This study investigated survival of the pathogens Phytophthora ramorum, P. alni and P. kernoviae as zoospores or sporangia in response to an important water quality parameter, electrical conductivity (EC), at its range in irrigation water reservoirs and irrigated cropping systems. Experiments with different strengths of Hoagland’s solution showed that all three pathogens survived at a broad range of EC levels for at least 3 days and were stimulated to grow and sporulate at ECs > 1·89 dS m−1. Recovery of initial populations after a 14-day exposure was over 20% for P. alni subsp. alni and P. kernoviae, and 61·3% and 130% for zoospores and sporangia of P. ramorum, respectively. Zoospore survival of these pathogens at ECs < 0·41 dS m−1 was poor, barely beyond 3 days in pure water; only 0·3% (P. alni), 2·9% (P. kernoviae) and 15·1% (P. ramorum) of the initial population survived after 14 days at EC = 0·21 dS m−1. The variation in rates of survival at different EC levels suggests that these pathogens survive better in cropping systems than in irrigation water. Containment of run-off and reduction in EC levels may therefore be non-chemical control options to reduce the risk of pathogen spread through natural waterways and irrigation systems.
Zoosporic oomycetes, so-called water moulds, can be spread through natural waterways or irrigation water. In fact, P. ramorum was demonstrated to spread through an irrigation system and was detected in streams and effluents from plant production areas (Werres et al., 2007; Tjosvold et al., 2008; Chastagner et al., 2009; Sutton et al., 2009). Phytophthora alni has also been reported to grow and sporulate in river water (Chandelier et al., 2006). Phytophthora kernoviae is biologically and ecologically similar to P. ramorum, producing caducous sporangia (Brasier et al., 2005), and may be dispersed by rain, irrigation water splash or by recycled irrigation water.
The extent to which these species disperse through water depends upon their aquatic biology and ecology. Populations of Phytophthora species have been shown to decline with increasing distance from run-off entrance in irrigation water reservoirs (Hong et al., 2003), suggesting the presence of suppressive factors in the water. Studies on survival of human bacterial pathogens in water implies that water quality parameters such as pH, dissolved oxygen, electrical conductivity (EC) and turbidity may all contribute at certain levels to suppressing or killing these pathogens in retention ponds (Curtis et al., 1992; Fallowfield et al., 1996; Cirelli et al., 2008). For Phytophthora species, efforts have been made to dissect water quality parameters that may play a role in phytophthora decline, initially focusing on pH (Hong et al., 2009; Kong et al., 2009, 2011). Phytophthora alni, P. kernoviae and P. ramorum survived a wide range of pH for more than 14 days in a diluted Hoagland’s solution (Kong et al., 2011), which indicates that pH may not be a suppressive factor for survival of these pathogens.
Effects of other water quality factors on survival of Phytophthora species remain to be elucidated. As a major water quality factor, EC has been associated with disease severity (MacDonald, 1982, 1984; Blaker & MacDonald, 1986; Rasmussen & Stanghellini, 1988; Swiecki & MacDonald, 1988; Bouchibi et al., 1990; Nachmias et al., 1993). EC represents the ability of water to conduct an electric current, which in turn is associated with the concentration of total salt in solution. EC is not the same as salinity, which is the concentration of a specific dissolved salt such as NaCl. High levels of salinity (e.g. over 10 dS m−1) have been shown to promote mycelial growth of P. capsici, P. cryptogea and P. nicotianae, but suppress zoospore activity (Blaker & MacDonald, 1985; Swiecki & MacDonald, 1991; Sanogo, 2004). High to moderate levels of EC can occur in the field, although the effect on Phytophthora species is not clear. In the rhizosphere of irrigated plants, EC can reach as high as 5·0 dS m−1 (J. D. Lea-Cox, unpublished data). These root-zone EC levels could be even higher if poor-quality (alkaline or saline) irrigation water is used, or excessive rates of fertilizer are applied. On the other hand, EC levels in an irrigation reservoir are typically lower, between 0·16 and 0·87 dS m−1 depending on season and water resource. EC levels in pristine water are at the low end but those in nearby run-off containment ponds are high (Hong et al., 2009). Many Phytophthora species survived well in 10–15% Hoagland’s solution at EC = 0·21–0·31 dS m−1 (Kong et al., 2009, 2011), indicating that these species may be tolerant of lower EC aquatic environments. However, some Phytophthora species cannot germinate in pure water (Kong & Hong, 2010), which may be the result of a hypotonic response linked to osmoregulation, i.e. the active regulation of the osmotic pressure of an organism’s cellular solute concentration to maintain homeostasis. The responses of other species, including the three quarantine pathogens in the present study, to the extremely low EC levels which occur in pristine water is not known.
The objective of this study was to examine the survival of P. ramorum, P. alni and P. kernoviae in response to low to moderate EC levels (0–3·6 dS m−1). This range represents EC levels in irrigation reservoirs, in container production of ornamental species and run-off. As the major dispersal agents, zoospores of P. alni and P. kernoviae, and zoospores and sporangia of P. ramorum were tested in different strengths of Hoagland’s solution corresponding to a range of EC levels. Hoagland’s solution contains total salts required for plant growth, and was the medium used for testing the survival of Phytophthora in response to pH (Kong et al., 2009, 2011). Survival rates of zoospores or sporangia were determined by counting colony-forming units on agar media and microscopic observation of these propagules in response to different EC levels over a 14-day period. These results may be used for risk assessment of pathogen spread through water and identification of suppressive EC levels that could potentially be used to control pathogen populations in irrigation water.
Materials and methods
Sporangium and zoospore preparation
Four isolates of three Phytophthora species: P. alni subsp. alni, P. kernoviae and P. ramorum, were used in this study (Table 1). To produce zoospores or sporangia, agar plugs of 1-week-old or younger cultures were grown in 10% clarified V8 juice (CV8) broth for 7 days at 20°C. The mycelial mats were rinsed twice with chilled sterile distilled water (SDW) after the medium was removed. For zoospore induction, the mats were flooded with sterile soil water extract (SSWE) and incubated at 20°C overnight in the dark for P. alni, overnight under light for P. kernoviae, and 2 h under light for P. ramorum. Zoospores were harvested by passing suspensions through a layer of sterile Miracloth®. Only sporangia were prepared for P. ramorum 33F2 because zoospores of this isolate failed to grow after plating on medium. To obtain sporangia at >3000 mL−1, the mycelial mats were rinsed in SDW to facilitate sporangium detachment then filtered through a 125-μm sieve. Concentrations of zoospores or sporangia were determined with a haemocytometer. Zoospore suspensions at 5 × 103–105 or sporangium suspensions at 3100–3700 mL−1 were used as stocks for working suspension preparation.
Table 1. Phytophthora species used in this study
33F2 (Pr-102;ATCC MYA2440)
Solution preparation for electrical conductivity (EC) treatments
To exclude effects of other factors in natural irrigation water samples and generate reproducible results, Hoagland’s solution was used throughout this study. Full- and double full-strength solutions were prepared using Hoagland’s modified basal salt mixture (MPBio), adjusted to pH 7 with NaOH and filter-sterilized. The full-strength solution was then diluted to 5%, 10%, 20% and 50% with SDW at pH 7. EC levels of these solutions were determined by measuring aliquots with a water quality meter (Horiba U10, Environmental Equipment & Supply). Salt concentration of the full-strength solution was analysed at the JR Peters Laboratory (Table 2).
Table 2. Component concentrations of the full-strength Hoagland’s solutions at pH 7 in this study
Concentration (ppm) of componentsa
aElectrical conductivity 1·93 dS m−1.
Zoospore/sporangium survival was rated by colonization of propagules on agar medium and germination and further development in the test solutions. To determine colonization, the number of colony-forming units (CFU) of treated propagules was recorded after addition of a small amount of a freshly prepared stock suspension into a series of 100-mL EC test solutions in 175-mL tissue culture containers (Greiner Bio One). The volume of stocks to be added was based on the CFU value obtained after plating 1 mL of the final concentration of zoospores or sporangia in 10% Hoagland’s solution at pH 7, which resulted in 20–50 colonies on PARP-V8 agar (Ferguson & Jeffers, 1999) in a 90-mm Petri dish. This final concentration of zoospore suspension varied between species/isolates. For P. alni and P. kernoviae, it was about 60 zoospores mL−1, and for P. ramorum 32G2 it was 100 mL−1. The final concentration of sporangium suspension of P. ramorum 33F2 was only about 20 mL−1 as almost 100% of the sporangia colonized. For each treatment, 1-mL samples were taken at six exposure times, with the first sampling immediately after addition of the propagules (day 0), and then subsequently at 1, 3, 5, 7 and 14 days. These samples were spread on PARP-V8 agar and incubated at 20°C in a growth chamber. Colonies were counted within 2–3 days. To examine propagule behaviour in response to each EC level, 1-mL aliquots from each treatment were placed in individual wells of a 24-well plate. Germination and development were examined at each time point under an inverted light microscope (Olympus IX71).
Each experiment included seven EC levels and six exposure time points, and each treatment combination included three replicate containers in a completely randomized design. Two 1-mL samples were taken from each container for plating and microscopy. For each species or isolate, the experiment was repeated at least twice. To allow comparison among treatments, relative survival was rated by dividing CFU from each plate by the highest average CFU of a treatment at the first exposure time. The relative rates (%) from repeated experiments were pooled after homogeneity analyses and then subjected to proc anova of sas (SAS Institute, Inc.), to determine impacts of EC level and exposure time on zoospore and sporangia survival. Mean survival rates were separated by the least significant difference (LSD) at α= 0·01 confidence limit. The relationship between zoospore survival and EC was determined by multiple regression analysis. The explanatory power of the regression was determined by R2, and the statistical significance was determined by anova and t-Stat at the 95% confidence level.
All the experiments were conducted with extreme caution in a restricted laboratory (BL-1) as per USDA-APHIS permit #P526P-10-00732 using stringent protocols. All used non-disposable containers and tools were treated with 10% bleach, washed and autoclaved before re-use. All biological wastes including used suspensions, cultures, washing wastes, disposable containers and pipette tips were autoclaved before disposal. All open-lid handling associated with the cultures, including transfer, inoculation, rinse, filtration and plating, was performed in a certified Fisher Hamilton Class II Biosafety cabinet (Thermo Scientific). All closed-lid plates and containers were placed in a locked growth chamber in the restricted-access laboratory.
Effects of EC levels on survival of P. alni zoospores
Phytophthora alni zoospores favoured higher ECs for survival. Despite the fact that the population of zoospores declined significantly irrespective of EC after overnight exposure, the relative survival rates at higher ECs were generally higher than at lower ECs. Survival dependence on EC was more obvious after overnight exposure. A reduction in EC from 3·58 to 1·89 dS m−1 resulted in a decline of relative survival rates (Table 3). The rates further reduced at ECs below 0·41 dS m−1; after 3 days of exposure very few colonies emerged. When EC was 0·21 or lower, the survival rate dropped to zero within 5 days. The survival of zoospores of P. alni was closely related to EC in the system, as shown by the R2 value of 0·99.
Table 3. Relative zoospore survival (%) of Phytophthora alni, P. kernoviae and P. ramorum as affected by electrical conductivity (EC) in different strengths of Hoagland’s solution at pH 7
EC (dS m−1)
Relative survival (%) by exposure time (days)a
aWithin each species, the relative zoospore survival rate is a mean of nine replicates with two observations (plates) per replicate from three independent experiments. Relative zoospore survival rate was calculated by dividing the number of colonies counted in each plate by the mean colony count from the EC treatment with the greatest survival at the immediate exposure time within an experiment.
bSignificant level of differences in relative survival rates among the EC levels at the same exposure time and among the exposure times at the same EC level within individual species assessed.
cRelative survival rates followed by a different letter in each column or different numbers of asterisks differ significantly according to LSD test at α= 0·01.
P. ramorum (32G2)
Microscopic observation showed the same trend. Zoospores at EC levels above 0·96 dS m−1 germinated well (Fig. 1). Higher EC also supported growth of mycelia and reproduction of sporangia to allow longer survival of the pathogen (Fig. 2).
Effects of EC levels on survival of P. kernoviae zoospores
Survival of P. kernoviae zoospores in response to EC was similar to P. alni. The highest survival rate was found with the highest EC tested. However, differences among survival rates at ECs between 0·11 and 3·58 dS m−1 were smaller. After 3 and 5 days of exposure, these differences generally became insignificant (Table 3). Higher survival rates were found after a 7-day exposure at ECs from 0·96 to 3·58 dS m−1. Notably, the survival rate doubled at an EC of 3·58 after day 5, compared to that at EC = 1·89. As with P. alni, the survival of P. kernoviae was related to increasing EC level, although the overall R2 value across all treatments was lower (0·89). Specifically, the relationship between zoospore survival and EC was not significant between exposure times.
Phytophthora kernoviae germinated at all EC values, except at EC = 0 dS m−1. However, its germination was delayed compared to P. alni (Fig. 1). Higher EC stimulated hyphal growth and reproduction (Fig. 2). Sporangium production was observed after 3 days of exposure, at ECs higher than 0·41 dS m−1.
Effects of EC levels on survival of P. ramorum zoospores and sporangia
Two isolates of P. ramorum were tested because zoospores of the California isolate 33F2 failed to grow on plates. To understand survival of P. ramorum in response to different ECs, sporangia of this isolate were tested and zoospores of another isolate (32G2) were examined simultaneously and compared. Both isolates gave similar results in terms of response to different ECs, irrespective of propagules used. Unlike the other two species, zoospores of P. ramorum 32G2 had higher survival rates at an EC of 1·89 dS m−1 than at 3·58 dS m−1, although survival after 3 days of exposure was better at EC = 3·58 dS m−1 (Table 3). ECs between 0·11 and 1·89 dS m−1 had less impact on 32G2 zoospore survival at shorter exposure times, but an EC of 0·11 dS m−1 resulted in good recovery of the population over extended exposure times. Similarly, sporangia of 33F2 survived the best at 1·89 dS m−1 and were less sensitive to different salt concentrations, except for ECs lower than 0·11 (Table 4). They survived well over a large range of ECs and comparative survival rates were very high. The population recovered or increased at ECs higher than 0·21 dS m−1 after 3 days of exposure. The relationship between survival of P. ramorum and EC was not significant, with an R2 of 0·73.
Table 4. Relative sporangial survival (%) of Phytophthora ramorum as affected by electrical conductivity (EC) in different strengths of Hoagland’s solution at pH 7
EC (dS m−1)
Relative sporangial survival (%) by exposure time (days)a
aThe relative sporangial survival rate is a mean of nine replicates with two observations per replicate (plate) from three independent experiments. Relative survival rate was calculated by dividing the number of colonies counted in each plate by the mean colony count from the EC treatment with the greatest survival at the immediate exposure time within an experiment.
bSignificance level of differences in relative survival rates among the EC levels at the same exposure time and among the exposure times at the same EC level.
cThe relative survival rates followed by a different letter in each column or different numbers of asterisks differ significantly according to LSD test at α = 0·01.
Both zoospores and sporangia germinated at ECs higher than zero (Fig. 1). Germination occurred at an early stage of exposure. Rapid early growth, hyphal swelling and differentiation were observed after 5 days of exposure, particularly with higher EC. A decrease in growth of zoospores but not sporangia was observed over 14 days of exposure (Fig. 2).
This study provides evidence that P. alni, P. kernoviae and P. ramorum can survive over a wide range of EC values. Adaptation to different ECs indicates that these pathogens may spread widely not only in irrigation water reservoirs and natural waterways where the EC can vary from 0·16 to 0·87 dS m−1, but also within irrigated cropping systems and root environments where EC can be as high as 5 dS m−1. The adaptation to a wide range of ECs is necessary for the survival and spread of invasive Phytophthora species, because ECs fluctuate in aquatic environments (Hong et al., 2009). The fact that higher ECs promoted the survival of these pathogens suggests that they are well adapted to these conditions, and their populations can increase by attacking stressed plants and spread through run-off. Thus, production practices that have a higher EC may have a higher risk of introducing and spreading these pathogens. In contrast, lower EC levels may reduce this risk because low EC, especially values below 0·2 dS m−1, can significantly reduce the survival rates of these pathogens over time.
The survival of P. alni, P. kernoviae and P. ramorum in response to EC was very similar, although survival of P. alni appeared more vulnerable to lower EC (Fig. 2). Survival of zoospores of these species was facilitated by higher ECs, which prevent lysis and promote germination and formation of structures for continuing the life cycle (Figs 1 and 2). However, such effect did not reverse the inherent ability of zoospores to survive. In a previous study, the Californian P. ramorum isolate 33F2 failed to form colonies at any concentrations on plates or rhododendron leaves, although it produced a huge number of zoospores (Kong et al., 2011). In this study it also failed to colonize at any tested EC levels (data not shown). On the other hand, survival of sporangia may be more adaptive. Both isolates of P. ramorum produced a huge number of caducous sporangia with high colonization and survival rates. Interestingly, it was found that fresh sporangia of P. ramorum were able to float on the water surface for at least 8 h, which could increase the likelihood of this propagule being dispersed by water flow. Other propagules such as chlamydospores may be superior to zoospores and sporangia as natural survival structures in water. However, they were not tested in this study because of inefficient isolation techniques for adequate purity and concentration.
The propagules focused on in this study could become threats to nursery production and natural forests downstream of the production area because of their strong adaption to a range of aquatic situations. The findings of this study provide two potential strategies for prevention and management of spread of these three pathogens in irrigation water systems. First, containment of high-EC run-off water that may be contaminated with these pathogens. Higher EC facilitates sedimentation as well as formation of structures that settle out of the water column more easily than zoospores or cysts. Sedimentation may reduce the risk of pathogen spread in the water by providing ecologically different factors within the water column such as bacteria and low dissolved oxygen levels. Another strategy is to reduce EC levels in irrigation water to reduce survival time of the pathogens. EC in pristine water is normally low, below 0·2 dS m−1, but that of recycled irrigation water is much higher. There are some integrated methods that could be used to reduce EC in recycled irrigation water. These methods include constructed wetlands (White et al., 2010) or using a sedimentation area prior to the recycling pond to contain all the run-off from a production facility. Additionally, run-off water in the containment pond can also be blended with pristine water or run through biofilters to increase the efficacy of these treatments. Investigation into additional water quality parameters which may affect survival of these pathogens is warranted, to identify further pathogen management strategies.
Using different strengths of Hoagland’s solution to investigate the responses of these three pathogens to different levels of EC may be artificial but is the best choice at present. Because the salt composition includes nutrients required for plant growth, and different strengths of the solution constitute corresponding levels of EC, this system facilitates comparison of different EC levels against the same background of, for example, pH, to generate reproducible results. pH variation can affect EC levels and zoospore survival. For example, the EC of 10% Hoagland’s solution at pH 7 is 0·22 dS m−1, but more than doubles at pH 3 (0·68 dS m−1) and is 50% higher at pH 11 (0·31 dS m−1). Despite the fact that they favour higher EC levels, zoospores of P. ramorum cannot survive at pH 3 and those of P. kernoviae were sensitive to pH 11 (Kong et al., 2011). On the other hand, many technical problems may be encountered if irrigation water is used. First, a single water sample would require artificial modification to create a range of EC levels for testing. Secondly, water samples taken at different times and locations may have different EC levels but the salt composition, pH and other aspects of composition may be different. Thirdly, there are many unknowns in natural water samples which could lead to different results and outweigh the impact of EC. It should be noted that the same EC may be produced from different concentrations of the same components. For example, EC = 0·22–0·23 dS m−1 in 10% Hoagland’s solution can result from Na concentrations of 0·396–5·92 ppm by pH adjustment between pH 5 and 9. Also, the effects of EC do not allow conclusions to be made about tolerance of salinity. Phytophthora kernoviae zoospores were tolerant of high salinity with Na levels as high as 24·9 ppm and Cl levels as high as 88·9 ppm (pH 3), while the EC level of the solution was relatively low (0·68 dS m−1) (Kong et al., 2011). On this point, Hoagland’s solution may be a suitable medium to elucidate the effect of individual components of irrigation water by manipulating their concentration in solution.
This study is supported in part by a grant from the National Institute of Food and Agriculture -Specialty Crop Research Initiative of United States Department of Agriculture (agreement no. 2010-51181-21140). The authors would like to thank Clive Brasier (Forest Research, UK), M. Garbelotto (University of California, Berkeley, USA) and Steven Jeffers (Clemson University, USA) for providing the cultures.