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The increase of human population and associated increasing demand for agricultural products lead to soil over-exploitation. Biofertilizers based on lyophilized plant material containing living plant growth-promoting microorganisms (PGPM) could be an alternative to conventional fertilizers that fits into sustainable agricultural technologies ideas. We aimed to: (1) assess the diversity of endophytic bacteria in sugar and sea beet roots and (2) determine the influence of osmoprotectants (trehalose and ectoine) addition during lyophilization on bacterial density, viability and salt tolerance. Microbiome diversity was assessed based on 16S rRNA amplicons sequencing, bacterial density and salt tolerance was evaluated in cultures, while bacterial viability was calculated by using fluorescence microscopy and flow cytometry. Here we show that plant genotype shapes its endophytic microbiome diversity and determines rhizosphere soil properties. Sea beet endophytic microbiome, consisting of genera characteristic for extreme environments, is more diverse and salt resistant than its crop relative. Supplementing osmoprotectants during root tissue lyophilization exerts a positive effect on bacterial community salt stress tolerance, viability and density. Trehalose improves the above-mentioned parameters more effectively than ectoine, moreover its use is economically advantageous, thus it may be used to formulate improved biofertilizers.
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
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Choosing source of microorganisms
and processing technology
for next generation beet
bioinoculant
Sonia Szymańska1, Marcin Sikora2, Katarzyna Hrynkiewicz1*, Jarosław Tyburski2,3,
Andrzej Tretyn2,3 & Marcin Gołębiewski2,3*
The increase of human population and associated increasing demand for agricultural products lead
to soil over-exploitation. Biofertilizers based on lyophilized plant material containing living plant
growth-promoting microorganisms (PGPM) could be an alternative to conventional fertilizers that ts
into sustainable agricultural technologies ideas. We aimed to: (1) assess the diversity of endophytic
bacteria in sugar and sea beet roots and (2) determine the inuence of osmoprotectants (trehalose and
ectoine) addition during lyophilization on bacterial density, viability and salt tolerance. Microbiome
diversity was assessed based on 16S rRNA amplicons sequencing, bacterial density and salt tolerance
was evaluated in cultures, while bacterial viability was calculated by using uorescence microscopy
and ow cytometry. Here we show that plant genotype shapes its endophytic microbiome diversity
and determines rhizosphere soil properties. Sea beet endophytic microbiome, consisting of genera
characteristic for extreme environments, is more diverse and salt resistant than its crop relative.
Supplementing osmoprotectants during root tissue lyophilization exerts a positive eect on bacterial
community salt stress tolerance, viability and density. Trehalose improves the above-mentioned
parameters more eectively than ectoine, moreover its use is economically advantageous, thus it may
be used to formulate improved biofertilizers.
Conventional agriculture practices negatively aect environment, e.g. by decreasing microbial diversity, soil
quality, water supply and plant productivity1,2. Wide adoption of sustainable agricultural technologies, e.g. biofer-
tilizers, may signicantly decrease the use of chemical fertilizers, reducing negative consequences of agriculture
on the environment2,3.
Biofertilizers are based on living plant growth-promoting microorganisms (arbuscular mycorrhizal fungi—
AMF, plant growth-promoting rhizobacteria—PGPR, nitrogen xing bacteria—NFB) and are key players in
sustainable agriculture4. ey can promote plant growth in several dierent ways (e.g. increasing availability
of nutrients, synthesizing phytohormones or siderophores, xing nitrogen), especially under unfavorable envi-
ronmental conditions (e.g. drought or salinity)57. Most of commercially available biofertilizers are based on
combination of two or more microbial benecial strains, which is called ‘consortium4. Compared to single
strains, consortia display increased spectrum of benecial eect of inoculum on plants. However, criteria of
strain selection are the crucial factor inuencing the inoculum success and should be considered not only based
on plant genotype compatibility but also environmental factors.
e methods of biofertilizers production, storage and application are diverse. Inoculation techniques are
based on microorganisms application in liquid (sprays and drenches) or solid form (lyophilizates delivered to
soil/growth substrate). e most important problem in the preparation and storage technology of biofertilizers is
maintaining high viability of microorganisms8. Lyophilization is well known and widely used technique extend-
ing microbial cell viability9. To alleviate negative eect of low temperature and desiccation on microorganisms
in this technology, several dierent stabilizers can be used e.g. nonreducing disaccharides, glicerolglycerol or
skim milk10.
OPEN
Department of Microbiology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University

             
        *email: hrynk@
umk.pl; mgoleb@umk.pl
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Trehalose (α--glucopyranosyl-(1 1)-α--glucopyranoside) is a disaccharide present in almost all prokary-
otic and eukaryotic organisms and exhibits high eciency in protection of cells against low temperature, drying
and osmotic stress1012. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is synthesized mostly
by halotolerant and halophilic microorganisms and responsible for regulation of osmotic pressure in microbial
cells, increasing their tolerance to osmotic stress (salinity)1316. Application of trehalose and ectoine in the process
of lyophilization of endophytic microbiomes was tested in our work for the rst time.
Biofertilizer eciency analyzed under laboratory conditions may not correspond to results obtained under
eld conditions2,17. is eect may be due to adverse eect of environmental conditions or autochthonic micro-
organisms on gene expression in microbial cells2 or low competitiveness of microorganisms used as biofertilizers,
i.e. they may be outgrown by the autochthonic ones. is is why “plant microbiome” was proposed as the new
generation of inoculants18. Inoculation of crops with microbiome and organic matter present in lyophilized
plant roots seems to be a better solution to enrich microbial biodiversity of soil and crops with new endophytes.
Endophytes are bacteria and fungi that colonize the internal plants tissues without causing pathogenic
symptoms19 and can directly (nitrogen xation, phosphate solubilization, siderophore and phytohormone syn-
thesis) and/or indirectly (biocontrol agents) promote plant growth and development, e.g. in crops20.
Recent data show that more than 7% of global land surface and 70% of all irrigated agricultural soils world-
wide is aected by salinity21, and the problem is exacerbated by inorganic fertilization as well as by climate
changes. Moreover, halophytes thriving in naturally saline environments are reservoirs of endophytes possessing
high tolerance to salt stress22,23 that may be useful in alleviation of salt stress in crops. Application of halotoler-
ant microbes in sustainable agriculture e.g. in the increasing salinity tolerance of non-halophytic crops, is well
known and was extensively studied6,2427.
Cultivated beets are one of the few crops whose direct ancestor (sea beet, Beta vulgaris ssp. maritima) still
grows in the wild. is feature enables comparison of traits in plants that are very close genetically (ca. 0.5%
dierence28), but whose ecology diers considerably. Moreover, as sea beet is as a halophyte growing in nature29,
it seems to be a good candidate for a source of microorganisms that could be useful for crop beets improvement.
We analyzed both rhizosphere soil and plant roots to determine if the plant inuence on the former is strong
enough to make it the source of microbes for bioinoculant formulation.
e goal of our study was twofold: (i) to characterize rhizosphere and root microbiomes of cultivated and wild
beet and choose the source of microorganisms for prospective bioinoculant formulation for growing beet in saline
soils and (ii) to check if addition of osmoprotectants during lyophilization changes root bacterial community
structure as well as microbiome salinity tolerance and viability. Specically, we formulated two general hypoth-
eses: (i) roots of dierent beet genotypes would lter rhizosphere microbes specically and therefore they would
be better source of microbes for bioinoculation than rhizosphere soils, (ii) addition of osmoprotectants would
allow formulation of a better inoculant because of increased bacterial viability and microbiome salinity tolerance.
Results
Rhizosphere as a source of microbes in endosphere. Rhizosphere soil physicochemical parameters
are dierent for sugar and sea beet. Majority of tested parameters was higher in sugar beet soil, but only in
cases of CaCO3 and Na+ the dierence was statistically signicant. On the other hand, OC, P, Ca2+, Mg2+ and Nt
were higher in sea beet soil and for the latter the dierence was signicant (Table1). To check if these changed
parameters inuenced bacterial communities in rhizosphere soils, we sequenced 16S rRNA amplicon libraries.
Bacterial diversity in sugar beet roots is lower than in its wild ancestor. Bacterial diversity, evenness and species
richness were the highest in 16S rRNA libraries coming from rhizosphere soil, regardless of plant genotype. Lyo-
Table 1. Physico-chemical rhizosphere soil parameters (mean and standard deviation) obtained aer three
months of cultivation of sugar- and sea beet. [] signicantly higher level based on Newman-Keuls test of
rhizosphere soil parameter observed between the plant species.
Parameter\plant genotype cv. ’Huzar’ B. maritima
OC (%) 4.97 (1,646) 5.66 (1,210)
Nt (%) 0.28 (0.032) 0.34 (0.025) []
CaCO3 (%) 1.88 (0.178) [] 1.6 (0.107)
Pcitr. [mg/kg] 1183,08 (116,312) 1253,29 (48.876)
pH 7.1 (0.077) 7.0 (0.074)
EC 1:5 [µS·cm−1] 176,93 (57,826) 142,28 (23,973)
Na+ [mg·dm−3] 16,80 (7,652) [] 7,35 (1,885)
K+ [mg·dm−3] 2,95 (0,644) 2,55 (1,111)
Ca2+ [mg·dm−3] 9,63 (2,291) 12,93 (2,916)
Mg2+ [mg·dm−3] 1,28 (0,306) 1,52 (0,223)
Cl [mg·dm−3] 46,02 (15,494) 38,94 (2,890)
SO4y2− [mg·dm−3] 39,35 (5,211) 38,58 (7,998)
HCO3 [mg·dm−3] 94,55 (14,306) 88,45 (29,063)
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philized sea beet roots harbored more diverse community than sugar beet (Fig.1). e number of OTUs was ca.
three times higher in the wild beet than in the crop (Fig.1A,B), while the diversity was around 1.5 times higher
(Fig.1C), and evenness was ca. 1.3 times greater (Fig.1D).
Both endophytic and rhizosphere soil bacterial community is dominated by Proteobacteria. ere were no sig-
nicant dierences in taxonomic composition of rhizosphere soil bacterial communities of sugar- and sea beet
at the level of phylum (Fig.2A). At the level of genus, three taxa were dierentially represented, all of them
belonging to Alphaproteobacteria: two Rhizobiales-belonging genera, Pedomicrobium and an unknown genus
of JG34.KF.361 family as well as Woodsholea (Caulobacteraceae) were more abundant in the crop (Fig.2C). Dif-
ferences in lyophilized roots communities were more pronounced, although still there were no taxa signicantly
dierentially represented between osmolyte treatments. At the level of phyla Proteobacteria-derived reads were
more abundant in libraries from sugar beet lyophilized roots, while Actinobacteria, Bacteroidetes, Acidobac-
teria, Verrucomicrobia and rare phyla were more abundant in its wild ancestor (Fig.2B). Among genera sig-
nicant dierences were observed for Stenotrophomonas and Bacillus that were more abundant in the crop and
for proteobacterial genera Novosphingobium, Devosia (Alphaproteobacteria), Hydrogenophaga, Polaromonas
(Betaproteobacteria), Rhizobacter and Tahibacter (Gammaproteobacteria) as well as for rare and unclassied
genera being more abundant in sea beet (Fig.2D).
Figure1. Species richness, evenness and diversity of bacterial communities in rhizosphere soils of sugar beet
(Bh) and sea beet (Bm) and lyophilized roots of these plants untreated (C), and treated with ectoine (E) or
trehalose (T). Means (n = 8–32) are presented, whiskers show standard error of the mean (SEM), and signicant
dierences (ANOVA, p < 0.05) are denoted with dierent letters. Observed number of OTUs (A), estimated total
number of OTUs (Chao1 index, B), Shannons diversity index (H, C), Shannon’s evenness (D).
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At the level of phyla , regardless of plant genotype, there were signicant dierences between soil and roots
in all taxa but Firmicutes. Proteobacteria and Firmicutes were more abundant in roots than in soil, while abun-
dance of the remaining phyla was lower in planta, and Gemmatimonadetes as well as Verrucomicrobia were
absent from roots. At the level of genus, regardless of genotype, Pseudomonas and Rhizobium were signicantly
more abundant in roots than in soil, while Sphingomonas, Pedomicrobium, rare and unclassied bacteria were
less frequent in roots than in rhizosphere. Novosphingobium, Pantoea, Hydrogenophaga, Polaromonas, Paeni-
bacillus, Hyphomicrobium and Rhizobacter were signicantly more abundant in wild beet roots than in soil,
while Stenotrophomonas was the only genus that was more frequent in sugar beet roots than in soil. Variibacter,
Chryseolinea, and Woodsholea were less abundant in wild beet roots than in soil, while Devosia and Hirschia
were less frequent in sugar beet than in soil.
Eect of osmolytes on diversity, viability, and tolerance to salinity of bacterial communities
in lyophilized beet roots. Bacterial cell density in lyophilized roots depends on host genotype but not on
Figure2. Taxonomic composition of bacteria communities in rhizosphere soils of sugar beet (Bh) and sea beet
(Bm) (A, C) and lyophilized roots of these plants (B, D) untreated (Bh_C and Bm_C) and treated with ectoine
(Bh_E, Bm_E) or trehalose (Bh_T, Bm_T) at the phylum (A, B) and genus (C, D) levels. Means (n = 8–32) are
presented, and signicant dierences between genotypes are marked either with m and h letters (panels A and
C, signicant dierences between rhizosphere and endosphere in sea (m) or sugar (h) beet) or with asterisks
(panels B and D, signicant dierences between genotypes, no dierences due to osmolytes were found).
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osmolyte. In total, 72 bacterial strains were isolated and identied, 35 coming from sugar beet and 37 from sea
beet. Proteobacteria were the most frequent phylum in fresh roots of both sugar and sea beet, followed by Act-
inobacteria in the crop and Firmicutes in the wild plant. Pseudomonas and Sphingomonas were characteristic for
fresh roots of sugar beet, while Bosea and Sphingopyxis were found exclusively in sea beet roots before lyophiliza-
tion (Table2). Density of culturable root endophytic bacteria was higher in sugar beet lyophilizates than in sea
beet (ANOVA, p < 0.05, Fig.3), regardless of the osmolytes addition. We observed no inuence of osmolytes on
sea beet endophytes density, while trehalose increased slightly, but signicantly (ANOVA, p < 0.05) the density
in sugar beet samples (Fig.3).
Sea beet endophytes are more salt tolerant than sugar beet ones. Increasing salinity negatively aected growth of
culturable fraction of microbiome regardless of origin (sea- vs. sugar beet), however stronger eect was observed
for sugar beet. In control treatment the growth was inhibited (nal cell density below the critical level of 0.2
OD600) at 200mM and 300mM NaCl concentration for sugar and sea beet, respectively. Addition of osmolytes
enhanced the growth in general and increased the inhibitory concentration to 400 and 700mM, respectively
(Supplementary Table1). Inuence of both osmolytes was similar, with trehalose performing slightly better at
high NaCl concentrations., e eect was greater for sea beet, than for sugar beet (Fig.4).
Bacterial viable cell density in lyophilized roots is associated with plant genotype and osmolyte. Cell viability in
lyophilized beet roots was assessed by means of three, complementary methods: via plate counts, uorescence
Table 2. Identication of cultivable endophytic bacteria associated with roots of sugar- and sea beet before
and aer lyophilization without addition of any osmolyte (C) or supplemented either with ectoine (E) or
trehalose (T).
Aer lyophilization Genotype
Treatment cv. ‘Huzar’ B. maritima
C
1Gordonia sp. BH1CTR8 (A) Bacillus sp. BM1CTR1 (F)
2Bacillus sp. BH1CTR2 (F) Bacillus sp. BM1CTR10 (F)
3Bacillus sp. BH1CTR5 (F) Bacillus sp. BM1CTR9 (F)
4Bacillus sp. BH3CTR4 (F) Bacillus sp. BM3CTR10 (F)
5Paenibacillus sp. BH3CTR10 (F) Bacillus sp. BM3CTR4 (F)
6Acinetobacter sp. BH2CTR5 (P) Psychrobacillus sp. BM3CTR11 (F)
7Pantoea sp. BH4CTR1 (P)
8Pseudoxanthomonas sp. BH3CTR6 (P)
9Pseudoxanthomonas sp. BH3CTR9 (P)
10 Shinella sp. BH1CTR1 (P)
E
1Bacillus sp. BH1EKT5 (F) Bacillus sp. BM1EKT11 (F)
2Bacillus sp. BH1EKT9 (F) Bacillus sp. BM1EKT2 (F)
3Bacillus sp. BH4EKT3 (F) Bacillus sp. BM2EKT5 (F)
4Bacillus sp. BH4EKT5 (F) Bacillus sp. BM4EKT1 (F)
5Pseudoxanthomonas sp. BH1EKT3 (P) Bacillus sp. BM4EKT10 (F)
6Pseudoxanthomonas sp. BH5EKT5 (P) Shinella sp. BM1EKT6 (P)
7Sphingobium sp. BH1EKT10 (P) Stenotrophomonas sp. BM4EKT2 (P)
8Sphingobium sp. BH2EKT1 (P) Stenotrophomonas sp. BM4EKT3 (P)
9Stenotrophomonas sp. BM4EKT5 (P)
10 Stenotrophomonas sp. BM4EKT8 (P)
T
1Bacillus sp. BH3TRE4 (F) Nocardiopsis sp. BM4TRE1
2Pantoea sp. BH4TRE2 (P) Bacillus sp. BM1TRE9 (F)
3Pantoea sp. BH4TRE3 (P) Bacillus sp. BM3TRE11 (F)
4Pseudomonas sp. BH4TRE5 (P) Bacillus sp. BM3TRE8 (F)
5Pseudomonas sp. BH4TRE1 (P) Bacillus sp. BM4TRE10 (F)
6Shinella sp. BH2TRE2 (P) Bacillus sp. BM4TRE4 (F)
7Shinella sp. BH2TRE3 (P) Pseudomonas sp. BM1TRE2 (P)
8Pseudomonas sp. BM3TRE2 (P)
9Pseudoxanthomonas sp. BM1TRE1 (P)
10 Shinella sp. BM3TRE3 (P)
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microscopy and ow cytometry (Fig.5). Bacterial viability in sugar beet was consistently higher than in roots of
its wild relative, regardless of osmolyte treatment, storage time and measurement methodology. Both trehalose
and ectoine increased the viability compared to control, regardless of genotype, but the eect of the former was
more pronounced (Fig.5).
Discussion
Bacterial diversity in beet rhizosphere. Dierences in rhizosphere soil physicochemical properties
observed in our study, may be due to greater nutritional demands of the two beet genotypes (TN, Na) or vary-
ing exudates composition (OC), as it was found that rhizodeposition is the primary organic carbon source in
the rhizosphere30. Alternatively, they might be caused by changes in microbial activity resulting from microbial
metabolic activity or interaction between microorganisms31,32.
Greater microbiome diversity in rhizosphere compared to endosphere was commonly observed, and resulted
from natural plant selection mechanisms3335. Accordingly, in our study, the higher bacterial diversity, evenness
and species richness were noted in rhizosphere soil of both investigated genotypes, than in roots. At the same
time, in spite of slightly dierent TN, OC and Na levels, microbiome composition and diversity were similar in
rhizosphere soils of both studied plant genotypes. is observation could be explained by the use of the same
starting substrate (garden soil) and short culture period (three months), not allowing the rhizosphere dier-
ences to fully manifest. Culture-independent analysis revealed that dominating bacterial phyla were the same
as those observed in rhizosphere of many plant species e.g. barley, alfalfa or wheat3638. Only a few dierences
between the genotypes were noted at the genus level, mainly concerning Alphaproteobacteria. Pedomicrobium as
well as JG34.KF.361_ge, more frequent in sugar beet, represent Rhizobiales, an order known for organisms that
establish benecial interactions with plants and comprises numerous bacteria with nitrogen-xing capability39.
e observed lower TN level in the sugar beet rhizosphere may indicate higher demand for nitrogen. Tsuru-
maru and colleagues40 indicated that Mesorhizobium and Bradyrhizobium, also belonging to Rhizobiales, play
an important ecological role in the taproot of sugar beet. Moreover, it was showed that higher levels of nitrogen
(N) and potassium (K) signicantly aect the growth parameters of sugar beet. Both elements were generally
recognized as crucial for obtaining higher yields of this crop, favorably aecting organic metabolites biosynthesis
and improving nutritional status41.
Bacterial diversity in beet roots. e higher diversity both in rhizo- and endosphere of the wild plant
compared to its crop counterpart was observed42,43. It was hypothesized that benecial endophytes associated
with wild plants were absent or fewer in domesticated crops43. Sugar beet as a cultivated plant grows under
more controlled conditions regulated by farmers, while sea beet grows mainly in highly saline and nutrients
poor coastal soil28. Growth under adverse environmental conditions requires support of microorganisms with a
wide range of benecial metabolic properties tailored for specic plant needs23. e loss of high tolerance to salt
stress during the process of sea beet domestication was demonstrated29 and might be associated with the loss of
Figure3. Density of endophytic bacteria (expressed as log10 CFU per g of dry weight) isolated from lyophilized
sugar- and sea beet roots. Means (n = 3) ± standard deviation are presented. Signicant dierences between
variants (ANOVA, p < 0.05, with Tukey’s HSD; C–control untreated with any osmolyte, ectoine (E) or trehalose
(T) treated) were marked with dierent letters.
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Figure4. Osmolytes eect on growth of LB cultures inoculated with lyophilized sugar and sea beet roots
untreated with any osmolyte (C), treated with ectoine (E) and trehalose (T). Means (n = 4–6) ± standard
deviation are presented. Signicant dierences between treatments (ANOVA, p < 0.05, with Tukey’s HSD) are
marked with asterisks.
Figure5. Bacterial viability in lyophilized beet roots. Viability measured with BD Cell Viability kit under
uorescence microscope (AB) and using ow cytometer (C). Means are presented and statistically signicant
dierences between treatments are marked with diering letters. Stars denote signicant dierences between
genotypes.
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microbes that increased tolerance of this plant to salinity. Concordantly, despite the lack of dierences in rhizos-
phere soil microbial composition, lower diversity of endophytes in sugar beet compared to its wild ancestor was
noted in our study. is dierence might be explained by varying root system architecture, with brous root
system of sea beet providing more opportunities for bacteria to enter the endosphere33, which aects stochastic
community assembly. On the other hand microbe selection can be driven by the genetic makeup of two studied
subspecies. We observed that sea beet caused decrease in the soil Na level, suggesting accumulation of Na ions
in wild plant tissues. Accordingly, there was an increase in community salinity resistance in this plant, which
pointed at higher level of halotolerant and halophytic microorganisms.
In general, endophytic microbiome diversity and composition is related to soil properties as well as plant ecol-
ogy and physiology44. Members of only three phyla (Proteobacteria, Actinobacteria and Firmicutes) were cultured
in our experiment, this may be related to their high ability to grow on commercially available media5,6,44,45. It was
emphasized that Proteobacteria distinctly predominate among culturable plant endophytes, then the presence of
Firmicutes and Actinobacteria is common, and Bacteroidetes occur slightly less frequently44.
16S rRNA gene libraries generated in our study were dominated by the four phyla (Proteobacteria, Actino-
bacteria, Firmicutes, Bacteroidetes) commonly found in endosphere of glycophytes including maize (Zea mays
L.46), Dactylis glomerata L., Festuca rubra L. and Lolium perenne L.47 as well as in halophytes such as Salicornia
europaea23 or para grass (Urochloa mutica48). Sea beet was characterized by signicantly higher frequency of
Actinobacteria, Bacteroidetes, Acidobacteria, Verrucomicrobia and rare phyla compared to sugar beet, where
Proteobacteria were observed more oen. Zachow etal. observed greater frequency of Actinobacteria, Bacteroi-
detes and Verrucomicrobia in rhizosphere of wild beet cultivated in coastal soil than in sugar beet rhizosphere42.
is fact, together with our results, may point at these bacterial taxa being preferred by sea beet regardless of soil.
Our 16S rRNA gene sequencing results also revealed signicantly higher abundance of certain genera in sea
beet endosphere, including: Novosphingobium, Devosia (Alphaproteobacteria), Hydrogenophaga, Polaromonas
(Betaproteobacteria), Rhizobacter and Tahibacter (Gammaproteobacteria) as well as certain rare and unclassi-
ed bacteria. is set of microorganisms comprises extremophiles, e.g. Polaromonas49 or Hydrogenophaga50 and
organisms modulating plant stress response, such as Novosphingobium25. In our study, only Stenotrophomonas and
Bacillus genera were more frequent in roots of sugar beet than of sea beet. Stenotrophomonas and Pseudomonas
sp. were identied in rhizospheric soil of sugar and sea beet, while the former together with Staphylococcus sp.
were mainly observed in crop rhizosphere. Sea beet microbiome was found to be more diverse than that of sugar
beet, which is explained by greater number of rare taxa. It was found that sugar beet rhizosphere was more fre-
quently colonized by strains with antagonistic activity against plant pathogens and/or stress protection activity,
while abiotic stress-releasing ones were more oen found in sea beet’s rhizosphere42. ese facts together with
our results suggest that pre-adaptation to stress observed in sea beet transcriptome51 may also take place at the
level of microbiome serving as a helper.
Osmoprotectants enhance bacterial viability and diversity in lyophilized beet roots. Signi-
cantly higher cell density of culturable bacteria observed in sugar beet lyophilized roots can be attributed to high
content of sucrose. is sugar acts as a natural osmoprotectant, allowing better viability of microorganisms dur-
ing lyophilization52. Another explanation of obtained results can be associated with higher ability of sugar beet
endophytes to grow on solid medium.
Sea beet endophytic microbiome was found to be more resistant to salinity. Microorganisms present in a
more saline sea beet tissue most likely developed mechanisms of adaptation to high salt level, which provided
them ability to grow in higher NaCl concentrations compared to the sugar beet microbiome. is fact may be
related to higher sodium accumulation in this plant tissues51, which caused decrease of soil sodium concentra-
tion observed in our study.
Salinity-induced changes in community structure and adverse eects on microbial density, activity, biomass
were reported by many scientists53,54. e decrease in number of culturable microorganisms related to increas-
ing NaCl concentration was noted even in the case of endophytes associated with halophytes (Aster tripolium,
Salicornia europaea)5,6,55. Obtained results were in line with the above trend, but apart from negative eect of
salinity on sugar and sea beet bacterial density, a benecial impact of trehalose and ectoine on salt stress mitiga-
tion was demonstrated. Although ectoine is a major osmolyte in aerobic chemoheterotrophic bacteria and is
considered as a marker for halophytic bacteria15, a slightly better eect of trehalose, was conrmed by the results
of microscopic analyzes, ow cytometry and culture tests. Protective eect of trehalose is explained by “water
replacement hypothesis” that states that the compound lowers the phase transition temperature of membrane
phospholipids, by replacement of water molecules occurring around the lipid head groups56, thus protecting
membrane structure57. is suggests that the use of trehalose is a better and more economic solution providing
high viability of bacterial cells aer lyophilization. In the case of sugar beet the above mentioned positive sucrose
impact was enhanced by trehalose addition. Similar eect was observed for rhizobial strains, where trehalose
worked better than sucrose/peptone mixture58. In general, 16S rRNA gene sequencing results considering diver-
sity of endophytes associated with sea and sugar beet root did not show any eect of applied osmoprotectants
neither on alpha nor beta diversity of bacteria. is observation can be explained by the presence of ‘relic DNA,
i.e. DNA coming from non-viable cells59 in lyophilized samples.
Bacillus sp. was the only species identied among the strains representing the Firmicutes phylum isolated
from the lyophilized osmolytes-treated roots of both investigated genotypes. In the control variant the presence
of Psychrobacillus sp. and Paenibacillus sp. inside sea and sugar beet root was additionally found, respectively.
e viability of the above-mentioned bacteria aer lyophilization was probably associated with their commonly
known ability to form endospores and higher tolerance to environmental changes6062. Actinobacteria proved
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to be sensitive to lyophilization, while Proteobacteria remarkably well tolerated it, and additional osmolytes
promoted the incidence of culturable bacteria belonging to the latter phylum.
Conclusions
Our research revealed that plant genotype played a pivotal role in the shaping of its endophytic microbiome
diversity and physicochemical rhizosphere soil properties, aecting soil sodium content, but not soil bacterial
community structure. Bacterial diversity was lower in sugar beet roots than in its wild ancestor tissues. At the
same time sea beet endophytic microbiome was more salt resistant and consisted of genera characteristic for
extreme environments.
Supplementing osmoprotectants during root tissue lyophilization had a positive eect on bacterial salt stress
tolerance, viability and density. Trehalose proved to improve these parameters more eectively than ectoine,
moreover its use was economically advantageous.
Materials and methods
Experimental design. Sea beet (Beta vulgaris L. subsp. maritima L.) seeds were obtained from National
Germplasm Resources Laboratory, Beltsville, MD, USA, while in the case of sugar beet (B. vulgaris subsp. vul-
garis cv. ’Huzar’) commercial seeds were bought from WHBC Poznań, Poland. Healthy and uniform-sized seeds
were placed in 5l pots lled with 2.5kg of garden soil. From twenty plants, ve representative ones (with two
pairs of true leaves and similar in size) were chosen for analysis. Pot experiment was conducted from mid-March
through mid-May 2017 in a greenhouse (Nicolaus Copernicus University in Toruń, Poland). Plants were grown
under natural lighting conditions and temperature was maintained at 22–24°C throughout the growth period.
All plants were arranged randomly on the green house benches. e plants were watered with tap water every
two days, amount depended on the plants demand. Aer three months plants and rhizosphere soil samples were
collected and analyzed as shown in Fig.6.
Soil analysis. Soil parameters (TOC, TN, CaCO3, Pcitr, pH, EC, Na, K, Ca, Mg, Cl, SO42−) were analyzed as
described earlier in Furtado etal.27.
Plant and soil samples preparation. Plants were carefully uprooted, and 10g of soil adhering to roots
(rhizospheric soil) was collected, frozen at − 80°C and lyophilized before DNA isolation for metagenomic analy-
sis. Roots were washed with tap water to remove soil and were separated from shoots and leaves. en, they were
surface sterilized with 70% ethanol and 15% hydrogen peroxide mixture (1:1 v:v) for 5min and subsequently
rinsed six times with 0.9% NaCl. Eciency of the sterilization process was evaluated by plating the last rinse on
LuriaBertani (Difco LB Agar, Miller) and potato dextrose extract (Lab A Neogen Company) media. Only prop-
erly sterilized plant material was used for subsequent analyzes. Approximately 100g of fresh root material was
homogenized in 100ml of 0.9% NaCl by using surface sterilized (rinsed with 70% ethanol and UV-irradiated)
blender. Homogenates were used to evaluate bacterial density and to prepare lyophilizates.
Roots lyophilization. Homogenized sugar and sea beet roots were used to prepare three variants of lyo-
philizates including (1) no osmolytes addition (control—C) (2) trehalose (T) and (3) ectoine (E) supplemented.
ree biological replicates were prepared for each tested plant species (9 samples per plant species, in total 18
samples were used for downstream analyzes). Either 1ml of 0.9% NaCl (control) or 1.0mg of trehalose (Tre)
or 1.0mg of ectoine (Ect) were mixed with 50g of homogenized roots. e mixtures were lyophilized in Telstar
LyuQues (DanLab) until completely dry (approximately 24h).
Estimation of bacterial density. Serial dilutions were prepared directly from the homogenized fresh
roots and lyophilizates re-suspended in 0.9% NaCl (1:9m:v). e dilutions (10−3 to 10−8) were plated in tripli-
cates on LB plates supplemented with nystatin (Sigma, 100µg/ml) to prevent fungal growth, and the plates were
incubated for 5days at 26°C. Colony counts (expressed as CFU per 1g of fresh or dry weight for homogenates
and lyophilizates, respectively) were based on plates with 30–300 colonies. At least six bacterial isolates were
puried per experimental variant.
Bacterial viability assessment: uorescence microscopy and ow cytometry. Ten-miligram
samples of ground lyophilized roots were mixed with 10ml of PBS (pH = 7.4) and incubated for 2days at 26°C
with mixing. e mixtures were ltered through a 40µm cell strainer (Biologix) and 2ml were centrifuged
for 3min at 1000 × g at RT to pellet the residual plant debris. Cells in the supernatant were stained with Cell
Viability kit (BectonDickinson) as per the manufacturer’s protocol, than bacterial viability was analyzed using
uorescence microscopy (aer 6 and 12months of storage) and ow cytometer (aer 12months storage). Prepa-
rations were photographed in red and green channel under 40 × magnication upon uorescence excitation with
433nm light on Axiostar plus uorescence microscope (Zeiss) equipped with Delta Optical camera. Percentage
of live cells was based on counts from at least 30 view elds per sample. Flow cytometric analysis was performed
on samples stained as described above with FACS Aria III (BectonDickinson) using 488nm laser for excitation.
Fluorescence was collected at 530 ± 30nm (for thiazole orange—TO) and 616 ± 26nm (for propidium iodide—
PI) bands and seventy-micrometer nozzle was used. Parameters were optimized basing on pure environmental
strains and their mixtures analyses and autoclaved lyophilizate samples served as negative controls.
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Salt tolerance assessment. Salt tolerance of root bacterial communities was measured as OD600 aer
5days incubation at 26°C using 96-wells microtiter plate reader (Biolog Micro Station). 140µl of LB medium
supplemented NaCl to obtain nal concentrations of 0, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900mM
were used per well. Inoculates were prepared by suspending 2g of mortar-ground lyophilized roots in 18ml of
0.9% NaCl and diluting the mixture ten times. e inoculates were ltered through 40µm cell strainer (Biologix)
to remove plant debris. Six test and two control wells were inoculated with 10µl of ltered inoculate or 0.9%
NaCl, respectively.
Isolates identication by 16S rRNA gene sequencing. Genomic DNA was isolated from puried
strains using GeneMatrix Bacterial and Yeast Genomic DNA Purication Kit (EurX) according to the manufac-
turer’s protocol with modied homogenization step (FastPrep-24 bead-beater, one cycle of 20s at 4.0m/s). e
DNA was analyzed spectrophotometrically (NanoDrop 2000). 16S rRNA gene fragment was amplied using
27F and 1492R primers63, following the procedure described in Szymańska etal.6. e products were puried
with GeneMatrix PCR/DNA Clean-Up DNA Purication Kit (EurX) according to the manufacturer’s protocol.
Sanger sequencing was performed with BrightDye Cycle Sequencing kit (Nimagen), using 40ng of template
DNA, 1.5pmol of primer and 1µl of kit and 1.5µl of BD buer in 10µl volume. e reactions were EtOH/NaAc
precipitated and read out at IBB PAS, Warsaw, Poland.
16S rRNA gene fragment library construction and sequencing. Metagenomic DNA was isolated
and V3-V4 16S rRNA gene fragment libraries for Illumina sequencing were prepared as described earlier64. ey
were sequenced on Illumina MiSeq using 600 cycles v.3 kit at CMIT NCU.
Figure6. Experimental design.
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Statistical analysis and bioinformatics. Bioinformatics analyses of Illumina reads was performed as
described earlier64. Briey, the reads were denoised, merged and chimeras were removed with dada265, then
amplicon variant sequences were exported together with abundance information and processed in Mothur
v.1.3966: aligned against SILVA v.132 database, screened for those covering the 6428-22400 positions of the align-
ment, ltered to remove gap-only and terminal gap-containing positions, pre-clustered to remove residual noise
and clustered into 0.03 dissimilarity OTUs. Representative OTU sequences were classied using naïve Bayesian
classier67 and SILVA database68. Sanger reads were manually inspected in Chromas to remove obvious errors,
the corrected sequences were merged with CAP369, and classied using naïve Bayesian classier with SILVA
v.132 reference les.
Signicance of dierences between means was assessed with ANOVA test with Tukey’s post-hoc analysis
implemented in Statistica 10.0 (StatSo). Normality of data was tested with Shapiro–Wilks test and homogeneity
of variance was assessed with Levene’s test. When the assumptions were violated, non-parametric Kruskal–Wallis
test with Dunns test as a post-hoc analysis was used. Signicance level of 0.05 was assumed.
Data availability
Sequences generated during this study were deposited in the SRA repository and are accessible via BioProject
no. PRJNA606174.
Received: 2 March 2020; Accepted: 12 January 2021
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Acknowledgements
is study was nanced through grant from National Science Centre, Poland number 2016/21/B/NZ9/00840 to
MG. e funder had no role in study design, analyzing data and writing the manuscript. We would like to thank
Ada Błaszczyk and Anita Kowalczyk for their help in maintaining the plants for the experiments.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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Authors contributions
S.S.: performed experiments, analyzed data, draed the manuscript, M.S.: performed experiments, participated in
writing the manuscript, K.H.: conceptualized the study, participated in writing the manuscript, J.T.: participated
in writing the manuscript, A.T.: participated in writing the manuscript, M.G.: supervised the project, conceptual-
ized the study, analyzed data, participated in writing the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at (https ://doi.
org/10.1038/s4159 8-021-82436 -5).
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