Phytophthora infestans is a hemibiotroph oomycete that primarily infects potato and tomato. It infects stems, leaves, and tubers and fruits of potato and tomato. High throughput and reproducible infection assays are prerequisites to find sources of resistance in any crop. In this protocol, we describe a detached leaf assay (DLA) for conducting the virulence assay of P. infestans in potato leaves. A late blight infection assay using a potato detached leaf is a semi-high throughput assay in which hundreds of plants can be screened in a laboratory setting. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVY^O strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVY^O and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVY^O and PVY^N strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCPro^O to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.

RB is a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogen Phytophthora infestans. RB belongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. The RB protein detects the presence of class I and II IPI-O effectors from P. infestans to initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or expand the resistance capability of this protein against P. infestans in both Nicotiana benthamiana and potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.[ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

In July 2024, soybean (Glycine max) with symptoms of red crown rot, caused by Calonectria ilicicola, were observed in a commercial field with a history of corn-soybean rotation in Marion County, Missouri. Soybean were planted on May 12 and foliar symptoms including interveinal chlorosis and premature senescence, were visible on plants at the R3 growth stage with incidence estimated at 5% of the field. Reddish coloration was observed on the lower stem of symptomatic plants, along with reddish-orange globular perithecia measuring 300-500 µm in height and 250-350 µm in diameter, typical of C. ilicicola infection. Twelve symptomatic plants were collected, and stems were cleaned, followed by immersion in 70% ethanol for 30 seconds, 1.25% NaOCl for 5 minutes, and rinsing three times. Surface disinfected stems were dried at 24°C. After 24 hours, stems were split and placed on water agar (WA) amended with 1% penicillin-streptomycin solution (P4333; Sigma Aldrich). After 4 to 5 days, conidiophores characteristic of C. ilicicola, were observed. Conidia from nine stems were transferred to WA and serial transfers of conidia were made until pure single spore cultures of isolates were obtained and transferred to potato dextrose agar (PDA). On WA, the isolates produced hyaline mycelium that developed conidiophores and conidia as well as reddish brown chlamydospores. Single spore isolates transferred to PDA produced fluffy white mycelium that turned reddish. Three isolates were used for DNA extraction with the Zymo DNA extraction kit (ZD6005). Parts of the internal transcriber region (ITS) (ITS-F2: 5'-TTTACAACTCCCAAACCCCATGTGAAC-3'and ITS-R2: 5'-CTACCTGATTCGAGGTCAA CCAGAA-3') histone 3 (HIS3) (Crous et al. 2004), translation elongation 1α (EF1α) (Carbone and Kohn 1999; O'Donnell et al. 1998) and β-tubulin (TUB2) (Crous et al. 2004; O'Donnell and Cigelnik 1997) genes were amplified and their DNA sequenced. Sequences were processed in Geneious Prime 2024.0 and deposited at NCBI GenBank under the accession numbers PQ390253, PQ390254 and PQ390255 (ITS), PQ519580, PQ519581 and PQ519582 (HIS3), PQ507479, PQ507480 and PQ507481 (EF1α) and PV092563, PV092564 and PV092565 (TUB2). These DNA sequences show 100% identity to C. ilicicola sequences. To fulfill Koch's postulates, eight pots containing three plants (cultivar Williams 82) each were grown for two weeks in a growth chamber at 26°C with a 14-hour photoperiod and watered daily. Six pots were inoculated with 4 mm plugs of mycelium from the distal growing edge of the fungal isolate on PDA plates. The other two pots, treated with 4 mm plugs of PDA-only, served as negative controls. The planting medium at the base of each plant was gently removed, and inoculum was placed on the primary root, approximately 7 mm below the soil level. Five days after inoculation, plants started exhibiting reddish/blackish discoloration at the base of the stems, typical of red crown rot. Then, C. ilicicola was re-isolated from symptomatic plants three weeks post-inoculation and identified by conidial morphology. Together, these results confirmed the presence of C. ilicicola in Marion County, Missouri. Although C. ilicicola was first confirmed in the United States in 1965 on peanuts, it has gained renewed interest due to its presence in commercial soybean production, where symptoms can resemble sudden death syndrome. The pathogen poses a significant threat to >2,000,000 ha of soybean and approximately 8,000 ha of peanut grown in Missouri.

Red crown rot (RCR) of soybean, caused by Calonectria ilicicola, is new to the Midwest region of the United States, first identified in 2018 in western Illinois, and later in the neighboring states of Kentucky (2021), Indiana (2022) and Missouri (2024). Illinois has seen the most reported cases—various fields in 34 counties as of the end of the 2024 growing season. To determine the diversity and spread of C. ilicicola in Illinois, RCR-symptomatic soybean plants were collected across 44 fields from 18 Illinois counties and 79 C. ilicicola isolates were purified from lower stems. To examine the genetic diversity of these isolates, seven genetic regions were targeted for PCR amplicon sequence comparisons. The DNA sequences of the amplicons from act, cmdA, his3, tef1 and tub2 were identical to all. However, the ITS and IGS amplicons each revealed one single nucleotide polymorphism (SNP). Additionally, the PCR amplicon sequences were compared to amplicons from historical isolates collected between 1970 and 1988 in the Southeastern United States from peanut, blueberry, and soybean hosts. Sequence polymorphisms were found for all PCR amplicons (except cmdA) from the Southeastern U.S. isolates compared to the Illinois isolates, and these amplicon sequences were identical in all Southeastern U.S. isolates regardless of collection location or year. These results show that Illinois isolates collected from 2018 to 2024 showed low diversity and are most likely not due to the spread of the isolate(s) previously found in the Southeastern U.S.

Plants encounter diverse pathogens and have evolved a two-layered innate immune system to detect pathogen molecules and activate defense mechanisms that restrict infection. Most cloned plant Resistance ( R ) genes encode NLR immune receptors. NLR genes are often found in clusters of paralogs with sequence and copy number variation; whether these NLR clusters evolve in response to single or multiple pathogens has been unclear. We report here the isolation of a Phytophthora capsici resistance gene, Rpc2 , along with a novel P. infestans resistance gene, Rpi-amr5 , from two Solanum americanum accessions. These orthologous genes reside in the Rpi-amr1 cluster, which has previously been associated with resistance to P. infestans . By screening RXLR effector libraries of P. infestans and P. capsici , we identified multiple effectors recognized by both NLRs. Our findings highlight the complexity of NLR clusters and evolution driven by interactions with multiple pathogens. This work will underpin efforts to elevate resistance against Phytophthora pathogens and enhances our understanding of NLR evolution.