First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

Development of a Standardized Soybean Cyst Nematode Screening Assay in Pennycress and Identification of Resistant Germplasm.

The prospect of incorporating pennycress as an oilseed cover crop in the Midwest's corn-soybean rotation system has drawn researcher and farmer attention. The inclusion of pennycress will be beneficial as it provides an excellent soil cover to reduce soil erosion and nutrient leaching while serving as an additional source for oilseed production and income. However, pennycress is an alternative host for soybean cyst nematode (SCN), which is a major biological threat to soybean that needs to be addressed for sustainable pennycress adoption into our current production systems. To develop a standardized SCN resistance screening strategy in pennycress, we tested and optimized five parameters: (i) germination stimulants, (ii) inoculation timing, (iii) inoculation rate, (iv) experimental incubation time, and (v) susceptible checks. The standardized SCN resistance screening protocol includes the following: (i) treating pennycress seeds with gibberellic acid for 24 h, (ii) transplanting seedlings 12 to 15 days after initiating germination and inoculating 10 to 12 days after transplantation, (iii) inoculating at a rate of 1,500 eggs/100 cc soil (1,500 eggs per plant), (iv) processing roots at 30 days after inoculation, and (v) using susceptible pennycress accession Ames 32869 to calculate the female index. The standardized protocol was used to quantify the response of a diverse set of pennycress accessions for response against SCN HG type 1.2.5.7 and HG type 7. While there were no highly resistant pennycress lines identified, 15 were rated as moderately resistant to HG type 1.2.5.7, and eight were rated moderately resistant to HG type 7. The resistant lines identified in this study could be utilized to develop SCN-resistant pennycress cultivars. The study also opens a new avenue for research to understand SCN-pennycress interactions through molecular and genomic studies. This knowledge could aid in the successful inclusion of pennycress as a beneficial cover/oilseed crop in the United States Midwest.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Loss-of-function of an α-SNAP gene confers resistance to soybean cyst nematode.

Plant-parasitic nematodes are one of the most economically impactful pests in agriculture resulting in billions of dollars in realized annual losses worldwide. Soybean cyst nematode (SCN) is the number one biotic constraint on soybean production making it a priority for the discovery, validation and functional characterization of native plant resistance genes and genetic modes of action that can be deployed to improve soybean yield across the globe. Here, we present the discovery and functional characterization of a soybean resistance gene, GmSNAP02. We use unique bi-parental populations to fine-map the precise genomic location, and a combination of whole genome resequencing and gene fragment PCR amplifications to identify and confirm causal haplotypes. Lastly, we validate our candidate gene using CRISPR-Cas9 genome editing and observe a gain of resistance in edited plants. This demonstrates that the GmSNAP02 gene confers a unique mode of resistance to SCN through loss-of-function mutations that implicate GmSNAP02 as a nematode virulence target. We highlight the immediate impact of utilizing GmSNAP02 as a genome-editing-amenable target to diversify nematode resistance in commercially available cultivars.

Epistatic interaction between Rhg1-a and Rhg2 in PI 90763 confers resistance to virulent soybean cyst nematode populations.

KEY MESSAGE: An epistatic interaction between SCN resistance loci rhg1-a and rhg2 in PI 90763 imparts resistance against virulent SCN populations which can be employed to diversify SCN resistance in soybean cultivars. With more than 95% of the $46.1B soybean market dominated by a single type of genetic resistance, breeding for soybean cyst nematode (SCN)-resistant soybean that can effectively combat the widespread increase in virulent SCN populations presents a significant challenge. Rhg genes (for Resistance to Heterodera glycines) play a key role in resistance to SCN; however, their deployment beyond the use of the rhg1-b allele has been limited. In this study, quantitative trait loci (QTL) were mapped using PI 90763 through two biparental F3:4 recombinant inbred line (RIL) populations segregating for rhg1-a and rhg1-b alleles against a SCN HG type 1.2.5.7 (Race 2) population. QTL located on chromosome 18 (rhg1-a) and chromosome 11 (rhg2) were determined to confer SCN resistance in PI 90763. The rhg2 gene was fine-mapped to a 169-Kbp region pinpointing GmSNAP11 as the strongest candidate gene. We demonstrated a unique epistatic interaction between rhg1-a and rhg2 loci that not only confers resistance to multiple virulent SCN populations. Further, we showed that pyramiding rhg2 with the conventional mode of resistance, rhg1-b, is ineffective against these virulent SCN populations. This highlights the importance of pyramiding rhg1-a and rhg2 to maximize the impact of gene pyramiding strategies toward management of SCN populations virulent on rhg1-b sources of resistance. Our results lay the foundation for the next generation of soybean resistance breeding to combat the number one pathogen of soybean.