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.

Comparative Analysis of Novel Lytic Phages for Biological Control of Phytopathogenic Xanthomonas spp.

Xanthomonas is an important genus of plant-pathogenic bacteria that affects agronomic and economically important crops, causing serious economic losses. In fact, several Xanthomonas species are considered regulated quarantine pests. Due to the lack of effective control measures to treat plant-pathogenic bacteria, innovative control tools are needed to carry out integrated disease management. In this regard, bacteriophages (phages), viruses of bacteria, constitute a promising biocontrol tool. In this work, we report the isolation and characterization of 11 novel Xanthomonas arboricola pv. juglandis phages belonging to different families and genera of the class Caudoviricetes. Infectivity matrix in more than 60 isolates of different xanthomonads and other phytopathogenic bacteria suggests that these phages are specific to the Xanthomonas genus, with different host ranges depending on the isolates tested. Interestingly, some of these phages showed relevant features to be used as biocontrol tools to combat pathogenic Xanthomonas spp. as important as X. oryzae or X. citri. IMPORTANCE Phytopathogenic bacteria represent serious losses worldwide. The lack of current treatments has focused the spotlight on phages, viruses of bacteria, as very promising biocontrol tools. Phages are very specific and can help to control bacterial infections in crops, as is the case of xanthomonads-associated diseases. The discovery of new environmental phages with lytic capacity that can help to combat these pathogens is of special relevance, and it is necessary to implement phage isolation and characterization techniques to determine their host range and their genomic properties. The establishment of phage collections worldwide will allow their use as preventive, diagnostic, or therapeutic tools. Although there is still a long way to go, this work is a step forward in the implementation of new ecofriendly techniques to combat key pathogens in the field.

A Pan-Global Study of Bacterial Leaf Spot of Chilli Caused by Xanthomonas spp.

Bacterial Leaf Spot (BLS) is a serious bacterial disease of chilli (Capsicum spp.) caused by at least four different Xanthomonas biotypes: X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, X. hortorum pv. gardneri, and X. vesicatoria. Symptoms include black lesions and yellow halos on the leaves and fruits, resulting in reports of up to 66% losses due to unsalable and damaged fruits. BLS pathogens are widely distributed in tropical and subtropical regions. Xanthomonas is able to survive in seeds and crop residues for short periods, leading to the infections in subsequent crops. The pathogen can be detected using several techniques, but largely via a combination of traditional and molecular approaches. Conventional detection is based on microscopic and culture observations, while a suite of Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) assays are available. Management of BLS is challenging due to the broad genetic diversity of the pathogens, a lack of resilient host resistance, and poor efficacy of chemical control. Some biological control agents have been reported, including bacteriophage deployment. Incorporating stable host resistance is a critical component in ongoing integrated management for BLS. This paper reviews the current status of BLS of chilli, including its distribution, pathogen profiles, diagnostic options, disease management, and the pursuit of plant resistance.

Taxonomic Refinement of Xanthomonas arboricola.

Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of nonpathogenic and taxonomically unvalidated strains are also designated as X. arboricola, leading to a complicated taxonomic status in the species. In this study, we have evaluated the whole-genome resources of all available Xanthomonas spp. strains designated as X. arboricola in the public databases to refine the members of the species based on DNA similarity indexes and core genome-based phylogeny. Our results show that, of the nine validly described pathovars within X. arboricola, pathotype strains of seven pathovars are taxonomically genuine, belonging to the core clade of the species regardless of their pathogenicity on the host of isolation (thus the validity of pathovar status). However, strains of X. arboricola pv. guizotiae and X. arboricola pv. populi do not belong to X. arboricola because of the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, we propose to elevate the two pathovars to the rank of a species as X. guizotiae sp. nov. with the type strain CFBP 7408T and X. populina sp. nov. with the type strain CFBP 3123T. In addition, other mislabeled strains of X. arboricola were scattered within Xanthomonas spp. that belong to previously described species or represent novel species that await formal description.

Resistance to diseases of sour, sweet and wild passion fruit genotypes

The cultivation of passion fruit is important for Brazil, since the country is currently the largest producer and consumer of fruit in the world. However, the fields of passion fruit still face important problems due to the incidence and severity of diseases in the field. Thus, the present study aimed to assess resistance to bacterial and fungal diseases in 13 genotypes of sour, sweet and wild passion fruit, in field conditions in the Distrito Federal, Brazil. For this, a field experiment was installed in a randomized block design, with four replications and 13 treatments (genotypes). The characteristics of incidence, severity and degree of resistance for bacteriosis, septoriosis, scab and anthracnose diseases were evaluated in 5 fruits per plot of each genotype. Genetic parameters of the evaluated traits were also estimated. High heritability values and CVg/Cve ratio were observed for most of the evaluated characteristics. The genotypes presented mean values of incidence and severity of bacteriosis, septoriosis, scab and anthracnose different among them, and the one that presented the best results in the degree of resistance for all diseases was F1 (MAR20 # 24 x ECL7 P1 R4).

Bacteriophage-Mediated Control of Phytopathogenic Xanthomonads: A Promising Green Solution for the Future.

Xanthomonads, members of the family Xanthomonadaceae, are economically important plant pathogenic bacteria responsible for infections of over 400 plant species. Bacteriophage-based biopesticides can provide an environmentally friendly, effective solution to control these bacteria. Bacteriophage-based biocontrol has important advantages over chemical pesticides, and treatment with these biopesticides is a minor intervention into the microflora. However, bacteriophages' agricultural application has limitations rooted in these viruses' biological properties as active substances. These disadvantageous features, together with the complicated registration process of bacteriophage-based biopesticides, means that there are few products available on the market. This review summarizes our knowledge of the Xanthomonas-host plant and bacteriophage-host bacterium interaction's possible influence on bacteriophage-based biocontrol strategies and provides examples of greenhouse and field trials and products readily available in the EU and the USA. It also details the most important advantages and limitations of the agricultural application of bacteriophages. This paper also investigates the legal background and industrial property right issues of bacteriophage-based biopesticides. When appropriately applied, bacteriophages can provide a promising tool against xanthomonads, a possibility that is untapped. Information presented in this review aims to explore the potential of bacteriophage-based biopesticides in the control of xanthomonads in the future.

Quorum Sensing Regulation in Phytopathogenic Bacteria.

Quorum sensing is a type of chemical communication by which bacterial populations control expression of their genes in a coordinated manner. This regulatory mechanism is commonly used by pathogens to control the expression of genes encoding virulence factors and that of genes involved in the bacterial adaptation to variations in environmental conditions. In phytopathogenic bacteria, several mechanisms of quorum sensing have been characterized. In this review, we describe the different quorum sensing systems present in phytopathogenic bacteria, such as those using the signal molecules named N-acyl-homoserine lactone (AHL), diffusible signal factor (DSF), and the unknown signal molecule of the virulence factor modulating (VFM) system. We focus on studies performed on phytopathogenic bacteria of major importance, including Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella,Dickeya, and Pectobacterium spp. For each system, we present the mechanism of regulation, the functions targeted by the quorum sensing system, and the mechanisms by which quorum sensing is regulated.

Tea Tree Oil Induces Systemic Resistance against Fusarium wilt in Banana and Xanthomonas Infection in Tomato Plants.

The essential tea tree oil (TTO) derived from Melaleuca alternifolia plant is widely used as a biopesticide to protect crops from several plant-pathogens. Its activity raised queries regarding its ability to, not only act as a bio-fungicide or bio-bactericide, but also systemically inducing resistance in plants. This was examined by TTO application to banana plants challenged by Fusarium oxysporum f. sp. cubense (Foc, Race 1) causing Fusarium wilt and to tomato plants challenged by Xanthomonas campestris. Parameters to assess resistance induction included: disease development, enzymatic activity, defense genes expression correlated to systemic acquired resistance (SAR) and induced systemic resistance (ISR) and priming effect. Spraying TTO on field-grown banana plants infected with Foc and greenhouse tomato plants infected with Xanthomonas campestris led to resistance induction in both hosts. Several marker genes of salicylic acid, jasmonic acid and ethylene pathways were significantly up-regulated in parallel with symptoms reduction. For tomato plants, we have also recorded a priming effect following TTO treatment. In addition to fungicidal and bactericidal effect, TTO can be applied in more sustainable strategies to control diseases by enhancing the plants ability to defend themselves against pathogens and ultimately diminish chemical pesticides applications.

Development of Real-Time and Colorimetric Loop Mediated Isothermal Amplification Assay for Detection of Xanthomonas gardneri.

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/µL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls.

Comparative Proteomic Analysis of Three Xanthomonas spp. Cultured in Minimal and Rich Media.

Bacteria change their gene expression when exposed to different nutrient conditions. The levels of proteins do not always correlate with those of RNAs, hence proteomic analysis is required for understanding how bacteria adapt to different conditions. Herein, differentially abundant proteins from Xanthomonas oryzae pv. oryzae (Xoo), X. campestris pv. vesicatoria (Xcv), and X. axonopodis pv. glycines (Xag), which were cultured in rich media and in minimal media, were determined using label-free shotgun proteomic analysis and clusters of orthologous groups classification. The detected proteins from all three species ranged from 1190 to 1187. Among them, 702, 584, and 529 proteins from Xoo, Xcv, and Xag, respectively, were more (> twofold) abundant depending on the media, indicating that about 11.4-13.8% of proteins from the three species were differentially expressed. The levels of abundant proteins in minimal media were significantly higher than those in rich media for all three species, demonstrating how Xanthomonas species actively change their protein expression in different nutrient conditions. These results will lead to new insights in elucidation of cellular mechanisms involved in virulence and adaption of bacteria to harsh environments for further studies. The MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD006310.

Pathotypes of Bacterial Spot Pathogen Infecting Capsicum Peppers in Korea.

Sixty-seven isolates of bacterial spot pathogen (Xanthomonas spp.) collected from six provinces of Korea were tested for the identification of their pathotypes and determination of their distribution throughout Korea in an effort to genetically manage the disease. Near isogenic lines of Early Calwonder (Capsicum annuum) pepper plants carrying Bs1 , Bs2 and Bs3 , and PI235047 (C. pubescens) were used as differential hosts. Race P1 was found to be predominant, followed by race P7, and races P3 and P8 were also observed. This is the first report of races P7 and P8 in Korea. The races P7 and P8 were differentiated from the former races P1 and P3, respectively, on the basis of their ability to elicit hypersensitive reactions to PI235047.

Development of a Species-specific PCR Assay for Three Xanthomonas Species, Causing Bulb and Flower Diseases, Based on Their Genome Sequences.

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of 1 pg/µl per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

In vitro control of plant pathogenic Xanthomonas spp. using Poncirus trifoliata Rafin.

The secondary metabolites such as essential oil and pure compounds (limonin and imperatorin) from Poncirus trifoliata Rafin were tested for in vitro control of phytopathogenic bacteria of Xanthomonas spp. In vitro studies showed that the oil had inhibitory effect on Xanthomonas campestris pv. compestris KC94-17-XCC, Xanthomonas campestris pv. vesicatoria YK93-4-XCV, Xanthomonas oryzae pv. oryzae KX019-XCO and Xanthomonas sp. SK12 with their inhibition zones and minimum inhibitory concentration (MIC) values ranging from 13.1~22.1 mm and 62.5~125 µg/ml, respectively. Limonin and imperatorin also had in vitro antibacterial potential (MIC: 15.62~62.5 µg/ml) against all the tested Xanthomonas spp. Furthermore, the SEM studies demonstrated that limonin and imperatorin caused morphological changes of Xanthomonas sp. SK12 at the minimum inhibitory concentration (15.62 µg/ml). These results of this study support the possible use of essential oil and natural compounds from P. Trifoliata in agriculture and agro-industries to control plant pathogenic microorganisms.

Avaliação do cloreto de dodecil dimetil amônio para o controle da mancha bacteriana do tomateiro / Evaluation of didecyl dimethyl ammonium chloride for the control of tomato bacterial spot

O objetivo deste trabalho foi avaliar a sensibilidade de isolados do complexo Xanthomonas spp. associado à mancha bacteriana do tomateiro ao cloreto de dodecil dimetil amônio e a sua eficiência no controle da doença. Este produto apresentou maior inibição in vitro aos isolados avaliados em relação ao hidróxido de cobre e aos cloretos de benzalcônio. Entretanto, não se observou redução da severidade da doença quando da utilização deste produto in vivo.

The objective of this study was to evaluate the efficiency of didecyl dimethyl ammonium chloride in vitro inhibition of Xanthomonas spp. and the control of tomato bacterial spot. This product showed a higher in vitro inhibition of the pathogen, compared to copper hydroxide and benzalkonium chloride. However, there was no reduction in disease severity when the product was sprayed on the plants.