Nodule inception (NIN)-like proteins (NLPs) have a central role in nitrate signaling to mediate plant growth and development. Here, we report that OsNLP2 negatively regulates ferroptotic cell death and immune responses in rice during Magnaporthe oryzae infection. OsNLP2 was localized to the plant cell nucleus, suggesting that it acts as a transcription factor. OsNLP2 expression was involved in susceptible disease development. ΔOsnlp2 knockout mutants exhibited reactive oxygen species (ROS) and iron-dependent ferroptotic hypersensitive response (HR) cell death in response to M. oryzae. Treatments with the iron chelator deferoxamine, lipid-ROS scavenger ferrostatin-1, actin polymerization inhibitor cytochalasin A, and NADPH oxidase inhibitor diphenyleneiodonium suppressed the accumulation of ROS and ferric ions, lipid peroxidation, and HR cell death, which ultimately led to successful M. oryzae colonization in ΔOsnlp2 mutants. The loss-of-function of OsNLP2 triggered the expression of defense-related genes including OsPBZ1, OsPIP-3A, OsWRKY104, and OsRbohB in ΔOsnlp2 mutants. ΔOsnlp2 mutants exhibited broad-spectrum, nonspecific resistance to diverse M. oryzae strains. These combined results suggest that OsNLP2 acts as a negative regulator of ferroptotic HR cell death and defense responses in rice, and may be a valuable gene source for molecular breeding of rice with broad-spectrum resistance to blast disease.

The reactive oxygen species (ROS) burst is the most common plant immunity mechanism to prevent pathogen infection, although the exact role of ROS in plant immunity has not been fully elucidated. We investigated the expression and translocation of Oryza sativa respiratory burst oxidase homologue B (OsRBOHB) during compatible and incompatible interactions between rice epidermal cells and the pathogenic fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We characterized the functional role of ROS focal accumulation around invading hyphae during P. oryzae infection process using the OsRBOHB inhibitor diphenyleneiodonium (DPI) and the actin filament polymerization inhibitor cytochalasin (Cyt) A. OsRBOHB was strongly induced during incompatible rice–P. oryzae interactions, and newly synthesized OsRBOHB was focally distributed at infection sites. High concentrations of ROS focally accumulated at the infection sites and suppressed effector biotrophy-associated secreted (BAS) proteins BAS4 expression and invasive hyphal growth. DPI and Cyt A abolished ROS focal accumulation and restored P. oryzae effector BAS4 expression. These results suggest that ROS focal accumulation is able to function as an effective immune mechanism that blocks some effectors including BAS4-expression during P. oryzae infection. Disruption of ROS focal accumulation around invading hyphae enables successful P. oryzae colonization of rice cells and disease development.

The order Lysobacterales consists of three families (Rhodanobacteraceae, Lysobacteraceae and Marinicellaceae), many members of which are important pathogenic and beneficial bacteria. Previous classifications of members within order Lysobacterales have relied heavily on 16S rRNA gene sequences, leading to taxonomic ambiguities at the familial level. With the advancement of sequencing technologies, an increasing number of whole-genome sequences have been available, providing an opportunity to revisit the taxonomy of families in Lysobacterales. In this study, we revisited the taxonomy of Lysobacterales by focusing on family-level reclassification based on phylogenomic frameworks. A total of 218 genome sequences, including 217 strains from Lysobacterales and 1 from Nevskiales (used as an outgroup), were collected for phylogenetic analysis. Phylogenetic relationships were inferred based on UBCG (up-to-date bacterial core gene) approach using 92 core genes and a concatenated protein phylogeney based on 227 single-copy orthologous proteins. Additionally, genomic similarity metrics, including average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), average amino acid identity (AAI) and core-proteome average amino acid identity (cpAAI), were employed to assess the taxonomy of order Lysobacterales. Our results support the proposal of one novel family and the reassignment of six genera across different families within Lysobacterales, emphasizing the need for a refined family-level taxonomy. In addition, four novel species belonging to the family Lysobacteraceae were also confirmed. This study provides an updated familial framework for Lysobacterales, laying a robust foundation for future detailed taxonomic revisions at the genus and species levels.

Reductive soil disinfestation (RSD) significantly alters soil characteristics, yet its combined effects with bacterial inoculation on subsequent rhizospheric microbial community composition remains poorly understood. To address this knowledge gap, we investigated the effects of RSD and endophytic Brevibacillus laterosporus inoculation on the composition, network, and predicted function of peanut rhizospheric bacteria and fungi. Our results demonstrated that RSD and B. laterosporus inoculation substantially increased rhizospheric bacterial diversity while reducing fungal diversity. Specifically, B. laterosporus-enhanced RSD significantly reshaped the bacterial community, resulting in increased relative abundances of Chloroflexi, Desulfobacterota, and Myxococcota while decreasing those of Firmicutes, Gemmatimonadota, and Acidobacteriota. The fungal community exhibited a more consistent response to RSD and B. laterosporus amendment, with reduced proportions of Ascomycota and Gemmatimonadota but an increase in Chytridiomycota. Network analysis revealed that B. laterosporus inoculation and RSD enhanced the bacterial species complexity and keystone taxa. Furthermore, canonical correspondence analysis indicated strong associations between the soil bacterial community and soil properties, including Eh, EC, NO₃⁻-N, and SOC. Our findings highlight that the shifts in bacterial taxa induced by B. laterosporus inoculation and RSD, particularly the keystone taxa identified in the network, may contribute to the suppression of soil-borne pathogens. Overall, this study provides a novel insight into the shifts in rhizospheric bacterial and fungal communities and their ecological functions after bacteria inoculation and RSD treatment.

Daphne tangutica Maxim. (Thymelaeaceae) is widely cultivated in China for its medicinal properties including promoting blood circulation, relieving pain, and dispelling wind to eliminate dampness (Liu et al. 2009). In August 2022, stem rot disease was observed on 1-to 5-year-old D. tangutica (Gansu Ruixiang) plants, with a disease incidence rate of 10% in a 40 m2 greenhouse located in Shiyan City, Hubei Province, China. The average temperature ranged from 28°C to 33°C, with relative humidity was 60% to 80%. The disease occurred during the fruiting period and affected plants regardless of age. White mycelia appeared at the crown of the stem at the soil line, and extended upward along the stem. Both young and mature leaves curled, wilted, and fell prematurely. The xylem of infected plants turned dark brown and softened progressively, eventually leading to plant death. A suspected fungal pathogen was isolated from two symptomatic plants. Fragments of mycelia were transferred onto potato dextrose agar (PDA) plates with a sterile dissecting needle and incubated at 28°C for 2 to 3 days. Colonies were purified using the hyphal-tip method and transferred to fresh PDA plates for morphological observation. White mycelia grew at a rate of 17.7 to 23.7 mm/day (n=16), and hyphae were hyaline with typical clamp connection under ×40 magnification. Light yellow sclerotia began forming after five days and gradually turned dark brown. On average, 65.65 ± 4.33 (n = 50) spherical, dark brown sclerotia, each with a mean diameter of 1.64 ± 0.29 mm (n=50), developed on each Petri dish after 18 days of incubation. Based on morphological characteristics, the isolates were tentatively identified as Agroathelia (=Sclerotium) rolfsii (Sacc.) Redhead & Mullineux (Amylocorticiaceae) (Redhead and Mullineux). Two representative isolates, HJBJ-5 and HJBJ-9, were selected for molecular identification. Genomic DNA was extracted using a DNA extraction kit (TSP101-200, Tsingke Biotech Co., Ltd.), and PCR amplification was performed using the internal transcribed spacer ITS primers (ITS1 and ITS4) (White et al. 1990) and the large subunit ribosomal LSU primers (LR0R and LR5) (Xu et al. 2010). The PCR products were sequenced and analyzed using BLAST with the current available sequences in NCBI. The ITS sequences of HJBJ-5 (OR578406/667bp) and HJBJ-9 (PP231038/659bp) shared 99.1% and 98.8% similarity, respectively, with A. rolfsii A8.2 (GU080230/685bp) and A. rolfsii NB2 (MN071108/658bp). The LSU sequences of HJBJ-5 (OR591284/913bp) and HJBJ-9 (PP231039/897bp) showed 99.8% and 99.9% similarity with A. rolfsii Penampang (OP809607/942bp) and A. rolfsii MY17B-1 (OP658955/922bp). Phylogenetic analysis using the maximum likelihood method confirmed that the two isolates (HJBJ-5 and HJBJ-9) clustered with other known A. rolfsii strains. To confirm pathogenicity, healthy, asympomatic eight-month-old D. tangutica (Gansu Ruixiang) plants were transplanted into pots containing sterilized soil. Ten plants were inoculated with five dark brown sclerotia of HJBJ-5 collected from PDA, while another ten plants, inoculated with PDA plugs without fungal materials, were used as control. Either sclerotia or PDA plugs were placed base of the stem in the each pot (Nie et al. 2020), and all plants were maintained in a greenhouse at 28°C with 80% relative humidity. At 10 days post -inoculation (dpi), white mycelia and dark brown sclerotia had spread across the soil surface and up the stems. By 15 dpi, the stem base became soften and rot, followed by leaf defoliation. One week later, the mycelia had reached the upper stem, leading to plant death, symptoms consistent with those observed in the field. In contrast, no symptoms developed in the control group. The pathogenicity test was repeated three times. Pathogenic fungi were re-isolated from infected plants and confirmed through morphological characteristics and ITS sequencing (ITS1 and ITS4), fulfilling Koch's postulates. Agroathelia rolfsii has previously been reported as the causal agent of root or stem diseases in various crops, including in Amorphophallus muelleri (Gao et al. 2014), Artocarpus heterophyllus (Yi et al. 2023), Basella alba (Khoo et al. 2023) and hemp (Mersha et al. 2020) in China, Malaysia, and the United States. However, this is the first report of southern blight caused by A. rolfsii on D. tangutica Maxim. (Thymelaeaceae) in Hubei, China. These findings highlight the need for effective disease management strategies during cultivation, such as drip irrigation and chemical protection on branches before cuttage.

The rhizospheric microbial community plays a crucial role in the growth and ecological adaptation of truffles. Although extensive research has been conducted on bacterial communities in truffle habitats, the spatial variation and functional implications across different regions and soil compartments remain poorly understood in the current literature. In this study, soil bacterial communities were analyzed using 16S rRNA high-throughput sequencing across truffle-producing (Tuber sinense) and non-producing sites in Quercus aliena forests in Panzhihua, China. To capture microhabitat-level variation, soils were classified into three compartments: rhizosphere soil tightly adhering to ectomycorrhizal roots (TRS), rhizoplane soil loosely attached to roots (TRE), and bulk soil from truffle zones without visible roots (TBS), with corresponding controls (RS, RE, BS) collected from truffle-free forests. An alpha diversity analysis revealed that truffle-producing soils harbored significantly higher bacterial richness than control soils, while beta diversity indicated more clustered community composition in truffle-associated soils. A taxonomic analysis showed that T. sinense occurrence was associated with enrichment of specific bacterial taxa, including Chloroflexi, Anaeromyxobacteraceae, and Bradyrhizobium, whereas widespread taxa such as Firmicutes and Bacteroidota were more abundant in control soils. To further identify microbial indicators closely associated with truffle presence, we employed random forest modeling, which highlighted Pseudomonas, Streptomyces, and Bradyrhizobium as key genera distinguishing truffle-associated rhizospheres. These findings suggest that T. sinense may influence the composition of rhizospheric microbial communities, thereby constructing a favorable rhizospheric microenvironment. This work provides new insights into the microbial ecology of T. sinense and lays a foundation for future truffle domestication and cultivation efforts.