J Appl Phycol DOI 10.1007/s10811-014-0507-z
5TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR APPLIED PHYCOLOGY
Observations on pests and diseases affecting a eucheumatoid farm in China Tong Pang & Jianguo Liu & Qian Liu & Hu Li & Junpeng Li
Received: 27 July 2014 / Revised and accepted: 11 December 2014 # Springer Science+Business Media Dordrecht 2015
Abstract Pests and diseases of eucheumatoid farming were studied in Lian Bay, Hainan Province, China, from March 2009 to December 2013. Neosiphonia savatieri, ice-ice disease, and the herbivorous fish Siganus fuscescens were found to be the dominant pests and disease. Infections of N. savatieri increased severely in May 2009 and especially so in August 2009. Then, they remained severe the whole year round, until all the Kappaphycus spp. in the bay was wiped out by October 2010. The outbreak pattern of ice-ice disease was the same as that for N. savatieri. Based on our results, the period mostly from May to August and also into October was the period of frequent outbreak of the epiphyte and ice-ice disease at this Kappaphycus spp. farm. Different from the Kappaphycus spp., Eucheuma denticulatum showed more resistance to N. savatieri and ice-ice disease at the same time and location. A reduced occurrence of N. savatieri and ice-ice disease infections in Kappaphycus spp. was observed when Kappaphycus spp. were co-cultured with E. denticulatum during July–August but not during September–October. The fish S. fuscescens swarmed into the bay from the outside sea from April to May, when the seawater temperature increased to above 26 °C. Interestingly, the S. fuscescens usually prefer N. savatieri to Kappaphycus alvarezii, and they grazed on K. alvarezii only after the amount of N. savatieri became insufficient to meet their needs. From these observations, we T. Pang : J. Liu : Q. Liu : H. Li : J. Li R&D Center of Marine Biotechnology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China T. Pang : J. Liu (*) : Q. Liu : H. Li : J. Li National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China e-mail:
[email protected] H. Li : J. Li University of Chinese Academy of Sciences, Beijing, China
suggest managing the S. fuscescens with different nets at different seasons but not bombs and poison. Keywords Kappaphycus . Eucheuma . Ice-ice disease . Epiphyte . Herbivores . Co-cultivation
Introduction Eucheumatoid species, including Eucheuma denticulatum, Kappaphycus alvarezii, and Kappaphycus striatum are primary sources of the commercially valuable hydrocolloid carrageenan (Bixler and Porse 2011). The cultivation efforts were begun by Dr. Maxwell Doty of the University of Hawaii in conjunction with Marine Colloids (purchased by FMC in 1977) in the mid-1960s and the Philippine Bureau of Fisheries and Aquatic Resources in the early 1970s (Ronquillo and Gabral-Llana 1989). The farming was further introduced to over 20 countries for commercial cultivation purposes and over 50,000 families are currently engaged in cultivating the commercial eucheumatoid species in Southeast Asia, in the Pacific, and the Western Indian Ocean (Ask 2001; Hurtado et al. 2014; Msuya et al. 2014). However, 40 years on, problems related to both the quality and quantity of the raw material still exist in the carrageenan industry (Ask and Azanza 2002; Loureiro et al. 2010). Pests and diseases, including epiphytic algae, herbivores, and ice-ice disease are currently the main reasons behind the decrease in the quality and quantity of carrageenan-producing seaweeds. Hurtado et al. (2006) and Vairappan (2006) reported that the outbreaks of Polysiphonia in the Philippines and Neosiphonia savatieri in Malaysia caused a decrease of K. alvarezii production. To obtain further information, the
J Appl Phycol
infected algae from carrageenophyte farms in Philippines, Indonesia, Malaysia, and Tanzania were collected and studied to establish the baseline information on epiphyte identity and density and any adverse symptoms and secondary infection on their host seaweeds (Vairappan et al. 2008). They found that the dominant epiphyte in these four culture areas was Neosiphonia apiculata. The seasonality of the prevalence of epiphyte infestation in cultivated K. alvarezii in Malaysia had also been noted by Vairappan (2006). Moreover, the epiphytic filamentous algae (EFA) had also caused a large-scale die off of K. alvarezii in China since March of 2009 (Pang et al. 2011). Our morphological studies revealed that the main epiphyte on K. alvarezii in China was N. savatieri (Pang et al. 2011). The main protocols for managing EFA are to note the seasonal patterns and when they appear, manually remove them as early as possible before they reproduce and spread (Ask and Azanza 2002). However, this protocol is so laborious and time consuming that it does not work well for sudden epiphyte outbreaks especially when they occur on a large scale. Therefore, some effective chemicals, such as Ascophyllum nodosum extract and glyphosate, to eliminate epiphytes from K. alvarezii were investigated recently (Loureiro et al. 2010, 2012; Borlongan et al. 2011; Pang et al. 2012). Like the pest weeds, herbivores have been noted as a major problem for farmers since the beginning of eucheumatoid cultivation (Doty 1973; Parker 1974; Doty and Alvarez 1981; Ask and Azanza 2002); Ask (1999) categorized the herbivores into four groups based on the type of damage: (1) “tip nippers,” which are various fish, including adult siganids, which eat the thalli tips; (2) “pigment pickers,” which are solely juvenile siganids that eat the outer pigmented layer of cells; (3) “thalli planers,” which are particular sea urchins, especially Tripneustes gratilla; and (4) if the entire propagule is missing, except for a bit around the tie, one can suspect the green turtle, Chelonia midas. The fish S. fuscescens are the dominant herbivore at the eucheumatoid farm of China. If stabbed by the dorsal fin of S. fuscescens, an injured animal feels serious pain from an uncharacterized venom released from glands in the fin. Probably for this reason, no natural enemy of S. fuscescens has been found at the eucheumatoid farm. Currently, barriers (gill nets, cages, and barrier nets) are used as an effective method to manage the impacts of herbivores. Ice-ice disease has been identified as a major problem for carrageenophyte farms since 1975 (Doty and Alvarez 1975); Largo et al. (1995a) reported that light intensity of less than 50 μmol photons m−2 s−1, salinity of 20 or less and temperature of 33–35 °C induced ice-ice disease in K. alvarezii. Moreover, Largo et al. (1995b, 1999) showed that certain bacteria appeared capable of inducing ice-ice disease, especially in stressed propagules. Loureiro et al. (2012) used to report that AMPEP could protect K. alvarezii from bleaching.
To elucidate the effect of ice-ice disease on the carrageenan quality, the chemical profile of carrageenans extracted from healthy and diseased K. striatum “sacol” strain was investigated using 13C and 1H NMR, FT-IR, and GPC methods of analysis (Mendoza et al. 2002). However, there were no effective ways to eliminate ice-ice disease now. However, an interesting chance observation made during the other experiments by us was that the green K. alvarezii was growing well among the E. denticulatum during the epiphyte and ice-ice disease outbreak in June 2009. Pests and diseases have been recognized as the main problems since the beginning of eucheumatoid cultivation (Ask and Azanza 2002). However, limited information was available on the eco-interactions in a eucheumatoid farm (Russell 1983; Woo et al. 2000). The outbreak of ice-ice disease in May 2008 followed by the EFA outbreak since March of 2009 on a Chinese eucheumatoid farm was reported by Liu et al. (2009) and Pang et al. (2011). Here, the occurrences of pests and diseases as well as their eco-interactions at a eucheumatoid farm in Lian Bay, Hainan province, China are presented.
Materials and methods Red K. alvarezii has been cultured year-round in Lian Bay, Hainan province, China (18°27′N, 110°5′E) for more than 25 years. Green mutants of K. alvarezii, red E. denticulatum, and green K. striatum were introduced and cultivated in this bay after the summer of 2008. These species of algae were cultured using four floating rafts (each raft about 10-m wide and 10-m long, thus about 100 m2) in the same sea area. Seedlings were tied to polyethylene ropes (4 mm in diameter, at a depth of about 0.3 m) which were then tied to the rafts. The seaweed was grown at a stocking density of about 1,000 g m−2 for about 30 days. To avoid grazing, the rafts were encircled by net. The occurrences of epiphytic algae, herbivores, and ice-ice disease were monitored at the eucheumatoid farm from March 2009 to December 2013. Seawater salinity and temperature (at a depth of about 0.5 m) near the seaweed culture location were measured at 8 a.m., 12 noon, and 6 p.m. of a single day, every 3 days at most, during each month of the year 2009. Epiphytic algae Red K. alvarezii, green K. alvarezii, green K. striatum, and red E. denticulatum (each sample about 250 g, n=10) were chosen haphazardly from their respective rafts each month from March 2009 to December 2013. Ten points, 4 mm2 in area and about 5 cm away from the branch tip of each sample, were haphazardly selected. Then, the numbers of points where N. savatieri existed were recorded and expressed as the
J Appl Phycol
proportion of EFA infection. The presence of epiphytes was estimated as percent cover using a scale of 1–10 (1=10 %, 2= 20 %…10=100 %) (Hurtado et al. 2006). Based on the data obtained from 10 samples, the percentages of epiphyte N. savatieri existing on plants of Kappaphycus spp. and E. denticulatum were calculated. Epidermises from healthy red and green K. alvarezii, green K. striatum, and red E. denticulatum were removed using a razor blade and transferred onto microscope slides. Then, the epidermises were viewed microscopically at ×400 magnification, and the images were recorded using an attached Canon digital camera to investigate the characteristics of cortical cells. Ice-ice disease The red and green K. alvarezii, green K. striatum, and red E. denticulatum (each sample about 1,000 g, n=10) were chosen haphazardly from the rafts on each month from March 2009 to December 2013. The length of all the white parts on each branch of each sample was recorded using a tape measure.
ropes at a depth of 0.3 m. The cultivation periods were 45 days (from July 6 to August 20 and from August 22 to October 6). The distance between ropes was 0.3 m and planting density was 10 plants per m2. The red K. alvarezii, green K. alvarezii, green K. striatum, and red E. denticulatum (n=10) were selected haphazardly from the co-culture rafts and the normal culture rafts when they were harvested at the end of the cultivation period. The levels of N. savatieri and ice-ice disease were assessed as described above when the algae were harvested on August 20 and October 6 2009. Siganus sp. Both red and green K. alvarezii, red E. denticulatum, green K. striatum (each sample 50 g, n=5, 250 g per species), and 20 S. fuscescens (about 3 cm in length) were co-cultured using a 9-m2 raft encircled by nets. The changes in the algae were recorded by a Canon digital camera after 2 days. Twenty Siganus sp. (about 3 cm in length) and about 25 kg of N. savatieri-infected green K. alvarezii were co-cultured using another net-encircled 9-m2 raft. The gut contents of the Siganus sp. were surveyed with a microscope after 2 days of co-cultivation.
Co-cultivation Statistics Three rafts (10 m×10 m) were prepared for co-culturing. The red K. alvarezii, green K. alvarezii, and green K. striatum were co-cultured separately with the red E. denticulatum in separate rafts as shown in Fig. 1. Four other rafts (10 m×10 m) were prepared for normal culturing with just one species per raft as described earlier in “Materials and methods” section. Healthy propagules of about 150 g were individually attached to the Fig. 1 Model utilized for coculturing Kappaphycus spp. with the E. denticulatum
Statistical analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, USA). Independent sample t test at p<0.05 was used to test the significant differences of the occurrence percentage of N. savatieri and the total lengths (mm) of all the white parts on the Kappaphycus spp. and E. denticulatum.
J Appl Phycol
Results Epiphytic algae EFA have caused large-scale death of K. alvarezii and K. striatum in Lian Bay, Hainan province, China, since the spring of 2009. Many types of EFA including Neosiphonia sp., Polysiphonia sp., Gracilaria sp., Chladophora sp., Hypnea sp., and Acanthophora sp. were found growing on Kappaphycus spp. at the eucheumatoid farm in China. Based on the morphological observations, the main species of epiphyte found on K. alvarezii and K. striatum in China was N. savatieri (Pang et al. 2011). The other species of epiphytes were not usually found or had little effect on the eucheumatoid species. N. savatieri was first noticed at the Kappaphycus spp. farm in China in March 2009. Then, it increased severely from May 2009 and especially in August 2009, until all the Kappaphycus spp. at the farm had died off in October 2010 (Table 1). Healthy propagules were re-introduced to the bay from Indonesia and Vietnam in October 2011. The percent cover of N. savatieri on the Kappaphycus spp. and E. denticulatum was very low, 1–3 % (Table 1), from November 2011 to February 2012. However, it increased severely during May 2012 until the Kappaphycus spp. had again died off during August 2012 (Table 1). Figure 2 shows the fluctuations of average seawater temperature and salinity for each month during the study period in 2009. The average salinity decreased from 31.9 to 30.6 psu, and the average
temperature increased from 29.2 to 30.5 °C in August 2009. The serious outbreak of N. savatieri in August 2009 coincided with significant changes in salinity and temperature. The Kappaphycus spp. including red K. alvarezii, green K. alvarezii, and green K. striatum were all affected seriously by N. savatieri, but the E. denticulatum was not (Table 1). Significant differences between Kappaphycus spp. and E. denticulatum were detected during the March 2009 to October 2010 and March to August 2012 (Table 1) periods. E. denticulatum showed more resistance to N. savatieri than the red K. alvarezii, green K. alvarezii, and green K. striatum at the same time and location. In order to study the reason for the difference between Kappaphycus spp. and E. denticulatum, the epidermises were viewed microscopically at ×400 magnification to investigate the characteristics of the cortical cells. The cell sizes of red K. alvarezii (8–10 μm), green K. alvarezii (6–8 μm), and green K. striatum (8–10 μm) are much larger as compared to the 5–6 μm of the red E. denticulatum (Fig. 3). The intercellular space between the outer cortex cells of red K. alvarezii (1–3 μm), green K. alvarezii (1–3 μm), and green K. striatum (1–4 μm) is also larger than the 0.5–1 μm of red E. denticulatum (Fig. 3). Ice-ice disease The lengths of all the white and decayed parts on the red E. denticulatum were much less as compared with the red K. alvarezii, green K. alvarezii, and green K. striatum
Table 1 The occurrence percentage of N. savatieri on red K. alvarezii (RA), green K. alvarezii (GA), green K. striatum (GS), and red E. denticulatum (RD) measured each month from year 2009 to 2013 Month 2009
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
2010
2011
RA
GA
GS
RD
RA
GA
GS
– – 27 28 45 44 43 83 78 81 76 75
– – 28 27 47 45 45 85 78 81 74 75
– – 28 32 46 44 46 83 76 82 77 76
– – 0 %abc 0 %abc 0 %abc 0 %abc 0 %abc 1%abc 0 %abc 0 %abc 0 %abc 1%abc
79 78 81 81 77 78 76 83 82 81 / /
79 79 78 76 73 79 76 77 82 77 / /
76 78 77 79 76 81 81 77 78 83 / /
% % % % % % % % % %
% % % % % % % % % %
% % % % % % % % % %
% % % % % % % % % %
% % % % % % % % % %
% % % % % % % % % %
2012
RD
RA
GA
GS
RD
2%abc 0 %abc 0 %abc 1 %abc 0 %abc 1 %abc 0 %abc 0 %abc 0 %abc 1%abc 0% 0%
/ / / 0 / / / 0 / / / 1 / / / 0 / / / 0 / / / 1 / / / 1 / / / 2 / / / 2 – – – 0 1% 1% 2% 0 2% 2% 1% 0
% % % % % % % % % % % %
2013
RA
GA
GS
RD
RA GA GS RD
2% 3% 21 % 25 % 43 % 49 % 52 % 76 % / / / /
3% 3% 24 % 31 % 51 % 47 % 56 % 78 % / / / /
2% 2% 24 % 28 % 43 % 44 % 56 % 78 % / / / /
0 0 0 0 0 0 0 0 1 2 0 0
/ / / / / / / / / / / /
% % %abc %abc %abc %abc %abc %abc % % % %
/ / / / / / / / / / / /
/ / / / / / / / / / / /
0 0 1 0 0 1 0 1 0 0 0 1
% % % % % % % % % % % %
Values represent the % cover based on 100 points from 10 individual samples; en dash indicates that data were not collected; solidus indicates that no one was culturing this species in the bay a
Significant difference at p<0.05 between red K. alvarezii (RA) and red E. denticulatum (RD)
b
Significant difference at p<0.05 between green K. alvarezii (GA) and red E. denticulatum (RD)
c
Significant difference at p<0.05 between green K. striatum (GS) and red E. denticulatum (RD)
36
35
32
30
28
25
24
20
20
Salinity
Fig. 2 Fluctuations of average seawater temperature (○) and salinity (□) of each month during the study period—2009, at seaweed culture location in Lian Bay, Hainan, China. Seawater salinity and temperature (at a depth of about 0.5 m) near the seaweed culture location were measured at 8 a.m., 12 noon, and 6 p.m. of a single day, every 3 days at most, during each month of the year 2009, error bars show the SD
Temperature (°C)
J Appl Phycol
15 Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sept
Oct
Nov
Dec
Month
(Table 2). Significant differences between Kappaphycus spp. and E. denticulatum were found from March 2009 to October 2010 and June to August 2012 (Table 2). The results showed that the resistance of E. denticulatum to ice-ice disease was stronger than Kappaphycus spp. The outbreak pattern of iceice disease was the same as for N. savatieri. Fig. 3 Epidermal structure of different eucheumatoids (×400 magnification). a Red K. alvarezii. b Green K. alvarezii. c Green K. striatum. d Red E. denticulatum
Co-cultivation The co-cultivation of Kappaphycus and Eucheuma reduced the levels of N. savatieri and ice-ice significantly during the July to August period. Compared to the heavy epiphyte load and serious ice-ice damage in K. alvarezii and K. striatum
J Appl Phycol Table 2 The total lengths (mm) of all the white parts of red K. alvarezii (RA), green K. alvarezii (GA), green K. striatum (GS), and red E. denticulatum (RD) measured each month from 2009 to 2013 Month
2009
2010
2011
2012
2013
RA
GA
GS
RD
RA
GA
GS
RD
RA
GA
GS
RD
RA
GA
GS
RD
RA
GA
GS
RD
Jan
–
–
–
–
166
148
147
0abc
/
/
/
0
0
0
0
0
/
/
/
4
Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
– 22 12 73 80 76 234 263 255 169 163
– 13 11 79 79 77 213 208 207 186 189
– 10 13 67 71 69 137 175 143 154 149
– 0ab 0ab 2abc 0abc 0abc 0abc 0abc 3abc 0abc 3abc
154 179 182 204 202 251 322 286 270 / /
162 163 176 183 210 232 282 240 293 / /
162 127 147 173 179 188 224 228 280 / /
0abc 0abc 0abc 0abc 0abc 0abc 1abc 0abc 0abc 0 0
/ / / / / / / / – 2 0
/ / / / / / / / – 2 0
/ / / / / / / / – 0 2
2 0 0 0 0 2 0 0 2 0 0
2 2 11 85 127 121 229 / / / /
0 2 12 86 98 162 213 / / / /
0 0 5 26 70 118 204 / / / /
0 0 0b 22b 23ab 15abc 3abc 0 0 2 0
/ / / / / / / / / / /
/ / / / / / / / / / /
/ / / / / / / / / / /
0 2 0 0 0 0 0 3 0 0 0
Values represent the total lengths of all the white parts of 10 individual samples; en dash indicates that data were not collected; solidus indicates that no one was culturing this species in the bay a
Significant difference at p<0.05 between red K. alvarezii (RA) and red E. denticulatum (RD)
b
Significant difference at p<0.05 between green K. alvarezii (GA) and red E. denticulatum (RD)
c
Significant difference at p<0.05 between green K. striatum (GS) and red E. denticulatum (RD)
growing alone, much less damage from N. savatieri and iceice infection was found both for K. alvarezii and K. striatum when the seaweeds were co-cultured with E. denticulatum during the July to August period (Tables 3 and 4). The average percentage of N. savatieri and total lengths of all the white/ decayed parts existing on red K. alvarezii green K. alvarezii and green K. striatum were 84 % and 290 mm, 82 % and 290 mm, and 72 % and 87 mm, respectively, during the July to August period when they were cultured individually (Tables 3 and 4). However, the average percentage of N. savatieri and total lengths of all the white/decayed parts existing on red K. alvarezii, green K. alvarezii, and green K. striatum were 15 % and 17 mm, 13 % and 27 mm, and 13 % and 5 mm, respectively, when they were co-cultured at the same time and location (Tables 3 and 4). However, different results were observed during the August to October period. The red K. alvarezii, green K. alvarezii, and green K. striatum were all seriously affected by the epiphyte and ice-ice disease no matter whether they were cultured alone or co-cultured with
E. denticulatum (Tables 3 and 4) during the August to October period. The average salinity was 31.9 to 30.6 psu during the July to August period (Fig. 2). However, the salinity was 30 psu through the whole month of September and up to October 8 (data recorded but not shown here). The additional decrease of the seawater salinity from 30.6 to 30 from August to September and into October may have acted as the trigger for the serious outbreak of the epiphyte and ice-ice disease during the August to October period. Herbivores Many types of herbivores such as fish, turtles, sea urchins, and sea hares were observed at the eucheumatoid farm in Lian bay, Hainan, China. The harmful effects of these herbivores varied greatly. Based on field observations, the dominant herbivore at the eucheumatoid farm was S. fuscescens, whose grazing usually led to massive losses, especially during the hot period in South
Table 3 The percent cover of N. savatieri on red K. alvarezii, green K. alvarezii, green K. striatum, and red E. denticulatum under normal and coculture conditions on August 20 and October 6 2009 Month
Jul–Aug Aug–Oct
K. alvarezii (red)
K. alvarezii (green)
K. striatum (green)
E. denticulatum (red)
Normal (%)
Co-culture (%)
Normal (%)
Co-culture (%)
Normal (%)
Co-culture (%)
Normal (%)
Co-culture (%)
84 71
15 70
82 76
13 74
72 72
13 76
4 5
0 6
J Appl Phycol Table 4 The lengths (mm) of all the white parts on red K. alvarezii, green K. alvarezii, green K. striatum, and red E. denticulatum under normal and coculture conditions on August 20 and October 6 2009 Month
Jul–Aug Aug–Oct
K. alvarezii (red)
K. alvarezii (green)
K. striatum (green)
E. denticulatum (red)
Normal
Co-culture
Normal
Co-culture
Normal
Co-culture
Normal
Co-culture
290 234
17 187
290 185
27 142
87 122
5 100
13 5
2 3
of China. S. fuscescens are a celadon green color with a brown spot beside each gill. The dorsal fin of S. fuscescens rises up and their color changes when they are frightened. Schools of juvenile S. fuscescens (about 3 cm in length) swarmed into the eucheumatoid farming bay from the outside open sea in April to May when the surface seawater temperature increased to above 26 °C. The juveniles stayed inside the bay for about a half year and grew into adult fish (about 20 cm in length) then left the farm in November when the surface water temperature dropped to 26 °C (Fig. 2). During the period when S. fuscescens remained in the bay, the seawater temperature
Fig. 4 Thalli of eucheumatoids grazed by Siganus. a Red K. alvarezii. b Green K. alvarezii. c Red E. denticulatum. d Green K. striatum
varied between 26 and 30 °C, which also is the best temperature for Kappaphycus growth (Trono and Ohno 1989). It was observed that the juveniles prefer to graze on the outer pigmented layer of cells and the adults prefer to eat the thalli tips. When the seaweed was co-cultured with 20 S. fuscescens per raft for only 2 days (described in the “Materials and methods” section), most of the outer pigmented layer of cells of green K. alvarezii, red K. alvarezii, and red E. denticulatum were taken by S. fuscescens. The green K. striatum (Fig. 4) was less grazed.
J Appl Phycol
In the 2-day grazing experiment involving N. savatieriinfected green K. alvarezii (25 kg) with 20 S. fuscescens (3 cm in length), mostly Neosiphonia sp. and Obelia sp. filaments rather than epidermis of K. alvarezii were found in the intestines of S. fuscescens. Most of the N. savatieri and Obelia sp. biomass was consumed by fish, and surprisingly little damage was found on the green K. alvarezii. Moreover, the seriously N. savatieri-infected green K. alvarezii appeared clean. These results suggest that S. fuscescens prefers N. savatieri and Obelia sp. to K. alvarezii. Moreover, S. fuscescens consumed only N. savatieri and Obelia sp. until there were insufficient algae remaining to satisfy their needs.
Discussion The results strongly suggest that the changes in abiotic factors act as a triggering mechanism for the epiphytes to infect Kappaphycus spp. Moreover, our results showed that the period from May to August, and even into October, is the period of frequent outbreak of epiphytes at this location in China. The average temperature was 27.2 to 29.2 °C during the April to May period and up to October. The average salinity decreased from 31.9 to 30.6 psu, and the salinity was 30 through the whole month of September and up to October 8 (data recorded but not shown here). Hence, there could be a correlation between the fluctuations in salinity and temperature with the emergence of epiphytes, as described by Vairappan (2006). Besides seawater salinity and temperature fluctuations, other physical factors such as seawater nutrient levels and photoperiod could also play important roles in epiphyte germination and outbreak. However, the culture location was in the subtidal area far from anthropogenic sources of nutrients, and there was no significant difference in photoperiod in Lian Bay. Vairappan (2006) considered that the tetraspores of N. savatieri became embedded between the outer cortex cells as the first step of N. savatieri infection. The cell sizes of red K. alvarezii, green K. alvarezii, and green K. striatum were much larger than those of the red E. denticulatum. The intercellular space between the outer cortex cells of red K. alvarezii, green K. alvarezii, and green K. striatum is also larger than in the red E. denticulatum. This suggests that the smaller cortex cells of the red E. denticulatum generate a much denser epidermis than any of the Kappaphycus spp. It may be that the embedment of the tetraspore of N. savatieri is relatively easier, in the outer cortex of both K. alvarezii and K. striatum, as compared to E. denticulatum. The difference in the size of the outer cortex cells and intercellular space between cells may explain why Kappaphycus spp. was more seriously infested by N. savatieri than E. denticulatum. The size of the outer cortex cells and intercellular space between
the cells may be one important index for the next seed selection considering the resistance to N. savatieri. Epiphytes occupied the surface of eucheumatoids and shaded their host from getting enough light. Moreover, it can be assumed that the filaments of N. savatieri covering the surface of the seaweed would reduce water movement over the thalli, thereby reducing the host’s access to the nutrients (mineral elements, O2, and CO2) normally exchanged between seaweed thalli and the external environment. Glenn and Doty (1992) reported that reef farming of eucheumatoids required high levels of water motion, provided by strong and consistent trade winds. Dense epiphytes generate severe stresses for the metabolism of their host seaweeds. Decay (i.e., ice-ice disease) in Kappaphycus was usually initiated after the seaweeds were heavily infected by N. savatieri. Then, the decaying parts of the thalli turned white and these parts were easily fractured. The serious outburst of ice-ice in August 2009 coincided with the substantial changes in salinity and temperature (the average salinity decreased from 31.9 to 30.6 psu and the average temperature increased from 29.2 to 30.5 °C, Fig. 2). Hence, there could be a possible correlation between the fluctuations in the salinity and temperature with the prevalence of ice-ice disease. All these outdoor results confirmed the lab results of Largo et al. (1999) and Largo et al. (1995a). However, the triggering condition for ice-ice disease was a salinity of 20 psu or less in the experiments of Largo et al. (1995a). The salinity decrease of the bay was usually induced by torrential rain. The rainfall in this bay is often more than 100 mm in several hours especially from the middle of August to middle of October (data from the China Weather Bureau). This indicates that the salinity of the surface layer of seawater in the bay may be very low, even approaching fresh water for several hours during a torrential rain. The algae were planted in the surface layer of seawater in our experiments. Because the average seawater salinity was only determined at a depth of 0.5 m, the lowest surface layer seawater salinity of the culture location would likely be much lower than what was measured. From this perspective, it was suggested that the culture depth of the algae be increased, just as Borlongan et al. (2011) reported, to reduce ice-ice disease triggered by low salinity. The results of this study showed that E. denticulatum was not infected by epiphyte and ice-ice disease when the Kappaphycus spp. were infected seriously at the same time and location. The results might be explained by a more effective defense mechanism in the E. denticulatum compared with the Kappaphycus spp. Mtolera et al. (1996) reported that E. denticulatum produces volatile halocarbons (VHCs) especially under strong light and in CO2-deficient environments. In marine macroalgae, the increased production of iodinated, brominated, or chlorinated organic compounds is associated with many types of stress induced by excess light,
J Appl Phycol
ultra-violet (UV) exposure, temperature changes, and grazing pressure (Mtolera et al. 1996; Laturnus et al. 2004; Abrahamsson et al. 2003; Nightingale et al. 1995). As many of the halogenated compounds are toxic, they could be part of the algal defense system (Pedersén et al. 1996). The production of a very toxic bromamine by Eucheuma has been shown by Collén and Pedersén (1992). This compound has been reported to have algicidal effects on the microalga Chlorella pyrenoidosa Chick at 0.4 mg L−1 (Kott et al. 1966). Moreover, it has been well established that VHCs as part of a defense system could be against microorganism infections, herbivore grazing, space competitors (allelopathy), detrimental fouling by different kinds of epiphytes, or excess of self-generated hypochlorite and hydrogen peroxide (Pedersén et al. 1996; Goodwin et al. 1997; Dworjanyn et al. 1999; Weinberger et al. 1999). These all reported results may provide some explanation for the co-cultivation of Kappaphycus and Eucheuma reducing the probability of N. savatieri and ice-ice disease infection. E. denticulatum might have an active defense system earlier or more effective than Kappaphycus spp. Moreover, E. denticulatum might excrete the VHCs related with allelopathy to reduce the infection of N. savatieri and iceice disease on itself and Kappaphycus spp. However, the salinity was 30 through the whole month of September and up to October 8 (data recorded but not shown here). The additional decrease of the seawater salinity from 30.6 to 30 psu from August to September and into October may have acted as the trigger for the serious outbreak of the epiphyte and ice-ice disease during the August to October period. Although the results in this study also showed that the a further decrease in the seawater salinity triggered serious epiphyte infestation and ice-ice disease regardless of whether Kappaphycus spp. were co-cultured with E. denticulatum. Such inter-algal allelopathic interaction seems to be a universal phenomenon and an interesting subject that needs to be investigated further. A few methods (avoidance, barrier, auditory, visual, electrical, and chemical) that had been developed to lessen or eliminate the impact of herbivores were reported by Ask and Azanza (2002). Barrier methods (gill nets, cages, and barrier nets) are currently the main method to eliminate the impact of herbivores in China, but the barrier method also reduces water movement through the eucheumatoid farm. It has been reported that epiphyte infestations and ice-ice disease break out when water movement is limited (Hurtado et al. 2006; Largo et al. 1999). Therefore, the barrier method is likely to increase the probability of epiphyte and ice-ice disease outbreaks. However, if the seaweeds were not protected by the nets, schools of juvenile S. fuscescens would graze on the epidermis of the seaweed in the eucheumatoids farm of China. Not surprisingly, the seaweed farmers detest S. fuscescens. Many operators of eucheumatoid farms in Indonesia and China even resort to pipe bombs or poison to kill S. fuscescens. These are effective but are destructive to the ecosystem. The size of the
juvenile S. fuscescens is different at different seasons. Therefore, we suggest the use different nets with different mesh sizes at different seasons. S. fuscescens prefer the red K. alvarezii, green K. alvarezii, and red E. denticulatum to green K. striatum. Compared with other species, K. striatum is relatively tough and lacks delicate shoots, which may be one reason why S. fuscescens appeared to dislike grazing on green K. striatum when another eucheumatoid was available. Moreover, S. fuscescens prefer grazing on N. savatieri and Obelia sp. over K. alvarezii. Though S. fuscescens is one dominant pest in the eucheumatoid farm, these herbivorous fish still perform an important role in clearing epiphytes from the host. The N. savatieri and Obelia sp. would be a much more significant problem without the host cleaning by S. fuscescens. The outbreak of N. savatieri can be very destructive to the culture of eucheumatoids (Hurtado et al. 2006; Vairappan 2006; Pang et al. 2011). Therefore, although S. fuscescens is a dominant pest at the eucheumatoid farm, these herbivorous fish nevertheless have an important role to play in clearing epiphytes from the cultivated seaweeds. S. fuscescens should not be destroyed but rather managed to maximize benefits and minimize damage to the crop. Acknowledgments This work was supported by the National Natural Science Foundation of China (41306154), Marine economy innovation and development fund of Qingdao and Special Project for Marine Public Welfare Industry (201105008). The authors would like to thank Dr. John van der Meer (Pan-American Marine Biotechnology Association) for his assistance with proofreading.
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