Improved Method for Injecting Fungal Inoculum into Corn Ears

Anderson, N. R., Ravellette, J. D., and Wise, K. A. 2016. Improved method for injecting fungal inoculum into corn ears. Plant Health Prog. 17:163-166. Corn (Zea mays L.) ear rot pathogens reduce yield and grain quality annually, and research on these pathogens and their interactions with the host can require inoculation of fungal material into the ears. A new system of mechanically inoculating corn ears was designed using a hydration backpack and auto-filling vaccinator with a needle attached. The efficiency of the new system was compared to a previously established inoculation method using a manual syringe method with an 18-gauge blunt end needle attached to a 60-ml syringe, with inoculum carried in a plastic beaker. Inoculation methods were tested by comparing the time to inoculate 100 corn ears with separate conidial suspensions of Fusarium verticillioides and F. graminearum in a replicated field trial. The new mechanical inoculation system reduced inoculation time by 42% (P = 0.0015) when compared to the manual syringe and needle method. Additional benefits of the new method include reduced risk of inoculum contamination, consistent inoculum volume per ear, and increased safety for personnel doing the inoculations. IMPORTANCE OF EAR ROT RESEARCH Corn (Zea mays L.) ear rot diseases result in several hundred millions bushels of lost yield every year in the United States (Mueller and Wise 2014). This yield loss is compounded by the ability of some ear rot fungi to produce mycotoxins, which adversely affect grain quality and marketability. Some of these pathogens include Aspergillus flavus (Link), Fusarium graminearum (Schwabe), F. verticilliodes (Sacc.), and Stenocarpella maydis (Berk.). The associated mycotoxins aflatoxin, deoxynivalenol, fumonisin, and diplodiatoxin, respectively, can cause significant health effects when ingested by animals and humans (Gelderblom et al. 1988; IARC 1993; Richard 2007). Field-based research on these ear rot fungi aims to understand fungal infection mechanisms, isolate variability or differentiation, and the factors affecting mycotoxin production in grain, to name a few (Gendloff et al. 1986; Pope and McCarter 1992; Reid et al. 1996; Reid and Sinha 1998; Steyn et al. 1972; Vigier et al. 2001). Screening germplasm or commercial hybrids for ear rot resistance is also common; as is research to better manage diseases caused by ear rot fungi (Afolabi et al. 2007; Presello et al. 2005; Reid et al. 1993; Robertson et al. 2005; Sutton and Beliko 1981). Many of these fungi infect through natural openings in the corn plant or wounds caused by insects or adverse environmental conditions, or by fungal transportation through the silk channels (Koehler 1942). Therefore, research to examine isolate variability, mycotoxin production, and germplasm resistance often involves introducing inoculum to corn plants at a susceptible growth stage. Standard inoculation methods include spreading colonized grain over the soil surface of the research area, spraying a conidial suspension over the top of the plants, wounding plants or ears and then introducing inoculum to the injury site, or directly injecting fungal material into silk channels (Fig. 1) (Boling et al. 1969; Chungu et al. 1996; Clements et al. 2003; Drepper and Renfro 1990; King and Scott 1982; Schaafsma et al. 1997; Mario et al. 2011). Inoculating with grain inoculum or spraying conidial suspensions over plants simulates natural infection, but can result in inconsistent disease levels across different environments. Wound injection methods (Gulya et al. 1980; Chungu et al. 1996) can be time consuming, cause unintended destruction to the plant, and require considerable practice to ensure consistent inoculum delivery. Silk channel injections provide a consistent and reproducible way to get fungal inoculum into the ear. Several studies have evaluated the effectiveness of inoculation methods, including Clements et al. 2003, that suggested that an injection technique was the most suitable for producing the desired disease severity. This information led us to develop a simple, selfcontained fungal injection system, which improves upon current