Zymoseptoria tritici - a global threat for wheat production
STB diseased wheat leaves. Photo: Petteri Karisto
Text contributed by: Andrea Sanchez-Vallet, Carolina Sardinha, Javier Palma-Guerrero, Lukas Meile, Julien Alassimone, Petteri Karisto, Anik Dutta, Alexey Mikaberidze, Xin Ma, Simone Fouché, Ziming Zhong and Bruce McDonald
Introduction Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), the main leaf disease of wheat in temperate regions (Fones and Gurr 2015) and a major threat for wheat production globally. It causes chlorotic lesions after a latent period of between 9 and 14 dpi that develop into necrotic tissue where the asexual fruiting bodies (pycnidia) develop. STB is especially damaging in humid and temperate areas where yield losses may reach up to 50%. Control of STB relies mainly on fungicide treatments. It is estimated that approximately 70% of fungicides used on wheat in Europe are aimed at controlling Z. tritici (Torriani et al. 2015). Z. tritici (syns. Mycosphaerella graminicola, Septoria tritici) is an Ascomycete fungus (Quaedvlieg et al. 2011) that is thought to have originated from closely related Zymoseptoria species colonizing wild grasses in the Fertile Crescent approximately 10000-12000 years ago during wheat domestication (Stukenbrock et al. 2006, Stukenbrock et al. 2010).
STB lesions on a naturally infected wheat leaf. Photo: Petteri Karisto, Alexey Mikaberidze, Bruce A. McDonald
Pathogen Biology The asexual stage of Z. tritici was first identified on wheat in 1842 by Desmazières (Desmazière, 1842, Shipton et al., 1951). The sexual stage was identified 130 years later by Sanderson in New Zealand (Sanderson, 1972). After sexual or asexual spores land on a leaf, they germinate and grow as filamentous hyphae that enter the host through stomata, other natural openings or wounds. The infection then enters a long asymptomatic phase, typically lasting 8-11 days after stomata penetration. During this phase the fungus colonizes the sub-stomatal cavities and apoplastic spaces without penetrating host cells. This asymptomatic phase is often called a biotrophic phase, although no feeding structures typical of biotrophic pathogens, such as haustoria and arbuscules, have been reported. This is followed by a switch to necrotrophy characterized by a collapse of the host mesophyll cells and the onset of plant cell death between 12-18 days after penetration (Kema, et al., 1996, Duncan & Howard, 1999, Steinberg, 2015). The combination of a prolonged initial asymptomatic phase, followed by a relatively rapid necrotrophic phase led to the suggestion that Z. tritici should be called a latent necrotroph instead of a hemibiotroph (Sanchez-Vallet et al., 2015).
Six individual lesions of Septoria tritici blotch developing on a wheat leaf. Photo: Susanne Dora, Petteri Karisto
Asexual phase of disease development The asexual pycnidiospores are slender, elongated, hyaline, and enclosed within a pycnidium. The pycnidia are embedded in the epidermal and mesophyll tissue on both sides of the infected leaf with an opening (ostiole) on the top. The pycnidiospores can be present in two distinct morphologies within the pycnidium: a multicellular form called macropycnidiospores (35-98 x 1-3 µm and including 3-5 septa) and a unicellular form called micropycnidiospores or spermatia (8-10 x 0.8-1 µm). A mass of pycnidiospores is exuded from a pycnidium in a gelatinous matrix called a cirrhus that contains a high concentration of sugars and proteins that wrap the pycnidiospores (Fournet, 1969). The pycnidiospores are dispersed by rain-splash to neighbouring leaves over a short distance (Holmes & Colhoun, 1975) averaging approximately 20 cm (A. Mikaberidze and P. Karristo, unpublished). Many cycles of asexual reproduction can take place during a growing season.
Sexual phase of disease development Z. tritici has a heterothallic mating system, which requires two compatible partners of opposite mating types (mat1-1 and mat1-2) to come together to produce the sexual spores. Sexual fruiting bodies called pseudothecia form beneath the host epidermis. Each ascus contains eight ascospores, the products of meiotic division followed by mitotic division, that are enclosed within each ascus. The ascospores are hyaline and consist of two cells of unequal size, measuring 2.5-4 x 9-16 µm. When the pseudothecia are mature the ascospores are ejected from the asci and can be transported by wind, enabling long-distance dispersal of the pathogen. 1-2 cycles of sexual reproduction can take place during a growing season, but the majority of sexual reproduction is thought to occur on decayed crop stubble between growing seasons.
Dimorphism Z. tritici is a dimorphic fungus that displays environmentally regulated morphogenic transitions between filamentous hyphae and blastospores (also called yeast-like form) (Motteram et al., 2006, King et al., 2017, Yemelin et al., 2017). Filamentous growth is required for stomata penetration and host colonization and is essential for pathogenicity. The natural biological role of blastospores remains unclear. The morphological growth transitions are easily induced in vitro. Pycnidiospores placed onto growth media will preferentially replicate as blastospores via budding on nutrient-rich medium incubated at a temperature ranging from 15°C to 18°C. Pycnidiospores incubated at a high temperature (> 22°C) and/or in a nutrient-poor medium will differentiate into filamentous hyphae (Mehrabi et al., 2006, Motteram et al., 2006, King et al., 2017, Yemelin et al., 2017).
Wheat-Zymoseptoria tritici interactions To cause infection, Z. tritici needs to suppress or evade the wheat immune system. The best understood method of evasion is the tight binding of chitin by the secreted Mg3LysM protein (Marshall et al. 2011) which efficiently blocks chitin perception by the host receptors CEBiP and CERK1 (Lee et al. 2014). Tight transcriptional regulation of pathogen effectors during different phases of infection are also thought to play a major role in evading host surveillance, especially during the early asymptomatic stage (Brunner et al. 2013, Rudd et al. 2015).
The existence of gene-for-gene interactions (Flor 1971) in Z. tritici has been known for many years (Brading et al. 2002, Brown et al. 2015), but the first avirulence gene of Z. tritici, AvrStb6, was cloned only recently. Variation in the coding sequence of AvrStb6 was shown to prevent recognition (Zhong et al. 2017). Until now, major resistance gene-mediated defense was not shown to involve a hypersensitive response and the mechanisms of successful defense remain poorly understood. Another unanswered question is how the necrotrophic phase is triggered. Although several effectors were shown to induce necrosis in wheat or other plants, their role for infection of Z. tritici remains elusive (Motteram et al. 2009, M’Barek et al. 2015).
Genomics of Zymoseptoria tritici The genome of Z. tritici consists of thirteen core chromosomes and up to eight accessory chromosomes that can be lost without a noticeable effect on pathogen fitness (Goodwin et al., 2011; Wittenberg et al., 2009). Z. tritici has the highest number of accessory chromosomes identified to date. These accessory chromosomes exhibit extensive absence/presence polymorphisms in experimental crosses and in natural field populations and differ from the core chromosomes in both gene and repetitive sequence content (Goodwin et al., 2011). Although their role in the biology of Z. tritici remains unclear, a recent study showed a correlation between the presence of some of the accessory chromosomes and increased virulence (Stewart et al., 2017). Z. tritici has frequent chromosomal rearrangements that may accelerate evolution (Plissonneau et al. 2016).
Compared to other fungal pathogens, the Z. tritici genome encodes very few genes involved in breaking down plant cell walls. This led to the hypothesis that “stealth pathogenesis” (Goodwin et al. 2011) was the strategy that enabled Z. tritici to emerge as a wheat pathogen from its mainly endophytic ancestors ~11000 years ago (Stukenbrock et al., 2007). When compared to its closely related sister species that infect wild grasses, it is evident that positive selection has altered secreted proteins and putative effectors in the Z. tritici genome, (Stukenbrock et al., 2011), suggesting that the co-evolution of the pathogen on its domesticated host greatly affected pathogen evolution.
Recent studies provided detailed descriptions of the transcriptomes of Z. tritici during its infection of wheat (Kellner et al., 2014; Palma-Guerrero et al., 2016; Rudd et al., 2015). A recently published project provided a comparative transcriptome analysis that included four strains of Z. tritici during the entire infection cycle on wheat (Palma-Guerrero et al., 2017). This analysis provided the expression profiles of several gene families involved in virulence, including proteases, PCWDEs, lipases, SSPs and secreted peroxidases and showed that differences in gene expression may be major determinants of virulence variation among Z. tritici strains.
From Goodwin et al 2011. Figure 1. Chromosome 2 of Zt. A: GC content. B: Repetitive regions. C: Single-copy (red) regions. D: Locations of genes for proteins containing signal peptides. E, Locations of homologs of pathogenicity genes of other pathogens. F: Approximate locations of quantitative trait loci (QTL) for pathogenicity to wheat. G: Alignments between the genomic sequence and two genetic linkage maps of crosses involving isolate IPO323. CC0.
Population genetics Population genetics studies have included Z. tritici populations sampled from approximately 30 wheat fields distributed across five continents. Comparisons of these field populations using neutral genetic markers led to identification of the center of origin and the most likely routes of gene flow among continents (CHEN & McDonald 1996; Linde et al. 2002; Zhan et al. 2003; Jurgens et al. 2006; Kabbage et al. 2009; Abrinbana et al. 2010; Chartouni et al. 2011; Drabesova et al. 2013; Naouari et al. 2016; Stukenbrock et al. 2007; Boukef et al. 2012). Neutral genetic markers have included restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), microsatellites (SSRs), random amplified polymorphic DNA (RAPDs) and genomic sequences (McDonald 1990; McDonald & MARTINEZ 1990; McDonald & Martinez 1991; McDonald & MARTINEZ 1991; Bahkali et al. 2012; Gautier et al. 2014; Kema et al. 1996).
Z. tritici originated in the Fertile Crescent where the genetic diversity is the highest compared to other populations (Zhan et al. 2003; Stukenbrock et al. 2007). Nearly all field populations exhibit high gene and genotype diversity within populations and low genetic differentiation among populations (Linde et al. 2002; Zhan et al. 2003; Jurgens et al. 2006; Abrinbana et al. 2010; Kabbage et al. 2009; Chartouni et al. 2011; Drabesova et al. 2013; Naouari et al. 2016), consistent with high gene flow among field populations and a high level of sexual recombination within each field population. The few field populations found to have low genetic diversity were usually shown to result from inoculation by one or a few strains.
The population structure of Z. tritici, which includes high genetic diversity distributed over a small spatial scale, large effective population size, long-distance gene flow and regular sexual reproduction, facilitates rapid evolution in these populations at the field scale. This high evolutionary potential explains the rapid development of fungicide resistance and the rapid failure of major STB resistance genes like Stb6 (Torriani et al. 2009; BRUNNER et al. 2008; Estep et al. 2013; Estep et al. 2015). Intragenic recombination was proposed to play an important role in the evolution of new CYP51 alleles that provide high levels of resistance to azole fungicides (Brunner et al. 2008). QTL (quantitative trait locus) mapping led to identification of candidate genes involved in local adaptation, including for thermal adaptation, fungicide resistance and virulence on different wheat cultivars (Lendenmann et al. 2016; Stewart et al. 2016; Lendenmann et al. 2015; Zhong et al. 2017). A recent review summarizes how knowledge of Z. tritici population genetics can be used to improve management of STB (McDonald & Mundt 2016).
Combination of genetic and chemical control: wheat cultivars on experimental plots treated with fungicides. Photo: Petteri Karisto
Epidemiology and control STB epidemic development is a result of three processes: infection, spore production and spore dispersal. Epidemics usually start in the autumn on the emerging winter wheat seedlings. The initial source of infection is usually sexual spores (ascospores), which are dispersed by wind (spore dispersal) from infected crop debris leftover in the field from the previous season. Asexual spores are splash-dispersed and cause new infections on emerging leaves. There are usually several (4-6) asexual cycles of infection and at least one sexual cycle of infection during a growing season.
STB epidemics can be controlled by suppressing any of the three processes. Chemical control with fungicides aims to prevent initial infection and remove already established infections. Genetic control with wheat varieties carrying qualitative or quantitative resistance to STB can reduce infection and/or spore production. To date, 20 major genes showing qualitative resistance and 167 genomic regions containing quantitative trait loci (QTLs) conferring quantitative resistance to Z. tritici have been mapped (Brown et al., 2015). Crop rotations and planting crop mixtures can reduce dispersal of spores within fields and between seasons.
As a result of high levels of sexual reproduction, combined with large population sizes and long-distance dispersal, Z. tritici populations are highly diverse. This makes control of STB using genetic resistance more challenging. Breeding for qualitative resistance is not sustainable because it can be broken down relatively quickly by the rapid evolutionary potential of Z. tritici. A classic example of the breakdown of qualitative resistance to Z. tritici after four years of commercial cultivation in the USA during mid 90’s can be found in the wheat variety “Gene” (Cowger et al., 2000). The most sustainable control measures are likely to involve an integrative approach that combines quantitative resistance (several genes with small additive effects), multi-target fungicides and agronomical practices that limit the survival of Z. tritici between growing seasons.
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