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Cassava Root Necrosis Disease (CRND): A New Crop Disease Spreading in Western Democratic Republic of Congo and in Some Central African Countries

Authors:
  • Institut National pour l'Etude et la Recherches Agronomiques / INERA

Abstract and Figures

Cassava is consumed in the Democratic Republic of Congo (DRC) as a staple food for the majority of the Congolese population. This crop is used in several forms: as fufu, chikwangue and pondu; cassava leaves are the most consumed vegetable in the country. In 2002, cassava root symptoms similar to cassava brown streak disease (CBSD) were reported for the first time in western DRC. PCR assays, using primers specific to Cassava brown streak virus (CBSV), failed to detect or identify any viral pathogens in diseased cassava samples from western DRC. Therefore, next generation sequencing (NGS) techniques were used as they are able to sequence full organism genomes and are widely used for the identification of pathogens responsible for new diseases. The main objective of this study was to identify the pathogens causing root necrosis in western DRC. Whatman®FTA™ cards were used to collect 12 cassava leaf samples from plants with symptoms indicative of very severe root necrosis, as well as two asymptomatic samples. These 12 samples were sent to Australia at the University of Western Australia in Perth for next generation sequencing (NGS) using the Illumina HiSeq platform. Additional bioinformatics tools included Geneious, CLC workbench, ParaKraken and Kaijou software for short DNA sequences. No viruses (including CBSV) were found in any of the DRC samples. These preliminary results confirm all the previous negative results obtained using PCR and CBSV primers. However, NGS analyses did reveal the presence of a number of bacterial and fungal taxa. These will require further investigation and tests such as the Koch Postulates, to establish their specific pathogenic role in cassava. This is the first scientific evidence that no currently known virus is responsible for the disease which had been referred to previously as ‘CBSD-like disease’. Consequently, the disease found in DRC cassava samples has been designated ‘Cassava Root Necrosis Disease’ or CRND.
Content may be subject to copyright.
Journal of Agricultural Science; Vol. 12, No. 3; 2020
ISSN 1916-9752 E-ISSN 1916-9760
Published by Canadian Center of Science and Education
105
Cassava Root Necrosis Disease (CRND):
A New Crop Disease Spreading in Western Democratic Republic of
Congo and in Some Central African Countries
Bakelana Zeyimo
1,5,6
, Laura M. Boykin
3
, Monica Kehoe
7
, Justin Pita
5
, Monde Godefroid
5,6
,
Mahungu Nzola
2
, Lema Munseki
6
, Tshilenge Kanana
6
& Kalonji Mbuyi
6
1
National Institute for Agricultural Research Studies (INERA), Democratic Republic of Congo
2
International Institute of Tropical Agriculture (IITA), Democratic Republic of Congo
3
School of Molecular Sciences and Australian Research Council Centre of Excellence in Plant Energy Biology,
University of Western Australia, Crawley, Perth, WA, Australia
4
Institute Faculty of Agricultural Science, Yangambi, Democratic Republic of Congo
5
West African Virus Epidemiology (WAVE), Abidjan, Ivory Coast
6
Agriculture Faculty, Kinshasa University, Democratic Republic of Congo
7
Department of Primary Industries and Regional Development Diagnostic and Laboratory Services, South Perth,
WA, Australia
Correspondence: Bakelana Zeyimo, National Institute for Agricultural Research Studies (INERA), Democratic
Republic of Congo. Tel: 243-81-190-8783. E-mail: bakelanatony@hotmail.com
Received: November 27, 2019 Accepted: January 2, 2020 Online Published: February 15, 2020
doi:10.5539/jas.v12n3p105 URL: https://doi.org/10.5539/jas.v12n3p105
This research is financed by the WAVE/BMGF program.
Abstract
Cassava is consumed in the Democratic Republic of Congo (DRC) as a staple food for the majority of the
Congolese population. This crop is used in several forms: as fufu, chikwangue and pondu; cassava leaves are the
most consumed vegetable in the country.
In 2002, cassava root symptoms similar to cassava brown streak disease (CBSD) were reported for the first time
in western DRC. PCR assays, using primers specific to Cassava brown streak virus (CBSV), failed to detect or
identify any viral pathogens in diseased cassava samples from western DRC. Therefore, next generation
sequencing (NGS) techniques were used as they are able to sequence full organism genomes and are widely used
for the identification of pathogens responsible for new diseases. The main objective of this study was to identify
the pathogens causing root necrosis in western DRC.
Whatman
®
FTA™ cards were used to collect 12 cassava leaf samples from plants with symptoms indicative of
very severe root necrosis, as well as two asymptomatic samples. These 12 samples were sent to Australia at the
University of Western Australia in Perth for next generation sequencing (NGS) using the Illumina HiSeq
platform.
Additional bioinformatics tools included Geneious, CLC workbench, ParaKraken and Kaijou software for short
DNA sequences. No viruses (including CBSV) were found in any of the DRC samples. These preliminary results
confirm all the previous negative results obtained using PCR and CBSV primers. However, NGS analyses did
reveal the presence of a number of bacterial and fungal taxa. These will require further investigation and tests
such as the Koch Postulates, to establish their specific pathogenic role in cassava.
This is the first scientific evidence that no currently known virus is responsible for the disease which had been
referred to previously as ‘CBSD-like disease’. Consequently, the disease found in DRC cassava samples has
been designated ‘Cassava Root Necrosis Disease’ or CRND.
Keywords: NGS, PCR, Illumina HiSeq, CBSD-like, CRND
jas.ccsenet.
1. Introd
u
Cassava (
M
a staple f
o
In Africa,
2004).
Cassava (
M
(DRC). It
highest in
with Rep
u
Storage r
o
consumed
Thresh, 2
0
highly res
i
Cassava
disease (
C
annual lo
s
In 2004,
C
above sea
Uganda
w
Burundi (
B
(Alicai et
a
CBSD is
c
Two gene
t
shown to
streak vir
u
Both CBS
(Mware e
t
endemic i
n
western T
a
Burundi a
n
cause eco
n
In the ear
l
western
pr
PCR has
b
to now, th
e
In
N
ovem
b
of the rec
o
Necrosis
D
org
u
ction
M
aniho
t
escul
e
o
o
d
crop for a
p
cassava is th
e
M
aniho
t
escu
l
is one of the
the world (M
b
u
blic of Congo
o
ots are used
as an everyd
0
02). Recent
r
i
lien
t
to clima
t
roduction of i
n
C
MD) and cas
s
ses of US$1
b
C
BSD, which
h
level (Alicai
e
w
ere confirme
d
B
igirimana et
a
l., 2017) and
c
aused by a s
i
t
ically-distinc
t
be two distin
u
s (UCBSV) (
M
V
and UCBS
V
t
al, 2019) an
d
n
coastal East
a
nzania and
K
n
d in isolated
n
omic losses
o
l
y 2000s, cas
s
r
ovinces of D
R
b
een unsucces
s
e
disease has
b
F
b
e
r
2018, in
K
o
mmendations
D
isease (CRN
D
e
nta Crantz,
fa
p
proximately
8
e
second mos
t
l
enta)
p
roduc
t
country’s
p
ri
n
b
ago & Loto
m
ranked secon
d
as a fresh so
u
ay-food, sold
r
esearch has s
t
e change and
n
East and C
e
sava brown s
t
b
illion (IITA,
2
h
ad been thou
g
et
al., 2007). I
n
d
by RT-PCR
(
al., 2011),
R
Mayotte Islan
i
ngle-stranded
t
strains of C
B
c
t
species, na
m
M
onge
r
et al.,
V
are transmit
t
d
through the
p
Africa and i
n
K
enya. Other
c
parts of the
D
o
f up to $100
m
s
ava root nec
r
R
C (Kinshasa
s
ful in detecti
n
b
een referred t
o
F
igure 1. Typi
c
K
inshasa durin
g
was that the t
D
) should be
u
Journal of
A
fa
mily Euphor
b
8
00 million
pe
t
important fo
o
t
ion is import
n
cipal crops,
w
m
be, 2017). Z
a
d
in 1996 (Du
f
u
rce of carbo
h
in local mar
k
uggested that
,
could provide
e
ntral Africa i
s
t
reak disease
2
014b) and a
d
g
h
t
to be conf
i
n
fections of c
a
(
Alicai et al.,
2
R
wanda (FAO,
d
(Roux-Cuv
e
RNA virus,
fa
B
S
V
were rec
o
m
ely Cassav
a
2010).
t
ed by the wh
i
p
ropagation o
f
n
parts of Mal
a
c
ountries whe
r
D
RC (
N
dung
u
m
illion USD a
n
r
osis (Figure
1
and Kongo C
n
g any known
o
as ‘CBSD-li
k
c
al root necros
i
g
the drafting
t
er
m
‘CBSD-l
u
sed instead.
A
gricultural Sc
i
106
b
iaceae)
p
rod
u
e
ople worldwi
d
od
staple in t
e
an
t
to the ec
o
w
ith per capita
a
ire, now DR
C
f
ou
r
et al., 19
9
h
ydrates and
t
k
ets or used i
n
,
in comparis
o
food security
s
severely co
n
(CBSD). Tog
e
d
versely affect
i
ne
d
to coastal
a
ssava plants
s
2
007). There
h
2011), easte
r
e
lie
r
et al., 201
fa
mily Potyvir
i
o
gnize
d
in Eas
t
a
brown
s
trea
k
i
tefly species
c
f
infected cutti
n
a
wi until rece
n
r
e CBSD has
u
ru et al., 201
5
n
nually (Ndun
1
) similar to t
h
C
entral) by M
a
n
potential cau
s
k
e disease’ (B
i
s of CRND o
b
meeting of th
e
i
ke’ should no
i
ence
u
ces carbohyd
r
d
e (Food and
e
rms of per c
ap
o
nomy of the
consumption
C
, was the wo
r
9
6).
the flour deri
v
n
several indu
o
n to other st
a
opportunities
n
strained by t
w
e
ther, it is est
i
food security
lowlands, wa
s
s
howing CBS
D
h
ave since
b
e
e
r
n DRC (Mul
i
4).
i
dae; genus
Ip
t
Africa (Mba
n
k
virus (CBS
V
c
omplex Bem
i
ngs used for
p
n
tly when out
b
been reporte
d
5
). The strain
s
n
guru et al., 20
h
a
t
caused by
a
hungu et al. (
2
s
al agent for t
h
akelana et al.,
b
served in we
s
e
DRC cassav
a
longer be use
d
r
ate-rich stora
g
Agriculture
O
a
pita calories
c
Democratic
R
of 353 kg
p
e
r
r
l
d
’s largest c
o
v
ed from the
strial food
p
r
o
a
ple food cro
p
fo
r
Africa (Ja
r
w
o viral disea
s
i
mated that t
h
in the region
s
found at alti
t
D
symptoms a
t
e
n additional
C
i
mbi et al., 2
0
p
omovirus (M
o
n
zibwa et al.,
V
) and Ugan
d
i
sia tabaci Ge
n
p
lanting. CBS
D
b
reaks were r
e
d
include Mo
z
s
of Cassava
15).
CBSD was
fi
2
003).To date
,
h
e observed s
y
2019a).
s
tern DRC
a
viral disease
d
, but that the
Vol. 12, No. 3;
g
e roots, whi
c
O
rganization, 2
c
onsumed (N
w
R
epublic of
C
r
year, which
i
o
nsume
r
of ca
s
processed ro
o
o
ducts (Hilloc
p
s, cassava m
a
r
vis et al., 201
2
s
es, cassava
m
h
ese diseases
c
(Patil et al., 2
t
udes above 1
0
t
higher altitu
d
C
BSD reports
0
12), South
S
o
ngeret al., 20
2009). These
d
an cassava
b
n
nadius in the
was known
e
porte
d
in Ug
a
z
ambique, Rw
a
brown streak
fi
rs
t
reported i
n
,
diagnosis th
r
y
mptoms. Th
u
response
p
la
n
term Cassava
2020
h
are
0
13).
w
eke,
ongo
i
s the
s
sava
o
ts is
k
s &
a
y be
2
).
o
saic
c
ause
0
15).
0
00
m
d
es in
from
S
udan
01a).
were
rown
field
to be
a
nda,
a
nda,
virus
n
the
r
ough
u
s, up
n
, one
Root
jas.ccsenet.
This reco
m
DRC. Thi
s
This nam
e
evidence
o
Several at
t
using cas
s
(Bakelana
Molecula
r
CBSVs (
C
disease i
n
Conventi
o
real-time
P
virus spe
c
sequencin
g
has now
be
Despite t
h
potential
c
CBSD vir
u
strains of
C
this study
specific o
r
In this
p
a
p
samples f
r
for the sy
m
necrosis d
i
2. Materi
a
2.1 Field
S
Cassava f
i
were surv
e
A total of
to the ma
n
Cassava l
e
2016.
Leaves sa
m
samples
fo
storage ro
o
org
m
mendation
w
s
scientific da
y
e
change was
b
o
f any virus (i
n
t
empts have
b
s
ava leaf samp
et al., 2019a).
r
diagnosis re
s
C
BS
V
and UC
B
n
western D
R
o
nal molecular
P
CR may be
t
c
ies and thus
g
platforms (
N
ecome a wide
l
h
e absence of t
y
c
ausal viral ag
e
u
ses were spr
e
C
BSD—unid
e
was to searc
h
r
known target
p
er, we repor
t
r
o
m
DRC. Ba
s
m
ptoms exhi
b
i
sease’ and its
a
ls and Meth
o
S
ample Collec
Figu
r
i
elds (with
p
la
n
e
yed. Leaf ma
t
12 leaf sampl
e
n
ufacturers’
p
r
o
e
af samples
w
m
ples were s
a
for
CBSV-det
e
ot
tissue. The
C
w
as also remi
n
y
was organiz
e
b
ase
d
on the r
e
n
cluding kno
w
b
een undertak
e
les (including
s
ults from fiv
B
SV),
p
roduc
R
Congo mig
h
methods hav
e
t
oo specific o
r
resulting in
N
GS), the me
t
l
y-used metho
d
y
pical CBSD
f
e
nts in diseas
e
e
ading from E
a
e
ntified to dat
e
h
for the caus
s.
t
the first use
s
ed on the
NG
b
ited in our c
a
acronym ‘CR
N
o
ds
tion for NGS
A
r
e 2. Cassava
n
ts more than
t
erial from
p
l
a
e
s (Table 1)
w
o
tocol in orde
r
w
ere collected
a
a
mpled accor
d
e
ction were y
C
BSD viruses
Journal of
A
n
de
d
during th
e
e
d on August
3
e
sults of
N
G
S
w
n CBSD viru
s
e
n since 2004
those from
pl
e different la
b
ed negative r
e
ht
be differe
n
e
their limitati
o
r
not broad e
n
false negativ
t
agenomic se
q
d
(Adams et a
l
folia
r
and ste
m
e
d plants in w
e
a
s
t
to Central
e
—might be r
e
al agent usin
g
of next gene
G
S results, we
a
a
ssava plant s
a
N
D’ within th
i
A
nalysis
root of plant
s
16 months ol
d
a
n
t
with root n
e
w
ere collected
f
r
to extract the
at
Mvuazi res
d
ing to Rweg
oung tender
l
were not dete
A
gricultural Sc
i
107
e
scientific d
a
3
rd by the Pla
n
S
analysis und
e
s
es) in our sy
m
to identify th
e
l
ants showing
b
oratories, us
i
e
sults. This su
g
nt
from those
o
ns—as indic
a
n
ough to succ
e
es. With the
q
uencing of di
l
., 2009; Kreu
z
m
symptoms a
n
e
stern DRC, t
h
Africa and ca
u
e
sponsible for
g
a broad dia
g
ration sequen
c
a
lso propose t
h
a
mples. Cons
e
i
s manuscript
t
s
used for lea
f
d
) in the 2 loc
a
e
crosis was co
f
ro
m
plants.
T
i
r
DNA.
earch center
a
asira et al. (2
l
eaves, young
cted from roo
t
i
ence
a
y on diseases
nt
Clinic of Ki
n
e
rtaken in this
m
ptomatic cas
s
e
causative a
g
very severe s
y
i
ng PCR
p
ri
m
g
geste
d
that t
h
known at
pr
a
ted by Adam
s
e
ssfully detect
advent of n
e
sease
d
cassav
a
z
e et al., 2019
)
n
d a failure o
f
h
e project con
s
u
sing this dis
e
these disease
g
nostic tool—
t
c
ing to analy
z
h
a
t
non-viral
c
e
quently, we a
t
o refer to this
f
sampling on
a
lities consider
llected (Figur
e
T
he
y
were cru
s
a
nd Lukuakua
011) who fo
u
es
t
symptom
a
t
necrotic tiss
u
and pests of
c
n
shasa.
study. This a
n
s
ava samples.
g
en
t
for CRN
D
y
mptoms)wit
h
m
ers specific
f
h
e causal agen
t
r
esen
t
(Bakel
a
s
et al. (2009)
all the know
n
e
x
t
generatio
n
a
plants to id
e
)
.
f
existing test
m
s
idere
d
that it
w
e
ase. It seeme
d
symptoms. T
h
t
ha
t
is, one n
o
z
e the sympt
o
c
ausal agents
m
r
e using the
n
disease.
FTA™ cards
r
ed hotspots (r
e
e
s 2) on FTA
s
he
d
onto FT
A
village on 29
u
nd that the
a
tic leaves an
d
u
es.
Vol. 12, No. 3;
c
ultivate
d
p
la
n
n
alysis did no
t
D
in western
D
h
no success t
o
f
o
r
the two k
n
t
of the CBS
D
a
na et al., 2
0
who indicate
d
n
variability
n
, high-throu
g
e
ntify plant vi
m
ethods to id
e
was still likel
y
d
feasible that
h
erefore, the a
i
ot
designed a
g
o
matic cassav
a
m
ay be respo
n
n
ame ‘Cassav
a
e
f) in western
cards.
A
™ cards acco
r
and 30
N
ove
m
os
t
suitable
t
d
the non-ne
c
2020
n
ts in
t
find
D
RC,
o
date
n
own
D
-like
19a).
d
that
w
ithin
h
put,
r
uses
e
ntif
y
y
that
o
the
r
im
of
g
ains
t
a
leaf
n
sible
a
root
D
RC
r
ding
m
be
r
issue
c
rotic
jas.ccsenet.
Tab le 1. L
The 12 F
T
Perth to c
o
2.2 Sympt
o
A sympto
m
described
root; 2 =
l
necrotic ti
s
2.3 RNA
E
RNA was
bromide).
2.0% PV
P
μl of the
e
shaking v
i
isoamyl a
l
rpm for 1
0
which 0.6
followed
b
supernata
n
org
F
eaf samples d
e
Sample ID
1
2
3
4
5
6
7
8
9
10
11
12
T
A™ sample
c
o
mplete the D
N
o
m
A
ssessmen
m
severity sco
in Hillocks a
n
l
ess than 2%
n
s
sue.
E
xtraction (Nd
u
extracted fro
m
The leaves w
e
P
, 0.5M EDTA
,
e
xtrac
t
was tr
a
i
gorously sev
e
l
cohol (24:1);
0
min at 4 °C
vol (300 μl)
c
b
y centrifugat
i
nt
was discard
e
F
igure 3. Hots
p
e
tails
Genotype
Mputa
Mputa
Mputa
Mputa
Mputa
Mvuazi
Mvuazi
RAV
RAV
Mputa
RAV
TME 419
c
ards,
p
reviou
s
NA
extraction
t
re was then r
e
n
d Thresh (20
0
n
ecrotic tissue
;
u
nguru et al.,
2
m
approxima
t
e
re ground in
a
,
1 M Tris-HC
a
nsferred into
e
ral times. Th
vortexed
b
ri
e
. The top aqu
e
c
old isopropa
n
i
on (Hettich
C
e
d.
Journal of
A
po
t
disease lo
c
Lo
c
Mv
Mv
Mv
Mv
Mv
Lu
k
Lu
k
Lu
k
Lu
k
Lu
k
Lu
k
Lu
k
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ly labeled, w
e
and for subse
q
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corded for th
e
0
0). Root necr
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;
3 = 2-5% ne
2
015)
t
ely 100 mg
o
a
mortar conta
i
l and 0.2% β-
m
a 1.5 ml mic
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e extract was
e
fly and centr
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ous solution
n
ol was adde
d
C
entrifugen, D
-
A
gricultural Sc
i
108
c
ations where
c
ation
uazi
uazi
uazi
uazi
uazi
k
uakua
k
uakua
k
uakua
k
uakua
k
uakua
k
uakua
k
uakua
e
re shipped to
q
uen
t
NGS
p
r
o
e
root of each
p
o
sis severity
w
crotic tissue;
4
o
f cassava le
a
i
ning 1 ml ext
r
m
e
r
-captoetha
n
r
o-centrifuge
t
then mixed
w
i
fuge
d
(Hettic
(500 μl) was
d
. The content
-
78532, Germ
a
i
ence
samples were
Status
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Diseased
p
Apparentl
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Apparentl
y
the Universit
y
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cessing.
p
lan
t
sampled
,
w
as assessed a
4
= 5-50% ne
c
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f using the C
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ube and incu
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h Centrifuge
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transferred in
t
was then inc
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ny) at 13,00
0
collected
lants
lants
lants
lants
lants
lants
lants
lants
lants
lants
y
healthy plants
y
healthy plants
y
of Western
A
,
using the 1-t
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s
follows: 1 =
c
rotic tissue;
5
TAB (cetyltri
m
(2.0% (w/v)
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b
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ated at 65 °
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volume (750
n
, D-78532,
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0
rpm for 10
m
Vol. 12, No. 3;
A
ustralia (U
WA
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apparently h
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5
= more than
m
ethyl ammo
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TAB, 2.0 M
N
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n
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for 15 min
w
μl) of chloro
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ermany) at 1
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centrifuge tu
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0
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2020
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thod
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50%
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0
min
d
the
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
109
The RNA pellet was then washed in 700 ml of 70% ethanol and the tubes vortexed briefly before being
incubated at -20 °C for at least 10 min. The tubes were then centrifuged for 5 min at 13,000 rpm. The ethanol
was then removed and the pellet was air-dried. Finally the dried RNApellet were re-suspended in 100 μl
1XTE/sterilized double distilled H20 on ice for about 30 min and stored at -20 °C before use.
2.4 cDNA Library Preparation and Illumina Sequencing (Ndunguru et al., 2015)
Total RNA extracts that presented 260/280 and 260/230 purity indices equal to or greater than 2.0 and integral
RNA in electrophoresis and Bioanalyzer measurements (RIN > 8) were selected. The cDNA libraries were
prepared from 1 μg of total RNA using the IlluminaTruSeq Stranded Total RNA Sample Preparation kit with
Ribo-Zero
TM
Plant according to the manufacturer’s instructions (Illumina, San Diego, California). Briefly, after
rRNA depletion and RNA fragmentation, first and second strand cDNA was synthesized, adapters were ligated to
the 50 and 30 ends of the fragments and the fragments enriched by PCR. cDNA libraries final size and
concentration of each library was estimated using a Bioanalyzer (Agilent, Santa Clara, CA, USA) and the Qubit
(Invitrogen, Carlsbad, CA, USA), respectively. Ten nM library pools were prepared by mixing the libraries to
achieve an equal molar concentration of each. Libraries were normalized, pooled and sequenced using a 2 × 300
cycle PE V3 Illumina kit. Paired end reads were generated using the Illumina MiSeq System at the Biosciences
Eastern and Central Africa-International Livestock Research Institute (BECA-ILRI) Hub in Nairobi, Kenya.
2.5 De Novo Sequence Assembly and Mapping (Ndunguru et al., 2015)
For each sample, reads were first trimmed using CLC Genomics Workbench 6.5 (CLCGW) (CLC Bio) with the
quality scores limit set to 0.01, maximum number of ambiguities to two and removing any reads with < 30
nucleotides (nt). Contigs were assembled using the de novo assembly function of CLCGW with automatic word
size, automatic bubble size, minimum contig length 500, mismatch cost two, insertion cost three, deletion cost
three, length fraction 0.5 and similarity fraction 0.9. Contigs were sorted by length and the longest subjected to a
BLAST search (blastn and blastx). In addition, reads were also imported into Geneious 6.1.6 (Biomatters) and
provided with reference sequences obtained from Genbank.
2.6 Library Preparation and Illumina Sequencing
Total RNA and DNA extractions was carried out in the UWA from FTA samples and were sent to the Australian
Genome Research Facility of the UWA for library preparation and sequencing on an Illumina HiSeq 2500.
2.7 Sequences Analysis
For each sample, reads were first trimmed using CLC Genomics Workbench 6.5 (CLCGW) (CLC Bio) with the
following parameters: quality scores limit set to 0.01, maximum number of ambiguities set to twoand removal of
any reads with < 30 nucleotides. Contigs were assembled using the de novo assembly function of CLCGW with
automatic word size, automatic bubble size, minimum contig length 500, mismatch cost two, insertion cost three,
deletion cost three, length fraction 0.5 and similarity fraction 0.9. Contigs were sorted by length and the longest
subjected to a BLAST search (blastn and blastx) (Altschul et al., 1990). In addition, reads were also imported
into Geneious 6.1.6 (Drummond et al., 2010) (Biomatters) and provided with reference sequences obtained from
Genbank (NC012698 for CBSV, GQ329864 for CBSV-T and NC014791 for UCBSV). These methods have been
used previously for the successful recovery of whole CBSV and UCBSV genome sequences (Ndunguru et al.,
2015; Alicai et al., 2016; Ateka et al., 2017).
Mapping was performed using Kaiju software with minimum overlap 10%, minimum overlap identity 80%,
allow gaps 10% and fine tuning set to iterate up to 10 times.
While recent taxonomic classification programs achieve high speed by comparing genomic k-mers, they often
lack sensitivity for overcoming evolutionary divergence; these results in large fractions of the metagenomic
reads remaining unclassified. Kaiju is a novel metagenome classifier, which finds maximum (in-) exact matches
on the protein level using the Burrows-Wheeler transform (Menzel et al., 2016).
It has been shown that that Kaiju classifies reads with higher sensitivity and similar precision compared with
current k-mer-based classifiers, especially in genera that are under-represented in reference databases. It has also
been demonstrated that Kaiju classifies up to 10 times more reads in real metagenomes. Kaiju can also process
millions of reads per minute and can run on a standard PC (Menzel et al., 2016).
3. Preliminary Results and Discussion
After trimming and assembling NGS data outputs using CLC workbench and Geneious software, sequences were
processed using the Kaiju and outputs are presented in Figures 4 and 5 below. The bioinformatic processes and
jas.ccsenet.
analyses
d
samples.
H
Samples 1
which we
r
asympto
m
org
d
id not find
e
H
owever, a nu
m
-10, which w
e
re collected
o
m
atic plants.
Figure 4. Ex
a
e
vidence of a
n
m
be
r
of
b
acte
r
e
re collected
o
o
n apparently
a
mple of a sa
m
Figure 5. Lac
Journal of
A
n
y virus (inc
l
r
ial and fungal
o
n diseased
p
l
asymptomati
c
m
ple results sh
identified
u
ck
of viruses i
n
A
gricultural Sc
i
110
l
uding known
taxa were rec
o
ants,
p
resente
d
c
plants
p
res
e
owing list of
m
u
sing Kaiju so
f
n
all tested sa
m
i
ence
CBSD virus
e
o
rded.
d
fungi and
ba
e
nted only
b
a
m
icroorganis
m
f
tware
m
ples (Kayju s
o
e
s) in our sy
m
a
cteria while
s
a
cteria. No fu
n
m
s (bacteria an
d
o
ftware)
Vol. 12, No. 3;
m
ptomatic ca
s
s
amples 11 an
n
gi were fou
n
d
fungi)
2020
s
sava
d
12,
n
d in
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
111
The figure 5 shows that viral sequences were quantified at 0.5%.
The list of all microorganisms identified in all 12 samples and those suspected to play a pathogenic role in plant
diseases according to the literature are presented in Tables 2 and 3 below.
Table 2. Bacteria and fungi identified through NGS in all 12 samples
Microorganisms identified Classification
Acremonium chrysogenum Fungus
Aspergillus niger Fungus
Aspergillus sp. Fungus
Aspergillus sydowii Fungus
Aspergillus versicolor Fungus
Diaporthehelianthi Fungus
Diaportheampelina Fungus
Diaporthehelianthi Fungus
Dickeya zeae Fungus
Diplodia sp. Fungus
Diplodia orticola Fungus
Diplodia serata Fungus
Erwinia sp. Fungus
Fusarium sp. Fungus
Macrophomina parvum Fungus
Macrophominaphaseolina Fungus
Neofusicoccum parvum Fungus
Pseudomonas fluorenscens Bacteria
Pseudomonas libanensis Bacteria
Pseudomonas aeruginosa Bacteria
Pseudomonas tolaasii Bacteria
Penicillium brasiliarum Fungus
Penicillium chrysogenum Fungus
Penicillium decumbens Fungus
Penicillium digitatum Fungus
Penicillium expansum Fungus
Penicillium marneffei Fungus
Penicillium steckii Fungus
Pestalotiopsis sp. Fungus
Pestalotiopsisfici Fungus
Pseudomonas aeroginosa Bacteria
Pseudomonas brassicacearum Bacteria
Pseudomonas dioxanivorans Bacteria
Pseudomonas fluorecens Bacteria
Pseudomonas fuscovaginae Bacteria
Pseudomonas mendocina Bacteria
Pseudomonas pseudoalcaligenes Bacteria
Pseudomonas syringae Bacteria
Pseudomonas tolaasii Bacteria
Pseudoxanthomonas sp. Bacteria
Pseudoxanthomonas spadix Bacteria
Sordariomycetidae Bacteria
Xanthomonas sp. Bacteria
Xanthomonas citri Bacteria
Xanthomonas euvesicatoria Bacteria
Xanthomonas phaseoli Bacteria
Xanthomonas sacchari Bacteria
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
112
Table 3. Plant pathogenic microorganisms among list of bacteria and fungi identified through NGS—according
to literature review
Microorganisms
Diplodiaseriata
Diplodiacorticola
Macrophominaphaseolina
Neofusicoccum parvum
Diaporthehelianthi
Diaportheampelina
Pestalotiopsis
Neofusicoccum parvum is the predominant species within the Botryosphaeriaceae. Several Botryosphaeriacea
species are important grapevine pathogens causing dieback and decline worldwide, and in recent years they have
been recognized as causing serious problems in New Zealand vineyards (Baskarathevan et al., 2012).
Diplodia corticola A.J.L. Philips, Alves et Luque is a well-known canker pathogen of oak (Quercus spp.) that is
contributing to the decline of oaks in the Mediterranean region (Alves et al., 2004). Recently, the pathogen has
been affecting Quercus spp. in California, Vitis vinifera in California and Texas (Lynch et al., 2010; Úrbez-Torres
et al., 2009; Úrbez-Torres et al., 2010), and live oak (Q. virginiana Mill.) in Florida (Dreaden et al., 2011).
Diplodia seriata (= Botryosphaeriaceaeobtusa) and Neofusicoccum parvum (Pennycook & Samuels) Crous, are
the most common pathogens associated with grapevine dieback worldwide (Auger et al., 2004; Larignon et al.,
2001; Phillips, 2002, Taylor et al., 2005; Úrbez-Torres et al., 2006; Úrbez-Torres et al., 2006; Van Niekerk et al.,
2004).
Species of Diaporthe and their Phomopsis asexual states have broad host ranges and are widely distributed,
occurring as plant pathogens, endophytes or saprobes, but also as pathogens of humans and other mammals
(Webber & Gibbs, 1984; Carroll, 1986; Boddy & Griffith, 1989; Rehner & Uecker, 1994; Garcia-Reyne et al.,
2011; Udayanga et al., 2011).
Diaporthe sp. are responsible for diseases on a wide range of plants hosts, some of which are economically
important worldwide, causing root and fruit rots, dieback, cankers, leaf spots, blights, decay and wilt (Uecker,
1988; Mostert et al., 2001a; van Rensburg et al., 2006; Santos et al., 2011; Thompson et al., 2011).
More researches are currently ongoing and each suspected microorganisms above needs to be confirmed by the
Koch Postulates assays as causative pathogen(s) of CRND in western DRC.
Isolations of bacteria and fungi are currently ongoing with the partnership of the Plant Clinic of Kinshasa.
Microorganisms that will be isolated from cassava roots necrotic tissues will be genetically characterized and
sequenced.
Koch Postulates trials will be done with the involvement of the DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen) in Germany.
It is possible that the CRND root necrosis disease could be caused by the action of a bacterium-fungus complex.
The disease could be initiated by an initial attack of bacteria and root necrotic symptoms externalized by a
secondary attack of fungi. Further studies are required to confirm or refute this hypothesis.
4. Conclusion and Perspectives
This study points to the apparent absence of CBSV in western region of DRC and suggests that CRND could be
caused by other microorganisms such as bacteria, fungi or a combination of both. There appear to be two distinct
diseases, namely CRND and CBSD which have similar root symptoms but different stem and foliar symptoms.
Since 2004, CBSD has been spreading from East Africa to Central Africa and was confirmed in 2012 in eastern
DRC; it is expected to spread to western DRC and on to West Africa. At the same time, CRND is spreading from
western DRC towards West Africa and eastern DRC.
If no control measures (quarantine, etc.) are put in place, there is a strong possibility that both diseases will
spread to West Africa. Should this event cause cases of infections of both diseases, the results are likely to mean
devastating cassava root crop losses and significant economic impacts on farmers’ livelihoods. Ultimately, this
has serious implications for food security in Central Africa.
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
113
We consider that further research on CRND pathogens identification is paramount. Koch’s Postulates on isolated
microorganisms from diseased plants and other biological assays will help to elucidate the causal pathogens of
this disease. Information on disease etiology will allow for future disease epidemiology and genetic disease
resistance research.
Acknowledgements
We would like to thank the WAVE program and BMGF (Grant number OPP1082413) for funding this study.
We would also like to thank the farmers of the Lukuakua village for providing us with cassava samples which
were sent to Australia for analysis.
References
Adams, I. P., Glover, R. H., Monger, W. A., Mumford, R., Jackeviciene, E., Navalinskiene, M., … Boonham, N.
(2009). Next-generation sequencing and metagenomic analysis: An universal diagnostic tool in plant
virology. Mol. Plant Pathol., 10(4), 537-45. https://doi.org/10.1111/j.1364-3703.2009.00545.x
Alicai, T., Omongo, C. A., Maruthi, M. N., Hillocks, R. J., Baguma, Y., Kawuki, R., … Colvin, J. (2007).
Re-emergence of cassava brown streak disease in Uganda. Plant Dis., 91, 24-29. https://doi.org/
10.1094/PD-91-0024
Alicai, T., Ndunguru, J., Sseruwagi, P., Tairo, F., Okao-Okuja, G., Nanvubya, R., … Boykin, L. M. (2016).
Cassava brown streak virus has a rapidly evolving genome: implications for virus speciation, variability,
diagnosis and host resistance. Scientific Resports, 6, 36164. https://doi.org/10.1038/srep36164
Altschul, S. F, Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. J.
Mol. Biol., 215(3), 403-10. https://doi.org/10.1016/S0022-2836(05)80360-2
Ateka, E., Alicai, T., Ndunguru, J., Tairo, F., Sseruwagi, P., Kiarie, S., … Boykin, L. M. (2017). Unusual
occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector
transmission. PLoS ONE, 12(11). https://doi.org/10.1371/journal.pone.0187883
Auger, J., Esterio, M., Ricke, G., & Perez, I. (2004). Black dead arm and basal canker of Vitis vinifera cv. Red
globe caused by Botryosphaeriaobtusa in Chile. Plant Dis., 88, 1286. https://doi.org/10.1094/PDIS.
2004.88.11.1286A
Baskarathevan, J., Jaspers, M. V., Jones, E. E., Cruickshank, R. H., Ridgway, H. J., & Spatafora, J. W. (2011).
Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards. Fungal Biology,
116(2), 276-288. https://doi.org/10.1016/j.funbio.2011.11.010
Bakelana, Z., Musben, Z., Boykin, L., Pita, J., Mvila, A., Monde, G., … Tshilenge, K. (2018). First Report and
Preliminary Evaluations of Cassava Brown Streak-Like Root Necrosis in Congo Republic. International
Journal of Development Research, 8(08), 22400-22407.
Bakelana, Z., Magembe, E., Boykin, L., Macharia, M., Mahungu, N., Tata, H., … Tshilenge, K. (2019a).
Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo. Journal of
Agricultural Science, 11(2), 31. https://doi.org/10.5539/jas.v11n2p31
Bakelana, Z., Boykin, L., Mahungu, N. M., Mavila, N., Matondo, M., Lufuankenda, M., … Tshilenga, K.
(2019b). First report and preliminary evaluation of cassava root necrosis in angola. International Journal of
Agriculture, Environment and Bioresearch, 4(03), 37-46. https://doi.org/10.35410/IJAEB.2019.3746
Bigirimana, S., Barumbanze, P., Ndayihanzamaso, P., Shirimana, R., & Legg, J. P. (2011). First report of Cassava
brown disease and associated Ugandan cassava brown streak virus in Burundi. New Dis. Rep., 24(26).
https://doi.org/10.5197/j.2044-0588.2011.024.026
Chalupowicz, L., Dombrovsky, A., Gaba, V., Luria, N., Reuven, M., Beerman, A., ... Manulis-Sasson, S. (2019).
Diagnosis of plant diseases using the Nanopore sequencing platform. Plant Pathol., 68, 229-238.
https://doi.org/10.1111/ppa.12957
Dreaden, T. J., Shin, K., & Smith, J. A. (2011). First report of Diplodia corticola causing branch cankers on live
oak (Quercus virginiana) in Florida. Plant Dis. 95:1027.corticola causing grapevine (Vitis vinifera) cankers
and trunk cankers and dieback of canyon live oak (Quercus chrysolepis) in California. Plant Dis., 94, 785.
https://doi.org/10.1094/PDIS-02-11-0123
Dombrovsky, A., Reingold, V., & Antignus, Y. (2014). Ipomovirus—An atypical genus in the family Potyviridae
transmitted by whiteflies. Pest Manag Sci., 70(10), 1553-67. https://doi.org/10.1002/ps.3735
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
114
Drummond, A. J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., & Heled, J. (2010). Geneious v5.1. Retrieved
from http://www.geneious.com
Dufour, D., O’Brien, G. M., & Rupert, B. (1996). Cassava flour and starch: Progress in research and
development. CIAT.
FAO (Food and Agriculture Organization). (2011). Cassava Virus on Verge of Epidemic in East Africa: Expert
Urge funding, Swift Action to Protect Staple Food Crop. Rome: Food and Agricultural Organization.
FAO (Food and Agriculture Organization). (2013). Save and Grow Cassava: A Guide to Sustainable Production
Intensification. Rome: Food and Agriculture Organizationof the United Nations.
Garcia, B. J., Labbé, J. L., Jones, P., Abraham, P. E., Hodge, I., Climer, S., & Jacobson, D. A. (2018).
Phytobiome and Transcriptional Adaptation of Populus deltoides to Acute Progressive Drought and Cyclic
Drought. Phytobiomes Journal, 2(4), 249-260. https://doi.org/10.1094/pbiomes-04-18-0021-r
Hillocks, R. J., & Thresh, J. M. (2002). Cassava Biology, Production and Utilization. Wallingford, CT: CABI.
https://doi.org/10.1079/9780851995243.0000
IITA (International Institute of Tropical Agriculture). (2014b). IITA-led SCP project reports great strides in
regional exchange of improved cassava varieties. IITA News, Ibadan.
Jarvis, A., Ramirez-Villegas, J., Herrera Campo, B. V., & Navarro-Racines, C. (2012). Is cassava the answer to
African climate change adaptation? Trop. Plant Biol., 5, 9-29. https://doi.org/10.1007/s12042-012-9096-7
Kreuze, J. F., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I., & Simon, R. (2009). Complete viral
genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for
diagnosis, discovery and sequencing of viruses. Virology, 388(1), 1-7. https://doi.org/10.1016/j.virol.
2009.03.024
Larignon, P., Fulchic, R., Cere, L., & Dubos, B. (2001). Observations of Black dead arm in French vineyards.
Phytophathol. Mediterr., 40, 336-342.
Le May, C., Potage, G., Andrivon, D., Tivoli, B., & Outreman, Y. (2009). Plant disease complex: Antagonism
and synergism between pathogens of the ascochyta blight complex on pea. Journal of Phytopathology,
157(11-12), 715-721. https://doi.org/10.1111/j.1439-0434.2009.01546.x
Lynch, S. C., Eskalen, A., Zambino, P. J., & Scott, T. (2010). First report of Bot canker disease caused by
Diplodia corticola on coast live oak (Quercus agrifolia) in California. Plant Dis., 94, 1510.
https://doi.org/10.1094/PDIS-04-10-0266
Mahungu, N. M, Bidiaka, M., Tata, H., Lukombo, S., & N’luta, S. (2003). Cassava brown streak disease-like
symptoms in Democratic Republic of Congo. ROOTS, 8, 8-9.
Markowitz, V. M., Chen, I. M. A., Palaniappan, K., Chu, K., Szeto, E., Grechkin, Y., … Kyrpides, N. (2012).
IMG: The integrated microbial genomes database and comparative analysis system. Nucleic Acids Res., 40,
D115-D122. https://doi.org/10.1093/nar/gkr1044
Masumba, E. A., Kapinga, F., Mkamilo, G., Salum, K., Kulembeka, H., Rounsley, S., … Ferguson, M. (2017).
QTL associated with resistance to cassava brown streak and cassava mosaic diseases in a bi-parental cross
of two Tanzanian farmer varieties, Namikonga and Albert. Theor Appl Genet, 130, 2069-2090.
https://doi.org/10.1007/s00122-017-2943-z
Mbago, B. S., & Lotombe, B. G. (2017). Democratic Republic of Congo: Improving cassava production and
supply systems. SNV Netherlands Development Organisation.
Mbanzibwa, D. R., Tian, Y. P., Mukasa, S. B., & Valkonen, J. P. (2009). Cassava brown streak virus (Potyviridae)
encodes a putative Maf/HAM1 pyrophosphatase implicated in reduction of mutations and a P1 proteinase
that suppresses RNA silencing but contains no HC-Pro. Journal of Virology, 83, 6934-6940. https://doi.org/
10.1128/JVI.00537-09
Mbanzibwa, D. R., Tian, Y. P., Tugume, A. K., Patil, B. L., Yadav, J. S., Bagewadi, B., … Valkonen, J. P. (2011).
Evolution of cassava brown streak disease-associated viruses. J. Gen. Virol., 92(4), 974-87. https://doi.org/
10.1099/vir.0.026922-0
Menzel, P., Ng, K. L., & Krogh, A. (2016). Fast and sensitive taxonomic classification for metagenomics with
Kaiju. Nature Communications, 7, 1-9. https://doi.org/10.1038/ncomms11257
Mohammed, I. U., Abarshi, M. M., Muli, B., Hillocks, R. J., & Maruthi, M. N. (2012). The symptom and genetic
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
115
diversity of cassava brown streak viruses infecting cassava in East Africa. Adv Virol., 2012, 795697.
https://doi.org/10.1155/2012/795697
Monger, W. A., Seal, S., Isaac, A. M., & Foster, G. D. (2001a). Molecular characterisation of Cassava brown
streak virus coat protein. Plant Pathology, 50, 527-534. https://doi.org/10.1046/j.1365-3059.2001.00589.x
Monger, W., Alicai, T., Ndunguru, J., Kinyua, Z. M., Potts, M., Reeder, R. H., ... Smith, J. (2010). The complete
genome sequence of the Tanzanian strain of Cassava brown streak virus and comparison with the Ugandan
strain sequence. Archives of Virology, 155(3), 429-433. https://doi.org/10.1007/s00705-009-0581-8
Monger, W. A., Alicai, T., Ndunguru, J., Kinya, Z. M., Potts, M., Reeder, R. H., ... Smith, J. (2010). The
complete genome sequence of the Tanzanian strain of Cassava brown streak virus and comparison with the
Ugandan strain sequence. Arch. Virol., 155, 429-33. https://doi.org/10.1007/s00705-009-0581-8
Mulimbi, W., Phemba, X., Assumani, B., Kasereka, P., Muyisa, S., Ugentho, H., … Thom, F. E. F. (2012). First
report of Ugandan cassava brown streak virus on cassava in Democratic Republic of Congo. New Dis. Rep.,
26(11). https://doi.org/10.5197/j.2044-0588.2012.026.011
Mware, B., Narla, R., Amata, R., Olubayo, F., Songa, J., Kyamanywa, S., & Ateka, E. M. (2009). Efficiency of
Cassava brown streak virus transmission by two whitefly species in coastal Kenya. J. Gen. Mol. Virol., 1,
40-5.
Ndunguru, J., Sseruwagi, P., Tairo, F., Stomeo, F., Maina, S., Djinkeng, A., … Boykin, L. M. (2015). Analyses of
Twelve New Whole Genome Sequences of Cassava Brown Streak Viruses and Ugandan Cassava Brown
Streak Viruses from East Africa: Diversity, Supercomputing and Evidence for Further Speciation. PLoS
ONE, 10(10), e0139321. https://doi.org/10.1371/journal.pone.0139321
Nweke, F. (2014). New challenges in the cassava transformation in Nigeria and Ghana. Int. Food Pol. Res.
Inst, .118.
Ogwok, E., Alicai, T., Rey, M. E. C., Beyene, G., & Taylor, N. J. (2015). Distribution and accumulation of
cassava brown streak viruses within infected cassava (Manihot esculenta) plants. Plant Pathol., 64(5),
1235-1246. https://doi.org/10.1111/ppa.12343
O’Leary, N. A., Wright, M. W., Brister, J. R., Ciufo, S., Haddad, D., McVeigh, R., & Pruitt, K. D. (2016).
Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional
annotation. Nucleic Acids Research, 44(D1), D733-D745. https://doi.org/10.1093/nar/gkv1189
Patil, B. L., Ogwok, E., Wagaba, H., Mohammed, I. U., Yadav, J. S., Bagewadi, B., … Fauquet, C. M. (2011).
RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown
streak disease. Mol. Plant Pathol., 12(1), 31-41. https://doi.org/10.1111/j.1364-3703.2010.00650.x
Patil, B. L., Legg, J. P., Kanju, E., & Fauquet, C. M. (2015). Cassava brown streak disease: a threat to food
security in Africa. J. Gen. Virol., 96, 956-968. https://doi.org/10.1099/vir.0.000014
Roux-Cuvelier, M., Teyssedre, D., Chesneau, T., Jeffray, C., Massé, D., Jade, K., … Lett, J. M. (2014). First
report of Cassava brown streak disease and associated Ugandan cassava brown streak virus in Mayotte
Island. New Dis. Rep., 30(28). https://doi.org/10.5197/j.2044-0588.2014.030.028
Rwegasira, G. M., Rey, M. E. C., & Nawabu, H. (2011). Approaches to diagnosis and detection of cassava brown
streak virus (Potiviridae: Ipomovirus) in field-grown cassava crop. African Journal of Food, Agriculture,
Nutrition and Development, 11(3). https://doi.org/10.4314/ajfand.v11i3.66626
Salzberg, S. L., & Wood, D. E. (2014). Kraken: Ultrafast metagenomic sequence classification using exact
alignments. Genome Biology, 15. https://doi.org/10.1126/science.1093857
Taylor, A., Hardy, G. E. St. J., Wood, P., & Burgess, T. (2005). Identification and pathogenicity of
Botryosphaeria species associated with grapevine decline in Western Australia. Aust. Plant Pathol., 34,
187-195. https://doi.org/10.1071/AP05018
Úrbez-Torres, J. R., Leavitt, G. M., Voegel, T., & Gubler W. D. (2006). Identification and distribution of
Botryosphaeria species associated with grapevines cankers in California. Plant Dis., 90, 1490-1503.
https://doi.org/10.1094/PD-90-1490
Úrbez-Torres, J. R., Adams, P., Kamas, J., & Gubler, W. D. (2009). Identification, incidence, and pathogenicity
of fungal species associated with grapevine dieback in Texas. Am. J. Enol. Vitic., 60, 497-507.
Van Niekerk, J. M., Crous, P. W., Groenewald, J. Z., Fourie, P. H., & Halleen, F. (2004). DNA phylogeny,
jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 3; 2020
116
morphology and pathogenicity of Botryosphaeria species on grapevines. Mycologia, 96, 781-798.
https://doi.org/10.1080/15572536.2005.11832926
Winter, S., Koerbler, M., Stein, B., Pietruszka, A., Paape, M., & Butgereitt, A. (2010). Analysis of cassava brown
streak viruses reveals the presence of distinct virus species causing cassava brown streak disease in East
Africa. J. Gen. Virol., 91(5), 1365-72. https://doi.org/10.1099/vir.0.014688-0
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