The root-knot nematode (RKN) disease is a highly destructive soilborne disease that significantly affects peanut yield in Northern China. The composition of the soil microbiome plays a crucial role in plant disease resistance, particularly for soilborne diseases like RKN. However, the relationship between the occurrence of RKN disease and the structure and diversity of bacterial communities in peanut fields remains unclear. To investigate bacterial diversity and the community structure of peanut fields with severe RKN disease, we applied 16S full-length amplicon sequencing based on the third high-throughput sequencing technology. The results indicated no significant differences in soil bacterial α-diversity between resistant and susceptible plants at the same site. However, the Simpson index of resistant plants was higher at the site of peanut-wheat-maize rotation (Ro) than that at the site of peanut continuous cropping (Mo), showing an increase of 21.92%. The dominant phyla identified in the peanut bulk soil included Proteobacteria, Acidobacteria, Actinobacteria, Planctomycetes, Chloroflexi, Firmicutes, and Bacteroidetes. Further analysis using LEfSe (Linear discriminant analysis effect size) revealed that Sulfuricellaceae at the family level was a biomarker in the bulk soil of susceptible peanut compared to resistant peanut. Additionally, Singulisphaera at the genus level was significantly more enriched in the bulk soil of resistant peanut than that of susceptible peanut. Soil properties were found to contribute to the abundance of bacterial operational taxonomic units (OTUs). Available phosphorus (AP), available nitrogen (AN), organic matter (OM), and pH made a positive contribution to the bacterial OTUs, while available potassium (AK) made a negative contribution. The metabolic pathway of novobiocin biosynthesis was only enriched in soil samples from resistant peanut plants. Eleven candidate beneficial bacteria and ten candidate harmful strains were identified in resistant and susceptible peanut, respectively. The identification of these beneficial bacteria provides a resource for potential biocontrol agents that can help improve peanut resistance to RKN disease. Overall, the study demonstrated that severe RKN disease could reduce the abundance and diversity of bacterial communities in peanut bulk soil. The identification of beneficial bacteria associated with resistant peanut offered the possibility for developing biocontrol strategies to enhance peanut resistance to RKN disease.

Seed coat color in peanut (Arachis hypogaea L.) is a critical agronomic trait that affects both nutritional quality and market appeal. In this study, we identified two bHLH transcription factor genes, AhWSC1a and AhWSC1b, homologues of Arabidopsis TRANSPARENT TESTA 8, as indispensable gatekeepers of basal flavonoid pigmentation. QTL-seq analysis of a recombinant inbred line population derived from a black-testa parent (S3) and a white-testa parent (S2) revealed that recessive loss-of-function mutations in both AhWSC1a/1b abolish proanthocyanidin biosynthesis, resulting in a white testa. Integrated metabolomic and transcriptomic profiling confirmed the absence of proanthocyanidins and a strong repression of late anthocyanin-pathway genes (DFR, LDOX) in the mutants. Molecular assays further demonstrated that AhWSC1 physically interacts with the R2R3-MYB regulator AhTc1 to form a functional MBW complex that activates AhDFR and AhLDOX transcription. In this research, we also found that the black testa phenotype may arise from elevated AhTc1 expression associated with a structural variant (SV); however, in the SV background, the introduction of ahwsc1a/1b mutant leads to a significant suppression of AhTc1 expression. Notably, because AhWSC1 is transcriptionally silent in hairy-root systems, overexpression of AhTc1 alone failed to induce these late-stage anthocyanin biosynthesis genes, highlighting AhWSC1 as an indispensable, rate-limiting hub of anthocyanin biosynthesis pathway regulation. Collectively, our findings establish AhWSC1a and AhWSC1b as master regulators of peanut testa pigmentation, elucidate the molecular basis of classical white testa inheritance, and provide genetic targets for precision-breeding of nutritionally enhanced cultivars.

Nitrogen is an indispensable nutrient for plant growth and crop production, but it is not directly accessible to plants without the help of nitrogen-fixing bacteria. Legume plants can form root nodules in symbiosis with rhizobia. NODULE INCEPTION (NIN), a founding member of the NIN-like protein (NLP) family, is essential for nodulation in legume species. However, the knowledge of functional characteristics of the NLP family members in peanuts is limited. In this study, a genome-wide analysis of the NLP genes was carried out. A total of 16 NLP genes were identified in the peanut genome, including 2 AhNIN and 14 AhNLP, which were unevenly distributed on nine chromosomes of the peanut genome. Furthermore, transcriptomic profiles and expression pattern analysis showed that both AhNINa and AhNINb genes were specifically expressed in root nodules. Subcellar localization and transcriptional activity analysis revealed that both AhNINa and AhNINb encode transcriptional activators. In addition, the roots that down-regulated the expression of AhNINa and AhNINb genes failed to form nodules. These findings provide significant insights into the molecular functions of AhNINa and AhNINb genes in regulating peanut nodule development.