Root cortical aerenchyma in maize hybrids in Pennsylvania and interaction with mycorrhiza and soil-borne pathogens

Root cortical aerenchyma in maize hybrids in Pennsylvania and interaction with mycorrhiza and soil-borne pathogens

Summary

In this project we study arbuscular mycorrhizal colonization (AMC), and root pathogen infection (PI) in relation to root cortical aerenchyma (RCA) of hybrids commercially planted in Pennsylvania. Two field seasons and two greenhouse experiments were proposed. Results from the first field harvest conducted in summer 2013 (see annual report 2013), indicate substantial variation in RCA and other anatomical variables among corn hybrids.  RCA varied from 0 – 30 %, with six lines showing RCA values > 20%, and three lines < 5% RCA. Other anatomical variables evaluated were cortical cell size (CCS) and metaxylem vessel number (MXV), with variations of 2x and 2.5x respectively, between the different hybrid lines. Variation in PI, measured as percent rotten root, was observed in the second and third crown whorls. PI ranged 5 – 75%, with most of the lines presenting intermediate values (~50% rotten root). By the end of 2014, we have completed the field experiments and are making progress on the sampling processing in the lab in order to determine RCA, AMC and PI, and the correlations between these variables.  We have also completed three greenhouse studies; one with the mychorrhizal forming fungi Rhizophagus intraradices and two with the pathogen Fusarium verticillioides.  We continue to analyze the samples taken in 2014 in three different locations in Pennsylvania. The samples have been prepared for anatomical and microbiological measurements. The contribution of this project to sustainable agriculture is focused on better understanding of anatomical traits in root systems of corn that confer resistance to drought and decreased nutrient availability and how such anatomical characteristics may affect interactions with soil microbes.

Objectives/Performance Targets

The overall objective of this project is to evaluate the interaction of the anatomical trait RCA of corn hybrids with soil microorganisms in Central Pennsylvania.  The soil microbes we are studying in this project are arbuscular mycorrhizae forming fungi and root pathogens.  The former is detected by measuring AMC, and the latter can be quantified as PI. AMC and PI will be used herein to denote the two soil microbe groups in reference to their interaction with roots.

We completed the activities planned by the end of 2014 which comprise:

1. 1. Field harvest and sample preparation of roots collected at different locations in Pennsylvania,

1. 1. Greenhouse study planning and preliminary experiments with the pathogen F. verticillioides,

1. 1. Greenhouse experiment with the mycorrhizal forming fungi R. intraradices;

1. 1. Data analysis of preliminary results.

 
We are currently performing laboratory analysis of root anatomy and AMC on the field samples collected in Summer 2014. Also, by means of two greenhouse pilot experiments in 2014, we have defined methods and techniques for addressing the specific objectives that include greenhouse with F. verticillioides  in 2015.  We will continue our collaboration with Dr. Gretchen Kuldau, plant pathologist, who have been helping with the root pathogen experiments in the greenhouse.  Dr. Gregory Roth is also collaborating with this project as Agronomist and Extension specialist for the field experiments and seed request. The detailed activities per objective executed this year are listed below:

2.1. Evaluate the variation of RCA of corn plants normally planted by farmers in Pennsylvania.

• • Root crowns were collected from 30 – 40 experimental plots of the 2014 Grain and Silage Hybrid Corn Test at three sites across Pennsylvania (Figure 1).  Three plants per plot (for a total of 900 crowns) were shoveled and washed, and two representative crown roots per plot were photographed and root segments preserved for further analysis.

• • Root segments from the crowns were obtained according to described methods (Burton et al., 2013). The segments were then laser-ablated and images of the cross-root sections were acquired and edited for the subsequent analysis with RootScan. This is the software in use for obtaining anatomical traits like RCA, developed by Burton et al. (2012)

2.2. Correlate RCA levels with AMC of corn plants normally planted by farmers in Pennsylvania.

• • Representative root subsamples from the plants described above were prepared for AMC assessment with the gridline-intersect method. The samples will be cleared and stained according to Vierheilig et al. (1998) before the measurements could be performed.  

• • A greenhouse experiment with nine corn genotypes showing contrasting levels of RCA was conducted. Plants were equally inoculated with a commercial source of R. intraradices and grown for 7 weeks. Samples were collected and prepared for further anatomical and mycorrhizal analysis.

2.3. Correlate RCA levels with PI of corn plants normally planted by farmers in Pennsylvania.

• • The field samples described in 2.1. were scored for root rots in the second and third whorls (in the inner part of the crown root, see Figure 2).

• • Two preliminary experiments were conducted in 2014 to confirm root infection by the fungal species F. verticillioides in corn seedlings. In the first experiment, two inoculation methods (soil spore infestation and seed inoculation), at two different concentrations (5.81×10⁵ and 4.65×10⁶ cells*mL^-1) were tested.  We confirmed fungal growth in the root tissue 4 weeks after planting, as shown by re-isolation of the fungus from root tissue collected from the pots after the growth period.  However, the extent of colonization in the cortex and on the epidermis was low, as shown by microscopic observations (Figure 3).  A second pilot experiment, in which the best inoculation technique and the best inoculant concentration, obtained in the first experiment, were used for a longer growth term (7 weeks). We expect to observe profuse fungal colonization in these roots. The samples will being analyzed in January, 2015 in order to define the best conditions for the final experiment in April 2015.

• • Part of this project is the quantification of the pathogen root infection with qPCR, for which a literature review was completed early in 2014. A protocol was drafted based on previous reports (Bottalico and Perrone, 2002; Mulè et al., 2004; Kristensen et al., 2005; Brandfass and Karlovsky, 2006; Niessen, 2007; Rahjoo et al., 2008; Nicolaisen et al., 2009; Kurtz, 2010; Kurtz et al., 2010; Faria et al., 2011; Nutz et al., 2011; WU et al., 2011). The draft includes: Plant and fungal DNA extraction, calibration curve with pure and combined DNA (from the fungus and the plant), and qPCR conditions. We will be evaluating this protocol in February 2015.

Fungicide-free (untreated) seeds have been requested by Dr. Gregory Roth, who is the direct contact with the seed providers in this project. These seeds, the greenhouse protocol, and the qPCR method will be used in the final experiment with F. verticillioides to be conducted in April 2015.

• Figures and references

Accomplishments/Milestones

With the results obtained from the field harvest in 2013, we identified 15 hybrid lines with contrasting levels of RCA in a set of 30 lines.  Although we originally proposed to sample these lines in a more comprehensive manner in 2014 at four different sites across Pennsylvania, we learned that the exact lines are not usually planted two consecutive years under commercial conditions by farmers in the U.S., as seed companies change varieties offered every year. So, we modified our plans accordingly and sampled 30 – 40 hybrid lines in 2014, in order to ensure enough variation in the root traits.  Also, we decreased the number of sites to three in order to compensate for the extra effort of sampling twice the number of genotypes originally proposed.

Another unexpected situation was the difficulty in getting untreated seeds for pathogen and mycorrhizal studies.  By the time we finished the root phenotyping of the 2013 samples, the seed companies had already harvested and treated their seeds with fungicide. So, we decided to use untreated corn seeds that we had available in our laboratory in the preliminary greenhouse experiments. The lines were inbred lines that we use for root studies as described previously (Burton et al., 2013).  A description of the activities in sequence throughout the year is as follows:

January – February 2014

• • Analyzed field data obtained from 2013. 15 corn lines with contrasting RCA levels identified.

• • Conducted preliminary experiment in the greenhouse to determine best inoculation technique and spore concentration for future experiments with F. verticillioides.

March – April 2014

• • Preformed literature review about qPCR for root pathogen colonization.

• • Analyzed data from the green house experiment conducted in January.

April – May 2014

• • Coordinated logistics for field sampling in July 2014.

• • Extracted DNA for qPCR protocol validation.

June – July 2014

• • Evaluated DNA quality extracted in May.

• • Sampled field experiments at three sites in Pennsylvania.

August – October 2014

• • Field samples laser-ablated and images acquired.

• • Greenhouse experiment with mycorrhizal forming fungi R. intraradices conducted.

• • Second preliminary greenhouse experiment with F. verticillioides conducted.

October – December 2014

• • Harvest of the two greenhouse experiments described above.

• • Started image analysis of the root cross sections acquired from field and greenhouse.

• • Prepared annual report.

Impacts and Contributions/Outcomes

The results obtained through this project will contribute to the development of sustainable agriculture by providing better understanding of root traits that could be used for selecting corn varieties with greater stress tolerance and reduced input requirements and how these traits interact with soil-borne microbes.   Specifically this year, we have advanced in the description of root rots and anatomical traits of 32 hybrids commercially planted in Pennsylvania in 2013.  We have finished the second and final field harvest at three sites in Pennsylvania, and completed the greenhouse experiment with a mycorrhizal inoculant. Additionally, we have explored methods and conditions for the pathogen experiments to be done at the greenhouse in Spring 2015.

Significant variation was found in anatomical traits and root rots in the field experiment harvested in 2013. For root rots, values ranged from 0 to 80 %, with 83% of the lines between 50% – 80%.  The root anatomical trait RCA varied 0 – 30%.  Accordingly, living cortical area (LCA) ranged 70 – 100 %. Metaxylem vessel number (MXV) presented values 10 – 25, and cortical cell files (CCF) were 9 – 16. These traits (RCA, LCA, CCF) have been proved, or are being studied (MXV) as influencing factors of maize performance, specially under stress conditions (reviewed in Lynch (2014)).  Studying these traits in hybrids commercially planted in Pennsylvania will inform us about the potentialities of the root traits as breeding targets to improve current corn materials. A combined study of root traits with soil-borne root pathogens extends the biological tradeoffs of selecting specific states of the root characteristics.

Collaborators: