Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: recent progress and future prospects

Summary

Diseases caused by Fusarium pathogens inflict major yield and quality losses on many economically important plant species worldwide, including cereals. Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is a cereal disease that occurs in many arid and semi‐arid cropping regions of the world. In recent years, this disease has become more prevalent, in part as a result of the adoption of moisture‐preserving cultural practices, such as minimum tillage and stubble retention. In this pathogen profile, we present a brief overview of recent research efforts that have not only advanced our understanding of the interactions between F. pseudograminearum and cereal hosts, but have also provided new disease management options. For instance, significant progress has been made in the genetic characterization of pathogen populations, the development of new tools for disease prediction, and the identification and pyramiding of loci that confer quantitative resistance to FCR in wheat and barley. In addition, transcriptome analyses have revealed new insights into the processes involved in host defence. Significant progress has also been made in understanding the mechanistic details of the F. pseudograminearum infection process. The sequencing and comparative analyses of the F. pseudograminearum genome have revealed novel virulence factors, possibly acquired through horizontal gene transfer. In addition, a conserved pathogen gene cluster involved in the degradation of wheat defence compounds has been identified, and a role for the trichothecene toxin deoxynivalenol (DON) in pathogen virulence has been reported. Overall, a better understanding of cereal host–F. pseudograminearum interactions will lead to the development of new control options for this increasingly important disease problem.

Taxonomy: Fusarium pseudograminearum O'Donnell & Aoki; Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreomycetidae; Order Hypocreales; Family Nectriaceae; Genus Fusarium.

Disease symptoms: Fusarium crown rot caused by F. pseudograminearum is also known as crown rot, foot rot and root rot. Infected seedlings can die before or after emergence. If infected seedlings survive, typical disease symptoms are browning of the coleoptile, subcrown internode, lower leaf sheaths and adjacent stems and nodal tissues; this browning can become evident within a few weeks after planting or throughout plant development. Infected plants may develop white heads with no or shrivelled grains. Disease symptoms are exacerbated under water limitation.

Identification and detection: Fusarium pseudograminearum macroconidia usually contain three to five septa (22–60.5 × 2.5–5.5 μm). On potato dextrose agar (PDA), aerial mycelia appear floccose and reddish white, with red or reddish‐brown reverse pigmentation. Diagnostic polymerase chain reaction (PCR) tests based on the amplification of the gene encoding translation elongation factor‐1a (TEF‐1a) have been developed for molecular identification.

Host range: All major winter cereals can be colonized by F. pseudograminearum. However, the main impact of this pathogen is on bread (Triticum aestivum L.) and durum (Triticum turgidum L. spp. durum (Dest.)) wheat and barley (Hordeum vulgare L.). Oats (Avena sativa L.) can be infected, but show little or no disease symptoms. In addition, the pathogen has been isolated from various other grass genera, such as Phalaris, Agropyron and Bromus, which may occur as common weeds.

Useful websites: https://nt.ars-grin.gov/fungaldatabases/; http://plantpath.psu.edu/facilities/fusarium-research-center; https://nt.ars-grin.gov/fungaldatabases/; http://www.speciesfungorum.org/Names/Names.asp

Introduction

Fusarium crown rot (FCR), caused by the fungal pathogen Fusarium pseudograminearum, is a chronic disease of wheat and barley in many arid and semi‐arid cropping regions of the world, including Australia (Akinsanmi et al., 2004; Burgess et al., 1975), the Pacific Northwest (e.g. Oregon, Washington, Montana and Idaho) of the USA (Cook, 1968, 1980; Smiley and Patterson, 1996; Smiley et al., 2005a), Canada (Fernandez and Zentner, 2005; Mishra et al., 2006), New Zealand (Cromey et al., 2006), South Africa (Ferreira et al., 2015; Lamprecht et al., 2006; Marasas et al., 1988), the Middle East (e.g. Turkey, Iran, Iraq and Syria) (Alkadri et al., 2013; Hameed et al., 2012; Pouzeshimiab et al., 2016; Saremi et al., 2007; Tunali et al., 2006, 2008), China (Ji et al., 2016; Li et al., 2012; Xu et al., 2015; Zhang et al., 2015), North Africa (e.g. Tunisia, Egypt, Morocco) (Gargouri et al., 2011; Kammoun et al., 2009) and South America (e.g. Argentina) (Castañares et al., 2012). Fusarium pseudograminearum often co‐exists in these regions with other FCR‐causing Fusarium species, including F. culmorum, F. avenaceum, F. poae and F. graminearum, as well as fungal or oomycete pathogens, including the common root rot pathogen Bipolaris sorokiniana, the take‐all pathogen Gaeumannomyces graminis and Pythium spp., which cause disease symptoms that are similar to those caused by FCR (Akinsanmi et al., 2004, 2006; Chakraborty et al., 2006; Gebremariam et al., 2017; Smiley et al., 2005a; Tunali et al., 2008, 2012). In Australia, F. pseudograminearum has been identified as the most frequent Fusarium species associated with FCR in several surveys conducted since the 1970s (Akinsanmi et al., 2004; Backhouse and Burgess 2002; Backhouse et al., 2004; Burgess et al., 1975; Khangura et al., 2013; McKnight and Hart, 1966). The predominant presence of F. pseudograminearum in Australia as the primary FCR pathogen was explained by the relatively warm and dry environmental conditions found throughout the Australian Wheat Belt, which spans the cereal growing states of Queensland, New South Wales, Victoria, South Australia and Western Australia (Chakraborty et al., 2006). Similarly, pathogen surveys conducted in the Pacific Northwest of the USA have shown that F. pseudograminearum is more common in warmer and drier regions than is F. culmorum, which is often found in cooler regions with higher rainfall (Poole et al., 2013).

Among cool season cereals, bread wheat and, particularly, durum wheat are susceptible to F. pseudograminearum, although there is a distinct range of reactions to FCR in bread wheat (reviewed by Liu and Ogbonnaya, 2015). Barley is considered to be tolerant (i.e. displays limited yield losses on infection). Fusarium pseudograminearum can also infect oats, resulting in only minor or no disease symptom development (Percy et al., 2012). Fusarium pseudograminearum has also been isolated from other grass genera, such as Phalaris, Agropyron and Bromus (Purss, 1969). Fusarium pseudograminearum can cause seedling death before or after emergence, especially if the seeds sown are collected from spikelets previously infected by various Fusarium spp. in the field (Simpfendorfer, 2013). Fusarium pseudograminearum can cause extensive browning of subcrown internodes and leaf sheaths shortly after infection and throughout plant development (Fig. 1A,B). Infected wheat plants can also develop white heads that contain either no or shrivelled grains (Fig. 1C,D). As a result of an effect on grain development, FCR incidence and severity are negatively correlated with grain yield, kernel number per head, kernel weight, tiller height and straw weight (Smiley et al., 2005b). Yield loss estimates in the Pacific Northwest of the USA have indicated that FCR can cause up to 35% reduction in wheat grain yield under natural inoculum levels (Smiley et al., 2005b). In Australia, FCR caused by F. pseudograminearum is estimated to routinely cause 10% yield loss in cereals (Murray and Brennan, 2009), although, in some years, much greater losses are encountered (Klein et al., 1991). The average annual cost of FCR‐induced crop losses to the Australian wheat industry is estimated to be 88 million Australian dollars, but the disease has the potential to cost much more than this (up to 434 million Australian dollars) under favourable conditions or if no control options are employed (Murray and Brennan, 2009). These figures clearly show that FCR is an economically important cereal disease.

Figure 1.

Fusarium crown rot (FCR) symptoms caused by Fusarium pseudograminearum on wheat. (A) Stem browning caused by F. pseudograminearum on field‐grown plants. The arrows point to disease lesions observed on the stem base of two infected plants (left) versus an uninfected plant (right). (B) Internodal sections from infected wheat plants showing pinkish mycelial growth (arrowed) of F. pseudograminearum. (C) White heads caused by F. pseudograminearum infection (arrowed). (D) Shrivelled grains (top) from wheat plants infected with F. pseudograminearum versus normal grains (bottom) from healthy plants.

Fusarium pseudograminearum can survive in stubble for up to 3 years (Burgess, 2014; Summerell and Burgess, 1988; Summerell et al., 1989, 1990). Therefore, the increased adoption of minimum tillage and stubble retention to preserve soil moisture seems to constitute the major reasons that have led to increased FCR incidence in many regions of the world (reviewed by Chakraborty et al., 2006; Paulitz, 2006; Paulitz et al., 2002). Certain agronomic practices, such as crop rotation, stubble management, tillage and soil fertility management, can reduce FCR incidence (Cook, 1980, 2001). However, such practices are not always compatible with economical and practical considerations. Therefore, the genetic improvement of host resistance to FCR is an important breeding objective for wheat and barley. However, no cultivar fully resistant or immune to this pathogen is currently available. Partial host resistance identified against F. pseudograminearum behaves as a quantitative trait controlled by many genes that have relatively small effects (Collard et al., 2005, 2006; Martin et al., 2015; reviewed by Liu and Ogbonnaya, 2015). Environmental factors, such as water stress, also strongly affect the expression of disease symptoms (Cook, 1980; Papendick and Cook, 1974). Therefore, a better understanding of pathogen biology and epidemiology, as well as host resistance and pathogen virulence, is key to the development of sustainable disease control options. In this pathogen profile, we provide an overview of recent research efforts on FCR and highlight potential future research strategies required to address this chronic disease problem.

Epidemiology of F. pseudograminearum

Fusarium pseudograminearum overwinters on crop residue as mycelia, or potentially as spores (e.g. chlamydospores) (Fig. 2), which germinate and/or grow to produce asexual macroconidia. The stubble of the previous year's crop acts as the source of the inoculum, but it is unclear what is the dominant infective propagule in the field. It is often assumed that both mycelia and spores (e.g. chlamydospores) contribute to infection, although it should be noted that the information on spores or conidia in the environment is sparse. The most common route of infection of host cereals by F. pseudograminearum in the field seems to occur at the coleoptile, progressing into the subcrown internode and leaf sheaths and, subsequently, into the stem epidermal tissues, frequently penetrating tissues via stomatal openings. The pathogen then moves into the hypodermis to induce typical browning of the stem and, subsequently, into vascular tissues (Knight and Sutherland, 2013a, 2013b, 2016). In addition, findings from glasshouse studies suggest that the pathogen can move from the stem base to the heads through the pith parenchyma (Mudge et al., 2006). The colonization of stems by F. pseudograminearum reportedly blocks the vascular tissues and restricts both water and nutrient translocation within the plant, which can potentially contribute to the formation of white heads (Burgess et al., 2001; Knight and Sutherland, 2016; Smiley, 2009). Histopathological analyses have shown that at least the three lower internodes of wheat and barley plants are colonized by the fungus (Knight and Sutherland, 2016). This colonization pattern seems to occur in both susceptible and partially resistant genotypes. However, the extent of colonization is often reduced in partially resistant genotypes (Knight and Sutherland, 2016), or the pathogen moves more slowly within the plant during the colonization of partially resistant genotypes compared with susceptible genotypes (Percy et al., 2012).

Figure 2.

Lifecycle of Fusarium pseudograminearum causing Fusarium crown rot (FCR) in cereal crops. The pathogen saprophytically survives as chlamydospores and mycelia in plant debris. Under suitable conditions, chlamydospores can form. However, their role as inoculum in the field has not been reported. Mycelia can produce asexual macroconidia. Both mycelia and macroconidia can induce disease symptoms in artificial inoculations. It is uncertain, however, what is the dominant infective propagule in the field. As seedlings become colonized, they develop visual browning symptoms on the subcrown internode and leaf sheaths of the lower nodes. As they grow, these symptoms spread to the stems, with potential whitehead formation during grain fill. Fusarium pseudograminearum can also undergo a complete sexual cycle on plant debris to produce sexual structures called perithecia containing ascospores that are discharged to the environment, although the relative importance of ascospores as an inoculum source is unknown. MAT1‐1 and MAT1–2 refer to different mating types.

Although mostly associated with FCR disease in dry regions, F. pseudograminearum can also incite Fusarium head blight (FHB), especially if warm and humid conditions exist during anthesis (Obanor et al., 2013). For instance, F. pseudograminearum contributed to the FHB epidemics that occurred in the 1980s (Burgess et al., 1987) and 1990s (Southwell et al., 2003), as well as in 2010–2011 (Obanor et al., 2013), in Australia. The FHB disease caused by F. pseudograminearum is characterized by the development of typical necrotic or bleached spikelets contaminated with the fungal toxin deoxynivalenol (DON) and is symptomatically similar to that caused by the principal FHB pathogen F. graminearum (Obanor et al., 2013).

Genetic Diversity of F. pseudograminearum

Fusarium pseudograminearum was formerly known as F. graminearum Group 1 (Dodman and Wildermuth, 1987; Klein et al., 1991; Summerell et al., 1900). Subsequently, this species was separated from F. graminearum based on morphological and molecular differences (Aoki and O'Donnell, 1999a, 1999b). It is estimated that F. graminearum and F. pseudograminearum diverged from a common ancestor 3.4 million years ago (range of 1.2–6.5 million years ago) (O'Donnell et al., 2000). Allelic genealogies of six single‐copy nuclear genes from isolates representing the global genetic diversity indicated that F. pseudograminearum is a reproductively isolated and phylogenetically distinct species. Multilocus sequence analyses of F. pseudograminearum isolates from different regions of the world (i.e. Australia, Canada, Turkey, New Zealand and the USA) support the conclusion that F. pseudograminearum is a single phylogenetic species without consistent lineage development (Scott and Chakraborty, 2006). Relatively high levels of genetic diversity are found amongst the isolates of this species. For instance, the amplified fragment length polymorphism (AFLP) analysis of 27 F. pseudograminearum isolates collected from a single field revealed 18 different AFLP haplotypes (Akinsanmi et al., 2006). Furthermore, nearly all isolates that originated from the two neighbouring Australian states of New South Wales and Queensland showed unique AFLP haplotypes (Akinsanmi et al., 2006). In another study, the AFLP analysis of eight F. pseudograminearum populations (217 isolates in total), which originated from three geographically distinct regions of the Australian wheat belt, revealed high levels of genetic diversity (Bentley et al., 2008a). This study identified two major groups that genetically separated the northern region isolates (Queensland and New South Wales) from the southern (South Australia) and western (Western Australia) region isolates (Bentley et al., 2008a). Similarly, Mishra et al. (2006) found high levels of genetic diversity within F. pseudograminearum populations that originated from Alberta and Saskatchewan in Canada. It appears that the level of genetic diversity within F. pseudograminearum populations exceeds that between populations, which is most probably a result of random mating and gene flow between different populations (Mishra et al., 2006). Indeed, although teleomorphs have rarely been documented in the field, the observation of F. pseudograminearum sexual structures (perithecia) found on infected stubble supports the notion that sexual recombination in this heterothallic species occurs in agricultural settings (Bentley et al., 2008b).

Fusarium Crown Rot Management

Agronomic practices

Both management practices and cereal genotypes can influence FCR development, and thus should be taken into account in risk management analyses. Given that crop debris or stubble from infected plants harbours the inocula (Summerell et al., 1989, 1990; Swan et al., 2000), stubble management can have a significant impact on the control of FCR (Wildermuth et al., 1997). As a result of its positive effect on the moisture content of surface soil, stubble provides the ideal conditions required for the initial infection of young seedlings (Liddell and Burgess, 1988; Swan et al., 2000). Indeed, a positive correlation was found between the level of soil moisture and the ability of F. pseudograminearum to infect seedlings. The optimum soil water potential that promotes the infection by F. pseudograminearum was found to vary between −0.3 and −0.7 MPa (Liddell and Burgess, 1988). Therefore, stubble burning or incorporating stubble into the soil can significantly reduce inoculum levels (Burgess et al., 1996; Summerell and Burgess, 1988). However, this practice may negatively influence grain yields in dry regions by increasing evaporation‐mediated moisture losses and removing moisture that would otherwise be available to the crop, especially during early developmental stages (Burgess et al., 2001).

Crop rotation is another agronomic practice used to manage FCR development. Non‐host cereal crops can be effective at reducing FCR in subsequent plantings. For instance, sorghum is not a host for F. pseudograminearum (Burgess, 2005; Simpfendorfer, 2005), and lower levels of FCR have been recorded in sorghum–wheat rotations than in continuous wheat (Burgess et al., 1996; Quazi et al., 2009). The use of oats in rotation with wheat has also been reported to have beneficial effects at reducing FCR (Nelson and Burgess, 1995). In contrast, cereals such as triticale can lead to increases in FCR inoculum levels (Wallwork, 2005). As expected, non‐cereal species used in rotations had more positive effects than cereal species in reducing FCR inoculum levels in the field (Evans et al., 2010). For instance, Brassica crops (i.e. canola and mustard) had a positive effect on the yield of FCR‐susceptible durum wheat in New South Wales when used in rotation (Kirkegaard et al., 2004). The rapid decomposition of cereal residues carrying FCR inocula under dense Brassica canopies was proposed to be a factor responsible for this effect (Kirkegaard et al., 2004). Chickpea (Cicer arietinum L.) is a food legume that reduces FCR when used in rotation with wheat (Felton et al., 1998), and wheat–lentil (Lens culinaris Medik.) rotations promote FCR in spring wheat in western Canada (Fernandez and Zentner, 2005).

The appropriate control of weed grasses that harbour FCR inocula is another agronomic practice that can reduce the disease incidence. Similarly, planting dates can influence FCR development. For instance, in northern Oregon, early plantings in September increase the incidence of FCR (Chen et al., 2003; Paulitz 2006). Therefore, if possible, planting dates should be selected in such a way that dry finishes (i.e. the occurrences of dry conditions during grain fill), which promote FCR, can be avoided during maturity.

The nutrient status of the soil has also been reported to affect FCR disease development. For instance, studies have suggested that nitrogen fertilizers in the field can increase the disease incidence and severity of root diseases caused by Fusarium pathogens in wheat (Cook, 1980; Papendick and Cook, 1974; Paulitz, 2006). In contrast, the availability of sufficient amounts of zinc is important to maintain the grain yields of Australian durum and bread wheat varieties under FCR (Al‐Fahdawi et al., 2014). A positive effect of zinc in restricting the colonization of wheat stems by F. pseudograminearum has also been reported (Grewal et al., 1996; Sparrow and Graham, 1988). However, additional experiments are needed to determine how the moisture and nutritional status of the soil, as well as the presence of non‐pathogenic or beneficial microbes, affect FCR inoculum levels and disease development. Inter‐row sowing is another agronomic practice that was found to reduce FCR and increase grain yield by 6% (Verrell et al., 2017). However, the effect of this treatment was dependent on crop sequence and was lost after two consecutive wheat crops.

Biocontrol organisms and fungicides

Biocontrol microbes can potentially offer options to minimize FCR. For instance, Trichoderma spp. show strong inhibitory effects on F. pseudograminearum when sprayed onto straw colonized by this pathogen (Wong et al., 2002), suggesting that this treatment might reduce FCR inoculum levels in the field. The beneficial effects of bacteria, such as Burkholderia cepacia, on FCR were observed when applied as a soil drench, but this effect was dependent on the soil type (Huang and Wong, 1998). Similarly, biocontrol agents, such as certain Bacillus strains and Trichoderma harzianum, provided significant protection against FCR in glasshouse assays (Moya‐Elizondo et al., 2016). The protection afforded by these biocontrol agents was correlated with the systemic expression of wheat genes encoding pathogenesis‐related (PR) proteins, such as endochitinases, β‐1,3 glucanases and peroxidases, suggesting that these biocontrol agents may act by stimulating or priming host defences (Moya‐Elizondo et al., 2016).

The treatment of seeds with fungicides or the application of fungicides to stem bases does not seem to provide sufficient protection from FCR (Evans et al., 2010; Simpfendorfer, 2013). However, more recently, the successful use of fungicides, either as a seed dressing or as a foliar spray (applied twice at Zadoks growth stages 31 and 45 with fluquinconazole, with tebuconazole, or with epoxiconazole and carbendazim), against FCR caused by F. culmorum has been reported (Akgul and Erkilic, 2016; Moya‐Elizondo et al., 2016). In addition, combinatorial applications of biocontrol agents and fungicides seem to provide additive protection against FCR (Moya‐Elizondo et al., 2016). It is unclear, however, whether these effects are also applicable to FCR caused by F. pseudograminearum and operate under field conditions.

Disease prediction

Given that F. pseudograminearum survives in colonized plant debris, the level of fungal inoculum already present in the field before sowing can be an indicator of the FCR incidence of the subsequent year. Several polymerase chain reaction (PCR) and quantitative PCR (qPCR) protocols have been developed to estimate the levels of FCR inoculum present in the soil or stubble (Evans et al., 2010; Hogg et al., 2007, 2010; Hollaway et al., 2013; Knight and Sutherland, 2017; Poole et al., 2015; Williams et al., 2002). In South Australia and Victoria, the levels of F. pseudograminearum and F. culmorum inocula found in the field prior to the planting season were positively correlated with FCR disease expression (e.g. stem browning and white head formation) in both wheat and barley. In addition, a negative correlation was found between FCR inoculum levels and grain yield (Hollaway et al., 2013). However, as expected, FCR inoculum levels prior to sowing are not the only factor influencing FCR disease incidence. The extent of the correlation between soil inoculum levels and symptom development or grain yield can be influenced by environmental factors and the types of cereals being grown (Hollaway et al., 2013). Indeed, the negative correlations found between FCR inoculum levels and grain yield were significant only in years with an average rainfall below the long‐term average during the grain‐filling period and not in relatively wet years (Hollaway et al., 2013).

A recent study examining the effect of prior inoculum levels of various soil‐borne pathogens on FCR concluded that prior inoculum levels, when combined with environmental factors, can exacerbate disease problems (Poole et al., 2015). However, the correlation found between pre‐sow and post‐harvest pathogen DNA levels was relatively weak for F. pseudograminearum compared with the correlations for other soil‐borne pathogens (Poole et al., 2015). The levels of pathogen DNA measured with PreDicta B commercial PCR assays (Ophel‐Keller et al., 2008) explained only a small portion of the overall root health (Poole et al., 2015). Therefore, the prediction of disease incidence solely on the level of FCR inoculum present before sowing may not be sufficient for all cereal‐growing regions.

Breeding for FCR Resistance and Tolerance

As indicated above, agronomic practices aimed at reducing FCR incidence may not be fully compatible with economical and practical considerations. Therefore, improving the genetic resistance of cereal cultivars to FCR is an important breeding objective. No absolute resistance is available against this pathogen, and the term ‘resistance’ used in this context refers only to ‘partial resistance’, which is a measurement of disease symptom development and/or fungal biomass. However, it is possible that certain cereal genotypes may be able to maintain their yield potential under infection or show reduced symptom development despite containing a high fungal load. Therefore, these latter genotypes can be called ‘tolerant’. Indeed, despite showing similar disease symptoms, barley is more tolerant to FCR than is wheat in terms of yield losses caused by F. pseudograminearum (Klein et al., 1989; Wildermuth and Purss, 1971). Therefore, growing tolerant cultivars might be more profitable to cereal growers than growing partially resistant cultivars, although it is possible that partially resistant cultivars can also be tolerant to FCR.

Assessment of FCR resistance and tolerance

Given the quantitative nature of FCR resistance, which is affected by environmental factors and plant development, the methods used for the assessment of FCR resistance need to be robust and preferably high throughput. Disease assays conducted in the glasshouse should reliably predict the field performance of the germplasm being tested. The establishment of a glasshouse disease assay for the assessment of the levels of FCR resistance or tolerance has been challenging, mainly because of the poor reproducibility and, in some instances, unreliability of such assays in predicting field performances. Therefore, several inoculation methods have been tested for germplasm evaluation. A durum wheat cultivar as a susceptible check and a number of wheat genotypes with known FCR field rankings are often used in some assays to determine whether the assay being developed could reliably predict the known FCR field performances (Li et al., 2008; Mitter et al., 2006; Wallwork et al., 2004; Yang et al., 2010). In most glasshouse and field assessments, disease symptom development observed under artificial or natural inoculum levels has been assessed for germplasm evaluation. The measurement of stem base browning is often used as a proxy for the evaluation of cereal germplasm for FCR resistance. Knight and Sutherland (2015) found positive correlations between stem base browning and fungal biomass measured using qPCR in wheat and barley in a 2‐year field study. However, these correlations were significant for different internodes of infected plants in different years. In addition, the time point of approximately 16 weeks after planting, which coincides with the post‐anthesis/early‐milk developmental stage under the climatic conditions of Queensland, seems to be the most suitable period for the detection of such correlations. However, other researchers were not able to show a direct correlation between the level of infection and resistance (Dodman and Wildermuth, 1987). Even if the extent of stem browning correlates with the level of fungal biomass present in the plant, (Knight et al., 2012) as discussed above, this measure itself is probably not a reliable indication of tolerance, which could be better assessed under field conditions through yield loss trials.

A recent study compared three inoculation methods for their suitability in germplasm evaluation (Erginbas‐Orakci et al., 2016). The seedling dip method originally developed by Li et al. (2008) produced the most severe infections, followed by the colonized grain (Nelson and Burgess, 1994; Wildermuth and McNamara 1994) and droplet inoculation (Mitter et al., 2006) methods. However, inoculations using the colonized grains produced results that were more consistent with the known field rankings of the tested wheat cultivars (Erginbas‐Orakci et al., 2016). It should be noted that the use of different field ranking procedures by different workers increases the challenge of finding glasshouse inoculation procedures that correlate with field rankings. It is also possible that different inoculation methods detect partial resistance mechanisms operating at different developmental stages. Indeed, some quantitative trait loci (QTLs) conferring partial resistance to FCR are only observed in seedlings, whereas others appear to be specific to adult plants (Martin et al., 2014).

QTLs and marker‐assisted selection

Breeding for FCR resistance requires the identification of useful variations in this trait within the available germplasm. Partial FCR resistance is found in existing cultivars, in wild relatives and in landraces of wheat and barley (Liu et al., 2012; Purss, 1966; Smiley and Yan, 2009; Wildermuth and Purss, 1971). For instance, various QTLs conferring partial FCR resistance have been identified in both Australian (e.g. Janz, Kukri, 2–49, Lang, Sunco, Ernie and EGA‐Wylie) and US (e.g. Macon) wheat cultivars (Bovill et al., 2006; Chen et al., 2013a, 2013b; Collard et al., 2005, 2006; Ma et al., 2010; Martin et al., 2015; Poole et al., 2012; Wallwork et al., 2004; reviewed by Liu and Ogbonnaya, 2015). Qcrs.cpi.3B, one of the strongest FCR QTLs, is derived from Triticum spelta, a wild relative of hexaploid or bread wheat (Ma et al., 2010). Qcrs.cpi.3B is located on the long arm of chromosome 3B (Ma et al., 2010), and resistant and susceptible near‐isogenic lines (NILs) with this locus have been generated to facilitate fine‐mapping and cloning of the gene underlying this QTL (Ma et al., 2012a; Zheng et al., 2014).

The FCR QTLs identified thus far are distributed across 13 of the 21 wheat chromosomes (Liu and Ogbonnaya, 2015). The analysis of the genomic locations of FCR QTLs given by Liu and Ogbonnaya (2015) also revealed that the A, B and D subgenomes contain 6, 25 and 11 FCR QTLs, respectively. This suggests that the B and D subgenomes contribute more strongly than the A subgenome to FCR resistance. Interestingly, a recent RNA‐sequencing (RNA‐seq) study has shown that the genes that are differentially expressed during infection by F. pseudograminearum are located more strongly on the B and D subgenomes of wheat (Powell et al., 2017a) than on the A subgenome, suggesting that a homoeolog expression bias (differential expression of homoeologous genes) operates in wheat during defence against this pathogen (Powell et al., 2017a).

The genetic relatedness of F. graminearum and F. pseudograminearum, which are pathogens that can cause both FCR and FHB, has led to the suggestion that the FHB‐resistant wheat germplasm currently available could be utilized to improve FCR resistance (Li et al., 2010). However, evidence suggests that the wheat germplasm known to be resistant to FHB (e.g. the 3BS QTL from the wheat cultivar Sumai‐3) does not confer resistance to F. pseudograminearum‐induced FCR (Li et al., 2010). Therefore, independent germplasm evaluations may be required to identify novel FCR resistance sources.

To date, only a limited number of QTL studies have been conducted in barley (Liu and Ogbonnaya, 2015). Interestingly, all three QTLs identified thus far from barley (i.e. 3HL, 1HL and 4HL) are derived from either Hordeum spontaneum (4HL), a wild relative of barley, or a barley landrace from East Asia (1HL and 3HL) (Chen et al., 2013a, 2013b). In barley, pyramiding all three FCR QTLs, 1H, 3H and 4H, into a single genetic background reduced FCR lesion development more strongly than the effect of a single locus or two separate loci (Chen et al., 2015). Readers interested in other aspects of FCR breeding in wheat and barley are referred to a recent review by Liu and Ogbonnaya (2015).

Associations between FCR resistance and physiological and developmental traits

Various physiological and morphological/developmental traits positively or negatively affect FCR resistance. For instance, associations between FCR severity and drought and heat stress have long been known (Papendick and Cook, 1974; Smiley 2009), although the potential mechanisms involved in this association remain largely unknown. Interestingly, NILs that carry the FCR QTL Qcrs.cpi‐3B contain lower malondialdehyde (MDA) content under both well‐watered and water‐deficit conditions than do those without this QTL (Ma et al., 2015). Given that low MDA content is correlated with stress tolerance, as MDA forms under stress through lipid peroxidation (Meloni et al., 2003), this finding raises the possibility that Qcrs.cpi‐3B or another locus that is tightly linked to this QTL is a mediator of stress tolerance. However, a previous study was unable to establish an association between partial FCR resistance and a putative osmoregulatory gene (Wildermuth and Morgan, 2004). Additional studies are required to better understand the genetic and molecular bases of the associations between abiotic stress and FCR tolerance and resistance.

Plant height also seems to influence FCR resistance. A QTL conferring increased FCR resistance is linked to the Rht1 gene, causing reduced height in wheat (Wallwork et al., 2004). In wheat, the analysis of FCR‐resistant and FCR‐susceptible NILs showed that short isolines were more resistant to FCR than were tall ones (Liu et al., 2010). In barley, loci affecting plant height are also located within the 3HL and 4H QTLs (Li et al., 2009). The findings from the studies employing NILs showed that short isolines have stronger FCR resistance than do tall isolines (Ma et al., 2010; Yang et al., 2010; Zheng et al., 2014). In addition, FCR infection seems to spread faster within tall plants, and this phenomenon was explained by the differences observed in cell densities between short and tall genotypes (Bai and Liu, 2015).

Host Defence Against F. pseudograminearum

Plant defence responses triggered by F. pseudograminearum infection can provide useful insights into the overall understanding of this host–pathogen interaction, and have been studied using transcriptome analyses in wheat. In a previous study, the transcriptomes of the partially FCR‐resistant wheat cultivar Sunco and the susceptible cultivar Kennedy were compared using an Affymetrix gene chip (Desmond et al., 2008a). These analyses showed that the genes involved in antimicrobial defence, oxidative stress and signalling, as well as those involved in primary and secondary metabolism, are differentially expressed in infected wheat tissues (Fig. 3). In addition, many of the F. pseudograminearum‐responsive genes are altered by the toxin DON and by plant defence‐related hormones, such as salicylic acid (SA) and jasmonates (JAs). The exogenous application of JAs and gibberellins (GAs) is also known to protect wheat plants against F. pseudograminearum (Desmond et al., 2006; Liu et al., 2010). In addition, a stronger and faster induction of defence‐related genes is observed in the partially FCR‐resistant cultivar Sunco than in the susceptible cultivar Kennedy (Desmond et al., 2008a). Additional analyses are required to determine the functions of the genes that are differentially expressed during infection by F. pseudograminearum. It is possible that some of the host genes induced by F. pseudograminearum encode factors that condition susceptibility rather than resistance by targeting plant defences (Kazan and Lyons, 2014).

Figure 3.

Molecular basis of cereal host–Fusarium pseudograminearum interactions. As indicated by orange boxes, F. pseudograminearum infects and colonizes host tissues by producing toxins (e.g. deoxynivalenol or DON), cell wall‐degrading and defence‐targeting enzymes and fungal effectors. The host plant defends itself from pathogen attack by producing phytoalexins, pathogenesis‐related proteins, defence hormones and proteins involved in toxin detoxification (blue boxes).

More recently, a combination of transcriptomics (using RNA‐seq) and targeted metabolomics has been used to investigate defence responses in the Australian bread wheat cultivar Chara (Powell et al., 2017b), for which a mutagenized population for functional genetic analyses is available (Fitzgerald et al., 2015a). This study showed that the genes associated with pathogen sensing and signalling, including transcription factors, cellular transport and detoxification genes (e.g. ABC transporters and UDP glucosyltransferases), are differentially expressed on pathogen infection (Powell et al., 2017b) (Fig. 3). In this study, several metabolites associated with host defence were also induced. Many responses observed in wheat during F. pseudograminearum infection show similarities to those induced by related Fusarium pathogens, such as F. graminearum and F. culmorum (Kazan and Gardiner, 2017; Tufan et al., 2017), suggesting that these related pathogens employ a common set of weaponry to colonize wheat. To the best of our knowledge, no transcription profiling of durum wheats and barley during FCR has been reported.

What makes a cereal genotype more or less resistant or susceptible to FCR is unknown. However, the analysis of NILs or available wheat genotypes that differ in resistance to FCR suggest that the pathogen infection proceeds at a faster rate in susceptible genotypes than in partially resistant ones (Bai and Liu, 2015; Percy et al., 2012). A comparative analysis of gene expression of NILs with and without the Qcrs.cpi.3B locus revealed the differential expression of several genes between these two genotypes (Ma et al., 2014). These genes are possible candidates responsible for the effects caused by this QTL. However, no gene underlying Qcrs.cpi.3B or any other FCR QTL has been cloned thus far.

Understanding Pathogen Biology

Responses to climate change

Climate change is expected to increasingly affect major wheat‐growing areas in the world and seems to be responsible for the recent stagnation of the wheat yield in Australia (Hochman et al., 2017). Climate change can have a dramatic effect, not only on plant growth and development, but also on pathogen biology (Chakraborty et al., 2000). The effects of climate change, and in particular both elevated CO₂ and altered temperatures, on the aggressiveness of F. pseudograminearum have been investigated (Khudhair et al., 2014; Melloy et al., 2010, 2014; Sabburg et al., 2015). Under glasshouse conditions, elevated CO₂ increases disease incidence (measured as the proportion of infected tillers) and severity (measured as stem browning and increased fungal biomass) (Melloy et al., 2014). If translated into field conditions, this finding may have implications in FCR inoculum build‐up under elevated CO₂ conditions. The data obtained from a free‐air CO₂ enrichment (FACE) facility, however, did not show any correlation between pathogen biomass and stem browning under different CO₂ levels. In addition, no correlation was evident between increased CO₂ levels and saprophytic fitness measured as the rate of colonization of wheat straw by the pathogen (Melloy et al., 2010). A recent glasshouse study conducted under moisture non‐limiting conditions found that cooler diurnal temperatures (e.g. 15/15 °C vs. 25/15 °C) increased the aggressiveness of F. pseudograminearum (Sabburg et al., 2015). Although this finding seems to be somewhat contradictory to the view that warmer and drier conditions, which often occur together, promote FCR, the unlimited availability of moisture during these experiments might explain the observed effects. The combined effects of altered temperatures and elevated CO₂ on the fitness of this pathogen are currently unknown.

Fusarium pseudograminearum genome

The advent of new technologies, such as next‐generation sequencing, has led to the generation of large amounts of sequence data from fungal genomes in recent years, significantly advancing our basic understanding of virulence‐related processes. The genomes of several Australian isolates of F. pseudograminearum have also been sequenced recently (Gardiner et al., 2012, 2017; Moolhuijzen et al., 2013). Not surprisingly, the F. pseudograminearum genome shows close resemblance to the previously sequenced genome of F. graminearum. The F. pseudograminearum genome is estimated to be 36.8 Mbp, which is similar in size to the F. graminearum genome (Gardiner et al., 2012). These two pathogens also contain similar numbers (approximately 12 000) of protein‐coding genes distributed across four chromosomes (Gale et al., 2005; Gardiner et al., 2012, 2017). The main differences between the two genomes were found in telomeric regions that undergo rapid evolution (Gardiner et al., 2012). More recently, a genetic map which revealed four chromosomes with nearly complete genome assembly was constructed for F. pseudograminearum (Gardiner et al., 2017). This map not only confirmed the previously observed synteny between F. pseudograminearum and F. graminearum, but also revealed various rearrangements affecting chromosome ends in these species (Gardiner et al., 2017). One of the rearrangements identified shows the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of phytoalexins by F. pseudograminearum (Gardiner et al., 2017).

Fusarium pseudograminearum virulence factors

The sequencing and comparative analyses of multiple F. pseudograminearum genomes have also provided new insights into the processes involved in pathogen virulence. For instance, novel virulence genes encoding an amidohydrolase and a dienelactone hydrolase, present in a few distantly related cereal pathogens and in plant‐associated bacteria, but mostly absent in other fungi, were discovered in F. pseudograminearum (Gardiner et al., 2012). This finding has led to the proposal that such virulence genes may have been acquired through horizontal gene transfer (Gardiner et al., 2012, 2013). Although the molecular mechanisms involved have yet to be deciphered, the finding that these virulence genes often encode fungal enzymes with relatively broad substrate specificities suggests that these genes may target or detoxify plant defences (Fig. 3). Indeed, other fungal genes involved in the detoxification of wheat defences have been characterized from F. pseudograminearum. The BOAs are defensive compounds produced by wheat, maize and rye, and have the ability to inhibit F. pseudograminearum growth (Kettle et al., 2015a). A comparative analysis of the F. pseudograminearum genome with the genomes of related Fusarium species revealed that this pathogen contains a gene cluster called ‘Fusarium Detoxification of Benzoxazolinone (FDB)’. The FDB cluster, the genes of which are shared by the maize pathogen F. verticillioides (Glenn et al., 2002), encodes several enzymes involved in the degradation of BOAs. The genetic analysis of this cluster using gene knockouts has revealed that the FDB1 gene encoding a lactamase is required for BOA detoxification, as the FDB1 knockout strains have an attenuated ability to detoxify this compound (Kettle et al., 2015a). FDB2, another gene found in this cluster, encodes an N‐maloyl transferase enzyme involved in BOA degradation. Fusarium pseudograminearum FDB2 knockout strains show reduced virulence towards wheat heads (Kettle et al., 2015b), suggesting that the ability to detoxify this compound is important for plant colonization. Finally, the FDB3 gene, encoding a zinc finger transcription factor, is required for 6‐methoxy‐benzoxazolin‐2‐one (MBOA) degradation by regulating the expression of other genes located in the FDB cluster (Kettle et al., 2016). Together, these studies suggest that F. pseudograminearum has evolved to successfully neutralize the potentially toxic effects of plant defensive compounds.

Toxins and secondary metabolites produced by F. pseudograminearum

Similarly to other Fusaria, F. pseudograminearum produces a number of secondary metabolites. These metabolites can act as pathogen virulence factors, but also have the potential to contaminate food and feed produced from infected plants. For instance, the type‐B trichothecene DON (3‐acetyl and 15‐acetyl‐deoxynivalenol, 3A‐DON and 15A‐DON) is an important metabolite produced by this pathogen. The majority of Australian F. pseudograminearum isolates produce 3A‐DON (Chakraborty et al., 2006; Obanor and Chakraborty, 2014), whereas some New Zealand F. pseudograminearum isolates reportedly produce nivalenol (NIV), another trichothecene (Monds et al., 2005). The fitness of F. pseudograminearum during stem base colonization is correlated with the level of DON production (Tunali et al., 2012). There is also evidence that F. pseudograminearum produces more DON during plant infection than during saprophytic growth (Tunali et al., 2012), suggesting that the host plant can elicit DON production in the pathogen. In a controlled environment study, the infection of wheat stems by F. pseudograminearum also led to the accumulation of DON in the heads of the infected plants, although the toxin levels were much lower than those commonly observed after F. graminearum infection of wheat heads (Mudge et al., 2006). Moreover, a role for DON in stem colonization was proposed based on the efficiency with which wild‐type and DON‐deficient tri5 mutants of F. graminearum could be recovered from the stems of inoculated wheat plants (Mudge et al., 2006). Furthermore, relatively low levels of fungal biomass are observed in plants inoculated with the tri5 F. graminearum mutant (Desmond et al., 2008b). In addition, Powell et al. (2017b) observed reduced pathogen virulence in the F. pseudograminearum tri5 mutant. Together, these findings suggest that DON can contribute to the virulence of F. pseudograminearum.

Many secondary metabolites in fungi are synthesized by non‐ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) (Ma et al., 2014). Recently, the lipopeptide W493 B, encoded by a gene cluster that contains NPRS32, was characterized from F. pseudograminearum (Sørensen et al., 2014). In contrast with DON, W 493 B does not seem to be required for pathogen virulence, as NRPS32 deletion mutants do not exhibit virulence‐associated phenotypes (Sørensen et al., 2014). The F. pseudograminearum genome is estimated to encode at least 16 NRPSs and 14 PKSs (Hansen et al., 2015), suggesting that this pathogen has the ability to produce many other secondary metabolites yet to be identified. Both horizontal and vertical gene transfer events have been proposed to be responsible for the generation of these metabolite‐encoding gene clusters (Ma et al., 2014). The number of such secondary metabolite‐encoding genes and gene clusters present in Fusaria is intriguing, and the analyses of these genes could reveal potential roles of metabolites in the saprophytic and/or pathogenic lifecycles of F. pseudograminearum.

Conclusions and Future Prospects

The studies reviewed in this article clearly show that significant progress has been made recently to better understand the interactions between F. pseudograminearum and cereal hosts. It is hoped that this knowledge will lead to the development of new strategies to tackle this disease problem in the future. In this section, attempts are made to identify potential future research areas to help manage this disease problem.

It is becoming evident that integrated strategies that employ suitable management options in addition to growing partially resistant or tolerant cultivars are important for the management of this disease, especially when high FCR risk is predicted. Some cultural practices, such as deep cultivation of soil, can be effective in reducing disease development, particularly in areas in which soil moisture is not a limiting factor. Changing the time of sowing could exploit weather conditions that are unfavourable to the pathogen. Given the strong influence of environmental factors on FCR resistance, the question of how a changing climate might affect FCR disease management will be increasingly relevant in the future (Chakraborty et al., 2000). Thus far, most efforts in this area have concentrated on the effects of elevated CO₂ on pathogen aggressiveness (Melloy et al., 2010, 2014). However, the predicted increase in global CO₂ levels is only a single aspect of climate change. Altered temperatures, reduced rainfall and altered soil moisture levels resulting from climate change could also directly or indirectly affect the incidence of FCR, as wheat‐growing areas (e.g. in Australia) gradually become warmer and drier (Hochman et al., 2017). A recent study has concluded that the effect of climate change on moisture availability will be greater than the effect of temperature change in the USA (Fei et al., 2017). Although the association between water stress and FCR disease symptom development has long been known (Papendick and Cook, 1974), it is unknown whether drought‐tolerant wheat and barley varieties would also be tolerant to FCR. Evaluation of the existing drought‐tolerant wheat and barley varieties for FCR resistance or tolerance would be useful to test this possibility.

The diversity observed in F. pseudograminearum isolates certainly has implications for breeding for FCR resistance. Although more extensive tests are required, there is at least one report indicating that FCR‐resistant wheat varieties from Australia can maintain their resistance status when tested against local isolates in the Pacific Northwest (Smiley et al., 2004). If confirmed for other germplasm and regions, this finding could make the use of global germplasm for FCR breeding possible. However, there is the possibility that increased aggressiveness may arise in pathogen populations if the same resistance genes or strong QTLs are widely deployed (Miedaner et al., 2008). Therefore, wherever possible, the use of various resistance sources, or the combination of QTLs with different modes of action, may constitute a useful risk management strategy.

Durum wheats are more susceptible to FCR than are bread wheats, but the underlying reason(s) for this are not clear. Improvement of FCR resistance in durum varieties may require the identification of novel resistance sources, which may be available in unadapted germplasm. Indeed, the wild relatives of durum wheat, including T. monococcum, T. timopheevii, T. turgidum var. dicoccum and T. turgidum var. carthlicum, show enhanced resistance to FCR (Davis et al., 2008). However, to the best of our knowledge, these resistance sources are yet to be utilized to improve FCR resistance in durum wheat. However, it should be noted that the incorporation of FCR resistance from wild relatives into adapted varieties may require time, especially if linkages are found between the genes conditioning FCR resistance and undesirable traits. The transfer of FCR resistance from bread to durum wheat can be another option to improve the resistance of durum varieties, and preliminary reports suggest that such transfer is technically possible (Ma et al., 2012b; Martin et al., 2013).

Thus far, many QTLs providing varying degrees of FCR resistance have been identified in wheat and barley (Bovill et al., 2006; Chen et al., 2013a, 2013b; Ma et al., 2010; Martin et al., 2015; Poole et al., 2012; Wallwork et al., 2004). Pyramiding multiple QTLs with different modes of action can increase the effectiveness and durability of FCR resistance (Bovill et al., 2010; Chen et al., 2015). Genomic selection, which employs multiple molecular markers distributed throughout the whole genome, can be a valuable tool to maximize the accuracy of crown rot breeding programmes (Agostinelli et al., 2012; Habier et al., 2009). Although a number of QTLs providing partial FCR resistance are available, the mechanism(s) by which they provide disease resistance are unknown, as none of the genes underlying these loci have been cloned. However, it is expected that the genetic and genomic resources developed for some large‐effect QTLs (Chen et al., 2015; Ma et al., 2013) should make the cloning task feasible. The availability of cloned resistance genes would facilitate the interspecific transfer of resistance genes from one cereal species to another and enhance our knowledge concerning the processes involved in host defence against this pathogen.

A better understanding of pathogen infection strategies can also facilitate the identification of disease susceptibility genes and the implementation of effector‐mediated germplasm screening strategies. Despite the availability of the genome sequence for this pathogen, only a few virulence genes/effector candidates have been functionally characterized (Gardiner et al., 2012; Kettle et al., 2015a, b, 2016; Powell et al., 2017b). Nevertheless, it is becoming evident that F. pseudograminearum has evolved to overcome existing host defences (Gardiner et al., 2012; Kettle et al., 2015b). Therefore, the incorporation of new defence pathways into cereal hosts or the modification of existing ones through genetic modification (GM) or breeding approaches can potentially enhance FCR resistance in cereals.

Currently, little is known regarding the proteomic and metabolomic changes that occur in the host during infection. However, as reviewed here, transcriptome analyses have revealed a number of candidate genes putatively associated with pathogen resistance or susceptibility in wheat. However, to the best of our knowledge, none of these host genes have been functionally characterized. The modification of such genes can provide new options for the enhancement of disease resistance. For instance, the transgenic expression of genes involved in DON detoxification, which was shown to confer increased resistance to F. graminearum (Li et al., 2015), can be tested for FCR resistance, given that DON is also a virulence factor in F. pseudograminearum. Recently, host‐delivered RNA interference (RNAi) targeting essential genes in the pathogen has been shown to be an effective method of reducing FHB infection in wheat (Cheng et al., 2015). However, GM approaches required to achieve these outcomes can potentially limit the utilization of such technologies in the short term. Non‐GM RNAi approaches, such as spraying long double‐stranded RNAs (dsRNAs) to target critical pathogen genes (Koch et al., 2016), can be an alternative to GM approaches. The removal of potential susceptibility genes through mutational (Fitzgerald et al., 2015a) or clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9)‐mediated genome editing approaches (Wang et al., 2014) can also be effective at enhancing disease resistance.

The model cereal Brachypodium distachyon has recently been shown to be susceptible to infection by F. pseudograminearum (Fitzgerald et al., 2015b), suggesting that Brachypodium, which has a fully sequenced and annotated genome, has a large number of available mutants and is easily transformed, may be a suitable model for the identification of host genes that affect resistance and susceptibility to this pathogen.

In conclusion, significant progress has recently been made to dissect the interactions between F. pseudograminearum and cereal hosts. The adoption of integrated strategies, from the application of improved management techniques to the deployment of resistant varieties, is required to reduce the losses caused by F. pseudograminearum under changing climatic conditions. The knowledge to be gained from the study of both host and pathogen genetics and genomics will certainly contribute to these endeavours.