MICROBIOLOGY LETTERS. Introduction RESEARCH LETTER. Ping Kong 1, John D. Lea-Cox 2, Gary W. Moorman 3 & Chuanxue Hong 1. Abstract

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1 RESEARCH LETTER Survival of Phytophthora alni, Phytophthora kernoviae, and Phytophthora ramorum in a simulated aquatic environment at different levels of ph Ping Kong 1, John D. Lea-Cox 2, Gary W. Moorman 3 & Chuanxue Hong 1 1 Virginia Tech, Hampton Roads Agricultural Research and Extension Center, Virginia Beach, VA, USA; 2 Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA; and 3 Department of Plant Pathology, The Pennsylvania State University, University Park, PA, USA MICROBIOLOGY LETTERS Correspondence: Ping Kong, Virginia Tech, Hampton Roads Agricultural Research and Extension Center, Virginia Beach, VA 23455, USA. Tel.: ; fax: ; pkong@vt.edu Received 7 February 2012; revised 9 April 2012; accepted 12 April Final version published online 3 May DOI: /j x Editor: Jan Dijksterhuis Keywords quarantine Phytophthora species; zoospore survival; water ph. Introduction Abstract Phytophthora alni (Brasier et al., 2004), Phytophthora kernoviae (Brasier et al., 2005), and Phytophthora ramorum (Werres et al., 2001) are three pathogens of forests and ornamental production areas. Phytophthora ramorum, known for Sudden Oak Death (Rizzo et al., 2002), has been found in Europe and United States since the late 1990s. Phytophthora alni, causing alder mortality in Britain (Brasier et al., 1995) is widespread across Europe (Brasier et al., 2004; Cerny et al., 2008; Solla et al., 2010) and has recently been found in the USA (Schwingle et al., 2007; Adams et al., 2008). Phytophthora kernoviae has been reported in the United Kingdom and shares symptoms and hosts with P. ramorum (Brasier et al., 2005; Ramsfield et al., 2009). The identification and spread of these pathogens has led to increasing concern about their threat to plant biosecurity and natural ecosystems. The horticultural trade has been identified as a major route for pathogen introduction to new areas, as was Phytophthora ramorum, Phytophthora alni, and Phytophthora kernoviae present significant threats to biosecurity. As zoosporic oomycetes, these plant pathogens may spread through natural waterways and irrigation systems. However, survival of these pathogens in aquatic systems in response to water quality is not well understood. In this study, we investigated their zoospore survival at ph 3 11 in a 10% Hoagland s solution over a 14-day period. The results showed that all three pathogens were most stable at ph 7, although the populations declined overnight irrespective of ph. Extended survival of these species depended on the tolerance of ph of their germinants. Germinants of P. alni ssp. alni and P. ramorum were more basic tolerant (ph 5 11), while those of P. kernoviae were more acidic tolerant (ph 3 9). These tolerant germinants formed compact hyphae or secondary sporangia to allow longer survival of these pathogens. Long-term survival at a broad ph range suggests that these pathogens, especially P. ramorum, are adapted to an aquatic environment and pose a threat to new production areas through water dispersal. demonstrated in the case of P. ramorum (Lane et al., 2003). However, some pathogens could also spread through wind, runoff, and irrigation water (Campbell, 1999; Hong & Moorman, 2005). Phytophthora ramorum has been detected in streams and effluents of irrigation systems and demonstrated to be spread through an artificial irrigation system (Werres et al., 2007; Tjosvold et al., 2008; Chastagner et al., 2009). Phytophthora alni also has been shown to be able to grow and sporulate in river water (Chandelier et al., 2006). Phytophthora kernoviae is biologically and ecologically similar to P. ramorum (Brasier et al., 2005), thus it may be capable of dispersal by water splash or by irrigation water recycling. Zoospores are believed to be relatively short-lived but how they manage to disperse in irrigation water and natural water ways within their life span is not clear. In fact, some Phytophthora species are continuously recovered from aquatic environments despite the fact that their populations decline with increasing distance from the entrance of runoff water in irrigation reservoirs

2 Survival of quarantine Phytophthora in response to ph 55 (Hong et al., 2003; Hong & Moorman, 2005; Werres et al., 2007; Tjosvold et al., 2008; Chastagner et al., 2009). It has been found that there are diurnal and seasonal fluctuations in ph from 6.5 to 10.3 in irrigation water reservoirs (Hong et al., 2009). Apparently, zoospores or other life stages have to adapt to a wide range of ph in order to survive in and be dispersed by water. Zoospores of some species, such as Phytophthora megasperma, can survive from ph 3 to 11 for a long period, while other species are sensitive to extreme acidic or basic conditions (Kong et al., 2009). For many other zoosporic pathogens, including P. alni, P. kernoviae, and P. ramorum, such information is missing. The objective of this study was to examine the survival of P. alni, P. kernoviae, and P. ramorum in response to different levels of ph. Specifically, zoospores were tested over a range of ph from 3 to ph 11 in 10% Hoagland s solution. Responses of these pathogens to ph were determined by relative survival rates measured as colonyforming units (CFU) and behaviors of zoospores measured as relative counts of swimming zoospores, encysted zoospores (cysts), and germinating zoospores. Materials and methods Preparation of ph solutions Ten percent Hoagland s solution (HS) used for tests was prepared as follows. The stock HS solutions were made using Hoagland s basal salt mixture (MP Bio, OH), ph-adjusted with NaOH or HCl, and then filtersterilized. Precipitation observed for stock solutions at ph 9 and 11 was removed through filtration. The ph solutions were used immediately or stored at 4 C until used. Sterile distilled water (SDW) to be used for dilution was also ph-adjusted with NaOH or HCl. To obtain 10% HS solution with appropriate ph at 3, 5, 7, 9, or 11, a stock solution was diluted with SDW with the same ph. Solutions were tested for the total concentration of salts and dissolved individual ions by JR Peters Laboratory (Allentown, PA) (Supporting Information, Table S1). Zoospore survival assay Zoospore survival of P. alni, P. kernoviae, and P. ramorum isolates (Table 1) was assessed with colony-forming units of zoospores (CFU ml 1 ) at each test ph and exposure time and zoosporic behavior at each test ph up to 24 h after exposure. Stock zoospore suspensions were prepared through a liquid culture for 7 days followed by sporangia induction and zoospore release as described previously (Kong et al., 2012). To determine CFU in response to ph, a volume of fresh zoospore stock was Table 1. Phytophthora species used in this study Species Isolates Host Origin Supplier* P. alni ssp. alni 32J6 Alnus glutinosa France C. Brasier (P834) P. kernoviae 38E3 Fagus sylvatica England C. Brasier (P1590) P. ramorum 32G2 Camellia SC, USA S. Jeffers (4398) *Original reference number of the isolates. added to diluted HS in a 175-mL tissue culture container (Greiner Bio One, Monroe, NC) to make 100 ml of 10% HS so that there were about 50 zoosporic colonies when 1 ml was placed in a 90-mm Petri dish. Each treatment included three replicate containers. Samples were taken immediately after the addition of the zoospore suspension stock solution (day 0) and after 1, 3, 5, 7, and 14 days incubation. At each time point, two 1-mL aliquots were taken from each container and spread onto two 90-mm Petri dishes containing PARP-V8 agar (Ferguson & Jeffers, 1999). Dishes were incubated at 20 C in a growth chamber for 2 3 days and emerging colonies were counted. To examine the behavior of zoospores in each ph treatment, 1-mL samples were also taken from each treatment container as noted previously at the first time point (day 0) and placed in wells of a 24-well plate. Zoospore motility, encystment, germination, and lysis were observed using an inverted light microscope (Olympus IX71) at 2 and 24 h after placement in three replicate wells. Two microscope fields at 1609 magnification were examined. Motile zoospores, cysts, and germinating spores in fixed fields were counted separately. All the experiments were conducted as federally required in a restricted laboratory under USDA-APHIS permit #: P526P as described in the previous work (Kong et al., 2012). Statistics To calculate relative survival rates of zoospores or sporangia of an isolate, CFU in each dish was divided by the highest average CFU of a treatment at the first exposure time. The rates from repeated experiments were pooled after homogeneity analyses and then subjected to PROC ANOVA (SAS Institute, Inc., Cary, NC). Mean survival rates were separated by the least significant difference (LSD) at a = These rates were used to calculate population survival or survival index (the sum of survival rates at each exposure time 9 corresponding exposure time divided by the longest exposure time, for example 14, in which exposure time on day 0, 1, 3, 5, 7, and 14 was FEMS Microbiol Lett 332 (2012) ª 2012 Federation of European Microbiological Societies

3 56 P. Kong et al. weighted as 1 2, 3, 5, 7, and 14, respectively). Survival index was used to assess the overall survival ability of each test species population. To determine the effect of ph on zoospore behavior, relative counts of swimming zoospores, cysts, and germinating cysts in six microscopic fields at 1609 magnification were recorded. The count from a field of each treatment was divided by the highest average cysts of a treatment among all ph treatments at 1 or 24 h. The relative count for swimming zoospores indicates only those present transiently in fixed microscopic fields during observation and is much lower than the actual population in the water column. Thus, the number of cysts present was used as the base for relative counts because it better indicates the population level in a treatment. The standard errors were calculated using Microsoft Excel. Results Effect of ph on colony formation from zoospores Effect of ph on CFU was dependent on species as indicated by the overall population survival (Fig. 1). Phytophthora ramorum survived in the narrowest range of ph with the highest rates, while P. alni and P. kernoviae Relative counts (%) P. alni ph3 ph5 ph7 ph9 ph11 2 h 24 h 2 h 24 h 2 h 24 h P. kernoviae 2 h 24 h 2 h 24 h 2 h 24 h P. ramorum 2 h 24 h 2 h 24 h 2 h 24 h Swimmer Cyst Germinant Fig. 1. Motility, encystment, and germination of zoospores of three quarantine pathogens in response to different ph levels in 10% Hoagland s solution. Data are indicated using relative percentages of swimmers, cysts and germinants in an observation microscopic field over the highest average cysts of a treatment in six fields. Each bar shows standard deviation from six observations. survived in wider ranges of ph with lower rates. Specifically, zoospores of P. alni formed colonies at ph 3 11 over the 14-day test period (Table 2). Higher relative survival rates were obtained among ph 5 11 but the rates decreased dramatically after overnight exposure. The difference of the rates among these phs diminished with increasing exposure time. At day 14, differences in survival rates were no longer statistically significant (Table 2). In addition, increased zoospore relative survival rates were found at day 5. Colony formation of P. alni was poor at ph 3. The relative survival rates were reduced by almost 17 times after brief exposure and more than 300 times after overnight exposure compared to those at ph 7. Zoospores of P. kernoviae did not tolerate ph 11 but survived well at lower phs, including ph 3. They survived at acidic ph 5 and 3 for at least 14 days and the relative rates were almost twice as high as P. alni (Table 2). They also survived at ph 7 and 9 over the 14-day period but at low rates. Like P. alni, the differences in response to different ph became less significant with increasing exposure time, and the number of colonies increased after 5 days at ph 5 9. Mycelia were observed in the treatment containers. However, they failed to form colonies at ph 11 after a 5-day exposure, indicating that they are sensitive to high ph. Colony formation by P. ramorum zoospores was relatively poor compared with P. alni and P. kernoviae. Normally, plating 1 ml 100 fresh zoospores of the suspension at ph 7 resulted in fewer than 20 colonies. However, their relative survival rates at immediate exposure were much higher because of rapid colony formation. At ph 5 9, relative survival rates declined much slower compared with P. alni and P. kernoviae but varied significantly over time (Table 2). Like P. alni, zoospores of P. ramorum also were tolerant of basic ph, surviving at ph 9 and 11 for at least 14 days. At ph 9, the survival was about 4 and 6 times higher than that of P. kernoviae and P. alni, respectively (Table 2). However, the best survival was at moderately acidic conditions (ph 5), although survival was very poor, not beyond 1 day, at ph 3. Effect of ph on zoospore behavior Zoospore motility, encystment and germination among P. alni, P. kernoviae, and P. ramorum responded differently to ph. Most zoospores of P. alni swam for more than 2 h at all phs except for ph 3. Many continued swimming over 24 h, although at ph 11 there were relatively fewer. The relative count for swimming zoospores (Fig. 1) represented only those present transiently in fixed microscopic fields during the observation, which was much lower than the actual number contained in the water column. The number of cysts was close to the

4 Survival of quarantine Phytophthora in response to ph 57 Table 2. Relative zoospore survival (%) of Phytophthora alni, Phytophthora kernoviae, and Phytophthora ramorum as affected by ph in 10% Hoagland s solution Relative zoospore survival (%) at following exposure time (days) Species ph Survival index P. alni 3 6.0d* 0.3c 0.5b 0.2b 0c 0.2a bc* 2.8b 0.5b 1.4ab 1.6bc 5.3a a* 4.3b 1.9ab 3.5a 5.5ab 4.6a b* 6.9a 1.6ab 1.3ab 1.3bc 2.6a c* 4.9ab 3.2a 3.0ab 7.2a 5.5a 14.9 P < < P. kernoviae c** 11.4a* 3.4b 2.1b 2.7b 0.7c b* 11.4a 12.2a 8.4a 7.5a 9.3a a* 6.2ab 4.3b 1.7b 2.6b 3.2bc b* 7.0ab 1.4b 2.5b 1.8b 5.5b c* 4.7b 1.7b 0.5b 0b 0c 2.8 P < < < < < P. ramorum b* 0c 0c 0c 0d 0b a*** 20.9b* 4.0bc 4.6bc 10.5bc 56.4a** a** 39.9a* 9.8b 12.4ab 12.0ab 20.3ab a* 30.5ab 22.1a 17.1a 21.9a 28.4ab b* 2.8c 1.2c 2.0c 1.7cd 3.2b 8.8 P < < < < < Zoospore survival rates were calculated by dividing the number of colony-forming units (CFU) counted at each ph and exposure time treatment by the maximum number of CFU observed within each species. Population survival of individual species was presented with the survival index: The sum of survival rates at each exposure time multiplies corresponding exposure time then is divided by 14 (the longest exposure time). Significance level of differences within each species: relative survival rate followed by different letters within each column differed according to LSD test at a = 0.01, and those followed by different numbers of asterisks differed according to LSD test at a = actual number of zoospores present. The cyst count at ph 3 was higher than at ph 5 11, suggesting that less lysis occurred at ph 3 than at other phs. Early cyst germination was observed for P. alni, starting as soon as 2 h after exposure at ph 5 11, while most of cysts lysed after 24 h exposure. Hypha growth and secondary sporangium production was observed after 5 days exposure at ph 5 11 (Fig. 2). However, the new hyphae at ph 11 appeared abnormal, forming beaded structures that were still able to grow on plates as indicated in Table 2. No germinants were observed at ph 3 (Fig. 2), consistent with their colonization on growth media (Table 2). Zoospores of P. kernoviae were less motile compared with P. alni at ph They encysted immediately after exposure to ph 3 (Fig. 1). A few swam at ph 7 11 briefly, but did not last overnight except at ph 7 where only a very few swimmers were occasionally observed in a field. More cysts lysed compared with P. alni, which occurred in all the treatments with the most at ph 5 9. In addition, germination of the cysts was later than that of P. alni, which occurred after 24 h. Germinants were observed at ph 3 9 with the most at ph 5 but none at ph 11. Germinants at ph 5 9 grew and formed massive hyphae and secondary sporangia as observed at exposure day 7 (Fig. 2). At ph 3, germinants or cysts had little further growth, although a small population of them still formed colonies when plated on media as shown in the Table 2. However, colonies from these cysts developed much more slowly, normally a 2 3 day delay, than those in ph 5 9. Behavior of P. ramorum zoospores in response to ph fell between P. alni and P. kernoviae. Like P. kernoviae, they lost motility immediately after exposure (Fig. 1), and most of them lysed before encystment. But the cysts that did form germinated early as did P. alni. Also, like P. kernoviae, the cysts formed compact swollen hyphae or mycelia after a 5-day exposure at ph 5 9. They also formed hyphae at ph 11 like P. alni, although their hyphae appeared much thinner and formed nipple-like swellings on branches of hyphae (Fig. 2). Hyphae and cysts at ph 3 were not viable, forming no colonies on culture media (Table 2). Water quality analysis The only significant differences in the water quality analyses (between solutions) were in EC, alkalinity, Na, Cl and Ca levels at extreme phs (Table S1). This is not surprising, because the ph levels were adjusted with NaCl and NaOH solutions. The difference in EC levels between FEMS Microbiol Lett 332 (2012) ª 2012 Federation of European Microbiological Societies

5 58 P. Kong et al. Fig. 2. Zoosporic response to ph of three quarantine pathogens in 10% Hoagland s solution. Images were captured under an inverted microscope at 1609 at 7-day exposure at 20 C in the dark. Bar = 50 lm for all images. treatments was relatively small, and the EC of all solutions ( ds m 1 ) was well within the range of ECs found in the root zone of fertilized ornamental plants in commercial nurseries. Variation in alkalinity was significant, especially at ph 11 (83.3 mg L 1 ) (Table S1). However, this value is much lower than the alkalinity (< 100 meq or 5004 mg L 1 ) associated with groundwater (and hence irrigation water) in many areas of the United States. Similarly, variation in Cl and Na concentrations was also significant at extreme phs. Na at ph 3 and ph 11 was elevated and 21.2-fold, respectively, compared with that at ph 7. At ph 9, the elevation was smaller (3.1-fold), and at ph 5, the level was reduced 4.9- fold. Significant elevation in Cl was only present at ph 3 and ph 11; and 2.4-fold, respectively, compared with ph 7. However, the maximum concentrations of each of these ions in solution (41.2 mg Na L 1 and 88.9 mg Cl L 1 ) (Table S1) are again well within root zone concentrations tolerated by most ornamental crop species. Significant variation in Ca occurred only at ph 11 where the level reduced by half compared with that at other phs. The difference in Ca levels did not affect cyst counts (Table S1, Fig. 1). Discussion Survival of P. alni, P. kernoviae and P. ramorum in response to ph has three things in common, and each has an important implication in managing these pathogens. First, their initial responses to ph at immediate exposure are very similar. They all survived best at neutral ph were favored by basic ph over acidic ph and were sensitive to ph 3 and 11. Second, like other previously studied species (Kong et al., 2009), these three pathogens all declined substantially within 24 h no matter how they responded to ph initially. Only a small population of these pathogens can survive longer. This suggests that populations of these pathogens may decline

6 Survival of quarantine Phytophthora in response to ph 59 depending upon the time required to spread. Thus, extending their time in water by locating the pump house far from the runoff entrance may mitigate the dispersal of these three pathogens via recycled irrigation water (Hong et al., 2003). Third, extended survival of a small population of all these pathogens occurred over a broad ph range through the formation of compact hyphae. These structures may be important for the survival of these pathogens in aquatic environments because they were long lasting and formed secondary sporangia that can lead to new cycles of zoospore production. On the other hand, these structures are likely to settle out of the water column over time because they probably are heavier than individual zoospores or cysts. During sedimentation, they could be subject to degradation by other microorganisms in the sediments. Based on this, the addition of a sedimentation or retention pond to recycling systems may be an additional means of preventing them from being dispersed to crops in recycled water. Differences in ph responses also are present among these three pathogens. First, P. alni had quite distinct zoospore behavior at initial exposure compared with P. kernoviae and P. ramorum. Its zoospores remained motile for at least 24 h at ph 5 9, which may allow sufficient time for it to spread actively. In contrast, zoospores of P. kernoviae and P. ramorum encysted rapidly irrespective of ph. Although they lose the advantage of spreading actively when they encyst, they may gain a form of resistance against environmental stress. Such resistance may allow these pathogens to survive longer or to be carried away effectively by water currents. Phytophthora nicotianae has been shown to survive better with cysts formed when pressurized CO2 was applied (Ahonsi et al., 2010). Secondly, the extended survival of these pathogens in response to ph is divergent from initial survival. Phytophthora alni and P. ramorum became more tolerant of basic phs. Basic ph is widespread in nursery irrigation water reservoirs, typically found during summer days because of photosynthetic activity of algae and other aquatic plants (Chen et al., 2003; Cirelli et al., 2008; Hong et al., 2009). Seasonal and diurnal fluctuation of ph in irrigation water ponds based on our most recent observations can range from low ph 6 to close to ph 11. However, such fluctuation is unlikely to become an issue for the survival of these pathogens in irrigation water systems because they can survive well at ph 5 7 despite the fact that only a small population of them can survive long. More concern should be given to P. ramorum as it can maintain higher population even at ph 11 over 14 day exposure times, thus it may have a high potential to spread via irrigation systems. In contrast, P. kernoviae is less tolerant of basic ph but tolerated acidic ph as low as ph 3. It survived < 1 week at ph 11, although it and other two species tested here survived at ph 9. Consequently, it may have a low potential to spread through irrigation water, especially during the summer months. Acknowledgements 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 #: ). The authors would like to thank Clive Brasier (Forest Research, UK) and Steven Jeffers (Clemson University, USA) for providing the cultures. References Adams GC, Catal M, Trummer L, Hansen EM, Reeser P & Worrall JJ (2008) Phytophthora alni subsp. uniformis found in Alaska beneath thinleaf alders. Online Plant Health Prog, doi: /PHP BR. 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