PCR-Based Detection of Cephalosporium gramineum in Winter Wheat

A polymerase chain reaction (PCR) assay was developed amplifying a 496-bp fragment of the internal transcribed spacer region of Cephalosporium gramineum genomic DNA at concentrations of 100 fg/μl. Winter wheat seed and seedlings were collected from field plots where C. gramineum was present. Seed was tested by PCR using 20-seed samples bulked for DNA extraction. Estimates of seed infection, based on isolation of the pathogen on semiselective medium and PCR, were comparable at 0.18 and 0.13% of winter wheat ‘Stephens’ (P = 0.6042), and 0.45 and 0.58% of experimental line WA7970 (P = 0.5636), respectively. PCR differentiated between plants with well-developed symptoms of Cephalosporium stripe and noninoculated plants. Positive PCR was obtained from 22% of asymptomatic leaf blades from inoculated plants. We found no false positives when PCR and C. gramineum isolation on a semiselective medium were performed using tissue from the same leaf. The PCR assay has potential to diagnose Cephalosporium stripe disease prior to the appearance of symptoms. Negative PCR for some samples from which C. gramineum was isolated suggests that C. gramineum may be present below the level of detection in some asymptomatic leaves. This PCR assay may be useful for investigations into C. gramineum infection of wheat.

Relationship between Incidence of Cephalosporium Stripe and Yield Loss in Winter Wheat

Cephalosporium stripe (caused byCephalosporium gramineum) can be a serious disease of winter wheat (Triticum aestivumL.) in the Pacific Northwest of the USA. Effects of Cephalosporium stripe on yield, test weight, protein, and kernel characteristics were examined using 12 winter wheat varieties in field plots inoculated and not inoculated with the pathogen. Averaged over varieties, inoculation decreased yield, test weight, kernel weight, and kernel diameter; grain protein and the standard deviations of kernel weight and kernel diameter were increased by inoculation. Grain yield of the susceptible check was reduced by as much as 41% with addition of inoculum. The most resistant and the most susceptible varieties performed similarly for yield in the two environments, while varieties with intermediate levels of resistance were sometimes inconsistent. There was a linear relationship between yield and % whiteheads (sterile heads caused by disease) in one environment and a curvilinear relation in the other.

First Report of Cephalosporium gramineum, Causal Agent of Cephalosporium Stripe of Wheat, in a Commercial Winter Wheat Field in Virginia

Cephalosporium stripe (CS) (2) was identified in a commercial field of winter wheat (Triticum aestivum) near Riner, Montgomery County, Virginia in May 2006. Nearly 15% of the field was severely affected. Broad, yellow-brown stripes were observed on the leaf blades of affected plants, and many plants were stunted and had ripened prematurely. Symptomatic plants were associated with low acidic (pH 5.2), wet spots of the field. Leaves and nodes of affected plants were surface disinfested for 1 min in 5% sodium hypochlorite, plated on corn meal agar (CMA), and incubated at 20°C for 5 days. Cephalosporium gramineum was isolated from numerous plants. Cultures of the fungus produced hyaline conidiophores approximately 5 μm long and unicellular conidia 3 to 7 μm long. Aqueous suspensions of mycelia and conidia were prepared from pure cultures. Several spring wheat cultivars were wounded by severing the root mass and were inoculated when the fifth stem node was detectable (35 on Zadoks scale). Noninoculated plants were wounded as controls. Plants were kept in the greenhouse at temperatures of 22 to 27°C. After 14 days, inoculated plants produced symptoms of CS, and the fungus was reisolated from the leaves of these plants. No symptoms were observed on noninoculated control plants. Though CS had been observed in Virginia in research nurseries (1), to our knowledge, this is the first confirmed report of the disease in a commercial wheat field in Virginia. References: (1) J. B. Jones et al. Plant Dis. 64:325, 1980. (2) C. M. Stiles and T. D. Murray. Phytopathology 86:177, 1996.

Seed Transmission of Cephalosporium gramineum in Winter Wheat

Although isolation of Cephalosporium gramineum from wheat (Triticum aestivum) seed has been reported, development of Cephalosporium stripe in plants from infected seed has not been demonstrated experimentally. Winter wheat seed was collected from three experimental field plots where Cephalosporium stripe was present, and C. gramineum was isolated from the seed following surface-disinfection and incubation on a semi-selective medium. C. gramineum was isolated from 0.10 to 0.88% of seed from 11 of 12 cultivars in a field experiment at Pullman, WA, and from 0.10 to 0.30% of seed from 3 of 4 genotypes in a field experiment at Fort Hall, ID; differences among cultivars were not significant in either experiment. C. gramineum was isolated from 0.35 and 0.55% of cv. Stephens plants with no symptoms and severe symptoms, respectively, from a uniform seeding in Pullman. Seed of the four genotypes from Fort Hall and Stephens from Pullman were grown under controlled environment in a soilless potting mix with no added inoculum and in which C. gramineum was not detected. Symptoms of Cephalosporium stripe developed in 0.08 and 0.17% of Stephens and breeding line 87-00314A plants, respectively, from Fort Hall, and from 0.18 and 0.55% of Stephens plants with no symptoms and severe symptoms, respectively. Although development of Cephalosporium stripe in plants grown from seed lots harvested from diseased plants was low, infected seed can provide an important source of inoculum for introducing the pathogen and initiating epidemics in areas where the pathogen did not occur previously.

Perennial Wheat Germ Plasm Lines Resistant to Eyespot, Cephalosporium Stripe, and Wheat Streak Mosaic

A perennial wheat cropping system on the Palouse Prairie of eastern Washington may provide an alternative to the Federal Conservation Reserve Program and reduce soil erosion while providing a harvestable crop for growers. Twenty-four perennial wheat germ plasm lines resulting from crosses between wheat and wheatgrass were evaluated under controlled environment conditions for resistance to Wheat streak mosaic virus (WSMV), Cephalosporium gramineum, and Tapesia yallundae (anamorph Pseudocercosporella herpotrichoides var. herpotrichoides). Perennial wheat lines SS452, SS103, SS237, MT-2, and PI 550713 were resistant to all three pathogens. Eight lines (33%) were resistant to WSMV at 21°C and 25°C; AT3425 was resistant to WSMV at 21°C but not at 25°C. Thirteen lines (54%) were highly to moderately resistant to C. gramineum. Thirteen lines (54%) were resistant to T. yallundae in each experiment, but the reactions of four lines differed between experiments. The wheatgrasses Thinopyrum intermedium (PI 264770) and Thinopyrum ponticum (PI 206624) are reported as new sources of resistance to T. yallundae. Perennial wheat must have resistance to these diseases in order to be feasible as a crop in the Pacific Northwest.

Sensitivity of Wheat Genotypes to a Toxic Fraction Produced by Cephalosporium gramineum and Correlation with Disease Susceptibility

Cephalosporium stripe is an important disease of winter wheat (Triticum aestivum) in several areas of the world, especially where stubble mulch and early seeding are practiced to maintain soil moisture and prevent erosion. We developed a procedure to mass-produce a toxic fraction produced by Cephalosporium gramineum through a modification of the method of Kobayashi and Ui. Exposure of excised wheat leaves to a concentration of 60 μl/ml of the toxic fraction for 72 h produced distinct wilting symptoms that allowed us to distinguish toxin-sensitive wheat genotypes in a repeatable manner. Twenty wheat genotypes belonging to four distinct germ plasm groups (common, club, durum, and synthetic) were evaluated. Variation in toxin sensitivity of wheat genotypes was mostly at the level of the germ plasm group, and all differences among the four germ plasm groups were highly significant (P < 0.001) based on linear contrasts. Seventeen winter wheat genotypes representing the common, club, and durum germ plasm groups were planted in C. gramineum-infested fields at two locations. The logarithm of the percentage of tillers showing whitehead symptoms at each of the two locations was significantly (P < 0.0001) correlated with wilting symptoms measured by the toxin assay (r = 0.80 and 0.84). The common wheat genotypes were all sensitive to the toxic fraction, but showed a substantial range of disease reactions in the field. However, we found no case of a toxin-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 are operative as well. The toxin assay may be useful as an initial screening procedure to reduce the number of genotypes to be tested in the field.

Infection of Winter Wheat by a β-Glucuronidase-Transformed Isolate of Cephalosporium gramineum

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 colonization 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 resistant than in susceptible cultivars.

A Hydroponic Seedling Assay for Resistance to Cephalosporium Stripe of Wheat

A procedure was developed to rate winter wheat seedlings approximately 20 days old for resistance to Cephalosporium stripe, a vascular wilt caused by the soilborne fungus Cephalosporium gramineum. Seedlings were inoculated after 12 to 15 days of growth in liquid culture in controlled-environment chambers, then assessed for disease symptoms at 7 to 8 days post-inoculation. Disease severity was assayed by measuring chlorophyll in the youngest fully expanded leaf, using a chlorophyll meter. Four replicated trials tested a total of 12 winter wheat cultivars, including both hard red cultivars from the U.S. Southern Plains and soft white winter cultivars from the U.S. Pacific Northwest. With one exception, the procedure consistently ranked cultivars correctly, according to field performance, as moderately resistant or susceptible. Jagger, a moderately resistant, hard red Kansas wheat, was ranked with susceptible cultivars in one of three trials.