Enhancement of Seaweed Rhizoid and Blade Formations by the Chlorophyte Codium fragile Extract

녹조류 청각 추출물에 의한 해조류 가근 및 유엽형성 촉진

Abstract

Living organisms can maintain or extend their territories by producing allelochemicals that influence the growth, survival, and reproduction of other organisms. To identify natural biostimulants of positive allelochemicals, we screened 18 common seaweed extracts for enhancement of rhizoid and blade production in a convenient Porphyra suborbiculata monospore assay. By addition of methanolic extract from the most potent green seaweed, Codium fragile, 100% and 50% enhancement doses reflecting the amount of C. fragile extract required to enhance rhizoid formation (in terms of number of spores with rhizoids per total spores tested) were approximately 100 and 50 μg/ml, respectively, in the P. suborbiculata monospore culture. The C. fragile extract quickly enhanced rhizoid formation, rhizoid numbers per rhizoid-holding spore, rhizoid length, blade formation (in number of spores with blade per total spores tested), and blade length from most monospores at early culture days. The extract enhanced rhizoid formation after 2 days of culture significantly, rhizoid numbers per rhizoid-holding spore after 3 days, rhizoid length after 3 days, blade formation after 2 days, and blade length after 1 day, respectively, from most monospores. The allelochemicals that enhanced favorite seaweed species may be efficacious for new seaweed management technologies, including the development of biostimulant agents based on natural products.

많은 생물체들은 다른 생물체들의 성장, 생존, 재생에 영향을 미치는 allelochemical 물질들을 생성함으로서 자신들의 영역을 유지 확장할 수 있다. 천연 생물촉진활성을 가진 allelochemicals을 찿기 위하여 18종의 흔한 해조류를 대상으로 실험실에서 편리하게 배양가능한 둥근돌김의 중성포자들로부터 가근 및 유엽의 형성 촉진효과를 탐색하였다. 그중 가장 활성이 높은 녹조류 청각의 추출물은 약 100 및 50 μg/ml 농도에서 최대 및 반 정도의 중성포자로부터 가근 형성 촉진효과를 나타내었다. 또한 청각 추출물은 중성포자 배양 초기에 가근형성, 가근갯수, 가근길이, 유엽형성, 유엽길이 등의 성장을 촉진시켰다. 이 같은 다른 해조류의 성장을 촉진시키는 allelochemicals 들은 천연 해조류 촉진제의 개발 및 유용 해조류 종들의 유지관리 등에도 활용될 가능성을 지닌다.

Introduction

The marine ecosystem is a community of living organisms with severe competition, cooperation, and regulation occurring between them. For competition or cooperation, some plants produce allelopathic substances－biochemicals that influence the growth, survival, and reproduction of other organisms－that facilitate growth of the producing or receiving organism [21]. Allelochemicals are either beneficial (positive allelopathy) or detrimental (negative allelopathy) to the target organisms and play an important role in plant induction or defense against herbivory [18]. Allelochemicals that suppress or eliminate competing plant species have received special attention due to their potential as natural herbicides in agriculture [9, 20]. This focus has shifted attention to alternative seaweed control technologies, such as algicidal or growth-stimulating agent development based on selective natural products. For example, the red seaweed Ceramium rubrum has anti-germination activity in Sargassum muticum, Enteromorpha intestinalis, and Ulva lactuca [8]. The green seaweed Monostroma nitidum produces a microalgal growth-enhancer levoglucosan [13]. The green seaweed Monostroma arctium has a positive effect on the growth and photosynthetic activity of Porphyra yezoensis in co-cultures, cultured medium filtrate, and dry powder assays [22]. Foliar application of eckol, from the brown seaweed Ecklonia maxima, enhances shoot and root length, leaf area and number, and aphid resistance capacity in cabbage plant [16]. For the development of environmentally friendly biostimulant products for seaweed growth, natural compounds from marine plant and animal sources are the best candidates.

The red seaweed Porphyra suborbiculata is a common wild seaweed that uses a discoidal holdfast to grow on rocks in the higher intertidal zone [1]. Monospores (blade archeospores) from juvenile blades can be produced year-round by adjusting culture conditions in the laboratory. Most monospores germinate to produce new juvenile blades, which themselves produce monospores under axenic culture conditions [2]. Thus, monospores of P. suborbiculata are used as a bioassay for rhizoid and blade formation. To search for natural biostimulant or seaweed growth-enhancing products, we prepared 18 common seaweed extracts and screened for enhancement of rhizoid and blade formation at the early stage using the monospore assay. Lead extracts were further optimized for treatment concentions, and the effects during culturing with the most potent enhancer, Codium fragile, were also measured.

 

Materials and Methods

Seaweed, extraction, and reagents

Seaweed thalli collected from 18 different species on the coast of Korea were dried for 3-7 days at room temperature. Thalli were then ground to a powder for 5 min using a coffee grinder (HMF-340; Hanil Co., Seoul, Korea). For each 20 g sample, one liter methanol was used for extraction at room temperature for 24 hr. For a stock solution of each methanol-soluble fraction, 1 ml dimethyl sulfoxide (DMSO) was added to every 40 mg dried extract. Most of reagents used in this study were of analytical grade from Sigma- Aldrich Co., St. Louis, MO, USA.

Spore collection

To obtain monospores, juvenile blades of P. suborbiculata, collected from Cheongsapo (35°9′46.47″ N, 129°11′43.76″ E), Busan, Korea, were sonicated (28 kHz) twice for 1 min in autoclaved seawater, and immersed in 1% betadine for 2 min to eliminate epiphytes. For each 24-well plate, five excised tissue pieces (each 5×5 mm2) were cultured in 1 ml Provasoli′s enriched seawater (PES) [15]. The blades were incubated at 18℃ with 40 μmol/m2/s light intensity on a 12 hr light:12hr dark cycle to obtain monospores.

Monospore culture and bioassay

Spore germination assays (for rhizoid and blade formation) were performed by adding approximately 100–200 monospores to a 200-μl aliquot of PES in a 96-well plate, which was placed in the dark at 18℃ for 1 day. After non-settled spores were removed by centrifugation (1,500 rpm, 15 min) in an inverted position, 200 μl fresh PES was added to each well with 1 μl extract (200 μg/ml final concentration). DMSO inhibited spore germination by a minimum at 0.5% (data not shown). Spore cultures were placed at 18℃ and 80 μmol/m2/s light intensity on a 12L:12D cycle for 1 week to facilitate spore development [3]. After 1 week, rhizoid formation (number of spores that produced rhizoids per total spores tested), number of rhizoids per rhizoid-holding spore, rhizoid length, blade formation (number of spores that produced blades per total spores tested), and blade length were measured using a microscope (200×). Lengths of rhizoid and blade were measured by a haemocytometer. The relative rate (%) of rhizoid or blade formation was determined by the following formula: (S/T)×100, where S = number of spores that produced rhizoids or blades, and T = total spores tested.

Statistical analysis

The experiments were repeated at least three times. Mean differences between extract and control assays were compared using Student′s t-test.

Fig. 1.Micrographs of monospores released from the juvenile blade of Porphyra suborbiculata (A) and their rhizoids and blades formed from monospores (B). The bars in A and B indicate 100 μm, respectively.

 

Results

To search for positive allelopathic or growth-enhancing agents in seaweeds, common seaweed extracts were tested for their ability to enhance rhizoid and blade production in a P. suborbiculata monospore assay. The 18 seaweed species tested included green seaweed (Codium fragile, Monostroma nitidum, Ulva linza, Ulva pertusa), brown seaweed (Ecklonia cava, Eisenia bicyclis, Hizikia fusiformis, Ishige sinicola, Saccharina japonica, Sargassum fulvellum, Sargassum hemiphyllum, Sargassum hornei, Sargassum thunbergii, Scytosiphon lomentaria, Undaria pinnatifida), and red seaweed (Chondrus ocellatus, Corallina pilulifera, Pachymeniopsis elliptica). Most seaweed extracts at 200 μg/ml suppressed the rhizoid and blade production (Table 1). Among the seaweed extracts tested, C. fragile exhibited significant enhancement, with 63% of monospores producing rhizoids compared with 41% of the control PES (p<0.01). Rhizoid numbers per rhizoid-holding spore also increased. In C. ocellatus and C. fragile, rhizoid length was enhanced significantly, with average rhizoid lengths of 40.2 and 34.6 μm, respectively, compared with 19.5 μm for the control (p<0.01). Regarding germinated spores, E. bicyclis extract enhanced blade formation significantly, with 27% of monospores germinating to juvenile blades in 1 week compared with 14% for the control (p<0.05). C. fragile showed blade formation similarly to the control. E. cava, U. linza, and U. pertusa extracts enhanced blade growth significantly, to an average of 14.3, 10.9, 11.9 μm, respectively, compared with 7.1 μm for the control (p<0.01). C. fragile increased blade length a little. Thus, the C. fragile extract was selected for further evaluation, based on overall enhancing activities of rhizoid and blade formation.

Table 1.Monospores were cultured in each extract (200 μg/ml) for a week. Seaweed number 1, Chondrus ocellatus; 2, Codium fragile; 3, Corallina pilufera; 4, Ecklonia cava; 5, Eisenia bicyclis; 6, Hizikia fusiformis; 7, Ishige sinicola; 8, Monostroma nitidium; 9, Pachymeniopsis elliptica; 10, Saccharina japonica; 11, Sargassum fulvellum; 12, Sargassum hemiphyllum; 13, Sargassum hornei; 14, Sargassum thunbergii; 15, Scytosiphon lomentaria; 16, Ulva linza; 17, Ulva pertusa; 18, Undaria pinnatifida; PES, Provasoli′s enriched seawater. Values are expressed as means ± SE (n>3). *p<0.05 and **p<0.01 as compared with PES control by student t-test.

Various concentrations of the C. fragile extract were added to the monospore culture to determine enhancement activity. The enhancement dose 100 (ED100) is expressed as the concentration of C. fragile extract required to produce rhizoids maximally from monospores after 7 days of culture. The enhancement dose 50 (ED50) is the extract concentration required to increase rhizoid production in 50% of monospores after 7 days. The ED100 and ED50 values reflecting the amount of C. fragile extract required to enhance rhizoid formation (in terms of number of spores with rhizoids per total spores tested) were approximately 100 and 50 μg/ml, respectively, in the monospore culture (Fig. 2). Next, growth of P. suborbiculata monospores was observed upon treatment with 100 μg/ml (approximate ED100 for rhizoid formation) C. fragile extract for 10 days. The C. fragile extract quickly enhanced rhizoid formation (Fig. 3A), rhizoid numbers per rhizoid-holding spore (Fig. 3B), rhizoid length (Fig. 3C), blade formation (Fig. 3D), and blade length (Fig. 3E) from most spores at early culture days in the monospore assay. The extract enhanced rhizoid formation after 2 days culture significantly, rhizoid numbers per rhizoid- holding spore after 3 days, rhizoid length after 3 days, blade formation after 2 days, and blade length after 1 day, respectively, from most P. suborbiculata monospores. However, after 10 days of culture, monospores began to reach almost maximum in rhizoid and blade formation.

Fig. 2.Effects of different concentrations of Codium fragile extract on the rhizoid formation from Porphyra suborbiculata monospores. Enhancing activities of rhizoid formation were expressed as % of spores with rhizoid per total monospores tested. No addition of C. fragile extract is the value for PES control. *p<0.05 and **p<0.01 as compared with PES control by student t-test.

Fig. 3.Effects of Codium fragile extract on the productions of rhizoids and blade from Porphyra suborbiculata monospores during 10 days of culture. Enhancing activities were measured using rhizoid formation (% of spores with rhizoids / total spores tested; A), number of rhizoids / rhizoid-holding spore (B), rhizoid length (C), blade formation (% of spores with blade / total spores tested; D), and blade length (E). ●; C. fragile extract (100 μg/ml). ○; PES control. *p<0.05 and **p<0.01 as compared with control by student t-test.

 

Discussion

Plant interactions in ecosystems are known to be mediated by plant active compounds, referred to as allelochemicals, which are receiving increasing attention in the context of sustainable plant management [4]. It should be paid to these chemical interactions for the development of new biostimulants. To search for such positive allelopathic agents in seaweed, common seaweed extracts were tested for their ability to enhance rhizoid and blade production in a monospore assay. Among the seaweed species tested, C. fragile showed the strongest enhancement activities. C. fragile is an edible and abundant aquaculturable green seaweed. The amount of C. fragile produced by farming in 2013 amounted to 2,045 t (wet weight), with an additional 132 t (wet weight) collected from natural populations in Korea [11]. Currently, it occurs in temperate regions worldwide as an invasive species [6]. In our previous study [10], C. fragile extracts showed in vivo anti-inflammatory, antipyretic, and analgesic activities. Siphonaxanthin having anti-angiogenic effect [7] and a sulfated galactan having immunostimulating effect [12] were also isolated from C. fragile.

The C. fragile extract quickly enhanced rhizoid formation, rhizoid numbers, rhizoid length, blade formation, and blade length at the early stages of the P. suborbiculata monospore germination. Rhodophyte Gracilaria gracilis tips and chlorophyta U. lactuca growth were increased by addition of a commercial kelp E. maxima extract [17]. The E. maxima triggered rooting in cucumber [14] and tomato plants [5]. Enhancing shoot length, root length, leaf area, and leaf number in cabbage plant was caused by the phlorotannin eckol compound from the E. maxima [16]. Some seaweeds contain plant growth hormones (such as auxin and cytokinin), and the additions to media increased growth of Gracilaria vermiculophylla [19, 23]. Even though active compounds from the C. fragile is not proved yet, the C. fragile extract demonstrated a positive effect on the rhizoid and blade formation of P. suborbiculata monospores. Thus, there is potential for using the C. fragile or its extract in the seedling of commercial Porphyra mariculture. Such positive allelochemicals that enhanced favorite seaweed species may be efficacious for new seaweed management technologies, including the development of biostimulant agents based on marine natural products.