Improved quantification of Fusarium pseudograminearum (Fusarium crown rot) using qPCR measurement of infection in multi-species winter cereal experiments.

Fusarium crown rot (FCR) causes significant grain yield loss in winter cereals around the world. Breeding for resistance and/or tolerance to FCR has been slow with relatively limited success. In this study, multi-species experiments were used to demonstrate an improved method to quantify FCR infection levels at plant maturity using quantitative PCR (qPCR), as well as the genotype yield retention using residual regression deviation. Using qPCR to measure FCR infection allowed a higher degree of resolution between genotypes than traditional visual stem basal browning assessments. The results were consistent across three environments with different levels of disease expression. The improved measure of FCR infection along with genotype yield retention allows for partitioning of both tolerance and partial resistance. Together these methods offer new insights into FCR partial resistance and its relative importance to tolerance in bread wheat and barley. This new approach offers a more robust, unbiased way to select for both FCR traits within breeding programs. Key message: Genetic gain for tolerance and partial resistance against Fusarium crown rot (FCR) in winter cereals has been impeded by laborious and variable visual measures of infection severity. This paper presents results of an improved method to quantify FCR infection that are strongly correlated to yield loss and reveal previously unrecognised partial resistance in barley and wheat varieties.

Interactions of Fusarium Crown Rot of Wheat with Nitrogen.

The cereal disease Fusarium crown rot (FCR), caused by the fungal pathogen Fusarium pseudograminearum (Fp), is a major constraint to cereal production worldwide. Nitrogen (N) fertilizer is estimated to be approximately 30% of the input costs for grain growers in Australia and is the primary driver of yield and grain protein levels. When targeting high yield and protein, generous nitrogen fertilizer applications are thought to result in large biomass production, which exacerbates FCR severity, reducing grain yield and quality. This research was undertaken to investigate the effect of temporal N availability in high-protein bread and durum wheat varieties on FCR severity. Laboratory and controlled environment experiments assessed the relationship between FCR and N at a mechanistic and plant level. An in vitro study demonstrated an increase in Fp mycelial growth under increased N availability, especially when N was supplied as urea compared with ammonium nitrate. Similarly, under controlled environmental conditions, increased soil N availability promoted FCR severity within infected plants. Stem N transfer efficiency was significantly decreased under FCR infection in both bread and durum wheat varieties by 4.5% and 10.2%, respectively. This new research demonstrates that FCR not only decreases yield and grain quality but appears to have previously unrecognised detrimental impacts on nitrogen-use efficiency in wheat. This indicates that the current impact of losses from FCR may also decrease N-use inefficiencies, as well as yield and quality penalties. An improved understanding of the interactions and restrictions of FCR infection may allow growers to better manage the disease through manipulation of the soil's temporal N availability.

Genetic variation for fusarium crown rot tolerance in durum wheat.

Tolerance to the cereal disease Fusarium crown rot (FCR) was investigated in a set of 34 durum wheat genotypes, with Suntop, (bread wheat) and EGA Bellaroi (durum) as tolerant and intolerant controls, in a series of replicated field trials over four years with inoculated (FCR-i) and non-inoculated (FCR-n) plots of the genotypes. The genotypes included conventional durum lines and lines derived from crossing durum with 2-49, a bread wheat genotype with the highest level of partial resistance to FCR. A split plot trial design was chosen to optimize the efficiency for the prediction of FCR tolerance for each genotype. A multi-environment trial (MET) analysis was undertaken which indicated that there was good repeatability of FCR tolerance across years. Based on an FCR tolerance index, Suntop was the most tolerant genotype and EGA Bellaroi was very intolerant, but some durum wheats had FCR tolerance indices which were comparable to Suntop. These included some conventional durum genotypes, V101030, TD1702, V11TD013*3X-63 and DBA Bindaroi, as well as genotypes from crosses with 2-49 (V114916 and V114942). The correlation between FCR tolerance and FCR-n yield predictions was moderately negative indicating it could be somewhat difficult to develop FCR-tolerant genotypes that are high yielding under low disease pressure. However, FCR tolerance showed a positive correlation with FCR-i yield predictions in seasons of high disease expression indicating it could be possible to screen for FCR tolerance using only FCR-i treatments. These results are the first demonstration of genetic diversity in durum germplasm for FCR tolerance and they provide a basis for breeding for this trait.

Fusaristatin A production negatively affects the growth and aggressiveness of the wheat pathogen Fusarium pseudograminearum.

Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.

Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens.

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

Using Yield Response Curves to Measure Variation in the Tolerance and Resistance of Wheat Cultivars to Fusarium Crown Rot.

The disease crown rot, caused predominantly by the fungal pathogen Fusarium pseudograminearum, is a major disease of winter cereals in many regions of the world, including Australia. A methodology is proposed, using response curves, to robustly estimate the relationship between grain yield and increasing crown rot pathogen burdens. Using data from a field experiment conducted in northern New South Wales, Australia in 2016, response curves were derived for five commercial wheat cultivars exposed to six increasing rates of crown rot inoculum, where the rates served to establish a range of crown rot pathogen burdens. In this way, the response curve methodology is fundamentally different from alternate approaches that rely on genetic or environmental variation to establish a range in pathogen burdens over which yield loss relationships are estimated. By manipulating only the rates of crown rot inoculum and, thus, pathogen burden directly, the number of additional confounding factors and interactions are minimized, enabling the robust estimation of the rate of change in yield due to increasing crown rot pathogen burdens for each cultivar. The methodology revealed variation in the rate of change in yield between cultivars, along with the extent of crown rot symptoms expressed by the cultivars. Variation in the rate of change in yield between cultivars provides definitive evidence of differences in the tolerance of commercial Australian wheat cultivars to crown rot caused by F. pseudograminearum, while variation in the extent of crown rot symptoms signifies differences in the resistance of the cultivars to this disease. The response curve methodology also revealed variation in how the different mechanisms of tolerance and resistance act to limit yield losses due to crown rot for different cultivars.

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana.

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12-kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici-repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non-synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non-ToxA isolate. This work demonstrates that proteins that confer host-specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.

Passage through alternative hosts changes the fitness of Fusarium graminearum and Fusarium pseudograminearum.

Species of the necrotrophic fungal pathogen Fusarium that cause head blight and crown rot of cereals including wheat also infect a number of alternative host plants. This raises the prospect of more damaging pathogen strains originating and persisting as highly successful saprophytes on hosts other than wheat. The immediate impact on pathogenic (aggressiveness) and saprophytic (growth rate and fecundity) behaviour of six isolates with low, moderate or high initial aggressiveness was examined in two species of Fusarium after their passage through 10 alternative plant hosts. One passage through alternative hosts significantly reduced the pathogenic fitness of most isolates, but this change was not associated with a concomitant change in their overall saprophytic behaviour. The overall weak association between aggressiveness, fecundity and growth rate both before and after passage through the alternative hosts indicate that pathogenic and saprophytic fitness traits may be independently controlled in both Fusarium species. Thus, there was no trade-off between pathogenic and saprophytic fitness in these necrotrophic plant pathogens.