Application of real-time PCR assay for detection and quantification of ...

Plankton Benthos Res 5(2): 56–61, 2010

Plankton & Benthos Research © The Plankton Society of Japan

Application of real-time PCR assay for detection and quantification of bloom-forming diatom Chaetoceros tenuissimus Meunier KENSUKE TOYODA, KEIZO NAGASAKI & YUJI TOMARU* National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi, Hatsukaichi, Hiroshima 739–0452, Japan Received 7 July 2009; Accepted 12 February 2010

Abstract: Viral infection of the bloom-forming diatom Chaetoceros tenuissimus was discovered in 2008, and is now assumed to have a significant influence on the dynamics of C. tenuissimus populations in natural environments. Enumeration of C. tenuissimus and its viruses is essential when examining the host-virus relationship in situ; however, the diatom species is so small in size that its identification and counting by optical microscope is almost impossible. To resolve this problem, we have developed a TaqMan-based real-time polymerase chain reaction (PCR) method for detection and quantification of C. tenuissimus. We designed primers and a TaqMan probe to target the D1 region of its 28S ribosomal RNA (rRNA) gene; the established real-time PCR was specific at the species level by testing 41 microalgal strains including C. tenuissimus. Tris–EDTA buffer-based boiling method was shown to be efficient for extracting DNA from filter-trapped C. tenuissimus cells in this study. The detection range of the established TaqMan-based real-time PCR method for C. tenuissimus was 101 to 106 cells collected on a filter; the method was applicable for C. tenuissimus cells accompanied with natural microorganisms. Key words:

Chaetoceros tenuissimus, detection, diatom, enumeration, TaqMan-based real-time PCR

Introduction Diatoms (Bacillariophyeae) are key players in the oceanic carbon cycles, accounting for a large part of marine primary production (Smetacek 1999, Falkowski et al. 2000). Hence, it is important to understand their dynamics in natural environments from the viewpoint of biogeochemistry and marine ecology. Phytoplankton population dynamics are the result of reproduction and losses; mechanisms for the latter include grazing, sinking, and natural mortality. There has been recent recognition of the importance of viral infection as one of the primary causes of phytoplankton mortality. Thus far, at least seven different viruses have been isolated and reported as infecting diatoms of the genera Rhizosolenia and Chaetoceros; they are assumed to have a significant effect on the dynamics of their host diatoms in natural environments (Nagasaki 2008, Eissler et al. 2009, Tomaru et al. 2009). The genus Chaetoceros is one of the largest diatom groups, with more than 400 species described (VanLandingham 1968, Rines & Hargraves 1988). Because of its * Corresponding author: Yuji Tomaru; E-mail, [email protected]

high abundance, it is important to understand the ecology of Chaetoceros in natural waters from the viewpoint of primary production. In the genus, Chaetoceros tenuissimus Meunier is well-known as a cosmopolitan species (Meunier 1913, Hasle & Syvertsen 1996, Sunesen et al. 2008, McQuoid & Godhe 2004) and our preliminary study shows that the species causes blooms in the coastal waters in Japan (unpubl.). Shirai et al. (2008) reported the existence of a virus infecting C. tenuissimus, however, the host-virus relationships in natural environments are only poorly understood due to the lack of data on the host dynamics. The main reason for the problem is that C. tenuissimus is one of the hardest species to count by optical microscopy because of the smallness of the cells (10 m m). Usually, transmission electron microscopy is the only way to achieve precise identification, but this is not feasible for counting. It is thus necessary to develop a new routine enumeration method for C. tenuissimus in natural plankton assemblages. Recent reports have described a new technique for monitoring microalgal cells, using a real-time polymerase chain reaction (PCR) assay that is based on rRNA gene sequences. The technique allows rapid and accurate detection

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Real-time PCR for Chaetoceros tenuissimus

and quantification of target cells (Bowers et al. 2000, Dyhrman et al. 2006, Kamikawa et al. 2006). As well as being a powerful tool, real-time PCR assays have the additional advantage of being applicable to field samples. In the present study, we developed a real-time PCR system to detect and enumerate C. tenuissimus. The method developed in the present study will help further understanding of the ecology of diatom host-virus systems in natural environments.

Table 1.

Materials and Methods Algal cultures and growth condition The main axenic clonal diatom strain used in the present study was Chaetoceros tenuissimus strain 2–10 which was isolated from the Maiko Coast, Seto Inland Sea, Hyogo, Japan, on 10 th August 2002 (Shirai et al. 2008). In addition, 40 clonal algal strains were used for testing the specificity of the real-time PCR system designed in this study

List of clonal algal strains, isolated from Japanese coastal waters, tested in the present study.

Sample code 2–10 2–6 AG07-C03 AR07-C03 Ch42* Ch48* Chae5* Chaet. sp.* D51* IT07-C09* IT07-C11* IT07-C34 IT07-C37 L-4* SS08-C03* SS08-C07* SS08-C09* TG07-C12* TG07-C28* SS08-0624-1* SS08-8-20 SS08-8-21 SS08-8-23* ITDia1* ITDia8 EzB* Rs2* SFBB TA0704Hama05 ACNG GC27-1 HU* Mz2 HCLG1 Ht GmH6 Pt1 ScKR CMKG-1* F96 H93616*

Species name Chaetoceros tenuissimus Meunier Chaetoceros tenuissimus Meunier Chaetoceros tenuissimus Meunier Chaetoceros tenuissimus Meunier Chaetoceros salsugineum Takano Chaetoceros debilis Cleve Chaetoceros cf. affinis Lauder Chaetoceros sp. Chaetoceros lorenzianus Grunow Chaetoceros sp. Chaetoceros setoensis Ikari Chaetoceros sp. Chaetoceros cf. pseudocurvisetus L.Mangin Chaetoceros socialis f. radians (Schütt) Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Chaetoceros sp. Thalassiosira rotula Meunier Ditylum brightwellii (T.West) Eucampia zodiacus Ehrenberg Rhizosolenia setigera Brightwell Nannochloropsis sp. Teleaulax amphioxeia (W.Conrad) Alexandrium catenella (Whedon & Kofoid) Gymnodinium catenatum Graham Heterocapsa circularisquama T.Horiguchi Heterocapsa circularisquama T.Horiguchi Heterocapsa circularisquama T.Horiguchi Heterocapsa triquetra (Ehrenberg) Karenia mikimotoi (Miyake & Kominami ex Oda) Prorocentrum triestinum Schiller Scrippsiella sp. Chattonella marina (Subrahmanyan) Fibrocapsa japonica Toriumi & Takano Heterosigma akashiwo (Hada)

Isolation locality Harimanada, Seto Inland Sea, Hyogo Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Ago Bay, Mie Pref. Ariake Sound, Northwest side of Kyushu Ariake Sound, Northwest side of Kyushu Ariake Sound, Northwest side of Kyushu Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Unknown Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Ago Bay, Mie Pref. Ago Bay, Mie Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Harimanada, Seto Inland Sea, Hyogo Pref. Seto Inland Sea, Hiroshima Pref. Unknown (Europe) Lake Hamana, Shizuoka Pref. Nagasaki Bay, Ngasaki Pref. Unknown Uranouchi Inlet, Kochi Pref. Maizuru, Kyoto Pref. Gokasho Bay, Mie Pref. Unknown Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref. Kure, Seto Inland Sea, Hiroshima Pref. Kagoshima Pref. Ehime Pref. Hiroshima Bay, Seto Inland Sea, Hiroshima Pref.

* Strains used for the preliminary specificity test for the primer pair (see Materials and Methods) Pref. indicates Prefecture.

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(Table 1). These algal strains were grown in modified SWM3 medium (Chen et al. 1969, Itoh & Imai 1987) enriched with 2-nM sodium selenite (Na2SeO3) using a 12-h light/12-h dark cycle with ca. 110 m mol photons m2 s1 provided as cool white fluorescent illumination; diatoms and flagellates were incubated at 15°C and 20°C, respectively. DNA extraction from algal cultures Cells in the late logarithmic phase of growth were collected onto 2.0-m m-pore-size Nucleopore filters, and preserved at 80°C until analysis. In order to optimize the DNA extraction protocol for the real-time PCR assay, the following three DNA extraction methods were compared. (1) TE-boiling method: 1103 cells on the filter were placed in a 0.5 mL tube and incubated in 100 m L of Tris–EDTA (TE) buffer (pH 8.0) at 100°C for 10 min, then stored at 4°C. After centrifugation at 17,000g for 5 min, 100 m L of the supernatant was used for further tests. (2) Cetyltrimethylammonium bromide (CTAB) method: 1103 cells on the filter were placed in a 1.5 mL tube and added with 500 m L of 2% CTAB solution and 2 m L of 2-mercap-

toethanol. The samples were incubated at 65°C for 1 h and then centrifuged at 17,000g for 5 min. Following this, DNA was purified using the phenol–chloroform extraction method, precipitated twice with ethanol, and redissolved in 100 m L of TE buffer. (3) DNeasy method: A DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) was used for extracting DNA from 1103 cells on the filter according to the manufacturer’s instructions. Finally, extracted DNA was dissolved in 100 m L of TE buffer. Each DNA extraction method was conducted in triplicate from the filter-collected cells, which were enumerated by optical microscopy using a hemocytometer (Erma INC., Tokyo, Japan). Efficiency of extraction was evaluated by means of the real-time PCR assay described below. Design of TaqMan probe and primer set The 28S rDNA D1 region of the 19 Chaetoceros clones (Table 1, Fig. 1) was amplified using primers D1F and D2R specific to the 28S rDNA D1/D2 region (Table 2) (Scholin et al. 1994). For the PCR reaction, 25 m L of reaction mixture contained the primers at 0.1 m M each; 1 m L of DNA template (containing 5–20 ng DNA); 0.1 mM each dATP,

Fig. 1. Alignment of the nucleotide sequences of partial D1 region of the 19 Chaetoceros species tested in the present study. Strains 2–10, 2–6, AR07-C03 and AG07-C03 are Chaetoceros tenuissimus; Ch42 is Chaetoceros salsugineum; Ch48 is Chaetoceros debilis; D51 is Chaetoceros lorenzianus; L-4 is Chaetoceros socialis f. radians; the other strains are Chaetoceros spp. apparently differing in morphology. Gray background areas correspond, respectively, to primers and a TaqMan probe designed for specific detection of C. tenuissimus. Table 2.

Primers and a TaqMan probe used in the present study.

Primer or probe Primer D1F Primer D2R Primer 2–10_EF Primer 2–10_ER 2–10_SetE_Probe

Target 28S rDNA D1/D2 region of diatoms 28S rDNA D1/D2 region of diatoms Chaetoceros tenuissimus C. tenuissimus TaqMan probe for 2–10 primer set

Sequence (5–3) ACC CGC TGA ATT TAA GCA TA CCT TGG TCC GTG TTT CAA GA TTG TGG AGA GGT ACG CTT GTC TT CCC TCA TAG GCA CCC TGT TC FAM-CCT TAG CTT AAA TCT CT-NFQ-MGB*

* FAM, carboxyfluorescein; 3-NFQ, non-fluorescent quencher; MGB, minor groove binder.

Real-time PCR for Chaetoceros tenuissimus

dCTP, dGTP, and dTTP; 2.5 m L of 10PCR Buffer for Blend Taq; and 1.25 units of Blend Taq polymerase (Toyobo, Osaka, Japan). The PCR conditions were as follows: an initial step at 94°C for 2 min, followed by 30 cycles with a denaturation temperature of 94°C for 30 sec, an annealing temperature of 50°C for 30 sec, and an extension temperature of 72°C for 1 min. This condition yielded PCR fragments of the expected size (592 to 641 bp) from each diatom DNA sample. Purified PCR products were analysed by bi-directional direct sequencing with the primers D1F and D2R. The resultant 19 sequences were aligned using Genetyx version 5.1.0 software (Genetyx Corporation, Tokyo, Japan). Based on the alignment, we designed two primers (2–10_EF, 2-10_ER) and a TaqMan MGB-Probe (2–10_SetE_Probe) to target the C. tenuissimus-specific region which is 64 bp in length (Table 2, Fig. 1). The TaqMan probe was labelled with carboxyfluorescein (FAM) at the 5-end, and non-fluorescent quencher (NFQ) and minor groove binder (MGB) at the 3-end.

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ducted. For the PCR reaction, 50 m L of reaction mixture contained the primers at 0.3 m M each; 1 m L of DNA template; 0.4 mM each dATP, dCTP, dGTP, and dTTP; 25 m L of 2PCR Buffer for KOD FX; and 1 units of KOD FX (Toyobo, Osaka, Japan). The PCR conditions were as follows: an initial step at 94°C for 2 min, followed by 25 cycles with a denaturation temperature of 98°C for 10 sec, an annealing temperature of 52°C for 30 sec, and an extension temperature of 68 °C for 30 sec. The resultant products were subjected to electrophoresis, stained with ethidium bromide, and visualized under UV. Furthermore, the strain-specificity of the designed realtime PCR was also examined. DNA was extracted from the 41 algal strains (Table 1) by means of the CTAB method; then, they were applied to the real-time PCR system at final concentrations of 0.1 and 0.5 ng m L1. The conditions for the real-time PCR assay and data analysis are mentioned above. Quantification of C. tenuissimus

Real-time PCR assay conditions Real-time PCR assays were performed with the abovementioned primer set and TaqMan probe using a StepOne™ Real-Time PCR System (Applied Biosystems Japan Ltd., Tokyo, Japan) according to the manufacturer’s recommendation. The following reagents were added to a 20 m L reaction mixture: forward and reverse primers (2–10_EF, 2–10_ER) each at a final concentration of 0.4 m M; fluorogenic probe (2–10_SetE_Probe) at a final concentration of 0.2 m M; 1 m L of template DNA; 10 m l of 2 TaqMan® Gene Expression Master Mix (containing PCR buffer, dNTPs, MgCl2, and AmpliTaq Gold DNA polymerase; Applied Biosystems, Japan), and PCR-grade water to a final volume of 20 m L. The thermal cycling conditions consisted of 2 min at 50°C and 10 min at 95°C followed by 39 cycles of 15 sec at 95°C and 1 min at 60°C. Fluorescence data were collected at the end of each cycle, and determination of the threshold cycle (CT) line was carried out automatically by StepOne™ Real-Time PCR System version 1.0 (Applied Biosystems Japan Ltd., Tokyo, Japan). For the resultant data, a statistical analysis of variance (ANOVA) test was performed using Origin 7 software (Origin Lab Co., Massachusetts, USA). Specificity test To check the specificity of the designed primer pair and the inhibitory effects of algal cell lysates on PCR, a preliminary test was conducted as mentioned below. First, each 1103 cells of the 21 algal strains (see Table 1: shown with asterisks) were added to two 1.5 mL tubes; then, one was added with 1103 cells of C. tenuissimus strain 2–10. After centrifugation at 6,500g for 5 min, each pellet was frozen at 80°C and then DNA was extracted according to the TE-boiling method as mentioned above. Then, PCR targeting the C. tenuissimus-specific fragment (64 bp) was con-

Standard curves were constructed using serial dilutions of C. tenuissimus 2–10 culture with known cell counts. Briefly, a cell quantity of 1106 was enumerated using the hemocytometer; then, 10-fold serial dilutions from 1101 to 1106 cells were prepared and the cells were harvested onto 2.0-m m-pore-size Nucleopore filters at each dilution (resulted in 101, 102, 103, 104, 105 and 106 cells filter1). DNA was extracted from the filter-collected cells using the TE-boiling method and was measured in duplicate by the real-time PCR assay. The measurements were then used to construct the standard curve. Then, cultured C. tenuissimus cells suspended in natural seawater were tested using the above-constructed standard curve. Natural seawater was collected from Hiroshima Bay (Hiroshima Pref., Japan) on 12 th December 2008, and filtered through 3-m m-nominal-pore-size filter (Nucleopore) to thoroughly remove natural C. tenuissimus cells. To each 50 mL of filtered natural seawater, we added C. tenuissimus cells to make the final cell number 101–106 cells based on hemocytometer enumerations, and the suspended cells were collected onto 2.0-m m-pore-size Nucleopore filters (101, 102, 103, 104, 105 and 106 cells filter1, respectively). Each filter was placed in a 0.5 mL tube and preserved at 80°C; thereafter, DNA was extracted by the TE-boiling method as described above, and measured by the real-time PCR assay. The experiments were conducted in triplicate. Results and Discussion The results of comparing the DNA extraction methods are shown in Fig. 2. The average CT values estimated for the three differently extracted DNA samples (by means of TE-boiling, CTAB, and DNeasy) were 28.93, 33.40, and 30.80, respectively (Fig. 2); the CT value for the TE-boiling method was significantly lower than that of the other two methods (ANOVA, p0.01). Hence, the TE-boiling method

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Fig. 2. Comparison of three DNA extraction methods, TE-boiling method (TE), CTAB method (CTAB) and DNeasy method (DNeasy). (a) Amplification plot with DNA sample extracted by each extraction method (r20.986). (b) Comparison of CT values calculated from the amplification plot of DNA extraction with the three methods. The measurements were conducted in triplicate. * p0.01, ANOVA.

was shown to be remarkably effective for extracting DNA from Chaetoceros tenuissimus cells in this study. This result was unexpected as DNA samples extracted by the TEboiling method are usually considered to be the least pure; i.e., containing unknown materials inhibiting PCR. In a real-time PCR system for dinoflagellate cysts, DNA extraction method using CTAB performed more effectively than using TE-boiling (Kamikawa et al. 2007); opposite to the present results. To this end, each algal sample is considered to have a most optimal combination of DNA extraction conditions; it is thus essential to perform an optimization experiment before embarking on a real-time PCR assay. Specificity of the designed primer pair and the inhibitory effects of algal cell lysates on PCR were checked by a simple PCR experiment. As a result, amplification of the PCR fragment (64 bp) was only observed in samples with C. tenuissimus cells added (data not shown). This indicated that the designed primer pair was highly specific to C. tenuissimus and that the cell lysate originating from the 21 algal strains did not inhibit the reaction. By the real-time PCR assay using the primers (2–10_EF, 2–10_ER) and the TaqMan Probe (2–10_SetE_Probe), all the tested C. tenuissimus strains (2–10, 2–6 AR07-C03 and AG07-C03) were positively detected; in contrast, the other 37 microalgal strains (including 19 Chaetoceros species) were not, indicating high specificity to C. tenuissimus. The result was consistent with the high homology in rRNA regions (18S, ITS-5.8S-ITS, 28S) and Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit gene among C. tenuissimus strains (99.9%, data not shown). The results of the quantification of C. tenuissimus cell number based on the real-time PCR method developed in this study are shown in Fig. 3. The linearity of the standard curve was high within the 5-log range tested, r20.998 (Fig. 3a). Figure 3b demonstrates the correlation between cell counts using the haemacytometer (cells) and estimates obtained by real-time PCR assay for C. tenuissimus cells suspended in the filtered natural seawater (cells filter1) (see Materials and Methods). There was a linear relation-

Fig. 3. Correlation between the cell counts and estimation results by real-time PCR assay of Chaetoceros tenuissimus cells (10-fold dilution between 101 and 106 cells). (a) Standard curve plotting log haemacytometer quantification of C. tenuissimus cells (X) versus CT values (Y); slope3.262, amplification efficiency102.572%, r20.998. (b) Scatter diagram of haemacytomater quantification (X) versus TaqMan Probe quantification (Y) of C. tenuissimus cells suspended in natural seawater. Y0.983 X0.085, r20.998, p0.0001, N18.

ship between them with a high correlation coefficient (Y0.983 X0.085, r20.998, p0.0001, N18), from 1101 to 1106 cells based on haemacytometer counts (Fig. 3b). These results indicate that the real-time PCR assay protocol we established allows sensitive detection and accurate quantification of C. tenuissimus cells accompanied with natural microorganisms. By means of the real-time PCR assay developed in this study, sensitive detection and accurate quantification of C. tenuissimus cells were shown to be possible. Just recently, the copy number of rRNA genes has been revealed to be changeable both with growth phase and among clones in dinoflagellates (Galluzzi et al. 2009); these are to be noted in translating the results of quantitative PCR for microalgae. Here, we compared the results of the real-time PCR assay for three different strains of C. tenuissimus, 2–10, 2–6, and AG07-C03; consequently, no statistically significant difference among the quantification results was detected (unpubl.). Still, it would be wise to keep paying attention to the variety in copy numbers of rRNA genes among newly-isolated clones to raise the reliability of the real-time PCR assay for C. tenuissimus. Not with standing, C. tenuissimus cells in natural seawater were also appropriately measured; thus, this method should be useful for examining the dynamics of C. tenuissimus in natural environments. Obviously, it should be noted that the microbial composition of

Real-time PCR for Chaetoceros tenuissimus

each seawater sample significantly differs; hence, careful translation of enumeration results would be necessary. Even then, this system should be very helpful for enumerating C. tenuissimus, as it obviates the need for troublesome and unreliable microscopic counting. Acknowledgements We express sincere thanks to T. Nagumo, Nippon Dental University, and H. Suzuki, Tokyo University of Marine Science and Technology for identifying several Chaetoceros spp. We are also grateful to S. Nagai (National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency) for providing the Teleaulax amphioxeia culture. This manuscript was improved by the valuable comments of the editor and two anonymous reviewers to whom we are indebted. This study was partially supported by Grants-in-Aid for Young Scientist (B) (20780149) from the Japan Ministry of Education, Science, Sports and Culture. References Bowers HA, Tengs T, Glasgow HB Jr, Burkholder JM, Rublee PA, Oldach DW (2000) Development of real-time PCR assay for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl Environ Microbiol 66: 4641–4648. Chen LCM, Edelstein T, McLachlan J (1969) Bonnemaisonia hamifera Hariot in nature and in culture. J Phycol 5: 211–220. Dyhrman ST, Erdner D, Du JL, Galac M, Anderson DM (2006) Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5: 242–250. Eissler Y, Wang K, Chen F, Wommack E, Coats W. (2009). Ultrastructural characterization of the lytic cycle of an intra-nuclear virus infecting the diatom Chaetoceros wighamii (Bacillariophyceae) from Chesapeake Bay, USA. J Phycol 45: 787–797. Falkowski P, Scholes RJ, Boyle E, Canadell J, Canfield D, Elser J, Gruder N, Hibbard K, Hogberg P, Linder S, Mackenzie FT, Moore B, Pedersen T, Rosenthal Y, Seitzinger S, Smetacek V, Steffen W (2000) The global carbon cycle: A test of our knowledge of earth as a system. Science 290: 291–296. Galluzzi L, Bertozzini E, Penna A, Perini F, Garcés E, Magnani M (2009) Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods. J Appl Phycol Online First DOI 10.1007/s10811009-9411-3. Hasle GR, Syvertsen EE (1996) Marine diatoms. In: Identifying marine diatoms and dinoflagellates. (ed Tomas CR). Academic

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