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2000 STEEP III Progress ReportRESEARCH PROJECT
TITLE: Improved Methods for Evaluating Resistance To Cephalosporium
Stripe of Wheat INVESTIGATORS: Chris Mundt, Tom
Wolpert, and Lynda Ciuffetti, Botany and Plant Pathology, OSU INTERIM REPORT: PROJECT OBJECTIVES:
KEY WORDS: Cephalosporium stripe, conservation tillage, fungal toxins, molecular mapping STATEMENT OF PROBLEM: Cephalosporium stripe has become a limiting factor for many Pacific Northwest wheat growers in erosion-prone areas, especially when early planting and/or trashy fallow are practiced. Burning or plowing stubble and delayed seeding can provide substantial control of Cephalosporium stripe. However, these cultural control methods conflict strongly with attempts to control soil erosion. Though no soft white winter wheat cultivar shows complete resistance to Cephalosporium stripe, there is considerable variation in the degree of resistance among cultivars. Further, higher levels of resistance may be available in exotic germplasm. However, identifying resistance in breeding programs remains problematic. Expression of resistance is incomplete and environmentally dependent. In addition, the disease tends to be aggregated within fields, thus requiring large plots to make useful comparisons, which is not possible in early generations of cultivar development. ZONE OF INTEREST: low and intermediate rainfall ABSTRACT OF RESEARCH FINDINGS: Protocols were developed
to further characterize and improve the production of a toxic fraction
produced by Cephalosporium gramineum, causal agent of Cephalosporium stripe.
A rapid assay was developed that resulted in wilting by 72 hours after
treatment of excised leaves with the toxic fraction. Comparison of laboratory
with field data showed that sensitivity of 17 wheat genotypes to the toxic
fraction was highly correlated with disease reaction in the field. As
a group, the common wheats were most sensitive/susceptible, the club wheats
were least sensitive/susceptible, and durum wheats were intermediate.
A synthetic wheat that derived its D genome from Aegilops taushii was
the least sensitive to the toxin. Though all common wheat genotypes studied
were very sensitive to the toxic fraction, some showed a moderate level
of resistance in the field. In no case, however, did a toxin-insensitive
genotype show a susceptible disease reaction in the field. It thus appears
that toxin insensitivity may be an important mechanism of resistance to
Cephalosporium stripe, but that other mechanisms may be operative as well.
We are currently studying the association of toxin sensitivity with Cephalosporium
stripe susceptibility in the progeny of a molecular mapping population.
Heritability of toxin insensitivity seems to be quantitatively inherited,
but highly heritable. We are also increasing seed stocks of progeny resulting
from crosses of wheat cultivars adapted to the Pacific Northwest. These
progeny will also be evaluated for toxin sensitivity and disease susceptibility.
Results of the project will enable us to determine the potential for using
the toxin assay to rapidly screen for resistance to Cephalosporium stripe. RESULTS AND INTERPRETATION: Objectives 1 and 2. Toxin Production and Characterization - Culture conditions and extraction of the toxic fraction were first done according to the methods of Kobayashi and Ui (1979. Physiol. Plant Pathol. 14:129-133). Thin layer chromatography indicated that the substance produced dark blue spots under UV radiation, consistent with the properties of Graminin A. Extraction methodology was subsequently modified to further refine the toxic substance and increase its yield, as described below. Broth medium was prepared, dispensed in 500 ml flasks (200 ml/flask), and inoculated with C. gramineum from a seed culture to attain 2 x 105spores per ml. At 24 hr after inoculation, flasks were shaken well and then incubated in standing culture for 35 days at 250C. After 35 days of incubation, broth was sieved, passed through filter paper, and then a 47 mm Millipore filter. With this modified method, the most time-consuming step of rotary evaporation of the culture filtrate was avoided. Culture filtrate was directly extracted four times with ethyl acetate in a 2 L separating funnel. Ethyl acetate was added at one tenth of the volume of culture filtrate each time. Pooled ethyl acetate extracts were then subjected to rotary evaporation to give an oily residue. The oily residue was then solubilized in 18 ml chloroform and left under a hood overnight to dry. After drying, the crude toxin was dissolved in 5.4 ml of ethanol. One third of the sample, 1.8 ml, was diluted to 10ml with water and loaded onto a C18 reversed-phase Sep Pak cartridge, which had been equilibrated in water. The cartridge was then progressively eluted, three times, with 10 ml each of water, 25%, 50%, 75%, and 100% acetonitrile in water (v/v). This entire process was repeated two more times with the remainder of the ethanol-solubilized crude toxin and all equivalent fractions pooled. Each pooled fraction was subjected to rotary evaporation, dissolved in 6ml of chloroform, dried under a hood overnight, and finally dissolved in 900 µl ethanol. Initial assays indicated that the majority of the toxic activity was contained in the 75% acetonitrile/water fraction. Consequently, only this fraction was used for the genotype evaluations described below. Symptomology depended on concentration of the toxic fraction. We observed chlorosis of wheat leaves at low concentration (20µl/ml), but this chlorosis did not reliably distinguish among wheat genotypes. At higher concentration (60µl/ml), we obtained distinct wilt symptoms that consistently distinguished wheat genotypes, and this concentration was thus used in our assays. Taken as a whole, our revised procedures have greatly increased the amount of the toxic fraction that can be derived from the fungus. Further, we have found that the toxic fraction can be frozen for future use, without loss of activity. Objectives 3 and 4. Genetics of Resistance - Our first step was to develop a reliable assay system for reaction of wheat genotypes to the toxic fraction. To do so, 7-ml Solvent Saver scintillation vials were numbered and marked for each wheat genotype in three replications per run, plus one control vial. Each of the three treatment vials received 840 ìl water and 60 ìl ethanol-dissolved toxic fraction; control vials received 60 ìl ethanol. Leaves were excised from 14-day-old plants with scissors and placed in the marked vials, one leaf per vial. To provide uniformity of leaves for all genotypes, only second leaves were chosen from robust plants in each pot, and leaves were cut to the same length. Vials containing the leaves were kept standing in a paperboard rack in a growth chamber at 210C and a 16-hr photoperiod. After 24 hrs, 1 ml of water was added to protect the leaves from desiccation. Leaves started showing wilting within 24 hour of exposure to the toxic fraction and sensitive genotypes were severely wilted within 72 hr of exposure, when data were recorded. In contrast, control leaves did not change in appearance during this time. Wilting was evaluated on a continuous scale of 1 to 5, with 1 indicating no change in leaf appearance and 5 indicating a fully wilted and dried or necrotic leaf. Twenty wheat genotypes were evaluated, representing four taxonomic groups: 1) Common wheat, Triticum aestivum L. (AABBDD). These included 9 soft white winter wheat cultivars and one soft white winter wheat breeding line that are adapted to the Pacific Northwest (PNW) region of the USA, and one hard red cultivar, Opata 85. Opata 85 is a spring wheat cultivar developed at the International Maize and Wheat Improvement Center (CIMMYT) and a parent of the International Triticeae Mapping Initiative (ITMI) mapping population. 2) Five winter club wheat cultivars adapted to the Pacific Northwest. Club wheats are hexaploid (AABBDD) T. aestivum types with compact heads, and originate from a different and substantially smaller gene pool than the common wheats. 3) Three durum wheat genotypes, T. turgidum (AABB). These included two advanced winter durum lines from the Oregon State University wheat breeding program and one CIMMYT spring durum wheat cultivar (Altar 84) that is a "grandparent" of the ITMI mapping population (see below). 4) The synthetic hexaploid wheat M6, which is the second parent of the ITMI population. M6 was produced by crossing Altar 84 with an accession of Aegilops tauschii, followed by doubling chromosome number with colchicine to obtain a synthetic hexaploid. Two runs of three replications each were conducted with these 20 genotypes, and the run x treatment interaction was non-significant. Thus, the analysis of variance was combined over runs. The common wheat
genotypes were all highly sensitive to the toxic fraction (Table 1), with
a wilting rating of 4 or greater. Fischer's protected LSD indicates that
there is significant variation among genotypes within the common and club
groups, but no overlap between these two groups. The three durum wheats
showed highly similar reactions, and were intermediate between that of
the common and the club groups. The synthetic wheat M6, which is a parent
of the ITMI population, showed the highest level of resistance. Fortuitously,
the other parent of the ITMI population (Opata 85) was the most susceptible
genotype evaluated, thus providing an opportunity for studying inheritance
of resistance to the toxic fraction among progeny of this cross (see below).
Linear contrasts were highly significant (P £ 0.001) for the six
pairwise combinations among the four germplasm groups. This association
of germplasm groups with toxin insensitivity should be interpreted with
caution, however, as only three durums and one synthetic wheat have so
far been evaluated in this study. There was more variation among genotypes within germplasm groups for the field data than for the toxin data (Table 1). Nonetheless, rankings of mean disease levels for the three germplasm groups tested in the field were the same as for the toxin assay, with the common wheats most susceptible, the club wheats most resistant, and durum wheats intermediate. Wilting symptoms measured in growth chambers were significantly (P = 0.0001) correlated with percent whiteheads estimated at each site (Figs. 3 and 4). In addition, a soft red winter wheat from France with very high yield potential in northcentral Oregon and a higher level of Cephalosporium stripe resistance than Madsen has shown reduced toxin sensitivity in preliminary studies. Some common wheats showed resistance in the field despite their toxin sensitivity to the toxic fraction (Table 1). It is important to note, however, that we found no case of an insensitive genotype being susceptible in the field. These results suggest that toxin insensitivity may be an important mechanism of resistance to Cephalosporium stripe, but that other mechanisms can be operative as well. To establish causal relationships between toxin sensitivity and disease susceptibility requires studying these two traits in segregating populations. This is now being addressed in two ways. First, we are evaluating 112 recombinant inbred lines from the cross between M6 and Opata 85. The detached leaf assay described earlier was used to obtain toxin sensitivity data from the individual mapping progeny. Frequency distribution of the wilting data (Fig. 3) suggests a continuous distribution skewed towards insensitivity. Seventeen of the progeny were numerically less sensitive than M6 and three were more sensitive than Opata 85, though none of these differences were significant at P = 0.05 based on either Duncan's Multiple Range or Fisher's Least Significant Difference Test. Analysis of variance indicated that difference among the progeny are highly significant (P £ 0.0001) with respect to toxin sensitivity. Based on variance components analysis, the genetic component of variance was very large, representing 88% of the phenotypic variance. Heritability of sensitivity to the toxic fraction estimated on a genotype mean basis was 0.88, with a 90% confidence interval of 0.85 - 0.91. A second run of this experiment is currently underway to determine repeatability of the reactions. In addition, we are currently working on studies to identify quantitative trait loci (QTLs) associated with the toxin reaction. We have begun efforts to evaluate mapping population progeny in direct assays with the pathogen. Unfortunately, the mapping population is in a spring background, whereas Cephalosporium stripe is mainly a disease of winter wheat. However, preliminary studies with exposure of these genotypes to the pathogen in a liquid culture system in the greenhouse showed M6, Altar 84, and Opata 85 to be resistant, intermediate, and susceptible to Cephalosporium stripe, respectively, which is consistent with their reaction to the toxic fraction. There also seems to be clear variation among the individual mapping population progeny. As an additional approach, we have planted and inoculated the mapping population at Pendleton in fall 2000, in the hope that a mild winter will allow survival and field evaluation of these spring genotypes. We will also study the association between toxin insensitivity and Cephalosporium stripe susceptibility in segregating populations of genotypes adapted to the Pacific Northwest. Progeny of crosses between susceptible and moderately resistant winter wheat parents have been increased in the 1999-00 field season, including cultivars grown in the Pacific Northwest. These progeny will be tested against the toxin and with the pathogen in the field to determine if there is a genetic association between toxin insensitivity and disease susceptibility. The ultimate goal
of the project is to produce wheat cultivars with resistance to Cephalosporium
stripe combined with favorable quality, yield, and other important agronomic
characteristics. Such cultivars will greatly increase ability of wheat
growers to successfully implement conservation tillage practices in erosion-prone
areas. In the short-term, we will identify and exploit useable levels
of resistance in adapted germplasm. In the longer term, we hope to develop
gene introgressions with potentially very high levels of resistance from
synthetic populations via molecular markers. INTERACTION (COOPERATION) WITH OTHER SCIENTISTS CONDUCTING RELATED ACTIVITY: Information regarding the biology and control of Cephalosporium stripe is exchanged with other pathologists in the Pacific Northwest, primarily Tim Murray (WSU) and Dick Smiley (OSU). In addition, contact is maintained with wheat breeding programs in Idaho, Oregon, and Washington to identify promising lines for evaluation, and to provide breeding programs with information regarding reactions of wheat cultivars and lines to Cephalosporium stripe. PUBLICATIONS AND
PRESENTATIONS:
Rahman, M., Mundt, C.C., Wolpert, T.J., and Riera-Lizarazu, O. 200_. Sensitivity of wheat genotypes to a toxic fraction produced by Cephalosporium gramineum and correlation with disease susceptibility. Phytopathology:internal review. Table 1. Mean wilting reaction of 20 winter and spring wheat genotypes to a toxic fraction produced by C. gramineum in the laboratory and percentage of whiteheads expressed by 17 winter wheat genotypes in naturally infested fields at two locations.
Fig. 1. Correlation of leaf wilting in response to a toxic fraction produced by Cephalosporium gramineum with percent whiteheads produced by 17 wheat genotypes in a naturally infested field in Condon, OR.
Fig. 2. Correlation of leaf wilting in response to a toxic fraction produced by Cephalosporium gramineum with percent whiteheads produced by 17 wheat genotypes in a naturally infested field in Dufur, OR.
Fig. 3. Frequency distribution of wilting for 112 recombinant inbred line progeny resulting from a cross between the sensitive parent Opata 85 and the insensitive parent M6 after exposure to a toxic fraction produced by Cephalosporium gramineum. |
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