Performing Department
Botany & Plant Pathology
Non Technical Summary
Stenocarpella ear rot has been a problematic disease for corn growers in the United States and worldwide for over a century, but the disease has become increasingly important in U.S. maize production over the past two decades. The disease reduces yield and grain quality, at a time when global demand for maize is high. The increase in demand is due to expanding global markets and the use of maize in alternative fuel markets, such as ethanol. To meet the growing demand, maize production has increased in the U.S., and there are now over 83 million acres in production each year since 2014 (USDA, NASS). Some producers ignore traditional cultural management methods that are effective at reducing the S. maydis inoculum potential in commercial production. One of these changes is the widespread adoption of conservation tillage practices, which increases the amount of infected crop residue on the field surface that can serve as a source of inoculum for the disease. Short rotations between maize and other crops, or planting maize continuously is also a common practice in the Midwest that allows producers to capitalize on high prices and demand for maize, but increases the risk for Stenocarpella ear rot development. At the same time, commercial maize breeders have not incorporated Stenocarpella ear rot resistance into many of the commercially popular high yielding hybrids. The lack of host resistance, coupled with an increased inoculum base has created a consistent risk of Stenocarpella ear rot. When environmental conditions are favorable for disease development, this can lead to widespread outbreaks of the disease. In fact, an increase in rain events during the silking period of corn development has resulted in conducive conditions for S. maydis infection over most of the Corn Belt in the last several years. (Coakley et al., 1999). These factors have increased the significance of Stenocarpella ear rot in U.S. maize production, and therefore a greater understanding of disease development is necessary to protect this economically important crop.Although the specific economic impact of Stenocarpella ear rot has not been calculated, it is now one of the major diseases affecting maize yield, grain quality and storability. Diseased grain is light-weight and contains low nutritional value. Producers receive a reduced price (dockage) when infected grain is sold, but it is never segregated from healthy grain. In storage, the damaged kernels attract insects and storage fungi, which results in shortened storability time, and an increased need for grain management. The pathogen produces the mycotoxin diplodiatoxin, which can affect animal health. The toxin, which affects cattle and sheep, causes a variety of neurological symptoms, such as tremors and ataxia (Riet- Correa et al. 2013), suggesting potential risk to food safety.Although the research proposed here does not directly define methods to remediate the problem of Stenocarpella ear rot, it will provide fundamental information about the pathogen. Experimental results will provide a better understanding about the environmental conditions and other factors that control the disease cycle, especially related to the pycnidium (primary inoculum) and the reaction at the host/pathogen interface (infection process). This work also will lead to new information about diplodiatoxin biosynthesis and regulation. Because both pycnidial development and diplodiatoxin have not received much attention, this work will provide valuable information relevant to other diseases and mycotoxins.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The goal of the proposed research is to increase the knowledge base on the under-researched pathogen S. maydis. The host-pathogen interaction appears to be distinctive, notably the vital role of pycnidia in the disease cycle and the infection process of a seed pathogen. Development of a molecular system to study S. maydis will provide novel information that will contribute towards our understanding of pathogenesis, mycotoxin production, host resistance mechanisms, and fungicide efficacy.Objective 1. Generate and characterize mutants defective in diplodiatoxin biosynthesis and pycnidial development. We will use REMI (Restriction Enzyme Mediated Integration) as well as specific gene disruptions to generate mutants of S. maydis that can be used to better understand toxin production and reproductive aspects of this fungus.Objective 2. Profile gene expression in distinct stages of pycnidial development. We will use next generation sequencing technology to generate data that can be used for gene discovery.
Project Methods
An Agrobacterium tumefaciens-mediated transformation (ATMT) system was developed for S. maydis, with an A. tumefaciens strain containing the binary vector, pBHt2_sGFP (Woloshuk and Bluhm, unpublished). The method is a slight modification of the protocol of Chen et al. (2011); transfer DNA fragment contains cassettes for hygromycin resistant and Green Fluorescent Protein (GFP) expression are randomly integrated in the S. maydis genome. A vector containing the gene forG418 resistance is also available fo complementation experiments.The initial goal is to obtain 5000 individual ATMTs.To screen for mutants affected in diplodiatoxin biosynthesis, 5 mm plugs of oatmeal agar cultures will be taken from each transformant (2 weeks of age) and placed into a microfuge tube containing chloroform/methanol (1:1 v/v) (Steyn et al., 1973, Rao, et al., 2003). After extracting overnight, the chloroform/methanol extracts of individual transformants will be spotted to thin-layer chromatography (TLC) plates. ATMT transformants that have no or barely detectable diplodiatoxin in TLC assays will be further analyzed by GC-MS and LC-MS to determine the level of diplodiatoxin production (Wicklow et al. 2011).To identify mutants defective in pycnidial development, the 24-well culture dishes containing the ATMT strains will be grown in a light/dark cycle and under dark only condition. For the wild type, mature pycnidia should be produced under the light/dark cycle within two weeks. No pycnidia will be produced in the continuous dark conditions. Cultures will be examined with a dissecting microscope to identify strains with altered pycnidial development. In addition to pycnidia production, all ATMT transformants will be examined for defects in conidiation and conidium morphology. In this screen, we expect to identify mutants that are defective in the production of pycnidia and conidiogenesis.To identify the insertion sites, the TAIL-PCR technique (Chen et al., 2011; Mullin et al. 2001) will be used. Subsequent sequence data will be BLAST analyzed against the S. maydis reference genome database and against the GenBank database. We will conduct in silico analysis on the putative gene sequences to obtain as much structural and functional information as possible. The wild-type gene will be cloned and then reintroduce into the mutant by ATMT with a G418 selection vector to determine if they can complement the mutant phenotypes.For the genes that are confirmed to be important for pycnidial development and toxin production, more detailed functional characterization will be conducted. General approaches include using qRT-PCR to determine their expression profiles in different developmental and infection processes and using GFP as the reporter to determine their subcellular localization. In addition, for each gene, specific experiments will be designed to determine their functional domains or promoter elements. It is impossible to predict what genes will be identified in ATMT mutagenesis. The goal will be to discover novel genes that are involved in either mycotoxin production or pycnidial development.To identify genes with expression specifically associated with pycnidial development, gene expression analysis will be done on the four stages of pycnidial development 1) mycelial, 2) initial, 3) mid-development and 4) mature - before conidia release. The wild-type strain will be grown on oatmeal agar medium. Tissue of each stage will be carefully collected from several culture plates and pooled into one tube for RNA extraction. This procedure will be repeated in a second time as an independent experiment. RNA isolation and preparation will be done as described by Bluhm et al. (2008). High quality RNA will be sent to the Purdue University Agricultural Genomics Center for sequencing on an Illumina HiScanSQ sequencer. Data will be analyzed with CLC Genomics Workbench software licensed to Woloshuk. Differentially expressed genes and fold-changes will be determined as described in Niu et al. (2015).