1286-1290Ice-ice disease occurs in cultivated algal seaweed Kappaphycus alvarezii due to pathogenic bacterial infections. This seaweed has rich source of carrageenan widely known as the kappa carrageenan. Generally, ice-ice disease leads to whitening of the branches initiated with colour changes of the thalli, which become transparent in the end. This study was aimed to isolate and identify the bacteria based on morphology and biochemical characterization on ice-ice diseased K. alvarezii from three different places, namely, Kottapatinam, Thondi and Rameswaram. The bacterium was isolated in Zobell Marine Agar (ZMA) and thiosulfate-citrate-bile saltsucrose (TCBS) agar. Morphological and biochemical characterizations revealed that the isolated bacteria causing ice-ice disease were closely related to the genera Bacillus in ZMA and Vibrio species in TCBS. Total viability count, physical and chemical properties of the bacteria by gram staining and morphological analysis were done for all species isolated from three places

Inbakandan, D.

Kavitha, G.

Nalini, S.

Riyaz, S.U. Mohammed

Sutha, S. Arul Delpin

English

NOPR

Indian Journal of Geo Marine Sciences Vol. 48 (08), August 2019, pp. 1286-1290       Characterization and identification of isolated bacteria from ice-ice disease infected seaweed Kappaphycus alvarezii S.U. Mohammed Riyaz1, S. Nalini1, G. Kavitha1, S. Arul Delpin Sutha2, & D. Inbakandan1* 1Centre for Ocean Research, MoES – Earth Science & Technology Cell, Sathyabama Institute of Science and Technology,  Chennai – 600 119, Tamil Nadu, India  2Department of Marine-Biotechnology, AMET University, Chennai, India. *[E-mail: inbakandan@gmail.com & inbakandan@sathyabama.ac.in] Ice-ice disease occurs in cultivated algal seaweed Kappaphycus alvarezii due to pathogenic bacterial infections. This seaweed has rich source of carrageenan widely known as the kappa carrageenan. Generally, ice-ice disease leads to whitening of the branches initiated with colour changes of the thalli, which become transparent in the end. This study was aimed to isolate and identify the bacteria based on morphology and biochemical characterization on ice-ice diseased  K. alvarezii from three different places, namely, Kottapatinam, Thondi and Rameswaram. The bacterium was isolated in Zobell Marine Agar (ZMA) and thiosulfate-citrate-bile saltsucrose (TCBS) agar. Morphological and biochemical characterizations revealed that the isolated bacteria causing ice-ice disease were closely related to the genera Bacillus in ZMA and Vibrio species in TCBS. Total viability count, physical and chemical properties of the bacteria by gram staining and morphological analysis were done for all species isolated from three places. [Keywords: Kappaphycus alvarezii; Vibrio sp.; Ice-ice disease] Introduction Solieriaceae is a red algal family in Order Gigartinales of Class Rhodophyceae. In this family, there are many valuable species including Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum which generally have multiaxial thalli and often inhabit the warm seas. K. alvarezii is often cultivated in the upper part of the sub littoral zone, from just below the low tide line to rocky substrates where water flow is slow to moderate. Kappaphycus is an important red alga cultivated in south-east Asia and India as raw material both for the extraction of the phycocolloid, carrageenan, and as food1. There are two kinds of diseases in seaweed: Infectious and non-infectious type. The former involves a transmissible infectious agent (bacteria, fungi, virus, etc.), while the latter is induced by physiogenic factors such as extremes of temperature, salinity, light intensity or pollution. Microorganisms have been identified as the colonizers of seaweeds, and bacteria have been regarded as the primary colonizer2. This bacteria-seaweed association often disturbed the production of K. alvarezii, which is often damaged by the occurrence of ice-ice disease. According to Mendoza3, ice-ice disease led to significant decrease in seaweed production and decrease in carrageenan yield ranging from 25-40% compared to the healthy crop. Bacteria are known to be the main component of seaweed epibionts with higher diversity and had implications in the development of the disease4.  It has been further found that non-pigmented bacterium and a yellow bacterium, Cytophaga-Flavobacterium complex and Vibrio-Aeromonas complex were causative microbes of the whitening of K. alvarezii. However, very little is known about seaweed disease and its status in India. The disease is spreading along with the extension of cultivation area and environmental degradation. Due to regular outbreaks and economic loss caused by bacterial community, several strategies have been attempted worldwide to stop spreading of seaweed diseases. However, those approaches were demonstrated ineffective and there were very little success in controlling the disease on large spatial scale. This study was conducted to determine the incidence and infection of ice-ice disease in Kappaphycus alvarezii, as well as the environmental factors that triggered the disease. To understand the causative agent of K. alvarezii ice-ice disease in Rameswaram (RH), Thondi (TH) and Kottaipatinam (KT), microbiological and biochemical techniques were applied to investigate the symptom and the causative bacteria.  RIYAZ et al: CHARACTERIZATION & IDENTIFICATION OF ISOLATED BACTERIA FROM ICE-ICE DISEASE   1287Materials and Methods Sampling site Infected samples of K. alvarezii were collected from cultivation farms at Kottapatinam (N 9° 58' 51.6842", E 79° 12' 9.4613"), Rameswaram (9° 17′ 16.8″ N, 79° 18′ 46.8″ E) and Thondi (9.7438°N, 79.0185°E) in August 2017 (Fig 1). After collection, the infected seaweed materials were stored into sterile borosilicate bottles containing autoclaved seawater and brought in chilled condition to the laboratory and stored at 4 °C.  Isolation of bacteria from infected seaweed One gram of infected seaweed sample was grinded using mortar and diluted in 10 mL of sterilized seawater. The sample was vortexed to homogenize the solution. Following this, 1 mL of the solution was diluted into 9 mL of sterilized seawater to make 10-1 seaweed dilution. The processes were repeated until 10-6 seaweed dilution. The 100 µL aliquots from  10-1to 10-7 dilution were spread onto Zobell marine agar (ZMA) and incubated at room temperature for  24 h. Based on morphological features, colonies  were randomly picked and purified by making  streak plates.  Identification of Isolated Bacteria by Gram staining method Thin smear of bacterial cultures was prepared on clear, dry glass slide and allowed to air dry and fixed by gentle heat. Gram staining was done for isolated bacterial cultures according to the protocol and procedures using Gram staining kit (Himedia K001). The slides were air-dried or blot-dried between sheets of clean bibulous paper and examined under oil immersion objective.  Identification of Isolated Bacteria by Biochemical test method Bacteria isolated from ice-ice disease infected K. alvarezii were subjected to biochemical test using KB001 HiIMViC Biochemical Test Kit which is a combination of 12 tests for identification of Gram negative species. It contains sterile media for Indole, Methyl red, Voges Proskauer’s, Citrate utilization tests and eight carbohydrates, namely, glucose, adonitol, arabinose, lactose, sorbitol, mannitol, rhamnose and sucrose, Reagents supplied with kit included: Kovac’s Reagent (R008) for Indole test Methyl Red Indicator (I007), Barritt Reagent A (R029) and Baritt Reagent B (R030) for VP test.   Results Sampling site Table 1 describes hydrological parameters namely, salinity, temperature, relative humidity and pH of three different sites. It shows that salinity, relative humidity and pH are higher in TH and KT sites than in RM site, whereas it's opposite with respect to temperature parameters, it's higher in RM site and lower in TH and KT sites.   Isolation of bacteria from infected seaweed The ice-ice disease infected K. alvarezii is shown in Fig. 1. Growth of white milky bacterial colonies is observed in RMZMA01, THZMA01, KTZMA01-Zobell Marine Agar plates (Fig. 2a) and yellow colonies in TCBS plates of RMTCBS01 and THTCBS01 plates, but no colonies are observed in  Fig. 1 — Collection of K. alvarezii sample from sampling site (A); (B & C) Ice-Ice disease infected K. alvarezii (B & C); Healthy sample of K. alvarezii (D). Table 1 — Physiological features and conditions of three sample sites S.No. Features Rameshwaram(RM) Thondi (TH) Kottapattinam(KT) 1. Salinity (ppt) 34 36 36 2. Temperature 38.5° C 33.1° C 32.7° C 3. Relative Humidity 60% 73% 69% 4. pH 8.1 8.3 8.3 INDIAN J. MAR. SCI., VOL. 48, NO. 08, AUGUST 2019   1288KTTCBS01 plates. Further plates are stored in 4 °C (Fig. 2b).  Identification of Isolated Bacteria by Gram staining method  The slides observed in light microscope showed rod-shaped bacteria with purple colour from ZMA culture plates for KTZMA01, THZMA01 plates and RMZMA01 cultures which elucidates that these cultures are Gram positive. The THTCBS01 and RMTCBS01 bacterial colonies from TCBS plates showed pink colour, with rod and comma shape, respectively which elucidates that these bacteria are Gram negative (Fig. 3).  Identification of isolated bacteria by biochemical test method The results of biochemical test are given in  Table 2. The test is based on pH change and substrate utilization. On incubation, the bacteria underwent metabolic changes which are indicated as colour change in the media that can be either interpreted visually or after addition of the reagent.  Discussion Diseases are generally caused by low salinity, high temperature and light intensity. When the plant is under stress whitening of the branches occurs, which results in crop loss. Using colony morphology on selected agars and Gram-stain morphology, a number of 1-step biochemical or enzymatic tests can be identified by the skilled microbiologists. These results are often available more quickly and are as accurate as those derived from conventional methods. To support the integrated prevention of ice-ice disease, information about bacterial pathogens and the availability of fast and accurate detection are required. The development of ice-ice disease in K. alvarezii depends on several factors to which the seaweed was exposed. Largo et al., 1995 found that the combined effect of stress and abiotic agents, such as opportunistic pathogens are primary factors of the ice-ice disease5. The infection of the seaweed by these pathogenic bacteria may depend on their ability to successfully attach to the seaweed surface.  The degree of infection during August was high in this study in TH and KT. This increase could be related to the changes of environmental conditions in the coast, mainly, increasing amount of pollution due to increasing human activities near the farms and increasing temperatures. This result confirms the findings of Largo et al., 20066, wherein high temperature during the summer months (sometimes reaching more than 30 °C in some places during low tides), coupled with high light intensity and low water movement are the key factors that trigger ice-ice development in the cultivation ground.   Fig. 2 — Isolation of bacteria in TCBS Agar from ice-ice disease infected K. alvarezii thalli (a); Isolation of bacteria in ZMA fromice ice disease infected K. alvarezii thalli (b).  RIYAZ et al: CHARACTERIZATION & IDENTIFICATION OF ISOLATED BACTERIA FROM ICE-ICE DISEASE   1289Conclusion Morphological and biochemical characterizations of the isolated bacteria from ice-ice disease infected branches of the seaweed Kappaphycus alvarezii from KT, TH and RH revealed that ice-ice disease is caused by bacteria closely related to the genera Bacillus in ZMA and Vibrio in TCBS. It can be concluded that isolates obtained are Gram positive and Gram negative bacteria areas. This study emphasises the need for further confirmation by molecular techniques with pathogenicity tests.  Acknowledgement The first author Dr. S.U. Mohammed Riyaz, National Post-Doctoral Fellow (PDF/2016/002741), is thankful to the SERB, Department of Science and Technology, Govt. of India and the management of Sathyabama Institute of Science of Technology   Fig. 3 — Identification of bacterial isolates from ice-ice diseased K. alvarezii by Gram staining method.  Table 2 — Identification of Isolated Bacteria by Biochemical test method S.No. Test KTZMA01 THTCBS01 THZMA01 RMTCBS01 RMZMA01 1.  Indole - - - - - 2.  Methyl Red + + - + - 3.  Voges Proskauers - - + - - 4.  Citrate Utilization + - + - - 5.  Glucose + + + + + 6.  Adonitol - - - - - 7.  Arabinose + - + - + 8.  Lactose - - - - - 9.  Sorbitol - - - - - 10.  Mannitol + + - + - 11.  Rhamnose - - - - - 12.  Sucrose - + - + -  INDIAN J. MAR. SCI., VOL. 48, NO. 08, AUGUST 2019   1290(Deemed University) for providing the facilities and support for carrying out this research work.  References  1 Vairappan, C.S., Chung, C.S., Hurtado, A.Q., Soya, F.E., Lhonneur, G.B., &Critchley, A., Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms, J Appl. Phycol., 20 (2008) 477–483. 2 Lachnit, T., Blümel, M., Imhoff, J.f., & Wahl. M., Specific epibacterial communities on macroalgae: phylogeny matters more than habitat, Aquat. Biol., 5 (2009) 181-186. 3 Mendosa, W.G., N.E, Montano, E.T. Ganzon-Fortes, and  R. D, Villanueva. Chemical and gelling profile of ice-ice infected carrageenan from Kappaphycus striatum (Schmitz) Doty “sacol” Strain (Solieriaceae, Gigartinales, Rhodophyta), J. Appl. Phycol., 14 (2002) 409-418. 4 Zablackis, E., West, J.A., Liao, M.L., and Bacic, A., Reproductive biology and polysaccharide chemistry of the red alga Catenella (Caulacanthaceae, Gigartinales), Bot. Mar., 36 (1993) 195-202. 5 Largo, D.B., Fukami, K., & Nishijima, T., Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae, Gigartinales, Rhodophyta), J. Appl. Phycol., 7 (1995) 545-554. 6 Largo, D.B. Disease in cultivated seaweeds in the Philippines: is it an issue among seaweed industry players? In: Siew-Moi, P., A.T. Critchley, and P.O. Ang Jr. (eds.). Advances in seaweed cultivation and utilization in Asia. (2006) 61-70.  

Characterization and identification of isolated bacteria from ice-ice disease infected seaweed Kappaphycus alvarezii

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Characterization and identification of isolated bacteria from ice-ice disease infected seaweed Kappaphycus alvarezii

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