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Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release, development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta)
FEMS Microbiology Letters 211 (2002) 253^257
www.fems-microbiology.org
Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release, development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta) M.K. Addepalli, Yuji Fujita
Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo Machi, Nagasaki 852-8521, Japan Received 23 March 2002; accepted 22 April 2002 First published online 22 May 2002
Abstract Formation of zoosporangia and cleavage of zoosporangial cytoplasm to zoospores in Pythium porphyrae is absolutely dependent on extracellular calcium. Calcium ion could be substituted neither by monovalent nor divalent cations tested. Increased concentrations of extracellular calcium did not affect the release of zoospores from zoosporangia but inhibited the zoospore motility. Chelating calcium ion by EGTA has decreased the ability of encysted zoospores to germinate and form appressoria. The increased external calcium-ion concentration has decreased the infectivity of Porphyra yezoensis thalli in a linear fashion apparently indicating a role of calcium in the signaling mechanism. - 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Marine Pythium ; Calcium ion; Zoospore; Motility; Porphyra yezoensis
1. Introduction Pythium porphyrae (Oomycota) is a microbial pathogen causes red rot disease in ‘nori’ (Porphyra spp.) cultivation farms in Japan [1]. The causative agent of the red rot disease is zoospore and its disease-initiation mechanism is similar to that of other oomycetes infecting higher plants. The initial process of host recognition involves activation of receptor molecules present on the surface of the zoospore membrane by the potential chemical signals of the host [2]. This activation of receptor molecules is believed to have a triggering e¡ect on secondary messenger-mediated signaling pathway involving calcium ions and inositol triphosphate for the encystment cum attachment of the zoospore to the host surface [3^5]. Attachment of the zoospore is followed by germination leading to the formation of germ tube and appressorium, a prelude to the invasion of the host membranes [2]. In Phytophthora cinnamomi cyst attachment calcium ion is an absolute requirement for the adhesive nature of the glycoprotein secreted [6] while in Pythium aphanidermatum cyst attachment and its germination are found to promote [7].
Studies on Phytophthora parasitica [8] and Phytophthora soja [9] showed that calcium has a central role in the regulation of zoospore infection and development respectively. In the case of Pythium marinum it is known that calcium plays an important role in the growth of vegetative hyphae [10]. However, until now there is no information available on in£uence of calcium ion during sporulation of Py. porphyrae. Due to marine habitat of Py. porphyrae it remains interesting to know how calcium regulates these processes in such extreme conditions where divalent ions calcium, magnesium and monovalent ions sodium, potassium etc. are abundant. Hence, the present study is aimed at understanding the role of calcium in the induction of zoosporangia, release of zoospores, their motility and germination, and further, to understand the infection ability of the zoospores in the presence of varying levels of calcium under in-vitro conditions.
2. Materials and methods 2.1. Culture and induction of zoospores
* Corresponding author. Fax: +81 (95) 844 1111 (ext. 3147). E-mail address : [email protected] (Y. Fujita).
Py. porphyrae strain C-1 [11] was maintained on Arasaki B medium [12]. Vegetative hyphal growth and culture
0378-1097 / 02 / $22.00 - 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 0 0 - 0
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conditions were performed as described previously [11]. The liquid culture grown strains of Py. porphyrae hyphae were washed with a volume of 500 ml, either in natural half-strength seawater (NHSS, contains Ca2þ of V5 mM) or in test solutions (arti¢cial half-strength seawater with various concentrations of calcium ion, AHSS) for 5 h on an orbital shaker with a change of wash medium every hour at 15‡C. The 5-h-washed hyphae were incubated for 14 h at 15‡C in the respective wash media. At the end of incubation time, hyphae were washed in the respective wash media for 1 h before incubating with wash medium at 15‡C for the synchronous release of zoospores. Maximum numbers of zoospores were usually released between 2 and 3 h of incubation. All the experiments contained vegetative hyphae grown from four agar discs in 40 ml of liquid culture medium. All throughout the experiments wherever applicable AHSS was used as a basal medium to which amendments were made as per requirement. Arti¢cial seawater contained NaCl, 24.60 g; KCl, 0.67 g; MgCl 2 6H 2 O, 4.66 g; MgSO 4 7H2 O, 6.29 g for 1 l of high-performance liquid chromatography grade water. All the solutions used were adjusted to pH 7.4 and sterilized by autoclave. 2.2. E¡ect of calcium on formation of zoosporangia and release of zoospores Mycelial mats of Py. porphyrae were washed in distilled water, AHSS with di¡erent calcium concentrations (0^90 mM), and incubated as above for the induction of zoosporangia. The mycelia mats were observed for zoosporangial formation using an inverted microscope with a 10U objective. The controls and cations included were NHSS, AHSS in which calcium was compensated with MgCl2 , KCl or NaCl separately. For the release of zoospores, mycelial mats bearing zoosporangia were washed with respective wash media, for 1 h with four changes of solutions each with a volume of 50 ml. Mycelial mats were incubated with 15 ml of fresh wash solution at 15‡C for 2 h to induce synchronous release of zoospores from zoosporangia in Petri dishes. The number of zoospores released from the zoosporangia was estimated using hemacytometer by withdrawing 1 ml of zoospore suspension and encysting the spores by vortexing in a Eppendor¡ tube for 1 min and ¢xing with 3% ¢nal concentration of glutaraldehyde for 10 min. 2.3. E¡ect of calcium on the motility of the zoospores Synchronous zoospore release was performed as above into the calcium-amended solutions. The dishes were observed for number of motile zoospores on the surface and at the bottom of the Petri dish with a microscope equipped with a digital video camera (Fujix digital camera HC-300/ 0L, Olympus, Japan). For each dish, video recordings were performed at three di¡erent locations for 25 s each.
By repeated playing and pausing, the numbers of motile spores were counted in each frame without repetition of the spores counted in the previous frame (monitor used was Multi scan G200, Sony Trinitron, Japan). The numbers of cysts were derived both from the video recordings and from the images at the bottom of the Petri dish. The formation of secondary zoospores from the encysted zoospores was observed by incubating 0.3 ml of vertex-encysted zoospores (a suspension containing 1U104 cells ml31 ) in a chambered glass slide for 2 h at room temperature. At the end of incubation, the cells were immobilized by the addition of glutaraldehyde to a ¢nal concentration of 3%. The number of encysted spores, cysts having germ tubes and zoospores were counted at ¢ve di¡erent microscopic ¢elds. 2.4. E¡ect of ethylene glycol-bis-(L-amino ethylene)N,N,NP,NP-tetraacetic acid (EGTA) on germination A 0.3 ml suspension of 1U104 zoospores ml31 induced in AHSS (5 mM) were taken in a 24-well plate bottom of which was covered by cover glass. A 100 Wl of EGTA stock solution prepared in dilute NaOH of ¢nal pH 8.0 was added to the wells to ¢nal concentrations ranging from 0.1 to 0.5 mM and incubated at 20‡C for 4 h. At the end of speci¢ed incubation period, glutaraldehyde of 3% ¢nal concentration was added to the wells and ¢xed for 5 min. The cover glasses were withdrawn from the wells, stained for 1 min in 0.1% (w/v) calco£uore white followed by brief rinsing in AHSS before observing by £uorescent microscope. The number of germinated zoospores was counted at ¢ve di¡erent microscopic ¢elds. 2.5. E¡ect of calcium on zoospores infecting Porphyra yezoensis thallus Culture and maintenance of P. yezoensis thalli were performed as described previously [11]. Zoospore suspension of 1 ml having 1U104 zoospores ml31 induced in AHSS containing 5 mM of CaCl2 /CaNO3 was inoculated into the wells of a 24-multi-well plate containing P. yezoensis thalli discs of 5 mm diameter. To the suspension, 100 Wl CaCl2 /CaNO3 solution prepared in AHSS was added to the ¢nal concentrations ranging from 10 to 90 mM. The plates were gently mixed and incubated at 20‡C for 4 h. At the end of incubation, glutaraldehyde of 3% ¢nal concentration was added to the wells and ¢xed for 5 min. The thallus discs were washed in AHSS by stirring for 1 min before staining with 0.1% (w/v) calco£uore white. The stained thalli discs were brie£y rinsed in AHSS and were observed by an epi£uorescent microscope (BHB, Olympus, Japan) to count the number of encysted and germinating zoospores at ¢ve di¡erent microscopic ¢elds using 20U objective. All the experiments were carried out twice and each in
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Fig. 1. A: Py. porphyrae zoosporangial vesicle formation from the septate vegetative hypha. Arrowhead indicates the movement of cytoplasm into the globular zoosporangial vesicle. Scale bar = 25 Wm. B: Reorganization of cytoplasm in zoosporangial vesicle after the completion of hyphal cytoplasm into the globular vesicle. Arrowhead indicates reorganization of the zoosporangial vesicle cytoplasm. In the absence of external source of calcium the zoosporangial vesicles gets arrested at this stage. Scale bar = 25Wm. C: Zoosporangial cytoplasm undergoing cytoplasmic cleavage before di¡erentiation into individual zoospores in the presence of external calcium. Arrowhead indicates cleavage furrow formed in the zoosporangial vesicle cytoplasm. Scale bar = 25 Wm. D: Actively moving zoospores inside the zoosporangial vesicle membrane before dissolution of vesicle membrane and their subsequent release into external medium. Arrowhead indicates zoospore. Scale bar = 25Wm. E: Pre-encysted zoospore which has developed into germinating cyst instead of developing into secondary zoospore. Arrowhead indicates the germ tube emerging from cyst. Scale bar = 10 Wm. F: Cysts attached to the surface of P. yezoensis thallus (stained by 0.1% (w/v) calco£uore white and observed by £uorescence microscope) before penetration and colonization of host membranes. Arrowhead indicates the penetration of the germ tube into the host membranes. Scale bar = 10 Wm.
quadruplicate and their standard deviations were calculated.
3. Results The 12^14 h incubated nutrient-depleted hyphae at 15‡C induced zoosporangia formation in test solutions in which calcium was amended. The morphogenetic changes associated with zoosporangia formation include the di¡erentiation of coenocytic ¢lamentous hypha into septate multicellular hypha followed by migration and reorganization of cytoplasm into the globular zoosporangial vesicle as shown in Fig. 1A p B respectively. Incubation of hyphae having zoosporangia in fresh NHSS or respective test solutions resulted in cleavage of zoosporangial cytoplasm (Fig. 1C) and subsequent formation of zoospores as shown in Fig. 1D. However, zoosporangia were not formed in the case of mycelia washed in calcium-free AHSS and in which calcium was supplemented with an equaling concentration of sodium chloride, potassium chloride or magnesium chloride. The mycelia washed in distilled water were plasmolysed. However, the mycelium incubated with 5 mM of calcium, the lower limit of test solutions, yielded zoosporangia and released zoospores upon incubation with fresh AHSS containing 5 mM calcium. Hyphae washed and incubated 12^14 h at 15‡C either in AHSS containing 5 mM calcium or in NHSS upon washing with 200 ml of calcium-free AHSS for 2 h followed by incubation at 15‡C in the fresh calcium free
AHSS did not release the zoospores in spite of the formation of the zoosporangial vesicles (Table 1). The zoosporangial vesicles (Fig. 1B) failed to form cleavage planes, as shown in Fig. 1C, to form zoospores in absence of external calcium source. The number of zoospores that had released from the zoosporangia incubated with di¡erent test solutions containing calcium from 5 to 90 mM did not vary markedly except at 90 mM, where there was an inhibition of number of zoospores released. O¡ calcium chloride and calcium nitrate, the calcium nitrate has a slightly inhibitory a¡ect on release of zoospores (Table 1). However, the motility of zoospores had decreased with the increase in the concentration of calcium (Table 2). Pre-encysted zoospores incubated at room temperature
Table 1 E¡ect of calcium on release of zoospores from zoosporangiaa Calcium concentration (mM)
Number of zoospores (U103 ) ml31 released
NHSS1 (V5) AHSS+Ca2þ
5.5 S 1.90 CaCl2
Ca(NO3 )2
5 10 30 50 70 90
4.0 S 0.50 4.5 S 1.50 4.3 S 1.00 4.6 S 1.50 4.0 S 1.00 2.0 S 1.00
6.3 S 1.00 5.0 S 2.00 3.0 S 0.50 5.0 S 2.60 3.0 S 1.00 0.6 S 0.00
a Zoospores were not released from the AHSS in which calcium was supplemented with cations Mg2þ , Kþ or Naþ .
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Table 2 E¡ect of calcium on the motility of Py. porphyrae zoospores Calcium concentration (mM)
Number of motile zoospores (value in parentheses indicates the percent of motile cells)
1
NHSS (V5) AHSS+Ca2þ
38.2 S 4.23 (85.12) CaCl2
Ca(NO3 )2
5 10 30 50 70 90
31.41 S 2.61 33.80 S 5.37 24.83 S 1.65 20.83 S 7.77 6.99 S 2.33 3.49 S 0.23
26.5 S 7.76 (74.80) 52.0 S 5.47 (67.80) 36.0 S 25.23 (53.06) 14.5 S 3.87 (38.82) 6.5 S 1.29 (22.80) 2.25 S 0.08 (5.00)
for 2 h had not formed the secondary zoospores. The preencysted zoospores have germ tubes (67.84 S 8.21), as shown in Fig. 1E, remain quiescent (17.92 S 2.29) or busted (14.26 S 4.20) out of 100 cells counted randomly for each. The number of zoospores that have germinated and possessed appressorium have decreased with the increase in the concentration of the EGTA from 0 to 0.4 mM as shown in Table 3. The co-incubation of P. yezoensis thallus discs with zoospore suspensions containing di¡erent concentrations of calcium resulted in adhesion of cysts to P. yezoensis thallus as observed by calco£uore staining (Fig. 1F). However, there was a decrease in the number of zoospores infecting the thallus with the increase of calcium concentration from 5 to 90 mM (Fig. 2).
4. Discussion In the present study, it has been found that calcium plays an important role in the zoospore biology of Py. porphyrae. In asexual reproduction of Py. porphyrae, external calcium regulates the formation and cleavage of zoosporangia and zoosporangial cytoplasm into individual zoospores. The lack of zoosporangia formation in the absence of external calcium and in the presence of other mono and divalent cations (sodium, potassium and magnesium respectively) as a substitute for calcium shows the absolute necessity of external calcium in the asexual reproduction of Py. porphyrae. This could be due to insu⁄cient quantity of internal calcium accumulated during growth
(86.20) (79.20) (59.06) (53.12) (25.58) (12.41)
and/or its inability to mobilize for sporulation. In aquatic fungus, Blastocladiella emersonii short exposure to calcium is found to be su⁄cient for the formation of zoosporangia and sporulation suggesting that an early calcium exposure is su⁄cient for the sporulation process to complete [13]. However, in the present study it was found that even though the zoosporangia were formed in the presence of calcium, sporulation does not take place in the absence of calcium. These results suggest that calcium plays an important role not only in the formation of zoosporangia but also for the cleavage of zoosporangial cytoplasm into individual zoospores. Similar responses were observed in the case of zoosporangia formation and their cleavage in different strains of Py. porphyrae proves that the calcium responses of Py. porphyrae were not strain-dependent but were of genetic in nature. In Phy. soja at high concentrations of calcium, zoospores germinate within the zoosporangial vesicles [9] whereas in this study at a concentration of 50 mM, Py. porphyrae zoospores were released from the zoosporangia and there was no zoospore germination observed within zoosporangia. This suggests that Py. porphyrae can tolerate higher levels of calcium ion in its vicinity not only during growth [10] but also for di¡erentiation into zoospores and their release from zoosporangia. Secondary zoospore formation by the cysts of Py. porphyrae was absent unlike that of in Phy. soja and Phy. parasitica where secondary zoospore formation is a default option if the zoospore fails to ¢nd an appropriate host [9,14]. In the present investigation with the increase in the concentration of the EGTA, calcium chelator, the
Table 3 E¡ect of EGTA on germination of pre-encysted zoospores EGTA concentration (mM)
0.0 0.1 0.2 0.3 0.4
Cell type (% S S.D.) germinated cysts
ungerminated cysts
79.17 S 5.80 76.64 S 6.48 57.01 S 5.71 29.59 S 7.34 24.53 S 5.58
20.83 S 5.80 23.35 S 6.48 42.99 S 5.69 70.40 S 7.34 75.47 S 9.92
Fig. 2. E¡ect of calcium on Py. porphyrae zoospore infectivity of P. yezoensis thallus.
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number of germinating cysts has decreased. This indicates that calcium is actively involved in the germination process of the encysted zoospore. Similar results were reported in Py. aphanidermatum [7]. However, with increase in the concentration of calcium, there was a decrease in the number of zoospores infecting P. yezoensis thalli. The decrease in infection levels with the increase in calcium-ion concentration might be due to activation of calcium-assisted signaling pathways resulting in early encystment and germination of the zoospores, which were independent from that of the host-related signaling mechanism. This proves that even though Py. porphyrae can tolerate higher levels of external calcium, the host-recognition mechanism gets remain impaired due to calcium-induced early signaling pathways resulting in failure to identify host-emitting signals. This might be the possible reason for the observation of frequent red rot disease occurrence at river mouths where calcium-ion concentration is less than that of in high-saline marine cultivation farms. In the present investigation we found that the Py. porphyrae requirement for calcium is absolute in zoosporangia formation, release of zoospores and their germination. Even though high-tolerance limits of calcium were observed in the present study, typical for marine-dwelling oomycetes, calcium-dependent signaling pathways remain sensitive to the external levels of calcium during the infection process in causing red rot disease of Porphyra spp.
Acknowledgements This work is partially supported by a grant-in-aid for Scienti¢c Research (No. 11660191) and a Scholarship to M.K.A. from the Ministry of Education, Science, Sports and Culture, Japan.
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References [1] Fujita, Y., 1990. Diseases of cultivated Porphyra in Japan. In: Introduction to Applied Phycology (Akatsuka, I., Ed.), pp. 177^190. SPB Academic Publishing, The Haugue. [2] Kerwin, J.L., Johnson, L.M., Oward, C., Whisler and Tuininga, A.R. (1992) Infection and morphogenesis of Pythium marinum in species of Porphyra and other red algae. Can. J. Bot. 70, 1017^1024. [3] Hardham, A.R., Cahill, D.M., Cope, M., Gabor, B.K. and Gubler, F. (1994) Cell surface antigens of Phytophthora spores biological and taxonomic characterization. Protoplasma 181, 213^232. [4] Hyde, G. (1998) Calcium imaging: A primer for mycologists. Fungal Genet. Biol. 24, 14^23. [5] Dean, R.A. (1997) Signal pathways and appressorium morphogenesis. Annu. Rev. Phytopathol. 35, 211^234. [6] Gubler, F., Hardham, A.R. and Duniec, J. (1989) Characterizing adhesiveness of Phytophthora cinnamomi zoospores during encystment. Protoplasma 149, 24^30. [7] Donaldson, S.P. and Deacon, J.W. (1992) Role of calcium in adhesion and germination of cysts of Pythium: a model toexplain infection of host plants. J. Gen. Microbiol. 138, 2051^2059. [8] Warburton, A.J. and Deacon, J.W. (1998) Transmembrane Ca2þ £uxes associated with zoospore encystment and cyst germination by the phytopathogen Phytophthora parasitica. Fungal Genet. Biol. 25, 54^62. [9] Xu, C. and Morris, P.F. (1998) External calcium controls the developmental strategy of Phyrtophthora sojae cysts. Mycologia 90, 269^ 275. [10] Kazama, F.Y. and Fuller, M.S. (1973) Mineral nutrition of Pythium marinum, a marine facultative parasite. Can. J. Bot. 51, 693^699. [11] Addepalli, M.K. and Fujita, Y. (2001) Serological detection of red rot disease initiation stages of microbial pathogen, Pythium porphyrae (Oomycota) on Porphyra yezoensis. J. Appl. Phycol. 13, 221^227. [12] Arasaki, S., Akitno, K. and Tomiyama, Y. (1968) A comparison of some physiological aspects in a marine Pythium on the host and on the arti¢cial medium. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12, 203^206. [13] Elisabete, C.C. and Luiz, C.C. (1999) The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium. FEMS Microbiol. Lett. 179, 353^359. [14] von Broemson, S.L. and Deacon, J.W. (1996) E¡ects of calcium on germination and further zoospore release from zoospore cysts of Phytophthora parasitica. Mycol. Res. 100, 1498^1504.
FEMSLE 10497 10-6-02
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Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release, development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta) M.K. Addepalli, Yuji Fujita
Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo Machi, Nagasaki 852-8521, Japan Received 23 March 2002; accepted 22 April 2002 First published online 22 May 2002
Abstract Formation of zoosporangia and cleavage of zoosporangial cytoplasm to zoospores in Pythium porphyrae is absolutely dependent on extracellular calcium. Calcium ion could be substituted neither by monovalent nor divalent cations tested. Increased concentrations of extracellular calcium did not affect the release of zoospores from zoosporangia but inhibited the zoospore motility. Chelating calcium ion by EGTA has decreased the ability of encysted zoospores to germinate and form appressoria. The increased external calcium-ion concentration has decreased the infectivity of Porphyra yezoensis thalli in a linear fashion apparently indicating a role of calcium in the signaling mechanism. - 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Marine Pythium ; Calcium ion; Zoospore; Motility; Porphyra yezoensis
1. Introduction Pythium porphyrae (Oomycota) is a microbial pathogen causes red rot disease in ‘nori’ (Porphyra spp.) cultivation farms in Japan [1]. The causative agent of the red rot disease is zoospore and its disease-initiation mechanism is similar to that of other oomycetes infecting higher plants. The initial process of host recognition involves activation of receptor molecules present on the surface of the zoospore membrane by the potential chemical signals of the host [2]. This activation of receptor molecules is believed to have a triggering e¡ect on secondary messenger-mediated signaling pathway involving calcium ions and inositol triphosphate for the encystment cum attachment of the zoospore to the host surface [3^5]. Attachment of the zoospore is followed by germination leading to the formation of germ tube and appressorium, a prelude to the invasion of the host membranes [2]. In Phytophthora cinnamomi cyst attachment calcium ion is an absolute requirement for the adhesive nature of the glycoprotein secreted [6] while in Pythium aphanidermatum cyst attachment and its germination are found to promote [7].
Studies on Phytophthora parasitica [8] and Phytophthora soja [9] showed that calcium has a central role in the regulation of zoospore infection and development respectively. In the case of Pythium marinum it is known that calcium plays an important role in the growth of vegetative hyphae [10]. However, until now there is no information available on in£uence of calcium ion during sporulation of Py. porphyrae. Due to marine habitat of Py. porphyrae it remains interesting to know how calcium regulates these processes in such extreme conditions where divalent ions calcium, magnesium and monovalent ions sodium, potassium etc. are abundant. Hence, the present study is aimed at understanding the role of calcium in the induction of zoosporangia, release of zoospores, their motility and germination, and further, to understand the infection ability of the zoospores in the presence of varying levels of calcium under in-vitro conditions.
2. Materials and methods 2.1. Culture and induction of zoospores
* Corresponding author. Fax: +81 (95) 844 1111 (ext. 3147). E-mail address : [email protected] (Y. Fujita).
Py. porphyrae strain C-1 [11] was maintained on Arasaki B medium [12]. Vegetative hyphal growth and culture
0378-1097 / 02 / $22.00 - 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 0 0 - 0
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conditions were performed as described previously [11]. The liquid culture grown strains of Py. porphyrae hyphae were washed with a volume of 500 ml, either in natural half-strength seawater (NHSS, contains Ca2þ of V5 mM) or in test solutions (arti¢cial half-strength seawater with various concentrations of calcium ion, AHSS) for 5 h on an orbital shaker with a change of wash medium every hour at 15‡C. The 5-h-washed hyphae were incubated for 14 h at 15‡C in the respective wash media. At the end of incubation time, hyphae were washed in the respective wash media for 1 h before incubating with wash medium at 15‡C for the synchronous release of zoospores. Maximum numbers of zoospores were usually released between 2 and 3 h of incubation. All the experiments contained vegetative hyphae grown from four agar discs in 40 ml of liquid culture medium. All throughout the experiments wherever applicable AHSS was used as a basal medium to which amendments were made as per requirement. Arti¢cial seawater contained NaCl, 24.60 g; KCl, 0.67 g; MgCl 2 6H 2 O, 4.66 g; MgSO 4 7H2 O, 6.29 g for 1 l of high-performance liquid chromatography grade water. All the solutions used were adjusted to pH 7.4 and sterilized by autoclave. 2.2. E¡ect of calcium on formation of zoosporangia and release of zoospores Mycelial mats of Py. porphyrae were washed in distilled water, AHSS with di¡erent calcium concentrations (0^90 mM), and incubated as above for the induction of zoosporangia. The mycelia mats were observed for zoosporangial formation using an inverted microscope with a 10U objective. The controls and cations included were NHSS, AHSS in which calcium was compensated with MgCl2 , KCl or NaCl separately. For the release of zoospores, mycelial mats bearing zoosporangia were washed with respective wash media, for 1 h with four changes of solutions each with a volume of 50 ml. Mycelial mats were incubated with 15 ml of fresh wash solution at 15‡C for 2 h to induce synchronous release of zoospores from zoosporangia in Petri dishes. The number of zoospores released from the zoosporangia was estimated using hemacytometer by withdrawing 1 ml of zoospore suspension and encysting the spores by vortexing in a Eppendor¡ tube for 1 min and ¢xing with 3% ¢nal concentration of glutaraldehyde for 10 min. 2.3. E¡ect of calcium on the motility of the zoospores Synchronous zoospore release was performed as above into the calcium-amended solutions. The dishes were observed for number of motile zoospores on the surface and at the bottom of the Petri dish with a microscope equipped with a digital video camera (Fujix digital camera HC-300/ 0L, Olympus, Japan). For each dish, video recordings were performed at three di¡erent locations for 25 s each.
By repeated playing and pausing, the numbers of motile spores were counted in each frame without repetition of the spores counted in the previous frame (monitor used was Multi scan G200, Sony Trinitron, Japan). The numbers of cysts were derived both from the video recordings and from the images at the bottom of the Petri dish. The formation of secondary zoospores from the encysted zoospores was observed by incubating 0.3 ml of vertex-encysted zoospores (a suspension containing 1U104 cells ml31 ) in a chambered glass slide for 2 h at room temperature. At the end of incubation, the cells were immobilized by the addition of glutaraldehyde to a ¢nal concentration of 3%. The number of encysted spores, cysts having germ tubes and zoospores were counted at ¢ve di¡erent microscopic ¢elds. 2.4. E¡ect of ethylene glycol-bis-(L-amino ethylene)N,N,NP,NP-tetraacetic acid (EGTA) on germination A 0.3 ml suspension of 1U104 zoospores ml31 induced in AHSS (5 mM) were taken in a 24-well plate bottom of which was covered by cover glass. A 100 Wl of EGTA stock solution prepared in dilute NaOH of ¢nal pH 8.0 was added to the wells to ¢nal concentrations ranging from 0.1 to 0.5 mM and incubated at 20‡C for 4 h. At the end of speci¢ed incubation period, glutaraldehyde of 3% ¢nal concentration was added to the wells and ¢xed for 5 min. The cover glasses were withdrawn from the wells, stained for 1 min in 0.1% (w/v) calco£uore white followed by brief rinsing in AHSS before observing by £uorescent microscope. The number of germinated zoospores was counted at ¢ve di¡erent microscopic ¢elds. 2.5. E¡ect of calcium on zoospores infecting Porphyra yezoensis thallus Culture and maintenance of P. yezoensis thalli were performed as described previously [11]. Zoospore suspension of 1 ml having 1U104 zoospores ml31 induced in AHSS containing 5 mM of CaCl2 /CaNO3 was inoculated into the wells of a 24-multi-well plate containing P. yezoensis thalli discs of 5 mm diameter. To the suspension, 100 Wl CaCl2 /CaNO3 solution prepared in AHSS was added to the ¢nal concentrations ranging from 10 to 90 mM. The plates were gently mixed and incubated at 20‡C for 4 h. At the end of incubation, glutaraldehyde of 3% ¢nal concentration was added to the wells and ¢xed for 5 min. The thallus discs were washed in AHSS by stirring for 1 min before staining with 0.1% (w/v) calco£uore white. The stained thalli discs were brie£y rinsed in AHSS and were observed by an epi£uorescent microscope (BHB, Olympus, Japan) to count the number of encysted and germinating zoospores at ¢ve di¡erent microscopic ¢elds using 20U objective. All the experiments were carried out twice and each in
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Fig. 1. A: Py. porphyrae zoosporangial vesicle formation from the septate vegetative hypha. Arrowhead indicates the movement of cytoplasm into the globular zoosporangial vesicle. Scale bar = 25 Wm. B: Reorganization of cytoplasm in zoosporangial vesicle after the completion of hyphal cytoplasm into the globular vesicle. Arrowhead indicates reorganization of the zoosporangial vesicle cytoplasm. In the absence of external source of calcium the zoosporangial vesicles gets arrested at this stage. Scale bar = 25Wm. C: Zoosporangial cytoplasm undergoing cytoplasmic cleavage before di¡erentiation into individual zoospores in the presence of external calcium. Arrowhead indicates cleavage furrow formed in the zoosporangial vesicle cytoplasm. Scale bar = 25 Wm. D: Actively moving zoospores inside the zoosporangial vesicle membrane before dissolution of vesicle membrane and their subsequent release into external medium. Arrowhead indicates zoospore. Scale bar = 25Wm. E: Pre-encysted zoospore which has developed into germinating cyst instead of developing into secondary zoospore. Arrowhead indicates the germ tube emerging from cyst. Scale bar = 10 Wm. F: Cysts attached to the surface of P. yezoensis thallus (stained by 0.1% (w/v) calco£uore white and observed by £uorescence microscope) before penetration and colonization of host membranes. Arrowhead indicates the penetration of the germ tube into the host membranes. Scale bar = 10 Wm.
quadruplicate and their standard deviations were calculated.
3. Results The 12^14 h incubated nutrient-depleted hyphae at 15‡C induced zoosporangia formation in test solutions in which calcium was amended. The morphogenetic changes associated with zoosporangia formation include the di¡erentiation of coenocytic ¢lamentous hypha into septate multicellular hypha followed by migration and reorganization of cytoplasm into the globular zoosporangial vesicle as shown in Fig. 1A p B respectively. Incubation of hyphae having zoosporangia in fresh NHSS or respective test solutions resulted in cleavage of zoosporangial cytoplasm (Fig. 1C) and subsequent formation of zoospores as shown in Fig. 1D. However, zoosporangia were not formed in the case of mycelia washed in calcium-free AHSS and in which calcium was supplemented with an equaling concentration of sodium chloride, potassium chloride or magnesium chloride. The mycelia washed in distilled water were plasmolysed. However, the mycelium incubated with 5 mM of calcium, the lower limit of test solutions, yielded zoosporangia and released zoospores upon incubation with fresh AHSS containing 5 mM calcium. Hyphae washed and incubated 12^14 h at 15‡C either in AHSS containing 5 mM calcium or in NHSS upon washing with 200 ml of calcium-free AHSS for 2 h followed by incubation at 15‡C in the fresh calcium free
AHSS did not release the zoospores in spite of the formation of the zoosporangial vesicles (Table 1). The zoosporangial vesicles (Fig. 1B) failed to form cleavage planes, as shown in Fig. 1C, to form zoospores in absence of external calcium source. The number of zoospores that had released from the zoosporangia incubated with di¡erent test solutions containing calcium from 5 to 90 mM did not vary markedly except at 90 mM, where there was an inhibition of number of zoospores released. O¡ calcium chloride and calcium nitrate, the calcium nitrate has a slightly inhibitory a¡ect on release of zoospores (Table 1). However, the motility of zoospores had decreased with the increase in the concentration of calcium (Table 2). Pre-encysted zoospores incubated at room temperature
Table 1 E¡ect of calcium on release of zoospores from zoosporangiaa Calcium concentration (mM)
Number of zoospores (U103 ) ml31 released
NHSS1 (V5) AHSS+Ca2þ
5.5 S 1.90 CaCl2
Ca(NO3 )2
5 10 30 50 70 90
4.0 S 0.50 4.5 S 1.50 4.3 S 1.00 4.6 S 1.50 4.0 S 1.00 2.0 S 1.00
6.3 S 1.00 5.0 S 2.00 3.0 S 0.50 5.0 S 2.60 3.0 S 1.00 0.6 S 0.00
a Zoospores were not released from the AHSS in which calcium was supplemented with cations Mg2þ , Kþ or Naþ .
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Table 2 E¡ect of calcium on the motility of Py. porphyrae zoospores Calcium concentration (mM)
Number of motile zoospores (value in parentheses indicates the percent of motile cells)
1
NHSS (V5) AHSS+Ca2þ
38.2 S 4.23 (85.12) CaCl2
Ca(NO3 )2
5 10 30 50 70 90
31.41 S 2.61 33.80 S 5.37 24.83 S 1.65 20.83 S 7.77 6.99 S 2.33 3.49 S 0.23
26.5 S 7.76 (74.80) 52.0 S 5.47 (67.80) 36.0 S 25.23 (53.06) 14.5 S 3.87 (38.82) 6.5 S 1.29 (22.80) 2.25 S 0.08 (5.00)
for 2 h had not formed the secondary zoospores. The preencysted zoospores have germ tubes (67.84 S 8.21), as shown in Fig. 1E, remain quiescent (17.92 S 2.29) or busted (14.26 S 4.20) out of 100 cells counted randomly for each. The number of zoospores that have germinated and possessed appressorium have decreased with the increase in the concentration of the EGTA from 0 to 0.4 mM as shown in Table 3. The co-incubation of P. yezoensis thallus discs with zoospore suspensions containing di¡erent concentrations of calcium resulted in adhesion of cysts to P. yezoensis thallus as observed by calco£uore staining (Fig. 1F). However, there was a decrease in the number of zoospores infecting the thallus with the increase of calcium concentration from 5 to 90 mM (Fig. 2).
4. Discussion In the present study, it has been found that calcium plays an important role in the zoospore biology of Py. porphyrae. In asexual reproduction of Py. porphyrae, external calcium regulates the formation and cleavage of zoosporangia and zoosporangial cytoplasm into individual zoospores. The lack of zoosporangia formation in the absence of external calcium and in the presence of other mono and divalent cations (sodium, potassium and magnesium respectively) as a substitute for calcium shows the absolute necessity of external calcium in the asexual reproduction of Py. porphyrae. This could be due to insu⁄cient quantity of internal calcium accumulated during growth
(86.20) (79.20) (59.06) (53.12) (25.58) (12.41)
and/or its inability to mobilize for sporulation. In aquatic fungus, Blastocladiella emersonii short exposure to calcium is found to be su⁄cient for the formation of zoosporangia and sporulation suggesting that an early calcium exposure is su⁄cient for the sporulation process to complete [13]. However, in the present study it was found that even though the zoosporangia were formed in the presence of calcium, sporulation does not take place in the absence of calcium. These results suggest that calcium plays an important role not only in the formation of zoosporangia but also for the cleavage of zoosporangial cytoplasm into individual zoospores. Similar responses were observed in the case of zoosporangia formation and their cleavage in different strains of Py. porphyrae proves that the calcium responses of Py. porphyrae were not strain-dependent but were of genetic in nature. In Phy. soja at high concentrations of calcium, zoospores germinate within the zoosporangial vesicles [9] whereas in this study at a concentration of 50 mM, Py. porphyrae zoospores were released from the zoosporangia and there was no zoospore germination observed within zoosporangia. This suggests that Py. porphyrae can tolerate higher levels of calcium ion in its vicinity not only during growth [10] but also for di¡erentiation into zoospores and their release from zoosporangia. Secondary zoospore formation by the cysts of Py. porphyrae was absent unlike that of in Phy. soja and Phy. parasitica where secondary zoospore formation is a default option if the zoospore fails to ¢nd an appropriate host [9,14]. In the present investigation with the increase in the concentration of the EGTA, calcium chelator, the
Table 3 E¡ect of EGTA on germination of pre-encysted zoospores EGTA concentration (mM)
0.0 0.1 0.2 0.3 0.4
Cell type (% S S.D.) germinated cysts
ungerminated cysts
79.17 S 5.80 76.64 S 6.48 57.01 S 5.71 29.59 S 7.34 24.53 S 5.58
20.83 S 5.80 23.35 S 6.48 42.99 S 5.69 70.40 S 7.34 75.47 S 9.92
Fig. 2. E¡ect of calcium on Py. porphyrae zoospore infectivity of P. yezoensis thallus.
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number of germinating cysts has decreased. This indicates that calcium is actively involved in the germination process of the encysted zoospore. Similar results were reported in Py. aphanidermatum [7]. However, with increase in the concentration of calcium, there was a decrease in the number of zoospores infecting P. yezoensis thalli. The decrease in infection levels with the increase in calcium-ion concentration might be due to activation of calcium-assisted signaling pathways resulting in early encystment and germination of the zoospores, which were independent from that of the host-related signaling mechanism. This proves that even though Py. porphyrae can tolerate higher levels of external calcium, the host-recognition mechanism gets remain impaired due to calcium-induced early signaling pathways resulting in failure to identify host-emitting signals. This might be the possible reason for the observation of frequent red rot disease occurrence at river mouths where calcium-ion concentration is less than that of in high-saline marine cultivation farms. In the present investigation we found that the Py. porphyrae requirement for calcium is absolute in zoosporangia formation, release of zoospores and their germination. Even though high-tolerance limits of calcium were observed in the present study, typical for marine-dwelling oomycetes, calcium-dependent signaling pathways remain sensitive to the external levels of calcium during the infection process in causing red rot disease of Porphyra spp.
Acknowledgements This work is partially supported by a grant-in-aid for Scienti¢c Research (No. 11660191) and a Scholarship to M.K.A. from the Ministry of Education, Science, Sports and Culture, Japan.
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