Home

About

Introduction

Pathogen Biology

Breeding

Resistance

Mechanisms

Evaluation

Disease Control

Conclusions

References

Researchers

Other Resources

Glossary

NAL

Search for information about enzymes and metabolic pathways in SoyBase

Agricultural Regions of China

Research on WHS resistance mechanisms began in China in the 1980s. The major focus has been histological study of structural differences and biochemical analysis of preformed and induced compounds in resistant and susceptible wheat cultivars.

Histology

F. graminearum infects wheat spike tissues primarily through anthers that protrude from the glumes, then spreads to the epidermis of the glumes and to the ovaries (Xu and Hideki, 1989). The fungus can also directly invade the external and internal epidermis of glumes, and can penetrate the external epidermis of anthers through natural openings (Chen and Xu, 1989; Xu and Hideki,1989). Generally, spread to the ovary is much faster than spread throughout the glume, which can result in partial infection of individual spikelets (Xu and Hideki,1989). From the infected floret, the mycelia of the fungus enter adjacent florets in the same spikelet, and then through the rachis enter adjacent spikelets in the same spike. Wheat cultivars differ greatly in resistance to spread of the fungus within the rachis. Comparative studies indicate that resistant cultivars are characterized by high density of vascular bundles in the rachis; smaller vessel diameters; greater and earlier thickening of the cortical sclerenchyma and its cell walls; closer internodes in the upper part of rachis joints; less mycelium in vessels of the rachis vascular bundles; and slower development of necrosis in the rachis (Zhang and Ye, 1993; Yu and Liu, et al., 1996). These characteristic structures in resistant cultivars are believed to limit the growth and spread of the fungal mycelium, and to reduce the effects of the fungal mycelial mass on water flow within the rachis which is considered to be one of the causes of spike blight symptoms (Yu and Liu et al., 1996). Thus, these structures have been histologically associated with head scab resistance mechanisms in wheat (Zhang and Ye, 1993).

Biochemistry

Preformed compounds and enzymes in resistant and susceptible cultivars

The preformed biochemical differences between resistant cultivars and susceptible cultivars may be one of the mechanisms for WHS resistance. The susceptible cultivar Nannong 824 was higher than Sumai 3 in level of chlorogenic acid, a phenolic compound that stimilates conidial germination and elongation of germ tubes of F. graminearum in culture (Ye and Xu et al., 1990). However, the level of total phenolic compounds in glumes or in spike tissues showed no significant difference between a resistant cultivar Sumai 3 and a susceptible cultivar Fan 6 (Wan and Ye, 1993). The percentage of scabbed spikelets was positively correlated with choline contents of spikes during anthesis (Li and Wu, 1994). Among the 13 cultivars tested, the level of choline in fresh spikes was 1,300 µg/g in the most susceptible cultivar Alondra ‘s', which is two-fold that in the most resistant cultivar Wangshuibai. Based on their results, Li and Wu (1994) suggested that low choline content during anthesis could be an indirect selection criterion for WHS resistance. This criterion, however, has not been adopted in wheat breeding programs, largely because analysis of choline is technically difficult and requires laboratory facilities that are not available to most wheat breeders in China.

The oxidative enzymes superoxide dismutase and catalase may be involved in WHS resistance. The activity of superoxide dismutase in spikes was significantly higher in resistant cultivar Wangshuibai (600 to 700 U/gfw) than in moderately resistant or susceptible cultivars Yangmai 4 and Ningmai 6 (300 to 500 U/gfw) (Chen and Song et al., 1997). Catalase activity was 20 to 35 U/gfw in Wangshuibai, and approximately 15 U/gfw in Yangmai 4 and Ningmai 6 (Chen and Song et al., 1997).

The enzyme phenylalanine ammonia lyase is generally accepted to be involved in plant disease resistance. In the case of WHS, however, phenylalanine ammonia lyase specific activity in top leaves at the jointing stage and in young spikes at the booting stage was much higher in the susceptible cultivar Nannong 824 than in Sumai 3 (Ye and Xu et al., 1990).

There are disagreements about the possible role of ascorbic acid in plant disease resistance. The content of ascorbic acid in plant tissues has been associated with resistance to some diseases, but with susceptiblity to others (Kiraly and Farkas, 1962; Arrigoni and Zacheo et al., 1979; El-Zahaby and Gullner et al., 1995). In the case of WHS, a study of four wheat cultivars showed that ascorbic acid content in spike tissues two days after heading in resistant cultivar Wangshuibai and moderately resistant cultivar Ning 8026 was two-fold that in the susceptible and moderately susceptible cultivars Ningmai 6 and Yangmai 3 (Chen and Ye et al., 1997). Further studies of F. graminearum showed that ascorbic acid stimulated germination of conidia and elongation of germ tubes at concentrations of one to five mg/L, and inhibited mycelial growth at 30 to 60 mg/L, but had no effect on mycelial growth at concentrations lower than 15 mg/L (Chen and Ye et al., 1997). Ascorbic acid content within mycelium of F. graminearum was not influenced by the concentration of ascorbic acid in growth medium (Chen and Ye et al., 1997).

Polyacrylamide gel electrophoresis analysis of soluble proteins in young spikes of wheat identified a unique polypeptide band in resistant cultivars Sumai 3, Wangshuibai, and Yangangfangzhu, but no detailed description of this protein was presented (Shi and Wang et al., 1998).

Induced compounds and enzymes in resistant and susceptible cultivars

Many biochemical responses occur in wheat following either infection by F. graminearum or treatment with the mycotoxins it produces. Superoxide dismutase activity in infected spikes increased in moderately resistant or susceptible cultivars Yangmai 4 and Ningmai 6, and decreased in resistant cultivar Wangshuibai (Chen and Song et al., 1997). Comparative analysis of superoxide dismutase isozyme profiles showed that among the six or seven SOD isozyme bands in infected tissues, four bands were from wheat and others were from F. graminearum. Therefore, the increased activity of superoxide dismutase in susceptible and moderately resistant cultivars was due to F. graminearum within the spike tissues (Song and Xu et al., 1995; Chen and Song et al., 1997). The contribution of the pathogen to superoxide dismutase activity increased as the disease severity increased. In addition, DON treatments increased superoxide dismutase activity of calli of both resistant cultivars and susceptible cultivars, but did not distinguish between these two kinds of cultivars (Song and Chen et al., 1997).

An increase in catalase activity in spikes after infection has been positively correlated with the resistance level of cultivars (Chen and Song et al., 1997). In resistant cultivar Wangshuibai, catalase activity in infected spikes increased by 0.6-23% as compared with uninfected spikes. In contrast, catalase activity of spikes after infection decreased by 1-42% in moderately resistant cultivar Yangmai 4 and susceptible cultivar Ningmai 6 (Chen and Song et al., 1997). When plantlets of susceptible and moderately susceptible cultivars Longfu 3 and Hei85-6497 were screened and regenerated under high DON selection pressure, they had higher catalase activities as compared with plantlets regenerated without DON treatment (Li and Li et al., 1995).

The relationship between phenylalanine ammonia lyase activity and WHS resistance is rather complicated. In three cultivars tested, the specific activity of phenylalanine ammonia lyase in infected spikes increased by 4 to 50% from two to four days after infection, and then decreased by 8 to 55% four to six days after infection, as compared to uninfected spikes. From six days to eight days after infection, the specific activity of phenylalanine ammonia lyase continued to decrease by 33 to 47% in resistant cultivar Sumai 3 and moderately resistant cultivar Yangmai 4, but increased by 34% in susceptible cultivar Nannong 824 as compared to the uninfected spikes (Ye and Xu et al., 1990). Another experiment showed that infected spikes of Sumai 3 reached their highest level of phenylalanine ammonia lyase activity (263% higher than its uninfected spikes) seven days after infection, and head scab symptoms did not appear until eight days after infection. In the susceptible cultivar Fan 6, phenylalanine ammonia lyase specific activity reached its highest level (352% higher than its uninfected spikes) eight days after infection, but symptoms appeared six days after infection (Wan and Ye, 1993).
Plantlets of Hei85-6497 and Longfu 3 that were selected and regenerated on DON-containing medium had higher phenylalanine ammonia lyase activity than plantlets regenerated on medium without DON (Li and Li et al., 1996). Recently, a heat-stable, bioactive glycoprotein isolated from hyphal cell walls of F. graminearum was demonstrated to enhance the activity of phenylalanine ammonia lyase in etiolated coleoptiles of the susceptible cultivar Ningmai 6. The carbohydrate portion of this glycoprotein was responsible for the bioactivity (Wu and Chen et al., 1997; Wu and Chen et al., 1999).

In addition to superoxide dismutase, catalase, and phenylalanine ammonia lyase, other enzymes, including peroxidase, ascorbic acid peroxidase, and ascorbic acid oxidase, may also play a role in WHS resistance. In resistant cultivars Sumai 3 and Wangshuibai, the specific activity of peroxidase in infected spikes increased until 16 days after infection. In susceptible cultivar Nannong 824 and moderately resistant cultivars Zheng 7495 and Yangmai 4, peroxidase specific activity increased until eight days, and then decreased until 16 days after infection (Xu and Ye et al., 1991). Zymograms of isoperoxidases present 10 days after infection showed that the three bands with isoelectric points (pI) of 0.3, 9.5 and 10.3, respectively, were more intense in Wangshuibai, Sumai 3, and Zheng 7495 than in other cultivars. In Nannong 824, the band with pI=6.3 were less intense, and the bands with pI=9.5 and 10.3 disappeared (Xu and Ye et al., 1991). Some peroxidase isozyme bands were enhanced in plantlets regenerated from calli treated with DON (Li and Li et al., 1995). When compared with uninfected spikes, ascorbic acid peroxidase activity in infected spikes increased by 274%, 85%, 47%, and -3% for resistant cultivar Wangshuibai, moderately susceptible cultivars Ning 8026 and Yangmai 3, and susceptible cultivar Ningmai 6, respectively. The ascorbic acid oxidase activity in infected spikes showed a pattern similar to ascorbic acid peroxidase activity, and increased by 277%, 122%, 155, and 14% for Wangshuibai, Ning 8026, Yangmai 3, and Ningmai 6, respectively (Chen and Ye et al., 1997).

Soluble protein profiles were determined by polyacrylamide gel electrophoresis in resistant cultivars and susceptible cultivars with or without DON treatment. After treatment with DON, three soluble protein bands disappeared in young spikes of susceptible cultivars Yiningxiaomai, Anhui 11, and Alondra ‘s', while no change occurred in protein profiles in resistant cultivars Sumai 3, Wangshuibai and Yangangfangzhu (Shi and Wang et al., 1998).

DON levels and wheat head scab resistance

DON is considered to be a virulence factor in F. graminearum (Wang and Miller, 1987; Lu and Wang et al., 1989; Xu and Chen, 1993; Desjardins and Proctor et al., 1996). When isolates of F. graminearum with various degrees of virulence were compared for DON production, a high positive correlation was observed between virulence and levels of DON produced in cultures on autoclaved wheat grain (Lu and Wang et al., 1989; Xu and Yao et al., 1990; Wei and Lu et al., 1990). Following incubation at 25 C for 14 days, and then at 18 to 20 C for another 14 days, culture extracts were analyzed for DON by thin layer chromatography. Among the 15 isolates tested, DON production was only 0.6 µg/g for a weakly virulent isolate M6-3 from Mexico, but 5 µg/g of DON for a highly virulent isolate JF-21 from Jiangsu Province, China (Xu and Yao et al., 1990). The virulence was significantly correlated with DON production, with a correlation coefficient of 0.9 (Xu and Yao et al., 1990). In another experiment, four isolates with different degree of virulence to wheat were cultured on autoclaved wheat grain at 25 C for 10 days, and then at 13C for another 10 days, and analyzed as above. Two highly virulent isolates F15 and F17 produced DON at 440 and 300 µg/g, respectively, and weakly virulent isolates F22 and F7 produced DON at 150 and 80 µg/g, respectively (Lu and Wang et al., 1989). High performance liquid chromatography analysis showed that isolates from different provinces in the Yangtze River region produced different levels of DON. When incubated with shaking at 150 rpm for 100 hours on Czepek's liquid medium with 1% peptone, average DON production was 1.4, 1.5, 2.3, 4.0, 4.2, 6.6 and 8.3 µg/ml, respectively, for isolates from Jiangxi Province (eight isolates), Hubei Province (10 isolates), the municipality of Shanghai (26 isolates), Hunan Province (nine isolates), Anhui Province (13 isolates), Zhejiang Province (10 isolates), and Jiangsu Province (nine isolates) (Wei and Lu et al., 1990). Whether these isolates can produce the related trichothecene, nivalenol, was not investigated, although nivalenol-producing isolates of F. graminearum have been found in several Asian countries.

In addition, wheat cultivars with various degrees of resistance to F. graminearum differ in the amount of DON accumulated in infected spike tissues. DON can be detected by gas chromatography in spike tissues as early as 24 hours after infection (Chen and Song et al., 1996). DON level was low (7.4 µg/gdw) in infected spikes of the resistant cultivar Wangshuibai and high (36 and 38 µg/gdw, respectively) in the susceptible cultivar Ningmai 6 and the moderately susceptible cultivar Yangmai 3. Moderately resistant cultivars, Yangmai 4 and Ning 8026, contained intermediate levels of DON (17 and 19 µg/gdw, respectively) (Chen and Song et al., 1996). DON level reached its peak eight days after infection and then declined steadily in Wangshuibai. In Yangmai 3 and Ningmai 6, DON level increased rapidly from four to 16 days after infection (Chen and Song et al., 1996). Although extracts from spikes have not been tested, extracts from wheat leaves of the resistant cultivars Sumai 3 and Fan 9 were found to degrade DON into a unknown compound that suppressed germination of F. graminearum macroconidia (Yao and Liu et al., 1996). This unknown compound had no effect on growth of wheat coleoptiles (Yao and Liu et al., 1996). Leaf extracts of the susceptible cultivars Ningmai 6 and Xuzhou 21 could not degrade DON (Yao and Liu et al., 1996). These preliminary results suggest that DON degradation in resistant cultivars may contribute to WHS resistance (Type III resistance, Miller and Young et al., 1986).

In summary, WHS resistance is a genetically quantitative trait, and resistance mechanisms are complex. Chinese scientists have been studying WHS resistance mechanisms for approximately 20 years and have made some progress, but no persuasive conclusions have been reached. Much additional research is needed for a better understanding of the nature of WHS resistance.

USDA, ARS, National Agricultural Library 10301 Baltimore Ave. Beltsville, MD 20705-2351 USA TEL (301)504-5755, TDD (301)504-6856, FAX (301)504-6927 E-mail: agref@nal.usda.gov, http://www.nal.usda.gov