232 PHYTOPATHOLOGY Etiology Infection of Winter Wheat by a β-Glucuronidase-Transformed Isolate of Cephalosporium gramineum G. W. Douhan and T. D. Murray Department of Plant Pathology, Washington State University, Pullman 99164. Accepted for publication 20 November 2000. ABSTRACT Douhan, G. W., and Murray, T. D. 2001. Infection of winter wheat by a -glucuronidase-transformed isolate of Cephalosporium gramineum. Phytopathology 91:232-239. Field-grown winter wheat was inoculated with a β-glucuronidase- transformed isolate of Cephalosporium gramineum in two field seasons to elucidate the mode of infection in resistant and susceptible cultivars. Colonization of viable root epidermis and cortical cells occurred as soon as 15 days postinoculation and the pathogen was found in the vascular tissues by 20 days postinoculation, well before freezing soil temperatures occurred. Penetration occurred directly through the root epidermis and through wounds adjacent to emerging secondary roots. The pathogen also penetrated through root cap cells and colonized meristematic tissues near root tips to gain access to the vascular system. Lower stem base coloni- zation was observed where the pathogen penetrated directly through the epidermis, wounds, or senescent tissues. Appressorium-like structures, which appeared to aid penetration of cell walls, were often found within cells of both roots and stems after initial colonization. The mechanisms of resistance were not apparent, but less colonization occurred in resis- tant than in susceptible cultivars. Cephalosporium stripe of winter wheat (Triticum aestivum L.) is caused by the fungus Cephalosporium gramineum Nisikado and Ikata (sporodochial stage, Hymenula cerealis Ellis and Everh). C. gramineum is an economically damaging vascular wilt pathogen of small grain cereals in the northwestern United States where yield loss of up to 80% can occur on winter wheat when condi- tions are favorable for disease development (6,12,14). When cool, wet conditions exist in the autumn, small (5 to 11 × 1.5 to 3 μm) hyaline conidia produced on infested plant residues (7,34) are washed into the soil and serve as primary inoculum (13,34). Al- though infection is thought to take place through wounds in roots created by freeze-thaw cycles that occur during winter and early spring (2,7,19,20) or by damage caused by other means such as root-feeding insects (27), direct evidence for this does not exist. Mathre and Johnston (19) hypothesized that infection occurs when conidia are “vacuumed” into xylem vessels of damaged roots. In greenhouse experiments, however, Anderegg and Murray (1) demonstrated that root breakage is not a prerequisite for disease development and that severe disease can occur without soil freez- ing under conditions of low pH and high soil moisture. Some researchers believe that C. gramineum is strictly a xylem- inhabiting pathogen incapable of penetrating living cells (33), but Mathre and Johnston (18) suggested the pathogen has a rudimen- tary ability to penetrate and grow through living root cells. Bailey et al. (2) found that the epidermis and root cortex did not appear to serve as barriers to colonization. They found fungal hyphae could penetrate the root epidermis both intracellularly and intercellularly on tissues that had been frozen to –10°C and thawed overnight, but little colonization was found on nonfrozen roots. The frozen tissues appeared to be intact, as viewed by scanning electron mi- croscopy (SEM), and they concluded that freezing stress may be an important factor in predisposing wheat plants to active pene- tration and infection by C. gramineum. Bonde (5) also observed C. gramineum colonizing root cortical cells but did not find evi- dence of vascular infection. No work to date has demonstrated the process of vascular infection under field conditions. Variability in occurrence of Cephalosporium stripe from year to year has made screening for disease resistance difficult (7,16,17). Bruehl et al. (8) found that over a 5-year period susceptible culti- vars were consistently susceptible whereas cultivars with some resistance varied widely in the percentage of diseased stems. Variation in resistance screening methodologies has also confused conclusions about resistance. For example, Bruehl (6) categorized four cultivars as resistant to Cephalosporium stripe when inocu- lated with a hypodermic needle, but the same cultivars were sus- ceptible when grown under field conditions (25). There is great variation in the amount of disease between and within cultivars, and highly effective resistance to C. gramineum does not exist in wheat (6,8,15,26). Although resistance in winter wheat cultivars is not highly effective, some wheat relatives are resistant (18) and progeny from a cross between the wheat relative Agropyron elon- gatum (Host) Beav. (now called Thinopyrum ponticum [Podp.] Barkworth and D. R. Dewey [35]) and Triticum aestivum are highly resistant (20,32). Determining how C. gramineum infects and colonizes wheat has been difficult because these events take place in the soil dur- ing the winter. In addition, C. gramineum has nondescript hyphae and does not produce distinct lesions, which has made under- standing the general epidemiology, as well as host resistance diffi- cult. To overcome this problem, C. gramineum was transformed with the β-glucuronidase (GUS) reporter gene (24). GUS is nor- mally not produced in fungi (31), which makes it an ideal marker to study host–pathogen interactions. The GUS gene is constitu- tively expressed and its activity can be visually detected by incu- bation in the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) (30). The reaction produces an intensely blue dichloro-dibromoindigo (CIBr-indigo) precipitate that is stable throughout normal histological preparations (30). GUS-transformed fungi have been used successfully to study various host–pathogen interactions (9,17,31). No studies, however, have been conducted under field conditions. The objectives of this research were to elucidate the timing and mode of penetration and infection of winter Corresponding author: T. D. Murray; E-mail address: tim_murray@wsu.edu Publication no. P-2001-0102-03R © 2001 The American Phytopathological Society