Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2023 Annual Report
Objectives
Objective 1: Characterize emerging and endemic plant pathogens/pests used for identifying sources of resistance;
Objective 2: Identify and characterize resistance to pathogens/pests in Glycine species and map resistance loci to allow transfer into soybean;
Objective 3: Transfer putative resistance/defense alleles into elite soybean germplasm to validate effectiveness in reducing disease severity.
Approach
The work will characterize two emerging soybean diseases and the pathogens that cause them (red crown rot by Calonectria ilicicola and red leaf blotch by Coniothyrium glycines), identify resistance against these two pathogens and root-knot nematode (Meloidogyne incognita), and transfer resistance into elite genetic material suitable for use in breeding programs.
For red crown rot, lower stems of plants from fields in Illinois will be sampled (15-20 stems per 10-15 locations), isolates purified from infected tissue, and tested for virulence. For red leaf blotch, diseased leaf samples will be collected from soybean fields by our collaborator in Africa and purified isolates tested for virulence. The initial screening for red leaf blotch will use two isolates, one at Fort Detrick and one in Kenya. The goal for both pathogens is to recover 150 plus isolates for phenotyping and genotyping. Genetic characterization of C. ilicicola and C. glycines populations will be achieved through sequencing and using DNA single nucleotide polymorphisms marker analysis to estimate the parameters of genetic diversity and population structure and identify signatures of selection in these populations.
To evaluate resistance, experiments will be arranged in the following priority of testing: 1) released cultivars consisting of 25 public soybean cultivars, 2) a set of 70 ancestral lines, 3) a panel of 350 soybean plant introductions that have been identified as a genetically diverse core set, 4) a set of 100 G. soja accessions, and 5) a set of 200 accessions of perennial Glycine species. Having genotypic and phenotypic data on all accessions will allow for a genome wide association studies to identify resistance loci.
The strategy for transferring resistance to breeder-friendly genotypes will be dependent on sources of resistance identified. Resistance in G. max and G. soja will be crossed to elite breeding material. If resistance is found only in perennial Glycine species, then intra and inter specific crosses and mapping will be made if possible. Resulting lines with partial resistance or specific resistance conditioned by single major genes will be selected.
For root-knot resistance, we have F2:5 recombinant inbred lines (RILs) developed and resistant and susceptible G. latifolia accessions genotyped; preliminary mapping showed most of the resistance was controlled by a single locus on chromosome 13. We will do deep sequencing to continue to fine map this region until a small enough interval is identified to allow for prediction of candidate resistance genes. For phenotyping additional RILs, seeds will be scarified, pregerminated, and then planted in sand in polyvinyl tubes. Three weeks after transplanting, the seedlings will be inoculated with freshly prepared egg suspensions at a rate of 2,000 M. incognita eggs per plant. Candidate genes showing polymorphisms between resistant and susceptible accessions will be designed into Agrobacterium transformation vectors for transformation into susceptible G. max cultivar Williams 82. Lines homozygous for the transgene will be evaluated for resistance as described above.
Progress Report
For the Red Leaf Blotch component of Objective 1, we were very successful in finding Red Leaf Blotch disease, collecting infected leaves, and purifying isolates of the pathogen, Coniothyrium glycines. Our Oak Ridge Institute for Science and Education (ORISE) postdoc traveled extensively to diseased areas to collect infected leaf tissue from Ethiopia, Kenya, Malawi, Mozambique, Uganda, Zambia, and Zimbabwe. Diseased leaves collected in the field (34 fields total) were divided to keep half at the lab at the International Institute of Tropical Agriculture (IITA) in Nairobi, and to send the other half to our collaborator at the USDA-ARS select agent-approved containment lab in Fort Detrick, Maryland. Microsclerotia were removed, surface sterilized and plated to obtain 137 isolates in pure culture, with the vast majority coming from Ethiopia (59), Uganda (37), and Zambia (36). The lab at Fort Detrick extracted DNA from 56 isolates, mailed the DNA to ARS scientists in Urbana, Illinois, and samples were shotgun sequenced to be analyzed later after we have whole genome sequences of over 100 isolates. The lab at Fort Detrick is conducting a phylogenic analysis based on the sequence of the internal transcribed spacer (ITS) region of 96 newly collected samples.
Likewise, we were also very successful in collecting samples of soybean plants in Illinois showing Red Crown Rot (RCR) symptoms and purifying Calonectria ilicicola isolates. Via field scouting or cooperator submissions, over 700 soybean plant samples were collected across 43 locations in eight Illinois counties (Champaign, Kankakee, Iroquois, Macon, Madison, Piatt, Pike, and Shelby). Field areas showing foliar chlorosis were investigated and plants were sampled from those areas, especially if dark reddish coloration and/or production of perithecia on basal stems, characteristic signs of RCR infection were evident. Plant samples were thoroughly rinsed, surface-disinfected and incubated for up to 4 weeks in moist boxes to enhance fungal growth and encourage sporulation. Fungal mycelia or spores were then plated on antibiotic growth medium in Petri dishes. When characteristic fungal structures were observed such as perithecia or conidia, transfers were made to antibiotic medium and multiple isolations were then made to obtain a pure isolate of C. ilicicola from that stem. Based on sporulation on incubated stems, disease was detected widely across Madison County where, in 14 fields sampled across nine townships, at least one plant was positive for RCR. The three fields sampled in Pike county were all RCR positive, and a few positive fields were also found in Macon, Piatt and Shelby counties. No presence of C. ilicicola was detected in incubated stems collected from Champaign, Iroquois, or Kankakee counties in 2022, but samples are still being collected and assayed in 2023. It was a challenge to obtain pure cultures of the slow growing C. ilicicola as many other microbes could outgrow it and overtake a culture, but we did succeed in purifying at least 45 isolates from diverse locations. DNA extraction protocols were worked out, and we had 38 of the isolates (from 19 locations across seven fields), plus two reference controls (one from Illinois, one from Louisiana; from the USDA-ARS NRRL collection) shotgun sequenced. Polymerase Chain Reaction (PCR) amplification of the genetic regions of interest verified that the purified cultures were C. ilicicola, and showed no variation between the Illinois isolates (i.e. appears to be the same isolate spreading).
For the Red Leaf Blotch (RLB) component of Objective 2, our ORISE post-doc continued to screen germplasm that he had previously received from ARS scientists. The group at the IITA in Nairobi screened 183 genotypes under greenhouse conditions, and/or via detached leaf assays in the lab. Of the 183, 126 (69%) were assayed at least twice. A new set of 480 diverse genotypes (identical to a set also sent to Ethiopia) was sent to the IITA on June 9th. The 480 seed packs arrived in Jimma, Ethiopia, at the end of May 2023 and were planted in mid-June 2023 in an ‘hot spot’ for RLB to allow field screening of the germplasm. From the 183 genotypes assayed at the IITA, the preliminary results are very promising as nearly 35 of the 183 (19%) showed some resistance (<2 on a 1-5 scale with 5 as highly susceptible, 1 little-to-no lesion). Of these 35, 30 (86%) were Plant Introductions (PI’s) from the USDA National Soybean Germplasm Collection. Additionally, 26 of the 183 (14%) had a disease rating of 1, showing little-to-no lesion development. Again, these were mostly PI’s, with only two of the 26 not being a PI from the USDA collection. We also trained the staff at Fort Detrick on how to conduct detached leaf disease assays. The ARS lab at Fort Detrick has conducted one preliminary test, repeating seven of the genotypes (two highly sensitive, five very resistant) previously tested by the group at the IITA in Nairobi and six of the seven showed very similar results. The one sample that did not agree had a serious secondary infection that might have affected the results. Although this was a single replicate, it was encouraging to see the consistency using different isolates. We will continue to screen with multiple replicates and verify resistance at both locations to be certain of any resistant phenotypes.
The Red Crown Rot component of Objective 2 also made advancements. As this is a new disease for us, we spent considerable time optimizing a reliable inoculation method and disease scoring procedure. We have also grown some genotypes in the greenhouse to have healthy seed, and now have an initial set of 70 diverse genotypes to conduct our initial screenings during the summer of 2023. Preliminary screening has yet to identify any promising resistance.
For Objective 3, we needed high quality whole genome sequence of the G. latifolia F1 plant that will be used for mapping resistance. Therefore, we used PacBio HiFi sequencing of DNA extracted from F1 leaves to obtain a whole genome assembly. We also attempted Trio-binning assembly, by using shotgun sequence of the parents of the F1, together with the F1 genome. Those results were inconclusive as the original parents of the F1 were too heterozygous. Fortunately, as the F1 whole genome sequence is of very high quality, we will be able to use that sequence for our mapping efforts.
Accomplishments
1. Red Leaf Blotch (RLB) is an important emerging soybean disease confined to Africa, but very little is known about the pathogen. At the start of this project in the summer of 2022, there were only three isolates of the RLB pathogen Coniothyrium glycines at the International Institute of Tropical Agriculture (IITA) and 12 isolates in storage at Fort Detrick that were collected decades ago. Therefore, little was known about the genetic diversity of the pathogen across Africa as it has become increasingly prevalent and yield limiting. ARS scientists in Urbana, Illinois, now have over 130 purified isolates, and were able to genotype 108 of them spanning six countries and found a high level of diversity that neatly associated with the different geographic regions. Therefore, it is now known that each separate geographic area contains its own unique population of the pathogen, indicative of a pathogen that must not spread readily over large distances. These types of discoveries will help with the management and hopefully eventual control of the spread of RLB.
2. Red Crown Rot is a new emerging disease in Illinois (a major producer of soybean for the U.S.) and nothing is known about the genetic diversity of the RCR pathogen, Calonectria ilicicola in Illinois. ARS researchers in Urbana, Illinois, collected diseased samples and purified 43 isolates of the RCR pathogen collected from eight counties in Illinois. Additionally, we obtained isolates from Louisiana, North Carolina, and Georgia, from national repositories. Analyzing 40 isolates for genetic changes that occurred in six different regions of the genome, we determined that the Illinois isolates showed no variation, and therefore are likely to be the same isolate spreading across the state. Likewise, the isolates from other states obtained from the national repository collections shared the same five genetic differences, showing that those isolates might all be the same or very similar to each other, and none were the likely source of the Illinois isolate.
Review Publications
Herman, T.K., Bowen, C.R., Mahan, A.L., Hartman, G.L. 2022. Evaluation of soybean germplasm for resistance to Fusarium virguliforme, the major pathogen causing sudden death syndrome of soybean in the United States. Crop Science. 63(3):1344-1353. https://doi.org/10.1002/csc2.20903.
Diers, B., Lagos-Kutz, D.M., Schultz, S.J., Cary, T., Wang, D., Hartman, G.L. 2023. Registration of 64 soybean germplasm lines with all combinations of five soybean aphid resistance genes in two genetic backgrounds. Journal of Plant Registrations. 17(2):416-425. https://doi.org/10.1002/plr2.20279.
Kleczewski, N.M., Bradley, C.A., Hartman, G.L., Kandel, Y., Mueller, D., Rodriguez-Salamanca, L. 2022. A diagnostic guide for red crown rot of soybean. Plant Health Progress. 24(1):123-129. https://doi.org/10.1094/PHP-04-22-0041-DG.
Lagos-Kutz, D.M., Pawlowski, M.L., Han, J., Clough, S.J., Hartman, G.L. 2023. Reduction in productivity of soybean plants infested with Neohyadatothrips variabilis (Thysanoptera: Thripidae) with and without soybean vein necrosis virus. Phytoparasitica. 51:437-445. https://doi.org/10.1007/s12600-023-01070-1.
