Research Article: Development and application of a quantitative bioassay to evaluate maize silk resistance to corn earworm herbivory among progenies derived from Peruvian landrace Piura

Date Published: April 16, 2019

Publisher: Public Library of Science

Author(s): Miriam D. Lopez, Tesia S. Dennison, Tina M. Paque, Marna D. Yandeau-Nelson, Craig A. Abel, Nick Lauter, Miguel Lopez-Ferber.


Corn earworm (CEW), Helicoverpa zea (Boddie), (Lepidoptera: Noctuidae), is a major insect pest of corn (Zea mays spp. mays L.). CEW larvae feed on silks, kernels and cobs, causing substantial yield and quality losses both through herbivory and by vectoring pathogens. The long-term goal of this work is to elucidate the genetic and biochemical basis of a potentially novel CEW resistance source discovered in silk tissue of Piura 208, a Peruvian landrace of maize (PI 503849). We developed a quantitative CEW bioassay and tested it on four populations that contrast alleles from Piura 208 with those from GT119, a CEW-susceptible maize inbred line. In replicated analyses of two populations of F1:2 families, corn genotype accounts for 84% and 68% of the variance in CEW larval weights, and up to 60% of the variance in CEW pupation percentage, demonstrating both the success of the quantitative bioassay and the strength of the Piura 208 resistance mechanism. Analyses of two corresponding populations of BC1:2 families revealed substantially diminished effects of corn genotype on CEW weight gain and pupation. This loss of Piura 208-derived CEW resistance during backcrossing suggests complex (multi-genic) inheritance of a threshold-dependent mechanism. Technical factors in bioassay performance were also assessed, often by analyzing the 1,641 CEW larvae that were raised on control diet (meridic with no corn silks added). Minor, but statistically significant impacts on CEW weight gain, pupation, and mortality were attributable to multiple technical factors in the preparation, incubation and evaluation phases of the bioassay, demonstrating the importance of randomization, stratification, replication, and variable-tracking across the many steps of this quantitative CEW bioassay. Overall, these findings indicate that this scaled-up, quantitative CEW bioassay is fundamentally sound and that Piura 208-derived resistance alleles are experimentally tractable for genetic and mechanistic research using this approach.

Partial Text

Corn earworm, Helicoverpa zea (Boddie), (Lepidoptera: Noctuidae), is a widespread and well-studied insect pest of corn [1–5]. The larval stage of the corn earworm (CEW) is responsible for damage to many additional crops, including cotton, tobacco, beans, tomato, soybean, pepper, and many more, although maize is the preferred host. Adult CEWs are capable of long-distance migration; for example, populations in Iowa migrate from Southern states during each growing season. As a multivoltine pest with a short developmental period, facultative diapause, and high female fecundity, CEWs cycle through several generations even within a single temperate growing season, allowing for annual population expansion to damaging levels following initial colonization [1,2]. Female moths oviposit on emerged maize silks where the larvae begin feeding, eventually tunneling down the silk channel to feed on kernel and cob tissues. Larval feeding leads to yield and grain quality losses through pollination interference, vectoring of bacterial and fungal pathogens, and herbivory. Maize silks vary in suitability as a food source for CEW development, with some varieties facilitating fast growth and complete development, and others causing larval growth retardation and even death in some cases [3]. Preventing or slowing down CEW growth and development can provide significant benefits to corn producers, particularly if the exponential phase of CEW local population expansions can be reduced or eliminated.

Measuring inhibition effects of corn silks on CEW herbivory in a laboratory bioassay presents several challenges of scale and scope. To assure agricultural relevance of results, a robust genetically diverse colony of CEWs must be used, necessitating that experimental diets each be tested using at least a moderate-sized cohort of CEWs. In turn, this requires both a large amount of silk tissue per entry and a large incubation space. As each phase of the effort scales up, achieving consistency or being able to account for unavoidable inconsistencies increases the scope of the effort. To assess 439 silk-based test diets in this study, ~20,000 corn plants were raised to maturity and monitored daily during flowering to control the duration of silk exposure to the environment, which is known to affect the surface lipid metabolome of silks [25,26] that has been implicated in CEW resistance in prior studies [27–29]. Silks that had emerged from encasing husk leaves were specifically collected because CEW moths oviposit on emerged silks and thus this is the tissue CEW neonates initially feed on. Emerged silks collected from ~14,000 of these plants were then processed into test diets that were fed to ~12,000 CEWs raised in individual cells within an incubation chamber. Genetic differentiation among silk samples was assessed using mean CEW weights from 24 insects per test diet and three independent test diets per genetic entry. Technical aspects of the bioassay were also assessed using metadata collected from all phases of the process. The analysis of technical factors was conducted on a per insect basis, in some cases using only the data from CEWs that were fed control diet. Additional data analyzed in this study include daily weight measurements of CEWs raised on control diet, flowering time (days-to-silk) for the 439 plots, and microclimate measurements at six positions within the incubator across the bioassay runs, all of which are used for interpretation of biological and technical results.

The long-term goal of this work is to elucidate the genetic and biochemical mechanisms underpinning the CEW resistance discovered in silk tissue of Piura 208, a Peruvian landrace of maize (PI 503849). In this work, the quantitative CEW bioassay identified entry 100, a (GT119 x 91001)F1:2 family, as least permissive of CEW weight gain, the principle measure of resistance (Fig 3). We have used the remnant progeny of entry 100 to generate >500 advanced intercross doubled haploid lines (DHLs), which will facilitate detailed quantitative genetic and biochemical analyses. Several specialized metabolites have been implicated in resistance to lepidopteran herbivory of maize, belonging to the broad biochemical classes of flavonoids [15,17,19], benzoxazinoids [37,38], terpenes [39,40], oxylipins [41,42], and cuticular surface lipids [27–29]. Identification of the most resistant and most susceptible DHLs will be useful for metabolomic and entomological investigations to connect mechanism and function with causal genetic factors. When using only tens of test diets, the CEW bioassay can be augmented by quantifying differential effects on the timing of adult emergence, which lengthens generation time and creates mate-finding challenges [43,44]. Further extension could allow measuring effects on mating success and female fecundity, both of which can reduce damage even in cases where larvae are able to persist into adulthood. Lastly, Piura 208 has shown resistance to herbivory by larvae from a range of lepidopteran pests [3,21,22]. Thus, nearly isogenic contrasts among particular DHL pairs will be useful for testing efficacy against multiple pests for the individual mechanisms identified. Together, these efforts will enhance deployment of natural alleles for insect resistance that could improve food security and/or reduce grower dependence on insecticides.




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