RES E A R C H A R T I C L E Open Access
Emergence of wheat blast in Bangladesh
was caused by a South American lineage of
Magnaporthe oryzae
M. Tofazzal Islam
1*
, Daniel Croll
2
, Pierre Gladieux
3
, Darren M. Soanes
4
, Antoine Persoons
5
, Pallab Bhattacharjee
1
,
Md. Shaid Hossain
1
, Dipali Rani Gupta
1
, Md. Mahbubur Rahman
1
, M. Golam Mahboob
6
, Nicola Cook
5
,
Moin U. Salam
7
, Musrat Zahan Surovy
1
, Vanessa Bueno Sancho
5
, João Leodato Nunes Maciel
8
,
Antonio NhaniJúnior
8
, Vanina Lilián Castroagudín
9
, Juliana T. de Assis Reges
9
, Paulo Cezar Ceresini
9
,
Sebastien Ravel
10
, Ronny Kellner
11,12
, Elisabeth Fournier
3
, Didier Tharreau
10
, Marc-Henri Lebrun
13
,
Bruce A. McDonald
2
, Timothy Stitt
5
, Daniel Swan
5
, Nicholas J. Talbot
4
, Diane G. O. Saunders
5,14
, Joe Win
11
and
Sophien Kamoun
11*
Abstract
Background: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in
Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in
Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed
transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields.
Results: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms
on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast
outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus
Magnaporthe oryzae.
Conclusion: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease
outbreaks and provide valuable information regarding the identity and origin of the infectious agent.
Keywords: Field pathogenomics, Wheat blast, Phylogenomic analysis, Eleusine indica, Oryza sativa
Background
Outbreaks caused by fungal diseases have increased in
frequency and are a recurrent threat to global food se-
curity [1]. One example is blast, a fungal disease of rice,
wheat, and other grasses, that can destroy enough food
supply to sustain millions of people [1–3]. Until the
1980s, the blast disease was not known to affect wheat, a
main staple crop critical to ensuring global food security.
In 1985, the disease was first reported on wheat ( Triti-
cum aestivum L.) in Paraná State, Brazil [4]. It has since
spread throughout many of the important wheat-
producing areas of Brazil and to neighboring South
American countries including Bolivia and Paraguay. In
South America, blast is now a major threat to wheat
production [ 5–7]. Currently, wheat blast affects as much
as 3 million hectares, seriously limiting the potentia l for
wheat production in the vast grasslands region of South
America.
Blast diseases of grasses are caused by fungal species
from the Pyriculariaceae [8] and can occur on 50 grass
species [9]. However, a high degree of host specificity ex-
ists among and within these fungal species [8, 10]. In
South America, wheat blast is caused by isolates of Mag-
naporthe oryzae (syn. Pyricularia oryzae) known as
pathotype Triticum [10–12]. The rice-infecting isolates
of M. or yzae are genetically distinct from wheat-
infecting isolates and generally do not infect wheat [11,
* Correspondence: tofazzalislam@yahoo.com; sophien.kamoun@tsl.ac.uk
1
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman
Agricultural University, Gazipur 1706, Bangladesh
11
The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK
Full list of author information is available at the end of the article
© 2016 Islam et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Islam et al. BMC Biology (2016) 14:84
DOI 10.1186/s12915-016-0309-7
13–20]. Typical symptoms of wheat blast on spikes are
premature bleaching of spikelets and entire heads [21–
23]. Severely infected wheat heads can be killed, result-
ing in severe yield losses [ 21, 22]. The disease is gener-
ally spread by infected seeds and airborne spores, and
the fungus can survive in infected crop residues and
seeds [14]. Little is known about the physiology and gen-
etics of the whe at blast pathogen, and our understanding
of the molecular interactions of this pathogen with
wheat remains limited.
In February 2016, wheat blast was detected for the first
time in Asia with reports of a severe outbreak in
Bangladesh relayed through local authorities and the
media [24]. Although wheat is not a traditional crop in
Bangladesh, its cultivation has expanded in recent years,
making it the second major food source after rice [25].
The outbreak is particularly worrisome because wheat
blast could spread further to major wheat-producing
areas in neighboring South Asian countries, thus threat-
ening food security across the region. Here, we report
our immediate response to this plant disease outbreak.
To rapidly determine the precise identity and likely ori-
gin of the outbreak pathogen, we applied field pathoge-
nomics, in which we performed transcriptome
sequencing of symptomatic and asymp tomatic leaf sam-
ples collected from infected wheat fields in Bangladesh
[26, 27]. To promote the proje ct and recru it experts, we
immediately released all raw sequence data through a
Fig. 1 Geographical distribution and severity of the wheat blast outbreak in eight southwestern districts of Bangladesh. The map depicts the
intensity of the 2016 wheat blast outbreak across Bangladesh. The percentage of affected area and the total area (hectares) under cultivation are
shown for each district based on the color chart
Islam et al. BMC Biology (2016) 14:84 Page 2 of 11
dedicated website Open Wheat Blast (http://www.wheat-
blast.net). Phylogenomic and population genomic ana-
lyses revealed that the Bangladesh wheat blast outbreak
was probably caused by isolates belonging to the South
American wheat-infecting lineage of M. oryzae. We con-
clude tha t the wheat blast pathogen was most likely in-
troduced into Asia from South America.
Results and discussion
Geographical distribution of the wheat blast outbreak in
Bangladesh
The total area of wheat cultivation in Bangladesh in
2016 was about 498,000 ha (Department of Agricultural
Extension, Bangladesh). Wheat blast was observed in
eight southwestern districts , viz., Pabna, Kushtia, Meher-
pur, Chuadanga, Jhenaidah, Jessore, Barisal, and Bhola
(Fig. 1). Out of a total 101,660 ha of cultivated wheat in
those eight districts, an estimated 15 % were affected by
wheat blast.
The severity of wheat blast and associated yield losses
varied among districts. The highest percentage of in-
fected wheat fields was observed in Meherpur (70 %)
followed by Chuadanga (44 %), Jessore (37 %), Jhenaidah
(8 %), Bhola (5 %), Kushtia (2 %), Barisal (1 %), and
Pabna (0.2 %) (Fig. 1). Yield losses in different affected
districts varied. The highest average yield loss was re-
corded in Jhenaidah (51 %) followed by Chuadanga
(36 %), Meherpur (30 %), Jessore (25 %), Barisal (21 %),
Pabna (18 %), Kushtia (10 %), and Bhola (5 %). Although
the average yield loss was lower than 51 % across dis-
tricts, yield losses in individual fields were as high as
100 %. Importantly, 100 % of government-owned
Bangladesh Agricultural Development Corporation
(BAD C) seed multiplication farms in the affected dis -
tricts (ca. 355 ha) were comple tely cleared by burning to
destroy pathogen inocula by decision of the Ministry of
Agriculture (see https://www.youtube.com/watch?v=Em-
L5YM0kIok). Farmer wheat fields that were severely af-
fected (~100 %) were also burned.
Wheat blast symptoms in the field
To examine disease symptoms in affected wheat fields,
we collected samples from the affected district s. Major
symptoms associated with the epidemic included com-
pletely or partially bleached (dead) spikes similar to
symptoms reported for Brazilian wheat blast epidemics
[21, 22] and symptoms reported from Bangladesh in
2016 [23]. The pathogen attacked the base or upper part
of the rachis, severely affecting spikelet formation above
the point of infection. Complete or partial bleaching of
the spike above the point of infection with either no
grain or shriveled grain was common in all areas af-
fected by wheat blast (Fig. 2a–c). We commonly ob-
served bleached heads with traces of gray, indicative of
fungal sporulation at the point of infection (arrows in
Fig. 2a–c and g). In severely infe cted fields, we also
found typical eye-shaped necrotic disease lesions with
gray centers in the leaves of some wheat plants (Fig. 2d)
[21, 28]. Head infections during the flowering stage re-
sulted in no grain production (Fig. 2g), whereas infection
at the grain filling stage resulted in small, shriveled, light
in weight, and discolored (pale) grains (Fig. 2e, f ) [22].
To determine whether the spike and leaf symptoms on
wheat were associated with infection by blast fungi (Pyr-
icularia and related genera from the Pyriculariaceae
sensu; see Klaubauf et al. [8]), we examined infected
plant samples using a light microscope. A hallmark of
blast fungi is the production of asexual spores that have
a specific morphology consisting of three-celled pyriform
conidia [8]. Microscopic analyses revealed that gray col-
ored lesions observed on both spikes and leaves carried
large numbers of three-celled pyriform conidia from aer-
ial conidiophores (Fig. 2h). This indicates that the fungus
present in the se lesions belongs to the Pyriculariaceae,
consistent with a previous report [22]. However, molecu-
lar taxonomy tools are needed to determine the species
identity.
Strains isolated from infected wheat sampl es cause
symptoms of wheat blast on artificially inoculated wheat
To confirm whether the fungus found on infected wheat
leaves is able to cause the observed symptoms, we iso-
lated ten strains (BTJP 3-1, BTJP 3-2, BTJP 3-3, BTJP 4-
1, BTJP 4-2, BTJP 4-3, BTJP 4-4, BTJP 4-5, BTJP 4-
6, and BTJP 4-7) using a single-conidia isolation method
(Fig. 3a). On potato dextrose agar (PDA) plates, the pre-
dominant morphology of the isolates was gray to white
aerial mycelia with an olive or brown center (Fig. 3b).
After 14–21 days of inoculation, the center of the cul-
ture became black (Fig. 3c). Artificial inoculation of
wheat seedling leaves using conidia of two isolates (BTJP
3-1 and BTJP 4-1) produc ed characteristic symptoms
five days after inocula tion (Fig. 3d–h). Initially, a
diamond-shaped, water-soaked lesion in green leaves
was observed (Fig. 3d), which gradually turned into an
eye-shaped lesion, with a tan or gray colored center
(Fig. 3e, f ). At a later stage, the spots enlarged, spread to
entire leaves, and killed the leaves (Fig. 3g, h). No differ-
ence in symptoms was observed on wheat seedlings of
the cultivars Shatabdi and Prodip and between the two
isolates (BTJP 3-1 and BTJP 4-1). Similar disease symp-
toms and sporulation were observed on leaves of artifi-
cially inoculated goosegrass (Eleusine indica) (Fig. 3k)
and barley (Hordeum vulgare) (Fig. 3l). Terminal infe c-
tion stages were characterized by a massive production
of hyaline to pale gray, pyriform, and asexual conidia on
aerial conidiophores. Conidia formation was observed
on all infected wheat (Triticum aestivum), barley (H.
Islam et al. BMC Biology (2016) 14:84 Page 3 of 11
vulgare), and goosegrass (E. indica) leaves (Fig. 3i–l).
Under the same conditions, no visible symptoms or
sporulation of conidia were observed microscopically on
leaves of artificially inoculated rice (Oryza sativa cv.
BRRIdhan 49; data not shown). These results are con-
sistent with those of Castroagudin et al. [29] showing
that wheat-infecting M. oryzae can infect seedlings of
barley but is largely asymptomatic on rice. The patho-
genicity of wheat blast on E. indica is also consistent
with reports that E. indica is a major alternate host in
South America [30, 31]. E. indica is also a common
weed in the highlands of Bangladesh and may similarly
serve as a alternate host of wheat blast. Understanding
the role of alternate hosts in disease cycles and
epidemics of wheat blast will be key in formulating ef-
fective disease management strategies.
Transcriptome sequencing of wheat leaf samples from
Bangladeshi fields
We used field pathogenomics [26] to ide ntify which
blast fungus spe cies was present in infe cted wheat
fields in B a ngladesh. We collected samples of both
symptomatic and a s ymptomatic leaves from wheat
fields in different regions of Bangladesh, including
Meherpur and Jhenaidah district s , and extracted total
RNA from four pairs of symptomatic (samples 2, 5, 7,
and 12) and a sy mptomatic samples (samples F2, F5,
F7, and F12) (Additional file 1: Table S1). We
Fig. 2 Symptoms of blast disease in spikes, leaves, and seeds of wheat in a farmer’s field in Jhenaidah in Bangladesh, and a micrograph showing
two conidia of Magnaporthe oryzae. a A completely bleached wheat spike with traces of gray from blast sporulation at the neck (arrow) of the
spike. b Complete bleaching of a wheat spike above the point (arrow) of infection. c Two completely bleached spikes with traces of gray (upper
arrow) and a lesion (lower arrow) from blast sporulation at the base. d Typical eye-shaped lesion (arrow) and dark gray spots on a severely dis-
eased wheat leaf. e Mild blast disease-affected slightly shriveled wheat seeds. f Severe blast-affected shriveled and pale wheat seeds.
g A severely infected rachis with dark gray blast sporulation at the neck (arrow) and severely damaged spikelets. h Micrograph of two conidia
isolated from the infected spike of wheat. Scale bars in e and f = 1 cm and in h =10μm
Islam et al. BMC Biology (2016) 14:84 Page 4 of 11
prepared and sequenced RN A-seq libraries using Illu-
mina tec hnology, yielding 68.8 to 125.8 million 101-
bp pa ir-end reads with an average insert size of
419 bp. Next , following data trimming, we aligned
high-quality reads to both the M. oryzae wheat blast
fungus BR32 and wheat genomes [19, 32]. Sequence
reads from all samples with disease symptoms aligned
to the BR32 genome, ranging from 0.5–18.6 % of the
total reads (Fig. 4a). By contrast, only a minor pro-
portion of the reads from the a symptomatic samples
aligned to the BR32 genome (range: 0.003 –0.037 %,
Fig. 4a–c). Between 37.7 % and 86.5 % of total reads
aligned to the wheat ge nome sequence (Fig. 4a). We
obtained similar numbers when considering the reads
aligning to M. oryz ae and wheat transcriptomes (Add-
itional file 2: Table S2). Variation in percentage
mapped reads of h ost and fungal transcripts among
symptomatic samples is most likely explained by dif-
ferences in the disea se severity and infe ction stage
among field collected leaves. The finding that on
average 6.8 % reads per sampled transcriptome
aligned to the wheat blast genome BR32 indicated
that M. or yzae is present in symptomatic (infe cted)
wheat samples from Bangladesh.
Fig. 3 Reinoculation of seedlings with fungal strains isolated from infected wheat seeds. Germinated conidia, growth of mycelia, infection, and
sporulation of strains used to artificially inoculate wheat, barley, and goosegrass. a A germinated three-celled pyriform conidia (arrow) with hyphal
growth on water agar medium. b, c Culture of isolate BTJP 3-1 on PDA plate; upper (left) and reverse side (right). d Photograph showing a
diamond-shaped, water-soaked lesion (initial stage of infection symptom, upper arrow) on a green wheat seedling leaf five days after conidial
inoculation. e, f Development of an eye-shaped lesion with a gray center (arrows in e and f) on wheat leaves. g, h A gradual progression of
symptoms (arrows) on wheat leaves. i–l Light micrographs showing massive conidia production (red arrow) on aerial conidiophores (black arrow)
on artificially infected leaves of wheat cultivars Prodip (i) and Shatabdi (j), goosegrass (k), and barley (l). Photographs were taken by a camera
attached to a microscope at 100× magnification. Scale bars in j, k, and l indicate 50 μm
Islam et al. BMC Biology (2016) 14:84 Page 5 of 11