Seed phytochemicals shape the community structures of cultivable actinobacteria‐inhabiting plant interiors of Thai pigmented rice

Abstract We examined abundance, bioactivity, and endophytism of cultivable actinobacteria isolated from plant interiors of two Thai pigmented rice cultivars: Hom Nin (HN) rice and Luem Pua (LP) glutinous rice. Both rice cultivars housed the same amount of endophytic actinobacteria (33 isolates each). Microbispora (76%) and Streptomyces (73%) were the predominant endophytic actinobacteria of LP glutinous rice and HN rice, respectively. Sphaerisporangium (9%) was found only in LP glutinous rice. Twelve percent of endophytic actinobacteria was the possibility of discovering novel species from both rice cultivars. Most endophytic actinobacteria exhibited plant growth‐promoting potentials, including antimicrobial activity against test bacteria and phytopathogenic fungi, solubilization of phosphate, and production of biostimulants (i.e., ammonia, indole‐3‐acetic acid, and siderophore) and biocatalysts (i.e., amylase, cellulase, chitinase, lipase, and protease). Our findings revealed that seed phytochemicals of pigmented rice (e.g., anthocyanin, γ‐oryzanol, phytate, antioxidants, and content of amylose) were effectors, shaping the community structures and biofunctions of endophytic actinobacteria. We conclude that pigmented rice is yet a challenging source for discovery of bioactive and novel actinobacteria. This study also provides new insights into the plant‐endophyte interactions by which seed phytochemicals act as a primary checkpoint in the natural selection for establishing unique plant endophytomes.

Schmid, van Tuinen, & Berg, 2009). Phytochemicals consisted in root exudates act as the sorting tools, being either attractants or repellents toward the rhizosphere microbial community. However, roles of in planta phytochemicals on the selection, subsistence, and distribution of endophytic microbes are rarely known.
The endophytes dwelling in such chemically bioactive niche within plant materials would indicate the microbial evolution through genetically resistant mechanisms, for example, sharing biosynthetic genes for bioactive metabolites or harboring cellular mechanisms and genes to resist foreign bioactive substances. There are many studies that have disclosed the bioactive functions of endophytes that act as the secondary defensive tools in plant immunology for optimizing plant growth and protecting plants from phytopathogens and harmful xenobiotic agents like herbicides (Golinska et al., 2015;Kampapongsa & Kaewkla, 2016;Qin et al., 2011;Rangjaroen et al., 2017;Strobel & Daisy, 2003;Tétard-Jones & Edwards, 2016;Tian et al., 2004). Out of such plant growth-promoting benefits, various metabolites from endophytes also reveal pharmaceutical and medical potentials, for example, antimicrobial, antiviral, and anticancer/antitumor activities (Golinska et al., 2015;Qin et al., 2011;Strobel & Daisy, 2003). Hitherto, endophytes are yet a promising source for discovering novel and effective medicines. Although diverse endophytes can synthesize bioactive compounds, we still keep focusing on actinobacteria (Golinska et al., 2015;Kampapongsa & Kaewkla, 2016;Qin et al., 2011;Tian et al., 2004) that are notable producers of various commercial metabolites and promising reservoirs for novel drug discovery.
For centuries, rice (Oryza spp.) is an important crop cultivated worldwide for human nourishment (Hardoim et al., 2011;Mano & Morisaki, 2008;Rangjaroen et al., 2017;Tian et al., 2004). The trend of pigmented rice consumption has been increasing because of its rich nutritive values, especially for antioxidants and vitamins, when compared to the other white rice (Goufo & Trindade, 2014;Sompong, Siebenhandl-Ehn, Linsberger-Martin, & Berghofer, 2011). In this study, we aim to isolate cultivable actinobacteria that live in plant interiors of

| Endophytic actinobacteria of Thai pigmented rice and their bioactivities
An equal number (33 isolates) of endophytic actinobacteria was isolated from each Thai pigmented rice cultivar (Table 1). All obtained endophytic actinobacteria were only derived from rice seedlings grown in agricultural soil. Based on morphological characteristics, all endophytic actinobacteria were categorized into three groups and identified with the phylogenetic analysis of their 16S rRNA gene sequences into three genera; Microbispora, Sphaerisporangium, and Streptomyces (  (Kampapongsa & Kaewkla, 2016;Tian et al., 2004). The chance to discover novel species of endophytic actinobacteria from any pigmented rice cultivar was estimated at 12.5%. With such level, we still believe that pigmented rice would be a promising source offering unique ecological niches for exploring novel actinobacteria.
Every endophytic actinobacterium obtained was evaluated for their bioactivities in promoting plant growth (Figure 2b- Morphological characterization was carried out after growing endophytic actinobacteria on HT agar medium for 14 days. b The 16S rRNA gene sequences of the group representatives were deposited in GenBank and used for phylogenetic analysis (Figure 1).
c The % identity refers to the percentage of the gene sequence similarity of the group representatives compared to their closest phylogenetic species.
bacteria tested ( Figure 2b). Twenty-four percent of endophytic actinobacteria derived from LP glutinous rice showed anti-grampositive and antifungal activities, which was higher than those (0%-9%) of HN rice. While, slightly higher number (48%-73%) of endophytic actinobacteria isolated from HN rice could produce ammonia, IAA, and siderophore, compared to those (42%-70%) of LP glutinous rice (Figure 2c). Only a few percentage (6%) of endophytic actinobacteria could solubilize phosphate, and all of them derived from LP glutinous rice. Interestingly, many isolates of endophytic actinobacteria were capable of producing biocatalysts ( Figure 2d).
The higher number (24-55%) of endophytic actinobacteria derived from HN rice could produce biocatalysts, compared to those (6-52%) of LP glutinous rice. These findings did not surprise us, as most endophytic actinobacteria are the best-known sources of bioactive metabolites (Golinska et al., 2015;Kampapongsa & Kaewkla, 2016;Qin et al., 2011;Tian et al., 2004). However, further studies for the structural elucidation and additional bioactivity screening of bioactive metabolites derived from such emerging endophytic actinobacteria would enhance the possibility of discovering novel metabolites for pharmaceutical and agricultural applications.

| Endophytic colonization and role of phytochemicals in endophytism
The highest number (24-26 isolates) of endophytic actinobacteria inhabited the seedlings' root tissues of both rice cultivars (Table 1).
Although we found abundant endophytic actinobacteria in rice roots, another study reports more found in rice leaf compartments (Kampapongsa & Kaewkla, 2016). It might be an influence of the difference in growth and developmental phases of rice, as we used 15-day-old seedlings, while another (Kampapongsa & Kaewkla, 2016) used mature rice plants (7-week-old planting in paddy fields).
In fact, plant growth during the seedling stage is highly dependent upon the root functions rather than the photosynthesis. Hence, roots of rice seedlings that have direct contact with soil, and would be rich in nutrients and minerals, were optimal and attractive for the endophytic colonization of actinobacteria (Hardoim et al., 2011;Hartmann et al., 2009). Some studies reveal that rice seeds house various bacterial endophytes, where they prolong their life in plant interiors during growth and development of rice and pass through the next generation of rice seeds (Hardoim, Hardoim, van Overbeek, F I G U R E 1 Maximum likelihood tree constructed with nearly full-length 16S rRNA gene sequences of the representative rice endophytic actinobacteria (see Tables 1 and 2 Table 3, while the seedling phytochemicals of both rice cultivars were analyzed and reported in this study (Table S1). The phytochemical profiles of rice seedlings were similar between both rice cultivars, while some of their seed phytochemicals were significantly different. We discovered that the difference in seed phytochemicals would be a pioneer set of effectors, inducing neighbor microbes to set up their life within plant interiors.
Although seeds of pigmented rice contained rich contents of bioactive phytochemicals, for example, anthocyanin, γ-oryzanol, phytate, and antioxidants (Goufo & Trindade, 2014;Sompong et al., 2011), we found that such phytochemical contents were highest in seeds of LP glutinous rice. We also observed some links between the richness of such bioactive phytochemicals and biofunctions of endophytic actinobacteria. For example, more isolates of endophytic actinobacteria derived from LP glutinous rice showed antimicrobial activities than those of HN rice, supporting the hypothesis that chemically active plant materials would offer a unique ecological niche for the discovery of bioactive endophytes. Another example was the richness of phytate (4800 mg·kg −1 ) in LP glutinous rice seeds. Phytate is a phosphate (P)-storage form of plants, and that is the reason why we found P-solubilizing endophytic actinobacteria only in this rice cultivar. Moreover, the low or negligible content of amylose is the bestknown characteristic of glutinous rice (Olsen & Purugganan, 2002;Sompong et al., 2011). With this notable, we, therefore, found fewer amylase-producing endophytic actinobacteria from LP glutinous rice F I G U R E 2 Generic diversity of endophytic actinobacteria and their plant growth-promoting potentials. The generic abundance (a) was determined using morphological and phylogenetic characteristics of endophytic actinobacteria (see Table 2). The plant growth-promoting potentials comprised of antimicrobial activity (b), soil nutrient and mineral conversion and biosynthesis of plant biostimulants (c), and production of some biocatalysts (d). White and black bars are results derived from Leum Pua glutinous rice and Hom Nin rice, respectively. The number in parenthesis refers to the isolate number of endophytic actinobacteria than those of HN rice (Figure 2d). Based on our findings, rice plants employed variable phytochemicals during seed germination as the natural selection tools for attracting and shaping the community structures of their pioneer endophytic actinobacteria.
We conclude that pigmented rice is yet a promising source for discovery of bioactive and novel actinobacteria, which are reservoirs of diverse biotechnological metabolites. This study also provides new insights into the plant-endophyte interactions by which plant seed phytochemicals would act as a primary checkpoint in the natural selection process for establishing plant endophytomes.

| Source and preparation of plant materials
HN rice and LP glutinous rice are local rice breeds cultivated routinely for commercial purpose at Khao Kho district, Phetchabun province, Thailand. The mature seeds of both rice breeds were purchased from their cultivating area mentioned above and maintained in the dry condition prior use (within 3 months after harvest). Plant materials used for isolation of endophytic actinobacteria comprised of 20 peeled-off mature seeds, six seedlings grown in an axenic moist chamber for ~10 days (divided into roots and shoots), and six seedlings planted in an agricultural soil for ~15 days (divided into roots, stems, and leaves).
The growing conditions of rice seedlings (i.e., dark-light cycle, moisture, soil, and watering) were controlled the same for both rice breeds.
The rice seedlings derived from soil cultivation were gently uprooted, and their roots were cleaned several times under running tap water.
After thoroughly washing plant materials with tap water, their surfaces were sterilized by soaking in 10% (w/v) sodium hypochlorite solution for 5 min and washing twice with sterile distilled water.

| Isolation of endophytic actinobacteria from plant materials
Surface sterilized plant materials were ground separately in the presence of 1 ml sterile distilled water, using aseptic mortar and pestle.
Starch Casein agar (Table S2) plus nalidixic acid (25 μg·ml −1 ) and cycloheximide (10 μg·ml −1 ) was an isolation medium for this study. The medium was spread over with a 100 μl of the ground plant suspension prepared above. Six seeded agar plates per each plant material were carried out and incubated at 30°C for 20 days, while the other agar plates seeded with the last washes of plant materials served as controls. Appeared colonies of actinobacteria were collected and subcultured on Hickey-Tresner (HT) agar (Table S2) until becoming pure cultures. The purified isolates of actinobacteria were conserved in 20% (v/v) glycerol for a long-term storage and further studies.

| Morphological and phylogenetic characterization of endophytic actinobacteria
All endophytic actinobacteria obtained were grouped based on some of their morphological characteristics (Table 1), following the standard of Bergey's Manual of Systematic Bacteriology (Goodfellow et al., 2012). The representatives of each morphological group were identified at the generic level, using their 16S rRNA gene sequence data.
Initially, we increased the biomass of endophytic actinobacteria by growing them in 50 ml International Streptomyces Project medium II (ISP2) (

| Bioactivity screening of endophytic actinobacteria
Plant growth-promoting potentials of all endophytic actinobacteria were determined with their bioactivities, comprised of antimicrobial activity, soil nutrient and mineral conversion, and production of some biostimulants and biocatalysts. The antimicrobial activity against a set of test microorganisms (i.e., bacteria and phytopathogenic fungi listed in The other plant growth-promoting activities, including the production of ammonia (NH 3 ) through peptone degradation, indole-3acetic acid (IAA), and siderophore, and the solubilization of phosphate, were evaluated by screening protocols described elsewhere (Nakaew, Rangjaroen, & Sungthong, 2015). We also estimated the capability to produce some biocatalysts, that is, amylase, cellulase, chitinase, lipase, and protease, using a set of specific agar medium screening assays.
Briefly, Nutrient agar medium (Himedia, India) supplemented with 2 g·L −1 soluble starch or 1% (v/v) tributyrin (Glogauer et al., 2011) was used for testing the amylase and lipase production, respectively. The agar medium was inoculated with the actinobacteria and incubated at 30°C for 7 days. For detecting amylase production, staining the seeded agar plates with 1% (w/v) Lugol's iodine reagent for 20 min was needed. The clear zone that appeared around the actinobacterial colony determined the positive amylase and lipase production. For the production of cellulase, chitinase, and protease, the screening assays were carried out following the protocols described by Nakaew et al. (2015).

| Phytochemical analysis of plant materials
The  Table S1.