Identification and assessment of two major QTLs for dwarf bunt resistance in winter wheat line 'IDO835'.

KEY MESSAGE: Two major dwarf bunt resistance QTLs were mapped to a known Bt9 locus and a novel locus. The associated KASP markers were developed and validated in other two populations. Dwarf bunt (DB), caused by Tilletia controversa J.G. Kühn, and common bunt (CB), caused by T. caries and T. foetida, are two destructive diseases that reduce grain yield and quality in wheat. Breeding for bunt-resistant cultivars is important in many wheat production areas, especially where organic wheat is grown. However, few molecular markers have been used in selection of bunt resistance. In the present study, a doubled haploid (DH) population derived from the bunt-resistant line 'IDO835' and the susceptible cultivar 'Moreland' was evaluated for DB resistance in a field nursery in Logan, Utah, for four growing seasons. The population was genotyped with the Illumina 90 K SNP iSelect marker platform. Two major QTLs were consistently identified on chromosomes 6DL (Q.DB.ui-6DL) and 7AL (Q.DB.ui-7AL), explaining up to 53% and 38% of the phenotypic variation, respectively. Comparative study suggested that Q.DB.ui-6DL was located in the same region as the CB resistance gene Bt9, and Q.DB.ui-7AL was located at a novel locus for bunt resistance. Based on Chinese Spring reference sequence and annotations (IWGSC RefSeq v1.1), both resistance QTLs were mapped to disease resistance gene-rich (NBS-LRR and kinase genes) regions. To validate the identified QTL and design user-friendly markers for MAS, five SNPs were converted to Kompetitive Allele-Specific PCR (KASP) markers and used to genotype two validation panels, including a DH population and a diverse winter wheat population from USDA-ARS National Small Grain Collection, as well as a Bt gene investigation panel, consisting of 15 bunt differential lines and 11 resistant lines.

A novel QTL associated with dwarf bunt resistance in Idaho 444 winter wheat.

KEY MESSAGE: A novel QTL, Q.DB.ui-7DS, and the PCR-based markers identified in the current study will accelerate variety development for resistance to dwarf and common bunt of wheat. Dwarf bunt [Tilletia controversa J.G. Kühn [as 'contraversa'], in Rabenhorst, Hedwigia 13: 188 (1874)] is a destructive disease of wheat (Triticum aestivum L.) that reduces grain yield and quality. A number of distinct genes conferring resistance to dwarf bunt have been used by breeding programs for nearly 100 years. However, few markers were identified that can be used in selection of dwarf bunt resistance. A recombinant inbred line (RIL) population derived from the bunt-resistant germplasm, Idaho 444 (IDO444), and the susceptible cultivar, Rio Blanco, was evaluated for phenotypic reaction to dwarf bunt inoculation in four trials in two locations (USU and USDA) over 3 years. The population was genotyped with the Diversity Arrays Technology (DArT) and the Illumina Infinium 9K iSelect marker platforms. A total of three QTL were detected, and resistant alleles were from IDO444. QTL Q.DB.ui-7DS on 7DS was determined based on the location of a DArT marker wPt-2565 (X116197), which was consistently detected and explained 32 to 56 % of phenotypic variation among the four trials. QTL Q.DB.ui-1A on 1A was detected in three Utah State University (USU) trials and explained 11-15 % of phenotypic variation. QTL Q.DB.ui-2B on 2B was detected in two USU and one United States Department of Agriculture (USDA) trials and explained up to 6 % of phenotypic variation. Two PCR-based markers were developed based on the sequence of wPt-2565 and validated in the RIL population and used in genotyping of dwarf bunt differential lines, known resistance sources, and resistant cultivars.

Identification of New Pathogenic Races of Common Bunt and Dwarf Bunt Fungi, and Evaluation of Known Races Using an Expanded Set of Differential Wheat Lines.

Pathogenic races of Tilletia caries and T. foetida, which cause common bunt of wheat (Triticum aestivum), and Tilletia contraversa, which causes dwarf bunt of wheat, have been identified previously by their reaction to 10 differential wheat lines, each containing single bunt resistance genes Bt1 through Bt10. The reactions of races to the differential wheat lines follow the classic gene-for gene system for host-pathogen interactions. The pathogens are closely related and resistance to both diseases in wheat is controlled by the same genes. To better define pathogenic races, six additional wheat lines containing the genes Bt11 through Bt15 and a wheat line with a resistance factor designated as Btp were added to the set of 10 differentials and tested with all named U.S. races of common bunt and dwarf bunt. In addition, new isolates of dwarf bunt, and common bunt from hybrids and field collections, were tested with all 16 differentials for race identification. Six new races of T. caries, five new races of T. foetida, and two new races of T. contraversa were identified. Races of common bunt virulent to Bt8 or Bt12, and dwarf bunt races virulent to the combinations of Bt11 and Bt12, and Bt8, Bt9, Bt10, Bt11, and Bt12, were identified for the first time. Comparison of the reactions of the common bunt races with the Bt14 and Bt15 differentials grown in different environments after initial infection showed that these genes are temperature sensitive, indicating they should be excluded from the set of differential lines to avoid ambiguity in determining virulent or avirulent reactions. In the previous list of bunt races, there were races that had the same reaction to the set of 10 differentials but were designated as different races. These races were not differentiated further with the six additional differentials, indicating that the duplicate races should be dropped from the list of pathogenic races. The new races of common bunt and dwarf bunt identified have unique patterns of virulence that allow specific targeting and elucidation of bunt resistance genes in wheat and will aid the development of bunt-resistant wheat cultivars.

Analysis of Induction and Establishment of Dwarf Bunt of Wheat Under Marginal Climatic Conditions.

Dwarf bunt caused by Tilletia contraversa is a disease of winter wheat that has a limited geographic distribution due to specific winter climate requirements. The pathogen is listed as a quarantine organism by several countries that may have wheat production areas with inadequate or marginal climate for the disease-in particular the People's Republic of China. Field experiments were conducted in the United States in an area of Kansas that is a climatic analog to the northern winter wheat areas of China to evaluate the risk of disease introduction into such areas. The soil surface of four replicate 2.8 × 9.75 m plots, planted with a highly susceptible cultivar, was inoculated with six teliospore concentrations ranging from 0.88 to 88,400 teliospores/cm2. A single initial inoculation was done in each of three nurseries planted during separate seasons followed by examination for disease for 4 to 6 years afterward. Any diseased spikes produced were crushed and returned to the plots where they were produced. One nursery had no disease during all six seasons. In two nurseries, the disease was induced at trace levels at the three highest inoculation rates. Disease carryover to the second year occurred during one year in one nursery in plots at the highest inoculation rate, but no disease occurred the following three seasons. A duplicate nursery planted in a disease conducive area in Utah demonstrated that the highest rate of inoculum used in the experiments was sufficient to cause almost 100% infection. This study demonstrated that in an area with marginal climatic conditions it was possible to induce transient trace levels of dwarf bunt, but the disease was not established even with a highly susceptible cultivar and high levels of inoculum. Our results support the conclusions of the 1999 Agreement on U.S.-China Agricultural Cooperation which set a tolerance for teliospores in grain, and supports the Risk Assessment Model for Importation of United States Milling Wheat Containing T. contraversa.

Survival of secondary sporidia of floret-infecting Tilletia species: implications for epidemiology.

Secondary sporidia of Tilletia horrida, T. indica, and T. walkeri initiate local infection of rice, wheat, and ryegrass florets, respectively, leading to disease in seed. Secondary sporidia are considered to be fragile and short lived. To examine this, secondary sporidia from agar cultures of these species were naturally discharged onto petri dish lids and were air-dried and maintained in the laboratory at 10 to 20% relative humidity (RH) at 20 to 22 degrees C, and at 40 to 50% RH at 18 degrees C. Lids were periodically inverted over fresh agar to determine viability of dried sporidia. Sporidia held 31 to 49 days at 10 to 20% RH and 56 to 88 days at 40 to 50% RH regenerated rapidly. Commonly, 18 h after lids with dried sporidia were inverted over agar, newly produced secondary sporidia had discharged onto the agar and produced extensive hyphal growth. There was no difference in the viability of sporidia that were initially dried rapidly or dried slowly over 10 h. Sporidia of T. horrida or T. indica dried on petri dish lids placed in the lower canopy of barley or wheat fields in Idaho and Arizona during early flag leaf to soft dough stages and held until crops were near or beyond maturity regenerated rapidly despite temperatures up to 46 degrees C and several days of RH < 20%. These results suggest that sporidia produced well prior to susceptible growth stages of the host can lay dormant in very dry field environments and then rapidly regenerate under humid rainy conditions associated with the diseases.

Effect of biofumigation with volatiles from Muscodor albus on the viability of Tilletia spp. teliospores.

Volatile organic compounds produced by the fungus Muscodor albus inhibit or kill numerous fungi. The effect of these volatiles was tested on dormant and physiologically active teliospores of the smut fungi Tilletia horrida, Tilletia indica, and Tilletia tritici, which cause kernel smut of rice, Karnal bunt of wheat, and common bunt of wheat, respectively. Reactivated rye grain culture of M. albus was used to fumigate dormant teliospores in dry Petri dishes and physiologically active teliospores on water agar for up to 5 days at 22 degrees C. Teliospores of all 3 species were incapable of germination when fumigated on agar for 5 days. When T. tritici on agar was fumigated only during the initial 48 h of incubation, viability was reduced by 73%-99%. Fumigation of dry loose teliospores of T. tritici caused a 69%-97% loss in viability, whereas teliospores within intact sori were not affected. Dormant teliospores of T. horrida and T. indica were not affected by M. albus volatiles. It appears that M. albus has potential as a seed or soil treatment for controlling seedling-infecting smuts where infection is initiated by germinating teliospores prior to seedling emergence. The volatiles were not effective for postharvest control of teliospores under conditions used in these experiments.

Nonsystemic bunt fungi--Tilletia indica and T. horrida: a review of history, systematics, and biology.

The genus Tilletia is a group of smut fungi that infects grasses either systemically or locally. Basic differences exist between the systemically infecting species, such as the common and dwarf bunt fungi, and locally infecting species. Tilletia indica, which causes Karnal bunt of wheat, and Tilletia horrida, which causes rice kernel smut, are two examples of locally infecting species on economically important crops. However, even species on noncultivated hosts can become important when occurring as contaminants in export grain and seed shipments. In this review, we focus on T. indica and the morphologically similar but distantly related T. horrida, considering history, systematics, and biology. In addition, the controversial generic placement and evolutionary relationships of these two species are discussed in light of recent molecular studies.

Susceptibility of Wheat to Tilletia indica During Stages of Spike Development.

ABSTRACT Karnal bunt of wheat is caused by the fungus Tilletia indica, which partially converts kernels into sori filled with teliospores. Despite minor overall yield and quality losses, the disease is of considerable international quarantine concern. Plant development stages reported susceptible to infection vary considerably. A study was designed to better define the susceptibility period by inoculating wheat spikes at different growth stages with naturally liberated secondary sporidia under optimal conditions for disease development. Spikes of a resistant and susceptible cultivar were inoculated at eight growth stages from awns emerging to soft dough. Spikes became susceptible only after emerging from the boot and continued to be susceptible up to soft dough stage at which low levels of disease occurred. Disease severity in both cultivars peaked when spikes were inoculated after complete emergence, but before the onset of anthesis. Disease levels tapered off gradually in spikes inoculated after anthesis. The results broaden the known susceptibility period of wheat to T. indica to include stages long after anthesis, and indicate that infection from airborne inoculum is not possible during boot or awns emerging stages, which are commonly referred to as the most susceptible stages.

Relationship Between Soilborne and Seedborne Inoculum Density and the Incidence of Dwarf Bunt of Wheat.

The incidence of dwarf bunt of wheat as a function of inoculum density was studied in a susceptible and a partially resistant cultivar at three disease-conducive locations for three seasons. Prior to seeding, plots were fumigated with methyl bromide to eliminate residual inoculum. Each cultivar was seeded into two 1.2-m rows in four replicates. The soil surface was inoculated with 0, 16 × 102, 16 × 103, 16 × 104, 16 × 105, and 16 × 106 teliospores of Tilletia controversa per row, or seed was inoculated with 0, 2 × 102, 2 × 103, 2 × 104, 2 × 105, and 2 × 106 teliospores per gram. To determine maximum possible infection, two 3.1-m rows of each cultivar were soil-surface inoculated at 10× the highest treatment rate. In the soil-inoculated plots, a minimum of 16 × 103 teliospores/row was needed to cause trace amounts of disease (0.6% maximum), even when the positive indicator treatment had up to 88% incidence. Only trace amounts or no disease occurred below the 16 × 105 rate. In the seed-inoculated plots, infection was rare and occurred only at inoculation rates of 2 × 105 teliospores/g or higher; the highest incidence was 0.4%.