Marine-derived fungi from Kappaphycus alvarezii and

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Article in press - uncorrected proof Botanica Marina 53 (2010): 587–594 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2010.071

Marine-derived fungi from Kappaphycus alvarezii and K. striatum as potential causative agents of ice-ice disease in farmed seaweeds

Michael Jay L. Solis1,2, Siegfried Draeger3 and Thomas Edison E. dela Cruz1,4,* 1

Graduate School, University of Santo Tomas, Espanã 1015 Manila, Philippines 2 Department of Natural Sciences, College of Science and Information Technology, Ateneo de Zamboanga University, La Purisima St. 7000 Zamboanga City, Philippines 3 Institute of Microbiology, University of Braunschweig, Spielmannstrabe 7, 38106 Braunschweig, Germany 4 Fungal Biodiversity and Systematics Group, Research Cluster for the Natural and Applied Sciences, University of Santo Tomas, Espanã 1015 Manila, Philippines, e-mail: [email protected] * Corresponding author

Abstract Ice-ice disease in cultivated seaweeds is often associated with environmental stress and infection by pathogenic marine bacteria. No studies have associated the disease with marine fungi. Our study aimed to isolate marine fungi from cultivated Kappaphycus species and assess their ability to induce the disease. Kappaphycus alvarezii and K. striatum were collected from Calatagan, Batangas, Philippines. Following washing with sterile artificial seawater and inoculation into culture media, 18 morphospecies of marine-derived fungi (MDF) were isolated. Fungal diversity (Hss2.4) in infected seaweeds was higher than in healthy specimens. K. striatum (orange variety) had the highest incidence of MDF with 67 isolates, while K. striatum (green variety) had the lowest incidence with only 17 isolates. The ability of MDF to produce carrageenolytic and cellulolytic enzymes and utilize algal components was also tested. Of the 18 MDF selected, three had carrageenolytic activity and ten had cellulolytic activity. Most isolates utilized carrageenan, agar, and cellulose. Among the 10 MDF assayed for their ability to induce ice-ice disease, three isolates (Aspergillus ochraceus, A. terreus and Phoma sp.) induced ice-ice disease symptoms (thallus bleaching) in healthy, non-axenic cultures of K. alvarezii. Keywords: cultivated seaweeds; ice-ice disease; marine fungi; pathogenicity.

Introduction Commercial seaweed production in the Philippines has decreased recently due to water pollution in farming areas,

peace and order conditions in some major seaweed-producing regions and the occurrence of diseases in cultivated seaweeds (Philippine Fish Profile 2003–2006). Consequently, this may result in great economic losses. One of the most commonly encountered disease among cultivated seaweeds is ice-ice, characterized by whitening and softening of the algal thallus (Doty and Alvarez 1975), which eventually lead to low yield and poor quality carrageenan (Mendoza et al. 2002). Previous studies have shown that environmental stress, e.g., high temperature, low irradiance and low salinity, induced the disease in cultivated seaweeds (Largo et al. 1995a). Aside from these abiotic factors, the marine bacteria Vibrio and Cytophaga-Flavobacterium groups have also showed pathogenic activity and produced the disease in Kappaphycus alvarezii Doty and Eucheuma denticulatum (N.L. Burmann) Collins et Hervey (Largo et al. 1995b). Other bacteria, e.g., Pseudomonas, Xanthomonas and Achromobacter, were also isolated from infected seaweeds (Uyenco et al. 1981). Several marine fungi have also been reported to cause diseases in green, brown and red algae (Raghukumar 1986, 1987, Hyde et al. 1998, Ramaiah 2006). However, there have been no studies so far on the role of these marine fungi in the induction of ice-ice disease. Thus, our study aimed to isolate, identify and assess the diversity of marine-derived fungi (MDF) from healthy and ice-ice-infected K. alvarezii and K. striatum (Schmitz) Doty. The ability of these MDF to produce seaweed-component degrading enzymes, utilize algal components as substrates and induce ice-ice-disease in vitro were also assayed.

Materials and methods Isolation, identification and diversity assessment of marine-derived fungi

A total of 48 mature thalli of cultivated Kappaphycus alvarezii and K. striatum (green and orange varieties) were randomly collected from August to December 2007 in Barangay Uno, Calatagan, Batangas in Southern Luzon, Philippines and segregated into healthy or ice-ice infected thalli. Following collection and transport to the laboratory, thalli were immersed in sterile artificial seawater (ASW) for 1 min to wash-off soil particles and epiphytic algae. The thalli were then aseptically cut into 1 mm explants and inoculated onto half-strength malt extract agar (MEAS) and potato carrot agar (PCAS) supplemented with marine salts (33 g l-1) and antibiotics (streptomycin and tetracycline at 300 mg l-1) (dela Cruz 2006). For healthy samples (non-bleached thalli), explants were derived from various sections (mid-branches

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and tips) of the thallus; for ice-ice infected samples, explants were only taken from bleached/white sections of the thalli. All culture plates were incubated at room temperature in the dark for two weeks and examined periodically for fungal growths. Fungal colonies were purified by subsequent subcultures using the spore-touch technique (Dogma 1995). Identification was based on morphocultural and molecular characterization. Molecular identification included extraction of genomic DNA and amplification of the internal transcribed spacers 1 and 2 (ITS 1 and 2) regions including the flanking 5.8S rRNA gene with the fungal specific primer combination: SR6R (59-AAG TAG AAG TCG TAA CAA GG-39) and LR1 (59-GTT TGG TTT CTT TTC CT-39) (White et al. 1990). The amplification cycle consisted of an initial denaturation step of 948C for 1 min followed by 30 cycles of (i) denaturation (948C for 1 min), (ii) annealing (508C for 1 min), and (iii) elongation (728C for 90 s), and a final elongation of 728C for 7 min (White et al. 1990). Species diversity, species richness and evenness of marinederived fungi (MDF) in healthy and ice-ice infected Kappaphycus alvarezii and K. striatum were also assessed using the Shannon-Wiener, Gleason and evenness indices, as previously described by Kumar and Hyde (2004).

Assessment of growth in the presence or absence of marine salts

Gleason index (HG)s Np-1/ln Ni where NpsThe total number of species NisThe total number of individuals in ith species

Production of extracellular enzymes and utilization of algal components

Shannon-Wiener index (HS)s-8i (pi ln pi) where pisThe total number of individuals in ith species Species evenness (E) EsHs/Hmax where HssShannon-Wiener index of diversity HmaxsThe maximun value of HS To determine similarities in the marine fungal species isolated among the different seaweed varieties, the number of fungal morphospecies common to the seaweed varieties was counted. The similarity index (Jaccard’s coefficient) was computed using NTSYSpc v.2.2 software (Exeter Software, Setauket, NY, USA). To assess colonization rate (CR), the number of explants with one or more fungal isolate/s for each seaweed variety was also determined, as described by Kumar and Hyde (2004). CR was computed as follows: CRs

Total no. of explants positive for MDF per seaweed species/variety Total no. of explants per seaweed species/variety =100

Finally, to assess the isolation rate (IR), the total number of fungal isolates for each seaweed variety was determined (Kumar and Hyde 2004). IR was computed using the formula below: IRs

Total no. of fungal isolates per seaweed species/variety =100 Total no. of explants per seaweed species/variety

The CR and IR were calculated to assess the susceptibility of the seaweed host/s to the MDF.

To determine the ability of 18 MDF morphospecies to grow in the presence or absence of marine salts, their colony extension rates were determined on malt extract agar with (MEAS) and without (MEA) 33 g l-1 marine salts. Initially, mycelial disk were cut aseptically approximately 5 mm from the colony margin of 7-day-old cultures using a flame-sterilized cork borer and inoculated into culture plates (in triplicates) containing MEA or MEAS. Culture plates were then incubated at room temperature in the dark. Colony radial growth (two readings per plate) was measured for each of the MDF isolates as the distance from the agar block to the margin of the growing fungal colony on the 3rd and 5th days of incubation. The mean colony extension rates (MCER) were then computed as previously described (dela Cruz et al. 2006): Mean colony radial growth (day 5) – mean colony radial growth (day 3) MCERs Number of days of incubation (2 days)

Eighteen morphospecies of MDF were selected and assayed qualitatively for their ability to produce seaweed-component degrading enzymes, e.g., carrageenase, agarase, gelatinase and cellulase. MDF were initially grown on potato dextrose agar (PDAS) supplemented with marine salts at room temperature for one week. For production of carrageenase, agarase and gelatinase, MDF were inoculated into culture tubes with 5 ml basal medium (composed of 5 g l-1 peptone, 5 g l-1 yeast extract and 33 g l-1 marine salts) supplemented with 1% (w v-1) kappa-carrageenan (Ricogel 83161, PCIW, Taguig City, Philippines), agar-agar (Hi-Media, Mumbai, India) and 5.4% (w v-1) gelatin (Hi-media). Liquefaction indicated the production of the respective enzymes. For cellulase production, MDF were also inoculated on culture plates pre-filled with Basal Medium with 0.1% (w v-1) carboxylmethylcellulose (CMC, Crismon Enterprises, Davao City, Philippines) and 1.5% (w v-1) bacto-agar (Hi-Media) as described by Pointing (2000). Following incubation, the fungal colonies were flooded with 0.02% (w v-1) aqueous Congo red solution for 10 min. The presence of yellow clearing zones around the fungal colony indicated positive cellulolytic activity. MDF were also assayed for their ability to utilize several algal cell and cell wall components as sole carbon sources, e.g., carrageenan, agar, alginic acid and cellulose. MDF cultures grown on PDAS were used to prepare a spore suspension of 1.0=103 ml-1. A spore suspension was then inoculated into 60-ml culture vials pre-filled with Czapex Dox Medium (CDMS) supplemented with marine salts and 0.1% (w v-1) of either one algal components as sole carbon source: kappa-carrageenan, agar-agar, CMC and alginic acid (Sigma Aldrich, St. Louis, MO, USA). CDMS with 0.1%



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(w v-1) sucrose served as a control. Culture vials were incubated on a shaker incubator at room temperature for seven days. Resulting fungal biomass from each of the culture media was filtered onto pre-weighed filter paper and ovendried at 758C for 48 h or until the weight remained constant. Mycelium dry weight biomass was then determined. Induction of ice-ice disease in non-axenic culture of Kappaphycus alvarezii

Healthy (non-bleached) thalli of Kappaphycus alvarezii were collected as previously described. They were rinsed with sterile natural seawater (NSW) and cut into approximately 10–12 cm long thallus fragments. Seaweed thalli were stocked using floating culture method into pre-disinfected glass aquaria (15.24 cm=15.24 cm=20.32 cm) filled with 3 l sterile NSW. The seaweeds were then cultivated and acclimated under 10-W cool-white fluorescent light (photon irradiance of 310–380 mm s-1 m-2) on a 16 h light: 8 h dark photoperiod for three days. Adequate and constant aeration was provided and temperature was maintained at 248C. Culture water was supplemented with 10 mM NaNO3 (as nitrogen source) and antibiotics. After acclimation, seaweed thalli (36 explants) in good condition were selected for disease induction. Ten MDF were selected based on their ability to produce seaweed-component degrading enzymes and/or utilize algal components as sole carbon source. Spore suspensions (1.0=106 spores ml-1) of MDF were prepared as previously described and 10 ml were inoculated into aquaria containing seaweed thalli. No MDF spores were added to the

negative control. For positive control, seaweed thalli were exposed to 10% salinity and without MDF. All cultures were incubated as previously described for nine days. During incubation, thalli were then observed for the induction of ice-ice disease, i.e., whitening and fragmentation of thalli. To determine whether the inoculated marine fungi indeed induced the disease, fragments of seaweed thalli with disease symptoms were cut aseptically from the main branch and inoculated onto MEAS plates supplemented with antibiotics to re-isolate the MDF in accordance with Koch’s postulate. All culture plates were incubated at room temperature for seven days. Re-isolated MDF were purified and morphologically compared with the original cultures for confirmation. The re-isolated MDFs were subsequently tested for ability to infect another set of healthy Kappaphycus alvarezii for disease development.

Results Fungal isolation, identification and diversity assessment

A total of 144 fungal strains belonging to 18 morphospecies was isolated from healthy and ice-ice-infected thalli of Kappaphycus alvarezii and orange and green varieties of K. striatum (Table 1). Among the 18 morphospecies, 13 had higher mean colony extension rates on MEAS than on MEA without marine salts (Figure 1). Only one species (Phoma nebulosa) grew better in the absence of marine salts. Four species

Table 1 Fungal morphospecies isolated from healthy and ice-ice-infected Kappaphycus alvarezii and Kappaphycus striatum (orange and green varieties). Taxon

No. of isolated MDF strains

Scopulariopsis brumptii Salv.-Duval Cladosporium sp. 1 Phoma nebulosa (Pers.) Mont. Cladosporium sp. 2 Phoma lingam (Tode) Desm. Aspergillus terreus Thom Eurotium sp. Phoma sp. Aspergillus sydowii (Bainier et Sartory) Thom et Church Curvularia intermedia Boedijn Cladosporium sp. 3 Fusarium sp. Fusarium solani (Mart.) Sacc. Aspergillus ochraceus G. Wilh. Aspergillus flavus Link Penicillium sp. Penicillium purpurogenum Stoll Engyodontium album (Limber) de Hoog Total

Kappaphycus alvarezii

Kappaphycus striatum (orange variety)

Kappaphycus striatum (green variety)

Healthy host

Infected host

Healthy host

Infected host

Healthy host

0 0 0 1 0 0 0 0 4 0 0 0 1 0 0 2 0 1 9

0 0 0 0 3 1 1 6 4 9 12 7 7 0 0 0 0 1 51

1 1 1 0 0 0 0 0 0 0 1 0 0 0 5 0 0 4 13

0 0 0 4 3 3 8 8 2 9 2 7 0 6 1 0 1 0 54

0 0 0 5 3 2 0 0 0 0 0 5 0 0 0 0 0 0 15


Total no. of strains

Infected host 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 2

1 1 1 10 9 6 9 14 10 18 15 19 9 6 7 2 1 6 144



Figure 1 Colony extension rates of marine-derived fungi (MDF) grown on MEA with or without marine salts (p-0.05). Values are meansqSEM; ns6.

(Cladosporium sp. 2, Cladosporium sp. 3, Fusarium solani, Penicillium purpurogenum) had equivalent colony extension rates in the presence and absence of marine salts, with no noticeable variations in colony morphology. Morphocultural and molecular identification showed that MDF isolates belonged to the genera Aspergillus, Cladosporium, Curvularia, Engyodontium, Eurotium, Fusarium, Penicillium, Phoma and Scopulariopsis (Table 1), all typical terrestrial taxa. Diversity assessment of MDF among the three seaweed hosts and between the healthy and infected seaweed explants showed highest number of fungal strains (67) and morphospecies (16) in the orange variety of Kappaphycus striatum (Table 2). Consequently, species diversity indices (HSs2.6, HGs3.6) and evenness (Es1.0) were highest for this K. striatum variety. In contrast, the lowest number of fungi was isolated from the green variety of K. striatum and, expectedly, lowest species diversity and evenness.

Our results also demonstrated higher fungal diversity in diseased seaweeds (Table 2), with 14 morphospecies compared to 10 in healthy seaweeds. The number of isolates also increased from 24 (healthy) to 120 (disease), indicating that infected seaweeds harboured more species and strains of marine-derived fungi. The diversity indices (HS, HG) and evenness values were higher in infected than in healthy seaweeds for all Kappaphycus species (Table 2). The highest CR was observed in Kappaphycus alvarezii (51%) and in infected thalli (64%) (Table 2). The rank order of IR was as follows: K. striatum (orange) )K. alvarezii )K. striatum (green). Among the 18 isolated fungi, 22% were found on all three seaweed hosts, suggesting that these MDF do not have any host preference (Table 3). Comparing the marine-derived fungi isolated from the three hosts, high similarities based on Jaccard’s coefficient were observed between K. alvarezii and K. striatum (orange variety) (Table

Table 2 Diversity indices, colonization rate and isolation rate of marine-derived fungi in healthy and ice-ice-infected Kappaphycus alvarezii and Kappaphycus striatum (orange and green varieties). Number of morphospecies (Np) Host seaweed Kappaphycus alvarezii 12 Kappaphycus striatum (orange variety) 16 Kappaphycus striatum (green variety) 6 Seaweed condition Healthy thalli 10 Infected thalli 4

Number of strains (Ni)

ShannonWiener index (Hs)

Gleason Species index evenness (Hg) (E)

Colonization Isolation rate (%) rate (%)

60 67 17

2.2 2.6 1.6

2.7 3.6 1.8

0.9 1 0.6

51 48 17

50 56 14

24 120

2.0 2.4

2.8 2.9

0.9 1.0

13 64

13 67




Table 3 Distribution of marine-derived fungi in Kappaphycus alvarezii and Kappaphycus striatum (orange and green varieties). Host seaweeds

Common fungal morphospecies

%

Kappaphycus Kappaphycus Kappaphycus Kappaphycus Kappaphycus Kappaphycus Kappaphycus

4 10 5 5 1 5 0

22 56 28 28 6 28 0

alvareziiqKappaphycus striatum (orange variety)qKappaphycus striatum (green variety) alvareziiqKappaphycus striatum (orange variety) alvareziiqKappaphycus striatum (green variety) striatum (orange variety)qKappaphycus striatum (green variety) alvarezii striatum (orange variety) striatum (green variety)

Table 4 Jaccard similarity index calculated from the presence of marine-derived fungi in Kappaphycus alvarezii and Kappaphycus striatum (green and orange varieties). Seaweeds

Kappaphycus alvarezii

Kappaphycus alvarezii Kappaphycus striatum (orange variety) Kappaphycus striatum (green variety)

1.00 0.50 0.31

Kappaphycus striatum (orange variety)

Kappaphycus striatum (green variety)

1.00 0.25

1.00

Table 5 Marine-derived fungi producing seaweed-component degrading enzymes. Enzyme

Fungal morphospecies positive for enzyme production

Carrageenase Cellulase

Aspergillus ochraceus, Aspergillus terreus, Phoma sp. Cladosporium sp. 1, Cladosporium sp. 2, Phoma nebulosa, Penicillium sp., Penicillium purpurogenum, Engyodontium album Aspergillus flavus, Aspergillus ochraceus, Aspergillus sydowii, Cladosporium sp. 2, Cladosporium sp. 3, Curvularia intermedia, Fusarium sp., Fusarium solani, Phoma sp., Phoma nebulosa, Phoma lingam None

Gelatinase Agarase

Table 6 Marine-derived fungi utilizing various algal components as sole carbon source. Substrate

Fungal morphospecies with mycelial growth

Carrageenan Cellulose

All except Fusarium sp. Aspergillus sydowii, Aspergillus terreus, Cladosporium sp. 1, Cladosporium sp. 2, Curvularia intermedia, Fusarium solani, Penicillium sp., Phoma sp., Phoma lingam Cladosporium sp. 1, Phoma lingam Aspergillus sydowii, Cladosporium sp. 1, Curvularia intermedia, Penicillium sp., Phoma lingam Phoma nebulosa, Scopulariopsis brumptii

Agar Alginic acid

4). This was supported by the high number (10) of morphospecies common to both seaweed hosts (Table 3).

Induction of ice-ice disease by MDF in non-axenic cultures of K. alvarezii

Production of extracellular enzymes and utilization of algal component

Of 10 selected fungi, seven isolates (A. ochraceus, A. terreus, Engyodontium album, Eurotium sp., Penicillium sp., Phoma lingam, and Phoma sp.) produced bleaching/whitening in mid branches and tips of the seaweed thalli within nine days of incubation (Table 7). Bleaching occurred as early as day 3 for those inoculated with A. ochraceus, while the other six isolates induced bleaching and fragmentation by days 6 or 9. Bleaching and fragmentation intensified as incubation time progressed. Incidence of ice-ice disease symptoms were highest in A. terreus. Negative control had no visible bleaching or fragmentation, while positive controls had the most pronounced bleaching. Out of the seven MDF producing iceice disease symptoms, only three fungi (A. terreus, A. ochra-

Only three fungi (Phoma sp., Aspergillus ochraceus, A. terreus) had carrageenase activity after 14 days (Table 5). Gelatinase activity was demonstrated in 14 isolates, and cellulase activity in seven isolates. None of the isolates had agarase activity. However, all MDF (except Fusarium sp.) grew with carrageenan as the sole carbon source (Table 6), with 10 isolates producing highest mycelial biomass (Figure 2). Cellulose was the second-most preferred carbon source among nine MDF. Seven MDF preferred alginic acid while only two MDF utilized agar as a sole carbon source.




Figure 2 Mycelial dry weight of marine-derived fungi (MDF) grown in various algal components as sole carbon source (p-0.05). Values are meansqSEM; ns3.

ceus, Phoma sp.) were re-isolated from diseased thalli in axenic culture. The reisolated MDF subsequently induced disease symptoms when inoculated into healthy K. alvarezii. Development of disease symptoms occurred in different sections of the seaweed thalli, i.e., tip, mid-branch and main branch.

Discussion Marine-derived fungi were isolated from healthy and ice-ice infected seaweeds (Table 1). The isolated marine-derived fungi belonged to nine genera: Aspergillus, Cladosporium, Curvularia, Engyodontium, Eurotium, Fusarium, Penicillium, Phoma and Scopulariopsis. All are typically terrestrial fungi. Their higher growth rates in the presence of marine

salts may indicate adaptation to the marine habitat (Figure 1). Jones (1994) noted that terrestrial fungi may have been carried into marine waters and evolved there as a result of selection pressures from the marine environment. No further tests have been done to corroborate adaptation of these MDF isolates to marine habitats. However, their higher growth rates in MEAS may indicate tolerance of the marine environment. In our research study, a higher number of fungi was observed in infected seaweeds, with Kappaphycus alvarezii and K. striatum (orange variety) most heavily colonized as indicated by their highest fungal diversity, and isolation and colonization rates (Table 2). The marine-derived fungi appeared to show no preference for any seaweed host (Table 3). High similarities in the fungi present were also observed between K. alvarezii and K. striatum (orange variety) (Table

Table 7 Disease development in Kappaphycus alvarezii inoculated with selected marine-derived fungi. Taxon

Aspergillus ochraceus Aspergillus sydowii Aspergillus terreus Cladosporium sp. 1 Eurotium sp. Engyodontium album Fusarium solani Penicillium sp. Phoma sp. Phoma lingam 1

No. of thalli with bleaching and fragmentation1 Day 3

Day 6

Day 9

3 0 0 0 0 0 0 0 1 0

3 0 4 0 2 1 0 0 3 3

3 0 5 0 3 4 0 2 3 3

Total number of cultivated thallis6.




4). Local seaweed farmers also observed that these two seaweeds were the most susceptible to ice-ice disease. However, to the best of our knowledge, no scientific studies have been done so far to corroborate this observation. But, in other studies, K. alvarezii and Eucheuma denticulatum had higher bacterial counts in ice-ice infected branches than in healthy seaweeds (Largo et al. 1995b). High incidence of epiphytes in seaweed farms were also recorded during periods of iceice disease infestation (Uyenco et al. 1981). Thallus bleaching in the red alga Chondracanthus chamissoi (C. Agardh) Ku¨tzing was also observed coincident with periods of high epiphytism (Vasquez and Vega 2001). The ability of some marine-derived fungi isolated in this study to produce algal component-degrading enzymes and utilize various algal components (Tables 5, 6, Figure 2) may possibly be contributory factors to the development of iceice disease in cultivated red seaweeds. Kappa-carrageenan is a polysaccharide cell wall component of Kappaphycus species (Santos 1989) and constitutes the bulk of the cell’s interstitial matrices. Mendoza et al. (2002) observed a significant decrease in carrageenan yield and average molecular weight of carrageenan extract in ice-ice-infected thallus of K. striatum. This depolymerization was attributed to the carrageenolytic activity produced and secreted by occasional bacteria. Cellulose is also a polysaccharide cell wall component of Kappahycus sp. (Trono 1997). Cellulase and carrageenase isolated from bacteria are able to release epidermal and medullary protoplasm in K. alvarezii (Zablackis et al. 1993). Largo et al. (1999) suggested that these hydrolytic enzymes might be a factor in the whitening/bleaching observed in seaweed thalli during ice-ice infection. During infection, carrageenan-lysing bacteria were able to penetrate the inner region of the seaweed thallus. This invasion is hypothesized to cause epidermal degradation and destruction of the cell’s pigment-containing plastids, resulting in the initial bleaching of the infected part. Perhaps the isolated marine fungi utilize a similar mechanism in the induction of ice-ice disease. Ice-ice disease is often associated with biotic and abiotic factors. Several bacterial genera have been isolated from infected Eucheuma sp., e.g., Pseudomonas, Flavobacterium, Vibrio, Xanthomonas, and Achromobacter (Uyenco et al. 1981). Agar-lysing bacteria of the Cytophaga-Flavobacterium and Vibrio groups were also isolated in infected K. alvarezii (Largo et al. 1995b). The diversity of bacterial species isolated from different sites supports the suggestion of Uyenco et al. (1981) that no single bacterial species is consistently associated with the disease and that the role of bacteria and perhaps other microorganisms is secondary to abiotic causative agents. Infection of bacteria in seaweeds is also dependent on their ability to successfully attach to the algal surface. Largo et al. (1999) observed that continuous stirring during in vitro cultivation decreased bacterial attachment to seaweed thalli. In our study, water motion (continuous stirring) was not introduced during disease induction. This may explain the possible success of higher number of fungal spores or mycelial fragments settling onto the seaweed surface and germinating, and thereby, inducing the disease (Table 7). Colonization and infection of marine-derived

fungi is also dependent on the host’s health (Yarden et al. 2007). A certain degree of stress is still expected for cultivated seaweeds even after acclimatization. The simple seaweed cultivation set-up used in this study can only partially replicate the true growth conditions of seaweeds in their natural environment. Marine-derived fungi may play roles similar to those previously reported ice-ice disease-causing bacteria. The abundance of these marine-derived fungi in infected seaweed thalli and their ability to produce seaweed-component degrading enzymes, i.e., carrageenase and cellulase, and their preference for carrageenan as a carbon source suggest their potential as causative agents of ice-ice disease. The induction of disease symptoms, i.e., thalli bleaching and fragmentation, by the isolated marine fungi again demonstrated the possible role of marine fungi in disease induction. Furthermore, the re-isolation of the inoculated marine fungi from diseased seaweed thalli was in accordance with Koch’s postulate of disease development.

Acknowledgements M.J.L. Solis would like to thank Ateneo de Zamboanga University and the Philippine Commission on Higher Education (CHED) for the graduate scholarship and research grants. We thank the Research Cluster for the Natural and Applied Sciences, University of Santo Tomas for research funds, and Dr. Irina Druzhinina, Technical University Vienna, Austria for her contribution to the molecular work.

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