plants

Article
Metagenomic Insights of the Root Colonizing
Microbiome Associated with Symptomatic and
Non-Symptomatic Bananas in Fusarium Wilt
Infected Fields
Manoj Kaushal 1, *, George Mahuku 1 and Rony Swennen 2,3,4
1 International Institute of Tropical Agriculture (IITA), Mikocheni B, Dar es Salaam-34441, Tanzania;
[email protected]
2 Bioversity International, Willem De Croylaan 42, B-3001 Leuven, Belgium; [email protected]
3 Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, KU Leuven,
B-3001 Leuven, Belgium
4 International Institute of Tropical Agriculture. c/o The Nelson Mandela African Institution of Science and
Technology (NM-AIST), P.O. Box 447, Arusha 23306, Tanzania
* Correspondence: [email protected]; Tel.: +255-758589012




Received: 23 December 2019; Accepted: 14 February 2020; Published: 18 February 2020


Abstract: Plants tissues are colonized by diverse communities of microorganisms called endophytes.
They are key determinants of plant production and health, for example by facilitating nutrient
exchanges or limiting disease development. Endophytic communities of banana plants have not been
studied until very recently, and their potential role in disease development has not been explored
so far. Roots from symptomatic and non-symptomatic banana plants were sampled from fields
infected by Fusarium oxysporum f.sp. cubense race 1. The goal was to compare the endophytic
microbiota between symptomatic and non-symptomatic plants through high throughput sequencing
of 16s rDNA and shotgun metagenome sequencing. The results revealed that the endophytic root
microbiome in bananas is dominated by Proteobacteria and Bacteroidetes followed to a lesser extent by
Actinobacteria. The development of disease greatly impacted the endophytic microbial communities.
For example, Flavobacteriales abundance was correlated with symptom development.

Keywords: banana; microbial diversity; rhizosphere; metagenomics; Fusarium oxysporum

1. Introduction
Plants live in close relationship with microbes that inhabit the rhizosphere region. The microbiome
plays a key role for plant health and productivity via nutrient acquisition, tolerance to stress conditions,
and inducing resistance against phytopathogens [1]. In addition, plants recruit microflora via the
production of root exudates. The endophytic microbiome corresponds to the microbes that colonize
and reside within the plant tissues [2]. These microbiomes have been exploited as biocontrol agents
promoting plant growth and health in various crops [3,4]. Despite this, endophytic microbiota are
poorly studied. Advanced technologies such as high throughput sequencing (HTS) allow for an
in-depth characterization of the functions of the plant endophytes [5–7].
Bananas contribute to a major portion of the diet and a key source of the income for small holder
farmers in Sub-Saharan Africa (SSA). Mchare is an important highland cooking banana, especially
in the highlands of Northern Tanzania and Great Lake regions of Kenya and Uganda [8], and Sukari
Ndizi is a popular dessert banana in Eastern Africa [9]. Banana plants are multiplied clonally through
suckers in the field or in a nursery (macropropagation) or by in vitro culture (micropropagation).

Plants 2020, 9, 263; doi:10.3390/plants9020263 www.mdpi.com/journal/plants

Plants 2020, 9, 263 2 of 18

The production of bananas has been limited by biotic and abiotic causes [10]. Various insects, pests,
and diseases constitute the biotic constraints to the banana plants, whereas the abiotic constraints
include heat, desiccation, salt, nutrient imbalance, and physical stress such as wind. Fusarium wilt
(FW), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc) is one of the most
devastating diseases [11]. In SSA, banana production is constrained by Fusarium wilt due to Foc race 1.
In addition, there is a threat posed by Foc race 4 [12–15], which is only found in Mozambique but
which could emerge in other countries [16] due to movement of planting materials. Foc invades the
vascular tissues of bananas through the roots and corm. The initial symptoms correspond to leaves
discolouration and wilting, which eventually turn into a bright yellow colour and dead leaf margins.
In the advanced stages of infection, more leaves become yellow, and necroses (brown lines) are evident
inside the pseudo-stem. The spores of Foc can persist in the soil for decades in the absence of a
host. Besides resistant varieties or disease-free planting material, no other effective disease control
methods exist for Foc [17]. Mchare and Sukari Ndizi are the most preferred varieties by smallholder
farmers in Tanzania that are susceptible to Foc race 1. Nevertheless, susceptible banana plants in
East African fields, cultivated with other banana varieties in association with other crops, can remain
non-symptomatic while neighbouring banana plants exhibit symptoms and succumb. This is in
contrast with large monocultured commercial plantations which are entirely wiped out once Foc has
entered. This phenomenon deserves a better characterization as these plants were clonally multiplied
and are growing in the same pedo-climatic conditions. Such fields were identified in Tanzania, and we
hypothesize that plant microbiota preserve the plant health and limit the disease development. More
specifically, part of the endophytic microbiota would colonize the inner tissues of the host and could
be in direct contact with Foc.
The level of microbiome diversity was directly correlated with the resistance to the Foc
invasions in bananas [18] and multiplication in the roots. Sometimes, host plants also recruit
specific beneficial microbiota after phytopathogen infections, which helps the plants to resist and
withstand the diseases caused by these pathogens [19]. We aimed to understand the composition and
distribution of the microbiome in symptomatic and non-symptomatic banana roots in two locations
and in two cultivars. We also aimed to analyse the banana root microbiome of Foc symptomatic
samples using sequencing-based functional metagenomics to characterize the gene repertoire of the
banana endophytes.

2. Results

2.1. General Characteristics of the Amplicon

After QIIME filtration and the removal of chimeric sequences, 308,784 seq out of 349,670 (S1H),
337,074 seq out of 381,417 (S2I), 242,504 seq out of 278,606 (S3H), 281,162 seq out of 307,503 (S4I),
464,508 seq out of 544,149 (S5H), 385,006 seq out of 457,699 (S6I), 199,861 seq out of 222,216 (S7H),
and 414,864 seq out of 467,607 (S8I) were obtained and taken forward for analysis (Supplementary
Table S1). Illumina sequencing for the pooled symptomatic samples resulted in a total of 99,692,954
high quality reads (average length of 312 bp) and about 13.97 Gb of base pairs from a mate-pair library.
The high-quality reads were assembled de novo with metaSPAdes, followed by gene prediction using
Prodigal at the metagenome mode wherein a total of 573,271 genes were identified.

2.2. Root Associated Microbiome Composition and Structure in Symptomatic and Non-Symptomatic Banana
After removal of singleton operational taxonomic units (OTUs), 21,897 OTUs were clustered
with a variable repartition among samples: 6571 (S1H), 5561 (S2I), 6782 (S3H), 5311 (S4I), 7450 (S5H),
8747 (S6I), 4867 (S7H), and 7811 (S8I) (Supplementary Table S2). The closely related microbiota
were found to be higher in symptomatic samples rather than in non-symptomatic root samples
at both locations. The results revealed that Proteobacteria were the predominant bacterial groups
in the non-symptomatic samples, followed by Bacteriodetes and Actinobacteria (Figure 1A–C).
Proteobacteria were observed more in the symptomatic root samples (59.60–81.80%) of Mchare than in
Plants 2020, 9, 263 3 of 18

the non-symptomatic samples (51.40–56.70%). In contrast, for Sukari Ndizi, in the non-symptomatic
banana samples (60.10–67.70%) more Proteobacteria diversity was observed than in the symptomatic
samples (53.50–58.20%). The members of Proteobacteria were relatively diverse and consisted mainly of
Flavobacteriales, Rhizobiales, Pseudomonadales, Burkholderiales, and Firmicutes. Bacteriodetes was
observed more in the non-symptomatic root samples (16.30–34.90%) of Mchare than in the symptomatic
samples (10.60–19.50%). In contrast, in Sukari Ndizi, the symptomatic banana samples (7.30–23.60%)
had a higher proportion of Bacteriodetes than the non-symptomatic samples did (9.20–17.10%).
Among the Actinobacteria, a higher diversity was observed in the non-symptomatic (12.20–13.30%)
than in the symptomatic samples (1.50–6.90%) of the Mchare cultivar. No clear differences were
observed with Actinobacteria among the symptomatic and non-symptomatic samples of Sukari Ndizi.
Actinomycetales and Alteromonadales were the predominant groups in the Actinobacteria.
The heatmap employing the relative abundance of the most common defined OTUs with more
than 5000 sequences in the banana root endophytes displayed a significant enrichment for the OTUs
belonging to Actinomycetales (mainly composed of Streptomycetales) of the phylum Actinobacteria,
and Alteromonadales and Pseudomonadales of the Gammaproteobacteria. Furthermore, we analysed
the most abundant 11,409 OTUs of the banana root-associated microbiome. The vast group of detected
OTUs were common (52.1%); however, 20.1% were identified only in the non-symptomatic and 27.8%
in the symptomatic banana root among the 11,409 that were classified as root endophytes (Figure 2).
The 52.1% overlap of OTU in the Venn diagram between the symptomatic and non-symptomatic
banana samples suggested that, despite the significant amount of separation in bacterial communities,
there is a presence of individual bacterial OTUs among samples. Non-symptomatic cultivars of Mchare
(33.90%) exhibited a higher percentage of species as compared to Sukari Ndizi (23.40%). Similarly,
symptomatic samples exhibited a higher diversity in Mchare (32.60%) when compared to Sukari Ndizi
(29.20%). With respect to the location, a higher number of species was observed in Akheri Kati than in
TACRI, both for the non-symptomatic and symptomatic samples of both Mchare (17.3% and 19.6%) and
Sukari Ndizi (7.9% and 19%), respectively. The beta diversity was calculated in a qualitative manner
using Euclidean, Jaccard, and Bray-Curtis’s metrics (Supplementary Table S3). The data of the banana
root-associated microbiome displayed a trend for the non-symptomatic samples of both Mchare and
Sukari Ndizi cultivars irrespective of the locations. Both the symptomatic and non-symptomatic root
endophytes harbored communities that are clustered together, as shown in the phylogenetic map
drawn from the UPGMA analysis (Figure 3). The closeness of each root microbiome samples of the
symptomatic and non-symptomatic banana plants was represented in terms of a dissimilarity matrix.
The alpha diversity of root endophytes calculated by the Shannon index displayed a significant
difference and variation between symptomatic and non-symptomatic banana roots of Mchare and
Sukari Ndizi (Supplementary Table S4). The root endophytes in the symptomatic plants had a higher
OTU richness and diversity than in the non-symptomatic banana samples (Figure 2; Supplementary
Table S4). Additional 10,809 OTUs were found in the root microbiome of bananas associated with
the symptomatic samples, which revealed the presence of other microbial species in banana roots
after pathogen infection. The beta diversity analysis reflected the difference in microbial composition
due to the impact of the Fusarium infection of banana roots. Symptomatic samples of Mchare
were dominated by Pseudomonadales (25.12%) from the samples of TACRI, and Rhizobiales and
Burkholderiales (15.20%) from the samples of Akheri Kati. However, the non-symptomatic samples of
Mchare were observed as having a higher diversity of Rhizobiales (28.50%) than the samples of Akheri
Kati and Flavobacteriales (30.30%) from the samples of TACRI. In the case of Sukari Ndizi, a higher
diversity was observed with Rhizobiales (21.10%) and Alteromonadales (18.50%) in the symptomatic
samples collected from TACRI and Akheri Kati, respectively. Non-symptomatic banana samples were
observed rich in Rhizobiales at both TACRI and Akheri Kati with a diversity of 26.10% and 19.50%,
respectively. In general, irrespective of the location and cultivars, microbes from symptomatic banana
roots were dominated by Pseudomonadales (25.12%), Rhizobiales (21.15%), Actinomycetales (19.76%),
Alteromonadales (18.55%), Burkholderiales (15.22%), and Flavobacteriales (13.66%) of Proteobacteria.
Plants 9, 263
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Figure 1. Cont.
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Figure
Figure 1. The
1. The compositionand
composition andrelative
relative abundance
abundance of
of major
majorbacterial
bacterialtaxa of of
taxa thethe
root-associated microbiome
root-associated in non-symptomatic
microbiome and symptomatic
in non-symptomatic banana. banana.
and symptomatic (A)
Endophytes inhabiting the non-symptomatic banana roots, (B) Endophytes inhabiting the symptomatic banana roots, and (C) Community composition. Where: Where:
(A) Endophytes inhabiting the non-symptomatic banana roots, (B) Endophytes inhabiting the symptomatic banana roots, and (C) Community composition. *S:
Sample;1–8:
*S: Sample; 1–8:Number;
Number; H: H: Non-symptomatic;
Non-symptomatic; I:I:Symptomatic.
Symptomatic.Samples
SamplesfromfromTACRI:
TACRI: S1H, S2IS2I
S1H, (Mchare); S3H,
(Mchare); S4IS4I
S3H, (Sukari Ndizi),
(Sukari andand
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samples Akheri
from Kati:Kati:
Akheri S5H,S5H,
S6I (Mchare);
S6I (Mchare); S7H,S7H,
S8I S8I (Sukari
(Sukari Ndizi).
Ndizi).
Plants 2020, 9, 263 6 of 18
Plants 2020, 9, x FOR PEER REVIEW 7 of 19

Figure 2. Venn diagram of the banana root microbiome: (A) Most abundant shared and unique OTUs
Figure 2. Venn diagram of the banana root microbiome: (A) Most abundant shared and unique OTUs
in non-symptomatic and symptomatic samples, (B) Shared OTUs in non-symptomatic samples, and
in non-symptomatic and symptomatic samples, (B) Shared OTUs in non-symptomatic samples, and
(C) Shared OTUs in symptomatic samples. Where: *S: Sample; 1–8: Number; H: Non-symptomatic;
(C) Shared OTUs in symptomatic samples. Where: *S: Sample; 1–8: Number; H: Non-symptomatic; I:
I: Symptomatic. The various colours corresponds to the sample location and cultivars in Venn digram.
Symptomatic. The various colours corresponds to the sample location and cultivars in Venn digram.
Samples from TACRI: S1H, S2I (Mchare); S3H, S4I (Sukari Ndizi) and samples from Akheri Kati: S5H,
Samples from TACRI: S1H, S2I (Mchare); S3H, S4I (Sukari Ndizi) and samples from Akheri Kati: S5H,
S6I (Mchare); S7H, S8I (Sukari Ndizi).
S6I (Mchare); S7H, S8I (Sukari Ndizi).
 Plants
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2020,
2020, FOR263PEER REVIEW 8 of 719of 18

Figure 3. Phylogenetic map for all the root samples. Where: *S: Sample; 1–8: Number; H:
Figure 3. Phylogenetic I:map
Non-symptomatic; for all the root
Symptomatic. samples.
Samples fromWhere:
TACRI:*S:S1H,
Sample; 1–8: Number;
S2I (Mchare); S3H, H:
S4INon-
(Sukari
symptomatic;
Ndizi) andI:samples
Symptomatic. Samples
from Akheri from
Kati: TACRI:
S5H, S1H, S2IS7H,
S6I (Mchare); (Mchare); S3H, Ndizi).
S8I (Sukari S4I (Sukari Ndizi) and
samples from Akheri Kati: S5H, S6I (Mchare); S7H, S8I (Sukari Ndizi).
It was also observed that the non-symptomatic banana roots were dominated by many specific
It was such
genera, also observed that the non-symptomatic
as Flavobacterium, banana and
Pseudomonas, Devosia, rootsPaenibacillus.
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accounted Proteobacteria and
for approximately
Bacteroidetes dominated the endophytic bacterial content of roots and accounted for approximately to
92.40% of the total microbiome community. When non-symptomatic roots were compared
92.40% of the total
symptomatic roots,microbiome
symptomaticcommunity. When abundance
roots had a higher non-symptomatic roots of
and diversity were compared
Rhizobiales to
(12.85%)
symptomatic roots, symptomatic
and Flavobacteriales (12.00%)roots had1C).
(Figure a higher abundance
Moreover, some andOTUdiversity
fractionsofofRhizobiales (12.85%)
Flavobacteriales were
and Flavobacteriales (12.00%) (Figure 1C). Moreover, some OTU fractions of Flavobacteriales were
also identified in one sample (S1H) of Mchare non-symptomatic banana roots collected from TACRI.
alsoActinobacteria
identified in one sample
(mainly (S1H) ofofMchare
composed non-symptomatic
Actinomycetales banana roots collected
and Streptomycetales) from
was another TACRI.
predominant
Actinobacteria
bacterial group, (mainly composed
with 9.30% of Actinomycetales
of the total identified OTUs among and Streptomycetales)
the endophytes, and wasit displayed
another a
predominant
significant bacterial
enrichment group, with 9.30% of
in symptomatic thenon-symptomatic
and total identified OTUs among
samples the endophytes,
of Sukari and from
Ndizi collected it
displayed
TACRI.aAsignificant
ubiquitously enrichment
identifiedingroup
symptomatic and non-symptomatic
with a persistent samples of was
community composition Sukari Ndizi
Rhizobiales,
collected
for bothfrom
the TACRI.
symptomaticA ubiquitously identified group
and non-symptomatic roots, with a persistent
irrespective of thecommunity
cultivars and composition
locations.
was Rhizobiales, for both the symptomatic and non-symptomatic roots, irrespective of the cultivars
In the present investigation, the comparative analysis of community profiles displayed a higher
anddiversity
locations. and OTU abundance of the root microbiomes in non-symptomatic and symptomatic plants.
In the presentclassifications
The functional investigation,revealed
the comparative analysis
that 269,046 (COG of community
database), profiles
245,805 (KEGG displayed
pathway a higher
database),
diversity
287,043and OTU
(Pfam abundance
database), of the(FIGfam
160,806 root microbiomes
database), inandnon-symptomatic and symptomatic
173,335 (GO database) plants.
genes were annotated.
The functional classifications revealed that 269,046 (COG database), 245,805 (KEGG pathway
database), 287,043 (Pfam database), 160,806 (FIGfam database), and 173,335 (GO database) genes
Plants 2020, 9, 263 8 of 18

The topmost taxonomic findings from the Kaiju results showed Proteobacteria with 168,867 genes as
the top phylum, Alphaproteobacteria with 75,473 genes as the top Class, Rhizobiales with 53,183 as
the top Order, Flavobacteriaceae with 26,166 genes as the top Family, Flavobacterium with 18,663 as
the top genus, and Fluviicola taffensis with 8640 genes as the top species. In symptomatic samples, we
identified genes encoding enzymes within microbiome that are involved in the degradation of host
cell walls. Most of them were related to the oligosaccharide-degrading enzymes with few encoding
endo-β-1,4-xylanase and polygalacturonase.

2.3. Growth Ameliorating Endophytes and Gene Function

The functional categories were compared to investigate the enriched functions among banana root
endophytes. The COGs database was used on a phylogenetic classification of the proteins encoded in
21 complete genomes of bacteria. A different COG database was identified with the genes associated
with different functional categories of the identified root microbiome (Figure 4A,B). A high relative
abundance of genes with a significant functional enrichment associated with growth promotion
(cell division) was observed. Several categories, including carbohydrates, protein, RNA, and DNA,
which related to metabolism in plants, were observed and existed with a high proportion. Moreover,
the reads that classified into virulence, disease, defense, and stress responses may contribute to disease
suppression in the banana samples with Fusarium infection. These results were in accordance with
the COG analysis mentioned above (Figure 4). Genes annotated under different functional categories
also displayed a high abundance of pathogenicity-related genes (cell signaling and regulation such
as quorum sensing) and membrane transport in Flavobacteriales. Apart from other endophytes,
Flavobacteriales and Actinobacteriales were clustered together, displaying their close functional
similarity (Figure 4B).
Plants 2020, 9, 263 9 of 18

Figure 4. (A) The distribution of genes in the COG functional annotation. (B) Heatmap showing the relative abundance of functional categories of the major
taxa-based assemblies.
Plants 2020, 9, 263 10 of 18

3. Discussion
Banana roots samples were collected from symptomatic and non-symptomatic banana plants
of two Fusarium susceptible varieties, Mchare and Sukari Ndizi, from two different locations. The
eight samples (S1H, S2I, S3H, S4I, S5H, S6I, S7H, and S8I) were analyzed together with a QIIME
suite of tools for a microbial analysis. A metagenomic analysis of the microbial community involves
problems of plant DNA intrusion which adversely affect the analysis [20,21]. In the present study, we
used a well-defined protocol to eliminate the intervention of banana host DNA that inhabits Fusarium
symptomatic roots. We clustered the similar sequences into one representative taxonomic unit called
OTU. The basis of this sequence clustering was a 97% sequence similarity and was implemented
through a UCLUST algorithm. These sequences were used to detect OTU in the de novo method.
Following OTU detection, a representative sequence was assigned to each OTU, facilitating the
taxonomic annotation using the Greengene database. Each sample of banana roots was observed
with a significant difference in the community composition and species abundance. Irrespective of
the cultivars and locations, Proteobacteria and Actinobacteria were consistently enriched in the roots
of both symptomatic and non-symptomatic samples, as observed by other researchers for the plant
species of banana, rice, maize, wheat, and sugar cane [22–24], which are generally known for their
good response to labile carbon sources with the fast growth of the plants [25,26]. Taken together, our
results reveal that Proteobacteria and Actinobacteria are adapted to banana roots. Further analyses of
most abundant banana-associated OTUs in root samples reflect a community variation with OTUs’
quantity and not their species in both Mchare and Sukari Ndizi cultivars. These most abundant OTUs
of endophytes identified in the roots are consistent with earlier reports which further hypothesize that
the endophytic microbiome was acquired from Rhizobacteria [5].
The host-microbiome interactions might change when a plant is attacked by phytopathogens.
We found a significant fraction of variation and differentiation in the endophytic diversity (both α- and
β-diversity) from the infection by Fusarium in both Mchare and Sukari Ndizi cultivars. This could be
attributed to the host genetics of symptomatic samples when compared to those from non-symptomatic
banana roots. More dispersion of OTUs among the symptomatic samples displayed that other microbes
were present in the root samples associated with pathogen infections. In the non-symptomatic roots
of Mchare and Sukari Ndizi cultivars, Rhizobales and Flavobacteriales were present in very small
fractions (less than 1%), but a significantly enhanced fraction (from 12% to 21%) was noticed in the
symptomatic root microbiome of both cultivars. The different root microbiome of symptomatic and
non-symptomatic samples is mostly due to variation in the members of Proteobacteria (observed in
Mchare and Sukari Ndizi), as confirmed by a paired metastatistical analysis.
In the non-symptomatic banana root microbiome of Sukari Ndizi (16.30%) and Mchare (12.00%)
cultivars, Rhizobiales are associated with a high abundance of the diverse group of symbiotic
nitrogen-fixers Devosia. Samples collected from non-symptomatic banana roots from Akheri Kati of
Mchare and Sukari Ndizi cultivars also exhibited a lower but significant abundance of Bradyrhizobium,
at 3.70% and 4.60%, respectively. However, Agrobacterium, a very common plant pathogen, was the
predominant bacterial group (2.20–5.30%) present with Rhizobiales in both cultivars at both locations.
Producers of active metabolic compounds against phytopathogens such as Actinomycetales were
also present in the root microbiome [27,28] and predominant in the non-symptomatic (6.70%) and
symptomatic (14.90%) samples of Sukari Ndizi. This could demonstrate the biocontrol response against
Fusarium infection in bananas via the variation in the microbial community between non-symptomatic
and symptomatic plant roots. Our work is supported by the previous study on Pseudomonadales,
the third-most abundant bacterial group in non-symptomatic banana roots. A high abundance of
Pseudomonadales was observed in both the Mchare (16.80%) and Sukari Ndizi (7.50%) cultivars of
non-symptomatic banana root samples, as compared to symptomatic samples. Pseudomonadales has
been widely investigated for its growth promotion and phytopathogen suppression efficacies, and also
in order to induce systemically induced resistance in plants [29,30]. Variation in the composition and
Plants 2020, 9, 263 11 of 18

abundance of the endophytic microbial community in symptomatic root samples provides the clue for
the effective response by beneficial microbes in root tissues during fungal pathogen attacks.
Endophytes have multiple strategies to overcome competition. Our results are supported by the
microbial community analysis of banana, sugar cane, and rice plants, which harbored an identical
microbiome profile dominated by Actinobacteria and Proteobacteria groups [31–33]. Before attacking
plants, phytopathogens secrete a number of lytic enzymes (pectinase, xylanase, and cellulase) in order
to break the cell wall, which is mainly composed of lignocellulose and in order to potentially protect
the plant from invasion by foreign microbes [34]. Endophytes also break the lignocellulosic barrier
for their entry but do so without causing any negative symptoms in the host tissue, as the bacterial
entry into the root tissue of hosts is the pre-requisite to become an endophyte [35]. It is also evident
that the endophytic colonization in host root tissues involves cell wall degradation enzymes such as
pectinases and cellulases such as endoglucanase and polygalacturonase, which are responsible for
bacterial entry and colonization [36]. The native endophytic microflora of plant root tissue might secrete
various enzymes for cell wall penetration that enable the outsider microflora to reside and replicate
within the host. To analyse the endophytic capabilities to produce lytic enzymes, we investigated an
assembly of metagenomic data for the existence of gene sequence codes for lignocellulose-degrading
enzymes. The functional annotation was done with COGNIZER to analyse the number of genes
assigned an annotation from each type of metagenomic database viz., COG, Pfam, FIG, KEGG, GO,
GenBank, PATRIC, and eggNOG. The presence of genes associated with oligosaccharide degradation
elucidated that endophytes degraded a small amount of polysaccharides (plant-associated) and used
oligosaccharides as a nutrient source with no damage or negative impact on the host tissue [36].
Endophytes have been widely studied in relation to benefitting plant health through various
kinds of growth promoting, disease suppression, and RIDER mechanisms [30]. The diversity of
microbiota associated with roots involved complex plant-microbe interactions which are crucial for
plant health. In our study of the metagenomic analysis of the banana root microbiome, we identified
endophytes like Bacillus and Pseudomonas in the non-symptomatic root samples of Sukari Ndizi and
Mchare cultivars. The endophytes like Bacillus have been effectively proven to possess a large number
of plant-growth promoting (nutrient solubilization and uptake, production of phytochromes) and
disease control (acting as biocontrol agents) attributes. Endophytes are widely known to produce IAA
(indole-3-acetic acid) to promote plant growth and development [37,38]. In addition to the diversity,
the taxonomic composition revealed a predominant colonization by Pseudomonadales (in both Mchare
and Sukari Ndizi), which are known plant colonizers (particularly the genus Pseudomonas) and are
responsible for beneficial plant-microbe interactions [39]. The metagenomic analysis of banana root
endophytes also revealed that it could be possible to get a potential candidate which serves as a
biocontrol agent against Fusarium wilt and other phytopathogens in bananas. The abundance of
Pseudomonadales was observed as showing a drastic increase (24.70%) in the symptomatic samples of
Mchare collected from TACRI. In the meantime, the high frequency presence of alpha-, beta-, gamma-,
and delta-Proteobacteria groups in Proteobacteria in symptomatic samples could be considered as
an important bacterium in the degradation of all kinds of glucoses, and could partially contribute
to the symptom observed after Fusarium infection [40]. Finally, all these enriched root microbiomes
advocated that the root endophyte microbiotas are selected based on the innate immune system of the
plant. Some of them might have performed as pathogenic complexes during the infection of the host
as it was also evident that non-antagonistic microbial strains can become antagonistic when they grow
with other specific strains [41].
The taxonomic classification and functional annotation in this study were carried out using
different pipelines. Using comparative pipelines made our results more reliable and allowed us better
chances to explore information that may otherwise have been left undiscovered.
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4. Materials and Methods

4.1. Sample Collection and DNA Extraction

Banana root samples were collected from TACRI (N” 36.4821◦ ; E” 3.235◦ ; 1230 m), in the Kilimanjaro
and Akheri Kati (N” 36.77541◦ ; E” 3.35579◦ ; 1452 m), in Arusha, Tanzania. Samples were collected
from Mchare and Sukari Ndizi cultivars from eight year old plants targeting Foc symptoms and
non-symptoms (lack Foc symptoms in plants) in the same field (Figure 5).
Both the cultivars are susceptible to Foc infection and are widely grown by smallholder farmers in
the region. Root samples were collected from a 15–30 cm depth from three random locations around
a single plant and were directly attached to the corm. For each cultivar (Mchare and Sukari Ndizi)
and type (Foc symptomatic and non-symptomatic), three plants (each from five different fields) were
targeted for the collection of root samples and made as one composite sample. In total, we collected
eight composite samples from two different sites representing non-symptomatic (S1H, S3H, S5H, and
S7H) and symptomatic (S2I, S4I, S6I, and S8I) bananas (Supplementary Figure S1). The samples were
immediately transferred to polythene bags and kept in ice until arrival at the laboratory. The samples
were kept at 4 ◦ C until processed. DNA was extracted using the Plant DNA Isolation Kit (Zymo
Research Corp., Irvine, CA, USA).
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2020, 9, x FOR PEER REVIEW 14 of 19 13 of 18

Figure 5. Banana plants used for sampling with typical symptoms of Fusarium wilt and with no symptoms.
Figure 5. Banana plants used for sampling with typical symptoms of Fusarium wilt and with no symptoms.
Plants 2020, 9, 263 14 of 18

4.2. PCR Amplification of 16S rRNA Gene

The primers 460F (50 -CCTACGGGNBGCASCAG-30 ) and 460R (50 -GACTACNVGGGTAT
CTAATCC-30 ) were used to amplify the V3-V4 hyper-variable region of the 16S rDNA gene of
bacteria and archaea [42]. The amplicons were amplified using i5 and i7 primers as per the standard
Illumina protocol [42]. The amplicon library was prepared with Nextera XT Index Kit (Illumina Inc.,
Hayward, CA, USA) as per the 16S Metagenomic Sequencing Library preparation protocol with a
2 × 150 read length. The amplicon libraries were purified by 1X AMpureXP beads, checked on Agilent
DNA1000 chip on Bioanalyzer2100, and quantified by Qubit Fluorometer 2.0 using Qubit dsDNA HS
Assay kit (Life Technologies, Carlsbad, CA, USA).

4.3. Bioinformatic Analysis of 16s Results

High quality paired-end reads were first stitched using a FLASH tool with a minimum of 10 bp
overlap to recreate the V3-V4 (forward and reverse hypervariable) regions and were then taken for
analysis. Furthermore, chimeras generated during the PCR process were first removed, using the
usearch61 algorithm. Then, similar sequences were clustered using the UCLUST algorithm and brought
together into one representative taxonomic unit called the Operational Taxonomic Unit (OTU) at 97%
sequence similarity criteria.

4.4. Sequencing, Assembly and Binning of the Pathogenesis-Associated Metagenome

The paired-end sequencing library was prepared with Truseq Nano DNA Library Prep kit (Cat. No.
20015965). The library preparation process for all the eight samples (four symptomatic and four
non-symptomatic) was initiated with 200 ng of gDNA [43].

4.5. Metagenome Assembly and Gene Prediction

The de novo assembly of high quality paired-end reads was accomplished with metaSPAdes
at default parameters including auto kmer. The genome assembler of metaSPAdes is a de Bruijn
graph-based assembler that performed well for metagenome data [43]. The statistical elements (number
of scaffolds, size of scaffolds, maximum and minimum size of scaffold, and their N50 value) of the
assemblies were calculated by Perl scripts. These generated scaffolds were subjected to genes prediction
with Prodigal (v2.6.3) at the metagenome mode and at default parameters. These predicted genes were
then taken further for a taxonomic and functional analysis.

4.6. Taxonomic Classification

The high-throughput sequencing data were classified from the whole metagenome sequencing
with the Kaiju program. First, the sequences were translated into the six possible reading frames, and
the resulting amino acid sequences were split into fragments at their stop codons. The fragments were
then sorted by their BLOSUM62 score (Greedy mode). This sorted list of fragments was then searched
against the reference protein database (NCBI non-redundant) with the backwards search algorithm on
the BWT (Burrows–Wheeler Transform algorithm). Once the remaining fragments in the list could
not achieve a better score (Greedy), the search stopped, and the taxon identifier of the corresponding
database sequence was retrieved.

4.7. Functional Annotation

The functional annotation was done with COGNIZER against a multiple database. COGNIZER
(v0.9b) was used at default parameters to assess the functional capacities of the microbial communities,
and to simultaneously provide Clusters of Orthologous Groups of proteins (COGs), KEGG (Kyoto
Encyclopedia of Genes and Genomes), Pfam, GO (Gene Ontology) and FIGfams annotations to
individual sequences. The predicted genes were taken as an input in COGNIZER for assigning
annotations using multiple databases.
Plants 2020, 9, 263 15 of 18

4.8. Annotation from COG Database and KEGG

The COG database is an attempt for a phylogenetic classification of the proteins encoded in
21 complete genomes of bacteria, archaea, and eukaryotes. The COGs were constructed by applying
the criterion of consistency of genome-specific best hits to the results of an exhaustive comparison
of all protein sequences from these genomes. This annotation aids in relating the identified genome
to other bacterial taxonomic lineages for both functionality and phylogeny. Genes annotated with
different functional attributes were displayed under different orthologous groups of the COG database.
The KEGG database was used to associate metabolic pathways with respective genes. The functional
heatmap showed a taxonomic assignment for each OTU (operational taxonomic unit).

4.9. Nucleotide Sequence Accession Numbers

The sequence data were deposited in NCBI with SRA accession PRJNA493728 and PRJNA494072.

5. Conclusions
In the present investigation, root-associated microbiomes were investigated in symptomatic
and non-symptomatic banana plants with the aim to investigate the responses of the endophytic
community during Fusarium infection and their functional attributes in endophyte-plant-fusarium
interactions. In our study, the non-symptomatic plants might have been infected due to the nature of
Foc that persists in soil for decades, even without a host and without the movement of farmers and
equipments between farms. However, the non-symptomatic plants were showing no symptoms of
disease development. Furthermore, Proteobacteria, followed by Bacteriodetes and Actinobacteria,
were the predominant bacterial groups. A comparative community analysis of non-symptomatic and
symptomatic banana roots also revealed the presence of Pseudomonadales and Streptomycetaceae,
widely known to produce antagonist compounds against phytopathogens. The manipulation of
pseudomonads and streptomyceae populations in rhizosphere soils could help us to reduce/diminish
the disease development in plants, even in the presences of the pathogen. Root microbiomes of both
Mchare and Sukari Ndizi cultivars were observed with an increased abundance of Flavobacteriales and
Rhizobiales endophytic groups, known to be involved in the carbohydrate metabolism. A metagenomic
analysis also revealed that several bacterial communities are associated with key infection processes
that include symbiotic relationships for the sake of nutrients, cell wall destruction at potential entry
sites of pathogens, as well as infection processes in banana roots. Future studies will focus on the
investigation of root pathogenesis-associated microbial communities, including activities and functions
of the microbiome, in order to understand complex microbe-plant-pathogen interactions that will
provide new opportunities to understand how microbiomes control plant health and that will open
new avenues to increase crop production.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/263/s1,

Figure S1. Layout plan for the banana root sample collection. Table S1. Summary of the QIIME filtration and
chimeric sequences removal. Table S2. Summary of clustering the similar sequences into one representative
taxonomic unit as the Operational Taxonomic Unit (OTU). Table S3. Summary of the beta diversity for each
sample. Table S4. Summary of the alpha diversity for each sample.
Author Contributions: M.K. and G.M. conceived the program. M.K. conducted the experiments, analyzed the
data and drafted the manuscript. M.K. and G.M. carried out field sampling. G.M. and R.S. helped interpret the
results of the research and correct mistakes in grammar. All authors have read and agreed to the published version
of the manuscript.
Funding: The authors thank all donors who supported this work through their contributions to the CGIAR Fund
(http://www.cgiar.org/who-we-are/cgiar-fund/fund-donors-2/), and in particular to the CGIAR Research Program
Roots, Tubers and Bananas (RTB-CRP).
Conflicts of Interest: The authors declare no conflict of interest.
Plants 2020, 9, 263 16 of 18

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