.3 0.2 0.1 0.0
a
a
a
a
a
0
1
2
3
6
Number of decontaminant sprays
Fig. 5. . Incidence (percent leaves infected A,B) and severity (percent area infected per leaf, C,D) on leaves of grapefruit plants spray-inoculated with bacteria of Xanthomonas citri subsp. citri and immediately passed through a personnel decontamination sprayer 1, 2, 3 or 6 times. Data shown are for the two experiments, II (A,C) and III (B,D). Treatments with the same letter within each experiment are not significantly different from each other based on Tukey’s HSD test. The standard error of the mean for each treatment is indicated, and was calculated as the sample estimate of the population standard deviation divided by the square root of the sample size.
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observations). In conclusion, these personnel decontaminant systems using quaternary ammonium compounds are an effective way of decontaminating at least some surfaces of Xcc. Other common materials should be tested using this system to confirm its efficacy in different situations. Acknowledgements This research was supported by the Florida Citrus Production and Research Advisory Council. Any mention of a trademark or proprietary product does not constitute an endorsement or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. References Amass, S.F., Vyerberg, B.D., Ragland, D., Dowell, C.A., Anderson, C.D., Stover, J.H., Beaudry, D.J., 2000. Evaluating the efficacy of boot baths in biosecurity protocols. Swine Health Prod. 8, 169e173. Beattie, G.A., Lindow, S.E., 1995. The secret life of foliar bacterial pathogens on leaves. Annu. Rev. Phytopathol 33, 45e172. Bock, C.H., Graham, J., Gottwald, T.R., Cook, A.Z., Parker, P.E., 2010a. Wind speed effects on the quantity of Xanthomonas citri sub sp. citri dispersed downwind from canopies of grapefruit trees infected with citrus canker. Plant Dis. 94, 725e736. Bock, C.H., Graham, J.E., Gottwald, T.R., Cook, A.Z., Parker, P.E., 2010b. Wind speed and wind associated injury affect severity of citrus canker on Swingle citrumelo. Euro. J. Plant Pathol. 128, 21e38. Bock, C.H., Parker, P.E., Gottwald, T.R., 2005. The effect of simulated wind-driven rain on duration and distance of dispersal of Xanthomonas axonopodis pv citri from canker infected citrus trees. Plant Dis. 89, 71e80. Brown, G.E., Schubert, T.S., 1987. Use of Xanthamonas campestris pv. vesicatoria to evaluate surface disinfectants for canker quarantine treatment of citrus fruit. Plant Dis. 4, 319e323. Campo del, R., Russi, P., Mara, P., Mara, H., Peyrou, M., de Leon, I.P., Gaggero, C., 2009. Xanthomonas axonopodis pv. citri enters the VBNC state after copper treatment and retains its virulence. FEMS Microbiol. Lett. 298, 143e148. Chase, A.R., 1992. Bacterial Disease Control on Ornamentals Using Aliette, Kocide, Greenshield and ASC-66825. Research Report RH-92e10. Central Florida Research and Education Center, Apopka, University of Florida, Fl. Christiano, C., Dalla Pria, M., Jesus, W.C., Amorim, L., Bergamin-Filho, A., 2009. Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness. Euro. J. Plant Pathol. 124, 1e7. Christiano, C., Dalla Pria, M., Jesus, W.C., Parra, J.R.P., Amorim, L., Bergamin-Filho, A., 2007. Effect of leaf-miner damage, mechanical damage and inoculum concentration on severity of symptoms of Asiatic citrus canker in Tahiti lime. Crop Prot. 26, 59e65. Dalla Pria, M., Christiano, R.C.S., Furtado, E.L., Amorim, L., Bergamin Filho, A., 2006. Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker. Plant Pathol. 55, 657e663. Draper, M.A., Burrows, R., Mills, J., 2003. Fireblight of Apples, Pears and Other Species. FS916. South Dakota State University, Cooperative Extension Service, South Dakota, 4 pp. Gamage, B., 2003. A Guide to Selection and Use of Disinfectants. British Columbia Centre for Disease Control, British Columbia, Canada, 18 pp. Goeres, D.M., Palys, T., Sandel, B.B., Geiger, J., 2004. Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model. Water Res. 38, 3103e3109. Gottwald, T.R., Graham, J.H., 1992. A device for precise and nondisruptive stomatal inoculation of leaf tissue with bacterial pathogens. Phytopathology 82, 930e935. Gottwald, T.R., Graham, J.H., Bock, C.H., Bonn, G., Civerolo, E.L., Irey, M., Leite, R.P., et al., 2009. The epidemiological significance of post-packinghouse survival of Xanthomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Prot. 28, 508e524. Gottwald, T.R., Graham, J.H., Egel, D.S., 1992. Analysis of foci of Asiatic citrus canker in a Florida citrus orchard. Plant Dis. 76, 389e396. Gottwald, T.R., Irey, M., 2007. Post-hurricane analysis of citrus canker II: predictive model estimation of disease spread and area potentially impacted by various eradication protocols following catastrophic weather events. Online. Plant Health Prog.. doi:10.1094/PHP-2007-0405-01-RS. Graham, J.H., Gottwald, T.R., Civerolo, E.L., McGuire, R.G., 1989. Population dynamics and survival of Xanthomonas campestris in soil in citrus nurseries in Maryland and Argentina. Plant Dis. 73, 423e427. Graham, J.H., Gottwald, T.R., Riley, T.D., Achor, D., 1992. Penetration through leaf stomata and strains of Xanthomonas campestris in citrus cultivars varying in susceptibility to bacterial diseases. Phytopathology 82, 1319e1325. Graham, J.H., Gottwald, T.R., Riley, T.D., Cubero, J., Drouillard, D.L., 2000. Survival of Xanthomonas campestris pv. citri on various surfaces and chemical control of
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