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    A seagrass harbours a nitrogen-fixing bacterial partner

    NEWS AND VIEWS
    03 November 2021

    A seagrass harbours a nitrogen-fixing bacterial partner

    How underwater seagrasses obtain the nitrogen they need has been unclear. Evidence has now emerged of a partnership with a bacterium that might be analogous to the system used by many land plants to gain nitrogen.

    Douglas G. Capone

     ORCID: http://orcid.org/0000-0002-3968-736X

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    Douglas G. Capone

    Douglas G. Capone is in the Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA.

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    Seagrass meadows are a prominent feature of many shallow coastal areas of the temperate through to the tropical ocean. Seagrasses provide a crucial habitat for invertebrates and juvenile fish, stabilize sediments and buffer the shoreline against erosion1. Moreover, they contribute directly and positively to the ‘blue economy’ of the oceans through their long-term storage of carbon2. Lush and highly productive seagrass beds often thrive in nutrient-deficient waters, and attempts to solve the enigma of how they accomplish this feat have driven considerable research over the years. Writing in Nature, Mohr et al.3 provide crucial evidence indicating that the success of a seagrass called Posidonia oceanica (Fig. 1), which proliferates throughout the warm waters of the Mediterranean Sea (and elsewhere), might be attributed to the development of a highly integrated partnership with a bacterium. This system is reminiscent of those found in some terrestrial plants.

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    doi: https://doi.org/10.1038/d41586-021-02956-y

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    The author declares no competing interests.

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    Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium

    Etymology‘Candidatus Celerinatantimonas neptuna’ (nep.tu’na L. fem. n.), pertaining to Neptunus (L. masc. n. Neptune), the Roman god of the seas and the Neptune grass, Posidonia oceanica.SamplingA P. oceanica meadow at 8 m water depth and nearby sandy sediments in Fetovaia Bay, Elba, Italy13 were sampled between June 2014 and September 2019; individual sampling months and years are indicated in the sections below and/or in the figures and tables. In May 2017, a P. oceanica meadow at the island of Pianosa, Italy was also sampled. All of the samples were obtained via SCUBA diving.Complete plants of P. oceanica were carefully separated from the meadow by hand and stored in seawater-filled containers until arrival at the shore-based laboratory. Sediment for use in the laboratory-based aquaria was scooped into containers from nearby sandy patches. Seawater was pumped through a hose (placed at about 0.5 m above the P. oceanica meadow) into several 50 l barrels onboard the boat and was later used in the laboratory for the aquarium and the incubation experiments.The sediment within the seagrass meadow was sampled with stainless steel core tubes (length, 50 cm), which were drilled into the sediment by divers, and the cores were briefly stored at 22 °C (ambient temperature, September 2019) in a seawater-filled barrel until further processing at the shore-based laboratory.Porewater nutrient samples were obtained using stainless steel lances41 at intervals of around 10 cm. Water column nutrient samples were obtained from above the seagrass meadow at the start or end of sampling. Nutrient samples were collected in 15 ml or 50 ml centrifuge tubes and were stored in a cooler box until further processing.Nutrient measurementsWater column nutrients were measured during several sampling campaigns as indicated in Extended Data Table 1a. Ammonium (NH4+) concentrations were measured fluorometrically42 in the nearby shore-based laboratory, and the remaining water was frozen (−20 °C) for later analyses of nitrate (NO3−), nitrite (NO2−), phosphate (PO43−) and silicate (SiO44−) using an autoanalyser (QuAAtro, Seal Analytical). Porewater samples were obtained in June 2019 and were processed the same as the water column nutrient samples with the exception that ammonium was not measured on site but at the home laboratory at the same time as the other nutrients. Dissolved inorganic nitrogen (ammonium plus NOx−) concentrations in the porewater were averaged for the upper 20 cm (Extended Data Table 1b).Net primary production measurements using the EC methodNet carbon dioxide (CO2) fluxes were calculated on the basis of oxygen (O2) fluxes determined using the aquatic eddy covariance (EC) method. In this non-invasive approach, turbulence-induced transport is resolved using high-frequency current meters combined with fast O2 microsensors. Under the assumption of stationarity, the instantaneous turbulent flux contributions are calculated by correlating vertical current fluctuations to oxygen fluctuations. Our EC system was equipped with an acoustic Doppler velocimeter (ADV, Nortek) and ultra-fast responding optode microsensors with a tip diameter of 430 µm (t90  More

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    Changes in microbial community and enzyme activity in soil under continuous pepper cropping in response to Trichoderma hamatum MHT1134 application

    Field control effect of strain MHT1134 on Fusarium wilt of pepperBefore the investigation of strain MHT1134 control effect, pepper plants with the same wilt symptoms were collected from CC9, TR1 and TR2 fields. The same wilt symptom is that the lower leaves of the plant turn yellow or fall off, and the whole seedling plant wilt and die in the later stage. The pepper root neck can be seen with obvious water-stained brown disease spots. When the root and stem are cut open, the vascular bundle turns brown and has a trend of upward stretching (Fig. 1A–C). We isolated a strain in the root, which colony color is purple (Fig. 1E,F), On the sixth day after inoculating healthy pepper with the spore suspension, the plants showed lower leaf shedding and plant wilting (Fig. 1D). And the pathogen was isolated in the root with the same colony characteristics and micromorphology. The main classification features are as follows: the conidiophores are colorless, with bottle-shaped spore-producing cells at the top (Fig. 1G). There are two kinds of conidias. The small conidia are monocytic, oval or kidney shaped, colorless and are 5–12 × 2–3.5 μm in size. Large conidia are multicellular, sickle-shaped, slightly curved, with slightly pointed cells at both ends, colorless and are 19.6–39.4 × 3.5–5.0 μm in size (Fig. 1H). The morphological characteristics of the strain were consistent with Fusarium oxysporum. The strain DNA was extracted and ITS sequence was amplified by PCR to obtain a DNA fragment with a length of about 500 bp. The sequencing results were compared with the gene sequences in Genbank, and the highest homology was found in Fusarium, and the sequence homology with Fusarium oxysporum reached 100%. The pathogen of pepper wilt was Fusarium oxysporum by means of morphological and molecular identification.Figure 1Typical symptoms and identification of pathogen strains of pepper Fusarium wilt in experimental sites. (A) At the late stage of Fusarium wilt, the whole plant withered and died; (B) the lateral root and taproot of the pepper turn brown and rot; (C) discoloration of vascular bundle in pepper stem after cutting; (D) after the isolated F. oxysporum was inoculated on the pepper, which showed the initial symptoms of wilt disease; (E) positive characteristics of F. oxysporum colony; (F) negative characteristics of colony; (G) sporulation peduncle in bottle shape; (H) large and small conidia.Full size imageCompared with CC9 treatment without biocontrol fungi MHT1134, the disease rate and disease index of pepper Fusarium wilt in TR1 and TR2 treatment were decreased. In TR1, the disease rate and disease index of pepper wilt decreased by 8.44% and 3.76%, respectively. In TR2, the disease rate and disease index of pepper wilt decreased by 57.69% and 63.02%, respectively. However, in the TR2 plots over 2018 and 2019, the disease rate and disease index decreased to 7.13% and 3.03%, which were 64.26% and 70.20%, respectively, less than in the CC9 plots. The control effect of MHT11341 on pepper wilt was 63.03% and 70.21% after one and two years of continuous cropping field, respectively (Table 1). The results indicated that the continuous application of a biocontrol strain further consolidated and improved the control effect.Table 1 Control effects of strain MHT1134 on Fusarium wilt in continuous pepper cropping fields.Full size tableEffects of strain MHT1134 on the physical and chemical properties of pepper rhizosphere soilSoil samples from different planting years showed differences in their physical and chemical properties. In particular, the contents of available phosphorus, available potassium and organic matter were significantly different between the soil planted for the first year and the soil continuously planted for 9 years (available phosphorus: F = 4.38 p = 0.03; available potassium: F = 2.94 p = 0.009; organic matter: F = 5.45 p = 0.02). With the increase in planting years, the organic matter and alkali-hydrolysable nitrogen contents in the soil showed decreasing trends. The organic matter content in the CC9 soil samples was 23.64% less than in the CC1 soil samples, and the alkali-hydrolysable nitrogen content was 45.2% less. The available phosphorus and available potassium levels did not show regular change trends, but the available potassium content in the CC9 soil was lower than in the CC1 soil.Compared with the CC9 soil samples, the alkali-hydrolysed nitrogen, organic matter, available phosphorus and available potassium contents in TR1 soil samples increased by 46.82%, 6.26%, 5.09% and 47.06%, respectively. The available potassium content increased most obviously, followed by alkali-hydrolysable nitrogen. The alkali-hydrolysable nitrogen, organic matter and available phosphorus contents decreased slightly in TR2, but were still higher than those in the CC9 soil samples. In addition, the available potassium content continued to increase by 20% after the application of biocontrol bacterium MHT1134 in the second year (Table 2).Table 2 Effects of MHT1134 on physical and chemical properties of the pepper rhizosphere soil.Full size tableEffects of strain MHT1134 on enzymatic activities in pepper rhizosphere soilBy comparing the activities of six kinds of enzymes in the five groups of soil samples, we found that all the activities, except for that of acid phosphatase, in the CC9 soil were lower than those in the CC1 soil. In TR1 and TR2, the activities of the six enzymes in the soil increased. The urease, dehydrogenase, acid phosphatase, catalase, invertase and acid protease activities increased by 9.04%, 4.42%, 29.02%, 9.35%, 17.83% and 6.83% in TR1, respectively, and by 18.60%, 20.26%, 22.86%, 18.87%, 16.59% and 14.30% in TR2, respectively (Fig. 2A–F). The results indicated that MHT1134 applications could improve the enzyme activities in the soil to different degrees. Moreover, the urease, dehydrogenase, catalase and acid protease activities in soil significantly increased after the continuous application of MHT1134.Figure 2Differences in the enzyme activities in the continuously cropped pepper rhizosphere soil after the application of strain MHT1134. Activity levels of (A) urease; (B) dehydrogenase; (C) acid phosphatase; (D) catalase; (E) invertase; and (F) acid protease. CC1, CC5 and CC9, represent the plots where pepper had been continuously planted for 1, 5 and 9 years, respectively, and TR1 and TR2 represent CC9 plots in which the MHT1134 biocontrol fermentation broth had been applied 1 and 2 years in advance, respectively.Full size imageMicrobial diversity and richnessThe sample dilution curve tended to be flat, and the fungal and bacterial diversity index table (Table 3) shows that the library coverage levels were greater than 99% and 98%, respectively. Together, they indicate that the OTU coverage of the soil samples is basically saturated; therefore, the OTUs reflect the species and structures of the fungal and bacterial communities in the samples. High-throughput sequencing results showed that 765,747 16S rRNA sequences and 1,012,237 ITS sequences were obtained from 15 samples of pepper rhizosphere soil subjected to five treatments. After data quality control, there were 35,362–72,498 bacterial 16S rRNA sequences and 54,007–74,562 fungal ITS sequences. In addition, using the 97% standard, the bacterial and fungal OTU numbers were 17,444–47,775 and 50,876–71,236, respectively.Table 3 Alpha-diversity indexes of fungi and bacteria in different continuous pepper cropping soils.Full size tableAlpha-diversity analysis of fungi and bacteriaThe changes in fungal and bacteria diversity are shown in Table 3. According to the Shannon index analysis, the species richness of fungi in CC1 was the highest (2.88). As the planting years increased, the Shannon index decreased gradually (2.71 in CC5 and 2.69 in CC9). Although ACE and Chao indexes, representing the species abundance of the community, did not show obvious increasing trends, in CC9, the values of the two indexes were significantly higher than in CC1, which indicated that as the planting years increased, the diversity of fungi in the pepper soil decreased, while the species abundance increased. As shown in Table 3, in TR1, the Simpson index, representing species dominance, and the Sobs index, representing species richness, increased significantly, and the Shannon index also increased. In TR2, the Shannon index increased significantly, while the values of other indexes decreased slightly. We hypothesised that after the first year of application, the strain MHT1134 colonised in large numbers, resulting in it being the dominant community species. After continuous application, the soil ecology had adjusted, and the diversity of soil fungi continued to increase. In general, the application of the biocontrol fungal MHT1134 increased the diversity of fungi in the pepper rhizosphere soil and decreased the dominance of some species.The changes in bacterial diversity and abundance in the pepper rhizosphere soil after different periods of continuous cropping are shown by the decreases in the Shannon and Sobs indexes decreased as the planting years increased, indicating that bacterial diversity and bacterial community richness decreased. Although ACE and Chao indexes representing the species abundance of the community did not show regular decreasing trends, in CC9, the values of the two indexes were significantly lower than in CC1, indicating that as the planting years increased, the diversity and richness of bacteria in the pepper soil decreased. Strain MHT1134 had no significant effect on the alpha-diversity index of soil bacteria in TR1, but Simpson, ACE and Chao indexes increased in TR2.Effects of MHT1134 on the microbial community structure in pepper rhizosphere soilAll the bacteria were classified into 352 genera and 23 phyla according to their 16S rRNA sequences, and all the fungi were classified into 6 phyla and 194 genera according to their ITS sequences. The top five phyla in terms of bacterial abundance were Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes and Nitrospirae. The top six phyla in terms of fungal abundance were Ascomycota, Zygomycota, Basidiomycota, Glomeromycota, Chytridiomycota and Rozellomycota.Effects of MHT1134 on fungal community structure in pepper rhizosphere soilThe effects of the biocontrol treatment on fungal phyla are shown in Fig. 3A. After treatment with MHT1134, the relative abundance of Ascomycota decreased significantly from 77.9 to 70.99%. The abundance of Basidiomycota increased significantly after the treatment, whereas it decreased with the continuous cropping time before the MHT1134 application. However, Zygomycota increased in abundance with the continuous cropping time. The abundance of strain MHT1134 increased significantly and then decreased by 1 year after treatment.Figure 3Fungal clustering accumulation map in pepper rhizosphere soil at the phylum (A) and genus (B) levels. CC1, CC5 and CC9, represent the plots where pepper had been continuously planted for 1, 5 and 9 years, respectively, and TR1 and TR2 represent CC9 plots in which the MHT1134 biocontrol fermentation broth had been applied 1 and 2 years in advance, respectively.Full size imageBy analysing the relative abundance of fungi of different genera in the soil, it was found that the fungi of several genera showed similar change trends in different soil treatments. The relative abundances of Fusarium, Gibberella and the alkali-resistant fungus Pseudallescheria in the soil increased along with continuous cultivation years (CC1  TR2). In addition, the trend was found for Trichoderma, Chaetomium and Mortierella, which declined as the planting years increased, but their relative abundance levels significantly increased in TR1 and significantly increased again in TR2 (Fig. 3B).Using Fusarium as the control, we analysed the variation trends of microorganisms in CC9, TR1 and TR2 soil samples. As shown in Fig. 4, the levels of three genera were positively correlated with the Fusarium change trend, Gibellulopsis, Giberella and Pseudallescheria, while three genera, Trichoderma, Chaetomium and Mortierella, were negatively correlated with Fusarium. Thus, the abundance levels of fungi in Gibellulopsis, Gibberella and Pseudallescheria were reduced after the MHT1134 application. Some species of Gibellulopsis are the pathogenic fungi that cause Verticillium wilt, and some species of Gibberella are the pathogenic fungi that cause gibberellic diseases. The abundance levels of Trichoderma, Chaetomium and Mortierella significantly increased after the application of strain MHT1134.Figure 4The relative abundances of the first 15 genera after the MHT1134 application. *0.01  CC5  > CC9), whereas the abundance of Actinobacteria in the soil increased significantly after the application of MHT1134 fermentation broth (CC9  More

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