The Actinobacteria phylum comprises Gram-positive and high guanine+cytosine (G+C) content bacterial taxa. Actinobacterial species are the main producers of active microbial natural products, are ubiquitously distributed among environments, and have been widely used in industrial and agricultural applications (Bèrdy, 2005; Qin et al., 2016). Recently, endophytic actinobacteria have particularly attracted significant attention, with increasing documentation of isolates from a wide range of plants, including various crop plants like wheat, rice, banana, apple, and tea plants, in addition to medicinal plants such as Artemisia annua, Tripterygium wilfordii Hook, Glycyrrhiza inflata, and Jatropha curcas (Conn and Franco, 2003; Tian et al., 2007; Qin et al., 2011, 2015b; Li et al., 2012; Miao et al., 2015; Álvarez-Pérez et al., 2017; Wei et al., 2018; Zhao et al., 2018). Streptomyces, Micromonospora, Micrococcus, Pseudonocardia, and Microbacterium are the most predominant endophytic actinobacterial genera cultivated, and many novel species from these genera have been identified in diverse host plants (Kaewkla and Franco, 2013; Golinska et al., 2015; Dinesh et al., 2017). Further, recent cultivation-independent analyses using 16S rRNA gene-based methods like denaturing gradient gel electrophoresis (DGGE), clone library analyses, and high-throughput sequencing have also revealed abundant endophytic actinobacterial communities within plants, including novel and uncultured taxa (Qin et al., 2012, 2018; Purushotham et al., 2018). For example, the high-throughput sequencing (HTS) of 16S rRNA genes from banana shoot tips yielded the detection of more than 50 known actinobacterial genera, which included many genera rarely identified as endophytes such as Actinomycetospora, Actinoallomurus, Kibdelosporangium, and Kitasatospora (Du et al., 2018).

In addition to the high species diversity of endophytic actinomycetes, increasing attention has been directed toward their ability to produce abundant bioactive metabolites. Several endophytic actinobacteria from medicinal plants produce novel bioactive compounds that have favorable antimicrobial and antitumor activities, besides showing a good potential for pharmaceutical development and biotechnological applications (Qin et al., 2011; Golinska et al., 2015; Dinesh et al., 2017). For example, the recently identified hamuramicins A and B obtained from the endophytic actinomycete Allostreptomyces sp. K12-0794 exhibited both antimicrobial activity and human cell line toxicity (Suga et al., 2018). In addition to their therapeutic uses, endophytic actinomycetes have a broad potential and value in agricultural application. The endophytes can promote plant growth via direct and indirect mechanisms, including phosphorus solubilization, production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophores, induction of pathogen resistance, and aiding resistance to abiotic stresses like those imposed by high salinity and drought (Conn et al., 2008; Qin et al., 2014, 2017; Trujillo et al., 2015; Zeng et al., 2018).

When compared with the well-studied crop plants and endophytic actinobacteria within common environments, little is known about naturally occurring plant-associated actinobacteria within harsh habitats like hot and cold deserts, saline and acidic soils, and marine habitats. Coastal salt marshes are one of the most biologically productive habitats on Earth, and are one such example of a unique and harsh environment (Pennings and Bertness, 2001). Recent studies from coastal sediments and mangrove forests have revealed high levels of actinobacterial diversity (Hong et al., 2009; Hamedi et al., 2013; Jiang et al., 2018). Surprisingly, however, there is still little information related to the isolation, diversity and their biological activity of endophytic actinobacteria within coastal salt marsh ecosystems, despite the fact that such environments are likely to contain unique and phylogenetically diverse endophytes. Our recent analyses have indicated that there were abundant Actinobacteria and novel taxa in the rhizosphere soils associated with halophytes collected from coastal salt marshes within the Jiangsu Province of China (Wang et al., 2015; Bai et al., 2016; Ding et al., 2018). In particular, the phylum Actinobacteria comprised 2.8%–43.0% of rhizosphere bacterial communities, as evidenced by HTS on the Illumina MiSeq platform (Gong et al., 2018). Importantly, rhizosphere soils can be a source of endophytes (Long et al., 2010). There are abundant halophyte resources in the Jiangsu coastal region. Consequently, we speculated that plants within the Jiangsu coastal zone also contained abundant endophytic actinobacteria.

Thus, the aims of this study were to (i) survey the diversity of culturable endophytic actinobacteria from plants within coastal salt marshes in the Jiangsu province, (ii) analyze root endophytic actinobacterial communities from representative plants using 16S rRNA gene-based HTS technique, (iii) evaluate the anti-phytopathogenic fungal and fibrinolytic activity and analyze secondary metabolite biosynthetic gene profiles of isolates. We expect to provide a preliminary basis for the further discovery of biological active metabolites from coastal salt marsh plants endophytic actinobacteria and their further utilization for biocontrol of crops and thrombosis treatment for human beings.

Diverse endophytic actinomycetes have been found in many crops, model plants (e.g., Arabidopsis thaliana), and other plants from numerous typical environments (Conn and Franco, 2003; Qin et al., 2009; Golinska et al., 2015; Dinesh et al., 2017). The method used to isolate endophytic actinobacteria is an important factor that affects the acquisition and diversity of pure cultures. For example, plant surface sterilization, use of different selective media types, and plant treatment methods can all influence cultivation-dependent outcomes. In this study, we used a previously described five-step sterile sample processing protocol, longer incubation times, and various selective media to obtain pure endophytic actinobacterial cultures (Qin et al., 2009). Starch-casein agar and sodium propionate agar that were supplemented with 3% (w/v) NaCl yielded the greatest diversity of endophytic actinobacterial isolates, which comprised 10 different genera, including those that have rarely been reported as endophytes such as Glycomyces, Modestobacter, and Pseudokineococcus (Table 1). Starch-casein agar has previously been suggested to be effective in the isolation of Actinobacteria from saline environments (Abbas, 2006). Similarly, sodium propionate medium is seen to be effective for isolating endophytic actinobacteria from various plant hosts (Qin et al., 2009; Li et al., 2012). Our recent investigation also found that these two types of media supplemented with 3% NaCl were suitable for isolating rare actinomycetes from rhizosphere soils associated with coastal halophytes (Gong et al., 2018). CMC medium has also been suggested as suitable for isolating rare endophytic actinobacteria (Kaewkla and Franco, 2013). Although only eight strains were obtained in this study using the CMC medium, they were distributed among five genera, which included Dietzia, Glycomyces, and Kocuria, and are all rarely reported as endophytes. These results suggest that further investigations of endophytic actinobacteria should strongly consider the in-depth exploration of the cultivation methods that are to be employed.

Marine environments host a diversity of rare actinobacteria (Pennings and Bertness, 2001; Hassan et al., 2017). Because coastal salt marshes are characterized by both terrestrial and marine characteristics, abundant and diverse endophytic actinobacterial resources are likely present in these environments. A total of 23 different actinobacterial genera were identified in the present study, including many novel species, suggesting a high diversity of actinobacteria within the native plants of the salt marsh investigated here. To our knowledge, such high diversity of culturable endophytic actinobacteria is yet to be demonstrated from such habitats. Tian and Zhang (2017) analyzed the endophytic and rhizosphere bacterial diversity of the Chinese coastal halophyte Messerschmidia sibirica using high-throughput sequencing method, and they revealed that the relative abundance of the phylum Actinobacteria accounts for more than 6.2% across all leaf, stem, and root tissues. Yamamoto et al. (2018) also found that Actinobacteria in the coastal halophyte Glaux maritima root tissues collected from the eastern part of Japan was extremely high using the Illumina MiSeq sequencing technique. However, none of these studies reported pure culture isolation of endophytic actinobacteria and their diversity. Recently, Jiang et al. (2018) obtained diverse endophytic actinobacterial strains (101 isolates from 15 families and 28 genera) from five mangrove plants collected from the Guangxi Zhuang Autonomous Region of China. Taken together with the above results, our study confirms that coastal plants contain abundant and diverse endophytic actinomycete resources. Interestingly, in this study, most of the culturable endophytic actinobacterial genera, such as Streptomyces, Saccharopolyspora, Pseudonocardia and Micromonospora, were also successfully cultured from their corresponding host plants rhizosphere soils previously (Gong et al., 2018). This result supports the previous conclusion that rhizosphere microorganisms are the main origin of endophytic bacteria (Mora-Ruiz Mdel et al., 2016).

A total of 72 known actinobacterial genera were detected from seven root samples using a combination of cultivation-dependent and -independent techniques (Figure 6). To our knowledge, this is the first investigation of coastal plant-associated endophytic actinobacterial diversity using both cultivation- and culture-independent methods. The majority of the isolates were obtained from plant roots (Table 2), which is consistent with the results of other studies, indicating that endophytic actinomycetes primarily colonized root tissues (Qin et al., 2009; Kaewkla and Franco, 2013; Tian and Zhang, 2017). The community composition of actinobacterial taxa at the phylum, family, and genus levels varied among different root samples (Figure 4, 5). Recent investigations of plant microbiomes have suggested that plant genotypes significantly affect the composition of symbiotic root microbiota (Wintermans et al., 2016; Li et al., 2018). Therefore, the root-associated actinobacterial compositional differences observed here could be explained by differences in host plant species. At the genus level of actinomycetes, differences were observed in the number of genera detected by cultivation-dependent and -independent methods (Figure 6). Specifically, high-throughput 16S rRNA gene sequencing resulted in the detection of 63 known genera, including many genera that have rarely been reported as endophytes such as Herbiconiux, Actinoplanes, Ilumatobacter, and Actinophytocola (Pérez et al., 2016). Furthermore, HTS within root samples resulted in the detection of much higher levels of taxa, which could not be classified into known taxonomic groups. These results indicated that coastal salt marsh plants harbored a high level of rare endophytic actinobacteria and novel taxa. Only 11 of the 63 genera detected by 16S rRNA gene sequencing were also detected by cultivation, while nine cultivated genera were not detected by sequence analysis (Figure 6). However, some genera that are widely distributed among different environments and that display diverse ecological functions associated with plants, including Arthrobacter (Prum et al., 2018; Qin et al., 2018), were not detected by cultivation. This result highlights the impetus to continue developing cultivation methods and strategies to culture greater diversity in future investigations. These disparities in the detected taxa could potentially be explained by the variable efficiency of genomic DNA extraction among species, taxonomic biases of primers used for PCR amplification, and the recalcitrance of certain taxa to cultivation under laboratory conditions. These explanations are consistent with the previous reports (Qin et al., 2012; Chen et al., 2016; Eevers et al., 2016). Therefore, it is critical to use a combined cultivation-dependent and -independent approach to accurately evaluate the composition of endophytic actinobacterial communities.

Streptomyces are the most prevalent genus observed among endophytic actinobacteria (Conn and Franco, 2003; Kaewkla and Franco, 2013; Qin et al., 2015b; Dinesh et al., 2017; Passari et al., 2017). In addition, many rare endophytic actinomycetes have also been recently reported (Qin et al., 2011; Dinesh et al., 2017). Our observation that Streptomyces were the most dominant species within coastal salt marsh plants is consistent with these previous studies. Endophytic Streptomyces spp. are well-known for their capacity to produce many types of bioactive metabolites, and several species have been reported as beneficial toward plant development and health (Mitra et al., 2008; Vurukonda et al., 2018). The highest proportions of secondary metabolite biosynthetic genes observed in this study were in Streptomyces spp. strains (63.2%), with at least 12 strains harboring PKS, NRPS, and/or Halo genes (Table 2). This indicates that the strains screened possess diverse secondary metabolite gene clusters, as evidenced by the presence of these genes. PCR-based screening of biosynthetic genes is a useful and efficient method to conduct bioprospecting of certain type of natural products producing actinobacteria. For example, based on the FADH₂-dependent halogenase genes screening from the mangrove-derived actinomycetes, Li et al. (2013) obtained a new enduracidin producer, Streptomyces atrovirens MGR140, which was further identified and confirmed by gene disruption and HPLC analysis. The presence of halo genes here is particular interesting, because more than 4,700 halogenated compounds have been reported, which are important sources for new drugs, due to their high diversity in structure and activity (Liao et al., 2016). Moreover, Streptomyces strains exhibited significant antagonistic activities toward fungal pathogens, with 52.6% strains displaying antifungal activities against at least one of the pathogentic fungi that were tested. One isolate, KLBMP 1357, exhibited broad-spectrum antifungal activity and contained both the PKS II and NRPS genes (Supplementary Table S1), meriting further research of its bioactive secondary metabolites. Nevertheless, antifungal activities and the presence of certain secondary metabolite functional genes did not appear to be directly correlated in this study. For example, functional secondary metabolite genes were not detected in the Pseudonocardia strain KLBMP 1272, despite the fact that it exhibited broad-spectrum antifungal activity. This result could potentially be due to one of two reasons. First, the fermentation medium used to cultivate the strain may not promote the production of antifungal secondary metabolites. Second, the degenerate primer pairs that were used to amplify the functional genes may not be suitable for amplification of these genes within certain strains. Alternatively, the strains considered here may actually lack the biosynthetic genes tested, but possess other secondary metabolite biosynthetic genes (Schneemann et al., 2010). Moreover, we have only carried out antifungal screening, and the results of gene screening may be more relevant to antibacterial and anti-tumor activity. The results reported here are consistent with those from a recent screening of culturable actinomycetes from hot springs, lichens, and tea plants (Liu et al., 2016, 2017, Wei et al., 2018). Considering that many actinomycetes were isolated from root tissues, and showed strong antagonistic activity against plant pathogenic fungi, we may speculate that these endophytic actinomycetes have potential for biological control and potential contribution to their ecological adaptation within roots.

We also observed that some strains of Pseudonocardia and Saccharopolyspora that are rarely described as endophytes exhibited good antifungal activity, including strains KLBMP 1272, KLBMP 1413, and KLBMP 1287. Pseudonocardia have been previously observed as dominant endophytes within plants; in addition, some new endophytic species have been discovered recently (Qin et al., 2009; Li et al., 2012; Zhao et al., 2012). In contrast, endophytic Saccharopolyspora spp. isolates have been rarely reported (Qin et al., 2008; Li et al., 2009). Recently, endophytic Pseudonocardia isolates were identified that were associated with mangrove trees and sea cucumbers. The isolates displayed antimicrobial and cytotoxic activities, and subsequently yielded novel bioactive compounds (Mangamuri et al., 2015; Ye et al., 2016). Microorganism is an important source of thrombolytic agents for thrombosis treatment, and lots of fibrinolytic enzymes were successively discovered from different microorganisms, including marine actinomycetes (Verma et al., 2018). However, non-Streptomyces rare actinobacteria and endophytes origin fibrinolytic enzymes were rarely reported (Lu et al., 2010; Mohanasrinivasan et al., 2017). Thus, we carried out the fibrinolytic activity screening for the endophytic actinobacterial isolates. Interestingly, we found that more than 40% of the strains secreted extracellular proteases that exhibited fibrinolytic activity in this study. It is noteworthy that some new species (KLBMP 1269, KLBMP 1483, KLBMP 1305, and KLBMP 1278) displayed strong and moderate fibrinolytic activity (Supplementary Table S2), meriting further purification and characterization of their fibrinolytic enzymes and exploring the thrombolytic effect. In addition, to our knowledge, this is the first time that strains of endophytic actinobacteria and the rare actinobacteria genera Glycomyces, Saccharopolyspora, Amycolatopsis, Pseudonocardia, and Tamaricihabitans have been reported to have fibrinolytic activity. Taken together, these results indicate the coastal marsh halophytes-associated endophytic actinobacterial yet-to-be discovered bioactive metabolites and functions warrant further exploration.

Endophytic actinobacterial community structure was analyzed among coastal salt marsh halophyte plants within the Jiangsu Province of Eastern China using both culture-dependent and -independent methods. Coastal salt marsh plants harbored abundant and diverse communities of endophytic actinobacteria, including many rare actinobacteria and novel taxa. These results also indicate that halophyte plants have an important role in shaping the community composition of endophytic microbiota within roots. Taken together, the results of this study suggest that coastal salt marsh plants-associated endophytic actinobacteria represent a promising, yet underexplored, new resource for discovering bioactive natural products with potential as biocontrol agents and for fibrinolytic enzyme production.

CZ, KX, and SQ designed the experiments. PC, XJ, YX, and SQ performed the experiments. PC, CZ, YX, and SQ analyzed the data and prepared the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.