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Culture Dependent and Independent Analysis of Potential Probiotic Bacterial Genera and Species Present in the Phyllosphere of Raw Eaten Produce

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Culture Dependent and Independent Analysis of Potential Probiotic Bacterial Genera and Species Present in the Phyllosphere of Raw Eaten Produce

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The plant phyllosphere is colonized by a complex ecosystem of microorganisms. Leaves of raw eaten vegetables and herbs are habitats for bacteria important not only to the host plant, but also to human health when ingested via meals. The aim of the current study was to determine the presence of putative probiotic bacteria in the phyllosphere of raw eaten produce. Quantification of bifidobacteria showed that leaves of Lepidium sativum L., Cichorium endivia L., and Thymus vulgaris L. harbor between 103 and 106 DNA copies per gram fresh weight. Total cultivable bacteria in the phyllosphere of those three plant species ranged from 105 to 108 CFU per gram fresh weight. Specific enrichment of probiotic lactic acid bacteria from C. endivia, T. vulgaris, Trigonella foenum-graecum L., Coriandrum sativum L., and Petroselinum crispum L. led to the isolation of 155 bacterial strains, which were identified as Pediococcus pentosaceus, Enterococcus faecium, and Bacillus species, based on their intact protein pattern. A comprehensive community analysis of the L. sativum leaves by PhyloChip hybridization revealed the presence of genera Bifidobacterium, Lactobacillus, and Streptococcus. Our results demonstrate that the phyllosphere of raw eaten produce has to be considered as a substantial source of probiotic bacteria and point to the development of vegetables and herbs with added probiotic value.
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International Journal of
Molecular Sciences
Article
Culture Dependent and Independent Analysis of
Potential Probiotic Bacterial Genera and Species
Present in the Phyllosphere of Raw Eaten Produce
Sascha Patz 1,2, Katja Witzel 2,*, Ann-Christin Scherwinski 2and Silke Ruppel 2 ,*
1Algorithms in Bioinformatics, ZBIT Center for Bioinformatics, University of Tübingen, Sand 14,
72076 Tübingen, Germany
2Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1,
14979 Großbeeren, Germany
*Correspondence: witzel@igzev.de (K.W.); ruppel@igzev.de (S.R.);
Tel.: +49-(0)33701-78220 (K.W.); Fax: +49-(0)33701-55391 (K.W.)
Received: 31 May 2019; Accepted: 16 July 2019; Published: 26 July 2019


Abstract:
The plant phyllosphere is colonized by a complex ecosystem of microorganisms. Leaves of
raw eaten vegetables and herbs are habitats for bacteria important not only to the host plant, but also to
human health when ingested via meals. The aim of the current study was to determine the presence of
putative probiotic bacteria in the phyllosphere of raw eaten produce. Quantification of bifidobacteria
showed that leaves of Lepidium sativum L., Cichorium endivia L., and Thymus vulgaris L. harbor between
10
3
and 10
6
DNA copies per gram fresh weight. Total cultivable bacteria in the phyllosphere of those
three plant species ranged from 10
5
to 10
8
CFU per gram fresh weight. Specific enrichment of probiotic
lactic acid bacteria from C. endivia,T. vulgaris, Trigonella foenum-graecum L., Coriandrum sativum L.,
and Petroselinum crispum L. led to the isolation of 155 bacterial strains, which were identified as
Pediococcus pentosaceus,Enterococcus faecium, and Bacillus species, based on their intact protein pattern.
A comprehensive community analysis of the L. sativum leaves by PhyloChip hybridization revealed
the presence of genera Bifidobacterium,Lactobacillus, and Streptococcus. Our results demonstrate that
the phyllosphere of raw eaten produce has to be considered as a substantial source of probiotic
bacteria and point to the development of vegetables and herbs with added probiotic value.
Keywords:
fresh produce; lactic acid bacteria (LAB); MALDI-TOF MS biotyping; phyllosphere
microbiota; PhyloChip®; probiotics
1. Introduction
Plants host a wide range of microorganisms, i.e., bacteria, fungi, archaea, oomycetes, and
viruses, which constitute their entire microbiome. Capturing the function and complexity of the
microbial community under diverse environmental impacts, the so-called phytobiome, has advanced
tremendously in the last decade. Using a range of microbial model organisms, a detailed picture
emerged on how microorganisms colonize plants, and how they influence the plant’s health and
productivity [
1
]. However, microorganisms residing as plant endophytes or epiphytes usually
form communities and our understanding of the influence of host genotype, host compartment,
environmental factors and microbiota relationships on the phytobiome needs to be enhanced in order
to use tailored microbiota for crop production [2,3].
Large eorts are being taken to illuminate the compositions and functions mainly of rhizosphere
microbiota with the aim to increase nutrient uptake eciency and resilience against soil-borne diseases
in crops [
4
]. Compared to the rhizosphere microbiota, phyllosphere microbiota is less diverse and
lower abundant [
5
,
6
]. Their ecological role, however, is equally significant for plant health and fitness
Int. J. Mol. Sci. 2019,20, 3661; doi:10.3390/ijms20153661 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019,20, 3661 2 of 16
and depends on the structure and function of the microbiome [
7
]. While community composition of
rhizosphere microbiota is governed by the soil type, the major driver for phyllosphere microbiota
seems to be the host genotype [8,9].
Leand Fierer [
10
] analyzed the bacterial communities found on the surface of vegetables and
fruits, and demonstrated that community composition and diversity is dependent on the produce type.
They also identified dierences between conventional and organically farmed produce, showing that
organic-labeled spinach, lettuce and tomatoes had a greater OTU richness, but peaches and grapes
had a lower OTU richness, when compared to the conventional produce type. Using both, a culture
dependent and an independent approach, leafy vegetables (spinach and lettuce varieties) were profiled
for their bacterial communities and revealed a distinct plant genotype-specific pattern [
11
]. Similar
results were found for fungal communities that also revealed a genotypes-specific variation in lettuce
phyllosphere [
12
]. Lopez-Velasco et al. [
13
] showed further that spinach leaves harbor a dierent
composition of bacterial populations as compared to seeds or cotyledons. Due to outbreaks of foodborne
illnesses caused by the consumption of vegetables, food safety and persistence of human pathogens on
crops became a focus in phyllosphere microbiota research [
8
]. Especially raw eaten leafy vegetables
and herbs are analysed to understand the eect of farming practice, postharvest processing and storage
conditions of fresh produce on microbiota composition [
14
]. Here, a recent study compared the bacterial
community composition of ready-to-eat rocket and spinach [
15
]. Authors demonstrated again that
community composition was dependent on the plant species and that refrigeration decreased bacterial
richness and led to the dominance of cold-adapted bacteria. Other studies investigated the colonization
and persistence of potential human pathogenic bacteria in leaf vegetable phyllospheres [16].
In contrast, probiotic bacteria, such as strains of the lactic acid bacteria (LAB), like Lactobacillus spp.,
Streptococcus spp., Enterococcus spp. or Pediococcus spp., and of bifidobacteria, are recognized for their
beneficial eects on the human intestinal microbiota and overall health for a long time [
17
20
]. Moreover,
recent studies give strongly evidence on their strain- and disease-dependent probiotic ecacy [
21
].
The eects are addressed through the production of antimicrobial compounds, high colonization
competence, immunomodulation of the host and inhibition of bacterial toxin production [
22
,
23
]. In
most cases, these bacteria are used as starter cultures in the production of fermented foods, such as
yoghurt, sauerkraut or rice wine [
24
]. Next generation probiotics have been proposed in the last decades,
like strains of Weissella spp. or Akkermansia muciniphila [
25
27
]. Beside the health-promoting and
antipathogenic eects of probiotics, there are reports on their risk to become a (opportunistic) pathogen,
as closely related virulent species are known or because of the risk of exchanging antibiotic resistance
genes [
28
32
]. The challenge here is to explore strains, like of the LAB group, especially Lactobacilli
and Enterococci, which are predestined to counteract for example foodborne pathogens [33].
However, while vegetable leaves are well populated with bacteria including potential probiotic
acting Lactobacillus strains [
34
37
], so far, plant-derived produce has only been considered as vehicles of
probiotic cultures [
38
,
39
]. A detailed understanding of how probiotic bacteria colonize leafy vegetables
or herbs would enable future approaches to enrich these tissues with the respective bacterial strains to
elevate vegetable leaves beyond their basic nutritional values.
The objective of the present study was to search for bacterial species which are known to contain
potential probiotic strains in the phyllosphere of raw consumed edibles and to bring them into
in vitro
pure cultures for further detailed studies. Therefore, we started with the quantification of the specific
bifidobacterial gene copy numbers in raw eaten produce. The investigated plant species were selected
due to their widespread raw consumption, economic value, and their aliation to dierent plant
orders, assuming that the plant genotype aects the bacterial community composition. Based on the
results we then used culture-dependent approaches and assessed the plant species-specific composition
of LAB. Finally, the cress phyllosphere microbiome was investigated comprehensively by applying the
PhyloChip G3 technology. Altogether, we confirm that the bacterial composition of the phyllosphere
of raw eaten edibles is highly dependent on the host genotype and we show that leaves harbor a wide
range of potential probiotic acting bacterial genera and species.
Int. J. Mol. Sci. 2019,20, 3661 3 of 16
2. Results
2.1. Quantification of Potential Probiotic Bifidobacteria in the Phyllosphere of Edible Plants
Lepidium sativum, family Brassicaceae, and plants belonging to two other families—Cichorium
endivia (Asteraceae) and Thymus vulgaris (Lamiaceae)—were grown under greenhouse conditions in
two independent experiments to collect phyllosphere samples. The aim was to search, in a culture
independent approach, for the presence of bifidobacteria and to quantify their specific gene copy
numbers. Using quantitative real-time PCR and Bifidobacterium genera-specific primers [
40
], between
10
3
to 10
6
copies per gram plant fresh weight were detected in all plant samples (Figure 1). The results
also reveal a considerable variability between biological replicates in all three investigated plant species.
Int. J. Mol. Sci. 2019, 20, x 3 of 16
2. Results
2.1. Quantification of Potential Probiotic Bifidobacteria in the Phyllosphere of Edible Plants
Lepidium sativum, family Brassicaceae, and plants belonging to two other families—Cichorium
endivia (Asteraceae) and Thymus vulgaris (Lamiaceae)—were grown under greenhouse conditions in
two independent experiments to collect phyllosphere samples. The aim was to search, in a culture
independent approach, for the presence of bifidobacteria and to quantify their specific gene copy
numbers. Using quantitative real-time PCR and Bifidobacterium genera-specific primers [40], between
10
3
to 10
6
copies per gram plant fresh weight were detected in all plant samples (Figure 1). The results
also reveal a considerable variability between biological replicates in all three investigated plant
species.
Figure 1. Abundance of bifidobacteria (log
10
copy number per gram fresh weight) in the phyllosphere
of three plant species determined via qPCR. Shown are mean values of five plants for the first (light
gray bars) and second (dark gray bars) experiment, with the standard deviation as error bars.
2.2. Amount and Diversity of Cultivable Bacteria in Lepidium sativum, Cichorium endivia, and Thymus
vulgaris
We showed that bacterial species and genera, which contain potential human probiotic acting
strains, are present in the phyllosphere of fresh consumed plants and that their abundance is
relatively high in all three selected plant species. Now, the question to be answered was, whether
these three selected plant species, originating from different plant families, harbor a similar
microbiota composition or differ significantly. Therefore, total cultivable bacteria were quantified on
a complex nutrient medium and their diversity was estimated using a conventional morphotyping
approach. Cultivable bacteria were considerable higher in L. sativum with up to 10
8
CFU g
1
fresh
weight as compared to C. endivia and T. vulgaris with up to 10
5
CFU g
1
fresh weight with a high
variability in C. endivia in the first experiment (Figure 2).
Figure 1.
Abundance of bifidobacteria (log
10
copy number per gram fresh weight) in the phyllosphere
of three plant species determined via qPCR. Shown are mean values of five plants for the first (light
gray bars) and second (dark gray bars) experiment, with the standard deviation as error bars.
2.2. Amount and Diversity of Cultivable Bacteria in Lepidium sativum, Cichorium endivia, and
Thymus vulgaris
We showed that bacterial species and genera, which contain potential human probiotic acting
strains, are present in the phyllosphere of fresh consumed plants and that their abundance is relatively
high in all three selected plant species. Now, the question to be answered was, whether these three
selected plant species, originating from dierent plant families, harbor a similar microbiota composition
or dier significantly. Therefore, total cultivable bacteria were quantified on a complex nutrient medium
and their diversity was estimated using a conventional morphotyping approach. Cultivable bacteria
were considerable higher in L. sativum with up to 10
8
CFU g
1
fresh weight as compared to C. endivia
and T. vulgaris with up to 10
5
CFU g
1
fresh weight with a high variability in C. endivia in the first
experiment (Figure 2).
Int. J. Mol. Sci. 2019,20, 3661 4 of 16
Int. J. Mol. Sci. 2019, 20, x 4 of 16
Figure 2. Viable counts (log
10
CFU g
1
fresh weight) of cultivable bacteria obtained from three plant
species. Shown are mean values of five plants for the first (light gray bars) and second (dark gray
bars) experiment, with the standard deviation as error bars.
Besides the different amounts of cultivable bacteria, also their morphotyping-based diversity
revealed pronounced differences. The high bacterial number of L. sativum community, compiled of
28 different morphotypes on standard nutrient agar plates, was shown to be very uneven distributed
between the different morphological colony types. That resulted in a lower Shannon diversity index
of only 1.95 compared to C. endivia with 2.87 and T. vulgaris with 2.72 (Table 1).
Table 1. Morphotype diversity index (Shannon index) of bacterial strains isolated from three plant
species.
Biodiversity Indices L. sativum C. endivia T. vulgaris
N 8018 56 276
S 28 26 30
H 1.95 2.87 2.72
H
max
3.33 3.26 3.40
E 0.59 0.88 0.80
N: colony-forming units count; S: number of morphotypes; H: Shannon index calculated based on the
distribution of N on S; H
max
: maximal Shannon index; E: eveness.
2.3. Isolation and Characterization of Phyllosphere Lactic Acid Bacteria (LAB)
We next attempted to isolate LAB, such as lactobacilli, and bifidobacteria from the phyllosphere
of edibles (C. endivia, T. vulgaris, Trigonella foenum-graecum, Coriandrum sativum, and Petroselinum
crispum). These bacteria are anaerobic or microaerophilic and many species do not grow well on the
surface of solid media incubated aerobically. Thus, these isolates were cultured under
microaerophilic conditions using a dedicated medium for LAB. A total of 155 bacterial isolates were
obtained: 42 from T. foenum-graecum, 33 from C. endivia, 27 from C. sativum, 18 from P. crispum, and
35 from T. vulgaris. For screening and identification of those isolates, matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied. Resulting mass spectra
were then matched to a bacterial reference database for identification. The isolates could be divided
into four major groups based on the similarity of the respective acquired mass spectra (Figure 3). One
major group was formed of isolates identified as Pediococcus pentosaceus, the second group consisted
Figure 2.
Viable counts (log
10
CFU g
1
fresh weight) of cultivable bacteria obtained from three plant
species. Shown are mean values of five plants for the first (light gray bars) and second (dark gray bars)
experiment, with the standard deviation as error bars.
Besides the dierent amounts of cultivable bacteria, also their morphotyping-based diversity
revealed pronounced dierences. The high bacterial number of L. sativum community, compiled of 28
dierent morphotypes on standard nutrient agar plates, was shown to be very uneven distributed
between the dierent morphological colony types. That resulted in a lower Shannon diversity index of
only 1.95 compared to C. endivia with 2.87 and T. vulgaris with 2.72 (Table 1).
Table 1.
Morphotype diversity index (Shannon index) of bacterial strains isolated from three
plant species.
Biodiversity Indices L. sativum C. endivia T. vulgaris
N8018 56 276
S28 26 30
H1.95 2.87 2.72
Hmax 3.33 3.26 3.40
E0.59 0.88 0.80
N: colony-forming units count; S: number of morphotypes; H: Shannon index calculated based on the distribution
of N on S; Hmax: maximal Shannon index; E: eveness.
2.3. Isolation and Characterization of Phyllosphere Lactic Acid Bacteria (LAB)
We next attempted to isolate LAB, such as lactobacilli, and bifidobacteria from the phyllosphere of
edibles (C. endivia,T. vulgaris, Trigonella foenum-graecum, Coriandrum sativum, and Petroselinum crispum).
These bacteria are anaerobic or microaerophilic and many species do not grow well on the surface of solid
media incubated aerobically. Thus, these isolates were cultured under microaerophilic conditions using
a dedicated medium for LAB. A total of 155 bacterial isolates were obtained: 42 from T. foenum-graecum,
33 from C. endivia, 27 from C. sativum, 18 from P. crispum, and 35 from T. vulgaris. For screening
and identification of those isolates, matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS) was applied. Resulting mass spectra were then matched to a bacterial
reference database for identification. The isolates could be divided into four major groups based on the
similarity of the respective acquired mass spectra (Figure 3). One major group was formed of isolates
Int. J. Mol. Sci. 2019,20, 3661 5 of 16
identified as Pediococcus pentosaceus, the second group consisted of Enterococcus faecium isolates, and
the two remaining groups contained Bacillus species as well as isolates with no reliable identifications.
Only 15% of the isolates were considered as not identified, indicating that the reference database
allowed identification at least to the genus level of most isolates (Supplemental Table S1). However,
the best score obtained for identification was 2.493 and the maximal score of 3.000 was never reached,
demonstrating the current lack of reference spectra from plant-associated samples.
Int. J. Mol. Sci. 2019, 20, x 5 of 16
of Enterococcus faecium isolates, and the two remaining groups contained Bacillus species as well as
isolates with no reliable identifications. Only 15% of the isolates were considered as not identified,
indicating that the reference database allowed identification at least to the genus level of most isolates
(Supplemental Table S1). However, the best score obtained for identification was 2.493 and the
maximal score of 3.000 was never reached, demonstrating the current lack of reference spectra from
plant-associated samples.
Figure 3. Cluster analysis of bacterial mass spectra obtained from selected reference strains and plant
isolates. The latter are labeled in color according to the plant host. The identification of major isolate
groups is shown on the right. Distance is displayed in relative units. For the full list of identified
isolates, please refer to Supplemental Table S1.
The bacterial communities found in the phyllosphere of edibles differed with respect to their
taxonomic composition. T. foenum-graecum leaves were harboring Bacillus sp., E. faecium, and one
Leuconostoc lactis strain; C. endivia leaves contained E. faecium and P. pentosaceus; the phyllosphere of
C. sativum was harboring P. pentosaceus, Staphylococcus sp. and one Lactobacillus plantarum; leaves of
P. crispum contained Bacillus sp. and P. pentosaceus; and T. vulgaris leaves were colonized by Bacillus
sp., P. pentosaceus, and Staphylococcus sp.
Interestingly, the mass spectra of isolates from the same species differed in their protein pattern,
depending on the host plant. An example is shown in Supplemental Figure 1 for isolates identified
as P. pentosaceus. This indicates that specific strains of P. pentosaceus colonize specific host plants and
that MALDI-TOF-based biotyping is capable of differentiate between those strains.
Figure 3.
Cluster analysis of bacterial mass spectra obtained from selected reference strains and plant
isolates. The latter are labeled in color according to the plant host. The identification of major isolate
groups is shown on the right. Distance is displayed in relative units. For the full list of identified
isolates, please refer to Supplemental Table S1.
The bacterial communities found in the phyllosphere of edibles diered with respect to their
taxonomic composition. T. foenum-graecum leaves were harboring Bacillus sp., E. faecium, and one
Leuconostoc lactis strain; C. endivia leaves contained E. faecium and P. pentosaceus; the phyllosphere of
C. sativum was harboring P. pentosaceus,Staphylococcus sp. and one Lactobacillus plantarum; leaves of
P. crispum contained Bacillus sp. and P. pentosaceus; and T. vulgaris leaves were colonized by Bacillus sp.,
P. pentosaceus, and Staphylococcus sp.
Interestingly, the mass spectra of isolates from the same species diered in their protein pattern,
depending on the host plant. An example is shown in Supplemental Figure S1 for isolates identified as
P. pentosaceus. This indicates that specific strains of P. pentosaceus colonize specific host plants and that
MALDI-TOF-based biotyping is capable of dierentiate between those strains.
Int. J. Mol. Sci. 2019,20, 3661 6 of 16
A typical application of this biotyping method is the dereplication of recurrent isolated
microorganisms [
41
]. However, we found only few isolates with identical mass spectra in our
culture collection (Figure 3), demonstrating a high level of biodiversity in the cultivable communities,
even if they were obtained on a highly selective medium under microaerophilic conditions.
2.4. Phyllosphere Microbiome Composition of Lepidium sativum
The L. sativum phyllosphere bacterial community was investigated using PhyloChip G3 technology
in two consecutive independent greenhouse experiments. The aim was to get insight into the entire
bacterial community composition and taxa richness of one of the fresh consumed edible plants
known for their health promoting eects in human diet. The bacterial genus richness ranged from
49 to 119, belonging to 61 identified and 58 unclassified genera, 63 families, and 17 phyla (Figure 4).
The most prominent phyla were Proteobacteria (47 genera) and Firmicutes (21). Other detected
phyla were Actinobacteria (9), Cyanobacteria (9), Bacteroidetes (7 genera), Chloroflexi (6), Tenericutes
(5), Acidobacteria (3), Planctomycetes (3), Verrucomicrobia (2), and with only one genus: Chlorobi,
Gemmatimonadetes, Nitrospirae, Saccharibacteria, (=TM7), Spirochaetes, and the candidate phyla
BRC1 and OP11. The presence-absence pattern of the eight biological phyllosphere samples represents
a quite high sample to sample variability in the bacterial community composition, although both
individual experiments form distinct clusters, except one outlying sample (Supplemental Figure S2).
This high variability between biological replicates was also detected earlier in the quantification of
bifidobacteria via qPCR (see Figure 1).
Within the highly diverse and variable community, hybridization signals were identified for
the genera Bifidobacterium,Lactobacillus, and Streptococcus (genus-specific OTUs: 111, 160, and 197),
that confirm the abundance of species known as probiotic acting bacteria, like two detected strains
of Streptococcus thermophilus (Table 2). Those potential probiotic strains are marked in Figure 4.
Additionally, a high proportion of hybridization signals revealed mostly unclassified taxa from various
genera (e.g., less probiotic strains containing Akkermansia, Bacillus, Clostridium, Propionibacterium,
and Staphylococcus), families or classes. In particular, Staphylococcus was represented by two
potentially skin-probiotic strains of S. epidermidis, among others.
Table 2.
Taxonomic aliation of genera and species, which are known to contain eective probiotic
strains, detected in L. sativum phyllosphere microbiome via PhyloChip analysis.
PHYLA ORDER GENUS SPECIES
Actinobacteria Bifidobacteriales Bifidobacteria unclassified
Firmicutes Lactobacillales
Lactobacillus unclassified
Streptococcus thermophilus
Streptococcus unclassified
Other genera containing potential probiotic genera found: Akkermansia,Bacillus,Clostridium and Propionibacterium,
and Staphylococcus (e.g., S. epidermidis).
Int. J. Mol. Sci. 2019,20, 3661 7 of 16
Int. J. Mol. Sci. 2019, 20, x 7 of 16
Figure 4. Taxonomic affiliation and absence-presence pattern of Lepidium sativum phyllosphere
microbiota bacterial genera detected by the PhyloChip G3 technology. Taxonomic assignment of the
genus-specific OTUs are displayed on the leaves and colored according their phyla (a–q). Spotted red
lines indicate uncertain assignments, whereby their last known taxonomic rank (P: Phyla; C: Class; O:
Order; F: Family) is listed behind the respective affiliation in the legend. The surrounding circles,
describing the four replicates for individual experiments (Repl_1, Repl_2), reveal the presence (black)
and absence (gray) of the appropriate genus. Genera with high probability of containing probiotic
species are marked with a green dot.
Figure 4.
Taxonomic aliation and absence-presence pattern of Lepidium sativum phyllosphere
microbiota bacterial genera detected by the PhyloChip G3 technology. Taxonomic assignment of the
genus-specific OTUs are displayed on the leaves and colored according their phyla (a–q). Spotted red
lines indicate uncertain assignments, whereby their last known taxonomic rank (P: Phyla; C: Class;
O: Order; F: Family) is listed behind the respective aliation in the legend. The surrounding circles,
describing the four replicates for individual experiments (Repl_1, Repl_2), reveal the presence (black)
and absence (gray) of the appropriate genus. Genera with high probability of containing probiotic
species are marked with a green dot.
Int. J. Mol. Sci. 2019,20, 3661 8 of 16
3. Discussion
A healthy diet is usually closely linked with the recommendation to eat raw vegetables, herbs,
or sprouts [
42
]. Since Pharao’s time, herbs, and fresh produce are known for their healing value [
43
].
It is assumed that these products are especially healthy to humans because of their high content
in vitamins, secondary metabolites, essential oils, or specific mineral compositions. Nowadays,
dierent herb-specific ingredients are known, for example, to suppress harmful microorganisms [44].
Overlooked so far is the additional beneficial eect on the improved balance of intestinal bacterial
flora by bacterial communities inhabiting the highly diverse herbs and fresh produce, that probably
also includes bacterial species already known as probiotics. Probiotics have recently been defined
as “live microorganisms which when administered in adequate amounts confer a health benefit on
the host” [
45
]. Likewise, prebiotics, are non-digestible food fiber components that contribute to host
health by activating proliferation and function of beneficial intestinal bacteria [
46
]. Synbiotics describe
a combination of probiotics and prebiotics [
23
,
47
] and has been established as medical terms. Probiotic
bacteria are processed and enriched for example in yogurt drinks, lactobacillary beverages or other
fermented foods and have been medically recognized. Strongest evidence for beneficial probiotic
eects has been shown for Lactobacillus rhamnosus GG and Bifidobacterium lactis BB-12 in curing diarrhea
mainly caused by rotaviruses in children (FAO/WHO 2002). Recent publications indicate a much
broader range of novel probiotic bacteria, such as Weissella cibaria [26].
The presented results show that plant phyllosphere microbiota naturally harbor potential probiotic
bacteria, as summarized in Table 3. The well acknowledged, beneficially acting probiotics, containing
genera Bifidobacterium and Lactobacillus, were detected in culture independent molecular investigations.
Their population reached ~10
6
bifidobacteria-specific gene copy numbers per gram plant material
which could be translated into ~10
5
CFU g
1
plant fresh weight, assuming a mean 16S rDNA operon
copy number per cell [
48
]. Such a number is comparable to the magnitude as probiotic bacteria
Latobacillus spp. or Bifidobacterium longum and Streptococcus thermophilus were administered in specific
enriched functional food [
49
]. In these products it is even dicult to keep living cell numbers above
10
6
CFU g
1
of product for up to 21 days. Often the viable cell counts decrease earlier when foods
are kept without adding prebiotic material [
49
]. Probiotic cells easily survive in plant material since
they live in their native habitat and will be digested together with their prebiotic acting plant matrix.
Additionally, the investigated plant species revealed highly diverse bacterial communities in their
phyllospheres. To which extend these plant inhabiting bacteria survive the acidic stomach environment
during the digestion pathway, is still unclear. However, since these bacteria at least partly reside
intracellularly of fiber rich material, it can be assumed that they are protected by their plant hosts
during this very harsh treatment in the human intestinal tract. Such plant-based transport systems
have to be investigated before and probiotics enriched plant products may expand the functional
food list.
The constitution of phyllosphere microbiota is linked on the one hand to the developmental stage
of the plant [
13
], but is also influenced by environmental conditions [
15
]. The presented data indicated
a considerable variation between independent experiments, e.g., see Figure 1for L. sativum, or between
plant replicate samples within an experiment, e.g., see Figure 2for C. endivia. A certain deviation within
the latter has been shown before for raw produce microbiota and is not unexpected [
10
,
15
]. The range
of variation between independent experiments, especially for L. sativum, could be explained by the
relatively short time of cultivation. While T. vulgaris was cultivated for nine weeks, L. sativum was
grown only for two weeks until plants were harvested at a stage where they are typically consumed.
Hence, it could be possible that a stable microbial community is more likely to be found in plant species
that are able to shape their microbiota over a longer period of time, as compared to plant species with a
shorter production time. This hypothesis should be tested in analyzing a wider range of independently
performed experiments using raw eaten produce with dierent cultivation regimes.
Int. J. Mol. Sci. 2019,20, 3661 9 of 16
Table 3.
Potential probiotic bacterial species detected in the phyllosphere of raw eaten produce of the
present study. Related references demonstrate the verified probiotic character of the strains of the
same species.
Probiotic Group Species Phyllosphere
(Plant) Probiotic Reference
Lactobacillus 1(Bacteria;Terrabacteria group;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae)
Lactobacillus sp. LS [5055]
L. plantarum CS [20,5459]
Bifidobacteria (Bacteria;Terrabacteria group;Actinobacteria;Actinobacteria;Bifidobacteriales)
Bifidobacteria sp. LS [53,55,60]
Streptococcus 1(Bacteria;Terrabacteria group;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae)
Streptococcus sp. LS [20,54,55]
S. thermophilus LS [20,53,54,61,62]
Enterococcus 1(Bacteria;Terrabacteria group;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae)
E. faecium CE, TF [20,5355,60,6367]
Pediococcus 1(Bacteria;Terrabacteria group;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae)
P. pentosaceus CE, CS, PC, TV [20,53,55,59,60,6871]
Leuconostoc 1(Bacteria;Terrabacteria group;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae)
L. lactis TF [72]
Bacillus (Bacteria;Terrabacteria group;Firmicutes;Bacilli;Bacillales;Bacillaceae)
Bacillus spp. LS, PC, TF, TV [55,73,74]
Propionibacterium (Bacteria;Terrabacteria group;Actinobacteria;Actinobacteria;Propionibacteriales;
Propionibacteriaceae)
Propionibacterium sp. LS [20,54,55]
Akkermansia (Bacteria;PVC group;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;
Akkermansiaceae)
Akkermansia sp. LS [20,7577]
Staphylococcus (Bacteria;Terrabacteria group;Firmicutes; Bacilli;Bacillales;Staphylococcaceae)
S. epidermidis CS [7880]
S. hominis CS, TV [8083]
Clostridium (Bacteria;Terrabacteria group;Firmicutes;Clostridia;Clostridiales;Clostridiaceae)
Clostridium sp. LS [29,84]
1
LAB (lactic acid bacteria); CE: Cichorium endivia L.; CS: Coriandrum sativum L.; LS: Lepidium sativum L.; PC:
Petroselinum crispum L.; TF: Trigonella foenum-graecum L.; TV: Thymus vulgaris L.
The aim of culturing phyllosphere bacteria on medium designed for LAB was to analyse whether
potential probiotic strains could be isolated from raw eaten herbs and vegetables for future functional
studies. From the cultivated isolates, one L. plantarum and one Leuconostoc lactis isolate were present,
both being recognized as probiotic bacteria [
55
,
57
,
58
,
72
,
85
]. Enterococci are LAB that are naturally
associated with the gastrointestinal tract of humans and animals, but plant-associated E. faecium isolates
have been found previously [
64
,
86
]. Several E. faecium strains possess strong probiotic activity [
65
,
67
],
and since two of the five investigated phyllospheres harbored this species, a possible influence on
the intestinal flora of the consumer is assumable. P. pentosaceus also belongs to the Lactobacillaceae.
Its probiotic activity is documented [
70
] and bacteriocins produced by its strains are able to inhibit the
growth of pathogens like Listeria monocytogenes [
68
,
69
]. Pediococci have been found residing in or on
plants [
71
,
87
] and the occurrence of this species in phyllosphere samples of four plant species points to a
wide-spread distribution with an extensive host range. Previous findings of S. hominis in human breast
milk, similarly to E. faecium and P. pentosaceus [
83
], and its location in two plant phyllospheres point to
a probiotic function of this species, although S. hominis does not belong to the LAB group and it has
not been reported as plant-associated before. Another species of Staphylococcus,S. epidermidis, found in
two plant species, was previously reported as most abundant Staphylococcus strain within the human
skin microbiome and as a skin probiotic against Cutibacterium acnes, even if applied as encapsulated
product to avoid bloodstream infections due to its opportunistic pathogenic activity [
78
,
79
]. Also the
last major group of bacteria identified in this study for three plant hosts, Bacillus spp., contains species
and strains with probiotic activity, such as B. coagulans [
55
,
88
]. Surprisingly, no bifidobacterial strains
could be cultured although they were detected in two culture independent techniques—the PhyloChip
Int. J. Mol. Sci. 2019,20, 3661 10 of 16
and real-time qPCR. Therefore, future studies should apply newly developed plant-based cultivation
media that promise a better chance of cultivating endophytic, plant adapted bacterial strains [89].
Major research eorts are directed to the exploration of plant microbiota with the aim to improve
crop performance [
1
]. However, the functionality and application of these microbiota with respect to
human nutrition and well-being is largely untapped. The fact that some novel probiotic bacterial strains
appear as opportunistic pathogens on the one hand, but also share antipathogenic properties, even
against closely related strains [
90
], demonstrates the complexity of microbial interactions. This study
shows that the phyllosphere of edibles is a substantial source of potential probiotic bacteria, albeit strain-
and disease-specific probiotic activity of the isolates has to be resolved in detail in the future. Thus,
next studies should focus on investigating the isolated strains functionally, on how these communities
can be enriched in the phyllosphere by means of plant production and whether they can be stably
transmitted via seeds.
4. Materials and Methods
4.1. Plant Species and Cultivation
The plant species Lepidium sativum, Cichorium endivia var. crispum,Thymus vulgaris,Petroselinum
crispum,Coriandrum sativum, and Trigonella foenum-graecum were grown in two replicated experiments
under greenhouse conditions: 16
C and 21
C night and day temperature and 60% relative air humidity
under natural light during April to July. The greenhouse location was 522101600 N 131802200 E.
L. sativum and T. foenum-graecum seeds (2 g seeds per tray) were sawn directly into growth
trays filled with Perligran G (Kanuf Perlite GmbH, Dortmund, Germany) and covered five biological
replicates. L. sativum was grown for two weeks. All other plant species were germinated in quartz sand
(
2 mm), seedlings were transplanted into pots (12 cm in diameter, filled with Fruhstorfer substrate
Type P, Germany) and randomly set on trivets to avoid water mediated microbial transfer.
C. endivia, one plant per pot, ten pots per biological replicate, with four biological replicates
were grown for seven weeks. Ten seedlings of T. vulgaris were transplanted into one pot; three pots
per biological replicate were established in four biological replicates. T. vulgaris plants were grown
for nine weeks. Four seedlings of P. crispum and C. sativum were transplanted per pot, five pots
per biological replicate and four biological replicates. Both plant species were grown for six weeks.
Above ground plant material was aseptically collected in a growth stage which is representative for
usual consumption.
4.2. Determination of Bifidobacterial-Specific Gene Copy Numbers Using Quantitative Real-Time PCR
To demonstrate the presence of potential probiotic acting bacteria in leaf material, the
well-known human probiotic acting bacterial genus Bifidobacterium was used as model.
One microliter (concentration 50 ng
µ
L
1
) of total DNA samples was extracted from lyophilized
plant tissue using DNeasy Plant MiniKit (Qiagen) was investigated in a bifidobacteria
genus-specific qPCR. Therefore, primers g-Bifid-F (5
0
-CTCCTGGAAACGGGTGG-3
0
) and g-Bifid-R
(5
0
-GGTGTTCTTCCCGATATCTACA-3
0
) were used [
40
]. Quantitative real-time PCR (qPCR) was
performed with iQ
TM
SYBR Green Supermix (Biorad, Hercules, CA, USA), the amplification protocol
of five minutes initiation step at 94
C and 30 cycles of 20 s at 94
C, 20 s 55
C, and 30 s 72
C followed
by 5 min 72 C reassociation and a melting curve incrementing 0.5 C in 85 cycles starting at 55 C to
verify the product purity and specificity. The DNA amplified in technical triplicates was used in the
original concentration, which was isolated from plant material and in a dilution 1:10. The reaction
was performed in a CFX96 Touch real-time PCR detection system (Biorad, USA). A standard dilution
series of a purified PCR product of Bifidobacterium breve (DSM 20213) was performed to calculate copy
numbers according to a regression curve. Copy numbers were calculated via this regression curve.
Int. J. Mol. Sci. 2019,20, 3661 11 of 16
4.3. Bacterial Cultivation
4.3.1. Total Cultivable Bacterial Numbers and Diversity on Complex Nutrient Agar
Fresh plant material (5 g) was ground, 1:10 diluted in 0.05 M NaCl buffer solution, and shaken on a
horizontal shaker (180 rpm) with 6 glass beads (5 mm diameter) at 4
C for one hour. A serial dilution (5
times 1:10 diluted) was prepared, 100
µ
L of each dilution were spread petri dishes of standard nutrient
agar (Carl Roth GmbH, Karlsruhe, Germany) (three replicates per dilution and sample) and incubated
at 37
C for 72 h. Colony forming units (CFUs) were counted and classified according their morphology
(color, side face, circumference, surface, diameter, and transparency). Different morphotypes and
numbers were used to calculate the Shannon diversity index (Shannon 1948) H =
P
(pi*lnpi), and the
evenness of morphotype occurrence E =H/H
max
, where pi =ni
×
N
1
, N =the total number of CFUs,
ni =number CFUs per colony type, S =number of different colony types, Hmax =lnS.
4.3.2. Specific Enrichment for Bifidobacteria and Lactobacilli
MRS-Agar (deMan-Rogosa-Sharpe-Agar, MERCK, Germany), which favors the growth of lactic
acid bacteria, was used to cultivate LABs, and TOS-Propionate agar with lithium mupirocin (MERCK,
Germany) was applied to cultivate bifidobacteria. Bacterial washes from phyllosphere plant samples
(as described in total cultivable bacterial numbers detection part) and their first three diluted samples
(10
1
, 10
2
und 10
3
) were spread in triplicates on media. They were incubated in anaerobic jars
(MERCK, Germany) with Oxoid AnaeroGen satchets (Thermo Scientific, Schwerte, Germany) for
generating a microaerophilic atmosphere in the jars for 72 h at 37
C. All emerging colonies were counted
and collected for protein pattern composition analysis using the MALDI-TOF biotyper approach.
No colonies were detected on TOS-agar.
4.4. Protein Extraction from Bacterial Colonies for MALDI-TOF Biotyper Analysis
For the extraction, the bacteria were suspended in 150
µ
L of MilliQ water and vortexed. Next,
450
µ
L of 100% ethanol was added, vortexed, and centrifuged (13,000
×
g, 2 min). The supernatant
was discarded, pellets were dried at room temperature, resuspended in 5
µ
L of 70% formic acid and
vortexed, then 5
µ
L of acetonitrile was added to the mix, strongly vortexed till the pellet completely
dissolved and centrifuged as above. Supernatants (1
µ
L) were spotted onto the MALDI-TOF polished
steel target plate (Bruker, Bremen, Germany) and dried at room temperature. Subsequently, 0.5
µ
L
of MALDI-TOF matrix (a saturated solution of
α
-cyano-4-hydroxycinnamic acid (Bruker) in 50%
acetonitrile and 2.5% trifluoroacetic acid) was applied onto the colony and allowed to dry before testing.
A bacterial test standard (Bruker) was used to calibrate the MALDI-TOF method prior to each run.
4.5. MALDI-TOF Mass Spectrometric Measurements and Isolate Identification
Automated acquisition of the mass spectra (2000–20,000 Da) was done using an UltraFlex
MALDI-TOF mass spectrometer (Bruker, Germany), equipped with a nitrogen laser, working in linear
positive mode and controlled by FlexControl software (v3.4, Bruker). Spectra were inspected using the
FlexAnalysis software (v3.4, Bruker). Processing of raw spectra, search against the bacterial database
(7,014 entries of reference strains) and similarity clustering was performed using MALDI BioTyper
software (v3.1, Bruker). For bacterial identification, a log score is generated by the software based
on the similarity to database entries, ranging from 0.00 to 3.00. A score of 2.30–3.00 was considered
as highly probable species identification, a score of 2.00–2.29 as secure genus and probable species
identification, and a score of 1.70–1.99 as probable genus identification.
4.6. Plant Microbiome Analysis
For the comprehensive microbiome composition of above ground usually fresh indigested herbs,
Lepidium sativum leaves were investigated applying the PhyloChip Array method (PhyloChip G3,
Int. J. Mol. Sci. 2019,20, 3661 12 of 16
Second Genome, San Bruno, CA, USA). Plant material was freeze-dried. Total DNA was extracted
using DNeasy Plant MiniKit (Qiagen, Germany) and moved forward for hybridization. After adding
PhyloChip Control Mix
TM
to each amplified product, these products were fragmented, biotin labeled
and hybridized to the PhyloChip
TM
Array. Data analysis was performed using PhyCA-Stats
TM
analysis software (Second Genome, USA). Significant present genus-specific OTUs per sample were
summarized in an OTU table. The absence-presence pattern of the genera were illustrated as heatmap
exerting the heatmap.2() function of the R package ggplot2. The map was further arranged according
the genera absence-presence pattern similarity, applying the binary method of the dist() function in
R on rows, and according the sample specific UPGMA cluster (columns). Taxonomic aliation of
the genus-specific OTUs, assigned within the OTU table, were parsed with an in-house script into a
Newick tree, where each node/furcation towards the leaf node can be equated with a new taxonomic
level, starting from the kingdom Bacteria at root towards the genus OTU at each leaf. The tree was
visualized with iTol [
72
]. Non-allocable taxonomic paths towards the leaves were plotted as spotted
lines. The genus OTU identifiers were used as leaf labels and their background colored according its
phylum. Their absence (gray box) and presence (black box) across all eight samples, encompassing the
two independent experiments with four replicates, is displayed in the surrounding circles. Genera
including potential probiotic strains were emphasized with a green dot behind the leaf label.
Supplementary Materials:
Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/15/
3661/s1. Supplemental Table S1: Identification of 156 bacterial isolates derived from the phyllosphere of Trigonella
foenum-graecum L., Cichorium endivia L., Coriandrum sativum L., Petroselinum crispum L., and Thymus vulgaris
L., using MALDI-TOF mass spectrometry. A score of 2.30–3.00 was considered as highly probable species
identification (dark green), a score of 2.00–2.29 as secure genus and probable species identification (bright
green), and a score of 1.70–1.99 as probable genus identification (yellow). Scores below 1.70 were accepted for
probable genus identification, when at least the top two database matches revealed the same genus (orange).
Supplemental Figure S1: Exemplary MALDI-TOF MS profiles obtained from strains that all were identified as
Pediococcus pentosaceus. The strains were isolated from the phyllosphere of four dierent plant species, as shown
in the figure. Some strain-specific peak patterns are indicated by boxes. Supplemental Figure S2: Heatmap of
genus absence-presence patterns (respectively white and black) is shown for the microbiome of Lepidium sativum
phyllosphere of two independent experiments (Repl_1, Repl_2) comprising four replicates each. Genus-specific
OTUs were clustered regarding their binary absence-presence pattern, using the binary method of the R dist()
function, whereas the samples were grouped based on the UPGMA clustering algorithm.
Author Contributions:
S.P., K.W., and A.-C.S. performed the experiments and data analysis. S.R. conceived the
research design. S.P., K.W., and S.R. wrote the paper.
Acknowledgments:
We thank Mandy Heinze and Birgit Wernitz for excellent technical assistance. Funding by
the Federal Ministry of Research and Education (SproutMO, FKZ 01DH14009) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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... Les bactéries des genres Bacillus (Bacillaceae) et Staphylococcus (Staphylococcaceae) sont présentes chez toutes les espèces d'isopodes, et ce quels que soit l'origine des individus et le tissu considéré. Ce sont des bactéries ubiquistes dans la nature, elles sont présentes dans beaucoup d'écosystèmes marins, d'eau douce et terrestres(Chen et al., 2019;Fernandes et al., 2019;Jin et al., 2020;Kamimura et al., 2019;Patz et al., 2019;Rahman et al., 2019). Les Bacillus sont fréquentes dans le sol, et certaines d'entre elles jouent un rôle important dans le cycle du carbone, de l'azote, et dans la dégradationde la ...
Thesis
Les isopodes représentent un modèle de choix pour l’étude des interactions au sein de l’holobionte car ils hébergent un microbiote riche et diversifié, dont la composition est très variable, sous la dépendance de l’environnement et des traits d’histoire de vie de l’hôte. Certains de leurs endosymbiotes tels que la bactérie féminisante Wolbachia et leurs conséquences sur la trajectoire évolutive des hôtes ont été bien caractérisés. Une perspective plus globale des interactions au sein de l’holobionte et leurs impacts sur la valeur adaptative de l’hôte n’a été que peu envisagée. Dans ce cadre, nous avons utilisé une combinaison d’approches expérimentales, de métagénomique, de transcriptomique et d’analyse de l’expression de gènes, afin de mieux comprendre les mécanismes et les conséquences de ces interactions sur la valeur adaptative de l’hôte. Dans un premier temps, cette thèse explore les interactions hôtes-microbiotes au travers du processus de dégradation de la lignocellulose, essentiel dans la nutrition des isopodes. Le répertoire enzymatique lié à la dégradation de la lignocellulose de l’hôte et du microbiote du cloporte Armadillidium vulgare ont été identifiés. La comparaison de ces deux répertoires enzymatiques a permis de mettre en évidence leur complémentarité, suggérant ainsi une étroite collaboration entre l’hôte et son microbiote pour la dégradation de la lignocellulose. La contribution de l’hôte et de son microbiote dans ce processus a ensuite été quantifiée en fonction de plusieurs régimes alimentaires avec des approches de métabarcoding et d’expression de gènes de CAZymes, enzymes spécialisées dans la dégradation de la lignocellulose. Dans un second temps, l’identification du répertoire de CAZymes dans 64 transcriptomes d’isopodes aquatiques et terrestres a permis d’apporter de nouveaux éléments sur les processus évolutifs qui ont favorisé la conquête des écosystèmes terrestres par les isopodes. Ces travaux ont été approfondis par l’obtention des métagénomes de cinq espèces d’isopodes permettant de caractériser les stratégies de déconstruction de la lignocellulose dans l’holobionte. La reconstruction des génomes bactériens à partir des métagénomes a permis d’identifier d’autres symbiotes chez les isopodes. Nous avons parallèlement caractérisé un premier virome des isopodes, qui montre que les phages constituent une part importante du microbiote. Ils pourraient jouer un rôle crucial comme régulateurs des communautés bactériennes au sein de l’holobionte. L’ensemble de ces travaux illustre la richesse des interactions entre le microbiote et l’hôte chez les isopodes pour la dégradation de la lignocellulose, et ouvre la voie à de nouvelles implications du microbiote au sein de l’holobionte des isopodes.
... , supporting the stability and reproducibility of RCS enriched consortium. Uninoculated reactor showed that the substrate endogenous bacterial diversity was low (less than 200 species), mainly composed of Enterococcaceae (32.6%) (Firmicutes), Enterobacteriaceae (62.7%) and Moraxellaceae (2.4%) (Proteobacteria), described as phyllosphere taxa(Williams and Marco, 2014;Patz et al., 2019).Others phyla, particularly Bacteroidetes and Fibrobacteres, were completely absent in the control reactor. Some taxa that were not detected on the substrate or were rare at the beginning of the incubation in this control reactor, increased with time; for example, Lachnospiraceae found at 0.09% on day 0 reached 30.9% on day 21 and Ruminococcaceae, found at 0.04% on day 0, increased to 24.4% abundance on day 21. ...
Thesis
Full-text available
La lignocellulose, principal composant des parois végétales et sous-produit agricole très abondant, est une des sources de carbone renouvelable les plus prometteuses pour la production d'énergie, de produits chimiques ou de biocarburants. L’utilisation de la lignocellulose pour la production de carboxylates repose sur l'utilisation de consortia microbiens. Pour améliorer les rendements de production de carboxylates, ces travaux de thèse se sont intéressés à i) caractériser le fonctionnement des consortia sélectionnés pour leur potentiel lignocellulolytique à partir de différents écosystèmes naturels et ii) à mieux comprendre leurs réponses fonctionnelles face à des changements dans la composition et la structure physicochimique du substrat. Pour cela, des approches d’écologie microbienne et des outils multi-omiques ont été appliquées.Ainsi, des consortia dérivés de rumen bovin ou d'intestin de termites ont été étudiés par des approches de métabarcoding 16S et de métaprotéomique comparative label-free. Leurs réponses fonctionnelles, enzymatiques et taxonomiques ont été évaluées en utilisant la paille de blée prétraitée par des méthodes mécaniques et/ou chimiques. Ces recherches ont permis de mettre en lumière le rôle et la complémentarité des différentes populations composant les consortia microbiens, ainsi que l’effet des modifications induites par le prétraitement du substrat sur la régulation de l’expression des enzymes impliquées dans la bioconversion des carbohydrates (CAZymes) en carboxylates. De plus, un consortium issu de rumen bovin et enrichi sur des résidus de maïs a également été étudié. L’étude de la dynamique des populations bactériennes au cours de l’enrichissement et de la bioconversion de la lignocellulose a permis de mettre en lumière le rôle des microorganismes libres et attachées au substrat, et de leurs enzymes, fournissant un aperçu inédit du fonctionnement des communautés lignocellulolytiques en bioréacteur.
... Additionally, bacteria belonging to genera, viz. Enterococcus, Pediococcus, Leuconostoc, Bacillus, Propionibacterium, Akkermansia, Staphylococcus, and Clostridium were also reported from phyllosphere of different plant species utilised as herbs and eaten raw (Patz et al. 2019). ...
Chapter
Plant microbiome refers to the diverse microbial counterparts that are associated with plants and plays a crucial role in host biology, ecology, and evolution. Though plant microbiomes have history of co-evolution with the host plants, certain members other than the core-microbiomes are shaped by various factors including plant genotype, plant age, associated host plant tissue or organ, other interacting microbial associates, arthropods, various environmental factors such as soil physio-chemistry, and human inference such as crop domestication, intensive and extensive cultivation, and use of agrochemicals especially in case of agro-ecosystems. Classical knowledge based on microbial culturing techniques and biochemical analysis prejudiced that when a plant interacts with a microbial partner the relationship could be detrimental as with pathogen interaction or promote plant growth in case of symbiotic associations. Advances in molecular techniques such as culture-independent approaches, next-generation sequencing, and high-throughput screening methods helped us to understand the robust nature of plant-associated microbiomes and their crucial role in plant fitness, environmental protection, and human health. This chapter gives a glimpse of patterns of plant microbiome associations and their importance in plant health and emphasise the importance of both basic and applied research which will enlighten us with deeper insights on the plant microbiomes. This will help us identify economical, eco-friendly, and effective strategies of manipulating the plant-associated microbiomes which can open up new avenues in maintaining plant health and ecological fitness and sustain crop production in a clean green way preserving the nature’s serenity and human health.
... can occur on asparagus, the availability of a rapid and safe species-specific diagnosis tool would be useful. Nowadays, the identification of microbial pathogens is routinely performed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonik, Bremen, Germany ), especially for bacterial iso lates [36]. Additionally, mass-spectrometry-based approaches have also been applied successfully for the identification and differentiation of Fusarium species based on spore protein profiles [37,38]. ...
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