Soil selenium enhanced the resistance of oilseed rape to Sclerotinia sclerotiorum by optimizing the plant microbiome.

Plant can recruit beneficial microbes to enhance their ability to resist disease. Selenium is well established as a beneficial element in plant growth, but its role in mediating microbial disease resistance remained poorly understood. Here, we investigated the correlation between selenium, oilseed rape rhizosphere microbes and Sclerotinia sclerotiorum. Soil application of 0.5 and 1.0 mg/kg selenium significantly increased the resistance of oilseed rape to Sclerotinia sclerotiorum compared with no selenium application, and the disease inhibition rate was higher than 20%. The disease resistance of oilseed rape was related to rhizosphere microorganisms, and beneficial bacteria isolated from the rhizosphere inhibited Sclerotinia stem rot. Burkholderia cepacia, and synthetic community enhanced plant disease resistance through transcriptional regulation and activated plant-induced systemic resistance to protect plants. Besides, inoculation of isolated bacteria optimized the bacterial community structure of leaves and enriched beneficial microorganisms such as Bacillus, Pseudomonas and Sphingomonas. Bacillus isolated from the leaves were sprayed on the detached leaves, and it also performed a significant inhibition effect on Sclerotinia sclerotiorum. Overall, our results suggested that selenium drive plant rhizosphere microorganisms to increase resistance to Sclerotinia sclerotiorum in oilseed rape.

Visible-light-driven water-soluble zinc oxide quantum dots for efficient control of citrus canker.

BACKGROUND: Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a devastating bacterial disease that reduces citrus yield and quality, posing a serious threat to the citrus industry. Several conventional chemicals have been used to control citrus canker. However, this approach often leads to the excessive use of chemical agents, can exacerbate environmental pollution and promotes the development of resistant Xcc. Therefore, there is significant interest in the development of efficient and environmentally friendly technologies to control citrus canker. RESULTS: In this study, water-soluble ZnO quantum dots (ZnO QDs) were synthesised as an efficient nanopesticide against Xcc. The results showed that the antibacterial activity of ZnO QDs irradiated with visible light [half-maximal effective concentration (EC50) = 33.18 µg mL-1] was ~3.5 times higher than that of the dark-treated group (EC50 = 114.80 µg mL-1). ZnO QDs induced the generation of reactive oxygen species (•OH, •O- 2 and 1O2) under light irradiation, resulting in DNA damage, cytoplasmic destruction, and decreased catalase and superoxide dismutase activities. Transcription analysis showed downregulation of Xcc genes related to 'biofilms, virulence, adhesion' and 'DNA transfer' exposure to ZnO QDs. More importantly, ZnO QDs also promoted the growth of citrus. CONCLUSION: This research provides new insights into the photocatalytic antibacterial mechanisms of ZnO QDs and supports the development of more efficient and safer ZnO QDs-based nanopesticides to control citrus canker. © 2024 Society of Chemical Industry.

Toxicity risk assessment of flupyradifurone for the predatory pirate bug, Orius strigicollis (Poppius) (Heteroptera: Anthocoridae), a biological control agent of Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Diaphorina citri Kuwayama (Hemiptera: Liviidae), commonly known as the Asian citrus psyllid, is a prominent citrus tree pest that serves as a vector for Asian huanglongbing (HLB). The substantial costs incurred by the citrus industry as a consequence of this disease have spurred considerable interest in the combined control of D. citri using insecticides and natural enemies. However, the successful implementation of such integrated pest management strategies is dependent on ensuring the compatibility of using natural enemies in the presence of insecticides. In this regard, we evaluated the lethal and sublethal effects of flupyradifurone on Orius strigicollis (Poppius) (Heteroptera: Anthocoridae), an important predatory biological control agent, in which we assessed the risk of exposure to flupyradifurone under both in- and off-field scenario. The median lethal rate (LR50) value of flupyradifurone against O. strigicollis (9.089 g a.i. ha-1), was found to be significantly lower than the maximum field recommended rate (MFRR, 170 g a.i. ha-1). Additionally, at 0.254 g a.i. ha-1, flupyradifurone was established to significantly prolong the developmental duration of O. strigicollis from the first to third instar nymphs. Although we detected no significant difference in the survival of immature O. strigicollis subjected to 0.064 g a.i. ha-1 and control treatments, survival was significantly lower in 0.127 and 0.254 g a.i. ha-1 treatments. Moreover, whereas there were no significant differences in adult longevity between the 0.127 g a.i. ha-1 and control treatments, we recorded a significant reduction in fecundity. Furthermore, there were reductions in peak life expectancy, reproductive value, finite rate of increase, intrinsic rate of increase, and net reproduction rate in response to exposure to increasing flupyradifurone rate. Additionally, at 0.127 g a.i. ha-1, the mean generation time was significantly longer than that under control conditions. Following simulated exposure to flupyradifurone for 100 days, population of O. strigicollis in the 0.064 g a.i. ha-1 and control treatments were found to be significantly larger than those exposed to 0.127 g a.i. ha-1. On the basis on LR50 evaluations, whereas the risk of exposure risk was unacceptable for O. strigicollis under in-field scenario, it remained acceptable off-field. Nonetheless, the sublethal effect of prolonged exposure to residual flupyradifurone could pose an unacceptable off-field risk to O. strigicollis (e.g., in adjacent habitats). Consequently, the effects of different flupyradifurone exposure scenarios on O. strigicollis should be thoroughly assessed, and reducing the dosage of flupyradifurone could be advantageous for the control of D. citri when combine with augmentative release of O. strigicollis.

Selenium Improved Phenylacetic Acid Content in Oilseed Rape and Thus Enhanced the Prevention of Sclerotinia sclerotiorum by Dimethachlon.

Sclerotinia sclerotiorum is a broad-spectrum necrotrophic phytopathogen that can infect many plant species worldwide. The application of fungicides is a common measure for controlling Sclerotinia sclerotiorum. Due to the risk of developing resistance to fungicides, it is imperative to find ways to be environmentally friendly and even effective. Using bioactive compounds in plants to reduce the amounts of fungicides has become a clean and sustainable strategy of controlling Sclerotinia sclerotiorum. Our study found that selenium in soil mediated the phenylacetic acid-related metabolic pathway in oilseed rape and reduced the incidence rate of Sclerotinia sclerotiorum. The growth-inhibition rates of Sclerotinia sclerotiorum were observed at 25.82%, 19.67%, and 52.61% for treatments of 0.8 mg·L-1 dimethachlon, 0.1 mg·mL-1 phenylacetic acid, and dimethachlon (0.8 mg·L-1) + phenylacetic acid (0.1 mg·mL-1), respectively. Phenylacetic acid reduced the application amount of dimethachlon and enhanced the inhibition effect for Sclerotinia sclerotiorum. Results also suggested that phenylacetic acid severely damaged the morphological structure, changed the electrical conductivity, and reduced the capacity of acid production and oxalic acid secretion of Sclerotinia sclerotiorum mycelium. Further studies revealed that phenylacetic acid increased the gene-expression level of Ssodc1, Ssodc2, CWDE2 and CWDE10 in mycelium while decreasing the expression level of SsGgt1, and phenylacetic acid + dimethachlon reduced the relative expression level of SsBil. These findings verified that phenylacetic acid could partially replace the amount of dimethachlon, as well as enhance the prevention of Sclerotinia sclerotiorum by dimethachlon, which provides evidence for developing an environment-friendly method for Sclerotinia sclerotiorum control.

Selenium Combined with Methyl Jasmonate to Control Tomato Gray Mold by Optimizing Microbial Community Structure in Plants.

Tomato cultivation is seriously affected by infection from Botrytis cinerea. The safe and effective control of tomato gray mold remains elusive. Plant-related microbial communities regulate not only plant metabolism but also plant immune systems. In this study, we observed that Selenium application in soil combined with foliar spraying of methyl jasmonate could reduce Botrytis cinerea infection in tomato fruits and leaves and improve tomato fruit quality. The infection rate of leaves decreased from 42.19% to 25.00%, and the vitamin C content increased by 22.14%. The bacterial community structure of the tomato was studied by using amplicon sequencing technology. The leaf bacterial alpha diversity of tomatoes treated with Se plus methyl jasmonate was significantly higher than that of the control. Then we isolated five strains antagonistic to Botrytis cinerea in vitro from tomato leaves in the treatment of Se plus methyl jasmonate. The antagonistic strains were identified as Bacillus subtilis and Bacillus velezensis. Spraying mixed antagonistic strain suspension significantly inhibited the diameter of Botrytis cinerea with an inhibition rate of 40.99%. This study revealed the key role of plant-beneficial bacteria recruited by Se combined with methyl jasmonate in improving tomato plant disease resistance. These findings may benefit our understanding of the new regulation of microorganisms on Botrytis cinerea.

Substrate pH Influences the Nutrient Absorption and Rhizosphere Microbiome of Huanglongbing-Affected Grapefruit Plants.

The substrate pH directly affects nutrient availability in the rhizosphere and nutrient uptake by plants. Macronutrients such as nitrogen, potassium, calcium, magnesium, and sulfur are highly available at pH 6.0-6.5, while micronutrients become less available at higher, alkaline pH (pH > 7.0). Recent research has indicated that low pHs can enhance nutrient uptake and improve sweet orange (Citrus sinensis) tree health. We designed a study to understand the influence of a wide range of substrate pH values on plant size and biomass, nutrient availability, leaf gas exchange, and rhizosphere microbiome of grapefruit (Citrus paradisi) affected by Huanglongbing (HLB). Two-year-old "Ray Ruby" grapefruit plants grafted on sour orange (Citrus aurantium) rootstock were cultivated indoors in 10-cm wide × 40-cm tall pots with peat:perlite commercial substrate (80:20 v/v). We tested two disease statuses [HLB-free or healthy (negative, HLB-) and HLB-affected (positive, HLB+)] and six substrate pH values (4, 5, 6, 7, 8, 9) in a 2 × 6 factorial arranged on a complete randomized design with four replications. The canopy volume of HLB+ plants was 20% lower than healthy plants, with pHs 7 and 9 resulting in 44% less canopy volume. The root and shoot ratio of dry weight was 25.8% lower in HLB+ than in healthy plants. Poor root growth and a decrease in fibrous roots were found, especially in pH 5 and 6 treatments in HLB+ plants (p < 0.0001). The disease status and the substrate pHs influenced the leaf nutrient concentration (p < 0.05). High substrate pH affects nutrient availability for root uptake, influencing the nutrient balance throughout the plant system. pH values did not affect plant photosynthesis, indicating that pH does not recover HLB+ plants to the photosynthetic levels of healthy plants-even though high pH positively influenced internal CO2. There were collectively over 200 rhizobacterial identified by the 16S rRNA gene sequencing in individual phylogenetic trees. Most rhizobacteria reads were identified in pH 9. Our results indicated no effect of substrate pHs on the plant disease status induced by enhanced nutrient uptake.

An automatic identification system for citrus greening disease (Huanglongbing) using a YOLO convolutional neural network.

Huanglongbing (HLB), or citrus greening disease, has complex and variable symptoms, making its diagnosis almost entirely reliant on subjective experience, which results in a low diagnosis efficiency. To overcome this problem, we constructed and validated a deep learning (DL)-based method for detecting citrus HLB using YOLOv5l from digital images. Three models (Yolov5l-HLB1, Yolov5l-HLB2, and Yolov5l-HLB3) were developed using images of healthy and symptomatic citrus leaves acquired under a range of imaging conditions. The micro F1-scores of the Yolov5l-HLB2 model (85.19%) recognising five HLB symptoms (blotchy mottling, "red-nose" fruits, zinc-deficiency, vein yellowing, and uniform yellowing) in the images were higher than those of the other two models. The generalisation performance of Yolov5l-HLB2 was tested using test set images acquired under two photographic conditions (conditions B and C) that were different from that of the model training set condition (condition A). The results suggested that this model performed well at recognising the five HLB symptom images acquired under both conditions B and C, and yielded a micro F1-score of 84.64% and 85.84%, respectively. In addition, the detection performance of the Yolov5l-HLB2 model was better for experienced users than for inexperienced users. The PCR-positive rate of Candidatus Liberibacter asiaticus (CLas) detection (the causative pathogen for HLB) in the samples with five HLB symptoms as classified using the Yolov5l-HLB2 model was also compared with manual classification by experts. This indicated that the model can be employed as a preliminary screening tool before the collection of field samples for subsequent PCR testing. We also developed the 'HLBdetector' app using the Yolov5l-HLB2 model, which allows farmers to complete HLB detection in seconds with only a mobile phone terminal and without expert guidance. Overall, we successfully constructed a reliable automatic HLB identification model and developed the user-friendly 'HLBdetector' app, facilitating the prevention and timely control of HLB transmission in citrus orchards.

CsiLAC4 modulates boron flow in Arabidopsis and Citrus via high-boron-dependent lignification of cell walls.

The mechanisms underlying plant tolerance to boron (B) excess are far from fully understood. Here we characterized the role of the miR397-CsiLAC4/CsiLAC17 (from Citrus sinensis) module in regulation of B flow. Live-cell imaging techniques were used in localization studies. A tobacco transient expression system tested modulations of CsiLAC4 and CsiLAC17 by miR397. Transgenic Arabidopsis were generated to analyze the biological functions of CsiLAC4 and CsiLAC17. CsiLAC4's role in xylem lignification was determined by mRNA hybridization and cytochemistry. In situ B distribution was analyzed by laser ablation inductively coupled plasma mass spectrometry. CsiLAC4 and CsiLAC17 are predominantly localized in the apoplast of tobacco epidermal cells. Overexpression of CsiLAC4 in Arabidopsis improves the plants' tolerance to boric acid excess by triggering high-B-dependent lignification of the vascular system's cell wall and reducing free B content in roots and shoots. In Citrus, CsiLAC4 is expressed explicitly in the xylem parenchyma and is modulated by B-responsive miR397. Upregulation of CsiLAC4 in Citrus results in lignification of the xylem cell walls, restricting B flow from xylem vessels to the phloem. CsiLAC4 contributes to plant tolerance to boric acid excess via high-B-dependent lignification of cell walls, which set up a 'physical barrier' preventing B flow.

MicroRNA Sequencing Revealed Citrus Adaptation to Long-Term Boron Toxicity through Modulation of Root Development by miR319 and miR171.

Boron (B) toxicity in Citrus is a common physiological disorder leading to reductions in both productivity and quality. Studies on how Citrus roots evade B toxicity may provide new insight into plant tolerance to B toxicity. Here, using Illumina sequencing, differentially expressed microRNAs (miRNAs) were identified in B toxicity-treated Citrus sinensis (tolerant) and C. grandis (intolerant) roots. The results showed that 37 miRNAs in C. grandis and 11 miRNAs in C. sinensis were differentially expressed when exposed to B toxicity. Among them, miR319, miR171, and miR396g-5p were confirmed via 5'-RACE and qRT-PCR to target a myeloblastosis (MYB) transcription factor gene, a SCARECROW-like protein gene, and a cation transporting ATPase gene, respectively. Maintenance of SCARECROW expression in B treated Citrus roots might fulfill stem cell maintenance, quiescent center, and endodermis specification, thus allowing regular root elongation under B-toxic stress. Down-regulation of MYB due to up-regulation of miR319 in B toxicity-treated C. grandis roots might decrease the number of root tips, thereby dramatically changing root system architecture. Our findings suggested that miR319 and miR171 play a pivotal role in Citrus adaptation to long-term B toxicity by targeting MYB and SCARECROW, respectively, both of which are responsible for root growth and development.

A New Diagnostic System for Detection of 'Candidatus Liberibacter asiaticus' Infection in Citrus.

In this study, two polyclonal antibodies were produced against the Omp protein of 'Candidatus Liberibacter asiaticus'. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and ß-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against 'Ca. L. asiaticus'-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect 'Ca. L. asiaticus' in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to 'Ca. L. asiaticus' detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

Diversity and plasticity of the intracellular plant pathogen and insect symbiont "Candidatus Liberibacter asiaticus" as revealed by hypervariable prophage genes with intragenic tandem repeats.

"Candidatus Liberibacter asiaticus" is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of "Ca. Liberibacter" associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyv(I) and hyv(II)) were identified in the prophage regions of the Psy62 "Ca. Liberibacter asiaticus" genome. Sequence analyses of the hyv(I) and hyv(II) genes in 35 "Ca. Liberibacter asiaticus" DNA isolates collected globally revealed that the hyv(I) gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv(II) contains up to 2 NITRs and 4 partial repeats and shares homology with hyv(I). Frequent deletions or insertions of these repeats within the hyv(I) and hyv(II) genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of "Ca. Liberibacter asiaticus" bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single "Ca. Liberibacter asiaticus"-infected sample. This is the first evidence of different "Ca. Liberibacter asiaticus" populations coexisting in a single HLB-affected sample. The Florida "Ca. Liberibacter asiaticus" isolates contain both hyv(I) and hyv(II), while all other global "Ca. Liberibacter asiaticus" isolates contain either one or the other. Interclade assignments of the putative Hyv(I) and Hyv(II) proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple "Ca. Liberibacter asiaticus" populations in the world and a multisource introduction of the "Ca. Liberibacter asiaticus" bacterium into Florida.

Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.