Campestris Pv Research Articles

Antibacterial Polyketides from the Plant Endophytic Fungus Fusarium sp.

Background: Endophytic fungi have been recognized as new sources of natural products with a variety of biological activities, providing lead compounds for drug discovery and development. Objective: The objective of this study is to isolate and identify the secondary metabolites from the plant endophytic fungus Fusarium sp. HJY2 and evaluate their antibacterial activities. Methods: The compounds were isolated and purified by the methods of silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high performance liquid chromatography (HPLC). The structures of the isolated compounds were elucidated by comparing the NMR and MS spectroscopic data with those of pieces of literature. The antibacterial activities were evaluated by the broth microdilution method. Results: Seven polyketides were isolated from the fermented extracts of the fungus Fusarium sp. HJY2 and identified as sydowinol (1), dihydrolateropyrone (2), 13-oxo-9Z,11E-octadecadienoic acid (3), (E)-ferulic acid (4), 4-hydroxyphenylacetic acid (5), methyl 2-(2-hydroxyphenyl)acetate (6) and 4-hydroxy-3-methoxyacetophenone (7). Compound 3 exhibited moderate antibacterial activities against Bacillus subtilis, Mycobacterium smegmatis, Ralstonia solanacearum, and Xanthomonas campestris pv. campestris with MIC values of 40, 40, 80 and 40 μg/mL, respectively. Conclusion: Seven compounds were isolated from the plant endophytic fungus Fusarium sp. HJY2. Compound 1 was isolated from the Fusarium genus for the first time. Compound 3 showed moderate antibacterial activities.

Design, Synthesis, and 3D-QASR of Oxazoline Derivatives Containing an S-S Moiety as Potential Acaricide.

To mitigate the detrimental effects of agricultural pest mites on crop yield, an active substructure splicing strategy was employed to modify etoxazole by introducing an S-S moiety. The target products were obtained efficiently via a bilateral disulfurating reagent (DSMO), which was developed by our group. The leaf dip method was used to evaluate the activities of the designed target compounds against the eggs and larvae of the spider mite (Tetranychus cinnabarinus). Most of the target compounds exhibited good efficacy in controlling the larvae and eggs of T. cinnabarinus. Based on these results, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established to guide the construction of compound 7l. Notably, compound 7l exhibited a better activity against T. cinnabarinus eggs (LC50 = 0.0035 mg/L) compared to etoxazole (LC50 = 0.2990 mg/L). Greenhouse bioassays indicated that compound 7l exhibits excellent acaricidal activity against egg of T. cinnabarinus, which is better than the etoxazole at 1.0 mg/L. Additionally, some of the compounds showed inhibitory effects against Dickeya zeae (D. zeae), Xanthomonas campestris pv campestris (Xcc), Xanthomonas oryzae pvoryza (Xoo), and Xanthomonas oryzae pvoryzicola (Xoc). Furthermore, compounds 7l not only exhibited relatively potent against Plutella xylostella activities (LC50 = 24.0 mg/L) but also had low toxicity (LC50 > 11.0 μg/bee) to Apis mellifera. In conclusion, the current experimental results suggest that oxazoline derivatives containing an S-S moiety have the potential to serve as lead compounds for the development of novel acaricide agents.

Biotechnological potential of silver nanoparticles synthesized by green method to control phytopathogenic bacteria: contributions from a proteomic analysis.

Silver nanoparticles (AgNPs) synthesized through green synthesis routes are widely used as antimicrobial agents due to their advantages such as biocompatibility, stability, sustainability, speed and cost-effectiveness. Although AgNPs appear to be more potent than silver ions, the mechanisms related to their antibacterial activity are not yet fully understood. The most common proposed mechanism of AgNPs' toxicity so far is the release of silver ions and/or specific functions of the particles. In this context, the present study aimed to investigate the mechanisms of action of AgNPs synthesized using noni fruit peels (Morinda citrifolia) against the phytopathogen Xanthomonas campestris pv. campestris (Xcc) through proteomics. Xcc was treated with AgNPs (32 µM), AgNO3 (32 µM), or received no treatment (Ctrl - control condition), and its proteomic response was comprehensively characterized to elucidate the antimicrobial mechanisms of AgNPs in the phytopathogenic microorganism. A total of 352 differentially abundant proteins were identified. Most proteins were regulated in the AgNPs × Ctrl and AgNPs × AgNO3 comparisons/conditions. When Xcc treated with 32 µM AgNPs were compared to controls, the results showed 134 differentially abundant proteins, including 107 increased and 27 decreased proteins. In contrast, when Xcc treated with 32 µM AgNO3 were compared to Ctrl, the results showed only 14 differentially abundant proteins, including 10 increased proteins and 4 decreased proteins. Finally, when Xcc treated with 32 µM AgNPs were compared to Xcc treated with 32 µM AgNO3, the results showed 204 differentially abundant proteins, including 75 increased proteins and 129 decreased proteins. Gene ontology enrichment analysis revealed that most of the increased proteins were involved in important biological processes such as metal ion homeostasis, detoxification, membrane organization, metabolic processes related to amino acids and carbohydrates, lipid metabolic processes, proteolysis, transmembrane transport, and others. The AgNPs used in this study demonstrated effective antimicrobial activity against the phytopathogenic bacteria Xcc. Furthermore, the obtained results contribute to a better understanding of the mechanisms of action of AgNPs in Xcc and may aid in the development of strategies to control Xcc in brassica.

Host-Driven Selection, Revealed by Comparative Analysis of Xanthomonas Type III Secretion Effectoromes, Unveils Novel Recognized Effectors.

Xanthomonas species are specialized plant pathogens, often exhibiting a narrow host range. They rely on the translocation of effector proteins through the type III secretion system to colonize their respective hosts. The effector arsenal varies among Xanthomonas spp., typically displaying species-specific compositions. This species-specific effector composition, collectively termed the effectorome, is thought to influence host specialization. We determined the plant host-derived effectoromes of more than 300 deposited genomes of Xanthomonas species associated with either Solanaceae or Brassicaceae hosts. Comparative analyses revealed clear species-specific effectorome signatures. However, Solanaceae or Brassicaceae host-associated effectorome signatures were not detected. Nevertheless, host biases in the presence or absence of specific effector classes were observed. To assess whether host-associated effector absence results from selective pressures, we introduced effectors unique to Solanaceae pathogens to X. campestris pv. campestris and effectors unique to Brassicaceae pathogens to X. euvesicatoria pv. euvesicatoria (Xeue) and evaluated if these introductions hindered virulence on their respective hosts. Introducing the effector XopI into X. campestris pv. campestris reduced virulence on white cabbage leaves without affecting localized or systemic colonization. Introducing the XopAC or XopJ5 effectors into Xeue reduced virulence and colonization on tomato but not on pepper. Additionally, XopAC and XopJ5 induced a hypersensitive response on tomato leaves when delivered by Xeue or through Agrobacterium-mediated transient expression, confirming recognition in tomato. This study demonstrates the role of host-derived selection in establishing species-specific effectoromes, identifying XopAC and XopJ5 as recognized effectors in tomato.

First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease on Broccoli in China.

Broccoli (Brassica oleracea L. var. italica) is one of the important cruciferous vegetables in China, known for its considerable economic and nutritional value (Li et al. 2021). In September 2023, leaf spot disease was observed on broccoli seedlings in the commercial fields in Weifang City, Shandong Province, China (119°15'E, 36°70'N). Further investigation revealed that the disease incidence was approximately 60% in 4.5 square hectometers (hm2) broccoli field, resulting in substantial economic losses. This disease primarily affects the leaves, manifesting distinct symptoms such as circular, dark necrotic spots that gradually lighten and are encircled by a chlorotic halo. Some lesions further develop a black or purplish border, exhibit concentric zonation, and eventually fall off, leaving behind holes, as shown in Figure S1B and C. In severe cases, decay originates from the central perforation and spreads outwards, as shown in Figure S1A. To identify the causal agent of this disease, infected leaf tissues were collected and surface disinfected by immersing in 75% ethanol for 30 seconds, followed by three rinses with sterile water. The samples were grinded in sterile deionized water, and the extract was plated on NA. After incubation at 28°C for 48 h, individual colonies were transferred to fresh NA plates. A total of 12 strains with the similar morphological characteristics were isolated from diseased samples collected from the three plots. After 48 hours of growth on NA medium at 28℃, each colony attained a diameter ranging from 3 to 5 mm. These colonies appeared yellow, slightly elevated, nearly circular in shape, with a smooth and moist edge. Three representative strains were selected for further investigation. All strains were Gram-negative, aerobic, and rod-shaped. The analysis of BIOLOG GENIII microplate system revealed the capability of three isolates using cellobiose, trehalose, glucose, mannose, galactose, and sucrose. Furthermore, the isolates were unable to hydrolyze arginine and utilize rhamnose and inositol. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Xanthomonas (OR772321-OR772323), followed by PCR amplification for 4 housekeeping genes atpD, dnaK, gyrB, and rpoD (Young et al. 2008; Saux et al. 2015). The obtained sequences were submitted to the GenBank under accessions OR789628-OR789630, OR785471-OR785473, OR789631-OR789633, and OR785468-OR785470. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). The phylogenetic analysis revealed that all the three isolates were clustered with Xanthomonas campestris pv. raphani strains 5055 and 576, respectively (Figure S2). (Dubrow et al. 2022). These results were consistent with those of the reported X. campestris pv. raphanin (Cruz et al. 2015). To verify the pathogenicity of these strains, we used a spray inoculation method. In detail, bacterial suspensions (30 mL per treatment) containing approximately 108 CFU/ml were sprayed onto healthy, four-week-old broccoli plants and incubated in a phytotron at 28°C and above 90% RH. Negative controls were performed using sterile distilled water. Each isolate underwent three trials and each treatment included 12 broccoli seedlings. Leaf spot symptoms were observed on 5 days post inoculation, as shown in Figure S1E, F and G. Negative control plants showed no symptoms (Figure S1D). We further re-isolated the bacterium from the symptomatic plants and verifying the bacteria as X. campestris pv. raphanin with the aforementioned sequence analysis, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of X. campestris pv. raphani causing leaf spot disease on broccoli in China. This study enhances our understanding of the pathogenic bacterium on broccoli and lays the groundwork for developing targeted management strategies.

The phytopathogen Xanthomonas campestris senses and effluxes salicylic acid via a sensor HepR and an RND family efflux pump to promote virulence in host plants.

Salicylic acid (SA) plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens. Following pathogen recognition, SA biosynthesis dramatically increases at the infection site of the host plant. The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood. The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv. campestris (Xcc) senses and effluxes SA during infection inside host plants. First, RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171, encoding a MarR family transcription factor HepR and an RND (resistance-nodulation-cell division) family efflux pump HepABCD in Xcc. Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR negatively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon, Phep. Second, isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor. SA binding released HepR from its cognate promoter Phep and then induced the expression of hepABCD. Third, the RND family efflux pump HepABCD was responsible for SA efflux. The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover. Finally, the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage. These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

Preliminary Exploration of Low Frequency Low-Pressure Capacitively Coupled Ar-O2 Plasma

Non-thermal plasma as an emergent technology has received considerable attention for its wide range of applications in agriculture, material synthesis, and the biomedical field due to its low cost and portability. It has promising antimicrobial properties, making it a powerful tool for bacterial decontamination. However, traditional techniques for producing non-thermal plasma frequently rely on radiofrequency (RF) devices, despite their effectiveness, are intricate and expensive. This study focuses on generating Ar-O2 capacitively coupled plasma under vacuum conditions, utilizing a low-frequency alternating current (AC) power supply, to evaluate the system’s antimicrobial efficacy. A single Langmuir probe diagnostic was used to assess the key plasma parameters such as electron density (ne), electron temperature (Te), and electron energy distribution function (EEDF). Experimental results showed that ne increases (7 × 1015 m−3 to 1.5 × 1016 m−3) with a rise in pressure and AC power. Similarly, the EEDF modified into a bi-Maxwellian distribution with an increase in AC power, showing a higher population of low-energy electrons at higher power. Finally, the generated plasma was tested for antimicrobial treatment of Xanthomonas campestris pv. Vesicatoria. It is noted that the plasma generated by the AC power supply, at a pressure of 0.5 mbar and power of 400 W for 180 s, has 75% killing efficiency. This promising result highlights the capability of the suggested approach, which may be a budget-friendly and effective technique for eliminating microbes with promising applications in agriculture, biomedicine, and food processing.

Host plant-derived benzoic acid interferes with 4-hydroxybenzoic acid degradation in the phytopathogen Xanthomonas campestris by competitively binding to PobR

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassica vegetables, which can induce the host plant to produce salicylic acid and 4-hydroxybenzoic acid (4-HBA) during infection. Xcc was previously shown to sense and degrade host plant-derived 4-HBA via the sensor PobR and a PobA-dependent pathway. The degradation of 4-HBA is associated with Xcc virulence in cabbage. The present study generated a reporter strain XC1::PpobA-gusA to monitor pobA transcription. 4-HBA-like compounds were screened for their ability to interfere with pobA transcription. Benzoic acid (BA) was found to efficiently decrease pobA transcription in a dose-dependent manner. Xcc neither produced nor degraded BA; however, the exogenous addition of BA to the 4-HBA-containing Xcc culture significantly decreased the 4-HBA degradation rate. Furthermore, addition of BA into the Xcc culture did not significantly affect the transcription of pobA or pobR; however, addition of BA into the 4-HBA-containing culture significantly decreased the transcription of both genes. Isothermal titration calorimetry and an electrophoretic mobility shift assay revealed that BA binds to PobR with a moderate affinity, which interfered with the binding of 4-HBA/PobR complex to the pobA promoter and thereby inhibiting pobA transcription and 4-HBA degradation. The endogenous BA level of the infected cabbage leaves increased in response to Xcc infection. In the presence of BA, the virulence of Xcc on cabbage decreased significantly. Taken together, these results suggest that cabbage utilizes BA to interfere with 4-HBA degradation, thereby reducing Xcc virulence. Thus, BA has the potential to be developed as a bactericide against Xcc infection.

Interactive regulation of immune-related resistance genes with salicylic acid and jasmonic acid signaling in systemic acquired resistance in the Xanthomonas–Brassica pathosystem

Pathogen-responsive immune-related genes (resistance genes [R-genes]) and hormones are crucial mediators of systemic acquired resistance (SAR). However, their integrated functions in regulating SAR signaling components in local and distal leaves remain largely unknown. To characterize SAR in the Xanthomonas campestris pv. campestris (Xcc)–Brassica napus pathosystem, the responses of R-genes, (leaf and phloem) hormone levels, H2O2 levels, and Ca2+ signaling-related genes were assessed in local and distal leaves of plants exposed to four Xcc-treatments: Non-inoculation (control), only secondary Xcc-inoculation in distal leaves (C-Xcc), only primary Xcc-inoculation in local leaves (Xcc), and both primary and secondary Xcc-inoculation (X-Xcc). The primary Xcc-inoculation provoked disease symptoms as evidenced by enlarged destructive necrosis in the local leaves of Xcc and X-Xcc plants 7 days post-inoculation. Comparing visual symptoms in distal leaves 5 days post-secondary inoculation, yellowish necrotic lesions were clearly observed in non Xcc-primed plants (C-Xcc), whereas no visual symptom was developed in Xcc-primed plants (X-Xcc), demonstrating SAR. Pathogen resistance in X-Xcc plants was characterized by distinct upregulations in expression of the PAMP-triggered immunity (PTI)-related kinase-encoding gene, BIK1, the (CC-NB-LRR-type) R-gene, ZAR1, and its signaling-related gene, NDR1, with a concurrent enhancement of the kinase-encoding gene, MAPK6, and a depression of the (TIR-NB-LRR-type) R-gene, TAO1, and its signaling-related gene, SGT1, in distal leaves. Further, in X-Xcc plants, higher salicylic acid (SA) and jasmonic acid (JA) levels, both in phloem and distal leaves, were accompanied by enhanced expressions of the SA-signaling gene, NPR3, the JA-signaling genes, LOX2 and PDF1.2, and the Ca2+-signaling genes, CAS and CBP60g. However, in distal leaves of C-Xcc plants, an increase in SA level resulted in an antagonistic depression of JA, which enhanced only SA-dependent signaling, EDS1 and NPR1. These results demonstrate that primary Xcc-inoculation in local leaves induces resistance to subsequent pathogen attack by upregulating BIK1-ZAR1-mediated synergistic interactions with SA and JA signaling as a crucial component of SAR.

Light- and Relative Humidity-Regulated Hypersensitive Cell Death and Plant Immunity in Chinese Cabbage Leaves by a Non-adapted Bacteria Xanthomonas campestris pv. vesicatoria.

Inoculation of Chinese cabbage leaves with high titer (107 cfu/ml) of the non-adapted bacteria Xanthomonas campestris pv. vesicatoria (Xcv) strain Bv5-4a.1 triggered rapid leaf tissue collapses and hypersensitive cell death (HCD) at 24 h. Electrolyte leakage and lipid peroxidation markedly increased in the Xcv-inoculated leaves. Defence-related gene expressions (BrPR1, BrPR4, BrChi1, BrGST1 and BrAPX1) were preferentially activated in the Xcv-inoculated leaves. The Xcv-triggered HCD was attenuated by continuous light but accelerated by a dark environment, and the prolonged high relative humidity also alleviated the HCD. Constant dark and increased relative humidity provided favorable conditions for the Xcv bacterial growth in the leaves. Pretreated fluridone (biosynthetic inhibitor of endogenous abscisic acid [ABA]) increased the HCD in the Xcv-inoculated leaves, but exogenous ABA attenuated the HCD. The pretreated ABA also reduced the Xcv bacterial growth in the leaves. These results highlight that the onset of HCD in Chinese cabbage leaves initiated by non-adapted pathogen Xcv Bv5-4a.1 and in planta bacterial growth was differently modulated by internal and external conditional changes.

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

An improved bacterial mRNA enrichment strategy in dual RNA sequencing to unveil the dynamics of plant-bacterial interactions

BackgroundDual RNA sequencing is a powerful tool that enables a comprehensive understanding of the molecular dynamics underlying plant-microbe interactions. RNA sequencing (RNA-seq) poses technical hurdles in the transcriptional analysis of plant-bacterial interactions, especially in bacterial transcriptomics, owing to the presence of abundant ribosomal RNA (rRNA), which potentially limits the coverage of essential transcripts. Therefore, to achieve cost-effective and comprehensive sequencing of the bacterial transcriptome, it is imperative to devise efficient methods for eliminating rRNA and enhancing the proportion of bacterial mRNA. In this study, we modified a strand-specific dual RNA-seq method with the goal of enriching the proportion of bacterial mRNA in the bacteria-infected plant samples. The enriched method involved the sequential separation of plant mRNA by poly A selection and rRNA removal for bacterial mRNA enrichment followed by strand specific RNA-seq library preparation steps. We assessed the efficiency of the enriched method in comparison to the conventional method by employing various plant-bacterial interactions, including both host and non-host resistance interactions with pathogenic bacteria, as well as an interaction with a beneficial rhizosphere associated bacteria using pepper and tomato plants respectively.ResultsIn all cases of plant-bacterial interactions examined, an increase in mapping efficiency was observed with the enriched method although it produced a lower read count. Especially in the compatible interaction with Xanthmonas campestris pv. Vesicatoria race 3 (Xcv3), the enriched method enhanced the mapping ratio of Xcv3-infected pepper samples to its own genome (15.09%; 1.45-fold increase) and the CDS (8.92%; 1.49-fold increase). The enriched method consistently displayed a greater number of differentially expressed genes (DEGs) than the conventional RNA-seq method at all fold change threshold levels investigated, notably during the early stages of Xcv3 infection in peppers. The Gene Ontology (GO) enrichment analysis revealed that the DEGs were predominantly enriched in proteolysis, kinase, serine type endopeptidase and heme binding activities.ConclusionThe enriched method demonstrated in this study will serve as a suitable alternative to the existing RNA-seq method to enrich bacterial mRNA and provide novel insights into the intricate transcriptomic alterations within the plant-bacterial interplay.

Effect of varying levels of achichi (Cannabis sativum L.) seed oil extract in the inhibition of bacteria spot disease of scotch pepper (Capsicum annum L.)

Negative food safety reports over the use of synthetic pesticides in controlling bacteria disease of pepper caused by Xanthomonas campestris pv is increasing in Nigeria, hence the need for friendly options. An experiment was carried out at the Teaching and Research Farm of Rufus Giwa Polytechnic, Owo, located on latitude 70 12N and longitude 50 35E at 350m above sea levels, to evaluate the effect of varying levels of achichi (Cannabis sativum L.) seed oil extract in the inhibition of bacteria spot disease of scotch pepper (Capsicum annum L.). Treatments include; Positive control (Synthetic Rindomil gold), Negative control (distill water), 2 ml ASO (Achichi seed oil), and 4ml ASO (Achichi seed oil) which were randomly distributed into plots, arranged in a Randomized Complete Block Design (RCBD). Results showed that the application of ASO had bactericidal inhibition potential against the bacteria spot diseases comparable to the synthetic ridomil powder. Application of between 2 – 4 ml / 1 liter of water of ASO was found to significantly influence the general performance of sprayed pepper in the study area. Further investigation should be carried out to determine the right volume between the ranges of 2 ml – 4 ml of ASO that can effectively control the bacteria spot disease in the study area.

Discovery of Anomer-Inverting Transglycosylase: Cyclic Glucohexadecaose-Producing Enzyme from Xanthomonas, a Phytopathogen.

Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized β-1,2-glucohexadecaose (CβG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CβG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CβG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear β-1,2-glucan to CβG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.

A large scale bacterial attraction assay: A new quantitative bacterial migration assay suitable for genetic screens.

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

ScAnalyzer: an image processing tool to monitor plant disease symptoms and pathogen spread in Arabidopsis thaliana leaves

BackgroundPlants are known to be infected by a wide range of pathogenic microbes. To study plant diseases caused by microbes, it is imperative to be able to monitor disease symptoms and microbial colonization in a quantitative and objective manner. In contrast to more traditional measures that use manual assignments of disease categories, image processing provides a more accurate and objective quantification of plant disease symptoms. Besides monitoring disease symptoms, computational image processing provides additional information on the spatial localization of pathogenic microbes in different plant tissues.ResultsHere we report on an image analysis tool called ScAnalyzer to monitor disease symptoms and bacterial spread in Arabidopsis thaliana leaves. Thereto, detached leaves are assembled in a grid and scanned, which enables automated separation of individual samples. A pixel color threshold is used to segment healthy (green) from chlorotic (yellow) leaf areas. The spread of luminescence-tagged bacteria is monitored via light-sensitive films, which are processed in a similar manner as the leaf scans. We show that this tool is able to capture previously identified differences in susceptibility of the model plant A. thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris. Moreover, we show that the ScAnalyzer pipeline provides a more detailed assessment of bacterial spread within plant leaves than previously used methods. Finally, by combining the disease symptom values with bacterial spread values from the same leaves, we show that bacterial spread precedes visual disease symptoms.ConclusionTaken together, we present an automated script to monitor plant disease symptoms and microbial spread in A. thaliana leaves. The freely available software (https://github.com/MolPlantPathology/ScAnalyzer) has the potential to standardize the analysis of disease assays between different groups.

Design, synthesis and bactericidal activity of novel coumarin derivatives containing pyridinium salts

Coumarin is a natural product known for its diverse biological activities. While its antifungal properties in agricultural chemistry have been extensively studied, there is limited research on its antibacterial potential. In this study, we developed several novel coumarin derivatives by combining coumarin with pyridinium salt through molecular hybridization and chemical synthesis. Our findings reveal that most of these derivatives exhibit promising antibacterial activity. Among them, derivative A25 has been identified as the most effective compound based on three-dimensional quantitative structure-activity relationships. It demonstrates significant in vitro and in vivo activity against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), and Xanthomonas campestris pv. citri (Xac), outperforming the commercially available thiediazole copper. Initial investigations into its mechanism of action suggest that A25 disrupts the cell membranes of Xoc and Xoo, thereby inhibiting bacterial growth. Additionally, A25 enhances the activity of defense enzymes in rice and modulates the expression of proteins related to the pyruvate metabolism pathway. This dual action contributes to rice's resistance against bacterial infestation. We anticipate that this study will serve as a foundation for the development of coumarin-based bactericides.

Functional Analysis of Type III Effectors in Xanthomonas campestris pv. campestris Reveals Distinct Roles in Modulating Arabidopsis Innate Immunity.

Xanthomonas campestris pv. campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Its virulence relies heavily on the type III secretion system (T3SS), facilitating effector translocation into plant cells. The type III effectors (T3Es) disrupt cellular processes, promoting pathogen proliferation. However, only a few T3Es from Xcc have been thoroughly characterized. In this study, we further investigated two effectors using the T3Es-deficient mutant and the Arabidopsis protoplast system. XopE2Xcc triggers Arabidopsis immune responses via an unidentified activator of the salicylic acid (SA) signaling pathway, whereas XopLXcc suppresses the expression of genes associated with patterns-triggered immunity (PTI) and the SA signaling pathway. These two effectors exert opposing effects on Arabidopsis immune responses. Additionally, we examined the relationship between the specific domains and functions of these two effector proteins. Our findings demonstrate that the N-myristoylation motif and N-terminal domain are essential for the subcellular localization and virulence of XopE2Xcc and XopLXcc, respectively. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to developing effective strategies for controlling bacterial disease.

Biological Control of Tomato Bacterial Leaf Spots and Its Impact on Some Antioxidant Enzymes, Phenolic Compounds, and Pigment Content.

Tomato bacterial spots, caused by Xanthomonas campestris pv. vesicatoria (Xcv1) and X. euvesicatoria (Xe2), as well as bacterial specks, caused by two strains of Pseudomonas syringae pv. tomato (Pst1 and Pst2), represent significant threats to tomato production in the El-Sharkia governorate, often resulting in substantial yield losses. The objective of this study was to evaluate the efficacy of various biocontrol culture filtrates, including bacteria and fungi agents, in managing the occurrence and severity of these diseases, while also monitoring physiological changes in tomato leaves, including antioxidant enzymes, phenolics, and pigment content. The culture filtrates from examined Trichoderma species (T. viride, T. harzianum, and T. album), as well as the tested bacteria (Bacillus subtilis, Pseudomonas fluorescens, and Serratia marcescens) at concentrations of 25%, 50%, and 100%, significantly inhibited the proliferation of pathogenic bacteria In vitro. For the In vivo experiments, we used specific doses of 5 mL of spore suspension per plant for the fungal bioagents at a concentration of 2.5 × 107 spores/mL. The bacterial bioagents were applied as a 10 mL suspension per plant at a concentration of 1 × 108 CFU/mL. Spraying the culture filtrates of the tested bioagents two days before infection In vivo significantly reduced disease incidence and severity. Trichoderma viride exhibited the highest efficacy among the fungal bioagents, followed by T. harzianum and T. album. Meanwhile, the culture filtrate of B. subtilis emerged as the most potent among the bacterial bioagents, followed by P. fluorescens. Furthermore, applying these culture filtrates resulted in elevated levels of chitinase, peroxidase, and polyphenol oxidase activity. This effect extended to increased phenol contents, as well as chlorophyll a, chlorophyll b, and carotenoids in sprayed tomato plants compared to the control treatment. Overall, these findings underscore the potential of these biocontrol strategies to effectively mitigate disease incidence and severity while enhancing plant defense mechanisms and physiological parameters, thus offering promising avenues for sustainable disease management in tomato production.

Biochemical characterization of a novel β-galactosidase from Pedobacter sp. with strong transglycosylation activity at low lactose concentration.

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917bp, encoding 638 amino acids with a predicted molecular mass of 62.3kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50°C, respectively, and it was stable within pH 4.5‒7.0 and up to 45°C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4°C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.