Bacterial Seed Endophytes of the Holoparasitic Cistanche Armena and C. Phelypaea (Orobanchaceae) from Saline Habitats: Semi-Deserts vs Flooded Coasts

The current study compares the bacterial seed endomicrobiomes of the endemic holoparasitic plant species Cistanche armena and C. phelypaea. Both species thrive in habitats that are saline but with a very different soil water status. The study aims to uncover how environmental conditions inuence the diversity of the bacterial communities of seeds of these holoparasites and also highlights the physiological activities, several enzymatic and PGP traits of culturable endophytes that may support the tolerance of their hosts to abiotic stresses. A combination of culture-dependent and molecular techniques was employed for the identication of the seed endomicrobiome (culturable and unculturable). From the seeds of both parasitic plant species closely related strains were isolated. Sixteen phyla, 323 genera and 710 bacterial species were identied, mainly Gram negative, halotolerant bacteria with an environmental origin. Most of the isolates were endospore forming, halotolerant and alkaliphile Bacillus spp. which suggests that the endophytic bacteria of C. armena and C. phelypaea seeds possess traits that are correlated with the natural habitat of their hosts. The Paenibacillus strains from both plant species demonstrate similar biochemical traits. Although the seed endophytic microbiomes of C. armena and C. phelypaea contain a high number of common bacterial taxa, also remarkable differences exist. We demonstrated that the diversity of the bacterial communities is related to the environmental conditions, water status or abiotic stresses. (pitted)/striate test sculpturing of inter periclinal walls. The seed coat of the polygonal cells more less isodiametric with different sizes, 101–227 × 75–160 µm with 1.0–2.22 length-to-width ratio. The number of cells along the seed longitudinal from 7 to 12 and in lateral view comprises from 35 to The anticlinal walls with remarkably depth with width 5.1–10.33 µm. (Fig. with sterile double distilled water, the samples were centrifuged for 30 sec at 12,000 rpm (13,400 x g). The washing process was repeated with decreasing the time of shaking (2 h, 1.5 h, 60 min, 45 min and 30 min). The samples were centrifuged 30 sec, then rinsed with sterile double distilled water, then kept at 4°C for 15 min. The rinsing procedure was repeated 3 times. To exclude the presence of DNA from bacteria present on the seed coat, PCR was performed on the last rinsing water. After verifying the effectiveness of the sterilization protocol, the sterile homogenous seed suspension was used for DNA extraction using the Genomic Mini AX Stool Spin (A&A Biotechnology) according to manufacturer’s instructions. The quality and quantity of the DNA was examined by electrophoresis on a 1% agarose gel. Amplied products were resolved in 1.0% (w/v) agarose gel in a Tris-acetate-EDTA buffer (TAE) and visualized under UV light (Syngene) after staining with ethidium bromide. amplication products were obtained. DNA was puried using Ampure XP magnetic beads (Beckman Coulter) and indexed by second-order PCR. The FASTP (github.com/OpenGene/fastp) tool used sequence ltration and analysis sequenced data.

The present study combined culture-dependent and molecular approaches. Molecular techniques were used to identify the culturable bacteria and to describe the diversity of the microbial communities in seeds of the two holoparasitic plant species. Some physiological, biochemical and PGP traits of the culturable endophytic bacterial strains were investigated. Also, the micromorphology of the seeds was studied. The micromorphological study of the examined seeds was crucial to select the appropriate method of surface sterilization, due to the unique structure of reticulated testa and the endothelium of the seed coat.
To the best of our knowledge that is the rst report about bacterial seed endophytes of the holoparasitic plant species C. armena and C. phelypaea.

Isolation of culturable endophytes from surface sterile seeds of Cistanche armena and C. phelypaea
The nutrient media that were used for the isolation of bacterial endophytes had clear effects on growth rate and diversity ( Table 2).
Growth rates of bacteria and morphological diversity of colonies were the highest on rich media. On LB, MPA, THB and TSB 3-4 types colonies were dominating, while in others (KB, MHA) less different colonies were observed. Even less growth and morphological diversity were observed on MHA and KB. In addition to organic compounds, they contain MgSO 4 , K 2 HPO 4 or Mg 2+ and Ca 2+ . However, the main difference with rich media was the concentration of sodium chloride. These media no contain NaCl. The slowest growth was observed on the SAA and SAB media that contained only mineral compounds and 10g l −1 NaCl.  34 and is highly similar to B. pumilus.

Physiological characteristics of isolated strains
The isolated strains were tested for their tolerance to higher temperatures (37°C, 42°C and 50°C), high concentrations of NaCl (10%, 15%) and broad pH ranges (4.0 to 11.0) ( Table 4). For all strains, substantial growth was observed at 42°C. In addition, strains CA12, CA4, CA3, CP4 and CP12 were growing at 50°C, which allows to consider them as thermophilic. Strains CA5, CA9, CA8, however, showed negative growth rates at 50°C. After 48 h of incubation at 25°C the bacterial colonies developed in all plates. This supports the endospore forming ability and real endophytic character of the isolated strains. The results of the seeds surface sterilization tests con rmed that the isolated strains were real endophytes and did not result from surface contamination of the seeds (Fig. 2). For the further experiments, 25°C was chosen as incubation temperature since this is the closest to a normal average soil temperature.
Abundant growth was observed at high concentrations of NaCl (10%, 15%). Strains CA3, CA5, CA12 and CP4 demonstrated growth in the range of 3-15% NaCl, with an optimum at 5-10%. Only strain CA9 did not develop at high concentrations of NaCl; its optimal NaCl concentration in the medium was 3%. Interestingly, strains CA4 and CP4 responded differently to high concentrations of NaCl. Wide ranges of adaptation and growth ability were observed at different pH (from pH 4.0 to pH 11.0). However, for all strains the best growth was observed at pH >7.0 (Table 4). Combining all results, we can conclude that the isolated strains were endospore forming, halophilic and alkaliphilic.

Selected biochemical activities of isolated strains
The isolated strains were examined for their in vitro ureolytic and catalase activities, plant cell-wall degradation capacity (hydrolytic enzymes) and potential plant growth promoting traits (Table 4). Only strains CA8 and CA9 tested positive for urease activity, while all strains were positive for catalase activity. Strains CA3 and CA8 showed amylase activity and might thus be involved in seed germination. Strains CA3, CA9, CP12 demonstrated pectinase activity. Interestingly, most of the strains, except CA5, were exhibiting protease activity. With the exception of strains CA5 and CA8, all other strains could hydrolyze cellulose.
The isolated bacterial strains were also tested in vitro for some potential plant growth promoting (PGP) traits. Strains CA4, CA5 and CA12 could solubilize phosphate in NBRIP medium. The Bacillus strains CP4 and CA4 showed different phosphate solubilization reactions. Interestingly, the majority of the strains were able to produce IAA. In general, the production levels were low; strains CA8 and CA9 demonstrated the highest productions: 0.29 mg L −1 and 0.26 mg L −1 respectively. The highest GA production was achieved by strains CA3, CA5, CA9, CA12. The strains CA4 and CP4 showed the lowest GA production. This is in contrast to Hao et al. 35 who reported that B. pumilus produced high concentrations of GA. Strains CA8 and CA9 produced high concentrations of both, IAA and GA.
Taking together all above mentioned data it is clear that strains CA3, CA8 and CA9 demonstrated the widest range of biochemical activities. The strains CA4, CA12 isolated from seeds of C. armena and strain CP4 from C. phelypaea seeds presented very similar biochemical activities. Strains CA4, CA5, CA12 possess PGP potential. Strain CA3, that has a high similarity to B. licheniformis DSM13 tested positive for all examined hydrolyzing enzymes. In contrast, strain CA5, isolated from seeds of C. armena and comparable to strain Oceanobacillus oncorhynchi subsp. incaldanensis 20AG, showed negative for all enzymes examined, except catalase. In addition, strain CA5 tested positive for phosphate solubilization and production of the phytohormones IAA and GA. To elucidate the effective bene ts of these endophytic bacteria for their host plants, particularly for the seeds, seed germination and development of the seedling, more research is required.

The seed-associated endophytic bacterial community compositions
The number of paired raw Illumina readings for C. armena seeds was 107443 and 105028 for C. phelypaea seeds. The obtained numbers of readings after the ltering procedure were su cient to carry out further stages of the analysis. It was anticipated to obtain minimal 100,000 paired readings for each of the tested samples. The readings obtained were ltered using the fastp tool (github.com/OpenGene/fastp) -the remaining sequences of the sequencing adapters and the low-quality readings were removed.
The Shannon-Wiener biodiversity indices for the seed endophytes of C. armena (CA) and C. phelypaea (CP) were 2.11 and 4.08 respectively (Fig. 3). 742 different OTUs were found in total: 352 OTUs for C. armena and 392 OTUs for C. phelypaea (Fig. 3). The taxonomy of the sequences was described primarily at the phylum level ( Fig. 4). For the seeds of the two examined Cistanche populations 16 phyla, 323 genera and 710 species were determined. Ten phyla, 187 genera, and 331 species were identi ed from C. armena and 15 phyla, 216 genera, and 379 species in the seeds of C. phelypaea.
The Proteobacteria were the predominating phylum in the seeds of both examined plant populations ( Table 5). The classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, as well as Acidithiobacillia were found but the Gammaproteobacteria clearly were the most abundant: 92.9% in seeds of C. armena and 61.8% in seeds of C. phelypaea. Also the phyla Actinobacteria, Bacteroidetes and Firmicutes were well represented. Other phyla constituted less than 0.22% ( Table 5). The phyla Chlamydiae, Cyanobacteria, Deinococcus-Thermus, WS6, Fibrobacteres, Fusobacteria were only detected in seeds of C. phelypaea. Likewise, the phylum Gemmatimonadetes was only identi ed in seeds of C. armena. The phylum Firmicutes represented just 0.22 % in seeds of C. armena but 9,75% in seeds of C. phelypaea. Nevertheless, only Firmicutes were isolated using culture medium: seven of the strains from the seeds of C. armena while only two Firmicutes strains could be isolated from the seeds of C. phelypaea.

Discussion
The heterotrophic lifestyle of parasitic plants from the Orobanchaceae family led to several morphological, physiological and molecular adaptations and makes it an exciting group for study. Examples of such adaptation mechanisms are the production of large numbers of seeds and very speci c conditions of germination 9 .
The seed surfaces of holoparasitic Cistanche species possess constantly alveolate ornamentation with perforated (pitted) sculpture formed by polygonal and isodiametric cells with different sizes. The quite coarse structure of the seed coat ( Fig. 1) can hamper the seed sterilization process. The results we obtained applying generally used sterilization protocols 39,40 was not always satisfying. We assume that the sterilizing agents could not always reach the deepest zones of the coarse seed surface. Finally, a combination of 70% ethanol and 10% H 2 O 2 with intense shaking showed to be adequate to remove all bacteria from the surfaces of C. armena and C.
To obtain cultivation of as many as possible of the endophytic bacterial strains, various rich and poor media were used ( Table 2). The results indicated that growth rates of bacteria and morphological diversity of colonies were the highest on rich media. According to earlier studies, most endophytes have a soil or environmental origin, and the diversity and richness of bacterial communities correlates with the composition and pH of the soil 41 . Hence, organic compounds in rich media are essential for the growth of most microorganisms. Thus, on cultivation media with only inorganic compounds the growth and diversity were very limited. Besides, like plants, bacteria need macro-and microelements for their growth i.e. manganese, zinc, cobalt, molybdenum, nickel and copper which are critical for many metabolic processes 42 .
Very similar bacterial strains were isolated from the seeds of both plant species (Table 3). We obtained nine strains from surface sterilized seeds of C. armena and C. phelypaea belonging to the Bacillaceae family. All isolated endophytes are motile, Gram-positive and endospore forming (Table 4). These traits allow bacteria to survive in harsh environmental conditions or in dry seeds for a long period of time. This is important in case of holoparasites, because these seeds have to stay viable in the soil for several decades 10 . Our results con rm that spore forming Bacillus species are frequently isolated endophytes from seeds of plants growing in saline soils, solar salterns and salt marshes [43][44][45][46] . That the isolated strains are well adapted to the growing conditions of their host plants was con rmed by their physiological traits. All isolated endophytes showed to be halotolerant and alkaliphilic. Strains CA3, CA4, CA12, CP4, CP12 were also thermophilic. The thermophilic features of CA3, that has a high similarity to B. licheniformis DSM13, and B. pumilus (CA4, CP4) were also isolated from geothermal springs in Armenia, which con rms their adaptability to high temperatures 47 and might have been marked as plant growth promoting 48 of these strains.
The production of several enzymes and PGP traits of the culturable endophytic bacterial strains were investigated (Table 4).
Interestingly, the production of one or more hydrolytic enzymes, especially cellulase and protease, was revealed for most of the isolated strains (Table 4). Cellulase and protease are involved in hydrolyzing cellulose and peptides and thus are 'instruments' for bacteria to get access to plant tissues and to colonize throughout the plant 49,50 . The production of amylase by strains CA3 B. licheniformis and CA8 B. gibsonii might be involved in seed germination and plant growth. During germination, amylase has a crucial role in the hydrolysis of endosperm starch, which provides energy for the developing roots and shoots 51 . According to Kaneko et al. 52 the amylase production depends on phytohormones, particularly GA. Thus, gibberellins (GA) stimulate the synthesis and production of α-amylase. These phytohormones can induce a range of genes, which are necessary for the production of amylases including α-amylase, proteases and β-glucanases 53,54 . Therefore, it is not unexpected that these strains show relatively high levels of GA production ( Table 4). Only for strain CA5 no activity of hydrolytic enzymes was detected, which concurs with existing literature. Production of enzymes involved in cell wall degradation was not reported before 55 . However, strain CA5, that shows a high similarity to Oceanobacillus oncorhynchi subsp. Incaldanensis AG20 tested positive for phosphate solubilization and production of the phytohormones IAA and GA. To elucidate the effective bene ts of these endophytic bacteria for their host plants, particularly for the seeds, seed germination and development of the seedling, more research is required. Phytohormone production (IAA and GA) was con rmed in all isolates, and we speculate that production of such compound by bacteria might be bene cial for C. armena and C. phelypaea, particularly for absorption of more water and/or nutrients from their host plants in harsh environments.
The strains were also tested for urease activity. It is well-known that urease is an important cytosolic enzyme that is synthesized by living organisms, including plants and microorganisms. Generally, urea is used as a source of nitrogen and has a key role in pathogenesis of microorganisms, participates in the germination process and the nitrogen metabolism of plants 56 . The strains Paenibacillus apiarius CA9 and P. taichungensis CP12 demonstrated very similar biochemical traits, which allows to predict that they have a similar role in C. armena and C. phelypaea respectively. However, they demonstrate different urease activity (Table 4). Bibi et al. 57 showed that the urease activity might depends on the different levels of gene expression.
Altogether, the results presented above do not allow to elucidate the real bene cial potential of seed endophytes for holoparasitic plants. The majority of plant associated bacteria are unculturable, and it is often assumed that only 0.001-1% can be grown in laboratory conditions 58 . In order to obtain more information about the composition of the microbial communities (culturable and unculturable) in seeds of C. armena and C. phelypaea, molecular techniques were also used.
The results of the next generation sequencing analyses on Illumina MiSeq showed that the gram-negative Proteobacteria, and speci cally the Gammaproteobacteria is an important taxon in seeds of both, C. armena and C. phelypaea. Many Gammaproteobacteria are halophilic 59 , thus their presence in the examined seeds is not surprising because of the natural habitats of both Cistanche species (Fig. 4, Table 5). Also Actinobacteria, Firmicutes and Bacteroidetes are well represented. These taxa were also isolated from seeds of Marama beans (Tylosema esculentum) growing in stressful environmental conditions and in poor-quality soils 60 . An overwhelming majority of seeds from different wild and commercial plant species, including some holoparasitic species are colonized by endophytes belonging to the phyla Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes 13,16,60−63 . The results about the seed endophytic microbiome of the parasitic Phelipanche ramosa 13 together with the outcomes from the current study allow to assume that Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes are also common and abundant taxa in seeds of species of the Orobanchaceae (  41 . Some studies demonstrated that Gemmatimonadetes are more adapted to arid and semiarid soils, and at the same time the survival of Gemmatimonadetes may be inhibited in wet conditions. Therefore, exposure to drought may result in rapid colonization of arid and desert plants by these bacteria that established the relationship between moisture and microbial community structure 64 .
Hence, considering the moisture requirements of C. phelypaea, Gemmatimonadetes are not expected to occur in the seeds of this species. Such information con rms our hypothesis, that water requirements and abiotic stresses in uence the development of the seed bacterial community. Other bacterial phyla like Fibrobacteres, Chlamydiae, Cyanobacteria, Chloro exi, Deinococcus-Thermus, WS6 were only present in the seeds of C. phelypaea. The phyla isolated from the seeds of C. phelypaea were recently also identi ed from sea water and anoxic marine sediments and were reported to have important ecological roles 65 . The Chloro exi is a phylum including ecologically and physiologically diverse bacteria reported to occur in sediments, hot springs and methanogenic anaerobic sludge digesters 66 , but not from plant seeds. Another bacterial phylum, Deinococcus-Thermus, comprises extremophiles highly resistant to abiotic stresses 67 and was recently found in seeds of different plant species 25,68 . The phylogenetic group WS6 is unexplored, widely spread in some environments, and detected mostly in anaerobic sediments. According to Dojka et al. 69 they may possess some undiscovered biochemical and metabolic novelties and important biogeochemical roles. Our prediction about the relationship between environmental conditions and endophytic bacterial diversity is supported by previous ndings 41,70 . Investigations of the desert plants Tribulus terrestris, Zygophyllum simplex, Panicum turgidum and Euphorbia granulata from the Namib Desert and Saudi Arabia showed that they harbor a very similar endomicrobiome where the dominating bacterial phyla are Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes also 41,71,72 . On the other hand, Eida et al. 71 reported that they possess various biochemical traits and salinity stress tolerance. The Actinobacteria of the genera Streptomyces, Micromonospora, Nocardia, and Amycolatopsis are mainly present in seeds of C. armena. Huang et al. 73 previously described these genera in arid plants. Although, the seed endophytic microbiomes of C. armena and C. phelypaea that originate from totally different geographical locations and environmental conditions comprise a high number of common bacterial taxa, there were, however, sizable differences between the compositions and diversities of both microbiomes. The Shannon-Wiener biodiversity index value was 2.11 for C. armena and 4.08 for C. phelypaea which means a higher diversity in seeds of C. phelypaea (Fig. 3). Considering that C. armena grows under more adverse abiotic stresses (salinity, drought and high temperature) it is predictable that the seeds are colonized by a less diverse microbiome. C. phelypaea is growing in saline but wet environmental conditions and its seeds harbor a more diverse microbiome, which could be expected. Generally, seeds do not provide favorable conditions for microbial growth and show low numbers of bacteria 76 . It was also shown that the composition of the seed endophytic community is changing during the seed maturation process and seeds are containing mainly bacteria adapted to survive in harsh abiotic conditions or that are able to stimulate the host plant metabolism for responses to abiotic stresses 77-79 .

Conclusion And Future Opportunities
We investigated and compared the endophytic bacterial communities of the seeds of two holoparasitic Cistanche species. A protocol for seed surface sterilisation was ne-tuned. Sixteen phyla, 323 genera and 710 bacterial species were identi ed, mainly Gram negative, halotolerant bacteria with an environmental origin. In the seeds of both species-speci c bacteria were identi ed that are adapted to the growing conditions of their respective host plants. However, also some unclassi ed and unexplored taxonomic groups were found in the seeds of both plants. The ndings allow us to assume that the bacterial endophytes of the seeds of both species show common physiological characteristics required to cope with the abiotic stress factors that are common to both locations -high temperatures and high salt concentrations.
The cultivable seed endophytes from C. armena and C. phelypaea were rather similar, notwithstanding the big distances between their growth habitats -Armenia and Portugal. Our results corroborate that Bacillus species are commonly isolated endophytes from plants growing on saline and arid soils. The Paenibacillus strains from both plant species demonstrate similar biochemical traits. Our results con rm the presence of Novosphingobium in holoparasitic plants.
Summarizing, the results obtained in the present work from the culturable seed endophytes indicate that some biochemical and enzymatic traits of the isolated strains might be favorable for supporting plant responses to abiotic stresses. In spite of that, the obtained results do not allow yet to designate the real bene cial potential and ecological signi cance of these seed isolates for the holoparasitic plant species C. armena and C. phelypaea, especially concerning their role in seed germination and the physiology of host plant infection by parasitic plants. However, genome sequencing, extensive bioinformatic analysis and identi cation of stress responding genes, and also identi cation of metabolic and enzymatic activity of the isolated bacteria can elucidate some aspects of interaction with their host, their role in seed germination and promotion of plant growth.

Species studied and plant material
For our study, we used seeds of two species from the genus Cistanche Hoffmanns. & Link (Orobanchaceae), from distant locations in Portugal vs Armenia, both from saline habitats, but differing in soil water status: ooded coasts vs semi-deserts. The study area is periodically ooded, and consists of coastal salt marshes, with sandy-silty zones, and a halophytic community dominated by chamaephytes, including host species of C. phelypaea, like Arthrocnemum macrostachyum, Sarcocornia fruticosa, Suaeda vera (Amaranthaceae), and Limoniastrum monopetalum (Plumbaginaceae) (Fig. 6a-b).
Seeds were collected from mature fruits, per 10 specimens for both species, and dried under natural conditions. Species were identi ed and seeds collected by Renata Piwowarczyk and herbarium materials were deposited in the Herbarium of the Jan Kochanowski University in Kielce (KTC), Poland. Field studies, including the collection of plant and seeds material was compiled with relevant local, institutional, national, and international guidelines, permissions and legislation.

Microscopic observation and morphometric analysis of seeds
General seed morphology was studied using an Axio Zoom.V16 Stereo Zoom system (Carl Zeiss, Germany) in bright eld illumination (objective lenses PlanApo Z 1.5x, FWD = 30 mm) and processed in ImageJ software using Fiji macros. The terminology of seed surfaces is taken from Barthlott 83 , and Piwowarczyk et al. 5 . For each species, at least 30 seeds were examined, and quantitative and qualitative morphological characteristics were determined several times for each seed.

Seed surface-sterilization method and cultivation conditions of culturable seed endophytic bacteria
The aim of the seed surface sterilization was to obtain only endophytic bacterial communities. 50 mg seeds of each sample were transferred into Eppendorf tubes and were submersed in 70% ethanol (EtOH) and vortexed for 60 sec (VELP Scienti ca), followed by a 10% H 2 O 2 aqueous solution with intense vortexing at 8000 rpm for 3-4 min. Finally, sterile seeds were rinsed 4-5 times with sterile deionized water. The surface sterile undamaged (marked F) and mechanically homogenized (marked H) seeds were plated on previously prepared Petri dishes (Fig. 2). Observations were done after 48 h of incubation at 25°C. Only bacterial colonies obtained from crushed seeds (marked H) were selected for further experiments and were placed onto growth media (autoclaved at 121°C for 20 min), with pH 7.0 ±0.1 (Fig. 2). In order to obtain an as complete overview of the culturable community as possible different bacterial growth media were used with peptones, corn starch, dextrose, papaic digest of soybean meal, enzymes, yeast extract, and components  Table 2). SAA medium aims to select for inorganic nitrogen using bacteria, MPA is used for isolation of organic nitrogen using bacteria 84 . Because of the particular natural habitats of the halophytes C. armena and C. phelypaea, media were selected with speci c compositions of micro-and macronutrients like they occur in the soils the host plants are growing on. C. armena grows in dry, saline, mainly clayey alkaline soils with pH 9-11 and ESP>20% (ESP-Exchangeable Sodium Percentage) 85 ; C. phelypaea is ourishing in sandy-silty zones of coastal salt marshes.
For further experiments single, morphological diverse colonies were picked. The chosen colonies were cultivated several times to get pure colonies with identical shape, color, size, and time of growth. These pure strains were used for DNA isolation. To verify purity of the colonies BOX PCR was used. The endophytic bacterial isolates were marked CA3, CA4, CA5, CA8, CA9, CA12, CA15 for C. armena and CP4, CP12 for C. phelypaea.

DNA isolation and polymerase chain reaction
DNA from the cultivable bacterial strains was isolated using Genomic Mini AX Bacteria Spin kits (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions. Genomic DNA was checked for quality using 1% gel electrophoresis agarose. Qualitative and quantitative assessment of the isolated DNA was provided. To assess the amount of matrix delivered, measurements were also made using a GelVue UV Transilluminator (Gbox Chemi16 Gel Imaging System Unit SYNGENE, UK, Cambridge).

Phylogenetic tree of bacterial endophytes
A maximum likelihood phylogenetic tree based on nearly complete 16S rRNA gene sequences was made (Fig. 7), showing the relationships between the nine bacterial strains isolated from seeds of C. armena and C. phelypaea and closely related strains from the database: Bacillus spp., Oceanobacillus, Gracilibacillus and Paenibacillus spp. The evolutionary history was inferred using the Neighbor-Joining method 86 . Evolutionary analyses were conducted in MEGA X 87 .

Identi cation of the total endophytic bacterial community
For identi cation of the total (cultivable and uncultivable) bacterial community, 50 mg of seeds was transferred to sterile 1.5 ml eppendorf tubes, and 1 ml 0.85 % sterile NaCl solution was added followed by shaking on a vortex (8,000 rpm) at 21°C for 2.5 h. Subsequently, the washed seeds were kept at 4°C for 15 min. Before rinsing with sterile double distilled water, the samples were centrifuged for 30 sec at 12,000 rpm (13,400 x g

Statistical analysis
The signi cance of differences in the numbers of OTU/genera was tested by using the Independent Sample T-Test by Jamovi Version 1.6 (2020, https://www.jamovi.org/). Due to the lack of normal distribution of the variables, comparisons among the C. armena and C. phelypaea for the phylum value were tested with the Mann-Whitney U test (p-value < 0.05 was considered signi cant).

Physiological traits of culturable bacterial seed endophytes isolated of C. armena and C. phelypaea
The culturable bacterial seed endophytes were investigated for their tolerance to temperature (25-50°C), salinity (0-20% NaCl) and pH 5.8. Biochemical activities of cultivable bacterial seed endophytes isolated of C. armena and C. phelypaea 5.8.1. Motility 89 and catalase activity 90 were tested according to the protocols mentioned in the papers.

Ureolytic activities:
The hydrolysis of urea was tested on modi ed Christensen medium. As a control Proteus vulgaris(ATCC13315) 91 strain was used.

Hydrolytic enzymes
Cellulose hydrolyzing ability: For determination of the cellulose-degrading ability of bacteria which use cellulose as carbon source, the cellulose Congo-Red agar medium 92 was employed. The cellulose Congo-Red agar medium without cellulose was used as a control.
Discoloration of Congo-Red demonstrates a positive cellulose-degrading reaction.

Protease
Protease activity was tested on skim milk agar plates 93 . After 72 h incubation at 25°C clean zones around colonies were observed. α-amylase: α-amylase activity was determined on Starch Agar. After 48 h, an iodine solution was used as an indicator. A clean zone around the colony indicated hydrolysis of starch and amylase production ability of bacteria 94 .

Pectinase
Pectinase activity was observed in basal medium. Clear zones diluted with Gram's iodine solution and dark blue background indicated pectinase production 95 .

Synthesis of plant growth promoting substances
Indole-3-acetic acid (IAA) synthesis Bacterial isolates were incubated in dark at 28°C in LB (Himedia™) liquid medium with tryptophan. The concentration of produced IAA was determined spectrophotometrically at 520 nm 96 .
Gibberellic acid (GA) production (Borrow method) Bacterial isolates were incubated in darkness onto Nutrient Broth medium at room temperature for 7 days. Absorbance was measured at 254 nm 96 .

Phosphate solubilization
The phosphate solubilization ability was tested in NBRIP (National Botanical Research Institute's Phosphate growth medium) medium for soil bacteria 97 . After incubation, the plates were checked for clean clear halos around the colonies. Pseudomonas sp. was chosen as a control.
All physiological, biochemical and PGP tests were performed at least two times.

Declarations
Data availability statement.
We declare that all data on the basis of which this manuscript was created are publicly available and disseminated in the manuscript itself or as supplementary materials. Figure 1 ZOOM microscopy micrographs of seeds: (a, b) Cistanche armena, (c, d) C. phelypaea.

Figure 2
Samples of seeds (Cistanche armena and C. phelypaea) planted intact (F) and after mechanical homogenization (H) on different growth media after surface sterilization. On the left side of the Petri dishes the intact (F) seeds (no bacterial growth) and on the right side crushed (H) seeds (growth of bacterial endophytes). Composition of the total seed endophytic communities of holoparasitic plant species Cistanche at phylum level.

Figure 5
Composition of the total seed endophytic communities of holoparasitic plant species Cistanche at genus level.

Figure 6
General habit of the studied species and their habitats: (a, b) Cistanche phelypaea in the coastal salt marshes in Portugal, (c, d) C. armena in the halophytic vegetation of semi-desert in Armenia. Phot. R. Piwowarczyk