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Universal Journal of Microbiology Research 6(2): 35-42, 2018 http://www.hrpub.org
DOI: 10.13189/ujmr.2018.060202
Bacterial Communities of Drosophila suzukii
(Matsumura, 1931) (Diptera: Drosophilidae)
Damaged in Strawberry in Turkeyi
Elif Tozlu1,*, Nasibe Tekiner1, Göksel Tozlu1, Recep Kotan1, Hatice Öğütçü2
1Plant Protection Department, Agricultural Faculty, Ataturk University, Erzurum, Turkey
2Field Crop Department, Agricultural Faculty, Ahi Evran University, Kırşehir, Turkey
Copyright©2018 by authors, all rights reserved. Authors agree that this article remains permanently open access under
the terms of the Creative Commons Attribution License 4.0 International License
Abstract Drosophila suzukii (Matsumura, 1931)
(Diptera: Drosophilidae) is an invasive species originating
from Southeastern Asia and spreads in a fast manner. It is
among major threats in soft-shell fruit cultivation in the
whole world. It was detected in 2014 in Turkey. According
to international criteria, it is considered that it has the
potential of threatening the fruit cultivation in Turkey
where garden plants are grown widely. In this study, a total
of 39 bacterial strains were isolated from 100 mature
Drosophila suzukii individuals. Gram staining
characteristics, catalase, oxidase and nitrate reductase
activities and chitinase enzyme activities and
hypersensitivity reaction of these strains were determined
by using microscopical and visual inspection. The bacterial
strains were identified according to their fatty acid methyl
esters (FAME) analysis by using Sherlock Microbial
Identification System (MIS). The identification test results
of the bacterial strains were also confirmed by
phylogenetic analysis and their closely related species
based on the 16S rRNA sequence. The most abundant
bacterial species were Paenibacillus alvei (31.57%) and
Bacillus amyloliquefaciens (47.36%) according to the MIS
and 16S rRNA sequence analysis results, respectively.
According to the MIS results, a total of 6 strains identified
as Paenibacillus alvei were identified as Bacillus
amyloliquefaciens according to the 16S rRNA sequence
analysis results. A total of three Paenibacillus macerans
strains identified in MIS system were also identified as
Bacillus amyloliquefaciens according to the 16S rRNA
sequence analysis. Morphological and biochemical
characteristics results of all of Bacillus amyloliquefaciens
strains showed the some results. According to the 16S
rRNA sequence analysis results, the other bacterial strains
consist of 1 Bacillus atrophaeus (5.2%), 1 Bacillus
safensis (5.2%), 1 Paenibacillus motobuensis (5.2%) and
1 Staphylococcus epidermidis (5.2%) strains. To our
knowledge, this is the first study characterizing the
bacterial communities of Drosophila suzukii.
Keywords Bacteria, Biological Control, Drosophila
suzukii, Microbiota
1. Introduction
Drosophila suzukii (Matsumura, 1931) (Diptera:
Drosophilidae), an indigenous species to the continent of
Asia, was first reported outside this continent in 1980 in
Hawaiian Islands of the North America [1]. Within two
years, it completely invaded the northern parts of Continent
of America from west to the east, and consequently reached
Canada at north and Mexico at south [2-5]. In the field
study in which European Drosophilidae species were
recorded, D. suzukii emerged as a predominant
Drosophilidae species in the highlands where it habitates.
From this date on, the first records in Italy [6], France [7],
Switzerland [8], Slovenia [9], Croatia [10], Austria [11],
United Kingdom, Portugal [12], Germany [13], Belgium
[14], Hungary [15], Serbia [16], Bosnia and Herzegovina
[17], Bulgaria [18], Greece (Crete Island) [19], Poland [20]
and Japan [21] were reported. The first record in Turkey
was in Erzurum in August-September 2014 [22].
Although the primary hosts of D. suzukii are cherry, sour
cherry, strawberries, blackberries, raspberries and
blueberries, a very wide spectrum of fruits can be affected.
It can cause also serious damage to fruits such as fig,
apricot, peach, plum, grape, medlar, greenhouse mandarin,
kiwi, persimmon, and fallen or cracked apple and orange [2,
4, 23, 24]. It is estimated that these losses have affected
14% of all potential fruit production worldwide [25].
Due to the wide range of host fruit selection and rapid
spreading, it is stated that this species is an important pest
that is likely to cause major losses to the European and
36 Bacterial Communities of Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) Damaged in Strawberry in Turkey
American fruit industry in the near future [3-4].
While other Drosophila species feed on rotten fruits, D.
suzukii prefers newly ripening fruits on the tree, and using
their saw-shaped ovipositor, female individuals lay their
eggs inside the fruits prior to their maturation for
harvesting, which all make this organism a very important
agricultural pest [2]. While the larvae feed on the rich
protein content of the flesh of the fruit, the synthesis of
inherent ethylene is increased in areas where tissue
integrity is compromised. Ethylene synthesis locally
accelerates maturity and causes collapse/softening (rotting)
of the flesh of the fruit. As a result, these products lose their
market value within a short period of time. In addition, the
wounds that these flies open on the fruit for laying their
eggs lead to additional losses caused by pathogens,
including fungi and bacteria [26].
Few studies have been carried out to identify the
microflora of this pest. The first study that was conducted
to identify the bacterial flora of D. suzukii revealed species
belonging to the genera Gluconobacter and Acetobacter
[27]. Another study reported that Wolbachia spp.
(Rickettsiales: Rickettsiaceae) could be used in biological
control of this pest [28-29]. To our knowledge, there is not
any study characterizing the microbial communities of D.
suzukii.
The present study aims to determine the microflora of
D. suzukii which continues to spread rapidly and cause
economic losses.
2. Materials and Methods
2.1. Pest Samples
The study was conducted on 100 healthy adult D.
suzukii individuals collected on July 25th-26th, 2016 in
Erzurum province of Turkey using traps prepared with
cider vinegar (Figure 1) in strawberry trial fields where
neither insecticide nor fungicide was used. Collected adult
individuals were put into tubes and brought to Atatürk
University Plant Protection Department, Plant Clinical
Laboratory.
Figure 1. The traps used for Drosophila suzukii
Universal Journal of Microbiology Research 6(2): 35-42, 2018 37
Figure 2. Serial dilutions were obtained from Drosophila suzukii adults
2.2. Isolation of Bacterial Strains
Superficial sterilization was applied to D. suzukii adults
in tens with 95% ethyl alcohol for 5 minutes. Adults were
homogenized by pulverizing in a sterile mortar with sterile
saline solution and serial dilutions were obtained from this
homogenate [30] (Figure 2). The dilutions prepared from
the adults were inoculated on Nutrient Agar (NA) for
bacterial growth. Then, Bacterial cultures were incubated
at 30°C, and at the end of 24-72 hours. The bacterial strains
with the dominant character were selected and purified [31].
Each of single colonies was prufied and streaked on agar
plate. For each pure culture was given a separate code
number, and information regarding the isolation conditions
(location, altitude, insect form, date, etc.) was noted. The
samples were kept at -86°C in stock growth media
containing 30% glycerol and Loria Broth (LB) for routine
use.
2.3. Determination of Morphological and Biochemical
Properties of Bacterial Strains
The colony morphology and color of the bacterial strains
were determined microscopical and visual inspection.
The Gram staining characteristics of the bacteria were
determined according to the method described by Sands
[32]. Presence of catalase and oxidase enzymes [33] and
nitrate enzyme was assessed by the method of Harley and
Prescott [34]. Chitinase enzyme activity was determined
according to the method reported by Ortucu [35].
2.4. Polymerase Chain Reaction (PCR) of the Bacterial
Strains
The strains were also identified in the molecular system.
Total 19 bacterial strains selected from 39 bacterial strains
obtained from D. suzukii adults according to high colony
density were identified by sequencing a fragment of
genome [36]. Bacterial DNA was amplified by a two-step
PCR targeting the 16S rDNA gene with primers 27F and
907R, designed to include Illumina adaptor and barcode
sequences. Sequencing was performed on an
IlluminaMiSeq at the UC Davis Genomics Core Facility
generating 963 basepair paired-end reads. Samples were
OTUs are identified by their closest hit in the SILVA SSU
Reference Database Release 111. Number of sequences is
after all quality-control steps.
2.5. Phylogenetic Relationship of the Bacterial Strains
The sequences obtained were used to perform BLAST
searches using the NCBI GenBank database to confirm
strain identification Altschul et al. [37]. Evolutionary
relationships of the 19 bacterial strains were evaluated.
Cluster analyses of the sequences were performed using
BioEdit (version 7.09) with Clustal W followed by
neighbor joining analysis on aligned sequences performed
with MEGA 6.0 software [38] Reliability of dendograms
was tested by bootstrap analysis with 1000 replicates using
MEGA 6.0.
The evolutionary history was inferred using the
Neighbor-Joining method [39]. The optimal tree with the
sum of branch length = 0.56341095 is shown. The
percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) is
shown next to the branches [40]. The tree is drawn to scale,
with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree.
The evolutionary distances were computed using the
Maximum Composite Likelihood method [41] and are in
the units of the number of base substitutions per site. The
analysis involved 16 nucleotide sequences. Codon
positions included were 1st+2nd+3rd+Noncoding. All
positions containing gaps and missing data were eliminated.
There were a total of 823 positions in the final dataset.
Evolutionary analyses were conducted [38].
3. Results
The identification test results of the isolated bacteria and
their morphological and biochemical characteristics were
given in Table 1.
38 Bacterial Communities of Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) Damaged in Strawberry in Turkey
Table 1. Identification test results of the isolated bacteria and their morphological and biochemical characteristics test results
Strains BLAST top hit
Identify
(%)
Colony
shape
Colony
color
Gram
staining
Catalase
test
Oxidase
test
Nitrate
reduction
Chitinase
activity
RK 1792
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1801
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1805
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1809
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1810
Bacillus
amyloliquefaciens
98 rod cream + + + + -
RK 1812
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1813
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1814
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1815
Bacillus
amyloliquefaciens
99 rod cream + + + + -
RK 1811 Bacillus atrophaeus 99 rod cream + + - + +
RK 1807 Bacillus safensis 93 rod cream + + + - -
RK 1770
Paenibacillus
motobuensis
99 rod white - + + + +
RK 1784 Proteus myxofaciens 99 rod cream - + - + -
RK 1785 Proteus myxofaciens 99 rod Cream - + - + -
RK 1787 Proteus myxofaciens 99 rod cream - + - + -
RK 1768
Staphylococcus
epidermidis
99 cocci white + + - + -
RK 1765 nd - rod cream - + + + -
RK 1767 nd - rod cream - + - - -
RK 1769 nd - rod cream - + - - -
Other a total of 12 bacterial strains nt nt nt nt nt nt nt
Undefined a total of 8 bacterial strains nt nt nt nt nt nt nt
SIM: Similarity index, nd: Not determined; +: Positive reaction, -: Negative reaction, nt: Not tested
It was observed that the RK 1768 strain had a rod
shaped colony, whereas other strains had a rounded
colony shape, and that RK 1768 and RK 1770 strains had
a white colony color while the other strains had cream
colony color (Table 1). The strains RK 1770, RK 1784,
RK 1785 and RK 1787 were gram-negative, while other
strains were gram-positive. All strains had positive
catalase and nitrate test results. Except RK 1770 and RK
1811 all other strains had negative chitinase test results.
Chitinase activity of RK-1811 and RK-1770 strains was
positive (Table 1) (Figure 3).
The bacterial strains with high colony density that were
obtained from D. suzukii adults were identified molecular
analysis. Molecular diagnostic results are given in Table 1.
According to the 16S rRNA sequence analysis results, a
total of 9 strains were identified as Bacillus
amyloliquefaciens. The other bacterial strains consist of 3
Proteus myxofaciens, 1 Bacillus atrophaeus, 1 Bacillus
safensis, 1 Paenibacillus motobuensis and 1
Staphylococcus epidermidis. Percent identify of all the
identified bacterial strains were 99%. But, a total of three
strains were not identified (Table 1).
Figure 3. Chitinase positive bacterial strains
Universal Journal of Microbiology Research 6(2): 35-42, 2018 39
Figure 4. Evolutionary relationships of taxa
These identifications were also confirmed by
phylogenetic analysis of the bacterial strains and their
closely related species based on the 16S rRNA sequence
(Figure 4).
4. Discussion
Chandler et al. [27], in their microbiota study on D.
suzukii, isolated the genus Tatumella from 99% of both the
adults and the larvae and Dunitz et al. [42] isolated
Tatumella sp. from the larvae. Although this genus is not
commonly found in Drosophila species, another study
identified it also in D. melanogaster, which feeds on apples
[43]. Again, Chandler et al. [27] reported that
Acetobacteraceae and Orbus species were also associated
with Drosophila population. However, Broderick and
Lemaitre [43] noted that Orbus species were not
associated with Drosophila. Brummel et al. [44]
conducted molecular analyses on strains from the whole
body of D. melanogaster adults, and they identified the
genera Lactobacillus, Gluconabacter, Enterobacter,
Anaerococcus, while Cox and Gilmore [45] identified
Wolbachia sp., Acetobacter aceti, A. cerevisiae, A.
pasteurianus, A. pomorum, Gluconobacter cerinus,
Enterobacter cloacae, Klebsiella oxytoca, Lactobacillus
plantarum, Leuconostoc mersenteroide and Enterococcus
faecalis species.
In this study, the most abundant bacterial species in
mature D. suzukii individuals were B. amyloliquefaciens
(47.36%), Proteus myxofaciens (15.78%), B. safensis, P.
motubensis, S. epidermis (%5.26) according to the 16S
rRNA sequence analysis results. This identification results
were supported with classical systems in this study.
Morphological and biochemical characteristics tests results
of all of B. amyloliquefaciens strains showed the some
results. According to the 16S rRNA sequence analysis
results.
It suggests that differences in species distribution in
microbial flora studies of the same insect species may be
due to the differences in the body part where strains were
obtained, the biological period of the pest, and dietary
differences. Indeed, it has been stated that, in comparison
to isolations made from the whole body, isolations from the
gut yielded less microorganisms, that the sample
preparation time may also influence variation due to the
shorter passage time in the gut [46-47], and that microbiota
could differ depending on the variations of consumed food,
and whether the individual fed in its natural environment or
in laboratory setting [47].
As it can be seen from all these studies, there is ongoing
research on microbiota of D. suzukii species. It is thought
that microbiota studies will guide biological pest control
studies. The discovery of new pathogens and parasites of
pest insects offers a chance to find organisms that may be
useful for biological control [48]. For this purpose it has
become increasingly important to identify microorganisms
which are present in the microbiota of harmful organisms,
and can be used in control of these pests, and to study them
in biological control research. Identification of
microorganisms that have the potential to be used in
biological pest control against the invasive species D.
suzukii will be possible detailed studies on this subject.
5. Conclusions
To our knowledge, this is the first study characterizing
the bacterial communities of Drosophila suzukii. In
conclusion, we think that expecially Bacillus
40 Bacterial Communities of Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) Damaged in Strawberry in Turkey
amyloliquefaciens strains can be used as biological control
agents against this economically important pest. Biological
control studies will be planned with bacterial strains stored
in the Atatürk University, Plant Protection Laboratory in
future.
Acknowledgments
We thank to Dr. Serkan ÖRTÜCÜ for helping
molecular identification of bacterial strains.
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1 This study submitted as abstract in International DNA Day and Genome Congress (IDDGC), 24-28 April 2017, Ahi Evran University, Kırşehir,
TURKEY.
