Upland cotton (Gossypium hirsutum L.) is a major global commodity crop whose production is threatened by the reniform nematode (Rotylenchulus reniformis Linford and Oliveira), a plant-parasitic pest that causes substantial yield losses. Host-plant resistance offers a sustainable management strategy, but currently available resistant cotton cultivars provide only partial protection and often require supplemental control methods. In this study, Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–CRISPR-associated 9 (Cas9) gene editing was used to generate targeted knockouts of Mildew Resistance Locus O (GhiMLO3) in cotton and assess its role in resistance to R. reniformis. Four independent knockout lines (A1, D3, E1, and P3) were developed, confirmed by sequencing, and evaluated for nematode resistance under controlled greenhouse conditions. Nematode reproduction was significantly reduced on lines D3 and E1, with lower egg counts and fewer vermiform life stages compared with the control genotypes, Coker 312 (WT), Delta Pearl, and Jin668. The edited lines also showed characteristic mesophyll cell-death phenotypes, suggesting potential pleiotropic effects associated with MLO-mediated resistance. Sequence analysis confirmed multiple homozygous and heterozygous mutations in MLO3 alleles from both the A and D subgenomes, with D3 and E1 lines displaying the strongest resistance profiles. These findings demonstrate that MLO3 gene editing is a promising approach for improving R. reniformis resistance in cotton.

Background/Objectives: Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum (FOV), is a destructive vascular disease that severely impacts cotton production. Among its variants, race 4 (FOV4) is especially aggressive, leading to early season stand losses and yield reductions. While resistant cultivars of Gossypium barbadense (pima cotton) have been developed, the molecular basis of this resistance remains unclear. This study aimed to characterize transcriptomic responses associated with FOV4 resistance in pima cotton. Methods: We conducted an in vitro infection assay using two G. barbadense cultivars with contrasting phenotypes: the highly resistant ‘DP348RF’ and the highly susceptible ‘GB1031’. Root tissues were sampled at multiple stages of infection, and RNA sequencing was performed to identify differentially expressed genes and pathways contributing to resistance. Results: Resistant plants ‘DP348RF’ showed strong induction of genes related to reactive oxygen species (ROS) metabolism, chitinase activity, and lignification compared to the susceptible cultivar. Notably, genes involved in the biosynthesis and reinforcement of the Casparian strip, a critical biochemical barrier limiting pathogen penetration into vascular tissues, were uniquely and significantly upregulated in resistant roots. These transcriptional responses suggest that fortification of cell wall barriers and enhanced antimicrobial defenses contribute to effective restriction of FOV4 colonization. Conclusions: Our findings identify a distinct molecular signature of resistance to FOV4 in pima cotton, with Casparian strip reinforcement emerging as a potential mechanism limiting vascular infection. These insights provide a foundation for breeding strategies aimed at improving Fusarium wilt resistance in cotton.

Pseudomonas syringae pv. syringae (Pss) is the causal agent of bacterial canker, a disease that can result in yield losses, aerial tissue damage, and tree mortality in stone fruits worldwide. Peach, one of the major stone fruit crops, experiences significant yield losses and tree mortality attributed to bacterial canker in the United States. As the second-largest peach-producing state, South Carolina faces direct and significant impacts due to Pss. Early evaluations of peach scion responses to Pss infection have relied primarily on circumstantial field observations in rootstock trials. Although laboratory evaluations in peach have been reported, these studies primarily focused on pathogen virulence testing or small accession sets and did not establish a standardized, scalable detached twig protocol for systematic germplasm phenotyping. The absence of a clearly described laboratory assay has limited reproducible and large-scale evaluation of bacterial canker tolerance in peach. To address this gap, a detached dormant twig assay, previously developed for cherry, was adapted and optimized for peach. Dormant shoots from nine peach accessions were cut into 10 cm segments, surface-sterilized, and inoculated with a Pss suspension prepared in 10 mM MgCl₂ buffer or with the buffer alone. After six weeks of incubation, inner bark lesion size was evaluated visually and quantified using ImageJ. A newly developed visual rating scale was established and compared with quantitative lesion measurements. Spearman correlation analysis showed strong positive correlations between visual disease scores and ImageJ-based lesion measurements across two independent replicates (ρ = 0.80–1.00, p < 0.01), while shoot segment diameter showed weak-to-moderate negative correlations with disease severity. This adapted and consolidated dormant twig assay provides a practical, reproducible, and scalable method for phenotyping bacterial canker tolerance in peach and supports future germplasm screening and breeding efforts.