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Evaluation of wheat cultivars for resistance is the basis of WHS resistance breeding programs. Chinese scientists have developed and utilized several evaluation techniques for testing the WHS resistance of wheat cultivars and breeding lines, and the virulence of pathogenic fusaria. Evaluation of wheat resistance to head scab should take into account the two well-established types of resistance to head scab: resistance to primary infection (Type I) and resistance to spread of scab in a spike (Type II) (Schroeder and Christensen, 1963). Resistance to head scab spread appears to be the major type of resistance in wheat cultivars studied to date (Xu and Chen, 1993). Field tests are the primary method used for evaluation of WHS resistance in China. Satisfactory results have also been obtained under greenhouse conditions. Most evaluation tests have been conducted on wheat spikes, but other wheat materials, such as leaves, mesophyllous protoplasts, and etiolated coleoptile tissues, may also be used for resistance evaluation to accelerate the resistance breeding process.

Spike Targeting Evaluation Techniques

Due to the sporadic nature of WHS epidemics, artificial inoculation is needed to secure stable selection pressure in breeding nurseries. Methods include the distribution of substrates that have been infested with F. graminearum on soil surface and the application of fungal spores to the wheat spikes. Among the techniques developed in China, scattering pathogen-infested wheat grain and injecting spores into single florets of spikes have been widely adopted.

Inoculation Methods

Entire spike inoculation
Wheat spikes can be infected by scattering pathogen-infested wheat grain in the field. In this method, autoclaved wheat grain is inoculated with F. graminearum, and incubated for five to seven days under 25 C. The F. graminearum infested grain is evenly scattered at a rate of 22 to 38 kg/hectare on the soil surface at the booting stage. A second application two weeks later, or 10 to 15 days before the heading stage, of the same amount of infested grain is necessary to produce enough inoculum at the flowering stage (Zhou and Xia, 1984; Bai, et al., 1989; Xu and Chen, 1993). Successful infection by this method depends on the temperature and moisture for ascospore development after inoculation. This method can be employed to evaluate both Type I resistance and Type II resistance, and is suitable for identifying resistant wheat cultivars and for preliminary screening of segregating populations for WHS resistance.

A variation of this method is to scatter stalks of corn and sorghum or other cereal plant residues in the wheat field in early spring. During the heading stage of wheat, perithecia develop in these residues, and release ascospores that infect wheat spikes in a manner similar to natural infection (Xu and Chen, 1993). With this method, however, it is difficult to control the ascospore dispersal time and to ensure an adequate level of inoculum at the flowering stage.

Spore suspensions can be sprayed over the wheat spikes at the flowering stage. Spraying in late afternoon gives a higher level of disease (Xu and Chen, 1993). This method is used to test for Type I resistance.

To inoculate spikes with pathogen infested wheat flour, wet wheat flour is inoculated with F. graminearum, and incubated for a few days, then air-dried and pulverized. The spikes are misted with water just before the powder is sprayed over the spike surface. Inoculation in late afternoon is advised in order to achieve a higher level of disease (Xu and Chen, 1993).

Single spikelet or single floret inoculation
Spore suspensions of 10 to 20 µl are carefully injected into a floret in a central spikelet of a spike (Wang and Yang et al., 1982). For laboratory tests, this method can also be applied to excised spikes that have been removed from plants by cutting below the top stem nodes during heading and early flowering stages (Wang and Yang et al., 1982; Xu and Fang, 1982; Xu and Chen, 1993). Inoculated spikes are put in a bottle half-filled with water, and kept in a moist chamber with 100% relative humidity at 25 C for two to three days. The bottles are then removed from moist chamber, and placed in the laboratory at 20 to 22 C under continuous light (Xu and Chen, 1993). This technique requires minimum space, and can be conducted under controlled conditions.

Alternatively, instead of injection of spore suspensions, a F. graminearum infested foxtail millet kernel can be placed within a single floret (Duan and Wang, 1989). This method can induce disease under either high or low humidity conditions (Duan and Wang, 1989).

Spikelets also can be infected by cutting glumes with scissors that are infested with spores. The glume tip, including the awn of a floret of a central spikelet, is cut off with scissors that have been dipped in a spore suspension. The spore suspension is thus carried by the scissors through the wound into the spikelet. The scissors should be dipped each time a new spikelet is inoculated (Wang and Yang et al., 1982; Xu and Chen, 1993). This method can be used to evaluate Type II resistance.

Time of Inoculation

All the spikelet inoculation methods are designed to evaluate Type II resistance, and can be applied at the heading stage, before the flowering stage, at the flowering stage, and at the grain filling stage (Duan and Wang, 1989; Cheng and Yang et al., 1994). Inoculation at milk stage, however, may induce disease in only 15% of inoculated spikelets (Duan and Wang, 1989). In most cases, inoculation at the heading and flowering stages is recommended.

Environmental Conditions

Successful induction of WHS requires efficient inoculation techniques, but also depends on suitable environmental conditions, including temperature and humidity. For 24 to 48 hours after inoculation, the temperature should be kept at 22 to 25 C and the relative humidity should be kept above 90%. In China, a variety of techniques are used to maintain high humidity (Xu and Chen, 1993). In the field or experimental plots, inoculated plants are misted by an overhead mist system for 10 to 15 minutes every hour from 7 a.m. to 10 a.m. and from 4 p.m. to 8 p.m. until symptoms appear on most of inoculated spikes. For small experimental plots or greenhouse tests, inoculated plants can be covered with transparent plastic bags or placed in a moisture chamber to maintain high humidity for at least 48 hours following inoculation.

Rating Scales

Several rating scales have been proposed in China for evaluation of WHS resistance. Evaluation using different rating scales may produce different results, thus it is very important to select a proper scale to get the most accurate and reproducible evaluation for WHS resistance.

The percentage of infected spikelets (disease severity) is the most frequently used criterion to evaluate Type II resistance, while the percentage of infected spikes (disease incidence) is often used to estimate Type I resistance (Bai and Chen et al., 1999). Several other modified rating scales have been proposed based on the percentage of infected spikelets. A more accurate scale for evaluation of Type II resistance was proposed to combine the number of scabbed spikelets and the number of diseased internodes on the rachis (Zhang and Pan et al., 1991). Cheng and Yang et al., (1994) compared four rating scales and suggested a "synthetic points" scale combining the number of scabbed florets and the degree of necrosis in rachis of each spike. The following formula was used to determine the synthetic points (D): D= F+ P. In this formula, F represents the number of diseased florets of a spike, P indicates the length (cm) of diseased rachis, and represents coefficient of disease severity ( =1 indicates that the lesion appears water-soaked or begins to turn brown, and =2 indicates that the lesion turns to deep brown, conidia form, or the spike shows blight symptoms). Since the synthetic point combines disease spread both on spikes and on the rachis, and reflects to a large extent the differences between various genotypes of wheat cultivars, the synthetic point scale has been recommended for WHS resistance evaluation (Cheng and Yang et al., 1994). This scale, however, has not yet been widely accepted in China because it is time-consuming and is not suitable for large-scale field evaluation.

Evaluation Using Leaves, Coleoptiles, and Protoplasts

To accelerate the process of breeding for WHS resistance, efforts have been made to develop new methods for evaluation of WHS resistance using materials other than spikes. These methods, however, have not received much attention from wheat breeders, since disease levels on tissues other than spikes may not reflect those in spikes. Clearly, there is still a need for development of easier, less expensive, more accurate, and more reliable methods for laboratory evaluation of WHS resistance.

Inoculation of leaves
In this technique, one of the top three, fully expanded leaves or any expanded leaves are excised, and are cut crosswise into three approximately equal pieces, of which only the basal piece is used for inoculation. The middle vein of the basal piece of the excised leaf is wounded with a needle, and inoculated with a small block of fungal mycelium. The inoculated leaf piece is kept in a Petri dish containing two layers of moist filter paper at 25 C for five days. The ratio of lesion width to leaf width was reportedly significantly correlated to percentage of scabbed spikelets in 19 wheat cultivars tested in the field following single spikelet inoculation (Peng and Yu, 1987; Yu and Peng et al., 1991). This technique can be carried out on leaves harvested at any time from seedling stage to heading stage, and was recommended for preliminary screening of breeding lines when only limited amounts of seeds are available. However, it has not been widely adopted in China because a correlation between resistance to leaf necrosis and resistance to head scab has not been well established.

Bioassay based on sensitivity to DON
A bioassay method based on the sensitivity of etiolated wheat coleoptiles to DON was suggested by the observation that the coleoptile response to DON differed among 21 wheat cultivars tested (Wang and Miller, 1989). WHS resistant cultivars Wangshuibai, Sumai 3, and Yangangfangzhu were less sensitive to DON, which was the most toxic among the seven trichothecenes tested (Wang and Miller, 1989). The sensitivity of etiolated coleoptiles of 18 wheat cultivars to culture extracts of F. graminearum was negatively correlated with their scab resistance in the field. Pure DON and the fungal culture extract had similar bioactivity to these 18 cultivars (Wang and Chen et al., 1989).

Another proposed bioassay method is based on sensitivity of mesophyllous protoplasts to DON (Huang and Liu et al., 1991; Huang and Liu et al., 1995). Protoplasts were treated with different concentrations of DON at 18 to 20 C for six hours. Then the protoplasts were treated with fluorescein diacetate and examined under a fluorescence microscope. Living protoplasts converted fluorescein diacetate to a substance that emitted yellowish green fluorescent light (Huang and Liu et al., 1991). For each specific cultivar, DON at an optimum concentration slightly, but significantly, increased the survival rate of treated protoplasts. Cultivars with different degrees of WHS resistance had different DON concentration optima for increasing protoplast survival. For moderately to highly resistant cultivars Suzhou 8113, Fanshanxiaomai, Sumai 3, Ning 7840, Wangshuibai, and Jianzimai, the optimum concentration of DON was 20 to 70 µg/ml. The optimum concentration of DON for moderately to highly susceptible cultivars Yangmai 5, Caijiexiaomai, Ningmai 6, Alondra's, Xuzhou 21, and 790-1 was lower than 20 g/ml (Huang and Liu et al., 1995). These bioassay techniques need to be improved and standardized before they can be used in WHS resistance breeding programs.

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