Many Phytomyxean species are obligate endophytic biotrophs that parasitize a wide range of organisms ranging from flowering plants, algae, diatoms to several oomycetes in both marine and terrestrial environments (Neuhauser et al., 2011). They may be direct agents of diseases, notable among them are Plasmodiophora brassicae and Spongospora subterranea which causes clubroot in crucifers (Dixon, 2014), and powdery scab disease in potato (Falloon et al., 2015), respectively. Phytomyxeans are also known vectors of at least 20 plant viruses, including Oat mosaic virus (Hebert and Panizo, 1975), Sorghum chlorotic stunt virus (Kanyuka et al., 2003), Soil-borne wheat mosaic virus (Canova, 1996) and Potato mop top virus (Jones and Harrison, 1969). These phytomyxid and phytomyxid-vectored diseases are responsible for major economic losses in vegetable, oil seed and cereal crops globally (Kanyuka et al., 2003; Dixon, 2014; Wilson, 2016).

Phytomyxid infections are initiated by motile zoospores released from thick-walled, uninucleate resting spores of the sporogenic phase of a biphasic life cycle (Bulman and Neuhauser, 2016). Soon after release, the zoospores propelled by their heterokont flagella move in soil water or the marine environment to locate their hosts (Harrison et al., 1997; Bulman and Neuhauser, 2016). Zoospore movement can be directed by several environmental stimuli, but chemical cues, such as root exudation products, are the most commonly used by zoospores for host location (Zentmyer, 1960; Rai and Strobel, 1966). These chemical cues are, however, unable to transcend the zoospore cell membrane, because they are either too large or too charged (Walrant et al., 2017). Thus, just as in more complex organisms, these unicellular organisms have evolved elaborate mechanisms to perceive signals (Renaud et al., 2004), comprising the transduction triad of receptors, transducers, and effectors (Trewavas and Malho, 1997). Signals or primary stimuli are first perceived by transmembrane receptors, the transducers decode and relay the signal intracellularly to the effectors for appropriate action (Trewavas and Malho, 1997).

Many signal transduction pathways utilize secondary messengers, small, non-protein molecules, as transducers (Demaurex and Nunes, 2016). Evidence from eukaryotes and prokaryotes show that many physiological responses including chemotaxis, spore development, migration, virulence, and host pathogen interaction are mediated by secondary messenger molecules (Dominguez, 2004; Campbell et al., 2007; Asmat et al., 2014). Several molecules have been identified to have secondary messenger function in eukaryotic organisms (Pollard et al., 2017), with intracellular free calcium ions (Ca²⁺) the only molecule known to serve as a universal messenger in all eukaryotes (Campbell, 1983; Dominguez et al., 2015) and prokaryotes (Dominguez, 2004). In these organisms Ca²⁺ is ubiquitous in most transduction pathways and involved in a wide range of physiological and cellular functions (Demaurex and Nunes, 2016). In Bacillus subtilis for instance, the rotational movement of the flagella has been reported to be controlled by cytosolic Ca²⁺ (Szurmant and Ordal, 2004) while in Escherichia coli the role of transient Ca²⁺ on bacteria motility is well established (Tisa et al., 2000). Indirect evidence also supports the role of Ca²⁺ signaling on chemotaxis, with isoflavone attractants of Phytophthora sojae zoospores to soybean (Morris and Ward, 1992), found to stimulate the influx of Ca²⁺ into the zoospore cytosol from intracellular stores and the extracellular environment (Connolly et al., 1999).

Generally, intracellular influx of Ca²⁺ ions occur through various transport systems including antiporters and voltage-gated Ca²⁺-selective channels which have high conducting capacity, of approximately one million Ca²⁺ ions per second per channel (Clapham, 2007). This influx results in high cytosolic loading of Ca²⁺. Because Ca²⁺ overload will result in cell dysfunction and death (Carafoli and Krebs, 2016), eukaryotes and prokaryotes trigger extensive homeostatic systems to restore the intracellular Ca²⁺ load (Berridge et al., 2000) to remarkably low (c. 100 nM) concentrations at 20,000-fold less compared to the extracellular matrix by pumping Ca²⁺ into endoplasmic reticulum or out of the cell (Clapham, 2007). For every Ca²⁺ ion pumped out one ATP is hydrolyzed (Niggli et al., 1981). Dominguez (2004), in reviewing earlier studies that continuously monitored cytosolic Ca²⁺ concentration in E. coli, reported that in the presence of extracellular Ca²⁺ concentration within the mM range, intracellular levels steadily rose peaking at c. 2 μM before slowly declining to the initial level. This shows the fluidity of cytosolic Ca²⁺ concentration on signaling and lays the foundation for the exergonic principle underpinning Ca²⁺ flux.

Inhibition of Ca²⁺ flux across cellular systems have been found to constrain various physiological processes including chemotaxis and motility. Blocking Ca²⁺ channels of E. coli with ω-conotoxin GVIA, gallopamil or verapamil is reported to inhibit E. coli chemotaxis and motility (Tisa et al., 2000). Similarly, in Spirochaeta aurantia, chemotaxis was constrained by a Ca²⁺ channel inhibitor, botulinum toxin A (Goulbourne and Greenberg, 1983). Ca²⁺ also impacts the pattern of motility with the chelating Ca²⁺ ion in EGTA causing zoospores of Pythium spp. to swim in a straight line rather than the typical extended helical pattern interspersed with abrupt changes in direction (Donaldson and Deacon, 1993). Conversely, in planta, intracellular influx of Ca²⁺ has also been reported to constrain disease through the activation of defense responses such as rapid production of reactive oxygen species (Stael et al., 2015), phytoalexin production and accumulation (Mithöfer et al., 1999), as well as programmed cell death (Petrov et al., 2015). There are no known reports of the role of Ca²⁺ signaling in the physiological processes of Phytomyxean spp., but given the significance in other species, identifying the role of Ca²⁺ signaling on phytomyxid zoospore chemotaxis could provide a potential target for the development of a novel management strategy for their diseases. Thus, the study aimed at determining the role of Ca²⁺ signaling in the homing response of S. subterranea zoospores to host tissues. Therefore, in this study, using S. subterranea as an example of an important phytomyxid pathogen, we examined the effect of various Ca²⁺ antagonists on zoospore swimming patterns and correlated their impact on zoospore chemotaxis, root attachment and root infection.

Many Ca²⁺ antagonists are known to constrain motility, chemotaxis, and other physiological processes in microbial organisms (Matsushita et al., 1988; Tisa et al., 2000; Islam and Tahara, 2001). These antagonisms have been exploited for medical treatment of several diseases including chlamydia (Ward and Salari, 1982) and neutrophils mediated atherosclerosis in humans (Shima et al., 2008). However, Ca²⁺ antagonism treatments are yet to be fully exploited for pathogenic plant disease control even though the role of Ca²⁺ signaling in various physiological processes of some plant pathogens are known. Here, we determined the role of Ca²⁺ signaling in S. subterranea zoospore chemotaxis, and proved Ca²⁺ antagonism to constrain motility, root attachment, and root infection. All four classes of Ca²⁺ antagonist tested (Ca²⁺ channel blockers, Ca²⁺ chelators, Ca²⁺ flux inhibitors and calmodulin antagonists) in this study were found to have antagonistic effects on zoospore chemotactic attraction to glutamine, root attachment and root infection in a dose dependent manner.

The cessation of chemotaxis of LaCl₃ or GdCl₃ treated zoospores at 50–150 μM concentration in this study is presumed to be due to their Ca²⁺ channel blocking activity. Other studies also found lanthanides such as La³⁺ and Gd³⁺ significantly constrain chemotaxis (Kinoshita et al., 2017; Wheeler, 2017). It was noted that by blocking Ca²⁺ channels in the plasma membrane and membranes of intracellular Ca²⁺ stores, the lanthanides limit the availability of free cytosolic Ca²⁺, the movement of which is required to phosphorylate ATP and drive motility and chemotaxis (Tisa and Adler, 1995). Conversely, the calmodulin proteins that bind Ca²⁺ and translate cytosolic Ca²⁺ transients associated with environmental stimuli also play crucial role in chemotactic response (Bothwell et al., 2006). Inhibiting calmodulin [“a multifunctional intermediate calcium-binding messenger protein” (Stevens, 1983)] function with trifluoperazine in this study reduced zoospore chemotaxis to glutamine along a concentration gradient. This agrees with Gauthier and O’Day (2001) who found a dose-dependent effect of trifluoperazine on Dictyostelium chemotaxis toward cAMP and folic acid.

The addition of EGTA generally reduced chemotaxis except at 100 μM which was not significantly different to the controls. It is conceivable that at 100 μM, EGTA was unable to chelate sufficient extracellular Ca²⁺ to impact on cytosol free Ca²⁺. In Dictyostelium discoideum, Schaloske et al. (2005) observed that 1 mM EGTA treatment was unable to produce perceptible change in the cytosol free Ca²⁺ concentration, however, 10 mM was enough to reduce the intracellular concentration significantly. Clapham (2007) argues that in low Ca²⁺ medium, plasma membrane Ca²⁺ ATPases pump Ca²⁺ out of the cell, to deplete the intracellular stores. It is this Ca²⁺ depletion that results in diminished chemotaxis. Inhibition of chemotaxis by amiloride hydrochloride such as we found with S. subterranea zoospores has been previously observed in the chemotactic response of neutrophils to N- formyl- methionyl- leucylphenylalanin (Simchowitz and Cragoe, 1986). Amiloride inhibits Ca²⁺ fluxes by blocking Na⁺-Ca²⁺ exchange (Teiwes and Toto, 2007) to limit cytosol free Ca²⁺.

Little is known of the physiological role of Ca²⁺ signaling in zoospore attachment, although recent evidence points to the regulatory role of Ca²⁺ in cell adhesion of pathogenic bacteria (Van Nhieu et al., 2018). The inhibition of cell-cell adhesion of human fibroblast with LaCl₃ (Ko et al., 2001) provided the first direct evidence of the role of Ca²⁺ signaling on adhesion. At the organism level, adhesion and encystment of the unicellular protist Phytophthora zoospore is reported to be mediated by the secretion of proteins (Zhang et al., 2013). The relationship between Ca²⁺ signaling and adhesive protein secretion remains unknown. However, based on the dose dependent response of Ca²⁺ antagonist treatment on S. subterranea zoospore root attachment, it is conceivable to presume a Ca²⁺ signal interference in the adhesive secretion cascade. This may have led to interference with chemotaxis and root attachment. The prevalence of zoosporangia root infection is directly associated with the level of zoospore attachment, and thus the similarity of response of antagonists to these pathogen-host interactions is expected. Similar observation was made by Adorada et al. (2000) whilst studying the attachment of P. capsici zoospores to wounded pepper roots.

Given delimiting cytosolic Ca²⁺ concentration with a Ca²⁺ antagonist led to impairment of swimming behavior and chemotaxis, we can assume that for S. subterranea zoospores, swimming behavior is critical to chemotaxis and the regulation of Ca²⁺ concentration is central to it. However, the optimum Ca²⁺ concentration for swimming and chemotaxis is yet to be determined.

Directly linked to basic quantitative swimming behavior, is swimming trajectory. We found the normal extended helical movement punctuated with changes in direction (Merz, 1992) was affected differently by each Ca²⁺ antagonist. These changes in qualitative swimming behavior mirrored the changes in quantifiable behaviors. Ca²⁺ flux inhibitor or amiloride treatments changed the S. subterranea zoospore swimming pattern into more oscillatory movement which alternated frequently with helical and zig-zag patterns. In contrast, with Phytophthora aphanidermatum, Donaldson and Deacon (1993) reported that amiloride caused an irregular swimming pattern with repeated directional change. On the other hand, the Ca²⁺ chelator EGTA, did not alter the helical swimming patterns observed in the control, rather it reduced the density of the loops and thus diminished the distance traveled.

With the calmodulin antagonist, trifluoperazine, zoospore movement pattern appeared more convoluted, combining multiple swimming patterns perhaps indicating a state of confusion which culminated in frequent changes of direction at 2 μM. As the concentration increased the level of agitation reduced along with slowing down in speed. The characteristic alternation of acceleration from high to low and back to high, gave a distinct hourglass swimming pattern at 5 μM. Trifluoperazine also induced slow and spiral movements in P. aphanidermatum (Donaldson and Deacon, 1993), but in zoospores of Achlya spp., 5 μM trifluoperazine was sufficient to cause instant cessation of motility (Thomas and Butler, 1989). Various channel blockers have been known to produce motility effects similar to calmodulin antagonists (Tanida et al., 1986). The observed swimming pattern disruption described here may have originated from changes in flagella activity induced by limitations on concentration of cytosolic Ca²⁺ imposed by the various antagonist treatments. Since Ca²⁺ is the most important intracellular regulator for modulating flagella movement (Inaba, 2015) a limitation on its availability leads to disruption in flagella modulation (Smith, 2002). Though the mechanism underlying the differences in pattern induced by the different Ca²⁺ antagonist classes is beyond the scope of this study, the inherent differences in mechanism with which they achieve Ca²⁺ antagonism could be responsible for the differences in qualitative and quantitative swimming behaviors.

Overall, the further swimming pattern deviated from the helical trajectory observed in the controls, the greater the inhibition of chemotaxis, root attachment and zoosporangia root infection. This is consistent with observation in P. aphanidermatum where perturbation of the typical helical pattern into circular or straight-line movement with Ca²⁺ antagonists was associated with cessation of chemotaxis (Donaldson and Deacon, 1993).

Chemotaxis of S. subterranea zoospores appear to be dependent on Ca²⁺ signaling. It is an important prerequisite for zoospore root attachment and zoosporangia root infection. Ca²⁺ channel blockers, chelators, flux inhibitors and calmodulin antagonists are all effective at inhibiting zoospore chemotaxis and reducing S. subterranea root attachment and root infection, but channel blockers, LaCl₃ and GdCl₃ at ≥ 50 μM provides the optimum inhibition. Ca²⁺ signaling affects both qualitative and quantitative swimming behaviors just as it affects chemotaxis, thus the rate of chemotaxis is proportional to the quantifiable zoospore swimming behaviors, notably speed, acceleration, and distance. Zoospores with helical swimming patterns, traveled longer distances at higher speed and acceleration and were the most chemotactically active. Detraction from the helical pattern to more oscillatory, twisted, or meandering patterns with the application of Ca²⁺ antagonists reduce all quantifiable swimming behaviors as well as chemotaxis. It remains, however, necessary to determine the effect of Ca²⁺ antagonists on the intracellular load of Ca²⁺ to confirm that Ca²⁺ antagonists operate by limiting the cytosolic concentration of Ca²⁺. The evidence above suggests Ca²⁺ antagonist as a potential treatment for limiting S. subterranea root infection.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

JA, RT, TT, and CW designed the experiments. JA performed the experiments, analyzed the data, and prepared the original draft. CW, RT, and TT reviewed and edited the manuscript. CW was recipient of financial support for the project. All authors provided critical feedback to the article and approved the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.