Indian Journal of Geo Marine Sciences Vol. 47 (06), June 2018, pp. 1208-1216

Occurrence of Ice-ice disease in seaweed Kappaphycus alvarezii at Gulf of Mannar and Palk Bay, Southeastern India

A. Arasamuthu1* &J.K. Patterson Edward1

1Suganthi Devadason Marine Research Institute, 44-Beach Road, Tuticorin - 628 001, Tamil Nadu, India [Email:kingpearl28@gmail.com]

Received 29 March 2016 ; revised 07 April 2017

Present study compares the diversity of culturable bacteria isolated from healthy and ice-ice disease infected branches of K. alvarezii using different farming methods. Incidence and degree of infection varied significantly between time (months) during January - April 2014 and farm sites as revealed in the two way ANOVA, Pearson correlation and microbiological analysis. The occurrence of ice- ice disease in K. alvarezii has higher incidence and infection, as well as the farm exposed mostly during the time low tide and high temperature. The disease has affected the short term additional income generation to few fisher folks, however the loss of cultivation helps possibilities of restoring ecological balance of the region where the key coastal habitats such as coral reefs and seagrass beds and associated biodiversity were much affected due to the cultivation of K. alvarezii.

[Key words: Kappaphycus, Incidence, Degree of infection.]

Introduction K. alvarezii (Rhodophyta: Solieriaceae) is a marine

macro algae cultivated in several Asian countries since 1960s1. It was introduced to the Gulf of Mannar, South east coast India in the year 20002.Cultivation has been recommended by the National Academy of Agriculture3 for commercial production of �-carrageenan, which has several pharmaceutical and industrial applications.

The ice-ice disease is a major problem in the cultivation of K.alvarezii and first reported in 1974 in Philippines4 and entire farms were wiped out5. This disease is non-infectious and could be triggered by unfavourable environmental conditions such as extreme temperature, irradiance and salinity, and opportunistic bacterial pathogens, like Vibrio sp. (P11) and Cytophaga sp. (P25)6-10. Ice-ice disease leads to a significant decrease in seaweed production and decrease in carrageenan yield compared to the healthy crop ranging from 25 to 40 %11. It also reduces the quality of carrageenan viscosity and gel strength12.

Local farmers particularly in Gulf of Mannar, attribute this disease results in temperature rise and slow water current in the farming sites, during months of March to April. Furthermore, pollution of anthropogenic activities origin occurs near the farms. On the other hand, reduced salinity during rainy periods, coinciding with low tide and coupled with slow water movement, is another perfect combination

leading to the ice-ice infection. These are as far as changes in physico-chemical factors are concerned13. In Gulf of Mannar, this seaweed cultivation has greater impacts over the marine ecosystem affecting the coral reefs by dominating and invading the area may be causing damage to the prevailing biotic factors2,14. Various researches have been focused on the impact of the red algae on coral ecosystem in the Gulf of Mannar, South east coast of India.

The present study specially focused on ice-ice disease is part of a comprehensive research program addressing the impact of K.alvarezii in Gulf of Mannar and Palk Bay. Detailed monitoring of the biological and environmental factors that might contribute to the onset of the disease with regard to season and farming sites has been conducted. The study was resolute on to determine the incidence and infection of ice-ice disease of K.alvarezii as well as the environmental factors that triggered the disease.

Materials and Methods

Two stations namely Mulukampi from Tuticorin (Fig.1) and Pamban from Ramanathapuram (Fig.2) were selected on the coast of Gulf of Mannar and Palk Bay, Southeastern India. Three different sites with the total coverage area of 500 m2 from each station were randomly chosen. In Tuticorin, K.alvarezii was cultivated by monoline culture method (Fig.3). Site-1 was located at 8°44'50.94"N 78°11'25.75"E which is

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about 100m away from the sea shore. Site-2 was located at 8°44'46.36"N 78°11'23.99"E that is about 250m away from the shore. Site-3 was located at 8°44'45.13"N 78°11'41.49"E that is about 500m away from shore; site-3 was always submerged within the water. The substrate at the three farming sites was sandy silt with sea grasses, Thallasia sp. and Syringodium sp., In Pamban three sites was selected. Each site covers an area of 500m2. At these sites floating raft culture method (Fig.4) was followed. Site-1, located in Gulf of Mannar at 9°16'45.96"N 79°11'19.54"E,with a distance from shore about 250m. Site-2, located in Palk Bay at 9°17'14.76"N 79°11'10.17"E, with a distance from shore about 500m. Site-3, located in Palk Bay at 9°17'1.87"N

79°11'22.98"E, with a distance from shore about

100m. In these three sites, substrate was rocky, silt with sea grasses, Thallasia sp.

Sampling was conducted during January to April 2014. In each sites (site 1, 2 &3) in Tuticorin and (site 1 2 &3) in Pamban, 10 nos. cultivating ropes were randomly chosen. A cultivating rope where bunches of seaweeds are being tied. Generally, a rope with 20 m length contains 60–80 bunches. The frequency of occurrence was observed every 10th bunch of seaweed per cultivating rope. For K. alvarezii, eight bunches were monitored in each line for 80 bunches.

There are 80 bunches from each site was monitored 15

In Tuticorin monoline culture method was practiced (Fig.3) and in Pamban floating raft culture method (Fig 4). The infection was identifying based on the standard symptoms of whitening of the branches and initiated with the colour changes of the thallai become transparent appeared.

To calculate the incidence, the number of infected bunches was divided by the total number of bunches observed at every site.

%No. ofinfectedbunchesTotalNo. ofbunches

100%

To calculate the degree of infection, the number of infected branches was divided by total branches in the bunch.

Fig. 1 — Study site in Tuticorin

Fig. 2 — Study site in Pamban

Fig. 3 — Monolineculture in Tuticorin

Fig. 4 — Floating raft culture in Pamban

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% No. ofinfectedbunchesTotalNo. ofbunches

100%

Data were then summarized using mean and standard error, by comparing the incidence and the degree of infection between time (month) and site. Two-Way Analysis of Variance was used to study the disease infestation on K. alvarezii16. The Pearson correlation was also used to correlate different physical parameters at Tuticorin and Pamban regions.

Physical parameters like salinity and temperature were recorded in each site during January - April 2014 every sampling of Water temperature was measured using digital thermometer and salinity using refract meter. The farming site was shallow and depth level was measured using a pole and tape.

Fortnight sampling was conducted at Tuticorin and Pamban farming during January to April, 2014. Samples of ice-ice disease infected K.alvarezii healthy tissue, sediment and water samples were taken for Bacterial analysis. In the field, about 1 cm long segments were cut from ice-ice-infected and healthy thalli were collected and kept in to the sterile container with sterilized natural seawater and transported to laboratory in an ice box. In the laboratory, samples were washed with sterile seawater to remove the epiphytic bacteria and were homogenized with sterile sea water by grinding using sterile mortar and pestle. The sample was serially diluted and pour plated on Zobell marine agar (Hi Media, Mumbai) and incubated at room temperature 37°C for 48 hours17. After the incubation period, colonies were noted as number of colonies and the microbes were selected and purified by making pure culture18. Purified microbial samples were classified as positive, negative, pigmented and non-pigmented through gram staining and visualization.

Result The temperature range started from 29.1 ºC at

site-3 of Tuticorin to 33.7 ºC at site-1 of Tuticorin. Almost in all the sites studied the highest temperature was recorded during April, 2014 and the lowest during January, 2014. There is gradual increase in temperature from January to April 2014 in both Tuticorin and Pamban (Fig. 5).

Salinity varies from 31ppt at site-1 of Pamban during January 2014 to 35ppt as highest. The highest salinity was recorded at different sites with different time. In Tuticorin the higher salinity was recorded at site-2 in April site-3 in March and April, 2014 and in Pamban in April, 2014 at site-3 recorded the higher salinity of 35ppt (Fig. 6).

The tide range and depth of the study area are reported in the table-1.

The thalli conditions K.alvarezii in farming areas. Fig. 7 shows healthy K.alvarezii thalli. Fig 8 shows whitening and hardening bleached thalli of K.alvarezii; Fig. 9 shows the infected branches of K .alvarezii.Fig.10 K.alvarezii thalli loaded with dirt, silt and epiphytes. Fig 11 shows moreover, wide-scale whitening leading to complete damage of the branches.

In Tuticorin, the incidence of ice-ice disease ranged between 11.25% and 41.25% in all the study sites. In Site 1 during the month of March and February, the disease was recorded the highest as 41.25% and 38.5% respectively, while during January the disease prevalence was recorded the lowest as 22.5%. At Site 2 also the months April and March recorded the highest incidence of ice-ice disease as 23.5% and 21.25% respectively and January the lowest incidence

Fig.5 — Temperature variation

Fig.6 — Salinity variation

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18.75%. At Site 3, April and March recorded the highest incidence of ice-ice disease with 27.5% and 38.75% respectively, while January the lowest incidence 11.25% (Fig. 12).

In Pamban, the disease prevalence is almost similar like in Tuticorin. The occurrence of the disease ranged between 13.75% and 37.5% in all the sites studied at Pamban. In Site 1 during April and

March the disease was recorded the highest as 23.75% and 20% respectively while in January the lowest incidence with 17.5%. In Site 2 April and March recorded the highest disease level with 25% and 23.75% respectively, while January the lowest level of 15%. In Site 3 April and March recorded the highest incidence of ice-ice disease with 37.5% and 28.75% respectively while January the lowest incidence 13.75 %(Fig.12).

The degree of infection is the variation in infectious rate. It represents whether the disease level is increased or decreased and is graphically represented in Fig.13.

In Tuticorin, the degree of infection ranged between 11.43 % and 45.83% in all the sites. In Site 1, February and March recorded the highest degree of infection of ice-ice disease with 31.4% and 45.8% respectively, while January the lowest degree of infection 22.2%. In Site 2, April and March recorded

Table 1 — Depth ranges in Tuticorin and pamban study sites during high and low tides

Depth Jan-14 Feb-14 Mar-14 Apr-14

Tuticorin site -1 site -2 site -3 site -1 site -2 site -3 site -1 site -2 site -3 site -1 site -2 site -3 low tide 0.24 0.48 0.82 0.21 0.39 0.61 0.23 0.42 0.72 0.24 0.48 0.82 high tide 0.72 1.10 1.68 0.81 1.32 1.83 0.95 1.49 1.96 1.00 1.53 2.10 Pamban site -1 site -2 site -3 site -1 site -2 site -3 site -1 site -2 site -3 site -1 site -2 site -3 Low tide 1.0 1.0 1.0 0.9 0.9 0.9 1.0 1.0 1.0 1.0 1.0 1.0 High tide 2.0 2.1 2.0 1.8 2.2 1.9 2.2 2.3 2.2 2.0 2.4 2.0

Fig .7 — Healthy thalli of K.alvarezii.

Fig.8 — Bleached thalli of K.alvarezii

Fig.9 (A) — Infected branches of K.alvarezii

Fig.9 (B) — Infected branches of K.alvarezii.

Fig. 10 — K.alvareziithalli loaded with dirt, silt.

Fig. 11 — Defragmentation of K. alvarezii

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the highest degree of infection with 24% and 23.07% respectively, while February the lowest degree of infection 20%. In Site-3, April and March recorded the highest degree of infection with 37.9% and 26.9% respectively, while January recorded the lowest degree of infection mean 11.42% (Fig.13).

In Pamban, the degree of infection ranged between 16.13% and 37.5%. In Site 1, April and March recorded the highest degree of infection of ice-ice disease with 30% and 24.13% respectively while January the lowest degree of infection 19.3%. In Site 2, April and March recorded the highest degree of infection with 26.93% and 23.07% respectively, while January the lowest degree of infection 17.24%. In Site 3, April and March recorded the highest degree of infection with 37.5% and 28.57% respectively, while January the lowest degree of infection 16.12 %(Fig.13).

In Tuticorin, highest range of microbes were recorded in diseased thalli in March 2014 in the site 1 as 142 strains, among them 106 strains are gram negative, 36 as gram positive and 79 as non pigment and 63 as pigmented. Lowest range was observed as 74 strains in the month of January 2014 in site 3,

gram negative strains are 59, 15 as gram positive and 61 as non pigmented, 13 as pigmented.

In sediment, highest ranges of microbes were recorded in March 2014 in the site 1 as 139strain, and among them 92 strains are gram negative, 47 as gram positive and 73 as non pigment and 66 as pigmented. Lowest range was observed as 61 strains in the month of January 2014 in site 3, gram negative strains are 37, 24 as gram positive and 42 as non pigmented, 19 as pigmented.

In water, highest ranges of microbes were recorded in March 2014 in the site 1 as 110strains; among them 86 strains are gram negative, 23 as gram positive and 92 as non pigment and 18 as pigmented. Lowest range was observed as 46 strains in the month of January 2014 in site 3, gram negative strains are 34, 12 as gram positive and 29 as non pigmented, 17 as pigmented.

In healthy thalli, the highest ranges of microbes were recorded in March 2014 in the site 1 as 67 strains; among them 42 strains are gram negative, 25 as gram positive and 39 as non pigment and 28 as pigmented. Lowest range was observed as 22 strains in the month of January 2014 in site 2, gram negative strains are 16, 6 as gram positive and 13 as non pigmented, 9 as pigmented (Fig.14).

In Pamban, highest range of microbes were recorded in diseased thalli in April 2014 at the site 1 as 182 strains, among them 129 strains are gram negative, 53 as gram positive and 142 as non pigment and 40 as pigmented. Lowest range was observed as 98 strains in the month of January 2014 in site 3, gram negative strains are 59, 39 as gram positive and 68 as non pigmented, 50 as pigmented.

In sediment, highest ranges of microbes were recorded in April 2014 in the site 1 as 143 strains; among them 119 strains are gram negative, 24 as

Fig. 12 — percentage of infected bunches

Fig.13 — Degree of infection

Fig. 14 — Number of colonies isolated from K.alvareziiice-icedisease at GoM

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gram positive and 121 as nonpigment and 22 as pigmented. Lowest range was observed as 76 strains in the month of January 2014 in site 3, gram negative strains are 59, 17 as gram positive and 49 as non pigmented, 27 as pigmented.

In water, highest ranges of microbes were recorded in April 2014 in the site 1 as 113 strains, among them 87 strains are gram negative, 26 as gram positive and 91 as non pigment and 22 as pigmented. Lowest range was observed as 55 strains in the month of January 2014 in site 3, gram negative strains are 30, 25 as gram positive and 37 as non pigmented, 18 as pigmented.

In healthy thalli, highest ranges of microbes were recorded in April 2014 in the site 1 as 109 strains; among them 79 strains are gram negative, 30 as gram

positive and 94 as non pigment and 15 as pigmented. Lowest range was observed as 31 strains in the month of January 2014 in site 3, gram negative strains are 19, 12 as gram positive and 16 as non pigmented, 15 as pigmented ( Fig.14).

Two way ANOVA tested between months and between regions showing (>p 0.05) no significant different in all parameters (Table 2). The Pearson correlation analysis for Temperature, salinity, % of incidence, degree of infection in Tuticorin show the positive correlation between Incidence of ice -ice disease at month with a degree of infection (Table: 3). This correlation coefficient in pamban study show positive correlation between percent of Incidence at month with a degree of infection (Table 4).

Table 2 — Two way ANOVA of disease infestation on K. alvarezii

No. of bunches affected SS df MS F P-value Remarks

Between region 77.04167 1 77.042 0.258 0.622 NS Between months 10685.13 11 971.375 3.253 0.031 NS Total 14046.63 23 % of Incidence Between region 120.3776 1 120.378 0.258 0.622 NS Between months 16695.51 11 1517.773 3.253 0.031 NS Total 21947.85 23 % of Incidence at month Between region 0.065104 1 0.065 0.001 0.977 NS Between months 1351.758 11 122.887 1.643 0.212 NS Total 2174.414 23 Total no of branches Between region 1.041667 1 1.042 0.020 0.890 NS Between months 454.4583 11 41.314 0.799 0.641 NS Total 1023.95 8 23 No .of branches affected Between region 0.166667 1 0.167 0.027 0.873 NS Between months 64.33333 11 5.848 0.935 0.544 NS Total 133.3333 23 Degree of infection Between region 3.081483 1 3.081 0.041 0.844 NS Between months 1089.953 11 99.087 1.305 0.333 NS Total 1928.235 23 Degree of infection a month Between region 7.423319 1 7.423 0.251 0.626 NS Between months 1249.084 11 113.553 3.841 0.017 NS Total 1581.682 23 “*”- Significant at 5% level, “NS” – Not significant

Table 3 — Pearson product – moment correlation of different parameters of Tuticorin region

Temperature (°C) Salinity

(ppt) % of Incidence at month degree of infection

Temperature (°C) 1 Salinity (ppt) 0.423 1 % of Incidence at month 0.230 0.077 1 degree of infection 0.257 0.140 0.960** 1 Note:*-P< 0.05;**-<0.01; NS- Not significant

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Discussion "Ice-ice" is generally caused by unfavourable

environmental conditions in the farming sites. This is a rather general statement because "unfavourable factors" in the farming sites refer either too high or low temperature, high or low salinity, high or low light intensity, and also to deficient nutrient. In Tuticorin, the incidence and degree of infection was found to be highest in Site 1 in all observations compared with other sites. This site is located 100m away from shore with shallow waters (depth – m). In low tide during February, Site l was directly exposed to the atmospheric air and sunlight, this combination elevated the temperature to thalli and lead in to ice ice disease. The combined effects of air and light exposure which increased the ambient temperature, stressed the seaweeds cause them to bleach. These observations are similar to the finding of Largo (2006)13 in Philippines.

Observations on the growth of K. alvareziiin the field for over a year revealed that commercial growth rates could not be obtained below 20°C and the optimum temperature range was 25–28°C. Temperature was playing a vital role to growth of K. alvarezii19. Where in high temperature during the summer months (sometimes attain more than 33.70°C in some places during low tides), coupled with high light intensity and low water movement are the key factors that triggering the prevalence of ice-ice disease. Moreover, wide-scale whitening that lead to complete damage of the branches was causedby temperature of up to 33-35°C20.

In tropical farm areas, however, Trono and Ohno (1989)21 reported that brisk growth and high biomass production by Kappaphycus and Eucheuma occur during months characterized by warmer temperature, i.e. 25–30°C. When a temperature below 30°C, in all the sites the incidence and degree of infection was minimum and the favourable temperature for the prevalence of ice ice infection is above 30°C. Hence, temperature is one of the main reasons for the occurrence of ice-ice disease in K. alvarezii.

In Tuticorin site 1 reached 33°C it may be a main reason the K. alvarezii affect that ice- ice disease.

In Tuticorin, the highest incidence was observed in site 1 (41.25%), subsequently in site 3 (38.75%) and site 2 (23%). This circumstance occurred in March and April with temperature level at 33.5°C and Site 1 were totally affected. In Pamban, the highest incidence was observed in Site 3 (37.5%), followed by site 1 (38.75%) and site 2 (23%). High incidence was occurred in April. When the degree of infection was correlated to environment conditions, the highest degree of infection occurred in April, when temperature was at maximum. The lowest degree of infection was observed in January, when all environmental factors were in the moderate ranges.

During low tides the incidence of disease and degree of infection was high because of slow water movement in the farming area. Some pathogens, especially bacteria, are highly motile and can very easily invade seaweed surfaces. Strong water current, aside from enhancing nutrient exchange, also prevents potential pathogen that comes from the surrounding water from establishing on the seaweed surface22.The rough thallus surface of K. alvarezii enables the dirt, epiphytes, parasites, and other microorganisms to easily attach to it in the low water motion. The more they can attach and establish themselves on the thalli surface, the more they can become a good medium for pathogens to grow and establish and later will penetrate to the cortex and medulary layers of thalli, making the thalli to become weaker. In addition of dirt, epiphytes can also decrease light absorption of the thalli. These conditions can make the thalli prone to bacterial infection. Therefore the low water movement lead to ice ice disease.

Uyenco et al (1981)5 noted high populations of bacteria on propagates with ice–ice, but they were secondary to the problem because the unfavourable environment leads to the ice-ice disease.

Microbiological studies provided further insights regarding the role of bacteria in ice-ice infections. It was reported that bacterial counts in ice-ice-infected

Table 4 — Pearson product – moment correlation of different parameters of Pamban region

Temperature (°C) Salinity (ppt) % of Incidence at month degree of infection

Temperature (°C) 1 Salinity (ppt) 0.710 1 % of Incidence at month 0.848 0.682 1 degree of infection 0.775 0.654 0.896* 1 Note:*-P<0.05;**-<0.01; NS- Not significant

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branches were 10 to 100 times greater than those in normal healthy K. alvarezii thalli23. Diseased and healthy K alvarezii thalli in all sites were microbiologically analyzed and the diseased K. alvarezii thalli have 114 numbers of microbes recorded in average at January to April 2014, whereas healthy thalli have 54 numbers of microbes recorded. In Pamban, diseased thalli have 128 numbers of microbes whereas healthy thalli have 64 numbers of microbes. Largo (1999)6 reported that the combined effect of stress and biotic agents, such as opportunistic bacteria are primary factors of the ice-ice disease. The infection of the seaweed by these pathogens was found to depend initially on the bacteria’s ability to establish them on the seaweed surface. He further stated that this ability, however, to be influenced by the members of the bacterial community or, possibly, with other co-existing micro-organisms, like epiphytes.

The Pearson correlation analysis for Temperature, salinity, % of incidence, degree of infection in both study locations (Tuticorin and Pamban) shows positive correlation between incidence of ice -ice disease at month with a degree of infection.

The short term income generation too few fisher folk in Gulf of Mannar and Palk Bay have been affected due to complete loss of cultivation caused by ice ice disease. However, the positive impact due to ice ice disease is possibilities of restoring ecological balance of the region where the key coastal habitats such as coral reefs and seagrass beds and associated biodiversity were much affected due to the cultivation of K. alvarezii. Over 6 Km2 coral reef area has been already affected in Gulf of Mannar due to invasion of K. alvarezii and vast of seagrass bed area has been degraded due to the shading by rafts and physical disturbance by cultivators.

Conclusion The study observed that the ice ice disease in

K. alvarezii is caused due to unfavourable environment conditions. High temperature, low salinity, and low depth play a crucial role the disease. March and April months in the study area have high temperature and the incidence and degree of infectivity of ice-ice disease was also high. Though, the loss of K. alvarezii cultivation due to ice ice disease in terms of short term additional revenue is minimal, the benefit to the ecological balance in particular protecting coral reef and seagrass habitats are high.

Acknowledgement The authors are thankful to Ministry of Environment,

Forests and Climate Change, Government of India for funding support; Principal Chief Conservator of Forests and Chief Wildlife Warden, Government of Tamil Nadu and Wildlife Warden, Gulf of Mannar Marine National Park for research permissions; and authorities of Suganthi Devadason Marine Research Institute for facilities.

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