An international wheat diversity panel reveals novel sources of genetic resistance to tan spot in Australia.

KEY MESSAGE: Novel sources of genetic resistance to tan spot in Australia have been discovered using one-step GWAS and genomic prediction models that accounts for additive and non-additive genetic variation. Tan spot is a foliar disease in wheat caused by the fungal pathogen Pyrenophora tritici-repentis (Ptr) and has been reported to generate up to 50% yield losses under favourable disease conditions. Although farming management practices are available to reduce disease, the most economically sustainable approach is establishing genetic resistance through plant breeding. To further understand the genetic basis for disease resistance, we conducted a phenotypic and genetic analysis study using an international diversity panel of 192 wheat lines from the Maize and Wheat Improvement Centre (CIMMYT), the International Centre for Agriculture in the Dry Areas (ICARDA) and Australian (AUS) wheat research programmes. The panel was evaluated using Australian Ptr isolates in 12 experiments conducted in three Australian locations over two years, with assessment for tan spot symptoms at various plant development stages. Phenotypic modelling indicated high heritability for nearly all tan spot traits with ICARDA lines displaying the greatest average resistance. We then conducted a one-step whole-genome analysis of each trait using a high-density SNP array, revealing a large number of highly significant QTL exhibiting a distinct lack of repeatability across the traits. To better summarise the genetic resistance of the lines, a one-step genomic prediction of each tan spot trait was conducted by combining the additive and non-additive predicted genetic effects of the lines. This revealed multiple CIMMYT lines with broad genetic resistance across the developmental stages of the plant which can be utilised in Australian wheat breeding programmes to improve tan spot disease resistance.

Genetic biofortification of wheat with zinc: Opportunities to fine-tune zinc uptake, transport and grain loading.

Zinc (Zn) is an important micronutrient in the human body, and health complications associated with insufficient dietary intake of Zn can be overcome by increasing the bioavailable concentrations in edible parts of crops (biofortification). Wheat (Triticum aestivum L) is the most consumed cereal crop in the world; therefore, it is an excellent target for Zn biofortification programs. Knowledge of the physiological and molecular processes that regulate Zn concentration in the wheat grain is restricted, inhibiting the success of genetic Zn biofortification programs. This review helps break this nexus by advancing understanding of those processes, including speciation regulated uptake, root to shoot transport, remobilisation, grain loading and distribution of Zn in wheat grain. Furthermore, new insights to genetic Zn biofortification of wheat are discussed, and where data are limited, we draw upon information for other cereals and Fe distribution. We identify the loading and distribution of Zn in grain as major bottlenecks for biofortification, recognising anatomical barriers in the vascular region at the base of the grain, and physiological and molecular restrictions localised in the crease region as major limitations. Movement of Zn from the endosperm cavity into the modified aleurone, aleurone and then to the endosperm is mainly regulated by ZIP and YSL transporters. Zn complexation with phytic acid in the aleurone limits Zn mobility into the endosperm. These insights, together with synchrotron-X-ray-fluorescence microscopy, support the hypothesis that a focus on the mechanisms of Zn loading into the grain will provide new opportunities for Zn biofortification of wheat.

Dimorphism in Neopseudocercosporella capsellae, an Emerging Pathogen Causing White Leaf Spot Disease of Brassicas.

White leaf spot pathogen: Neopseudocercosporella capsellae causes significant damage to many economically important Brassicaceae crops, including oilseed rape through foliar, stem, and pod lesions under cool and wet conditions. A lack of information on critical aspects of the pathogen's life cycle limits the development of effective control measures. The presence of single-celled spores along with multi-celled conidia on cotyledons inoculated with multi-celled conidia suggested that the multi-celled conidia were able to form single-celled spores on the host surface. This study was designed to demonstrate N. capsellae morphological plasticity, which allows the shift between a yeast-like single-celled phase and the multi-celled hyphal phase. Separate experiments were designed to illustrate the pathogen's morphological transformation to single-celled yeast phase from multi-celled hyphae or multi-celled macroconidia in-vitro and in-planta. Results confirmed the ability of N. capsellae to switch between two morphologies (septate hyphae and single-celled yeast phase) on a range of artificial culture media (in-vitro) or in-planta on the host surface before infection occurs. The hyphae-to-yeast transformation occurred through the production of two morphologically distinguishable blastospore (blastoconidia) types (meso-blastospores and micro-blastospores), and arthrospores (arthroconidia).

White Leaf Spot Caused by Neopseudocercosporella capsellae: A Re-emerging Disease of Brassicaceae.

White leaf spot can cause significant damage to many economically important Brassicaceae crops, including oilseed rape, vegetable, condiment, and fodder Brassica species, and recently has been identified as a re-emerging disease. The causal agent, Neopseudocercosporella capsellae, produces foliar, stem, and pod lesions under favorable weather conditions. N. capsellae secretes cercosporin, a non-host specific, photo-activated toxin, into the host tissue during the early infection process. The pathogen has an active parasitic stage on the living host and a sexual or asexual saprobic stage on the dead host. Where the sexual stage exists, ascospores initiate the new disease cycle, while in the absence of the sexual stage, conidia produced by the asexual stage initiate new disease cycles. Distribution of the pathogen is worldwide; however, epidemiology and disease severity differ between countries or continents, with it being more destructive in Subtropical, Mediterranean, or Temperate climate regions with cool and wet climates. The pathogen has a wide host range within Brassicaceae. Brassica germplasm show varied responses from highly susceptible to completely resistant to pathogen invasion and significant susceptibility differences are observed among major crop species. Cultural practices only provide effective disease control when the climate is not conducive. An increase in the susceptible host population and favorable weather conditions have together favored the recent rise in white leaf spot disease occurrence and spread. The lack of understanding of variation in pathogen virulence and associated resistant gene sources within brassicas critically limits the potential to develop efficient control measures.

Loci on chromosomes 1A and 2A affect resistance to tan (yellow) spot in wheat populations not segregating for tsn1.

KEY MESSAGE: QTL for tan spot resistance were mapped on wheat chromosomes 1A and 2A. Lines were developed with resistance alleles at these loci and at the tsn1 locus on chromosome 5B. These lines expressed significantly higher resistance than the parent with tsn1 only. Tan spot (syn. yellow spot and yellow leaf spot) caused by Pyrenophora tritici-repentis is an important foliar disease of wheat in Australia. Few resistance genes have been mapped in Australian germplasm and only one, known as tsn1 located on chromosome 5B, is known in Australian breeding programs. This gene confers insensitivity to the fungal effector ToxA. The main aim of this study was to map novel resistance loci in two populations: Calingiri/Wyalkatchem, which is fixed for the ToxA-insensitivity allele tsn1, and IGW2574/Annuello, which is fixed for the ToxA-sensitivity allele Tsn1. A second aim was to combine new loci with tsn1 to develop lines with improved resistance. Tan spot severity was evaluated at various growth stages and in multiple environments. Symptom severity traits exhibited quantitative variation. The most significant quantitative trait loci (QTL) were detected on chromosomes 2A and 1A. The QTL on 2A explained up to 29.2% of the genotypic variation in the Calingiri/Wyalkatchem population with the resistance allele contributed by Wyalkatchem. The QTL on 1A explained up to 28.1% of the genotypic variation in the IGW2574/Annuello population with the resistance allele contributed by Annuello. The resistance alleles at both QTL were successfully combined with tsn1 to develop lines that express significantly better resistance at both seedling and adult plant stages than Calingiri which has tsn1 only.

Draft Genome Sequence of the Plant-Pathogenic Soil Fungus Rhizoctonia solani Anastomosis Group 3 Strain Rhs1AP.

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼ 994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression.

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota.

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.

Comparative mycotoxin profiles of Gibberella zeae populations from barley, wheat, potatoes, and sugar beets.

Gibberella zeae is one of the most devastating pathogens of barley and wheat in the United States. The fungus also infects noncereal crops, such as potatoes and sugar beets, and the genetic relationships among barley, wheat, potato, and sugar beet isolates indicate high levels of similarity. However, little is known about the toxigenic potential of G. zeae isolates from potatoes and sugar beets. A total of 336 isolates of G. zeae from barley, wheat, potatoes, and sugar beets were collected and analyzed by TRI (trichothecene biosynthesis gene)-based PCR assays. To verify the TRI-based PCR detection of genetic markers by chemical analysis, 45 representative isolates were grown in rice cultures for 28 days and 15 trichothecenes and 2 zearalenone (ZEA) analogs were quantified using gas chromatography-mass spectrometry. TRI-based PCR assays revealed that all isolates had the deoxynivalenol (DON) marker. The frequencies of isolates with the 15-acetyl-deoxynivalenol (15-ADON) marker were higher than those of isolates with the 3-acetyl-deoxynivalenol (3-ADON) marker among isolates from all four crops. Fusarium head blight (FHB)-resistant wheat cultivars had little or no influence on the diversity of isolates associated with the 3-ADON and 15-ADON markers. However, the frequency of isolates with the 3-ADON marker among isolates from the Langdon, ND, sampling site was higher than those among isolates from the Carrington and Minot, ND, sites. In chemical analyses, DON, 3-ADON, 15-ADON, b-ZEA, and ZEA were detected. All isolates produced DON (1 to 782 microg/g) and ZEA (1 to 623 microg/g). These findings may be useful for monitoring mycotoxin contamination and for formulating FHB management strategies for these crops.

Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia.

A collection of isolates of Rhizoctonia solani anastomosis group (AG) 2 was examined for genetic diversity and pathogenicity. Anastomosis reactions classified the majority of isolates into the known subgroups of AG 2-1 and AG 2-2 but the classification of several isolates was ambiguous. Morphological characters were consistent with the species, with no discriminating characters existing between subgroups. Vertical PAGE of pectic enzymes enabled the separation of zymogram group (ZG) 5 and 6 within AG 2-1, but not the separation of ZG 4 and 10 within AG 2-2. PCR analysis using inter-simple sequence repeats (ISSR) and the intron-splice junction (ISJ) region supported the separation of ZG 5 and 6, while the AG 2-2 isolates were separated by geographic region. A comparison of distance matrices produced by the zymogram analysis and PCR indicated a strong correlation between the marker types. Pathogenicity studies suggested canola (Brassica napus) cultivars were most severely affected by AG 2-1, while cultivars of two species of medic (Medicago truncatula cv. Caliph and M. littoralis cv. Herald) were susceptible to both AG 2-1 and 2-2. The results indicate that AG 2 is a polyphyletic group in which the classification of subtypes is sometimes difficult. Further investigation of the population structure within Australia is required to determine the extent and origin of the observed diversity.

Nonpathogenic Binucleate Rhizoctonia spp. and Benzothiadiazole Protect Cotton Seedlings Against Rhizoctonia Damping-Off and Alternaria Leaf Spot in Cotton.

ABSTRACT Recent reports have shown induction of resistance to Rhizoctonia root rot using nonpathogenic strains of binucleate Rhizoctonia spp. (np-BNR). This study evaluates the biocontrol ability of several np-BNR isolates against root and foliar diseases of cotton in greenhouse trials, provides evidence for induced systemic resistance (ISR) as a mechanism in this biocontrol, and compares the disease control provided by np-BNR with that provided by the chemical inducer benzothiadiazole (BTH). Pretreatment of cotton seedlings with np-BNR isolates provided good protection against pre- and post-emergence damping-off caused by a virulent strain of Rhizoctonia solani (AG-4). Seedling stand of protected cotton was significantly higher (P < 0.05) than that of nonprotected seedlings. Several np-BNR isolates significantly reduced disease severity. The combination of BTH and np-BNR provided significant protection against seedling rot and leaf spot in cotton; however, the degree of disease reduction was comparable to that obtained with np-BNR treatment alone. Significant reduction in leaf spot symptoms caused by Alternaria macrospora occurred on cotyledons pretreated with np-BNR or sprayed with BTH, and the np- BNR-treated seedlings had significantly less leaf spot than BTH-treated seedlings. The results demonstrate that np-BNR isolates can protect cotton from infections caused by both root and leaf pathogens and that disease control was superior to that observed with a chemical inducer.

Persistence of DNA of Gaeumannomyces graminis var. tritici in soil as measured by a DNA-based assay.

There are an increasing number of assays available for fungal plant pathogens based on DNA technology. We have developed such an assay for Gaeumannomyces graminis var. tritici (Ggt) in soil, using slot-blot hybridisation. To ensure the validity of DNA-based soil assays for the fungus, it is important to determine the stability of Ggt DNA in soil. This study was undertaken to quantify the DNA degradation of dead Ggt in soil using a DNA-based assay. Mycelia were killed using various treatments, then DNA was extracted and estimated by a slot-blot hybridisation technique using the specific Ggt DNA probe, pG158. Mycelia were also killed using a fungicide (triadimefon) at a concentration of 150-250 microg ml(-1). The amount of detectable DNA of Ggt, killed using triadimefon, declined by 82-93%. Inoculum in the form of diseased wheat roots, artificially inoculated ryegrass seed, particulate soil organic matter and whole soil was killed using heat-treatment. The amount of detectable DNA of Ggt declined markedly (90%) in both heat-treated roots and inoculated ryegrass seeds, and declined by 50% in both treated soil and soil organic matter. The rate of DNA degradation of Ggt in soil varied with the type of inoculum. The amount of detectable DNA of Ggt in dead mycelia declined by 99.8% after 4 days of incubation in soil. No DNA was detected after 8 days of incubation. In contrast, Ggt DNA in live mycelia declined by 70% after 8 days of incubation and declined to 10% of original DNA level after 32 days. In ground ryegrass seed inoculum, DNA in both killed and live Ggt declined by 50% after 8 days. In diseased roots, DNA from both live and killed Ggt did not appear to decline over 16 days. Estimates of the amount of Ggt in the soil using a DNA-based assay reflect both live and dead populations of the fungus. The rate of breakdown of DNA of the dead fungus is very high and the presence of dead fungi in roots probably a rare event so the DNA from dead fungus probably contributes little to the total DNA level.