Diversity and plant growth promoting traits of the seed microbial endophytes of holoparasitic Orobanchaceae from extreme environments

Abstract

In extreme environments the plants, besides hereditary biochemical and morphological features, possess a unique interaction with bacteria that colonize various tissues. These microorganisms are important players in the growth and development of their host plants in various environmental conditions. The plant endophytes are involved in regulation of plant growth and defense mechanisms by means of nitrogen-fixation, production of plant hormones like auxins (IAA), solubilization of phosphate, siderophores and ACC deaminase activity and may decrease the availability of harmful metals in plant tissues. There exists a considerable body of evidence, that the plant associated microbial communities have an important role in the adaptation of their host to the environmental conditions or abiotic stresses of the plant growing habitats; their beneficial effects on plant physiology are well documented. However, the diversity of the endophytic bacterial communities in seeds of Orobanchaceae from extreme ecosystems, their potential plant growth promoting traits, and the bacterial diversity profiling shifts during seed maturation are not fully understood. In this study, we considered the diversity of the bacterial endophytic communities of seeds of the Orobanche and Cistanche growing in extreme habitats. Numerous factors such as environmental conditions, host species, individual characteristics of the holoparasitic plant, the microbiome of the host plants of the Orobanche and Cistanche, soil structure and physicochemical characteristics, soil microbiome etc., may impact on the final composition of the endophytic bacterial communities of the seeds of Orobanchaceae. In this study we considered the effects of environmental conditions such as salinity, different soil water status and metal stress on shaping the seeds endophyte communities of Orobanche and Cistanche. We hypothesize that the seeds of holoparasitic plants harbored the bacterial endophytes appropriate to the environmental conditions of the growing area and have potential PGP traits depending on the habitat. In view of this, we aimed to identify the seed bacterial endophytes of Orobanchaceae species growing in extreme habitats under salt or metal stress and their plant growth promoting potential. Meanwhile, the shifts in bacterial diversity profiling during seed maturation under metal stress have been evaluated as well. The current PhD work demonstrates the bacterial endophytic community in seeds of holoparasitic plant species from extreme environments in distinct geographical locations: (i)

two endemic holoparasitic species Cistanche armena and C. phelypaea belonging to the genus Cistanche, growing in extreme environments: high salinity but different in soil water status, (ii) Orobanche lutea belonging to genus Orobanche growing in a metal polluted mining area. To verify the stated hypothesis a range of interdisciplinary methods, such as microbiological, molecular, biochemical, spectroscopic, bioinformatic and statistics were used. The findings provide a scientific insight into the diversity of seed endophytic bacteria of holoparasitic plants in various harsh environmental conditions and their potential PGP traits in these extreme natural habitats. To the best of our knowledge it is the first report about the diversity of the bacterial seed endophytes of C. armena, C. phelypaea and O. lutea species from extreme ecosystems and the potential plant growth promoting traits of these bacteria. A crucial and primary stage was the optimization of the sterilization procedure for the seed surface that allowed me to obtain only the endophytic bacterial communities of the seeds. For both examined seed populations of the genus Cistanche, the phyla Firmicutes, Proteobacteria, Actinobacteriota were dominating. However, depending on the soil water status, changes in the ratio of bacterial phyla were observed. The seeds of C. armena inhabited mainly bacteria belonging to Firmicutes as the predominating and most abundant phylum in arid saline habitats. The seeds of C. phelypaea were colonized in high abundance by Actinobacteriota, a characteristic phylum in wetland ecosystems. The isolated strains demonstrated high levels of IAA, organic acids, siderophores and ACC deaminase production. Among those, the classes Bacilli, Actinomyceta and β- Proteobacteria demonstrated the most positive responses in both examined seed populations. In C. phelypaea seeds, the γ-Proteobacteria was the most diverse phylogenetic group. The genus Micromonospora seems to harbor the most beneficial bacteria in seeds of C. phelypaea. Pantoea spp., Bacilllus spp. and Stenotrophomonas spp. seem the most favorable PGP seeds endophytes of C. armena. These halotolerant bacteria probably have potential to mitigate the adverse effects of salt stress on the holoparasite seeds. Possibly, they may also protect the embryo of the holoparasite against salt stress and keep the seeds viable for a long time. The research performed in this doctoral dissertation constitutes an interdisciplinary approach to evaluate the bacterial diversity profiling of the

seeds of O. lutea during seed maturation under metal stress and PGP traits of these bacteria in metal polluted soil. There are only a few species that may accumulate heavy metals and keep active metabolism. Our research demonstrates that the highest concentrations of Cr and Zn were present in flowers of O. lutea. Our results indicated that the ripe seeds have selective accumulation tendency for minerals depending on the soil pollution which had not been reported before. We highlight that in polluted soil, the highest concentration of Ni was found in stems and gradually decreased from stems to seeds, demonstrating the filtering effect. However, Pb accumulation tendency by O. lutea ripe seeds regardless of soil contamination were reported. The current results provided essential support for further analyzing the diversity of the bacterial endophytic community of O. lutea seeds under metal stress. Our findings address the differences of the community diversity with regard to metal presence in the soil and are quite different from the previous reports of researchers in regard to diversity and structure of bacterial communities inside young and ripe seeds. The bacterial richness of seeds from polluted and non- polluted sites was similar and did not changes during seed maturation. Metabarcoding revealed that the young and ripe seed microbiome of O. lutea, was mainly composed of 11 bacterial phyla from which Proteobacteria, Actinobacteriota and Firmicutes as predominating groups. Our findings are not consistent with the results of earlier studies. It seems that the sterilization procedure was crucial for bacterial richness in young seeds. Instead of aggressive sterilizing agents we used the washing method with intensive shaking. We assumed that metal stress, environmental and climatic conditions, the holoparasitic plant species and host genotypes influence the seed endophytic community diversity. We defined that the bacterial genera Citrobacter, Erwinia and Kocuria are specific for ripe seed generations from polluted soil. The genera Brevibacterium, Pseudomonas, Priestia, Streptomyces and Microbacterium were found only in ripe seeds from both sites. However, the genera Peribacillus, Promicromonospora, Sphingomonas seemed to belong to the core microbiome of O. lutea seeds. Curtobacterium and Plantibacter were isolated only from the young seeds from both sites. These genera are also considered to be a core bacteria in O. lutea seeds and may be transferred from generation to generation. The accumulation tendency of lead (Pb) in ripe seeds may potentially influence the PGP ability of endophytes. Pseudomonas spp.,

Microbacterium spp., Risungbinella sp. isolated from the seeds from metal polluted soils displayed the highest numbers of ACC deaminase positive responses. The IAA, siderophore, organic acids and ACC deaminase activity were predominantly found in strains isolated from O. lutea seed from metal polluted soils. In contrast, the strains isolated from the seeds from the reference area that demonstrated significantly lower positive responses. This supported the hypothesis that endophytic bacteria possess the PGP traits correlated with the natural habitat or abiotic stresses of their hosts. We assume that these endophytic bacteria have potential to mitigate the adverse effects of salt or metal stress in the seeds and plants the holoparasite. To elucidate the effective benefits of these endophytic bacteria for their host plants, and specifically for the seeds, seed germination and development of the seedling, more research is required.