Download Transmission of rice blast from seeds to adult plants in a non

Plant Pathology (2013) 62, 879–887
Doi: 10.1111/ppa.12003
Transmission of rice blast from seeds to adult plants in a
non-systemic way
O. Faivre-Rampantab, L. Genièsb, P. Piffanellia and D. Tharreaub*
a
Parco Tecnologico Padano, Via Einstein, 26900, Lodi, Italy; and bCIRAD, UMR BGPI, TA A54/K, 34398, Montpellier 05, France
Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a serious threat to rice production worldwide. In temperate regions, where rice is not cultivated for several months each year, little is known about the initial onset of the disease in the field. The main overwintering and primary inoculum sources reported are infested residues and seeds, but the
subsequent steps of the disease cycle are largely unknown, even though a systemic infection has been proposed but not
demonstrated. The present work follows rice blast progression in infected seeds from germination to seedling stage, with
direct and detailed microscopic observations under both aerobic conditions and water seeding. With the use of GFPmarked M. oryzae strains, it was shown that spores are produced from contaminated seeds, infect emerging seedling tissues (coleoptile and primary root) and produce mycelium that colonizes the newly formed primary leaf and secondary
roots. Using different rice cultivars exhibiting distinct levels of resistance/susceptibility to M. oryzae at the 2/4-leaf stage, it
was observed that resistance or susceptibility of a considered genotype is already established at the seedling stage. The
results also showed that when plants are inoculated either at ripening stage (mature panicles), heading stage (flowering/
immature panicles) or even before heading (flag leaf fully developed), they produce infested seeds. These seeds produce
contaminated seedlings that mostly die and serve as an inoculum source for healthy neighbouring plants, which gradually develop
disease symptoms on leaves. The possible rice blast disease cycle was reconstructed on irrigated rice in temperate regions.
Keywords: contamination, Magnaporthe oryzae, microscopy, rice, seeds
Introduction
Rice is grown in a wide range of geographic and climatic
conditions. It constitutes the most important staple food
for half of the world’s population, especially in Asia
(http://www.fao.org). Its production has increased over
the last decades due to improvements in cultivation practices and the introduction of high-yielding cultivars
(Sakamoto & Matsuoka, 2004). However, rice is
exposed to diseases that contribute to limiting its production. Among them, rice blast, caused by the fungus Magnaporthe oryzae, is a major constraint for the
productivity of this crop worldwide (Wilson & Talbot,
2009). In the absence of rice blast control strategies, M.
oryzae can cause high annual yield losses (Oerke & Dehne, 2004; Skamnioti & Gurr, 2009). Rice blast disease
has been found in all rice-growing countries (Kato, 2001;
International Plant Protection Convention, 2011), covering a wide range of agro-ecosystems with very diverse
environmental conditions from rain-fed uplands in the
tropics to irrigated plains in temperate areas.
In field conditions, the fungus is able to infect all aerial
parts of rice, resulting in leaf, node, neck and panicle
*E-mail: [email protected]
Published online 1 November 2012
ª 2012 The Authors
Plant Pathology ª 2012 BSPP
blast. The fungal infectious process on its host is well
studied. Leaf infection by M. oryzae is initiated by
attachment of a spore that germinates and forms a
melanized appressorium on the rice cuticle (Wilson &
Talbot, 2009). This appressorium generates turgor pressure that ruptures the leaf cuticle, allowing M. oryzae to
infect leaf epidermal cells with a bulbous invasive hypha
(IH). Subsequently, filamentous IH are suggested to
invade neighbouring cells through plasmodesmata (Kankanala et al., 2007). Fungal progression within rice cells
results in the death of the infected tissues and the
appearance of necrotrophic lesions on leaves by 3–4 days
after inoculation (Wilson & Talbot, 2009).
In controlled conditions, and upon artificial inoculation with mycelium, M. oryzae is also able to colonize
roots by forming hyphopodia (Sesma & Osbourn, 2004).
However, little is known about the initial onset of the
disease and how rice blast disease spreads in the field. In
the tropics, overwintering is presumed not to be important because airborne conidia are present throughout the
year (Ou, 1985). This assumption may not hold true in
tropical regions of elevated altitude where rice is not
grown in winter. In temperate regions where rice is
absent for several months, overwintering and primary
inoculum sources have not yet been determined. Overwintering of mycelium and conidia on straw has been
proposed, but these inocula do not survive under moist
conditions (Ou, 1985). Harmon & Latin (2005) reported
that, in north-central Indiana (USA), M. oryzae can over-
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winter in infested residues of perennial ryegrass, but the
proportion of the surviving population is insufficient to
serve as effective primary inoculum for summertime epidemics. In addition, such survival was not demonstrated
for M. oryzae populations pathogenic to rice which have
previously been suggested to overwinter on an alternative
host (Ou, 1985) and which are different from populations pathogenic to perennial ryegrass.
Most of the studies carried out so far support the
occurrence of infested seeds as a source of primary inoculum. Long et al. (2001) showed that dead infested
grains could serve as primary inoculum when placed in
the field at the time of seedling emergence. Transmission
of M. oryzae from rice seeds to seedlings has already
been documented in different countries (Kuribayashi,
1928 and Suzuki, 1930 cited by Manandhar et al., 1998;
Lamey, 1970; Mayee, 1974 cited by Long et al., 2001;
Chung & Lee, 1983; Reddy & Bastawsi, 1989; Long
et al., 2001), but no cytological observations of the
infection process from the contaminated germinating
seed to the seedling have been performed so far. Using
naturally infested seeds as primary inoculum in field conditions in Nepal, Manandhar et al. (1998) showed that
panicle symptoms and seed contamination are correlated.
They observed that sporulation of M. oryzae on infested
seeds was preferentially found at the embryonic end of
germinating seeds, while occurring homogeneously all
over the surface for non-germinating seeds. Moreover, a
seed lot with 21% contamination led to <4% seedlings
with blast lesions. Tests employing different ways of covering seeds with soil (light or complete) and under water
seeding (no covering) pointed out that complete covering
or seeding under water induce a lower infection frequency (Manandhar et al., 1998). Guerber & TeBeest
(2006) reported similar experiments in the USA. They
showed that 23% of the seed lots examined, including
commercial lots, were contaminated and that the corresponding rate of diseased seedlings varied from 03 to
11% in greenhouse conditions. However, no disease was
observed when infested seeds were germinated under
water. When infested seeds were sown in the field, the
fungus was recovered from different seedling parts,
including roots, from plants with and without symptoms.
These results clearly show that the fungus can survive on
the grains used for seeding and that these contaminated
grains can serve as primary inoculum. However, Guerber
& TeBeest (2006) did not detect the fungus in seeds of
highly resistant rice varieties. Bernaux & Berti (1981)
reported that overwintering of mycelium and conidia
could occur between the glumella and caryopsis because
of the susceptibility of palea and lemma before anthesis.
The observation that inoculation of roots with mycelium could result in sporadic disease symptoms on the
leaves has led some authors to hypothesize that M. oryzae
could move systemically within a seedling (Sesma & Osbourn, 2004; Marcel et al., 2010). However, the same
authors did not check for the presence of M. oryzae
within the symptomless tissues. Similarly, systemic movement within a seedling from an infested seed has not been
demonstrated experimentally (Lamey, 1970; Mayee,
1974; Chung & Lee, 1983; Kingsolver et al., 1984; Ou,
1985; Manandhar et al., 1998). To summarize previous
reports, (i) seeds on panicles can be infested up to c. 25%
and give rise to up to roughly 10% of diseased seedlings;
(ii) infested seeds can serve as primary inoculum in the
field; (iii) complete covering of infested seeds or infested
seeds sown under water drastically reduces the frequency
of seedling infection in the field; and (iv) no fungus was
detected on seeds of resistant rice accessions.
Until now, no precise seedling infection time-course
from infested seeds has been documented, and in particular it remains unclear whether the fungal progression is
systemic. Here, the fungal progression has been followed
from contaminated seeds (harvested and then stored for
a few months) to seedlings by detailed cytological observations using two seeding conditions (aerobic/anaerobic).
Seeds were previously contaminated using green fluorescent protein (GFP)-tagged M. oryzae strains at different
plant development stages: (i) panicle (flag) leaf, (ii)
immature panicles, or (iii) mature panicles. Fungal progression was monitored from seed to seed using different
rice cultivars exhibiting diverse susceptibility levels.
Material and methods
Magnaporthe isolates, plant material and infection
assays
GFP-tagged M. oryzae strains were used to investigate seedling
infection. Magnaporthe oryzae isolate FR13-GFP was generated
using Agrobacterium-mediated transformation, as previously
described (Rho et al., 2001; Sesma & Osbourn, 2004). Magnaporthe oryzae strain CL3.6.7-GFP was engineered through
protoplast isolation and transformation using the plasmid
pfC2ORFGFP (CNRS-Bayer) and the protocol of Fudal et al.
(2007). These strains were selected because of their routine use in
this laboratory. Rice plants used in this study are temperate japonica cultivars: Sariceltik from Turkey, and Maratelli and Gladio
from Italy. Plants and M. oryzae strains were grown as described
by Faivre-Rampant et al. (2008). Inoculation was carried out by
spraying 50 000 conidia mL 1 of FR13-GFP and CL3.6.7-GFP
either on the panicle leaf (before heading) or on immature panicles
(heading stage) or mature panicles (ripening stage). Rice plants
were then placed in the dark with 100% relative humidity (RH)
for 16 h and transferred back to the greenhouse until seed harvest.
Seeds were then placed at 37°C for 2 days and stored at 20°C for
at least 6 months prior to cytological observations.
To measure seed contamination frequency, 50 seeds per
experiment were placed either in two Petri dishes on a Whatman
no. 5 filter paper moistened with sterile distilled water (SDW)
placed in a plastic box (‘aerobic conditions’), or in 15 mL tubes
containing 2 mL of SDW (‘seeding under water’). Plates or tubes
were then incubated at 25°C in 12 h light/dark cycles. Seed contamination percentage was calculated for each seed lot as the
number of seeds infested by the fungus divided by the total
number of seeds examined (50) and multiplied by 100. The final
contamination percentage for each seed lot was determined as
an average of at least three replicates ± standard error.
Sariceltik and Maratelli infected (with symptoms) and noninfected (without symptoms) seedlings grown in water were
transferred in the glasshouse when 2 weeks old. Seedlings with
Plant Pathology (2013) 62, 879–887
Rice seed infection by the blast fungus
and without symptoms were cultivated in the same tray. The
soil was not flooded permanently, but kept moist by frequent
watering. After 1 month, plants were placed 1 night per week in
a dew chamber with 100% RH for 16 h and brought back in
the glasshouse. This was repeated until seed harvest. Exposure
to high RH was used to mimic field conditions where moist conditions are necessary for sporulation. In the glasshouse, RH was
maintained below 70% to avoid sporulation and uncontrolled
infections.
Cytological analysis
Seed and plantlet infection by M. oryzae was monitored over
time using an Olympus BX60 microscope equipped with an UV
lamp. The progression of the fungus was followed on 25 seeds
per plate and a total of two plates, at 3, 6, 10, 13 and 15 days
after sowing (d.a.s.), and the experiment repeated independently
three times. For detailed inspection, rice tissues were observed
on a Zeiss LSM 700 confocal microscope using 9 10 N-Achroplan (numerical aperture 025) or 9 20 Plan-Apochromat
(numerical aperture 08) objective lenses. Fluorescence of GFP
was detected using an excitation wavelength of 488 nm. Signals
emitted were recorded from 555 to 639 nm. To check for the
presence of M. oryzae inside the root, sections of 50 lm from
embedded roots in 5% agarose were obtained using a vibratome
(Microm, Thermo Scientific).
Results
Seed contamination rates by M. oryzae
To determine how rice seeds are contaminated by
M. oryzae, adult rice plants were first inoculated at the
developmental stage of mature panicles (ripening stage),
with conidia of the green fluorescent protein (GFP)expressing M. oryzae strain FR13. Seeds were then harvested, stored for 6–9 months, and seed contamination
documented over a period of 13 days by fluorescence
and confocal microscopy. The seeds of three temperate
rice cultivars, showing different responses to the M. oryzae strain FR13 (Sariceltik, very susceptible; Maratelli,
susceptible; Gladio, resistant; Fig. S1), were placed on
moist filter paper (aerobic conditions) to examine the frequency of infested seeds. Among the three cultivars, seed
contamination percentages varied from 4% for Gladio
up to 21% for Maratelli (Fig. 1a), but all the seed samples germinated at high rates (75–98%) like the original
non-inoculated (control) seed lots (Fig. 1a). After germination, the percentage of seedlings with blast lesions ranged from 0 for Gladio to 14% (±9) for Maratelli
(Fig. 1a).
Adult plants of the susceptible (Maratelli) and the
resistant (Gladio) cultivars were then inoculated at an
earlier developmental stage (immature panicles) with
FR13-GFP. Harvesting of mature seeds and storage were
carried out as for mature panicle-inoculated plants. Seed
contamination percentages were similar for the two cultivars (Fig. 1a) and the seed lots germinated at 59% (±36)
for Gladio and 91% (±8) for Maratelli, like the original
non-inoculated seed samples (Fig. 1a). After germination,
Plant Pathology (2013) 62, 879–887
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the percentage of diseased seedlings ranged from 0 for
Gladio to 9% (±6) for Maratelli (Fig. 1a).
To determine seed contamination rate following inoculation of the panicle leaf, only the highly susceptible cultivar Sariceltik was tested. Seed infection rate reached
225% (±32) and 15% (±10) of seedlings exhibited blast
lesions (Fig. 1a). A last inoculation on adult Sariceltik
plants at the mature panicle stage was subsequently
performed with a less aggressive strain, CL3.6.7-GFP.
Thirty-three per cent (±17) of seeds were contaminated
and 27% (±11) of seedlings showed blast lesions
(Fig. 1a).
Seed contamination by rice blast was observed after
inoculation at panicle leaf, immature and mature panicle
stages. Moreover, in all tests M. oryzae was predominantly located on seed teguments, as shown in Figure 1b.
Time-course of seedling contamination by M. oryzae in
aerobic conditions
When infested seeds were placed on moist filter paper
(aerobic conditions), spores were visible at 3–4 d.a.s.
(Fig. 2a). Contamination occurred mostly at the embryonic end of the rice seeds for the three cultivars and
also at a lower rate (44%) on the awn in Sariceltik
(Fig. 2b). For the two susceptible cultivars (Sariceltik
and Maratelli), spores of FR13-GFP spread on the sterile lemmas (Fig. 2c) within 7 d.a.s. Magnaporthe oryzae
progression resulted in an infected coleoptile producing
mycelium and spores at 10 d.a.s. (Fig. 2d,e). From the
coleoptile, mycelium and spores were able to infect the
emerging leaf (Fig. 2f) within 13 d.a.s. Mycelium and
spores were also observed on the primary and secondary roots at this time (Fig. 2g,h). Observations with
confocal microscope confirmed the presence of fungal
hyphae in the root cells (Fig. 2i). The difference in the
infection process between Sariceltik and Maratelli was
quantitative, not qualitative, because the fungus progression was faster and seedling susceptibility was higher
for Sariceltik. After 2 weeks, plants of Maratelli and
Sariceltik that were infected/with symptoms were transplanted to soil and grown in the glasshouse. All Sariceltik plants did not recover from infection and died
within 2 weeks while 40% (±10) of Maratelli seedlings
survived. It was confirmed that plant death was due to
FR13-GFP infection in leaves as well as in roots
(Fig. 2j,k).
Regarding the resistant cultivar Gladio, spores and
mycelium were present on the seed and infected the
emerging coleoptile at 3–4 d.a.s. (Fig. 3a,b). Fluorescent
appressoria were visible on the coleoptile (data not
shown) within 5 d.a.s. resulting in an hypersensitive
response and cell wall autofluorescence of the rice epidermis (Fig. 3c). Spores were also observed on the primary root and mycelium spread to the secondary root
within 13 d.a.s. (Fig. 3d,e). However, no further infection occurred. After 2 weeks no fluorescence was
observed, indicating no living fungal material was pres-
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(a)
Germinaon %
Fluorescent seed %
Germinaon % of control seeds
Disease %
100
100
80
80
60
60
40
40
20
(b) 60
SK MP CL3.6.7
SK PL FR13
Gladio IP FR13
Maratelli IP FR13
Gladio MP FR13
Maratelli MP FR13
SK MP
CL3.6.7
SK PL
FR13
Gladio IP
FR13
Maratelli
IP FR13
Gladio
MP FR13
Maratelli
MP FR13
SK MP
FR13
0
SK MP FR13
0
20
Fluo. Tegument %
Fluo. Hulled seed %
50
40
30
20
*
10
0
SK MP CL3.6.7
Figure 1 (a) Germination rates and infection percentages of Sariceltik (SK), Maratelli and Gladio seeds by Magnaporthe oryzae (strains FR13 and
CL3.6.7) collected from inoculated mature panicles (MP), immature panicles (IP) or panicle leaf (PL) of rice adult plants. The percentages of blast
diseased seedlings are also shown. Three independent sowings were carried out for each seed lot and the mean with standard deviation was
calculated. On the right side of the main figure are the germination percentages of the original non-inoculated/control seed lot. (b) Infection
frequencies of rice seed teguments and rice hulled seeds. *Significant frequency difference between fluorescence from teguments and from hulled
seeds (v2 test, P < 005).
ent; seedlings grew normally and did not show any macroscopic lesions (data not shown).
Seed contamination by the blast fungus occurred irrespective of the level of susceptibility/resistance of the rice
cultivar and led to seedling colonization for the susceptible cultivars but not for the resistant one.
Time-course of seedling contamination by M. oryzae
under water seeding
The ability of the fungus to grow on infested seeds when
placed under water, mimicking seed germination conditions in the field, was examined. For this purpose, seeds
of Sariceltik (SK) were inoculated with CL3.6.7 at mature
panicle stage (MP). The germination rate of the tested
seed lot, 78% (±20), was similar to that obtained when
seeds were placed in aerobic conditions (85% ± 12;
Fig. 1a, SK MP CL3.6.7). Blast occurred on 30% (±33)
of the infested seeds, which is similar to the contamination frequency of the same seeds when placed on moist
filter paper (33% ± 17; Fig. 1a, SK MP CL3.6.7). Spores
and mycelium were visible on the infested seeds within
2 d.a.s., preferentially at the embryonic end. At 4 d.a.s.,
a mycelium layer covered the emerging coleoptile
(Fig. 4a), resulting in a total infection within 9 d.a.s.
(Fig. 4b) and spore production (Fig. 4c). Fluorescent
extended lesions then appeared on the growing primary
leaf due to new infections by spores and/or colonization
by fungal hyphae (Fig. 4d,e). At root emergence, the
mycelium cover colonized the developing primary root to
form a coat (Fig. 4f,g). Within 13 d.a.s., primary and secondary roots were infected by the fungus (Fig. 4h) and
produced spores outside the root epidermis (Fig. 4i).
Contamination of seeds sown under water followed by
colonization of seedlings by rice blast fungus occurred in
these experiments, in contrast to what has been previously reported.
Infection of symptomless plants
To follow the blast infection process on rice adult
plants, the 13-day-old Sariceltik plantlets with symptoms arising from germination of infested seeds under
water were then transferred into the glasshouse with
symptomless (non-fluorescent) Sariceltik and Maratelli
seedlings. Plants with and without symptoms were
grown together in the same tray and the soil was not
flooded permanently. The plantlets with symptoms did
Plant Pathology (2013) 62, 879–887
Rice seed infection by the blast fungus
(b)
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(c)
Lemma
Col.
(d)
(g)
(j)
(e)
(f)
(i)
(h)
(k)
Figure 2 Sariceltik seed infection by GFP-transformed Magnaporthe oryzae rice FR13 strain in aerobic conditions. (a,b) Spores on the embryonic
end of the seed (a), and on the awn (b), within 3 days after sowing (d.a.s.). (c) Spores infecting the lemma resulting in necrotic symptoms on the
coleoptile (white arrow) by 7 d.a.s. (d) Infected upper part of the coleoptile covered by fluorescent mycelium. (e) End of the coleoptile, shown in (d),
also infected by spores. (f) Infected primary leaf within 13 d.a.s. showing hyphal progression. (g,h) Infected primary (g) and partially infected
secondary (h) roots with mycelium; a non-infected root is shown inset in (g); white arrows indicate fungus progression within the root and red arrow
indicates external mycelium hyphae. (i) Longitudinal section of the infected root in (g) illustrating the presence of mycelium inside root cells; inset
shows a transversal section of the same root. (j,k) Dead infected plantlet by 25 d.a.s. with fluorescent spores on the leaf (j) and fluorescent root (k,
white arrow; the red arrow shows an uninfected root). Bar scale = 5 lm (magnification in i); 20 lm (i); 50 lm (b, magnification in e); 100 lm (a, c, d,
e, f, g, h, j, k). All pictures were taken by epifluorescence microscopy except (i) which was by confocal microscopy. Col., coleoptile.
not recover and died very quickly after transfer, while
the symptomless Sariceltik and Maratelli seedlings grew
normally (data not shown). After 2 months, typical
blast necrotic lesions (Fig. 4j) appeared on leaves of half
of the Sariceltik adult plants derived from symptomless
seedlings. When these lesions were placed on moist filter
paper overnight, fluorescent spores were clearly visible
and developed at the top of the necrotic symptoms
(Fig. 4k). After two additional weeks, 100% of the SariPlant Pathology (2013) 62, 879–887
celtik plants, symptomless when transferred in the glasshouse, exhibited necrotic lesions due to infection by the
GFP strain used. To check for a putative systemic colonization of the plant by the fungus via the stem vascular
system, the fungus was looked for in internodes and
nodes. Five Sariceltik plants showing typical blast
lesions were cut into 5 mm transverse sections and
placed on wet filter paper overnight to favour potential
sporulation from these cuttings. No fluorescence was
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O. Faivre-Rampant et al.
(a)
(b)
(d)
(e)
(c)
Figure 3 Gladio seed infection by GFP-transformed Magnaporthe oryzae rice FR13 strain in aerobic conditions. (a) Spores on the seed within 3 days
after sowing (d.a.s.). (b) Mycelium present on the seed infecting the emerging coleoptile at 4 d.a.s. (c) Hypersensitive response from epidermal cells
(red arrow) of the coleoptile and epidermal cell wall autofluorescence (white arrow) due to the presence of appressoria. (d,e) Spores and mycelium on
the primary (d) and secondary (e) roots at 13 d.a.s. Bar scale = 50 lm (a,b,c,e), 100 lm (d). All pictures were taken by epifluorescence microscopy.
visible on the cuttings (data not shown). When infected
leaves were cut into small pieces on both sides of the
macroscopically visible necrotic lesions, limited hyphal
progression from the lesion could be observed within
the leaf epidermis (Fig. 4l–o). Hyphae were observed in
the vascular tissues of the leaf, as previously shown by
Berruyer et al. (2006). After 3 months, 30% of the
Maratelli plants which were symptomless when transferred in the glasshouse exhibited typical blast disease
symptoms on the leaves fluorescing under the epifluorescence microscope (Fig. 4p). Two weeks later, 50% of
the Maratelli plants showed fluorescent blast lesions.
Panicles of the Sariceltik and Maratelli plants which
were gradually infected in the glasshouse were harvested
when mature. Full grains and empty seeds/secondary
branches were placed on moist paper to check if this
second generation of seeds could be infected. Results are
given in Fig. S2a. For Sariceltik full grains, 18% (±17)
exhibited fluorescent spores either on the tegument or
on the awn (Fig. S2b). Sariceltik empty seeds and panicle secondary branches were also infected at a higher
rate, i.e. 26% (±25; Fig. S2a,b). Maratelli full seeds
were infected at a lower rate (07% ± 11) while Maratelli empty seeds or secondary branches were not
infected (data not shown).
Discussion
In agreement with Lamey (1970), who previously
showed that only 2% of hulled seeds were contaminated
by M. oryzae, this study demonstrated with independent
experiments that the fungus is preferentially located on
the seed coat. This observation reinforces the hypothesis
that spores or mycelium of M. oryzae do not easily enter
seed tissues of rice. Rice blast occurred at the embryonic
end of rice seeds, as previously reported by Chung &
Lee (1983), Manandhar et al. (1998) and Long et al.
(2001). In this way, the rice blast fungus gets easy access
to the coleoptile emerging from the seed as well as to the
primary root, and consequently colonizes the first leaf
and the secondary roots. However, the present study
noted that M. oryzae was also frequently present on the
awn of Sariceltik, which is particularly long in this variety. From the awn, spores can attach to the growing
coleoptile and first leaf, or mycelium can grow from the
awn to reach and infect the seed.
Leaf blast severity on young rice plants (3/4-leaf stage)
is greater than on older plants (7/8-leaf stage; Hwang
et al., 1987; Roumen et al., 1992). Few studies have
addressed the importance of rice resistance in the early
stages of epidemics in younger plants. In previous
reports, the fungus was not detected on seeds of highly
resistant rice accessions (Guerber & TeBeest, 2006).
Using fluorescent M. oryzae strains enabled the rice blast
progression on infested seeds and seedlings to be followed easily and in detail. In the present work, different
rice cultivars with diverse levels of resistance to the
FR13 strain at an early vegetative stage were used, and it
was observed that this level of resistance or susceptibility
was already established at the developmental stage of the
seedling. Indeed, despite being present on the seeds and
the growing coleoptile, M. oryzae FR13 was not able to
infect Gladio, the most resistant accession of the study.
Gladio harbours at least one resistance gene, Pia, and
FR13 the corresponding avirulence gene (AvrPia;
D. Tharreau, unpublished data). Appressoria were formed
on the coleoptile, but an hypersensitive response as well
as autofluorescence at the plant cell wall were observed.
In contrast, M. oryzae FR13 was able to infect and penetrate into the coleoptile, leaf and root of Sariceltik and
Plant Pathology (2013) 62, 879–887
Rice seed infection by the blast fungus
(a)
(j)
(i)
(b)
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Confocal
(c)
(d)
(e)
(f)
(g)
(h)
seed
(k)
(l)
(m)
(n)
(o)
(p)
Figure 4 Sariceltik seed infection and Maratelli adult plant infection by GFP-transformed Magnaporthe oryzae rice FR13 strain under water seeding.
(a) Mycelium present on the seed forming a coat around the emerging coleoptile within 4 days after sowing (d.a.s.). (b) Infected coleoptile at
9 d.a.s.; inset is a non-infected coleoptile. (c) Magnification of (b) showing the presence of spores (white arrow) and mycelium at the coleoptile
surface. (d) Infected primary leaf at 10 d.a.s. (e) Cellular events in an infected leaf at 13 d.a.s. (f,g) Mycelium present on the seed forming a coat
around the emerging primary root at 6 d.a.s.; spores can also be observed on the root as shown in inset. (h,i) Infected root (h) producing spores (i)
within 13 d.a.s.; inset in (i) shows longitudinal section of the infected root in (h), illustrating the presence of hyphae inside root cells. (j,k) Leaf
necrotic lesion on 2-month-old plants before (j) and after (k) sporulation on moist filter paper overnight; inset in (j) is a macroscopic necrotic lesion
due to rice blast. (l–o) Adjacent 5 mm long cuttings of an infected leaf showing a necrotic lesion (l, circled with broken line) illustrating the mycelium
progression within the leaf from the necrotic lesion as indicated by the arrows in each picture; the end of the progression is shown in (o) as
indicated by the arrowhead. (p) Necrotic lesion of Maratelli 3-month-old leaf under epifluorescence microscope. Bar scale = 5 lm (longitudinal root
section in i), 50 lm (magnification in b and g, c, i) or 100 lm (a,b,d–h,j–p). All pictures were taken by epifluorescence microscopy except (e) and
inset in (i), which were by confocal microscopy.
Maratelli. The fungus carried on its progression within
the vascular tissues of the leaf to colonize neighbouring
tissues. The infection process led to the death of all Sariceltik seedlings, while some of the Maratelli seedlings survived. This difference of infection rate was consistent with
the known susceptibility of these two cultivars at older
stages (Vergne et al., 2010).
The results of these experiments also showed that rice
blast is found in seedlings arising from seeds harvested
from plants which were previously inoculated either at
ripening stage (mature panicles), heading stage (flowering/immature panicles) or even before heading (flag leaf
fully developed). Previous work on rice and M. oryzae
from different parts of the world (USA, Egypt, Nepal)
Plant Pathology (2013) 62, 879–887
reported that seeds can be infested in the field on mature
and flowering/immature panicles (Lamey, 1970; Bernaux
& Berti, 1981; Reddy & Bastawsi, 1989; Manandhar
et al., 1998). For some authors (cited in Guerber &
TeBeest, 2006), M. oryzae could infect rice grains via
three hypothetical routes of entry: (i) spikelet colonization from an infected boot leaf while the panicle is still
enclosed within the boot, (ii) seed infection after panicle
emergence through infected glumes, and (iii) fungus entry
via the hilar region from infected vascular tissues of the
mother plant. In this study, rice plant infection before
anthesis and early before the emergence of the panicle
tip from the flag leaf sheath led to seed contamination
by M. oryzae at a significant rate. These results show
886
O. Faivre-Rampant et al.
that the infected flag leaf contaminated the panicle when
emerging. This is consistent with neck blast symptoms
frequently observed in the field. Sporulating lesions are
often observed on the flag leaf in the field and the panicle stem often shows symptoms at the collar, probably
after infection by spores drained from the flag leaf.
In the present work, some of the germinating infested
seeds died because of blast colonization after a few days
of growth. Thus, two non-exclusive scenarios of rice
blast epidemics in the field can be proposed. The first
scenario is the systemic colonization of the plant from
the seed via the stem vascular system, without symptom
expression (as proposed by Guerber & TeBeest (2006)
and more recently by Marcel et al. (2010)). In this work,
in favourable conditions, blast sporulated on previously
symptomless seedlings, potentially supporting a systemic
colonization of the stem. But, when diseased adult rice
plants coming from symptomless seedlings were cut, no
fungus was detected within the main culm. Moreover,
blast is a hemibiotrophic fungus: an initial biotrophic
infection phase, during which the pathogen spreads in
living rice tissues, is followed by a necrotrophic phase
during which rice cell death is induced (Ou, 1985). Such
a necrotrophic phase makes a symptomless colonization
unlikely and most of the contaminated seeds gave rise to
infected seedlings that rapidly died.
The second scenario is the production of inoculum
from dead seeds or dead seedlings and the contamination
of healthy seedlings by transportation of mycelium or
spores. On dead seeds or seedlings on the soil surface,
spores and mycelium are produced and transported
through the irrigation water to neighbouring rice plants
and constitute a possible source of inoculum. This scenario requires that, in irrigated conditions, the fungus
grows under water. According to previous studies, blast
infection was never detected on seedlings raised under
water seeding conditions (Lamey, 1970; Manandhar
et al., 1998). However, in this study contaminated seed
lots placed within water resulted in diseased seedlings.
Although M. oryzae progression was slower than in aerobic conditions, it was present as mycelium or spores on
roots and coleoptiles/leaves. Therefore, it seems that M.
oryzae can survive, sporulate and infect the seedlings
under water and that dead seeds or seedlings produce
spores that contaminate healthy plants in irrigated conditions. In the field, rice plants are usually densely sown in
a very small area and disease can spread rapidly, resulting
in infection of all neighbouring rice plants (if very susceptible) or a proportion of them (if partially susceptible).
Long et al. (2001) showed that M. oryzae could sporulate for several weeks on inoculated seeds, previously
autoclaved, when placed on the soil surface and could
initiate epidemics in the field. In the same way, Guerber
& TeBeest (2006) found rice blast on seedlings from
planted or non-germinated seeds and seed coats on the
soil surface. Long et al. (2001) also indicated that the
development of symptoms was more correlated to inoculum time exposure than to plant age. This means that if
the inoculum is already present at the beginning of rice
growth, non-infected plants will have more chance to
become infected later; this was observed in the present
experiments, with symptom development between 2 to
3 months of cultivation. Therefore, a systemic invasion
of the stem is not required for blast epidemics to develop
from contaminated seeds and for infection of the panicle.
Marcel et al. (2010) proposed an alternative way of
overwintering. They presumed that M. oryzae is conserved in the soil as resting structures (microsclerotia) or
on rice residues. They speculated that the fungus could
enter via the root tissue and spread thereafter in a symptomless mode; this would allow the fungus to continuously feed on healthy rice tissue until reaching the aerial
parts for sporulating on leaves. This scenario seems unlikely because no resting structures have been described so
far for M. oryzae. Mycelium survival in the soil is also
unlikely because of competition with other microorganisms. In addition, no cytological proof of a systemic presence of M. oryzae in entire rice seedlings has been
reported until now.
Taken together, the present results and previous results
allow reconstruction of the rice blast disease cycle on
irrigated rice in temperate regions. Grains are infected by
sporulating lesions on the flag leaf probably during heading. The fungus survives on harvested seeds until the
next growing season. When seeded under water, infected
seeds are colonized by the fungus that, most of the time,
kills the seedling. Spores on dead seedlings then serve as
primary inoculum to infect healthy plantlets. Lesions on
leaves produce spores that will infect new leaves, and so
on until the infection of the flag leaf. In this cycle, sowing non-contaminated seeds appears to be the main way
to control the disease. Seed treatments have been shown
to reduce blast epidemics in temperate regions, supporting the idea that infested seeds are the source of primary
inoculum (Ou, 1985).
Acknowledgements
The authors thank Ane Sesma (JIC, Norwich, UK) for
kindly providing the FR13-GFP Magnaporthe strain,
Marc-Henri Lebrun (CNRS-Bayer, Lyon, France) for giving the plasmid pfC2ORFGFP, and Cécile Ribot and
Jean-Benoı̂t Morel (INRA, Montpellier, France) for supplying the CL3.6.7-GFP isolate. Part of this work was
supported by a grant from FranceAgrimer. The authors
are grateful to Jean-Benoı̂t Morel, Mathilde Sester and
Enrico Gobbato for critical reading of the manuscript.
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Supporting Information
Additional Supporting Information may be found in the online version of
this article.
Figure S1 Phenotypes of plant–pathogen interactions between rice
(temperate japonica) and Magnaporthe oryzae (strain FR13). Disease
symptoms were observed 7 days post-inoculation on leaves of 2-weekold plants (3/4-leaf stage) of the rice cultivars Gladio, Maratelli and
Sariceltik.
Figure S2 (a) Infection rates of Sariceltik (SK) and Maratelli full grains
and empty seeds as well as panicle secondary branches by Magnaporthe
oryzae strain CL3.6.7 harvested from plants coming from symptomless
infected seeds. Three independent sowings were carried out for each lot
and the mean with standard error was calculated. (b) Infection of Sariceltik full grain (top left), awn (top right) and secondary branch (bottom
left) of (a).