Broad-spectrum nano-bactericide utilizing antimicrobial peptides and bimetallic Cu-Ag nanoparticles anchored onto multiwalled carbon nanotubes for sustained protection against persistent bacterial pathogens in crops.

Worldwide crop yields are threatened by persistent pathogenic bacteria that cause significant damage and jeopardize global food security. Chemical pesticides have shown limited effectiveness in protecting crops from severe yield loss. To address this obstacle, there is a growing need to develop environmentally friendly bactericides with broad-spectrum and sustained protection against persistent crop pathogens. Here, we present a method for preparing a nanocomposite that combines antimicrobial peptides (AMPs) and bimetallic Cu-Ag nanoparticles anchored onto multiwalled carbon nanotubes (MWCNTs). The nanocomposite exhibited dual antibacterial activity by disrupting bacterial cell membranes and splicing nucleic acids. By functionalizing MWCNTs with small AMPs (sAMPs), we achieved enhanced stability and penetration of the nanocomposite, and improved loading capacity of the Cu-Ag nanoparticles. The synthesized MWCNTs&CuNCs@AgNPs@P nanocomposites demonstrated broad-spectrum lethality against both Gram-positive and Gram-negative bacterial pathogens. Glasshouse pot trials confirmed the efficacy of the nanocomposites in protecting rice crops against bacterial leaf blight and tomato crops against bacterial wilt. These findings highlight the excellent antibacterial properties of the MWCNTs&CuNCs@AgNPs@P nanocomposite and its potential to replace chemical pesticides, offering significant advantages for agricultural applications.

Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.

Antagonistic transcriptome profile reveals potential mechanisms of action on Xanthomonas oryzae pv. oryzicola by the cell-free supernatants of Bacillus velezensis 504, a versatile plant probiotic bacterium.

Bacterial leaf streak (BLS) of rice is a severe disease caused by the bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) that has gradually become the fourth major disease on rice in some rice-growing regions in southern China. Previously, we isolated a Bacillus velezensis strain 504 that exhibited apparent antagonistic activity against the Xoc wild-type strain RS105, and found that B. velezensis 504 was a potential biocontrol agent for BLS. However, the underlying mechanisms of antagonism and biocontrol are not completely understood. Here we mine the genomic data of B. velezensis 504, and the comparative transcriptomic data of Xoc RS105 treated by the cell-free supernatants (CFSs) of B. velezensis 504 to define differentially expressed genes (DEGs). We show that B. velezensis 504 shares over 89% conserved genes with FZB42 and SQR9, two representative model strains of B. velezensis, but 504 is more closely related to FZB42 than SQR9, as well as B. velezensis 504 possesses the secondary metabolite gene clusters encoding the essential anti-Xoc agents difficidin and bacilysin. We conclude that approximately 77% of Xoc RS105 coding sequences are differentially expressed by the CFSs of B. velezensis 504, which significantly downregulates genes involved in signal transduction, oxidative phosphorylation, transmembrane transport, cell motility, cell division, DNA translation, and five physiological metabolisms, as well as depresses an additional set of virulence-associated genes encoding the type III secretion, type II secretion system, type VI secretion system, type IV pilus, lipopolysaccharides and exopolysaccharides. We also show that B. velezensis 504 is a potential biocontrol agent for bacterial blight of rice exhibiting relative control efficiencies over 70% on two susceptible cultivars, and can efficiently antagonize against some important plant pathogenic fungi including Colletotrichum siamense and C. australisinense that are thought to be the two dominant pathogenic species causing leaf anthracnose of rubber tree in Hainan province of China. B. velezensis 504 also harbors some characteristics of plant growth-promoting rhizobacterium such as secreting protease and siderophore, and stimulating plant growth. This study reveals the potential biocontrol mechanisms of B. velezensis against BLS, and also suggests that B. velezensis 504 is a versatile plant probiotic bacterium.

Recovery of metagenome-assembled genomes from the phyllosphere of 110 rice genotypes.

The plant microbiota plays crucial roles in sustaining plant health and productivity. Advancing plant microbiome research and designing sustainable practices for agriculture requires in-depth assessments of microorganisms associated with different host plants; however, there is little information on functional aspects of many microorganisms of interest. Therefore, we enriched microorganisms from the phyllosphere of 110 rice genotypes and subjected them to shotgun metagenomic sequencing to reconstruct bacterial genomes from the obtained datasets. The approach yielded a total of 1.34 terabases of shotgun-sequenced metagenomic data. By separately recovering bacterial genomes from each of the 110 rice genotypes, we recovered 569 non-redundant metagenome-assembled genomes (MAGs) with a completeness higher than 50% and contaminations less than 10%. The MAGs were primarily assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. The presented data provides an extended basis for microbiome analyses of plant-associated microorganisms. It is complemented by detailed metadata to facilitate implementations in ecological studies, biotechnological mining approaches, and comparative assessments with genomes or MAGs from other studies.

Genome-wide association mapping for resistance to bacterial blight and bacterial leaf streak in rice.

MAIN CONCLUSION: Using genome-wide SNP association mapping, a total of 77 and 7 loci were identified for rice bacterial blight and bacterial leaf streak resistance, respectively, which may facilitate rice resistance improvement. Bacterial blight (BB) and bacterial leaf streak (BLS) caused by Gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), respectively, are two economically important diseases negatively affecting rice production. To mine new sources of resistance, a set of rice germplasm collection consisting of 895 re-sequenced accessions from the 3000 Rice Genomes Project (3 K RGP) were screened for BB and BLS resistance under field conditions. Higher levels of BB resistance were observed in aus/boro subgroup, whereas the japonica, temperate japonica and tropical japonica subgroups possessed comparatively high levels of resistance to BLS. A genome-wide association study (GWAS) mined 77 genomic loci significantly associated with BB and 7 with BLS resistance. The phenotypic variance (R2) explained by these loci ranged from 0.4 to 30.2%. Among the loci, 7 for BB resistance were co-localized with known BB resistance genes and one for BLS resistance overlapped with a previously reported BLS resistance QTL. A search for the candidates in other novel loci revealed several defense-related genes that may be involved in resistance to BB and BLS. High levels of phenotypic resistance to BB or BLS could be attributed to the accumulation of the resistance (R) alleles at the associated loci, indicating their potential value in rice resistance breeding via gene pyramiding. The GWAS analysis validated the known genes underlying BB and BLS resistance and identified novel loci that could enrich the current resistance gene pool. The resources with strong resistance and significant SNPs identified in this study are potentially useful in breeding for BB and BLS resistance.

Exploration of Intrinsic Microbial Community Modulators in the Rice Endosphere Indicates a Key Role of Distinct Bacterial Taxa Across Different Cultivars.

Microbial communities associated with the plant phyllosphere and endosphere can have both beneficial as well as detrimental effects on their hosts. There is an ongoing debate to which extend the phyllosphere and endosphere microbiome assembly is controlled by the host plant how pronounced cultivar effects are. We investigated the bacterial and fungal communities from the phyllosphere and endosphere of 10 different rice cultivars grown under identical environmental conditions in the frame of a targeted approach to identify drivers of community assembly. The results indicated that the endophytic bacterial communities were clearly separated into two groups. The α-diversity and microbial network complexity within Group I were significantly lower than in Group II. Moreover, the genera Nocardioides, Microvirga, and Gaiella were significantly more abundant in Group II and only present in the interaction networks of this group. These three genera were significantly correlated with α- and ß-diversity of the endophytic bacterial community and thus identified as major drivers of the endosphere community. We have identified keystone taxa that shape endophytic bacterial communities of different rice cultivars. Our overall findings provide new insights into plant-microbe interactions, and may contribute to targeted improvements of rice varieties in the future.

Scale-Dependent Effects of Growth Stage and Elevational Gradient on Rice Phyllosphere Bacterial and Fungal Microbial Patterns in the Terrace Field.

The variation of phyllosphere bacterial and fungal communities along elevation gradients may provide a potential link with temperature, which corresponds to an elevation over short geographic distances. At the same time, the plant growth stage is also an important factor affecting phyllosphere microorganisms. Understanding microbiological diversity over changes in elevation and among plant growth stages is important for developing crop growth ecological theories. Thus, we investigated variations in the composition of the rice phyllosphere bacterial and fungal communities at five sites along an elevation gradient from 580 to 980 m above sea level (asl) in the Ziquejie Mountain at the seedling, heading, and mature stages, using high-throughput Illumina sequencing methods. The results revealed that the dominant bacterial phyla were Proteobacteria, Actinobacteria, and Bacteroidetes, and the dominant fungal phyla were Ascomycota and Basidiomycota, which varied significantly at different elevation sites and growth stages. Elevation had a greater effect on the α diversity of phyllosphere bacteria than on that phyllosphere fungi. Meanwhile, the growth stage had a great effect on the α diversity of both phyllosphere bacteria and fungi. Our results also showed that the composition of bacterial and fungal communities varied significantly along elevation within the different growth stages, in terms of both changes in the relative abundance of species, and that the variations in bacterial and fungal composition were well correlated with variations in the average elevation. A total of 18 bacterial and 24 fungal genera were significantly correlated with elevational gradient, displaying large differences at the various growth stages. Soluble protein (SP) shared a strong positive correlation with bacterial and fungal communities (p < 0.05) and had a strong significant negative correlation with Serratia, Passalora, unclassified_Trichosphaeriales, and antioxidant enzymes (R > 0.5, p < 0.05), and significant positive correlation with the fungal genera Xylaria, Gibberella, and Penicillium (R > 0.5, p < 0.05). Therefore, it suggests that elevation and growth stage might alter both the diversity and abundance of phyllosphere bacterial and fungal populations.

Identification of HrpX regulon genes in Xanthomonas oryzae pv. oryzicola using a GFP visualization technique.

Xanthomonas oryzae pv. oryzicola is the causal agent of bacterial leaf streak in rice and injects repertoires of T3S effectors (T3SEs), which are normally regulated by a global regulator HrpX, into plant cells to suppress plant innate immunity for disease development. To establish a visualization technique to identify HrpX regulon genes in this pathogen, we chose six known or unknown T3SE genes of X. oryzae pv. oryzicola (strain RS105) as the targets in this report. The promoters of these candidates, whether or not containing a PIP-box, were fused with the gfp (green fluorescent protein) reporter gene, and the gfp reporters were introduced into the wild-type RS105 and the hrpX mutant RΔhrpX. GFP expression in the wild-type strain could be observed under fluorescence microscopy, but fluorescence was not observed in the hrpX mutant. Consequently, transcription and secretion detection demonstrated that these HrpX regulon members were repressed when hrpX was mutated and were not secreted into the hrp-inducing medium XOM3 because of a mutation in hrcV (T3SS deficient). These data suggest that the gfp reporter system is a feasible visualization tool for the identification of HrpX regulon genes and discovery of new T3SEs in the X. oryzae pv. oryzicola-rice pathosystem.

[Establishment of the hrp-inducing systems for the expression of the hrp genes of Xanthomonas oryzae pv. oryzicola].

The hrp genes of Xanthomonas oryzae pv. oryzicola (Xooc), which is the causal agent of bacterial leaf streak in rice, possesses the ability to elicit hypersensitive response on nonhost plants and the pathogenicity in host rice. In order to analyze the function of the hrp genes, we developed hrp-inducing systems using transcriptional hrp: :gfp fusions with the promoters of hrpX and hpa 1 of Xooc. The levels of GFP protein expression indicated that the hrp gene expression in Xooc was not efficiently induced in NB medium, but efficiently in XOM3 medium. Using the hrpG and hrpX mutants of Xooc as the controls, the results by RT-PCR demonstrated that in wild type strain the expression of the hpa1 gene was suppressed in NB medium, but was increased in XOM3 medium. When incubated in XOM3, the expression of the hpa1 gene was abolished in hrpX mutant, while the level of the hpa1 gene expression was lower in the hrpG mutant than that in wild-type strain. More importantly, it was found that the induction of the hrp gene expression was strongly increased in response to rice suspension cells and callus in this study. This suggests that the hrp-inducing systems, XOM3 or rice suspension cells or rice callus, for the induction of the hrp genes expression be useful for functionally analyzing the hrp genes, mining effectors secreted by the type III secretion apparatus and understanding pathogenicity determinats of Xooc.

Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice.

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.