(1) Screen adapted Kansas germplasm and KSVT entries for SDS resistance using three high-throughput methods.
A. Culture extract method. Soybean seedlings will be grown in vermiculite to the VI stage. Liquid cultures of the SDS pathogen, Fusarium virgui/iforme (strain Mont-I) will be grown for 7-10 days and strained to remove mycelium and spores. Seedlings will be uprooted from the vermiculite, washed, roots wounded, and placed in the culture extract. After 24 to 72 hours, wilting symptoms and leaf chlorosis will be measured on a relative scale to compare germplasm entries.
B. Rolled-towel method. Soybean seeds will be imbibed with a 2 x 105 spore suspension for 4 hrs and then placed in pre-moistened germination paper, rolled into a tube, secured by rubber bands, and then incubated in modified plastic Rubbermaid containers that act as humid infection chambers. After ten days of incubation at 23C, soybean seedling will be collected from rolled towels and measured for germination, seedling weight, and disease severity index to compare germplasm entries.
C. Laver cake method. A bottom layer of steamed soil, a layer of steamed soil mixed with F. virguiliforme inoculum, the seeds to be tested, and a top layer of steamed cover soil are arranged in a greenhouse tray to resemble a layer cake or lasagna. As seeds germinate, the roots grow through the middle inoculum layer, which introduces a heavy inoculum load into the seedling. At V3, seedlings are rated for symptoms (O = no symptoms, 3 = interveinal chlorosis, 5 = interveinal necrosis, 7+ = defoliated plants) in order to compare germplasm entries.
(2) Examine the interaction between ILeVO seed treatment and planting date for SDS.
A. This objective is continued from the previous grant cycle to obtain a second year of data. ILeVO is a seed treatment product produced by Bayer Crop Science that has activity against SDS and root nematodes. Briefly, planting dates will range from 05 May to 16 June, and the experiment will be an RCDB including ILeVO-treated and non-treated seed. Visual SDS ratings will be taken weekly after the onset of symptoms through R6. Yield will be measured for each treatment.
(3) Determine modes of residue and soilborne survival of the SDS pathogen in Kansas production fields.
A. Survival on corn residue. The survival of the SDS pathogen from season to season is a critical issue for the initiation of the disease during the following season. To assess this aspect of the pathogen's life cycle, corn stubble will be harvested from residue acquired from fields rotated to soybean (2017) from corn (2016). Field sites in Rossville, Manhattan, and Parsons will be selected for sampling. Corn residue will be dried and ground to a fine powder. F. virguiliforme colony forming units (CFUs) will be acquired from the residue powder and expressed regarding presence/absence and CFI-Js/g crop residue. Soybean residues and soil will be used as controls.
B. Survival in soil. Overwintering of the pathogen from one season to the next is also critical for the initiation of SDS. Clearly, some fields have higher populations that others. However, it is not understood what factors influence such survival. To examine this aspect, F. virguiliforme hyphae, conidia, chlamydospores, and colonized soybean residue fragments will be incorporated into the soil at three sites in Manhattan, Rossville, and Parsons in October 2017. Those sites will be marked with GPS and soil will be harvested in May 2018. Harvested soil will be tested for F. virguiliforme CFUs to compare treatments.
(4) Determine pathogenic variability of E virguiliforme isolates from multiple Kansas fields.
A. Collection of Kansas F. virguiliforme strains. The pathogen will be collected from soil, soybean residue (and potentially corn residue, see above), and symptomatic plants throughout the affected soybean production areas of Kansas. Strains will be isolated into pure culture and single-spored for long-term storage. An effort will be made to obtain isolates from as many counties as possible where the disease occurs. The goal will be to obtain approximately 90 strains of the pathogen. Each strain will be verified using morphology and PCR as to its species identity.
B. Pathoqenicitv assay. Initial screening work in 2012 indicated that KS3406RR was susceptible to seedling infection by F. virguiliforme. Therefore, after the best high-throughput screening technique is determined (see Obj. 1), all strains will be tested for their relative pathogenicity on KS3406. Additional pathogenicity tests on other susceptible genotypes will be conducted in time and resources allow. Also, testing these isolates for differential response to ILeVO would be possible after the collection phase and completing pathogenicity tests.
JUSTIFICATION: Sudden death syndrome (SDS) is a fungal disease of soybean that causes foliar spots, root decay, and yield loss of up to 100% in some fields with no pods visible at maturity, but normally yield losses average 5-15%. In Kansas, annual estimates provided by extension Row Crops Pathology range from trace levels to more than 250k bu of yield loss. However, SDS is a perennial cause of yield loss in the Kansas River Valley, for example. More recent data shows these numbers are on the rise. The disease is considered one of the top yield robbers of soybean and is now widespread across the Midwest soybean growing region. It is caused by the soilborne pathogen, Fusarium virgui/iforme. SDS is a good descriptor for the disease in that normal plants may abruptly turn yellow and die. SDS also can weaken the host making it more susceptible to abiotic stresses (e.g. drought), pest infestation (e.g. spider mites), and other diseases (e.g. charcoal rot).
KSC OBJECTIVE: This proposal addresses Obj. 1 (Breeding/Production/Environmental Programs) and integrates 1 B (Crop production/pest Management) and IC (High-throughput screening).
PROJECT LOCATIONS: (1) Kansas River valley Experimental Fields (KSU, Topeka/RossviIle); (2) Row crops pathology lab, KSU, Throckmorton Plant Sciences Center, Manhattan; (3) Environmental soil sciences and management lab, KSU, Throckmorton Plant Sciences Center, Manhattan; (4) Soil Testing Lab, Throckmorton Plant Sciences Center, Manhattan.

Update:
(Obj 1) Screen adapted Kansas germplasm and Kansas Soybean Variety Trial (KSVT) entries for SDS resistance using three high-throughput methods.

Germplasm is being screened using three methods: (i) culture extract/toxin assay method, (ii) rolled-towel method, and (iii) layer-cake method. Currently, KSVT entry seed has been obtained. Also, a new Fusarium virguiliforme (Mont-1) isolates was revived from the culture collection in Dr. John Leslie's (KSU) laboratory. Seedlings were grown in vermiculite and F. virguiliforme cultures were grown to test KSVT entries using the culture extract method for high throughput phenotyping (Table 1; Figure 1; see attachment). For the toxin assay, seedlings were bisected at the crown and incubated in F. virguiliforme culture extract and foliar symptoms were evaluated. In this test, germplasm was rated for both senescence and toxin reaction. Seedlings that wilted and showed foliar chlorosis/necrosis (3-6 on the SDS severity scale used) after stems were cut and placed in distilled water (control), were rated as senescent. However, those seedlings that remained relatively green after the same treatment (1-2 on the severity scale) were rated as non-senescent types. Likewise, germplasm was rated for tolerance or susceptibility to F. virguiliforme toxin. As a result of the toxin assay, 26 KSVT entries were rated as “non-senescent” and “toxin resistant” (Table 1). We are also using non-fungicide-treated seed to use for the rolled-towel (Figure 2) and layer cake (Figure 3) screens.

(Obj 2) Examine the interaction between ILeVO seed treatment and planting date for SDS.

The first, second, and third planting dates for the ILeVO/planting date study were completed at the Silver Lake ("Paramore") unit of the Kansas Research and Extension fields near Topeka. 2017 was the third year of this study and has contributed to a complete data set for analysis, publication, and extension & communication purposes. The goal is to confirm the effectiveness claimed by Bayer Crop Science for the ILeVO seed treatment product on SDS disease and to see whether seed treatment and planting date can have a positive interaction for SDS control. See Figure 4 for results. This season, as before, later planting resulted in reduced SDS severity. And, ILeVO did not have a significant effect on yield.

(Obj 3) Determine modes of residue and soilborne survival of the SDS pathogen in Kansas production fields.

This objective will be addressed in two ways: (i) corn residue and (ii) soil. Corn residue has been sampled to determine if this substrate is a source of F. virguiliforme survival in the Kansas River Valley. Working with the Co-PI, Eric Adee, we identified three sites that are in corn-soybean rotation and have a history of SDS disease. This year's soybean plots that were in corn last year, were sampled for corn residue. Corn residue samples were dried and ground to a powder. Using a culture-based method, colonies resembling F. virguiliforme were isolated from corn residue, which initially suggested a positive isolation of the pathogen (Figure 5). However, these isolates are likely F. solani and not F. virguiliforme based on Fusarium-specific PCR. Also, whole corn debris DNA, when amplified using F.v.-specific primers, did not produce a PCR product. This is a positive result since it suggest that F.v. is not surviving in corn residue. To check if F. virguiliforme survives in soil, fungal structures were incorporated into the soil at multiple sites and then checked for recovery after an overwintering period. This part of the project remains in progress. This experiment will be repeated during the 2018-2019 project year.

(Obj 4) Determine pathogenic variability of F. virguiliforme isolates from multiple Kansas fields.

As plants began to show symptoms of SDS, we worked with cooperators and extension personnel to identify and collect symptomatic plants for the isolation of F. virguiliforme from different parts of the state. Isolation of F. virguiliforme remains in progress from these samples. However, see Table 2 for a list of isolates obtained during this study so far. See Figure 6 for an example of pathogenic and less pathogenic F. virguiliforme isolates collected from Topeka and Belleville, KS, respectively.

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