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Male fertility in Pyricularia oryzae: microconidia are spermatia.
Authors
Alexandre Lassagne a,d, Sylvain Brun b, Fabienne Malagnac c, Henri Adreit a, Joëlle Milazzo a, Elisabeth
Fournier d and Didier Tharreau a
Affiliations
a. Plant Health Institute of Montpellier (PHIM), CIRAD, TA A120/K, 34398 Montpellier, France
b. Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France
c. Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 91118 gif-
sur-Yvette, France
d. Plant Health Institute of Montpellier (PHIM), University of Montpellier, CIRAD, INRAE, IRD,
Montpellier SupAgro, 34398 Montpellier, France
Originality and significance
This work is the first proof of the role of microconidia as male fertilizer element in Pyricularia oryzae
and improve comprehension of the sexual cycle of this phytopathogenic fungus. Furthermore, this study
is the first step in the understanding in the evolutionary process leading to a loss of fertility in this model
fungus.
Summary
Sexual reproduction in Ascomycetes is well described in several model organisms such as Neurospora
crassa or Podospora anserina. Deciphering the biological process of sexual reproduction (from the
recognition between compatible partners to the formation of zygote) can be a major advantage to better
control sexually-reproducing pathogenic fungi. In Pyricularia oryzae, the fungal pathogen causing blast
diseases on several Poaceae species, the biology of sexual reproduction remains poorly documented.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/1462-2920.16226
This article is protected by copyright. All rights reserved.
Besides the well-documented production of asexual macroconidia, the production of microconidia was
seldom reported in P. oryzae, and their role as male gamete (i.e. spermatia) and in male fertility has
never been explored. Here we characterized the morphological features of microconidia and demonstrate
that they are bona fide spermatia. Contrary to macroconidia, microconidia are not able to germinate and
seem to be the only male gametes in P. oryzae. We show that fruiting body (perithecium) formation
requires microconidia to get in contact with mycelium of strains of opposite mating type, to presumably
fertilize the female gametes.
Keywords: Pyricularia oryzae, male fertility, microconidia, sexual reproduction
Introduction
The reproductive system influences the apparition and the evolution of adaptive variants in response to
selective pressures. Asexual reproduction induces clonality which allows an adapted genotype to spread
quickly in a homogeneous environment. By contrast, sexual reproduction induces recombination which
generates new genotypic combinations that can be advantageous in heterogeneous environments (de
Meeûs et al., 2007; Stukenbrock and McDonald, 2008). Recombination also permits to slow down the
accumulation of deleterious mutations (Bruggeman et al., 2003). Some eukaryote taxa like Ascomycete
fungi exhibit a wide variety of reproductive systems, from strictly clonal to strictly sexual species,
including numerous species that alternate both (Billiard et al., 2012). To understand how reproduction
systems and genes involved in these processes evolved in Ascomycete fungi, a prerequisite is to
document how sexual reproduction takes place and what the biological determinants of fertility are.
Sexual reproduction implies the succession of haploid, dikaryotic and diploid phases implemented
during meiosis and syngamy (Billiard et al., 2012). In Ascomycete fungi, the reproduction cycle starts
by the encounter of a male gamete (either contained in antheridia, or present as individualized
specialized cells called spermatia) with a female differentiated gamete (the ascogonium), a step called
fertilization. The ascogonium is a multinucleate haploid cell produced by mitosis (Billiard et al., 2012)
which differentiates specialized hyphae called trichogynes (Debuchy et al., 2010). Hormonal attraction
directs the growth of trichogynes towards spermatia (Bistis, 1996). This hormonal chemotropism is
controlled by the mating type genes on a pheromone/receptor recognition basis and occurs only between
cells of opposite mating type in heterothallic species (Bistis, 1983). The contact between the trichogyne
and the spermatia triggers cell fusion, i.e. plasmogamy, after which the spermatia nucleus migrates
across the trichogyne into the ascogonium (Brun et al., 2021). Plasmogamy is controlled by mating type
genes (Bistis, 1996; Peraza-Reyes and Malagnac, 2016). Plasmogamy is followed by syngamy, meiosis
and differentiation of the ascus that will contain the ascospores produced after meiosis. Asci are
contained in fruiting bodies (perithecia in P. oryzae), which are composed of an envelope of maternal
origin that shelters the ascogonium. Hence, sexual reproduction in heterothallic Ascomycetes is an
intricate process which requires, on the one hand, mating type compatibility between partners, and on
the other hand, female and male fertile gametes.
Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus of Pyriculariaceae
(Klaubauf et al., 2014), causing blast disease on many cultivated cereals (rice, wheat, maize, millets…)
and others Poaceae (Couch et al., 2005; Gladieux et al., 2018a). This fungus is responsible for the rice
blast disease causing the loss of 3% of global rice yield (Savary et al., 2019). The genetic lineage of P.
oryzae pathogenic on rice likely emerged in South Asia, close to the Himalaya (Zeigler, 1998; Tharreau
et al., 2009; Saleh et al., 2014; Gladieux et al., 2018b) and is reported in more than 80 countries across
the world (Boddy, 2016). The asexual mode of reproduction, through asexual spores (macroconidia) or
mycelium multiplication, is commonly observed on rice host and most of the pathogen populations are
clonal (Zeigler, 1998). Although sexual reproduction of P. oryzae has never been observed in the field
on any host, evidences from biology, genetic and genomic studies of populations from rice suggest that
sexual reproduction took place or is still taking place in limited areas in the putative center of origin
(Saleh et al., 2012b; Gladieux et al., 2018b; Thierry et al., 2021). Pyricularia oryzae is a heterothallic
species with two idiomorphs at the mating type locus named Mat1.1 and Mat1.2 (Kanamori et al., 2007).
Sexual reproduction is easy to complete in vitro in this species. Formation of perithecia is easily
observed in laboratory by crossing fertile strains of opposite mating types, on artificial medium
(Notteghem and Silue, 1992). However, only some strains are able to form perithecia, and are then
defined as female fertile. Similarly, male fertility has so far been defined as the capacity to induce
perithecia formation in a female fertile strain of opposite mating type (Saleh et al., 2012a). However,
the mechanisms of fertilization have not yet been described. By analogy with other Ascomycetes,
microconidia have been supposed to be the male fertilizing elements (Fukumori et al., 2004).
Microconidia were observed for the first time by Kato et al. (1994). Microconidia as well as specialized
hyphae bearing them (phialides) were also observed by Chuma et al. (2009) and more recently by Zhang
et al. (2014). But the role of microconidia in fertilization was not demonstrated yet. Here we demonstrate
that microconidia are fertilizing cells, i.e. spermatia in P. oryzae. We propose a new and direct biological
definition of male fertility in P. oryzae, independent of the stimulation by a female fertile strain.
Experimental procedures
Biological materials
We used 12 Pyricularia oryzae strains isolated from six different host plants and collected in seven
different countries (Table 1). Strains of both mating types (Mat1.1 and Mat1.2) were used to measure
the production of microconidia. The strains CH0997 (Mat1.2) and CH0999 (Mat1.1) (Saleh et al.,
2012a), used for the fertilization and mating assays, were collected on Oryza sativa in China in 2008
and belong to lineage 1 (Gladieux et al., 2018b; Latorre et al., 2020; Thierry et al., 2021). All strains
were stored at -20°C on dried filter paper (Valent et al., 1986).
Growing media
Rice flour agar (RFA) medium was prepared with 20 g of organic rice flour, 15 g of Bacto agar, 2 g of
Yeast Extract (BactoTM) in 1 L water with 500,000 units of penicillin G added after autoclaving for 20
min at 120°C. Fresh homemade potato dextrose broth (PDB) was prepared with 200 g of sliced organic
potatoes boiled during 30 minutes in 800 mL sterile water. The preparation was filtrated through multi-
layer gauze. The filtrated solution was added with 20 g of glucose and replenished to 1 L.
Microconidia production
Microconidia were produced following a protocol modified from Zhang et al. (2014). Two circular plugs
of 5 mm diameter of actively growing mycelium on RFA medium were used to inoculate 40 mL of fresh
homemade PDB. The liquid culture was incubated during 3 days at 25°C then 6 days at 20°C with
permanent shaking at 150 rpm, and then filtrated on Miracloth (EMD Millipore Corp., 475855-1R) to
remove mycelium fragments, and centrifuged at 4500 g for 10 minutes. The pelleted microconidia were
re-suspended in 1 mL sterile distilled water. When microconidia production was too low, after filtration
on Miracloth, the liquid culture was filtrated on 0.45 µm and microconidia were collected from the filter
surface by washing the filter in 3 mL sterile water. To evaluate the capacity of each studied strain to
produce microconidia, two independent replicates were performed and microconidia were counted three
times for each replicate using a Malassez cell of 5 mm² and a limit threshold of 4000 units per mL.
Staining and microscopic observation
For staining of cell wall, a droplet of Uvitex 2B (biovalley, 19517-10) fluorescent dye was added directly
in the suspension of macroconidia or microconidia on a microscopic slide and incubated in the dark for
15 minutes. The preparation was observed under photonic microscope Nikon ECLIPSE Ni-E with 390
nm GFP excitation filter. Ascogonia were observed with an inverted microscope Zeiss axio observer
Z.1 (objective 63 X Plan Apo ON 1.4 Oil DIC) and photographed with an ORCA Flash4 LT CMOS
camera (Hamamatsu). Images were analyzed with the software Fiji (Schindelin et al., 2012).
Mating assays
Crosses between strains of opposite mating type were performed as described by Nottéghem and Silué
(1992) on RFA medium in 90 mm Petri dishes. Plugs of mycelia grown on RFA at 25°C for one week
were deposited on RFA medium following the design described in Saleh et al. (2012a). Cultures were
incubated under continuous light for two days at 25° C and then at 20°C. The reference strains CH0997
and CH0999 are known to be female-fertile when crossed with a broad number of genetically diverse
strains (Thierry et al., 2021). The production of mature perithecia was evaluated 21 days after
inoculation to determine the fertility phenotype according to Saleh et al. (2012b): the formation of two
rows of perithecia between a tested strain and a reference strain of opposite mating type indicated that
both strains were male-fertile and female-fertile (Figure 1A); the observation of a single row indicated
that only the reference strain produced perithecia, and that the tested strain was female sterile but male-
fertile (Figure 1B); the absence of perithecia indicated that the tested strain was both female-sterile and
male-sterile (Figure 1C). The cross CH0997 x CH0999 was used as positive control, the crosses CH0997
x CH0997 and CH0999 x CH0999 were used as negative controls.
Fertilization assays
To assess the fertilization capacity of microconidia, we first grew female fertile strains on RFA medium
in 90 mm diameter Petri dishes during 3 days at 25°C and 6 days at 20°C. The two reference strains
CH0997 (Mat1.2) and CH0999 (Mat1.1) were chosen as “female” thalli in this experiment. After
screening different strains, OG0002 (Mat1.1) was chosen as “male” thallus in this experiment for its
capacity to produce high quantities of microconidia. We prepared microconidia suspensions of OG0002
following the protocol described above, and adjusted them to 11 different concentrations: 400, 800,
1600, 3200, 4000, 6000, 8000, 12000 and 24000 microconidia.mL-1. For each suspension, 250 µL were
sprayed on the mycelium of CH0997 and CH0999 strains. The inoculated plates were then cultured for
3 more days at 20°C before observation of perithecia formation. Two replicates were performed for each
combination of female strain x concentration of microconidia suspensions.
The fertilization capacity of macroconidia was tested in a similar way, using the same strains grown as
described above. Macroconidia of strain OG0002 were harvested on aerial mycelium from cultures
grown on RFA medium during 2 weeks at 25°C. To release macroconidia from the mycelium, cultures
were gently scrapped after addition of 4 mL distilled water. Macroconidia suspensions were then
filtrated on a 40 µm filter to eliminate mycelium fragments. Female fertile strains CH0997 and CH0999
were inoculated with 250 µL of suspensions at 9.00x1004 macroconidia.mL-1 of OG0002.
We also tested the fertilization capacity of mycelium using the same strains as above (i.e. OG0002 as
“male”, and CH0997 and CH0999 as “female”). To produce mycelium fragments of OG0002, 40 mL of
commercial PDB (DifcoTM ) were inoculated with two plugs of 5 mm diameter of mycelium actively
growing on RFA medium. We observed that, in our conditions, microconidia are not produced in
commercial PDB. The liquid culture was incubated with a permanent shaking at 150 rpm during 3 days
at 25°C and then during 6 days at 20°C. The mycelium was then centrifuged at 4500 g for 10 minutes
and resuspended in 4 mL sterile water. Mycelium was sonicated during 1 minute at 12 W and 20 Hertz
to obtain fragments. The female fertile strains CH0997 and CH0999, grown as described above, were
then inoculated with 250 µL of mycelium fragment suspensions. The same volume of suspensions of
mycelium fragments were also inoculated on two plates of RFA medium to check their viability.
Quantification of perithecia on fertilized cultures
After three days of incubation, pictures of plates of fertilization assays were taken with a camera. The
proportion of the surface occupied by perithecia formed on plates was evaluated by image analysis with
the software IPSDK Explorer developed by Reactiv’IP (Grenoble, France). This software uses machine
learning techniques to classify pixels. The software was trained to classify pixels in three classes:
background, mycelium and perithecia (model in supplementary materials). Due to the difficulty to
precisely count individual perithecia, we quantified the number of pixels assigned to the “perithecia”
class. We then calculated the proportion of surface occupied by perithecia as the ratio of the number of
pixels assigned to the “perithecia” class on the number of pixels assigned to the “mycelium” +
“perithecia” classes. We used the “drc” R package (Ritz et al., 2015) to fit a log-logistic model with
lowest value fixed to 0 and three parameters (b, d, e) as in the following equation:
()=
[1+exp(∗(log ()−log())]
Time frame assay for production of fertilization competent female gametes
To determine the minimum time required for the female thallus to become competent for fertilization,
20 µL of a suspension of OG0002 microconidia of opposite mating type (1×106 /mL) were poured on
mycelial cultures of the female strain CH0997 of eight different ages (from three to ten days). Eight
plates of CH0997 were cultured on RFA medium at 25°C for two to nine days, respectively, before
being placed at 20°C one day before the inoculation with microconidia of OG0002. Perithecia formation
was assessed every day from the 10th to the 17th day of the experiment. The presence of perithecia was
assessed based on the observation of fungal structures with the morphological characteristics of
perithecia.
Results
Production of microconidia by P. oryzae strains
Ten out of the twelvestudied strains produced microconidia in fresh home-made PDB (Table 1). All ten
strains inducing the formation of perithecia in classical in vitro tests produced from 2.07×104 to 1.10×109
(significant effect of the strain tested with a nested ANOVA: F=1021.7, P=2×10-16, Df=11) microconidia
(Figure 2). A significant difference in microconidia production was also observed when considering
only the five strains isolated from the same host, rice (nested ANOVA: F=232.57, P= 2×10-16, Df=4).
The two strains producing the highest number of microconidia were OG0002 and OG0003, with
1.46×108 and 1.10×109 microconidia.mL-1, respectively, that is 100 to 10 000 times more than the other
producing strains. Based on its high microconidia production, OG0002 was chosen as “male” fertilizing
strain in the following assays. The number of microconidia produced was consistent between the two
biological replicates of the same strain, except for TH0012. Although microconidia were not observed
for strains CH0052 and BF0026 (Table 1), these strains induced formation of a low quantity of perithecia
on female reference strain. Furthermore, CH0052 and BF0026 induced perithecia formation with only
one of the two female reference strains (Figure 3). As expected from previous reports, microconidia
were produced regardless of the mating type of the strains, with four strains of each mating type
producing microconidia and one strain of each mating type not producing microconidia. It is important
to note that microconidia were obtained from cultures of single strains, confirming that the stimulation
by another strain is not necessary to induce their production.
Morphological characteristics of microconidia
The mean length of OG0002 microconidia was 7.36 µm (n=62, standard deviation=1.15). Hence
microconidia are smaller than macroconidia whose dimensions are 16–33 µm long and 6–13 µm wide
(Ou, 1985; Biju-Duval, 1994; Chuma et al., 2009). Microconidia are also clearly distinguishable from
macroconidia in their structure and shape (Figure 4): they are single-celled and crescent-shaped (Figure
4 B, D) whereas macroconidia are three-celled and pyriform with a characteristic light grey pigmentation
(Figure 4 A, C). Microconidia have a hyaline aspect (Figure 4 B), which makes them uneasy to observe
under microscope at low magnification and on cultures on solid medium. Both types of conidia have a
chitinous cell wall evidenced by Uvitex staining (Figure 4 C, D). Microconidia are worn by specialized
hyphae called phialides (Figure 5).
Fertilization assays
Spraying of pure suspension of microconidia of strain OG0002 (Mat1.1) on a “female” strain of opposite
mating type previously cultured during nine days induced the formation of perithecia (Figure 6).
Perithecia were formed on cultures of CH0997 (Mat1.2) for all concentrations of sprayed suspensions
of microconidia in both replicates. Perithecia were observed three days after spraying. No perithecium
was formed on cultures of CH0997 either sprayed with sterile water or unsprayed. As expected, no
perithecia was formed by CH0999 (Mat1.1) after spraying with OG0002 microconidia, since both strains
were of the same mating type.
We observed a significant positive relationship between the number of microconidia sprayed on the
“female” thalli and a proxy of the number of perithecia (i.e. the proportion of surface occupied by
perithecia on the fertilized culture). The relationship between the number of microconidia sprayed and
the proportion of surface occupied by perithecia was explained by a log-logistic model with minimum
value fixed at 0 and three parameters (b: slope, d: plateau, and e: ED50), with b=-1.59×10-3, d=0.39 and
e=2670 (Figure 7). A plateau seems to be reached around 0.39 corresponding to 39% of pixels assigned
to perithecia.
Unlike for microconidia spraying, no perithecia was formed three days after spraying with macroconidia
or with mycelium fragments of OG0002 on CH0997. However, perithecia started to appear two weeks
after spraying mycelium fragments or macroconidia. As expected, since they have the same mating type,
no perithecia was formed at all after spraying with macroconidia or with mycelium fragments of
OG0002 on CH0999.
Production of fertilization competent female gametes
Ascogonia were observed on female fertile strain thalli aged of 11 days (Figure 8). To determine a
putative minimum age for female receptivity, microconidia suspensions were sprayed on cultures of the
female fertile strain CH0997 from 4 to 11 days after the starting of the culture. When the female fertile
strain was grown during seven days or more on RFA medium before inoculation (i.e. six days or more
at 25°C and one day at 20°C), the formation of perithecia in those conditions occurred six days after
spraying microconidia suspension. For younger cultures, i.e. when the female fertile strain was grown
six days or less on RFA medium before inoculation (i.e. five days or less at 25°C and one day at 20°C),
it took more than six days for perithecia to be formed (seven to ten days for cultures aged of six to three
days, respectively; Figure 9). Hence, the age of the female thallus influences the ability to form
perithecia.
Discussion
The first aim of our study was to confirm that P. oryzae from different origins were capable of producing
microconidia. The other aim was to determine whether, in P. oryzae life cycle, microconidia could play
the role of spermatia, i.e. the biological elements inducing fertilization and perithecia formation on
compatible and competent strains.
Among the three previous studies describing microconidia, Chuma et al. (2009) and Zhang et al. (2014)
focused on a single strain (G10-1 and 70-15, respectively), whereas Kato et al. (1994) tested 45 strains
from different host plants, without quantifying microconidia production. In our experiments, ten out of
the 12 studied strains produced microconidia in quantities varying from 2.07 ×104 to 1.10 ×109 per mL.
It is worth to note that strains from both mating types were capable of producing microconidia.
Furthermore, the capacity of producing microconidia did not seem to depend on the original host of the
strain either, since strains collected from Hordeum vulgare, Eleusine coracana or Oryza sativa produced
microconidia. Morphological observations of P. oryzae microconidia confirmed previous studies (Kato
et al., 1994; Chuma et al., 2009): they are crescent-shaped hyaline and are two to four times smaller
than macroconidia, which makes their observation harder and could explain why they were so scarcely
reported in the literature.
Fertilizing cells in Ascomycetes can be either unspecialized cells like macroconidia or mycelial
fragments, or specialized cells, i.e. microconidia (Pöggeler et al., 2018). Microconidia were proved to
be spermatia in several Ascomycete species, including Botrytis cinerea (Fukumori et al., 2004) and
Neurospora crassa (Maheshwari, 1999). In these two species, however, microconidia designate the male
gametes, are able to germinate, and are distinct from macroconidia (often called conidia) which are
asexual spores but can also be fertilizing elements (Brun et al., 2021); hence both microconidia and
macroconidia are spermatia in B. cinerea and N. crassa.
In P. oryzae, the role of microconidia as spermatia is commonly accepted, but this had never been
demonstrated so far (Chuma et al., 2009; Zhang et al., 2014). Here we demonstrate that in P. oryzae,
microconidia are the only fertilizing elements, hence that they are spermatia. Contrarily to macroconidia
and mycelium fragments, only microconidia sprayed on a compatible female fertile strain led to the
formation of perithecia after only three days. In addition, the number of perithecia formed was positively
correlated to the number of microconidia sprayed, confirming that microconidia have the capacity to
induce perithecia formation. Reaching a plateau of perithecium formation despite growing numbers of
sprayed spermatia suggests that the number of female gametes competent for fertilization could be the
limiting parameter. Although no microconidia were observed for two strains (CH0052 and BF0056),
they induced the formation of perithecia on female fertile strains of the opposite mating type. We
hypothesized that the two strains produce quantities of microconidia that are below the detection
threshold of the counting method, ie 4000 microconidia per mL. The apparition of perithecia after
spraying macroconidia or mycelium fragments of the male strains on a female compatible strain was
clearly delayed (15 days compared to 3 days after spraying microconidia). We hypothesized that the
female strain has been fertilized by microconidia produced de novo by new thalli resulting from the
growth of mycelium fragments or macroconidia germination. However, we cannot exclude that a yet
unknown alternative way of fertilization may take place when macroconidia or mycelium fragments are
sprayed.
We observed that the age of the female thallus influenced its ability to produce perithecia when
inoculated with spermatia of a compatible strain (Figure 9). In our conditions, a minimum age of seven
days of culture is required for female thalli to be able to produce perithecia when sprayed with spermatia.
This minimum age could correspond to the time needed for female gametes to differentiate. In P.
anserina, male and female gametes start to differentiate after three days of growth on minimal medium
at optimal temperature (Coppin et al., 1997, Silar 2011). In P. oryzae, other parameters such as
temperature or nutrient supplies might probably also play a role on the time needed for female thalli to
become competent.
Before the present study, microconidia had never been observed in our laboratory conditions despite
successful in vitro crossing between strains. One explanation could be the low quantity of microconidia
produced on RFA medium (the medium routinely used for growing strains and performing in vitro
crosses; Nottéghem and Silué 1992). Here we used the modified protocol of Zhang et al. (2014) to obtain
high quantity of microconidia. Sporulation is a highly variable developmental process subjected to many
environmental parameters. In Neurospora crassa, several external factors such as humidity, temperature
or starvation have an effect on male gamete production (Debuchy et al., 2010; Maheshwari, 1999). For
instance, a low concentration of carbon source and a shift from high to low temperature also facilitates
production of microconidia in N. crassa (Ebbole and Sachs, 1990; Rossier et al., 1977). The production
of microconidia in P. oyyzae might also rely on specific conditions, which might be related to the fact
that sexual reproduction was seldom observed in nature.
The ten strains that produced microconidia in our conditions showed significant differences in the
quantity of microconidia produced. The strains OG0002 and OG0003, that were isolated from Eleusine
corocana in the same field, produced more microconidia than other. Confirming the difference of
production of microconidia between strains from different host specific groups will require additional
studies. However, differences within host specific groups of P. oryzae are also worth documented. For
example, in this study, we observed significant quantitative differences in the production of
microconidia between strains isolated from rice. Male fertility likely depends not only on the original
host but also on the reproduction mode of the populations or genetic groups sampled. If spermatia have
no other role than fertilization, their production is expected to decrease and to be lost in populations that
are no longer experiencing sexual reproduction. Pyricularia oryzae populations from rice allow to test
this hypothesis and to investigate the heritability and genetic bases of microconidia production.
The demonstration of the role of microconidia as spermatia allows to reconsider the definition of male
fertility in P. oryzae. Until now, male fertility was only defined as the capacity of a strain to induce
perithecia formation on a strain of opposite mating type in a cross (Notteghem, 1992; Saleh et al.,
2012a). There are several limitations to this initial definition. The use of a female auxiliary strain is
necessary to assess male fertility, which introduce an additional variable parameter. A second limitation,
is the potential existence of pre-zygotic barriers. Here, we define male fertility of P. oryzae strains as
the ability of a strain to produce male gametes (i.e. microconidia), and without crossing with a reference
strain. Counting the spermatia produced will in addition permit a quantitative phenotyping of male
fertility.
Acknowledgments
We want to thank the ImagoSeine facility, member of the France BioImaging infrastructure supported
by the French National Research Agency (ANR-10-INSB-04, « Investments for the future »). AL was
supported by grants of INRAE and CIRAD
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Accepted Article
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Accepted Article
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Accepted Article
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Accepted Article
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Strain
Synonymous
Reference
Genetic information
Mating
type
Isolated in
Isolated from
Lineage (rice
strains only)
Mating assays
Microconidia production.mL-1
Male
Fertility
Female
Fertility
Replicate 1
Replicate 2
Mean
Replicates
US0068
70-15
Dean et al. 2005
INSDC Assembly GCA_000002495.2
Mat1.1
USA
Hybrid lab strain
(pathogenic to rice)
1
Fertile
Fertile
2,36E+06
1,53E+06
1,94E+06
OG0002
UG771511,
KA3
Kato et al. 2000
Mat1.1
Ouganda
Eleusine coracana
Fertile
Fertile
8,57E+07
2,05E+08
1,46E+08
OG0003
UG771711,
KA4
Tanaka et al. 2009
Mat1.1
Ouganda
Eleusine coracana
Fertile
Fertile
7,03E+08
1,51E+09
1,10E+09
GY0006
This article
Mat1.2
French
Guyana
Oryza sativa
Fertile
Fertile
2,80E+04
1,73E+04
2,27E+04
GY0011
GUY11
Leung et al. 1988
NCBI database : ASM236848v1 (GY11 PacBio
Bao 2016)
Mat1.2
French
Guyana
Oryza sativa
1
Fertile
Fertile
1,04E+06
1,06E+06
1,05E+06
CD0141
This article
Mat1.2
Ivory
Coast
Leersia hexandre
Fertile
ND
2,00E+04
2,13E+04
2,07E+04
TH0012
Gladieux et al. 2018a
ENA repository : PRJEB8341 (TH12 GEMO)
Mat1.1
Thailand
Hordeum vulgare
1
Fertile
Fertile
6,13E+04
2,69E+06
1,38E+06
TH0016
Gladieux et al. 2018a
ENA repository : PRJEB8341 (TH16 GEMO)
Mat1.2
Thailand
Hordeum vulgare
1
Fertile
Fertile
3,60E+04
4,40E+04
4,00E+04
CH0997
Saleh et al. 2012
Mat1.2
China
Oryza sativa
1
Fertile
Fertile
5,33E+04
4,80E+04
5,07E+04
CH0999
Saleh et al. 2012
Mat1.1
China
Oryza sativa
1
Fertile
Fertile
4,52E+05
5,49E+05
5,00E+05
BF0026
This article
Mat1.2
Burkina
Faso
Eleusine indica
Fertile
Fertile
0,00E+00
0,00E+00
0,00E+00
CH0052
Gladieux et al. 2018a
Mat1.1
China
Oryza sativa
2
Fertile
Sterile
0,00E+00
0,00E+00
0,00E+00
Accepted Article
This article is protected by copyright. All rights reserved.
