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Mycorrhizae

Effect of Red and Green Algal Extracts on Hyphal Growth of Arbuscular Mycorrhizal Fungi, and on Mycorrhizal Development and Growth of Papaya and Passionfruit

^a Research Lab., Yamaki Co., Ltd., 1698-6 Kominato, Iyo, Ehime, 799-3194, Japan
^b Dep. of Horticulture, Jomo Kenyatta Univ. of Agriculture and Technology, PO Box 62000, Nairobi, Kenya
^c Faculty of Human Life, Okayama Gakuin Univ., Kurashiki, Okayama 710-8511, Japan
^d Graduate School of Agriculture, Kyoto Prefectural Univ., Kyoto 606-8522, Japan

^* Corresponding author (kkuwada{at}yamaki.co.jp)

Received for publication December 25, 2005.

	ABSTRACT

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Arbuscular mycorrhizal (AM) fungi enhance terrestrial plant growth by forming a symbiotic relationship with the roots of its host plant. Effects of red and green algal extracts on in vitro hyphal growth of AM fungi, Gigaspora margarita and Glomus caledonium, and on the mycorrhizal development and growth of papaya seedlings and passionfruit cuttings were investigated. Three kinds of red algae, Gracilaria verrucosa, Gelidium amansii and Eucheuma cottonii, and a green alga, Chlorella pyrenoidosa, were extracted with 75% methyl alcohol (MeOH), and these extracts were fractionated by a flash chromatograph equipped with an octa decyl silane column and eluted with 0, 10, 25, 50, and 100% MeOH. Twenty-five percent MeOH fractions (25% MeOH eluates) of each type of alga most significantly increased in vitro hyphal growth of AM fungi. Inhibition of AM hyphal growth was not observed in any of the fractioned MeOH eluates. Root colonization of papaya and of passionfruit were markedly stimulated by the application of the 25% MeOH eluates of red and green algal extracts. The growth of these plants were promoted in comparison to the control. These results suggest that red and green algae not only contain AM stimulatory substances, but also contribute to mycorrhizal development in higher plants.

Abbreviations: AM, arbuscular mycorrhizal • DW, dry weight • Exp, experiment • FW, fresh weight • JICA, Japan International Cooperative Agency • MeOH, methyl alcohol • ODS, octadecyl silane

	INTRODUCTION

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ARBUSCULAR MYCORRHIZAL FUNGI are found in many soils around the world, and they form an association with 80% of all terrestrial plant roots (Harley and Harley, 1987). The beneficial effects of AM symbiotic association on the growth of plants are well known (Powell and Bagyaraj, 1984; Safir, 1987). Nowadays, sustainable agricultural practices are attempted using these fungi. Applying the stimulants of AM fungi to the soil would contribute to the stimulatory growth of indigenous AM fungi and thus lead to less reliance on chemical fertilizers. Shrestha et al. (1996) reported that satsuma mandarin trees (Citrus unshu Marc.) inoculated with AM fungi grew larger and had better fruit quality as compared with non-AM control trees under low P concentration conditions. Ishii et al. (2000b) also reported that no reduction in tree vigor occurred in spite of using less chemical fertilizers when AM growth stimulants from citrus juice pomace extract was treated in a Citrus iyo orchard. Inocula of AM fungi are on sale, but they are very expensive because there are no effective methods for their fast multiplication and production. Furthermore, it is important to develop some AM growth stimulators to increase root colonization, to use indigenous AM fungi and the commercial AM inocula more effectively.

On the other hand, seaweeds and seaweed extracts have been used as fertilizers and soil conditioning agents for many years and are marketed for use in agriculture and horticulture (Blunden, 1991). Useful results from their use have been reported: increased nutrient uptake, promoted plant growth, deeper root development, and increased frost resistance (Abetz, 1980; Stirk and Van Staden, 1997; Zodape, 2001). We have reported that the several kinds of marine brown algal [Laminaria japonica Areschoug and Undaria pinnatifida (Harvey) Suringar] extracts can be used as an AM fungus growth stimulator. The 25% MeOH eluates of brown algal extracts fractionated with flash chromatography stimulated in vitro AM hyphal growth and increased root colonization by AM fungus on trifoliate orange [Poncirus trifoliate (Linn.) Raf.] seedlings (Kuwada et al., 1999). When liquid fertilizer containing tangle (L. japonica) stock ground extracts was applied by an overhead sprinkler system in a Miyauchi-iyokan (Citrus iyo Tanaka) orchard, root colonization by indigenous AM fungi was promoted by 27% and the number of indigenous AM spores in the soil was increased by 21% compared to control (Kuwada et al., 2000).

However, few studies have been conducted on the effects of other algae on AM fungi. Information on other algae, such as Gracilaria, Gelidium, Eucheuma, and Chlorella, needs further investigation because these algae are economically important as industrial materials and are therefore widely available in significant quantities. Gracilaria and Gelidium are mainly used in agar production (Critchley, 1993b), and Eucheuma are a major source of carrageenan, widely used as a food additive. These red algae inhabit tropical and subtropical seawaters and some of the algae have been cultivated. The genus Eucheuma is successfully cultivated in Philippines, Indonesia and Tanzania (Critchley, 1993b; Mshigeni, 1998). In addition, cultivation techniques of the genus Gracilaria have proceeded (Critchley, 1993a; Santelices and Doty, 1989). It has been well known that the genus Chlorella is used as a source of protein, and can be mass produced by artificial cultivation.

If these algae have stimulatory effects to increase AM root colonization, their use may contribute to sustainable plant production in tropical and subtropical regions. Papaya (Carica papaya Linn.) and passionfruit (Passiflora edulis Sims.) are economically important fruit trees in these regions. In orchards in such regions, such as in Kenya, there is little AM fungus in the soil (approximately 200 or less spores in 25 g of soil), while genus Glomus and Gigaspora were found, particuraly the genus Glomus was observed (Wamocho et al., 1997).

In this study, we report the effects of extracts from several kinds of red and green algae on growth of AM fungi in vitro. We also present the effects of the algal extracts on the AM colonization and growth of two species of tropical and subtropical fruits, papaya and passionfruit.

	MATERIALS AND METHODS

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Materials
Dried G. verrucosa (Hudson) Papenfuss, collected off the Pacific coast of Ooita, Japan, in April 1998, and G. amansii (Lamouroux) Lamouroux, collected off the East China Sea coast of Nagasaki, Japan, in April 1998, were the kind gift of Ina Food Industry Co., Ltd. Eucheuma cottonii Doty [Kappaphycus alvarezii Doty (Trono, 1993)] was collected off the Pacific coast of Kochi, Japan, in September 1998. Collected E. cottonii was washed with seawater, freeze-dried and stored at –20°C until used. Dried C. pyrenoidosa Chick was the kind gift of Chlorella Industry Co., Ltd. Dried L. japonica, harvested in Hakodate Japan in June, was purchased from Erimo Syokuhin Co., Ltd..

Preparation of Algal Extracts
Three hundred grams DW samples of the red and green algae (G. verrucosa, G. amansii, E. cottonii, and C. pyrenoidosa) were individually soaked in separate 6.0-L solutions of 75% (v/v) MeOH for 24 h at room temperature, to extract hydrophilic and hydrophobic carbohydrates in these algae. Each algal extract was concentrated by a rotary evaporator at 40°C, filtered with Advantec no. 5 C paper (Toyoroshi Co., Ltd., Tokyo), and filled up to 1.5 L with distilled water. The filtrate of each alga was fractionated with a flash chromatograph equipped with an ODS (Chromatorex ODS DM1020T purchased from Fuji Silysia Chemical Ltd.) column (45 by 400 mm). One hundred milliliters of the concentrated filtrate was applied to the column, and 700 mL of 0% (water), 10% (v/v), 25% (v/v), 50% (v/v), and 100% MeOH solutions were eluted successively (Kuwada et al., 1999). This separation of each algal extract was replicated 10 times. The column was rinsed with MeOH and equilibrated with water for the next separation. Each MeOH eluate was concentrated by the rotary evaporator at 40°C to remove MeOH, filled with distilled water, up to 200 mL, and stored at –20°C until use in biological experiments.

Experiment 1a: Effects of Red and Green Algal Extracts on Hyphal Growth of Arbuscular Mycorrhizal Fungi
All in vitro experiments were performed in asymbiotic conditions. To investigate the effect of these eluates (0, 10, 25, 50, and 100% MeOH eluates) from four kinds of algae (G. verrucosa, G. amansii, E. cottonii, and C. pyrenoidosa) on AM hyphal growth, AM spores of Gigaspora margarita Becker and Hall and Glomus caledonium (Nicol. and Gerd.) Trappe and Gerdeman (purchased from Central Glass Co., Ltd.) were used. The spores were surface-sterilized (Gianinazzi-Pearson et al., 1989) for 15 min in 5% chloramine T (Sigma) plus 0.04% streptomycin (Sigma) containing a few drops of Tween 80 (Sigma). After the spores were rinsed in sterile water, they were transferred onto Petri dishes (7-cm diam.) each containing a different MeOH eluate percentage, equivalent to 0.1 and 0.3 g DW, of alga respectively, and 10 mL of 1.5% sterilized agar (Wako Pure Chem. Ind., Ltd.) media. Four spores were placed in a Petri dish, with enough space between each spore, and three dishes per treatment were used. Control dishes contained only agar. After the spores were incubated at 25°C in the dark for 2 wk, the hyphal growth of germinated spores were measured individually using an image-processing system equipped with a light microscope and personal computer (Ishii and Kadoya, 1994).

Experiment 1b: Comparison between 25% MeOH Eluates of Red, Green and Brown Algae on Hyphal Growth Stimulation
Since all 25% MeOH eluates of algae promoted the hyphal growth of AM fungi in Exp. 1a, the effects of the 25% MeOH eluates of red, green, and brown algae on the hyphal growth stimulation were compared. Spores of Gi. margarita (Central Glass Co., Ltd.) were used in this in vitro experiment. The brown alga used was L. japonica, and the 25% MeOH eluate from dried L. japonica was obtained by the same method mentioned above. Each Petri dish contained 25% MeOH eluate equivalent to 0.3 g DW of each alga. Control dishes contained only agar. Hyphal growth was also determined by the same method as described in Experiment 1a.

Experiment 2: Effect of 25% MeOH Eluates of Algal Extracts Applied to the Soil on Root Colonization and Growth of Papaya and Passionfruit
One-year-old papaya (Carica papaya L. cv. Solo) seedlings and passionfruit (Passiflora edulis Sims. cv. No. 54039) cuttings were separately transplanted into plastic pots with a 4-L capacity, each filled with 1:1 mixture of sterile vermiculite and zeolite in a plastic greenhouse. A Murashige and Skoog micronutrient (100 mL) and a mixture of 0.25 g of CaCO₃ and 0.25 g of MgSO₄ were added to each pot. A week after transplanting, 1.0 g of N, 0.2 g of P, and 1.0 g of K per pot were added using (NH₄)₂SO₄, Ca₃(PO₄)₂, and K₂SO₄, respectively. Inoculation of AM fungi was performed 2 wk after transplanting. Half of the papaya seedlings were separately inoculated with approximately 300 spores of Gl. caledonium per pot, while the other half were inoculated with approximately 90 spores of Gi. margarita per pot. The passionfruit cuttings were inoculated with about 300 spores of Gl. caledonium per pot. One hundred milliliters of solution containing the concentrated 25% MeOH eluates equivalent to 1.0 g DW of each algae (G. verrucosa, G. amansii, E. cottonii, and C. pyrenoidosa) was added to the pots. Control plants were given 100 mL of water. There were four replicates for each treatment and the application was repeated once a week for a period of 8 wk.

Eleven weeks after transplanting, 10 pieces of the apical roots (3 cm) were collected from the passionfruit cuttings, and 10 pieces of the root zone (3–6 cm) behind the tips were collected from the papaya seedlings. The AM colonization was observed according to the procedures of Phillips and Hayman (1970) and Ishii and Kadoya (1994). At the same time, whole plants were harvested, and total and root dry weights were measured after drying at 70°C for 3 d.

Statistical Analysis
Statistical analysis was performed using ANOVA and means were compared by Duncan's multiple range test. Significant differences were determined at P < 0.05.

	RESULTS

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Experiment 1
Effects of MeOH eluates of G. verrucosa, G. amansii, E. cottonii, and C. pyrenoidosa obtained by flash chromatography, on in vitro hyphal growth of AM fungi are shown in Tables 1 and 2. All spores used were germinated in this experiment. As compared to the control, the 25% MeOH eluates of these algae significantly stimulated hyphal growth of Gl. caledonium. The hyphal growth of Gl. caledonium was most stimulated by 25% MeOH eluates from 0.3 g DW of G. amansii and E. cottonii (Table 1). The hyphal growth of Gi. margarita was enhanced by 10, 25 and 50% MeOH eluates of these algal extracts. In particular, the 25% MeOH eluates remarkably promoted hyphal growth (Table 2). No inhibition of AM hyphal growth by any of the MeOH eluates was observed.

View larger version (18K): [in this window] [in a new window]   Fig. 1. The comparison between 25% MeOH eluates (equivalent to 0.3 g DW of each alga per Petri dish) or red, green and brown algae on hyphal growth of Gi. margarita.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effect of soil application of 25% MeOH eluates from four kinds of algal extracts, on AM colonization of papaya and passionfruit. Papaya was inoculated with Gl. caldedonium or Gi. margarita, and passionfruit with Gl. caledonium only. Abbreviations: Gv, Gracilaria verrucosa; Ga, Gelidium amansil; Ec, Eucheuma cottonii; Cp, Chlorella pyrenoidosa; Cont, Control. AM colonization (%) = root length infected/root length observed x 100. In each graph, mean values followed by the same letters are not significantly different according to Duncan's multiple range test at 5% level (n = 4).

	DISCUSSION

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Several compounds are well known as growth stimulatory substances for AM fungi, i.e., ethylene (Ishii et al., 1996), polyamines (El Ghachtouli et al., 1995; Ishii et al., 2000a), and flavonoids (Aikawa et al., 2000; Gianinazzi-Pearson et al., 1989; Ishii et al., 1997; Muraleedharan et al., 1991; Siqueira et al., 1991). Aikawa et al. (2000) have identified two kinds of flavonoids, narirutin and hesperidin, in 25% MeOH eluates from citrus juice pomace extracts using a ODS flash chromatograph. Ishii et al. (2000a) also reported that alginate oligosaccharide, which is produced by enzymatic decomposition of alginic acid mainly extracted from brown algae, significantly stimulated the hyphal growth of AM fungi and their infectivity of citrus roots. Our results suggest that 25% MeOH eluates of the red and green algae we used contain certain AM stimulatory compounds. However, it is unclear what compounds are responsible for the AM stimulatory activity. Further research, identifying the growth stimulants for mycorrhizal fungi in the 25% MeOH fraction from algae, is under way in our laboratory.

Low values of in vitro hyphal growth of Gl. caledonium and Gi. margarita were observed in Exp. 1a (Tables 1 and 2). The hyphal growth of Gi. margarita obtained by each treatment in Exp. 1b (Fig. 1) was approximately three times longer than that of Exp. 1a (Table 2), although culture conditions in Exp. 1b were the same as that of Gi. margarita in Exp. 1a, and all these spores were germinated. The low development of hyphae and the considerable difference in the hyphal growth may have been caused by certain conditions such as timing of spore dormancy break, because the tendency observed in Exp. 1b was consistent with that of Exp. 1a (Gi. margarita); that is, the hyphal growth stimulated by every 25% MeOH eluate of algae (0.3 g DW per dish) was approximately three times higher than control; and there was no significant difference among each algal treatment. The spores used were purchased spores, and the conditions in distribution may have influenced the results.

The red and green algae in this study may be used as raw materials for "biostimulants," especially as an AM fungus stimulator. Ferrini and Nicese (2002) reported that applications of some commercial biostimulats (one of the biostimulants they used was a powder mixture of marine brown algae, AM fungi, vitamins, amino acid, and humic acid) to oak trees (Quercus robur Linn.) increase N concentration in leaf tissue. Nevertheless, they also indicated that these biostimulants seemed to be expensive in relation to its benefits. Kuwada et al. (1999) reported that marine brown algae, L. japonica and U. pinnatifida, contained growth inhibitory substances for mycorrhizal fungi as well as growth stimulatory compounds. The removal of the growth inhibitory substances from biostimulants would be necessary to enhance mycorrhizal activity. Interestingly, the growth inhibition for AM fungi was not observed in any MeOH eluates from red and green algae used (Exp. 1). The results indicated that using the red and green algae instead of brown algae may improve cost-perfomance of biostimulants.

Root colonization of papaya and passionfruit inoculated with Gi. margarita as well as Gl. caledonium was promoted by applying 25% MeOH eluates from red and green algae to the soil, and this would have resulted in an increase in the growth of these plants (Exp. 2). Although the natural habitats of the brown algae L. japonica and U. pinnatifida are in the temperate and frigid sea zone, the red algae used, range over tropical and subtropical zones of the sea, and C. pyrenoidosa, a green alga, is commercially cultivated in some freshwater environments. Given that papaya and passionfruit are economically significant in tropical and subtropical regions, the red algae (G. verrucosa, G. amansii and E. cottonii), found and cultivated in the seas of this region, would effectively contribute to sustainable plant production using indigenous mycorrhizal fungi.

	ACKNOWLEDGMENTS

 
We wish to thank Ina Food Industry Co., Ltd., for the donation of G. verrucosa and G. amansii. We also thank Chlorella Industry Co., Ltd. and Mr. Hidekazu Kawamura for the kind donation of C. pyrenoidosa and P. edulis cv. no. 54039, respectively.

	REFERENCES

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kuwada, K. Articles by Ishii, T. Search for Related Content PubMed Articles by Kuwada, K. Articles by Ishii, T. Agricola Articles by Kuwada, K. Articles by Ishii, T. Related Collections Sustainable Agriculture Soil Microbiology Symbiosis