Asexual reproduction induces a rapid and permanent loss of sexual reproduction capacity in the rice fungal pathogen Magnaporthe oryzae: results of in vitro experimental evolution assays

<p>Abstract</p> <p>Background</p> <p>Sexual reproduction is common in eukaryotic microorganisms, with few species reproducing exclusively asexually. However, in some organisms, such as fungi, asexual reproduction alternates with episodic sexual reproduction events. Fungi are thus appropriate organisms for studies of the reasons for the selection of sexuality or clonality and of the mechanisms underlying this selection. <it>Magnaporthe oryzae</it>, an Ascomycete causing blast disease on rice, reproduces mostly asexually <it>in natura</it>. Sexual reproduction is possible <it>in vitro </it>and requires (i) two strains of opposite mating types including (ii) at least one female-fertile strain (<it>i.e</it>. a strain able to produce perithecia, the female organs in which meiosis occurs). Female-fertile strains are found only in limited areas of Asia, in which evidence for contemporary recombination has recently been obtained. We induced the forced evolution of four Chinese female-fertile strains <it>in vitro </it>by the weekly transfer of asexual spores (conidia) between Petri dishes. We aimed to determine whether female fertility was rapidly lost in the absence of sexual reproduction and whether this loss was controlled genetically or epigenetically.</p> <p>Results</p> <p>All the strains became female-sterile after 10 to 19 rounds of selection under asexual conditions. As no single-spore isolation was carried out, the observed decrease in the production of perithecia reflected the emergence and the invasion of female-sterile mutants. The female-sterile phenotype segregated in the offspring of crosses between female-sterile evolved strains and female-fertile wild-type strains. This segregation was maintained in the second generation in backcrosses. Female-sterile evolved strains were subjected to several stresses, but none induced the restoration of female fertility. This loss of fertility was therefore probably due to genetic rather than epigenetic mechanisms. In competition experiments, female-sterile mutants produced similar numbers of viable conidia to wild-type strains, but released them more efficiently. This advantage may account for the invasion of our populations by female-sterile mutants.</p> <p>Conclusions</p> <p>We show for the first time that, in the absence of sexual reproduction, female-sterile mutants of <it>M. oryzae </it>rice strains can arise and increase in abundance in asexual generations. This change in phenotype was frequent and probably caused by mutation. These results suggest that female fertility may have been lost rapidly during the dispersion of the fungus from Asia to the rest of the world.</p

The variety mixture strategy assessed in a G × G experiment with rice and the blast fungus Magnaporthe oryzae

Frequent and devastating epidemics of parasites are one of the major issues encountered by modern agriculture. To manage the impact of pathogens, resistant plant varieties have been selected. However, resistances are overcome by parasites requiring the use of pesticides and causing new economical and food safety issues. A promising strategy to maintain the epidemic at a low level and hamper pathogen's adaptation to varietal resistance is the use of mixtures of varieties such that the mix will form a heterogeneous environment for the parasite. A way to find the good combination of varieties that will actually constitute a heterogeneous environment for pathogens is to look for genotype × genotype (G × G) interactions between pathogens and plant varieties. A pattern in which pathogens have a high fitness on one variety and a poor fitness on other varieties guarantees the efficiency of the mixture strategy. In the present article, we inoculated 18 different genotypes of the fungus Magnaporthe oryzae on three rice plant varieties showing different levels of partial resistance in order to find a variety combination compatible with the requirements of the variety mixture strategy, i.e., showing appropriate G × G interactions. We estimated the success of each plant-fungus interaction by measuring fungal fitness and three fungal life history traits: infection success, within-host growth, sporulation capacity. Our results show the existence of G × G interactions between the two varieties Ariete and CO39 on all measured traits and fungal fitness. We also observed that these varieties have different resistance mechanisms; Ariete is good at controlling infection success of the parasite but is not able to control its growth when inside the leaf, while CO39 shows the opposite pattern. We also found that Maratelli's resistance has been eroded. Finally, correlation analyses demonstrated that not all infectious traits are positively correlated. (Résumé d'auteur

Microsatellite markers for population studies of the rice blast fungus, Magnaporthe grisea

International audienceWe developed nine new microsatellite markers for rice blast (Magnaporthe grisea) population studies. These markers were used in addition to nine microsatellite markers previously developed by our group for mapping purpose. Altogether, the 18 markers were used in multiplex PCR (polymerase chain reaction) to characterize six populations from different geographical origins. The average number of alleles per locus across populations ranged from 1.2 to 7 and the total number of alleles detected from 2 to 19. Based on this large range of polymorphism, this set of markers is expected to be useful for different kind of population studies at different geographical scales

First Report of Rice Brown Spot Caused by Exserohilum rostratum in Mali

International audienceRice brown spot is an emerging disease of concern in many rice-growing countries. Different fungal species of the genera Bipolaris and Exserohilum were reported as the causal agents of this disease. These fungal pathogens cause similar necrotic lesions on leaves and infect grains with a significant effect on seed germination. In 2018, samples of rice seed and leaves with typical brown spot symptoms were collected from irrigated (Manikoura and Niono) and lowlands (M’pegnesso and Loulouni) rice fields in Mali and incubated for 5 to 7 days on wet filter paper at 25°C with 12 h photoperiod. Conidia observed under microscope were straight or slightly curved and light-brown or dark. They were also rostrate or obclavate and measured 31.4 to 275.6 x 7.3 to 18 µm (n=40). These morphological characteristics are identical to those of Exserohilum rostratum (Hernández-Restrepo et al. 2018). DNA from eight single-spored isolates was extracted by a CTAB-based protocol (Doyle and Doyle, 1987). Internal transcribed spacer (ITS) rDNA region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1 alpha (TEF1-α) genes were amplified by PCR with the primers ITS5/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999) and EF1 983/EF1 2218 (Rehner et al. 2005), respectively. The amplicons were sequenced and deposited in NCBI GenBank. Sequence similarity between Malian strain was 100% for ITS and GAPDH, and 99.8-100% for TEF1. Sequence similarity between Malian strains and reference E. rostratum sequences BRIP 11417 (GenBank acc. no. LT837836, LT882553 and LT896656) and CBS 128061 (GenBank acc. no. KT265240, LT715900 and LT896658) were 99.6-100%. The maximum-likelihood phylogenetic tree generated with ITS, GAPDH and TEF1-α concatenated sequences, using MEGA-X 10.1.7 grouped all eight strains from Mali in the E. rostratum clade with a bootstrap value of 100%. For pathogenicity test, four strains from leaves and seed were grown on rabbit food agar (50 g/liter steeped filtrate of rabbit food pellets, Kaytee Products, Inc. Chilton, WI, USA, and 15 g agar) for 14 days at 25°C with a 12 h photoperiod (Hau and Rush 1980). Spores were collected and the concentration of spore suspension adjusted to 1.5 x 105 conidia/ml with 0.5% gelatin. The rice varieties ADNY 11, ARICA 9 and Shwetasoké were grown in pots with peat soil and NPK 13-5-18 at 3.5 g/liter of soil for 21 days. Four pots of each variety (5 seedlings/pot) were placed in a tray (60 plants per tray) and the leaves were sprayed with 30 ml of the conidial suspension or water at 0.5% gelatin (negative control). Plants were kept at maximum humidity (100%) at 21°C for one night and then transferred to a phytotron at 27°C. Seven days after inoculation, circular or oval foliar lesions of less than 5 mm long, either brown or dark, sometimes whitish in their centers were observed . These lesions were identical to those observed in the field. E. rostratum was reisolated from these lesions. E. rostratum affects a wide range of plant species, particularly grasses and has been observed on rice in many countries (Cardona and Gonzàlez 2007; Majeed et al. 2016; Silva et al. 2016; Toher et al. 2016). However, to our knowledge, this is the first report of E. rostratum causing brown spot in rice in Mali