Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for Philippine seaweed farms

Technique improvement and cost reduction of branch culture, micropropagation, and callus production of carrageenan-yielding seaweeds Kappaphycus alvarezii and Eucheuma denticulatum is presented. Low cost branch culture is possible by enriching seawater with 0.1% coconut water with 1 mg l^–1 indole-3-butyric acid for 24 h wk^–1 or continuous culture with 0.01% Algafer, a Philippine fertilizer. Micropropagation of 0.5 cm explants had almost 100% new branch production demonstrating the viability of callus regenerated plants. The use of carrageenan as a media for callus production was not effective when compared to agar. Propagules of both species, transferred from the University of the Philippine Marine Science Institute (UPMSI) culture facility to the field, showed daily percent growth rates of 5 to 5.5% d^–1 over 84 days. Based on the costs of the UPMSI laboratory, a culture facility in the seaweed farming area is estimated to cost about U. S. $22000 during the initial year and 58% less the second year.     

Time-dependent attachment mechanism of bacterial pathogen during ice-ice infection in Kappaphycus alvarezii (Gigartinales, Rhodophyta)

The mechanism of infection by Vibrio sp. P11 promoting the ice-ice disease in Kappaphycus alvarezii was investigated in vitro. Its intensity of infection differs from that of another ice-ice promoter (Cytophaga sp. P25) by promoting the disease much faster. However, when secondary infection by other bacteria starts, its ability to compete with these bacteria gradually diminishes, whereas, infection by P25, although not displaying such drastic effects as P11, shows consistent competitive ability against other bacteria. Time-series infection experiments with application of polyclonal antibodies to specifically detect Vibrio sp. P11 revealed that this bacterium has a high affinity for the seaweed especially when the latter is stressed. It promotes the disease after a rapid increase in cell density of up to 10⁷ g⁻¹ (wet wt.) in the first 24 h. This bacterial cell build-up may take only 1–2 h on stressed thalli, but takes about 24 h on non-stressed thalli. Build-up is not sustainable in non-stressed thalli as high density is usually followed by a sudden decline in cell number believed to result from an algal defence against potential pathogens. Inoculation of the bacterium on thalli incubated in continuous culture system extends the time of bacterial attachment due to laminar flow and, possibly, competition by existing bacteria on the seaweed surface and in ambient seawater medium. Motility-driven cell attachment by this bacterium is suggested as an important factor for infection. This revised version was published online in June 2006 with corrections to the Cover Date.     

The farmed Eucheuma species (Gigartinales,Rhodophyta) in Danajon Reef,Philippines: carrageenan properties

Six cultured strains of Eucheuma denticulatum and E. alvarezii, from which stocks can be selected for the development of a Eucheuma seedling bank, were tested for their carrageenan quality from June to November 1988. Percent yield of all the varieties taken together was apparently higher in June, becoming lower in November (regression, r –0.785, probability, p 0.001). Stepwise regression analysis was done to determine the existence of any relationship between any of the following parameters: gel strength, viscosity, sulfate content, month of sampling, and yield, whether taken individually or in combination. Results show variations of the yield with the month of sampling. ANOVA was performed to test whether there are differences in sulfate levels, gel strength, and viscosity between the Eucheuma alvarezii morphotypes. There was no significant difference between the green and the brown types.     

Effect of ammonium on growth and carrageenan content in Kappaphycus alvarezii (Gigartinales,Rhodophyta)

Kappaphycus alvarezii, cultured in a region deficient in nitrogen with intermittent application of 10 mM ammonium at three-day intervals for 1 hour, attained a daily growth rate of 4.6%, which was considered to be the optimum growth rate. Under such conditions, the C/N ratio was 29 with a carrageenan content of 58% and gel strength of 45–70 g cm^–2. Gel strength of carrageenan was high when nitrogen content was high and vice versa.     

Non-enzymatic isolation of somatic cells from Kappaphycus spp. and Eucheuma denticulatum (Solieriaceae,Rhodophyta)

Cell culture technology is immensely useful in somatic hybridization, induction of mutations, cloning of specific isolates and maintenance of strains of defined genotypes. However, its application in strain improvement of some tropical red macroalgae has been limited due to the difficulty of isolating viable cells from their complex intercellular matrices. A simple, non-enzymatic technique of isolating somatic cells was developed for Kappaphycus spp. and Echeuma denticulatum. Surface-sterilized tissues (0.1 g fresh weight, 2.0 mm thick discs) from subcortical and medullary layers were treated with 3% NaOH, 3% KOH, or hydrogen peroxide in phosphate buffer solution (PBH₂O₂). Tissues of K. alvarezii treated with PBH₂O₂ softened after 5 h of treatment and completely dissociated after 12 h. Viable cell counts (VCC), determined through staining with Evan’s blue, were significantly higher (2.4 × 10⁵ cells g^?1 fresh weight tissue) in K. alvarezii (‘tambalang’ strain) treated with PBH₂O₂ compared with tissues treated with carrageenase from a marine bacterium.     

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

Systematics and genetic variation in commercial shape Kappaphycus and shape Eucheuma (Solieriaceae, Rhodophyta)

The systematics and taxonomy of Kappaphycus and Eucheuma (Solieriaceae) is confused and difficult due to morphological plasticity, lack of adequate characters to identify species and commercial names of convenience. These taxa are geographically widely dispersed through cultivation. Commercial, wild and herbarium sources were analysed; molecular markers provided insights into taxonomy and genetic variation, and where sources of genetic variation may be located. The mitochondrial cox2-3 and plastidal RuBisCo spacers were sequenced. There is a clear genetic distinction between K. alvarezii (“cottonii”) and K. striatum (“sacol”) samples. Kappaphycus alvarezii from Hawaii and some samples from Africa are also genetically distinct. Our data also show that all currently cultivated K. alvarezii from all over the world have a similar mitochondrial haplotype. Within Eucheuma denticulatum (“spinosum”) most African samples are again genetically distinct. Our data also suggest that currently cultivated E. denticulatum may have been “domesticated” several times, whereas this is not evident for the cultivated K. alvarezii. The present markers used do not distinguish all the morpho-types known in cultivation (e.g. var. tambalang, “giant” type) but do suggest that these markers may be useful to assess introductions and species identification in samples.     

Seasonal Occurrences of Epiphytic Algae on the Commercially Cultivated Red Alga Kappaphycus Alvarezii (Solieriaceae, Gigartinales, Rhodophyta)

Common problems faced in farming of the red algal genus Kappaphycus/Eucheuma are “ice-ice disease” and the occurrence of epiphytes. Considerable work has been documented on “ice-ice disease” and it's mode of infection but limited information is available on the emergence of epiphytes. The present study addresses the phenomenon of epiphyte infection, its prevalence in commercially cultivated red alga, Kappaphycus alvarezii, and their variability associated with seasonality. Cultured seaweed became susceptible to epiphytes in the dry seasons (1) between March – June and (2) September – November. Findings revealed Neosiphonia savatieri (Hariot) M. S. Kim et I. K. Lee, as the dominant infecting epiphyte, representing up to 80–85% of the epiphyte present during peak seasons. Besides N. savatieri, Neosiphonia apiculata, Ceramium sp., Acanthophora sp. and Centroceras sp. were observed in smaller quantities. SEM (Scanning Electron Microscope) micrographs revealed the epiphyte's attachment to the host. Further histological study showed the extent of penetration of epiphytes into the host's cortex tissues and condition of its surrounding tissues. The outbreak of epiphytic filamentous red algae also correlated with drastic changes in seawater temperature and salinity during March– June and September – November.     

Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny

A brown strain of Kappaphycus alvarezii from the Philippines produced tetraspores in the summer and autumn (December 1995 to May 1996) in cultivation experiments in the sea at Ubatuba, São Paulo State, Brazil. In vitro tetraspore release and germination experiments showed a mass mortality two to four days after release. Only 20 plants derived from tetraspores were grown successfully for over a year in the laboratory. Large differences in morphology, colour, size and growth rates were observed amongst these plants. The individual plants differed from one another in one or more characteristic. Differences appeared in the early developmental stages and persisted through time. After ten months, the plants that grew best in laboratory culture were transferred into the sea, but the others remained very small (3 to 5 mm), even after two years. In the sea, the plants also showed individual differences in their ability to survive and grow. These results emphasise the potential of the tetraspore progeny for strain selection in K. alvarezii. The results also suggest that the tetrasporophyte used in these studies is of hybrid origin.     

Growth rate of the carrageenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales) in vitro

Unialgal cultures were established trom a 2.5 g branch ot a brown variant ot Kappaphycus alvarezii (Doty) Doty ex P. Silva, with the main objective to produce branches for monthly outplanting in the sea. Ditterent conditions were tested to optimize production ot branches in the laboratory. The best growth was obtained under culture conditions of 25 ± 2°C, 170–210 μmol photons m² s^?1, 14:10 LD photoperiod and salinity 32–35‰. Three culture media (Provasoli,‘F/2’and von Stosch) were tested. Deleterious ettects were observed in branches incubated continuously in full‐ or halt‐strength Provasoli's enriched seawater medium (PES). Exponential growth rates ot about 3% day^?1 were obtained using PES, pulse‐fed 24 h per week, or other diluted media used continuously (one‐quarter strength ‘F/2’and half‐strength von Stosch). Laboratory‐grown branches with mean weights from 2.97 to 4.25 g were successfully introduced into the sea at Ubatuba, SP, Brazil (23°26.9′S, 45°0.3′W) an area with mean monthly seawater temperature from 20.3 to 28.5°C (extremes: 17.0–31.0°C). Transplantation of branches produced in unialgal culture, as done in the present study, avoids the risk of introduction of unwanted species into new areas.     

Introduction of non-native species of Kappaphycus (Rhodophyta, Gigartinales) in subtropical waters: Comparative analysis of growth rates of Kappaphycus alvarezii and Kappaphycus striatum in vitro and in the sea in south-eastern Brazil

We compared the growth rates of Kappaphycus alvarezii (Doty) Doty ex P. Silva and Kappaphycus striatum (Schmitz) Doty, both, in vitro under different conditions of light and temperature, and in the sea. Temperature was the most important factor controlling the growth of both species, in vitro and in the field. In the sea there was a clear seasonal pattern in growth rate, attributed to temperature and salinity variation. The lower growth rates were registered in winter and spring, and the highest in summer and autumn months for both species. Based on growth rate in the field, and the production of viable tetraspores during the summer in Kappaphycus striatum, we conclude that is more profitable, and ecologically safer, to only continue with the introduction program of Kappaphycus alvarezii.     

A Eucheuma (Solieriaceae,rhodophyta) cultivation test on the south-west coast of Madagascar

Well established populations of Eucheuma denticulatum and E. striatum exist on the coral reef of the south west coast of Madagascar but natural beds are not dense enough to support high harvest pressure. A cultivation test was conducted on a 250 m² module using the monoline method with 1560 seedlings of both species for over one year (weighings at 15 day intervals). E. denticulatum and E. striatum showed similar maximum growth rates (2.8 % d^–1) occurring respectively in fall and in summer. For both species, minimum growth was observed during the warmest months (February–March). Mean growth rates, although relatively low, showed a high standard deviation, suggesting wide possibilities of strain selection. The preliminary selection experiment led to strains more resistant to breakage and having a growth rate up to 3.3 % d^–1. The ice-ice disease is also present in this area and affects the algae mainly from January to March. Mean carrageenan yield for E. denticulatum was 42.5% ± 2.3 and rheological properties were above industrial standards, with a highly elastic gel and were thus suitable for commercial applications.     

Genetic diversity of Kappaphycus species (Gigartinales,Rhodophyta) in the Philippines

The mariculture of eucheumatoids (species of Kappaphycus and Eucheuma) in the Philippines has had a long history, dating back to the 1970s. Over this period, a number of varieties have been brought into domestication; some are now widely distributed and farmed in various regions of the country, but a significant number appear to have a more restricted distribution and are farmed only in certain areas. The taxonomy of many of these seaweed cultivars and their phylogenetic relationships still remain to be resolved at the specific and subspecific levels. In this study, two mitochondrial DNA markers, COI-5P region and cox2-3 intergenic spacer, were used to assess the genetic diversity of the farmed varieties and a few specimens collected from the wild. Analysis using haplotype networks revealed several new haplotypes for K. alvarezii, K. malesianus and K. striatus, mainly from specimens collected from eastern and southwestern regions of the Philippines. The inferred phylogenetic relationships based on both mtDNA markers resolved the identity of all the materials used in the study at the species level. We present molecular evidence that K. malesianus, in addition to K. alvarezii, K. cottonii, K. inermis, K. procrusteanus and K. striatus (and, hence, all currently recognized species of Kappaphycus) occurs in the Philippines. Collectively, these observations suggest that the Philippine archipelago has richer genetic diversity of farmed and wild Kappaphycus than do the other geographic regions, consistent with the hypothesis that the Philippines is, or is part of, the centre of Kappaphycus biodiversity in the world. These findings also reveal an untapped diversity that can potentially be exploited for improving the commercial production of these carrageenophytes.     

Germination and survival of tetraspores of Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) introduced in subtropical waters of Brazil

The carrageenophyte Kappaphycus alvarezii was introduced in 1995 and vegetatively propagated in Ubatuba, São Paulo State, Brazil, for the purpose of commercial cultivation. This species produces tetraspores mainly in the austral summer and fall. Tetraspore germination and survival were studied under different conditions of temperature, photon flux density, and photoperiod in the laboratory. Field experiments were also carried out. Although tetraspores of K. alvarezii germinated, they had low survival rates, most dying after 20 days. Recruitment of K. alvarezii tetraspores did not occur in experiments conducted in the field. The results indicated that the establishment of K. alvarezii via spore production in the natural environment of the south‐east coast of Brazil is rather remote.     

Recruitment of Eucheuma and Kappaphycus on a farm in Tawi-Tawi,Philippines

Vegetative regeneration is the only farming method for Eucheuma and Kappaphycus, the world's most important carrageenophytes. Lack of seedstock has been a problem following natural calamities, overharvesting, grazing and outbreak of "ice-ice" disease. Hence, studies to help develop an alternative method of generating Eucheuma and Kappaphycus sporelings on a seaweed farm in Tawi-Tawi, Philippines were carried out from May 1993 to July 1994. Mactan stone blocks were deployed in two positions at two sites in Tawi-Tawi, one of the biggest farming areas in the Philippines. Recruits were first observed five months after deployment. Nested ANOVA was used to investigate the effect of current speed (C), water movement (M), number of days with minus tide (D), salinity (S), temperature (T), area of deployment (A) and position of blocks (P) on the recruitment of sporelings (R). Since C, M, D, S, and T were always on the same mean level within an area, the effects of these variables were assumed by the effect of area and thus, the A and P variables were the ones fitted in the ANOVA model. Repeated measures ANOVA showed no significant difference in the effects of A and P on visible R from May to November 1993 and March to July 1994. The Helmert transformation showed that mean recruitment of Eucheuma and Kappaphycus between May 1993 and subsequent time points was significantly different. This was also true for recruitment in September 1993, December 1993, January 1994 and February 1994, which were highly significantly different compared to the means of subsequent time points. Multiple regression analysis showed that C, D, and S had significant effects on recruitment of Eucheuma and Kappaphycus in both areas while effect of T was significant only in area I.     

Isolation of protoplasts from tissue fragments of Philippine cultivars of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Solieriaceae,Rhodophyta)

Protoplasts were isolated from tissue fragments (<1 mm²) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 10³ g⁻¹ tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 10³ g⁻¹ tissue) and Bohol wild type (4.5 × 10³ g⁻¹ tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m⁻² s⁻¹, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars.