Abstract

Epiphytic filamentous algae (EFA) were noted as a serious problem to reduce the production and quality of K. alvarezii. The morphological studies revealed that the main epiphyte on K. alvarezii was Neosiphonia savatieri in China. Though the harmful effects of EFA on the production of K. alvarezii have been reported, the detailed mechanism of the N. savatieri in limiting the production of K. alvarezii has not been studied yet. The present paper studied the effects of N. savatieri infection on photosynthetic behaviors in K. alvarezii by detecting chlorophyll fluorescence transient in vivo. The results revealed that damage of oxygen-evolving complex (OEC), decrease of active reaction centers (RCs), and the plastoquinone (PQ) pool as well as significant reduction in the performance indexes (PI) of PSII were caused by the infection of N. savatieri. The influence of N. savatieri on photosynthetic activity of K. alvarezii should be one of the important reasons to reduce the production of K. alvarezii infected by N. savatieri.

1. Introduction

Kappaphycus alvarezii (Solieriaceae, Rhodophyta) have been farmed as raw materials for carrageenan production in many countries since 1970s [1]. However, the carrageenan industry was faced with raw material problems relating to quality and quantity [2]. Epiphyte infection was one of the main reasons causing the decrease of quality and quantity of raw materials.

Epiphytic filamentous algae (EFA) were noted as a serious problem since early K. alvarezii cultivation [3]. The outbreaks of EFA (Polysiphonia sp., Neosiphonia savatieri) in Philippines and Malaysia, which caused a decrease of K. alvarezii production, were reported by Hurtado et al. [4] and Vairappan [5], respectively. Vairappan [5] noted that the outbreak of EFA correlated with drastic changes in seawater temperature and salinity from March to June and September to November. For further information, the infected K. alvarezii from carrageenophyte farms in the Philippines, Indonesia, Malaysia, and Tanzania were collected and studied to establish baseline information on the epiphyte’s identity, density, symptoms, and secondary infection on the host seaweed [6]. Vairappan et al. [6] found out that the dominant epiphyte in these four culture areas was N. apiculata.

EFA comprise numerous species of filamentous algae that attach to the cortical layer of the host thalli. They leave the K. alvarezii stunted, rough, and poorly branched [7]. However, more detailed physiological mechanism of EFA on K. alvarezii has not been thoroughly studied yet. Anyway, EFA cannot do harm to host thalli without two channels: exchange of materials and energy metabolism. Photosynthesis is the basic anabolism and the only way light energy transfers into electric then chemical energy in plants. Thus, the process is vital for algal growth and survival. Chlorophyll a (Chl a) fluorescence analysis has been proved to be a very useful, noninvasive tool for plant study and more specifically the behavior of photosystem II [8–11]. Recent improvements in detecting the fluorescence signal through direct and time-resolved measurements could provide detailed information on the fast fluorescence rise. All oxygenic photosynthetic materials investigated so far show a polyphasic rise consisting of the basic steps from the “origin” (𝑂) through two “inflections” (𝐼1, designated as J, and 𝐼2, termed I) to a “peak” fluorescence [12]. The O-J-I-P polyphasic transient was found to change its shape according to changes in the environment conditions [11, 13, 14]. The analysis of the fast fluorescence rise according to the JIP test allows the derivation of several expressions leading to the actual description of a photosynthetic sample in a current physiological state [8]. Here, we presented the dominant EFA in China and its impacts on photosynthetic behaviors in K. alvarezii by using continuously Chl a fluorescence, which were recorded in vivo with high time resolution and analyzed according to JIP-test.

2. Materials and Methods

2.1. Materials

Both infected and healthy green K. alvarezii were collected from Lian Bay, Hainan province, China (18°27′N, 110°5′E). Detritus on the materials were cleaned by seawater. Sections and dominant epiphytes was removed by a razor blade and then transferred to microscope slides. Slides were viewed at 100x magnification under optical microscope. Images were taken using an attached Cannon digital camera to investigate the morphological characters of epiphyte.

EFA-infected green K. alvarezii were precleaned with a soft brush to remove all the epiphytes and contaminants and then were brought to our laboratory beside the bay accompanied with the healthy ones to carry out the physiological studies.

2.2. Chl a Fluorescence Measurement

Algal thalli, about 3-4 mm in diameter and 3 cm in length, were selected, respectively, from infected and healthy green K. alvarezii. Each thallus was transferred into one capped transparent glass vial filled with seawater, and subsequently the vial was incubated at room temperature in darkness for 15 min. Chl a fluorescence of dark-adapted sample was measured by a plant efficiency analyzer (Handy PEA, Hansatech UK) and a single vial adapter for liquid-phase samples (HPEA/LPA2 Hansatech, UK). Red light of 650 nm wavelength (1500 μmol m−2 s−1) was continuously provided for 1 s. The fluorescence transients were recorded in a time span from 10 μs to 1 s. For the first 300 μs, fluorescence was sampled at 10 μs intervals. The time resolution of digitization was then switched to slower acquisition rates as the kinetics of the fluorescence signal slow. Each group of experiments was done for four times.

2.3. Analysis of OJIP Chl a Fluorescence Induction Transient

Each transient was analyzed according to JIP-test [15–18] by utilizing the following data: the minimal fluorescence intensity (𝐹𝑂) when all RCs are open, the maximal fluorescence intensity (𝐹𝑚), assuming that excitation intensity is high enough to close all the RCs of PSII, and the fluorescence intensities at times 300 μs (𝐹𝐾), 2 ms (𝐹𝐽), and 30 ms (𝐹𝐼). Based on the above data, the following parameters were then calculated: the relative variable fluorescence intensity at the J-step, 𝑉𝐽≡(𝐹𝐽−𝐹𝑂)/(𝐹𝑚−𝐹𝑂); the relative variable fluorescence intensity at the 𝐾-step, 𝑉𝐾≡(𝐹𝐾−𝐹𝑂)/(𝐹𝑚−𝐹𝑂); the approximated initial slope of the fluorescence transient, 𝑀𝑂≡4(𝐹𝐾−𝐹𝑂)/(𝐹𝑚−𝐹𝑂); the total complementary area above the O-J-I-P transient, ∫Area=𝑡𝐹𝑚0(𝐹𝑚−𝐹𝑡)𝑑𝑡.

The normalized total complementary area above the O-J-I-P transient (reflecting single-turnover 𝑄𝐴 reduction events) is 𝑆𝑚≡(Area)/(𝐹𝑚−𝐹𝑂); the times of 𝑄𝐴 have been reduced to 𝑄𝐴− in the time span from 𝑡0 to 𝑡𝐹𝑚, 𝑁≡𝑆𝑚×𝑀𝑂×(1/𝑉𝐽).

The maximum quantum yield of primary photochemistry is𝜑𝑃𝑂≡TR𝑂/ABS=[1−(𝐹𝑂/𝐹𝑚)]; the probability that a trapped exciton moves an electron into the electron transport chain beyond 𝑄A− is 𝜓𝑂≡ET𝑂/TR𝑂=(1−𝑉𝐽); the quantum yield for electron transport is𝜑𝐸𝑂≡ET𝑂/ABS=[1−(𝐹𝑂/𝐹𝑚)]×𝜓𝑂.

The specific energy fluxes (per 𝑄A-reducing PSII reaction center (RC)) for the energy absorbed isABS/RC=𝑀𝑂×(1/𝑉𝐽)×(1/𝜑𝑃𝑂); the energy trapped isTR𝑂/RC=𝑀𝑂×(1/𝑉𝐽); the electron transported isET𝑂/RC=𝑀𝑂×(1/𝑉𝐽)×𝜓𝑂; and the energy dissipated isDI𝑂/RC=(ABS/RC)−(TR𝑂/RC).

Phenomenological energy fluxes (per excited cross-section (CS)) for absorption (ABS/CS), trapping (TRO/CS), electron transport (ETO/CS), and dissipation (DIO/CS) were calculated by the following equations: ABS/CS𝑂≈𝐹𝑂 (at 𝑡=𝑡0); ABS/CS𝑚≈𝐹𝑚 (at 𝑡=𝑡𝐹𝑚); TR𝑂/CS𝑂=𝜑𝑃𝑂×(ABS/CS𝑂) (at 𝑡=𝑡0); TR𝑂/CS𝑚=𝜑𝑃𝑂×(ABS/CS𝑚) (at 𝑡=𝑡𝐹𝑚); ET𝑂/CS𝑂=𝜑𝐸𝑂×(ABS/CS𝑂) (at 𝑡=𝑡0); ET𝑂/CS𝑚=𝜑𝐸𝑂×(ABS/CS𝑚) (at 𝑡=𝑡𝐹𝑚); DI𝑂/CS𝑂=(ABS/CS𝑂)−(TR𝑂/CS𝑂) (at 𝑡=0); DI𝑂/CS𝑚=(ABS/CS𝑚)−(TR𝑂/CS𝑚) (at 𝑡=𝑡𝐹𝑚).

The density of reaction centers per exited cross-section was computed by the equations below: RC/CS𝑂=𝜑𝑃𝑂×(𝑉𝐽/𝑀𝑂)×(ABS/CS𝑂) (at 𝑡=0); RC/CS𝑚=𝜑𝑃𝑂×(𝑉𝐽/𝑀𝑂)×(ABS/CS𝑚) (at 𝑡=𝑡𝐹𝑚).

The performance indexes for absorption (PIABS) and per excited cross-section (PICS) were calculated as follows: PIABS≡(RC/ABS)×[𝜑𝑃𝑂/(1−𝜑𝑃𝑂)]×[𝜓𝑂/(1−𝜓𝑂)]; PICS𝑂≡(RC/CS𝑂)×[𝜑𝑃𝑂/(1−𝜑𝑃𝑂)]×[𝜓𝑂/(1−𝜓𝑂)] (at 𝑡=𝑡0); PICS𝑚≡(RC/CS𝑚)×[𝜑𝑃𝑂/(1−𝜑𝑃𝑂)]×[𝜓𝑂/(1−𝜓𝑂)] (at 𝑡=𝑡𝐹𝑚).

2.4. Chlorophyll a Measurement

Cleaned algal thalli, 3-4 mm in diameter and 0.5 g fresh weight, were selected, respectively, from infected and healthy green K. alvarezii. The thalli were homogenized in 5 mL of 95% ethanol for 15 min then were centrifuged at 1000 rpm for 5 min. After centrifugation, 4 mL supernatant was transferred into a colorimetric tube and diluted to 25 mL with 95% ethanol. Absorbance was measured by 722 s spectrophotometer (Shanghai precision & scientific instrument CO., LTD) at 665 nm and 649 nm. Each group of experiments was done for 3 times. Pigment concentration was calculated according to Wintermans and de Mots [19],=Chla(𝜇g/g)13.7OD665nm−5.76OD649nm×dilutionrate.0.5g(1)

2.5. Phycobiliprotein Measurement

Cleaned algal thalli, 3-4 mm in diameter and 0.5 g fresh weight, were selected, respectively, from infected and healthy green K. alvarezii. The thalli were chopped into 3 mm3 and then homogenized in 3 mL of 10 mM CaCl2 solution, which was stocked in 4°C for 12 hours prior to the experiment, for 15 min. Subsequently, the homogenized solution was transferred into a colorimetric tube then diluted to 25 mL with 10 mM CaCl2. After that, the colorimetric tubes were incubated at 4°C in dark for 48 hours. Absorbance of the supernatant at 562 nm, 615 nm, and 652 nm was measured by 722 s spectrophotometer. Each group of experiments was done for 3 times. Phycobiliproteins were calculated according to Venkataraman [20] as below:=Phycocyanin(PC)(mg/g)OD615nm−0.474OD652nm×5.34dilutionrate,=0.5g(2)Phycoerythrin(PE)(mg/g)OD562nm−2.41PC−0.849APC×9.62dilutionrate,=0.5g(3)Allophycocyanin(APC)(mg/g)OD562nm−0.208OD615nm×5.09dilutionrate,0.5g(4)Phycobiliprotein(PEP)(mg/g)=PC+APC+PE.(5)

2.6. Statistics

Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, USA). Independent sample t-test at 𝑃<0.05 was used to test the significant differences between the infected and the healthy controls.

3. Results

3.1. Dominant Epiphytes on the K. alvarezii

The dominant epiphytes are brownish red and rigid and have percurrent main axes that reach 2–15 mm. The epiphyte thalli grow on the surface of K. alvarezii solitarily and close to each other in the peak season (Figure 1(a)). A basal attachment system of the axis is at first composed of a primary rhizoid only (Figure 1(b)), and later forms a tuft of rhizoids by the production of secondary rhizoids that cut off from the pericentral cells of lower segments (Figure 1(c)). The primary rhizoid often penetrates through the outer cortical cells of K. alvarezii to medullary layer (Figure 1(b)). The main axes are 60–250 μm in diameter, with segment length 0.5–1.0-fold of diameters. The axes abruptly taper at the apices. Each vegetative segment consists of 4 pericentral cells and lacks cortical cells. The axis produces vegetative trichoblasts or first-order branches from each segment in a spiral manner. Tetrasporangia are formed in the distal segments, one per segment, in a spiral manner. Mature tetrasporangia are 90–110 μm in diameter and protuberant (Figure 1(d)). Procarpial trichoblasts replace vegetative trichoblasts or lateral branches and appear on the distal portion of branches. Each procarpial trichoblast produces a single procarp on the suprabasal segment. The procarp consists of a three-celled carpogonial branch and initials of two sterile groups, one two-celled and lateral, and the other one-celled and basal (Figure 1(e)). Mature cystocarps are broadly ovoid or napiform with 200–350 μm × 200–300 μm in size. Spermatangia are produced on a lateral of fertile trichoblasts that issues from the suprabasal segment. Mature spermatangial branches are conical with 130–200 μm × 45–60 μm in size. They have a one-celled sterile suprabasal segment and the basal segment embedded in the parental branch (Figure 1(f)).

Rhizoids cut off from the pericentral cells of the lower segments, the production of lateral branch in a spiral arrangement, three-celled carpogonial branches, spermatangial trichoblasts with a sterile lateral, and spiralled tetrasporangia found in the epiphyte ally it with Neosiphonia than Polysiphonia [21]. In addition to these features, the morphology and size of the main axes, tetrasporangia, carpogonial, and spermatangial all ally it with N. savatieri than N. apiculata [22, 23]. Therefore, based on the results above the dominant EFA in Lian Bay, Hainan province, China are N. savatieri.

3.2. Fast Chl a Fluorescence Kinetics, O-JIP

Figure 2 showed the fast Chl a fluorescence induction kinetics of both the healthy and the infected K. alvarezii. When the thalli of K. alvarezii are exposed to saturating actinic light, the Chl a fluorescence curves start from the initial FO intensity and increase to a peak (𝑃 or 𝐹𝑚). When the curves were plotted on logarithmic scale, two intermediate steps 𝐹𝐽 (about 2 ms) and 𝐹𝐼 (about 30 ms) can be found between 𝐹𝑂 and 𝐹𝑚. To visualize the comparative effects of N. savatieri infection on each step, the curves were replotted as relative variable fluorescence, 𝑉𝑡=(𝐹𝑡−𝐹𝑂)/(𝐹𝑚−𝐹𝑂) in the insert chart of Figure 2. Based on the insert chart in Figure 2, certain increases in the peaks at 𝐾-, 𝐽-, and 𝐼-steps were found in the N. savatieri-infected K. alvarezii compared with the healthy seaweed.

3.3. Donor and Acceptor Side of PSII Reaction Center

Increase amplitude in K-step was used as a specific indicator of damage to the oxygen-evolving complex (OEC) [12, 17, 24, 25]. The amplitude in the K-step of K. alvarezii, expressed as the ratio 𝑉𝐾, was shown in Table 1. An obvious increase in 𝑉𝐾 was observed in N. savatieri-infected K. alvarezii, which reflected that the OEC of host was at least partly damaged. Meanwhile, the number of RCs per excited cross-section (RC/CS𝑂 or RC/CS𝑚) was reduced in K. alvarezii after N. savatieri infection. 𝑉𝐽 was used as an indicator of the proportion of active reaction centers (RCs) [12, 15, 17]. The increase of 𝑉𝐽 (Table 1) further indicated that the number of active RCs in the N. savatieri-infected seaweed obviously decreased.

The approximated initial slope of the fluorescence transient (𝑀𝑂), a profile of the maximal reduction rate of 𝑄𝐴, increased by 89.5% in N. savatieri-infected K. alvarezii (Table 1). However, the normalized total complementary area above the O-J-I-P transient (𝑆𝑚), the energy needed to reduce all the 𝑄𝐴, decreased by 29.5% (Table 1). The increase in 𝑀𝑂 and decrease in 𝑆𝑚 was one indicator of the decrease in the plastoquinone (PQ) pool [12, 15, 17, 25]. Therefore, the changes of the 𝑀𝑂 and 𝑆𝑚 in K. alvarezzi, after N. savatieri infection showed the plastoquinone (PQ) pool of the host decreased. What is more, 𝑁≡𝑆𝑚×𝑀𝑂×(1/𝑉𝐽), the negligible change (2.7%) in the turnover number of 𝑄𝐴(𝑁) were induced by the integrated effects of changes in 𝑀𝑂, 𝑉𝐽, and 𝑆𝑚.

3.4. Energy Distribution via PSII Reaction Center

After N. savatieri infection, the energy fluxes via PSII reaction centers (RCs) in K. alvarezii significantly changed. The light energy for absorption (ABS/RC) and trapping (TR𝑂/RC) in N. savatieri infected K. alvarezii-increased by 49.5% and 50% (Table 2). However, the specific energy fluxes (per 𝑄𝐴-reducing PSII reaction center (RC)) for the energy dissipated (DI𝑂/RC) increased significantly (Table 2), and the energy for electron transported per reaction center (ET𝑂/RC) in the N. savatieri-infected K. alvarezii did not change so significantly. Therefore, most of the energy trapped was not used for photosynthesis but dissipated by the reaction centers.

Similarly, the energy distribution was further expressed via excited cross-section. Regardless of Chl a fluorescence at 𝑡𝐹𝑂 or 𝑡𝐹𝑚, the phenomenological energy fluxes per excited cross section (CS) for absorption (ABS/CS), trapping (TR𝑂/CS), and dissipation (DI𝑂/CS) in K. alvarezii increased by 27% (Table 3) after N. savatieri infection. The increase in the DI𝑂/CS acted as a counterbalance to the increase of TR𝑂/CS. Therefore, the change of electron transport per excited cross section (ET𝑂/CS) in K. alvarezzii after N. savatieri infection was negligible (Table 3).

3.5. Performance Indexes (PI) and Quantum Yields

The probability that trapped exciton moves an electron into the electron transport chain beyond 𝑄A−(𝜓𝑂), and the quantum yield for electron transport (𝜑𝐸𝑂) decreased by 23.5% and 24.1% in N. savatieri-infected K. alvarezii comparing with the healthy control (Table 4). No significant changes in the maximum quantum yield of primary photochemistry (𝜑𝑃𝑂) were found in the infected K. alvarezii compared with the control. However, the comprehensive performance indexes (PI) significantly decreased (Table 4). The average PIABS,  PICS𝑂, and PICS𝑚 in N. savatieri-infected K. alvarezii decreased by 57.7%, 44%, and 42.9%, respectively, compared with the healthy control (Table 4).

3.6. Photosynthetic Pigments

Chl a and phycobiliprotein content in K. alvarezii changed significantly (Table 5) after the seaweed was infected by N. savatieri (𝑃<0.05). The content of Chl a, phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC), and phycobiliprotein (PBP) in N. savatieri-infected K. alvarezii increased about 56.4%, 104.5%, 146.2%, 139.4%, and 130.9% compared with the healthy control, respectively (Table 5). The pigments increase in N. savatieri-infected K. alvarezii (Table 5) was much higher than the increase of ABS/CS and TR𝑂/CS (Table 4). The above results indicated a relative decrease in the light energy absorbed per pigment.

4. Discussion

The changes in PSII performance of the photosynthetic apparatus caused by environmental stress or senescence have been explored widely by applying the chlorophyll fluorescence technique in higher plants [8, 26–31]. However, there is not detailed knowledge on the influence of epiphyte on the photosynthetic activity of its host. In the present study, we have demonstrated the response of PSII of K. alvarezii to N. savatieri infection. The Chl a fluorescence transient recorded with high time resolution was analyzed by the JIP-test in order to quantify the PSII behaviors in K. alvarezii after N. savatieri infection.

𝜑𝑃𝑂 changed slightly; however, PI decreased significantly in N. savatieri-infected K. alvarezii. The PI was calculated from three components, which depend on the reaction center density, the trapping efficiency, and the electron transport efficiency. The above changes of PI showed that photosynthesis in the infected K. alvarezii was inhibited which could partly explain the phenomenon of stunted, rough, and poorly branched carrageenan producing seaweed arisen by epiphyte infection [7]. Moreover, our results proved that PI is more sensitive to environmental change than 𝜑𝑃𝑂 and correlates well with plant vigor and performance again that agrees with the research by Hermans et al. [32].

Chlorophyll and phycobiliprotein content in N. savatieri-infected K. alvarezii was increased to 156% and 230% (Table 5). Therefore, the energy fluxes for absorption and trapping in N. savatieri-infected K. alvarezii were increased (Tables 2 and 3). However, the negligible changes of ET𝑂/CS (Table 3) and 𝜑𝑃𝑂 (Table 4) indicated that the trapped energy was not efficiently used for electron transport. The damage of OEC, decrease in RC number, and reduction of PQ pool could further explain why the light trapped in N. savatieri-infected K. alvarezii was not sufficiently consumed timely for photosynthesis.

The side impacts of epiphyte on K. alvarezii growth were not only limited to photochemical reactions. Largo et al. [33] reported that light intensity of less than 50 μmoL photon m−2 could induce the decay of K. alvarezii. N. savatieri occupied the outsurface of K. alvarezii and shielded the host from getting enough light. Moreover, both N. savatieri and K. alvarezii all belonged to Rhodophyta species and owned similar types of photosynthetic pigments that aggravated the competition of light absorption between them. The competition between N. savatieri and K. alvarezii seriously decreased the ambient light. However, the infected K. alvarezii tried to acclimate itself to the low-light conditions by increasing its photosynthetic pigments, especially phycobiliprotein (Table 5). Unfortunately, the adaptive regulation seemed to be meaningless for EFA-infected K. alvarezii because of the decrease in active RC number, damage of OEC, and reduction of PQ pool as mentioned above. Glenn and Doty [34] reported that culture of K. alvarezii required high levels of water motion provided by strong and consistent trade winds. N. savatieri, covered on the surface of K. alvarezii, were bound to reduce the water motion nearby the K. alvarezii as well as the materials exchange between K. alvarezii and external environment. Therefore, the production of oxygen by the photosynthesis of N. savatieri and K. alvarezii was easy to cause the surplus of oxygen during the daytime. Moreover, the consumption of oxygen by the respiration of N. savatieri and K. alvarezii was easy to cause the insufficiency of oxygen during the nighttime. In addition, the epiphyte N. savatieri competed with host K. alvarezii for absorbing nutrients (N, P, CO2, and other mineral elements). Most of the nutrients dissolving in the water body were first filtered by N. savatieri before reaching to K. alvarezii, and so nutrient deficiency inevitably occurred in K. alvarezii after EFA infection. Dense N. savatieri were severe stress for the metabolism of K. alvarezii by shading, high O2 concentrations in the light, anoxic conditions in the dark, and competition of nutrients. Therefore, heavy decay in K. alvarezii was usually found when the seaweed was infected by N. savatieri.

In conclusion, the dominant EFA on K. alvarezii in Lian Bay, Hainan province were N. savatieri. Damage of OECs, decrease of active RCs and the PQ pool and significant reduction in the performance indexes (PI) of PSII were caused by the infection of N. savatieri although the seaweed acclimated itself to the low-light condition by increasing its photosynthetic pigments to absorb more light energy. The influence of N. savatieri on photosynthetic activity of K. alvarezii was one of the important reasons to reduce the production of K. alvarezii.

Acknowledgments

The authors wish to thank Mr. Zhaoliang Zheng from Lingshui Haotian Company for the provision of algae. This work was supported by the National Natural Science Foundation of China (30771639), Special Project for Marine Public Welfare Industry (200705010).