Proteomics‐based identification of orchid-associated bacteria colonizing the Epipactis albensis, E. helleborine and E. purpurata (Orchidaceae, Neottieae)

Using proteomics-based identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we conducted the first analysis of the composition of endophytic bacteria isolated from different parts of selected Epipactis species, i.e. the buds, the inflorescences and the central part of the shoots, as well as the rhizomes. We identified aerobic and anaerobic bacteria, including such taxa as Bacillus spp., Clostridium spp., Pseudomonas spp. and Stenotrophomonas spp., which may be considered as promoting plant growth. Because most of the indicated bacteria genera belong to spore-producing taxa (spores allow bacterial symbionts to survive adverse conditions), we suggest that these bacteria species contribute to the adaptation of orchids to the environment. We found clear differences in the microbiome between investigated closely related taxa, i.e., Epipactis albensis, E. helleborine, E. purpurata and E. purpurata f. chlorophylla. Some of the analysed orchid species, i.e. E. albensis and E. purpurata co-occur in habitats, and their bacterial microbiomes differ from each other.


Introduction
The complex co-associations of plants with endophytic organisms, including bacteria, fungi, protists, nematodes and viruses have important roles in health of the plant, confer advantages including growth promotion, nutrient uptake, stress tolerance and resistance to pathogens (Trivedi et al., 2020). The concept that plants and the associated microbiota form a 'holobiont' has become popular and discussed (Vandenkoornhuyse et al., 2015). It should be mentioned that, the associations of microbiota with their host plants are varied and complex (Tadych et al., 2009). General structure of the bacterial and fungal communities depends on the plant compartment, environment, geographic location and host. The plant-associated microbiome is dynamic during lifecycle of the plant (Trivedi et al., 2020). Bacteria probably have a potential use/role as plant growth promoters, especially in acclimatizing seedlings obtained by micropropagation as well as nutrient uptake and pathogen resistance (Azizoglu, 2019;Dias et al., 2009;Trivedi et al., 2020) Beneficial microbiome protects the plant against pathogens by the production of antibiotics, lytic enzymes, volatiles and siderophores and can produce a range of enzymes that can detoxify reactive oxygen species (Vandenkoornhuyse et al., 2015). Moreover, plant-associated bacteria usually remain resistant to bacteriocines, so they might be a stable component of bacteria-plant symbiosis (Lee et al. 2016;Flores-Treviño, 2004;Liu et al., 2020). Edophytic bacteria and fungi, mainly yeast form a powerful consortium based on strong networks of coexistence and dependence (Villarreal-Soto et al., 2018).
The composition and role of orchid-associated bacteria (OAB) colonizing the underground tissues of terrestrial European orchids is relatively poorly understood. Tropical orchids have been scientif- ically well researched in this regard. Galdiano et al. (2011), based on partial sequencing of the 16S rRNA genes of bacteria cultures from root velamen of Cattleya walkeriana, identified four taxa of rhizobacteria, i.e. Bacillus sp., Burkholderia sp., Enterobacter sp. and Curtobacterium sp. These rhizobacteria can produce auxin, which favorably influences the growth C. walkeriana germinated in asymbiotic conditions and during the acclimatization process (Galdiano et al., 2011). Endophytic bacteria identified in the epiphytic orchid Dendrobium moschatum were recognized among the other genus: Rhizobium sp., Microbacterium sp., Sphingomonas sp., and Mycobacterium sp. They are responsible for plant growth promotion by producing indole acetic acid (IAA) and solubilizing inorganic phosphate (Malboodi et al., 2009). Additionally, it was proved that inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination (Tsavkelova et al., 2007, a, b;Zhang and Song, 2012). Research conducted on Thai species of the genus Cymbidium has shown that it follows a seasonal pattern of abundance that differed between orchid genera, especially on the morphological level of the endophyte-infected tissue (Vendramin et al., 2010;Chutima et al., 2011). Identified species of bacteria that can promote plant growth of Cymbidium sp. orchids are the following: Bacillus thuringiensis, Burkholderia cepacia, Burkholderia gladioli, Herbaspirillum frisingense, Pseudomonas stutzeri, Rhizobium cellulosilyticum, Rhizobium radiobacter, and Stenotrophomonas maltophilia (Gontijo et al., 2018). According to the literature data, some strains of OAB were able to promote the symbiotic germination of Cymbidium goeringii, Orchis militaris and Holcoglossum species (Sun et al., 2009;Vendramin et al., 2010;Tan et al., 2012). Therefore, it seems that bacteria play an equally important role in the orchid life cycle as mycorrhizal fungi.
The Epipactis species are well known in terms of the mycobiota that inhabits it, e.g. Ogórek et al. (2020), however, there is no data on endophytic bacteria, the presence of which may be of key importance in their adaptation to the environment.
In this in this preliminary studies the following issues have been raised for the first time: (i) isolation and identification the orchid-associated bacterial (OAB) endophytes of Epipactis species including vegetative plant organs, (ii) a comparison of the OAB between the species of the genus Epipactis including the taxonomy and habitat of the tested orchid and (iii) an attempt to explain their importance in the biology of the studied Epipactis taxa based on the literature data.

Plant materials
Plants belonging to three ecologically diverging species of the mixotrophic Epipactis genus, i.e. Epipactis helleborine (L.) Crantz, E. albensis Nováková et Rydlo, E. purpurata Sm. and rare intraspecific taxon E. purpurata Sm. f. chlorophylla (Seeland) P. Delforge were analyzed (Fig. 1). All the taxa were identified using their morphological character on the basis of the literature data, e.g. Baumann et al., 2006;Delforge 2006;Jakubska-Busse et al., 2017;. Plant samples were collected from three plant parts, i.e. closed flower buds, stem, rhizome fragments and adventitious roots. Plant material was collected in the summer of 2020, from natural populations of Epipactis purpurata, E. purpurata f. chlorophylla and E. albensis growing in modified Central European oak-hornbeam forest, Galio-Carpinetum in the Nieszczyce (SW Poland), as well as Epipactis helleborine (L.) Crantz from Trestno (SW Poland, Wrocław County) growing in the regenerative forest and bush communities referring to the riparian habitat or riparian woodland classified into the Sal-icetea purpureae class. GPS (Global Positioning System) coordinates are available from the authors upon request. All species of the genus Epipactis are legally protected in Poland, and only a small number of genets -genetic individuals (9) have been authorized to conduct research in this project. In the studied population, we examined 30 ramets classified to different Epipactis species. Material sampling was done with permission no. WPN.6400.24.2020. MH from the Regional Directors for Environmental Protection.

Isolation of endophytic bacteria
The bacterial microflora was isolated in aerobic conditions using the following media: Nutrient Broth (BioMaxima, Lublin, Poland) and Enriched LAB-AGAR (BioMaxima, Lublin, Poland) and in anaerobic conditions using the following media: Schaedler Broth (BioMaxima, Lublin, Poland) and Schaedler Anaerobe LAB-AGAR TM + 5% SB (Biomerieux, France). Incubation in aerobic conditions of the material was carried out at 30℃ for 24 h, while incubation in anaerobic conditions was carried out at 30℃ for 72 h. The flowers, leaves, stems, rhizomes and shoots were cleaned by rinsing in 70% ethanol and then rinsing twice with milliQ water. The plant material after crushing was introduced into 5 ml of Nutrient Broth medium and 10 ml of Schaedler Broth medium and left for incubation. Swabs collected from inside of the orchids flower orchids were introduced into liquid media and incubated. The soil was suspended in 20 ml of sterile milliQ water and vortexed until a homogeneous solution was obtained. Then 1 ml of the soil solution was introduced into the liquid medium of Nutrient Broth and Schaedler Broth and incubated. After incubation, the material was plated on solid Enriched LAB-AGAR (30℃/24 h) and Schaedler Anaerobe LAB-AGAR TM + 5% SB (30℃/72 h) (Faria et al., 2019).

Identification of bacterial isolates
All obtained bacterial isolates were analyzed using MALDI (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) Biotyper method as described before (Ksią _ zczyk, et al. 2016). Briefly, ribosomal proteins were extracted using 70% formic acid and acetonitrile method. Next, each sample was spotted on a 384 ground steel MALDI target plate. After drying, the applied sample, an equal volume of matrix (a-cyano-4-hydroxy-cinnamic acid (HCCA)) was spotted onto the spot. Then, the plate was left at room temperature for about 15 min to dry. Mass spectra of the extracted proteins was measured using the mass spectrometer MALDI-TOF ultrafleXtreme (Bruker Daltonics GmbH, Bremen, Germany). To identify bacterial mass spectra, Biotyper 3.1 software and database containing 6904 entries were used (Bruker Daltonics GmbH, Bremen, Germany). Based on 'Bruker Daltonik MALDI Biotyper Classification Results' protocol the following score criteria were applied to identification of bacteria: 2.300-3.000 -highly species identification, 2.000-2.299 -probable species identification, 1.700-1.999 -probable genus identification, 0.000-1.699 -not reliable identification.

Orchid associated bacteria
The used methods of orchid-associated bacteria (OAB) under aerobic and anaerobic conditions allowed us to obtain a total of 192 isolates. The MALDI Biotyper method assigned 103 isolates to species level. Analysis of the orchid associated microorganisms showed the presence of 20 different bacterial species within Epipactis albensis, Epipactis helleborine, Epipactis purpurata and Epipactis purpurata f. chlorophylla. The list of general identified bacteria from Epipactis species is presented in Table 1. Because the endophytic bacteria were isolated from different part of the orchid (ground and underground), a summary of the obtained data is presented in detail in Table 2. Percentage representations of each bacterial species contributing to the total isolates from every of Epipactis are summarized in Fig. 2. Moreover roots in some single genets were found -Epipactis helleborine ramet 3 and Epipactis purpurata f. chlorophylla and bacteria strains were isolated with differences between both individuals. In case of Epipactis helleborine ramet 3 the following bacteria strains were indicated: Buttiauxella agrestis, Pseudomonas putida and Raoultella ornithinolytica, while Bacillus mycoides and Bacillus cereus were selected from Epipactis purpurata f. chlorophylla.
Among the identified bacterial taxa in every tested orchid species, the following taxa were identified: Bacillus mycoides, B. weihenstephanensis and Lysinibacillus fusiformis. We found that the studied specimens of two related taxa, i.e. Epipactis purpurata and intraspecific taxon E. purpurata f. chlorophylla, despite their very close taxonomic relationship, differ in the composition of endophytic bacteria. In the case of the tested Epipactis spp., dominating groups of endophytic bacteria usually belong to spore forming genus such as Bacillus and Clostridium, and non-spore forming    Pseudomonas ( Table 3). All of the mentioned genera have good adaptability to the different, variable and extreme environmental conditions. This correlates with the high tolerance of environmental conditions among the tested orchids and may explain the varieties of strains within the Epipactis species. It seems there is no bacterial species specificity within the Epipactis species. We found some differences in the microbiome between closely related taxa, i.e. E. purpurata and E. purpurata f. chlorophylla. Unfortunately, due to the small number of studied individuals, we treat these results as preliminary. To attempt to explain the significance of bacteria in the biology of the studied Epipactis taxa based on the literature data, we analysed the selected biological function of identified bacteria species (Table 3). We analyzed not only parts of the plant, but also the soil from the close site of the orchid (Table 4). The soil's microflora of the all studied Epipactis spp. contain bacteria belonging to the following genera: Achromobacter, Acinetobacter, Bacillus, Clostridium, Citrobacter, Escherichia, Hafnia, Kluyvera, Lactococcus, Lysinibacillus, Pseudomonas, Raoultella, Serratia and Stenotrophomonas (identified bacteria species divided into each Epipactis species was summarized in Table 4. In total, the different bacteria species were endophytes belonging to the following genera: Achromobacter, Acinetobacter, Citrobacter, Escherichia, Hafnia and Raoultella.
A very interesting and unexpected result of our research was the finding that the soil microbiome differs from that of a specific ramets growing in the studied substrate. The co-occurrence of two plant species, i.e. Epipactis purpurata and E. albensis in close proximity does not confirm their common requirements for the presence of specific bacteria in the soil.

Discussion
The orchids classified to Epipactis genus are rather difficult to cultivate in vitro (Kunakhonnuruk et al., 2018), probably for this reason, no data is available on the bacterial microbiome and its potential contribution in the biology of these orchids.  Ewingella americana Gram-negative, cosmopolitan bacterial pathogen that has been isolated from many hosts, i.e. mushrooms, plant growth promoting, the strains encoded a set of common genes for type secretion, virulence effectors, CAZymes, and toxins required for pathogenicity in all hosts, antibiotic resistance, pigments to suppress or evade host defense responses, ability for adaptation to different environmental conditions, including temperature, oxidation, and nutrients (host adaptation strategies of Ewingella), and they also contribute to the development of effective control strategies Liu et al., 2020 Lysinibacillus fusiformis Gram-positive, isolated from factory wastewater and farming soil, can generate endospores, causes bacteremia, tropical ulcers

Sulaiman et al., 2018
Lysinibacillus sphaericus Gram-positive, insect pathogen, especially for mosquitoes, lethal effects on eggs of the nematode Trichostrongus colubriformis and effects on the grass shrimp Palaemonetes pugio, produce bacteriocins and toxins against some cockroach and mosquitocidal Berry, 2012 Paenibacillus amylolyticus Gram-positive, spore forming, aerobic or facultatively anaerobic bacteria, isolated from: soil, fresh and salt water, sewage, sediments, caves, humus, compost, rhizosphere, food, plants, insect larvae and clinical sample, rhizosphere soil of peanut, produce of siderophore, promote the iron absorption of plant in calcareous soil, thus promoting plant growth Liu et al., 2017;Sáez-Nieto et al., 2017 Pantoea agglomerans Gram-negative, plant-associated bacteria, occur commonly, usually as symbionts, in insects and other arthropods, occur in plants as an epi-or endophytic symbiont, often as mutualist, cause of diseases in a range of cultivable plants, such as cotton, sweet onion, rice, maize, sorghum, bamboo, walnut, an ornamental plant called Chinese taro (Alocasia cucullata), and a grass called onion couch (Arrhenatherum elatius)

Dutkiewicz et al., 2016
Pseudomonas fluorescens group Gram-negative, isolated from agricultural soil, well adapted to grow in the rhizosphere, rhizobacterium, biocontrol agent and promote plant growth ability, produce a wide spectrum of bioactive metabolites, i.e. antibiotics, siderophores, volatiles, and growth-promoting substances, responsible for the natural suppressiveness of some soilborne pathogen David et al., 2018 Pseudomonas sp. The results of our research, which we treat as preliminary to a further scientific project, turned out to be very interesting for a number of reasons. Firstly, we found that related orchid taxa, often co-occurring in habitats, i.e. Epipactis albensis, E. helleborine, E. purpurata and E. purpurata f. chlorophylla, differ in their microbiomes. These findings are surprisingly different from the results of our previous work on mycological evaluation of Epipactis helleborine and E. purpurata (Ogórek et al., 2020), where we showed that these two analyzed ecologically diverging Epipactis species, although growing in diverse habitats, did not differ significantly in terms of the composition of natural mycobiota (Ogórek et al., 2020).
Endophytes are defined as an important group of endosymbiotic microorganisms widespread among plants that colonize the intercellular and intracellular spaces of all known plant organs but do not cause any plant diseases or significant morphological changes (Miliute et al., 2015). This group also has been targeted as a valuable source of bioactive compounds and secondary metabolites important in the plant life cycle. Unfortunately, the species composition of endophytes inhabiting orchids and their biological role are very poorly understood.
Some of the bacterial strains, the genera Bacillus and Pseudomonas we isolated from Epipactis orchids, were previously found in the underground roots of Calanthe vestita var. rubro-oculata (Tsavkelova et al., 2001). These West Australian orchids and the genus Epipactis are classified in the same subfamily Epidendroidae. It is difficult to validate what role these bacteria can play in the biology of the Epipactis orchids without thorough research, but it is possible that they also support plant growth and/or plant development. Interestingly, we found these bacteria in various parts of the studied plants, i.e. in shoots, leaves and flower buds. Trivedi et al. (2020) reviewed that most of the endophytic bacteria belong to Proteobacteria but in case of our study significant number of isolates belong to Firmicutes (such as Bacillus and Clostridium genus).
Also, according to the literature, infection of Cattleya loddigesii with Paenibacillus macerans, orchid endophytic auxin-producing bacteria promoted seedling growth during the acclimatization process (Faria et al., 2013). Interestingly, in our research we identified related species, i.e. Paenibacillus illinoisensis as infected E. purpurata f. chlorophylla. It is possible that there is an association between Paenibacillus species and mycorrhizal fungi and the roots are the main penetration pathway for endophytic microorganisms (Faria et al., 2013).
Some bacterial genera identified in Epipactis orchids isolated during this work have been known for nitrogen or phosphor uptake in plants, e.g. Bacillus mycoides (Yi et al., 2018), Pseudomonas putida (Molina et al., 2020), P. koreensis (Gomesa et al., 2017;Sawada et al., 2019). This fact supports the hypothesis that they might be an important factor in Epipactis species growth promotion.
According to the literature, some soil bacteria identified in orchids, such as Pseudomonas, may promote the development of vesicular-arbuscular mycorrhizas (Azcon-Aguilar and Barea, 1985;Tsavkelova et al., 2001). It is well known that inoculation of the orchid seeds with an Azotobacter and the root-nodule bacterium Bacillus radicicola promote their germination (Knudson, 1922;Tsavkelova et al., 2001). The germination of orchid seeds is also enhanced by their bacterial infection by the genera Pseudomonas, Bacillus, Arthrobacter and Xanthomonas (Wilkinson et al., 1989;Wilkinson et al. 1994;Tsavkelova et al., 2001).
Moreover, the plant-associated bacteria species including Bacillus species (like Lysinibacillus sphaericus, Bacillus amyloliquefaciens B. cereus, B. mycoides and B. thuringiensis) or Pseudomonas species  Baba et al., 2009;Latour et al., 2013 Serratia liquefaciens Gram-negative, inhibition of the growth of pathogenic bacteria by the production of heliotropin, antifungal properties thanks chitinases enzyme production, plant growth promoting bacteria Kalbe et al., 1996;Cieniuch et al., 2019 Solibacillus sp. Gram-positive, round endospore-forming bacterium, isolated from a forest soil near Braunschweig, Lower Saxony, Germany, spore surface showed a cauliflower-like fine structure, contains lysine in its cell wall, plant protective bacteria Rheims et al., 1999;Lee et al., 2020 Stenotrofomonas sp.  (the most frequently a successful endophyte in many plants) promote their development via indirect control of phytopathogenic fungi growth (Hollensteiner et al., 2017;Nel et al., 2019, Table 3). Bacteria impact plants via different modes of action, such as ROS (Reactive Oxygen Species) production, fermentation product, enzymatic lysis of the structure components, presence of different antimicrobial and insecticidal compounds, cyclic peptides, siderophores, bacteriocins, molecules involved in beneficial plantbacteria interactions or the up-regulation of the expression of genes (Baba et al., 2009;Liu et al., 2010;Latour et al., 2013;David et al., 2018;Yi et al., 2018;Arrebola et al., 2019;Gautam et al., 2019;Liu et al., 2019;Molina et al., 2020). Moreover, Liu et al. (2019) observed that Bacillus amyloliquefaciens could inhibit mycelial growth, the germination of the cysts and the swimming of the motile zoospores of Phytophtora sojae. The other strains isolated by us belonging to the genus Pseudomonas (such as Pseudomonas orientalis, P. koreensis, P. chlororaphis) have been known for their antagonistic activity, especially in the rhizosphere, but also in the apple flower against sol borne plant pathogens (both fungi and bacteria) (Gomesa et al., 2017;Arrebola et al., 2019;Sawada et al., 2019).
It is possible that the ability to form biofilm within isolated bacteria genus (e.g. Bacillus, Pseudomonas) plays an important role in orchid growth promotion because of the protection of the vegetative cells against some the pathogenic strains (Yi et al., 2018).
Furthermore, it is interesting that some plant pathogens, considered previously as symbiotic bacteria, were recognized. We identified bacteria genera (including Erwinia, Pseudomonas, Bacillus and Clostridium) that may cause soft rots in living plant tissue as a consequence of the presence of a strong viral factor (Prod'homme et al., 2017;Liu et al., 2020), but the analyzed orchid did not show any disease symptoms.
Similarly, in our previous research on fungal communities of Epipactis helleborine and E. purpurata, we found that the plants were infected by three species of the genus Fusarium (F. oxysporum, F. sporotrichioides and F. tricinctum) (Ogórek et al., 2020). These fungi are recognized as pathogenic, but they may also have other functions in ecosystems. Interestingly, we also found that the presence in Helleborines of some others species of fungi, especially Alternaria tenuissima, Epicoccum nigrum, Penicillium biourgeianum and Trichoderma viride, which could be effective against both fungal and bacterial pathogens (Ogórek et al., 2020). Similarly, we did not observe any disease symptoms typical of infections with pathogenic fungi in the analyzed plants.
There are a lot of arguments proving that we should not marginalize the importance of the coexistence of bacteria and fungi in Helleborines, because these microorganisms most likely play an important role in the process of adaptation of orchids to a changing environment.
In the next planned research project, we would like to experimentally test the influence of the identified bacteria and seed germination of the tested orchid taxa of the Epipactis genus.

Conclusions
Analysis of the orchid-associated bacteria (OAB) showed the presence of 35 different bacterial species within Epipactis albensis, Epipactis helleborine, Epipactis purpurata and Epipactis purpurata f. chlorophylla. Most of the isolated OAB belong to spore-forming, Gram-positive bacteria (Bacillus and Clostridium). Moreover, a numerous group was represented by Pseudomonas species. Isolated bacterial endophytes are considered as growth-promoting factor and may be significant in plant growth and development. We indicated diversity of the bacterial microbiome between plants, that grew in different types of habitats. Analysis of the OAB isolated from the soil in which these ramets grew also confirmed the differences.
In our opinion, the presence of endophytic bacteria, especially classified to the group of prototrophic organisms, can stimulate the growth and development of Epipactis orchids, especially those plants that grow on nutrient-poor soils. This is of particular importance in the adaptation of plants to the new environmental conditions and in the process of colonizing new habitats and territories.

Funding
Publication of this article in open access was financially supported by the Excellence Initiative -Research University (IDUB) programme for the University of Wroclaw.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.