bioRxiv preprint doi: https://doi.org/10.1101/2021.06.30.450479; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Bacterial community composition of the sea grape Caulerpa lentillifera: a comparison 2 between healthy and diseased states 3 Germán A. Kopprioa*, Nguyen Dinh Luyenb,c, Le Huu Cuongb,c, Anna Fricked, Andreas 4 Kunzmanne, Le Mai Huongb,c, Astrid Gärdesf,g 5 a Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany 6 b Institute of Natural Product Chemistry, Vietnam Academy of Science and Technology, Hanoi, 7 Vietnam 8 c Graduate University of Science and Technology, Vietnam Academy of Science and 9 Technology, Hanoi, Vietnam 10 d Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren, Germany 11 e Leibniz Centre for Tropical Marine Research, Bremen, Germany 12 f University of Applied Sciences, Bremerhaven, Germany 13 g Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, 14 Germany 15 * Corresponding author at: Chemical Analytics and Biogeochemistry, Leibniz Institute of 16 Freshwater Ecology and Inland Fisheries, Müggelseedamm 301, 12587 Berlin, Germany. E- 17 mail: [email protected] 18 Key words: Vietnam, seaweed, aquaculture, green caviar, 16S rRNA diversity 19 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.30.450479; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Summary 21 The bacterial communities of the sea grape Caulerpa lentillifera were studied during a disease 22 outbreak in Vietnam. The Rhodobacteraceae and Rhodovulum dominated the composition of 23 healthy C. lentillifera. Clear differences between healthy and diseased cases were observed at 24 order, genus and Operational Taxonomic Unit (OTU) level. Bacterial diversity was lower in 25 healthy C. lentillifera, probably because of antimicrobial compounds from the macroalgae and/or 26 from Clostridium, Cutibacterium or Micrococcus bacteria. The likely beneficial role of 27 Bradyrhizobium, Paracoccus and Brevundimonas strains on nutrient cycling and phytohormone 28 production was discussed. The white coloration of diseased C. lentillifera may not only be 29 associated with pathogens but also with an oxidative response. Aquibacter, Winogradskyella 30 and other OTUs of the family Flavobacteriaceae were hypothesized as detrimental bacteria, this 31 family comprises some well-known seaweed pathogens. Moreover, Thalassobius OTU 2935 32 and 1635 may represent detrimental Rhodobacteraceae. Phycisphaera together with other 33 Planctomycetes and Woeseia were probably saprophytes of C. lentillifera. This study offers 34 pioneering insights on the co-occurrence of C. lentillifera-attached bacteria, potential detrimental 35 or beneficial microbes, and a baseline for understanding the C. lentillifera holobiont. Further 36 metagenomic and biotechnological approaches are needed to confirm functions of some 37 microbes on this macroalgae to enhance food security in the tropics. 38 Introduction 39 The sea grape or green caviar Caulerpa lentillifera is a green macroalgae widely cultivated in 40 tropical countries of the Indo-Pacific region, and its market as seafood delicacy has been 41 expanded drastically in the last decades. Not only C. lentillifera flavor, vesicular ramuli and 42 succulent texture which resembles caviar, but also elevated contents of polyunsaturated fatty bioRxiv preprint doi: https://doi.org/10.1101/2021.06.30.450479; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 43 acids, antioxidants, vitamins and trace elements (Saito et al., 2010; Paul et al., 2014; de 44 Gaillande et al., 2017; Yap et al., 2019), together with its marketing as functional food increased 45 exponentially its demand. Moreover, the combination of the high nutritional quality of C. 46 lentillifera with its simple and low-cost cultivation transforms this macroalgae in a promising 47 candidate to enhance food security of tropical countries (de Gaillande et al., 2017; Stuthmann et 48 al., 2021). However, one of the major factor contributing to the worldwide decline of macroalgal 49 populations are microbial diseases (Egan et al., 2014) and C. lentillifera aquaculture is currently 50 endangered by outbreaks. 51 Macroalgae provide a nutrient-rich niche for microbes and some bacteria are capable to invade 52 algal tissue degrading complex polymers causing disease. Agarases, carrageenases, alginases, 53 fucoidanases, fucanases, mannanases, cellulases and pectinases are depolymerizing enzymes 54 detected in several marine bacteria for the breakdown of algal cell walls (Goecke et al., 2010). 55 Moreover, the low genetic diversity of cultured algae and their tight distribution in aquaculture 56 settings facilitate the rapid spread of detrimental microbes. Several bacterial diseases have 57 been reported in commercial macroalgae such as the rote spot and hole-rotten diseases caused 58 by Pseudomonas spp. in Saccharina japonica, rotten thallus syndrome by Vibrio sp. in 59 Gracilaria verrucosa, or Anaaki by Flavobacterium spp. in Pyropia yezoensis (Goecke et al., 60 2010b; Egan et al., 2014; Ward et al., 2020). Nevertheless, the role of microbes is not only 61 detrimental but can also be beneficial. 62 Some bacteria are essential for algal health, growth and development. Beneficial bacteria 63 attached to macroalgae induce morphogenesis, fix nitrogen, produce phytohormones, release 64 antifouling and antimicrobial compounds, transport metabolites and nutrients, detoxify 65 pollutants, contribute to algal reproduction and are key for their adaptation to new environments 66 (Aires et al., 2013; Hollants et al., 2013; Singh and Reddy, 2014). The relation between bioRxiv preprint doi: https://doi.org/10.1101/2021.06.30.450479; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 macroalgae and bacteria is generally mutualistic, and bacteria are mainly benefited with organic 68 substrates for their metabolism and a stable micro-environment protected from predators and 69 changing environmental conditions. The relations between macroalgae and bacterial 70 communities are so tight and reciprocal that the whole entity is considered a holobiont (Egan et 71 al., 2013; Arnaud-Haond et al., 2017; Califano et al., 2020). Furthermore, the bioactive 72 compounds produced by microbes in the holobiont have an enormous potential for drug 73 discovery and biotechnological applications (Friedrich, 2012; Luyen et al., 2019). 74 Despite the commercial importance of C. lentillifera and its relevance for human nutrition under 75 future climate-driven and overpopulation scenarios, studies about their microbiome are very 76 limited. Liang et al., (2019) described a disease in C. lentillifera in Chinese aquaculture systems 77 characterized by a dark-green biofouling, pink-colorated and missing ramuli. In this particular 78 case, Bacteroidetes and Cyanobacteria dominated the bacterial communities of diseased C. 79 lentillifera. Although recent advances in the study of the macroalgal microbiome using next 80 generation techniques, the information is still limited and mostly derived from culture methods 81 with their intrinsic limitations. The 16S rRNA gene approach offers broader insights on the 82 natural composition of bacterial communities attached to algae and plays an important role in 83 understanding algal diseases (Egan et al., 2014; Kopprio et al., 2021). Aims of this work were: 84 1) to characterize the bacterial communities of C. lentillifera in Vietnamese aquaculture, and 2) 85 to assess the effect of an unknown disease on its bacterial community composition. We 86 hypothesize strong differences in bacterial communities of C. lentillifera between healthy and 87 diseased cases. 88 Results bioRxiv preprint doi: https://doi.org/10.1101/2021.06.30.450479; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 89 The order Rhodobacterales (class Alphaproteobacteria) and the genus Rhodovulum of the 90 mentioned order presented the highest relative sequence abundance in C. lentillifera at each 91 respective taxonomic level (Fig. 1). The Rhodobacteraceae was the only family detected within 92 the order Rhodobacterales. According to the Mann-Whitney test, the mean relative abundance 93 of Clostridiales (phylum Firmicutes) was significantly higher in healthy C. lentillifera, whereas the 94 abundances of Flavobacteriales (phylum Bacteroidetes), Phycisphaerales (phylum 95 Planctomycetes) and Steroidobacterales (class Gammaproteobacteria)