Foliar Inoculation of Phyllachora maydis into Corn Induces Infection and Local Spread in Field Environments.

Tar spot of corn (Zea mays L.) is a significant disease in the United States and Canada caused by Phyllachora maydis, an obligate biotroph fungus. However, field research critical for understanding and managing the disease has been hindered by a need for methods to inoculate corn with P. maydis in field environments. In this study, we developed and demonstrated the efficacy of a method to initiate tar spot in field settings using inoculations of corn leaves with P. maydis inoculum that had been stored at -20°C for 10 months. Stromata of P. maydis were observed 19 days after inoculations in two field experiments, and stromata resulting from secondary spread were initially observed 39 to 41 days after the initial inoculations. Tar spot was not present in the fields beyond the inoculated areas or localized spread area, signifying that the establishment of initial disease resulted solely from inoculations. This study enhances our understanding of inoculation and infection of corn with P. maydis and tar spot development in field environments. The results will aid new research into understanding the corn tar spot pathosystem and improving management strategies.

A new and effective method to induce infection of Phyllachora maydis into corn for tar spot studies in controlled environments.

BACKGROUND: Tar spot of corn is a significant and spreading disease in the continental U.S. and Canada caused by the obligate biotrophic fungus Phyllachora maydis. As of 2023, tar spot had been reported in 18 U.S. states and one Canadian Province. The symptoms of tar spot include chlorotic flecking followed by the formation of black stromata where conidia and ascospores are produced. Advancements in research and management for tar spot have been limited by a need for a reliable method to inoculate plants to enable the study of the disease. The goal of this study was to develop a reliable method to induce tar spot in controlled conditions. RESULTS: We induced infection of corn by P. maydis in 100% of inoculated plants with a new inoculation method. This method includes the use of vacuum-collection tools to extract ascospores from field-infected corn leaves, application of spores to leaves, and induction of the disease in the dark at high humidity and moderate temperatures. Infection and disease development were consistently achieved in four independent experiments on different corn hybrids and under different environmental conditions in a greenhouse and growth chamber. Disease induction was impacted by the source and storage conditions of spores, as tar spot was not induced with ascospores from leaves stored dry at 25 ºC for 5 months but was induced using ascospores from infected leaves stored at -20 ºC for 5 months. The time from inoculation to stromata formation was 10 to 12 days and ascospores were present 19 days after inoculation throughout our experiments. In addition to providing techniques that enable in-vitro experimentation, our research also provides fundamental insights into the conditions that favor tar spot epidemics. CONCLUSIONS: We developed a method to reliably inoculate corn with P. maydis. The method was validated by multiple independent experiments in which infection was induced in 100% of the plants, demonstrating its consistency in controlled conditions. This new method facilitates research on tar spot and provides opportunities to study the biology of P. maydis, the epidemiology of tar spot, and for identifying host resistance.

Assessment of symptom induction via artificial inoculation of the obligate biotrophic fungus Phyllachora maydis (Maubl.) on corn leaves.

OBJECTIVE: Tar spot is a foliar disease of corn caused by Phyllachora maydis, which produces signs in the form of stromata that bear conidia and ascospores. Phyllachora maydis cannot be cultured in media; therefore, the inoculum source for studying tar spot comprises leaves with stromata collected from naturally infected plants. Currently, there is no effective protocol to induce infection under controlled conditions. In this study, an inoculation method was assessed under greenhouse and growth chamber conditions to test whether stromata of P. maydis could be induced on corn leaves. RESULTS: Experiments resulted in incubation periods ranging between 18 and 20 days and stromata development at the beginning of corn growth stage VT-R1 (silk). The induced stromata of P. maydis were confirmed by microscopy, PCR, or both. From thirteen experiments conducted, four (31%) resulted in the successful production of stromata. Statistical analyses indicate that if an experiment is conducted, there are equal chances of obtaining successful or unsuccessful infections. The information from this study will be valuable for developing more reliable P. maydis inoculation methods in the future.