BACKGROUND
zoosporic plant pathogens in the phylum oomycota of the stramenopila kingdom include hundreds of devastating species that attack a broad range of economically important agricultural and ornamental crops as well as forest tree species  <cit> .
these oomycetes, including phytophthora and pythium species, use motile zoospores for dispersal and plant infection  <cit> .
plant infection by zoosporic pathogens is often effective in nature despite the fact that the population density in primary inoculum sources is relatively low  <cit> .
this has led to differing theories with regard to density-dependent zoospore behaviors and plant infection  <cit> .
a recent study with phytophthora nicotianae showed that plant infection may be regulated through zoosporic extracellular products in zoospore-free fluid  which can promote infection by a single zoospore  <cit> .
this indicates that the physical presence of the threshold density of zoospores at an infection site is not strictly required, and plant infection can be initiated efficiently through chemical communication by the population.
however, it is not clear how such a process is carried out by a pathogen at its naturally occurring low population density, which would be unlikely to produce adequate levels of functional signals unless these signals were also produced by other organisms and readily accessible in the environment.
ca2+ and autoinducer  <dig> , two widespread and non-specific signaling molecules, are known to be produced by zoosporic oomycetes  <cit> .
ca2+ plays a central role in autonomous encystment, adhesion and germination of cysts in zoosporic oomycetes  <cit> .
however, it is not considered to be an autoinducer because ca2+ does not directly trigger cooperative behaviors of zoospores and acts more like a secondary messenger  <cit> .
ai- <dig> was first detected in bacteria and is utilized for metabolism and quorum sensing in bacteria  <cit> .
in the latter process, bacteria respond to these released signaling molecules or autoinducers to coordinate their communal behavior.
eukaryotes including oomycetes can also produce ai- <dig> or ai-2-like activities  <cit>  although they do not use the luxs pathway that most bacteria use  <cit> .
instead, ai- <dig> is formed spontaneously from d-ribulose-5-phosphate that is synthesized in these eukaryotes from pentose-phosphates by ribose phosphate isomerase  in the pentose-phosphate pathway  <cit> .
ai- <dig> has been proposed as a universal signaling molecule in bacteria based on its role in inter-species signaling and postulated cross-kingdom communication  <cit> .
however, the function of ai- <dig> in eukaryotes has not been established.
the aim of this study was to investigate the nature of signal molecules in zff.
specifically, we identified inter-specific signaling activities of zff from four phytophthora species and one pythium species.
we also assessed the potential of ai- <dig> along with another known bacterial autoinducer as signal molecules for communication among zoosporic species.
RESULTS
zff interspecific stimulation of zoosporic infection
zoospore-free fluids were prepared from suspensions at a density of  <dig> zoospores ml- <dig> or higher of phytophthora nicotianae , p. capsici , p. hydropathica , p. sojae  and pythium aphanidermatum  and evaluated in three phytopathosystems.
inoculation of annual vinca  with suspensions containing an average of one zoospore of p. nicotianae in any of the four zffs resulted in significantly higher infection  compared to the control .
specifically, percentages of sites infected were 39%, 21%, 11%, and 15% for zffaph, zffhyd, zffnic, and zffsoj, respectively compared to 3% for sdw .
similarly, zffaph, zffhyd, zffnic and zffsoj stimulated infection of lupine  by p. sojae , while zffcap and zffsoj stimulated infection of soybean  by p. sojae .
these results indicate that zff from the different phytophthora species and py.
aphanidermatum contained one or more signals stimulating zoosporic infection by p. nicotianae and p. sojae that are active across species boundaries.
many plants are attacked by multiple oomycete species  <cit> .
the ability of oomycete pathogens to benefit from the presence of related  species is presumably a selective advantage, especially if the diverse pathogens are competing for a limited resource  and/or the initial population density of each individual pathogen population is low.
such self-interested cooperation may have further advantages if the effector molecules released by each pathogen species have complementary or synergistic capabilities for suppressing plant defenses.
zff inter-specific regulation of zoospore aggregation
to determine whether zff may also have cross-species activity in regulating zoospore aggregation, fresh zoospores of p. nicotianae and p. sojae at a concentration  below normal aggregation thresholds  were cross incubated in multiwell plates with zffsoj or zffnic and compared with those in sdw.
zoospores of p. nicotianae in zffsoj and those of p. sojae in zffnic aggregated  as if they were in zff produced by their own species.
as expected, zoospores of neither species aggregated in sdw .
zffcap and zffaph did not stimulate zoospore aggregation by p. nicotianae or p. sojae zoospores.
however, they did stimulate germination of cysts of both p. nicotianae and p. sojae , which may explain their activity in promoting plant infection .
it was interesting that zoospores of p. capsici did not aggregate even at a density of  <dig> zoospores ml- <dig>  these results indicate that the signal involved in aggregation are somewhat species-restricted and may be different from those mediating the infection process.
ai- <dig> is not involved in zoospore communication and promotion of plant infection
to test whether ai- <dig> may be involved in zoospore communication and promotion of plant infection, purified ai- <dig> was used in place of zff.
ai- <dig> was tested at a wide concentration range of  <dig>  μm - <dig> mm for its effects on p. nicotianae zoospore behaviors and plant infection; the concentration of ai- <dig> in zff was estimated to be less than  <dig> μm  <cit> .
under the microscope, an increased number of zoospores treated with ai- <dig> lysed before encystment and failed to germinate as the ai- <dig> concentration was increased .
zoospore aggregation was not observed at any concentration tested.
in infection experiments with annual vinca, ai- <dig> did not promote single zoospore infection at any concentration.
interestingly, ai- <dig> induced hypersensitive response -like micro-lesions on the inoculated sites at  <dig> μm and higher.
these results indicated that ai- <dig> was not responsible for any of the zoospore signals found in zff.
conc.
of
germinating
a difference between the total number of zoospores  in sdw and those countable in ai- <dig> at each concentration.
b m is the mean from  <dig> replicate fields  of three assays.
std is the standard deviation.
as a complementary test for the ability of ai-2-like molecules to mediate zoospore communication and promote plant infection, we cloned and silenced the ribose phosphate isomerase  gene of p. capsici.
rpi converts ribose-5-phosphate to ribulose-5-phosphate, which can spontaneously convert to ai-2-like molecules under physiological conditions  <cit> .
rpi was proposed to be responsible for production of ai-2-like molecules in zoosporic pathogens  <cit> .
to silence the rpi gene of p. capsici, protoplasts of p. capsici were treated with rpi dsrna.
if rpi had a role in production of zoospore signaling molecules, rpi-silenced lines would be expected to require much higher zoospore concentrations to infect plants than the wild type due to reduced or blocked ai- <dig> production by the inocula.
one third of the  <dig> t <dig> lines regenerated  <dig> days after dsrna exposure showed no or decreased expression with rpi compared to the endogenous control actin detected using rt-pcr.
half of these silenced or down regulated rpi lines retained the same reduced transcript levels two weeks after being transferred to fresh media  .
five t <dig> lines were simultaneously tested for zoospore threshold for infection.
the resulting disease incidences were very similar to those produced by wild type p. capsici at zoospore inoculum concentrations ranging from  <dig> to  <dig> ml- <dig>  .
these results indicate that rpi silencing had no significant impact on zoospore communication during infection.
the zff activity of the silenced lines was not evaluated due to the transient nature of dsrna-mediated silencing  <cit>  and insufficient numbers of t <dig> zoospores for zff production.
nevertheless, these findings are consistent with the conclusion that ai-2-like molecules that might be produced via the action of rpi are not required for infection at low inoculum densities.
the function of ai-2-like activities produced by zoosporic oomycetes remains unclear although it regulates bacteria quorum sensing  <cit> .
two-way communication has been observed between eukaryotes and bacteria such as leguminosae and bacterial rhizobia  <cit>  and between mycorrhiza and streptomyces  <cit> .
in the former case, plants release flavonoids that bind lysr-family transcriptional regulators in the bacteria, leading to the production of nod factor that facilitates nitrogen fixation.
in the latter case, fungal metabolites stimulate the bacteria to produce auxofuran which promotes growth of both the fungus and the host plants.
perhaps zoosporic oomycetes utilize ai- <dig> to attract quorum sensing bacteria which subsequently release factors that facilitate plant infection.
indeed, bacteria have been shown to benefit sporangium production by zoosporic oomycetes  <cit> .
involvement of other molecules in zff activity
acyl-homoserine lactones , or bacterial autoinducer  <dig>  are utilized by zoospores of the green seaweed enteromorpha  for communication in the search for settlement surfaces  <cit> .
a bioassay was performed using the agrobacterium tumefaciens reporter strain kyc55/pjz410/pjz384/pjz <dig>  <cit>  in plate and spectrophotometric tests to determine whether this molecule is present in zff.
lacz activity was detected in all four positive control plates at nm concentrations of ahl but not in zffnic or zffsoj prepared from zoospore suspensions at >  <dig> spores ml- <dig> nor in concentrated extracts from them obtained with ethyl acetate.
these results indicate that zoospores from these oomycete species do not produce ahls which therefore cannot be responsible for any zff activity.
temperature sensitivity of zff activities
to begin to characterize the signal molecules in zff we tested their temperature sensitivity.
zffnic did not stimulate zoospore aggregation after a freeze-thaw or heat treatment, suggesting that the molecule promoting this behavior may be a protein or lipoprotein that is sensitive to heat and freezing.
on the other hand, freeze-thaw did not affect the activity of zffnic in promoting plant infection by zoospores .
in addition, zffnic boiled for  <dig> minutes remained as active as the untreated in promoting infection , indicating that the molecule which stimulates plant infection is temperature insensitive and different from that involved in aggregation.
CONCLUSIONS
this study demonstrated inter-specific activities of zoospore extracellular products in promoting zoospore aggregation and plant infection by phytophthora.
zoosporic oomycetes contain hundreds of species and are widespread in irrigation water and plant production fields.
however, specific populations detected in primary inoculum sources are not in sufficient numbers to produce signals that could promote plant infection.
inter-specific chemical communication  as a strategy used by zoosporic pathogens for effective plant infection provides insights into the destructiveness of these pathogens and the importance of the microbial community and the environment in the infection process.
ai- <dig> was excluded as a signal for communal behavior in zoosporic oomycetes, despite its detection in zff and widespread presence in the environment.
ai- <dig> synthase rpi and purified ai- <dig> both were not required for regulation of zoospore aggregation and infection.
ahls also were excluded because of their absence in zff.
thus, zoosporic oomycetes may use completely different chemicals from bacteria for quorum sensing.
analysis of zff revealed that functional signals controlling zoospore aggregation and plant infection differ in molecular composition.
the former is not temperature labile and acts upon a restricted number of species while the latter is heat labile and non-species-specific.
identifying these molecules will facilitate our understanding of the mechanisms underlying natural plant infection by these pathogens and may lead to innovative control strategies.

 zoosporic plant pathogens in the phylum oomycota of the stramenopila kingdom include hundreds of devastating species that attack a broad range of economically important agricultural and ornamental crops as well as forest tree species. these oomycetes, including phytophthora and pythium species, use motile zoospores for dispersal and plant infection. plant infection by zoosporic pathogens is often effective in nature despite the fact that the population density in primary inoculum sources is relatively low. this has led to differing theories with regard to density-dependent zoospore behaviors and plant infection. a recent study with phytophthora nicotianae showed that plant infection may be regulated through zoosporic extracellular products in zoospore-free fluid which can promote infection by a single zoospore. this indicates that the physical presence of the threshold density of zoospores at an infection site is not strictly required, and plant infection can be initiated efficiently through chemical communication by the population. however, it is not clear how such a process is carried out by a pathogen at its naturally occurring low population density, which would be unlikely to produce adequate levels of functional signals unless these signals were also produced by other organisms and readily accessible in the environment. ca2+ and autoinducer, two widespread and non-specific signaling molecules, are known to be produced by zoosporic oomycetes. ca2+ plays a central role in autonomous encystment, adhesion and germination of cysts in zoosporic oomycetes. however, it is not considered to be an autoinducer because ca2+ does not directly trigger cooperative behaviors of zoospores and acts more like a secondary messenger. ai- was first detected in bacteria and is utilized for metabolism and quorum sensing in bacteria. in the latter process, bacteria respond to these released signaling molecules or autoinducers to coordinate their communal behavior. eukaryotes including oomycetes can also produce ai- or ai-2-like activities although they do not use the luxs pathway that most bacteria use. instead, ai- is formed spontaneously from d-ribulose-5-phosphate that is synthesized in these eukaryotes from pentose-phosphates by ribose phosphate isomerase in the pentose-phosphate pathway. ai- has been proposed as a universal signaling molecule in bacteria based on its role in inter-species signaling and postulated cross-kingdom communication. however, the function of ai- in eukaryotes has not been established. the aim of this study was to investigate the nature of signal molecules in zff. specifically, we identified inter-specific signaling activities of zff from four phytophthora species and one pythium species. we also assessed the potential of ai- along with another known bacterial autoinducer as signal molecules for communication among zoosporic species.  zff interspecific stimulation of zoosporic infection zoospore-free fluids were prepared from suspensions at a density of zoospores ml- or higher of phytophthora nicotianae, p. capsici, p. hydropathica, p. sojae and pythium aphanidermatum and evaluated in three phytopathosystems. inoculation of annual vinca with suspensions containing an average of one zoospore of p. nicotianae in any of the four zffs resulted in significantly higher infection compared to the control. specifically, percentages of sites infected were 39%, 21%, 11%, and 15% for zffaph, zffhyd, zffnic, and zffsoj, respectively compared to 3% for sdw. similarly, zffaph, zffhyd, zffnic and zffsoj stimulated infection of lupine by p. sojae, while zffcap and zffsoj stimulated infection of soybean by p. sojae. these results indicate that zff from the different phytophthora species and py. aphanidermatum contained one or more signals stimulating zoosporic infection by p. nicotianae and p. sojae that are active across species boundaries. many plants are attacked by multiple oomycete species. the ability of oomycete pathogens to benefit from the presence of related species is presumably a selective advantage, especially if the diverse pathogens are competing for a limited resource and/or the initial population density of each individual pathogen population is low. such self-interested cooperation may have further advantages if the effector molecules released by each pathogen species have complementary or synergistic capabilities for suppressing plant defenses. zff inter-specific regulation of zoospore aggregation to determine whether zff may also have cross-species activity in regulating zoospore aggregation, fresh zoospores of p. nicotianae and p. sojae at a concentration below normal aggregation thresholds were cross incubated in multiwell plates with zffsoj or zffnic and compared with those in sdw. zoospores of p. nicotianae in zffsoj and those of p. sojae in zffnic aggregated as if they were in zff produced by their own species. as expected, zoospores of neither species aggregated in sdw. zffcap and zffaph did not stimulate zoospore aggregation by p. nicotianae or p. sojae zoospores. however, they did stimulate germination of cysts of both p. nicotianae and p. sojae, which may explain their activity in promoting plant infection. it was interesting that zoospores of p. capsici did not aggregate even at a density of zoospores ml- these results indicate that the signal involved in aggregation are somewhat species-restricted and may be different from those mediating the infection process. ai- is not involved in zoospore communication and promotion of plant infection to test whether ai- may be involved in zoospore communication and promotion of plant infection, purified ai- was used in place of zff. ai- was tested at a wide concentration range of μm - mm for its effects on p. nicotianae zoospore behaviors and plant infection; the concentration of ai- in zff was estimated to be less than μm. under the microscope, an increased number of zoospores treated with ai- lysed before encystment and failed to germinate as the ai- concentration was increased. zoospore aggregation was not observed at any concentration tested. in infection experiments with annual vinca, ai- did not promote single zoospore infection at any concentration. interestingly, ai- induced hypersensitive response -like micro-lesions on the inoculated sites at μm and higher. these results indicated that ai- was not responsible for any of the zoospore signals found in zff. conc. of germinating a difference between the total number of zoospores in sdw and those countable in ai- at each concentration. b m is the mean from replicate fields of three assays. std is the standard deviation. as a complementary test for the ability of ai-2-like molecules to mediate zoospore communication and promote plant infection, we cloned and silenced the ribose phosphate isomerase gene of p. capsici. rpi converts ribose-5-phosphate to ribulose-5-phosphate, which can spontaneously convert to ai-2-like molecules under physiological conditions. rpi was proposed to be responsible for production of ai-2-like molecules in zoosporic pathogens. to silence the rpi gene of p. capsici, protoplasts of p. capsici were treated with rpi dsrna. if rpi had a role in production of zoospore signaling molecules, rpi-silenced lines would be expected to require much higher zoospore concentrations to infect plants than the wild type due to reduced or blocked ai- production by the inocula. one third of the t lines regenerated days after dsrna exposure showed no or decreased expression with rpi compared to the endogenous control actin detected using rt-pcr. half of these silenced or down regulated rpi lines retained the same reduced transcript levels two weeks after being transferred to fresh media. five t lines were simultaneously tested for zoospore threshold for infection. the resulting disease incidences were very similar to those produced by wild type p. capsici at zoospore inoculum concentrations ranging from to ml-. these results indicate that rpi silencing had no significant impact on zoospore communication during infection. the zff activity of the silenced lines was not evaluated due to the transient nature of dsrna-mediated silencing and insufficient numbers of t zoospores for zff production. nevertheless, these findings are consistent with the conclusion that ai-2-like molecules that might be produced via the action of rpi are not required for infection at low inoculum densities. the function of ai-2-like activities produced by zoosporic oomycetes remains unclear although it regulates bacteria quorum sensing. two-way communication has been observed between eukaryotes and bacteria such as leguminosae and bacterial rhizobia and between mycorrhiza and streptomyces. in the former case, plants release flavonoids that bind lysr-family transcriptional regulators in the bacteria, leading to the production of nod factor that facilitates nitrogen fixation. in the latter case, fungal metabolites stimulate the bacteria to produce auxofuran which promotes growth of both the fungus and the host plants. perhaps zoosporic oomycetes utilize ai- to attract quorum sensing bacteria which subsequently release factors that facilitate plant infection. indeed, bacteria have been shown to benefit sporangium production by zoosporic oomycetes. involvement of other molecules in zff activity acyl-homoserine lactones, or bacterial autoinducer are utilized by zoospores of the green seaweed enteromorpha for communication in the search for settlement surfaces. a bioassay was performed using the agrobacterium tumefaciens reporter strain kyc55/pjz410/pjz384/pjz in plate and spectrophotometric tests to determine whether this molecule is present in zff. lacz activity was detected in all four positive control plates at nm concentrations of ahl but not in zffnic or zffsoj prepared from zoospore suspensions at > spores ml- nor in concentrated extracts from them obtained with ethyl acetate. these results indicate that zoospores from these oomycete species do not produce ahls which therefore cannot be responsible for any zff activity. temperature sensitivity of zff activities to begin to characterize the signal molecules in zff we tested their temperature sensitivity. zffnic did not stimulate zoospore aggregation after a freeze-thaw or heat treatment, suggesting that the molecule promoting this behavior may be a protein or lipoprotein that is sensitive to heat and freezing. on the other hand, freeze-thaw did not affect the activity of zffnic in promoting plant infection by zoospores. in addition, zffnic boiled for minutes remained as active as the untreated in promoting infection, indicating that the molecule which stimulates plant infection is temperature insensitive and different from that involved in aggregation.  this study demonstrated inter-specific activities of zoospore extracellular products in promoting zoospore aggregation and plant infection by phytophthora. zoosporic oomycetes contain hundreds of species and are widespread in irrigation water and plant production fields. however, specific populations detected in primary inoculum sources are not in sufficient numbers to produce signals that could promote plant infection. inter-specific chemical communication as a strategy used by zoosporic pathogens for effective plant infection provides insights into the destructiveness of these pathogens and the importance of the microbial community and the environment in the infection process. ai- was excluded as a signal for communal behavior in zoosporic oomycetes, despite its detection in zff and widespread presence in the environment. ai- synthase rpi and purified ai- both were not required for regulation of zoospore aggregation and infection. ahls also were excluded because of their absence in zff. thus, zoosporic oomycetes may use completely different chemicals from bacteria for quorum sensing. analysis of zff revealed that functional signals controlling zoospore aggregation and plant infection differ in molecular composition. the former is not temperature labile and acts upon a restricted number of species while the latter is heat labile and non-species-specific. identifying these molecules will facilitate our understanding of the mechanisms underlying natural plant infection by these pathogens and may lead to innovative control strategies. 
zoospore-free fluids from phytophthora capsici, p. hydropathica, p. nicotianae, p. sojae and pythium aphanidermatum were cross tested for stimulating plant infection in three pathosystems. all zffs tested significantly increased infection of catharanthus roseus by p. nicotianae. only zffnic and zffsoj cross induced zoospore aggregation at a density of × ml- pure autoinducer-, a component in zff, caused zoospore lysis of p. nicotianae before encystment and did not stimulate plant infection at concentrations from to μm. when signal molecules are present at or above threshold level, single zoospores can infect plants. these pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. however, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. we found that some of these signal molecules could act in an inter-specific manner, though signals for zoospore aggregation were somewhat restricted. the signal molecules for both zoospore aggregation and plant infection are distinct from ai- and ahl. heat treatment by boiling for min also did not affect the infection-stimulating property of zffnic. p. capsici transformants with a transiently silenced ai- synthase gene, ribose phosphate isomerase, infected capsicum annuum seedlings at the same inoculum concentration as the wild type. after freeze-thaw treatments, zff remained active in promoting plant infection but not zoospore aggregation. oomycetes produce and use different molecules to regulate zoospore aggregation and plant infection. similar cross activities were observed in infection of lupinus polyphyllus and glycine max by p. sojae.
 oomycetes attack a huge variety of economically and ecologically important plants. these pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. when signal molecules are present at or above threshold level, single zoospores can infect plants. however, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. it is unclear whether these molecules are shared among related species and what their chemistries are.  zoospore-free fluids from phytophthora capsici, p. hydropathica, p. nicotianae, p. sojae and pythium aphanidermatum were cross tested for stimulating plant infection in three pathosystems. all zffs tested significantly increased infection of catharanthus roseus by p. nicotianae. similar cross activities were observed in infection of lupinus polyphyllus and glycine max by p. sojae. only zffnic and zffsoj cross induced zoospore aggregation at a density of × ml- pure autoinducer-, a component in zff, caused zoospore lysis of p. nicotianae before encystment and did not stimulate plant infection at concentrations from to μm. p. capsici transformants with a transiently silenced ai- synthase gene, ribose phosphate isomerase, infected capsicum annuum seedlings at the same inoculum concentration as the wild type. acyl-homoserine lactones were not detected in any zffs. after freeze-thaw treatments, zff remained active in promoting plant infection but not zoospore aggregation. heat treatment by boiling for min also did not affect the infection-stimulating property of zffnic.  oomycetes produce and use different molecules to regulate zoospore aggregation and plant infection. we found that some of these signal molecules could act in an inter-specific manner, though signals for zoospore aggregation were somewhat restricted. this self-interested cooperation among related species gives individual pathogens of the same group a competitive advantage over pathogens and microbes from other groups for limited resources. these findings help to understand why these pathogens often are individually undetectable until severe disease epidemics have developed. the signal molecules for both zoospore aggregation and plant infection are distinct from ai- and ahl. 