Real-time PCR analysis of maize seedlings for assessment of seed treatment efficacy and genetic resistance to infection by Sphacelotheca reiliana
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Head smut caused by Sphacelotheca reiliana (S. reiliana) is a devastating disease of maize that has been reported to account for up to 80% yield loss in fields. This disease exists in many major maize growing regions and the disease incidence is rising. It has become a major constraint in seed production areas as the spores it produces adhere to and contaminate seeds. The fungus infects systemically during the seedling stage and usually does not produce symptoms until the plant reaches its reproductive stages when the fungus replaces the inflorescences with large black masses of teliospores known as sori. When the sori rupture spores contaminate soil and seed which will then serve as a source of inoculum and a dispersal unit for the fungus. Disease management options include host resistance and fungicidal seed treatments but both of these strategies need improvement; resistance in most commercial hybrids is not complete and more seed treatments need to be tested for efficacy against this disease. The strategies for evaluating both of these options require considerable time expenditure, as the plant does not show symptoms of infection throughout the growing season. With this in mind a seedling assay for the evaluation of host resistance and seed treatment efficacy would be advantageous.
A seedling assay was developed using two novel inoculation techniques to consistently and efficiently infect maize seedlings with S. reiliana. Seeds were planted in a growth chamber under controlled conditions that were conducive to infection. Presence of the fungus within seedling tissue was detected by a sensitive and specific real-time polymerase chain reaction. Seed inoculation provided more consistent results than soil infestation.
Five commercially available fungicidal seed treatments were tested for their efficacy against head smut infection, using the seedling assay. Infection rates of inoculated, mock-treated controls varied between 60 - 80% Incidence of infection. Tebuconazole, fludioxonil and sedaxane seed treatments reduced incidence of seedling infection by S. reiliana (p=0.01) whereas azoxystrobin did not. Three rates, representing the ranges of rates on the product labels of both tebuconazole and sedaxane, were also tested. No differences in seedling infection were detected among the rates (p=0.01). Sedaxane (0.1 mg*seed-1) eliminated detectable seedling infection when teliospores of S. reiliana were applied at a rate of 10 spores*seed-1.The assay was effective for seed treatment efficacy testing and can be a useful tool to screen experimental fungicides for their efficacy against head smut.
The seedling assay was used to evaluate five maize inbred lines for their ability to resist infection by S. reiliana. Inbreds included A619, B68, B73, Mo17 and Va26. Mo17 had lower incidence of infection compared to the other four inbreds (p=0.01) but no other significant differences between inbreds were detected.
We also evaluated maize tissue from a susceptible hybrid in field trials (in California and Texas) with inoculated seed and non-inoculated seed planted into S. reiliana infested soil. Seedling and ear shoot tests showed that S. reiliana DNA was present at rates as high as 54% of seedlings in Texas and 11% in California and 30% of ear shoots in Texas and 8% in California; despite these rates of infection no symptoms were observed by field scouting. This assay may be useful to compare initial infection of among maize genotypes, but the lack of symptom development indicates that seedling and ear shoot infection are not predictive of disease incidence. A high percentage of maize plants infected with S. reiliana may not exhibit any symptoms and this knowledge is important for designing management strategies. Further work is needed to better understand the implications of this result on the epidemiology of this disease and the mechanisms for resistance.