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 specific conditions of germination9.
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 protocols39,40 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% H2O2 with intense shaking showed to be adequate to remove all bacteria from the surfaces of C. armena and C. phelypaea seeds (Fig. 2).
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 soil41. 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 processes42.
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 decades10. Our results confirm that spore forming Bacillus species are frequently isolated endophytes from seeds of plants growing in saline soils, solar salterns and salt marshes43–46. That the isolated strains are well adapted to the growing conditions of their host plants was confirmed 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 confirms their adaptability to high temperatures47 and might have been marked as plant growth promoting48 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 plant49,50. 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 shoots51. 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-glucanases53,54. 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 before55. 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 benefits 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 confirmed in all isolates, and we speculate that production of such compound by bacteria might be beneficial 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 plants56. 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 beneficial 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 conditions58. 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 specifically the Gammaproteobacteria is an important taxon in seeds of both, C. armena and C. phelypaea. Many Gammaproteobacteria are halophilic59, 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 soils60. 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 Bacteroidetes13,16,60−63. The results about the seed endophytic microbiome of the parasitic Phelipanche ramosa13 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 (Table 1). The phylum Gemmatimonadetes was only found in seeds of C. armena (0.03%). Different strains of Gemmatimonadetes were isolated from arid, semiarid and desert soils as well64. Besides of hereditary biochemical and morphological features desert plant show unique interactions with bacteria that colonize various plant parts. Well known, that the arid, semiarid and desert soils are nutrient poor, and the plants roots secrete organic compounds that attract bacteria which potentially can colonize the plant41. 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 structure64. Hence, considering the moisture requirements of C. phelypaea, Gemmatimonadetes are not expected to occur in the seeds of this species. Such information confirms our hypothesis, that water requirements and abiotic stresses influence the development of the seed bacterial community. Other bacterial phyla like Fibrobacteres, Chlamydiae, Cyanobacteria, Chloroflexi, Deinococcus–Thermus, WS6 were only present in the seeds of C. phelypaea. The phyla isolated from the seeds of C. phelypaea were recently also identified from sea water and anoxic marine sediments and were reported to have important ecological roles65. The Chloroflexi is a phylum including ecologically and physiologically diverse bacteria reported to occur in sediments, hot springs and methanogenic anaerobic sludge digesters66, but not from plant seeds. Another bacterial phylum, Deinococcus–Thermus, comprises extremophiles highly resistant to abiotic stresses67 and was recently found in seeds of different plant species25,68. 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 findings41,70. 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 also41,71,72. 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.
The seeds of C. phelypaea and C. armena are colonized by mostly unculturable, Gram negative, halotolerant bacteria that have an environmental origin. Some bacterial species were also isolated from other plants or soils, for instance, Serratia marcescens, Sphingomonas hylomeconis, Bacillus pumilus group, Bacillus cereus, Proteus mirabilis, Microbacterium halotolerans group, sp. nov. Kushneria spp, sp. nov. Halomonas stenophila Tamlana crocina, sp. nov.24,74,75. In seeds of C. armena Novosphingobium was determined. This genus was also identified by Iasur Kruh et al.11, in Phelipanche aegyptiaca and was reported as specific endophytes for this plant species.
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 bacteria76. 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 stresses77–79.