Strain selection of red algae Kappaphycus alvarezii (Doty) Doty farmed using different methods in Tablolong Waters, Kupang Regency-East Nusa Tenggara Province

The main problem is facing in farming red algae Kappaphycus spp. in Kupang Regency are decreasing quality of seeds and the infection of ice-ice disease. This research aimed to analyze the growth of different strains of K. alvarezii farmed using different methods, and analyze which strains of K. alvareziii have high resistance to ice-ice disease. This research was carried on August to November 2020 in Tablolong waters, Kupang Regency. The strains used were Sacol from Sulamu and Semau, Tambalang (tissue culture) from Rote Island, and Cottonii strain from Pasir Panjang. The farming methods used were the Longline and Off-Bottom. The main data taken were wet weight and incidence as well as infection intensity of the disease. The study found that growth rates varied based on the strains and farming methods. Based on the strain, the Sacol strain from Sulamu gave a higher growth than the others. This strain grew and reached an average weight of 1087.5 grams at week 7; with an average specific growth of 4.87% per day. Based on the method, the longline method gave better growth compared to the off-bottom method. Regarding ice-ice disease, the Sacol strain from Sulamu gave higher resistance to disease compared to the other strains, with an average incidence of 27.26% and an infection intensity of 2.81%. The longline method provides higher disease resistance than the off-bottom method.


Introduction
Seaweed is one of the leading fishery commodities in the Province of East Nusa Tenggara (NTT).For Kupang Regency, including Tablolong, the farming of this commodity is one of the main activities of coastal communities, with a significant contribution to increasing community income and local revenue.It was noted that the wet production of seaweed in Kupang Regency in 2020 reached 1,879,265.96tons [1].One of the main species being farmed is Kappaphycus alvarezii (trade name, Eucheuma cottonii).These species become the main choice for seaweed farming because of their secondary metabolic content which is in high demand in the international market, namely the Kappa carrageenan.Kappacarrageenan is currently used as an industrial and wastewater cleaner, acetic acid production, as well as a gelling and thickening agent in the food industry [2].
In the last decade, there has been a decline in the quality of farming red algae K. alvarezii caused by massive continuous propagation methods with fragmentation so that the thallus becomes sterile, stunted, and cannot withstand changes in aquatic environmental conditions, coupled with the emergence of the 1260 (2023) 012033 IOP Publishing doi:10.1088/1755-1315/1260/1/012033 2 phenomenon of ice-ice disease.that seasonally attacks massively K. alvarezii farming.These conditions caused production to drop drastically, reaching only about 10%.According to Trono (1993), Mendoza et al (2002), in [3] that the attacking of ice-ice disease can result in significant degradation of seaweed production, as well as degradation of carrageenan quality, viscosity, and gel strength of infected thalli compared to healthy plants, ranging from 25 to 40%.According to [4], ice-ice disease is caused by acting together with biotic and abiotic factors which are diagnosed by the presence of white and soft parts on the main talus of infected seaweed.Infection with this disease varies from species, place, and season.Control measures may include proper farm site management, diversification with other seaweeds, pre-soaking with antibacterial drugs and nutrient enrichment prior to planting, and possibly using genetic engineering.
The seaweed farming method being used in Kupang Regency, included in Tablolong Waters are dominated by the longline and off-bottom methods.The use of these methods is based on the coastal profile.The off-bottom method is usually used by farmers on shallow shores, placing the plants near the bottom of the sea; while the longline method is used on deeper shores, placing the plants near the surface of the waters.
The objectives of this research were to analyze the growth rate of different strains of K. alvarezii farmed using different methods; and to analyze which strains of farmed K. alvarezii have high resistance to ice-ice disease infestation.

Data Collection
This research was carried out for 3 (three) months, July -September 2021, at the farmed location in Tablolong village, Kupang District (figure 1).The farming methods used were the off-bottom and the longline method.The off-bottom method is a farming method that places the plant test near or at the bottom of the sea, with a depth of about 0.5-3 m. during low tide sometimes the plants are not submerged in the seawater.While the longline method is a method that places the plant test on the surface of the water, about 10-15 cm from the surface.
The growth and disease data were taken every 7 days (every week) for seven weeks (49 days).Sampling was carried out with 7 (seven) replications for each strain of seed by taking as many as 20 ties at random.For growth, data was taken in the form of wet weight data; while for disease, data was taken in the form of the number of plants infected and the number of infected branches, in the form of the white part of the thallus.
Data on the physical-chemical conditions of the waters were also measured, including temperature, salinity, current velocity, transparency, and pH.

Growth
Based on the results of the study, the weight gain of Kappaphycus alvarezii different strains farmed with different methods gave different values.For the off-bottom method, strain Sacol Sulamu gave the highest value, 844.5 grams on week seventh, followed by Sacol Semau (669.9 grams on week seventh).
For the longline method, still strain Sacol Sulamu gave the highest value, 1087.5 grams on week seventh, followed by Sacol Semau (996.2 grams on week seventh) (Figure 3).In terms of Specific Growth Rate (SGR), the results showed that SGR ranged between 0.38-9.60%per day for the off-bottom method, and 0.51-9.56%per day for the longline method.For the off-bottom method, strain Sacol Sulamu showed the highest value with a mean of 4.35% per day, followed by Sacol Semau (3.88% per day).For the longline method, still strain Sacol Sulamu showed the highest value with a mean of 4.87% per day, followed by Sacol Semau (4.69% per day) (Figure 4).
When a comparison is made between the two methods, the results showed that the longline method showed a higher SGR value than the off-bottom method, for all strains (Figure 5).The SGR value in the longline method ranged between 3.58-4.87%per day, with a mean of 4.22% per day; while in the off-bottom method, the SGR value ranged between 2.21-4.35%per day, with a mean of 3.41% per day.According to [7], in the study of Kappaphycus alvarezii, different strains/colors may give different reactions to biological parameters such as photosynthesis.In addition to color differences, the two varieties also have varying growth speeds.The green and brown strains of K. alvarezii are genetically different.The color pattern of the green and brown strains of K. alvarezii remains.However, the color performance may change according to ecological conditions.In the dry season, the green strain grows better than the brown strain.According to Dawes (1992) in [5], photosynthetic efficiency varied between the red, brown, and green color types of Eucheuma denticulatum.However, it is not stated which color confirms the higher photosynthetic efficiency.Since photosynthetic activity is an important determinant of growth rate, the different coloration of green and brown K. alvarezii varieties may contribute to different growth rates.In addition, [8] found that growth rates varied between strains, species, depths, and also between different farming sites of Kappaphycus alvarezii and E. denticulatum.There is a tendency that the growth rate decreases as the depth decreases.Plants placed on the surface area have a higher growth rate than other plants.This fact shows that nutrients, light, and current speed have a positive effect on growth rate.
[5] stated that on surface waters, cultured seaweed will get more light intensity for photosynthesis, and the current speed is faster (Kappaphycus and Eucheuma need medium current speed to stop fast).This condition makes plants absorb nutrients optimally.Plants that grow on the surface of the water have higher growth than those that grow below.The best-growing conditions it's in the surface area.This explains that nutrition and light have an influence on them.In terms of method, the longline method shows better growth performance than the off-bottom method.This is because Kappaphycus alvarezii farmed with the longline method has a stronger current than the off-bottom method.Moreover, [9], stated that water currents have a great influence on nutrient transport, aeration, and water agitation, thus influencing the growth rate of E. denticulatum.Currents that are too strong can also cause the thallus to break, so the location for farming E. denticulatum should be avoided from currents and waves that are too strong (above 0.5 m/sec).
The result of this study using the longline method had specific growth ranging from 3.58 -4.87%, slightly higher than the study conducted by [10] using the same farming method which obtained specific growth ranging from 2.22 -5.21%.

Ice-ice Disease
The main problem faced by seaweed farmers in Kupang Regency, included in Tablolong Village is the Phenomenon of Ice-ice Disease.According to Largo et.al., (1999) in [3], ice-ice disease in Kappaphycus/Eucheuma could be caused by extreme environmental conditions such as high temperature and irradiance, low salinity, and opportunistic bacterial pathogens, such as Vibrio sp.(P11) and Cytophaga sp.(P25) These findings suggested that the whitening phenomenon is caused by acting together simultaneously between biotic and abiotic factors.When stress occurs in seaweed, it produces a moist organic substance that attracts bacteria in the water to infect and in the end caused the "whitening" and "softening" of the seaweed thalli.Uninfected thalli remain healthy while infected ones undergo lost pigmentation and eventually lead to plant breakage by water movement.Similarly, the ability to produce kappa-carrageenase enzymes and degrade kappa-carrageenan by the bacterium Pseudoalteromonas carrageenovora in K. alvarezii indicated that this microorganism can cause symptoms of ice-ice disease such as whitening of the thalli [11].
The result also showed that all strains tested using off-bottom and longline farming methods were infected by bacteria caused ice-ice disease.In terms of incidence of disease, farming using the offbottom method showed incidence ranged between 7.70-90.56%.The strain that has more resistant to ice-ice disease was Sacol from Sulamu with a mean of 34.02%, while the susceptible one was strain Cottonii from Pasir Panjang, with a mean of 61.85%.for the longline method, the disease incidence ranged between 10.22-80.43%.The strain that has more resistant to ice-ice disease was Sacol from Sulamu with a mean of 27.26%, while the susceptible one was strain Cottonii from Pasir Panjang, with a mean of 49.08% (Figure 6).In terms of Intensity of Infection, the result showed that the intensity of disease ranged between 1.48-43.21%for the off-bottom method, and 1.44-16.86%for the longline method (Figure 7). Figure 7 also showed that for the off-bottom method, the strain that has more resistant to ice-ice disease was Tambalang Tissue Culture from Rote, with a mean of 3.88%; while the one with the highest intensity of disease was strain Cottonii from Pasir Panjang with the mean of 19.80%.For the longline method, the strain that has the lowest intensity of the disease, meaning have more resistant to ice-ice disease was Sacol from Sulamu, with a mean of 2.81%; while the one with the highest intensity of disease (more susceptible) was strain Cottonii from Pasir Panjang with the mean of 7.55%.
Figures 6 and 7 indicated that internally there were strains that are more resistant to ice-ice disease, while others are more susceptible.This showed that there are differences in internal conditions in dealing with stress due to unfavorable physical-chemical conditions of the waters, as well as against ice-ice disease.The differences in genetic diversity of strains belonging to K. alvarezii may be one of the causes.[12] have done genetic engineering in K. alvarezii for ice-ice disease controls using the Gα gene extracted from soybean into the callus of K. alvarezii using Agrobacterium tumefaciens and regenerated modified callus cells to transgenic plantlets.Results revealed that K. alvarezii transgenic plantlets with Gα genes were successfully created.[13] established a binary plasmid bearing chicken lysozyme gene and transferred it to K. alvarezii thalli using A. tumefaciens and also successfully produced a transgenic K. alvarezii.The Gα gene encodes for the heterotrimeric G protein α subunit is a gene that has an essential role in biotic and abiotic stress tolerance.Also, chicken lysozyme is a significant constituent of immune defense against pathogenic bacterial diseases that can boost seaweed tolerance against pathogens.According to Trono (1999), Largo (1999), and Uyenco (1981) in [3], high planting densities and the high presence of epiphytes at certain seasons covering the surface of the thallus can result in covering the plant surface from absorption of nutrients for growth and light intensity for photosynthesis.This condition can cause plant stress and trigger a high incidence of ice-ice disease.The effect of acting together between stress and biotic agents in the form of the presence of opportunistic bacteria is a major factor in the ice-ice disease.The infection of K. alvarezii and E. denticulatum by these pathogens was found by Largo to depend initially on the bacteria's ability to establish themselves on the surface of the thalli.He found this ability, however, to be influenced by the members of the bacterial community or, possibly, by other co-existing micro-organisms, like epiphytes.
When a comparison is made between the two methods, the results showed that the longline method showed a lower intensity of disease infection compared to the off-bottom method, for all strains (Fig. 8).The intensity of disease in the longline method ranged between 2.81-7.55%with the mean of 4.16%; while in off-bottom method, the intensity of disease ranged between 3.88-19.80%with the mean of 7.98%.This indicates that the off-bottom method is not suitable for farming K. alvarezii in terms of ice-ice disease, because it creates conditions that are less than optimal related to the physico-chemical conditions of the waters.During low tide, plants will be exposed to the intensity of sunlight, and experience the minimization of water movement (current).This condition will trigger stress on the plants and infection of bacteria caused the disease.According to [14], In Zanzibar-Tanzania, seaweeds, farmed in shallow intertidal lagoons nearly in direct contact with the seafloor bottom during low tides, received extremely high levels of temperature and light intensity causing ice-ice disease outbreak.

Water Quality
In this study, physical and chemical water parameters have also been taken at noon and afternoon time (table 1).The result of the study showed that all parameters were in the normal range for the growth of Kappaphycus alvarezii.According to [15], the optimal range for the growth of farming K. alvarezii is temperature ranging from 26-32 0 C, salinity ranging from 28-34 ppt, currents 0.25-0.40m/s, pH 7.5-8.5 and Transparency 1-5 m.

Conclusion
Based on the findings presented in this research, it can be concluded that the superior strain that has better growth and resistance to ice-ice disease was Kappahycus alvarezii strain green Sacol from Sulamu; the Longline Method gave better growth and more resistance to ice-ice disease compared to the off-bottom method, for all strains

Figure 7 .
Figure 7.The intensity of ice-ice disease of K. alvarezii different strains farmed using, A. off-bottom method, and B. longline method (TTC = Tambalang Tissue Culture).W = week 0

Table 1 .
Physical and chemical water parameters (W = week).