Satureja montana Essential Oil, Zein Nanoparticles and Their Combination as a Biocontrol Strategy to Reduce Bacterial Spot Disease on Tomato Plants

Tomato bacterial spot (Bs), caused by Xanthomonas spp., including X. euvesicatoria (Xeu) remains a major threat for tomato production. The emergence of copper resistance strains of Xeu calls urgently for eco-friendly phytosanitary treatments as sustainable green alternatives for disease control. Satureja spp. essential oil (EO) has antimicrobial activity against xanthomonads and combined with zein nanoparticles (ZNPs), might offer a viable option for field applications. This study aims to evaluate the effects of S. montana EO, of ZNPs, and their combination in a nanoformulation, on Xeu quantity, and how these compounds modulate molecular and physiological changes in the pathosystem. Uninfected and infected tomato plants (var. Oxheart) were treated with EO; ZNPs and nanoformulation (EO + ZNPs). Treatments reduced Xeu amount by a minimum of 1.6-fold (EO) and a maximum of 202-fold (ZNPs) and improved plants’ health. Nanoformulation and ZNPs increased plants’ phenolic content. ZNPs significantly increased GPX activity and reduced CAT activity. Overall treatments upregulated transcripts of the phenylpropanoid pathway in infected plants, while ZNPs and nanoformulation upregulated those transcripts in uninfected plants. Both sod and aao transcripts were downregulated by treatments in infected plants. These findings demonstrate that S. montana EO, ZNPs and their nanoformulation are suitable to integrate tomato bacterial spot management strategies, mainly due to their antimicrobial activity on Xeu, however further field studies clarifying the long-term action of these products are required. These results also support the prophylactic potential of ZNPs on tomato bacterial spot.

Attenuations of bacterial spot disease Xanthomonas euvesicatoria on tomato plants treated with biostimulants

Background The bacterial-spot disease caused by different Xanthomonas species is one of the major tomato diseases that reduce crop production and quality. Pesticides indiscriminate usage has resulted in an increase in resistant bacterial strains as well as contamination of farmers, consumers and the environment. Plant growth-promoting bacteria and humic acids can act as elicitors of plant defence mechanism causing extensive transcriptional and metabolic reprogramming which, in turn, produce a range of plant chemical defences. The purpose of this study was to study how humic acids and plant growth-promoting bacteria, when applied to the substrate, affected the severity of bacterial spot symptoms in tomato leaves. Materials and methods One-month-old Micro-Tom tomato (Solanum lycopersicum L.) were transferred to 3 L pots filled with a sterile mixture of sand and vermiculite (2:1, v:v) and treated or not (control) with 250 mL of 4.5 mmol C. L−1 of humic acids, Herbaspirillum seropedicae (108 CFU. mL−1) and the combination of humic acids plus H. seropedicae. One day after substrate treatment, the leaves were inoculated (or not) with X. euvesicatoria (Xe). The area below the disease progression curve based on severity scores and the number of symptomatic leaflets was used to assess phytopathogen virulence. The concentration of oxalic, citric and succinic acids in leaf extracts were determined using HPLC analysis. Results Sole or combined H. seropedicae (BAC) and humic acids (HA) application promoted shoot and root growth related to control when plants were challenged with Xe pathogen. For plants inoculated with Xe, more significant plant-growth promotion results were obtained for HA + BAC treatment. The first visible symptoms were observed 16 days after inoculation with 2 × 104 CFU. g−1 of Xe cells in leaves of control plants. HA and BAC applied alone or combined reduced disease severity. Only plants treated with HA were able to reduce disease incidence (number of the leaflets with symptoms). Organic acids, such as oxalic, citric and succinic acids, rose in Xe-inoculated leaves. The reduced amount of organic acids in diseased leaves treated with HA + BAC may be linked to a decrease in disease progression. Conclusion Humic acids and H. seropedicae increased growth by modulating the content of organic acids in leaf tissue, attenuating the symptoms of the bacterial spot disease. Graphic abstract

Reaction of Sour Passion Fruit Lineages and Hybrids to Bacterial Spot Caused by Xanthomonas axononopodis pv. passiflorae Under Protected Cultivation and Field Conditions

Passion fruit is infected by many plant pathogens, including Xanthomonas axonopodis pv. passiflorae, causing bacterial spot disease. This disease has been impaired orchards all over Brazil, resulting in low yield and production. In addition, it results in premature death of plants in the field. This study aimed to analyze the reaction of sour passion fruit genotypes to bacterial spot in leaves under protected cultivation (Experiment 1) and the bacterial spot severity in fruits under field conditions (Experiment 2). Experiment 1 was carried out as a randomized block design (RBD) with subdivided plots, composed of 24 treatments (genotypes), three replications, five plants per plot, and five evaluation dates. Bacterial spot incidence and severity were evaluated using a 0 to 5 grading scale. Evaluations were carried out at a 7-day interval after disease symptoms first appeared. Genotypes differed for mean disease severity and incidence. The bacterial spot disease evaluations showed that genotypes MAR20#46 P3 R4 X Rosa Claro R4 and MD 16 P3 X MAR20#39 P1 R4 presented the lowest scores for the disease incidence in the leaves. S2L AP R1, MAR20#19 ROXO R4 X ECRAM P3 R3, and MD 16 P3 X MAR20#39 P1 R4 were the genotypes with the lowest disease severity scores. Experiment 2 consisted of a RBD with 24 treatments, three replications, and seven plants per plot. Severity assessments were monthly performed on five fruits per plot, totaling three evaluation dates. In this experiment, the percentage of total fruit area with necrotic lesions was measured using a 1 to 4 grading scale. Bacterial spot severity assessments in fruits identified interactions between genotypes and evaluation dates (p ≤ 0.05). The lineages S2L MAR 20#15 R4 and S2L MAR20#19 R2 showed the lowest mean severity scores of bacterial spot in fruit. The genotypes with the lowest mean scores were selected to continue the breeding program.

Mitigation of bacterial spot disease induced biotic stress in Capsicum annuum L. cultivars via antioxidant enzymes and isoforms

Bacterial spot, caused by a group of Xanthomonads (Xanthomonas spp.), is a devastating disease. It can adversely affect the Capsicum annum productivity. Scientists are working on the role of antioxidants to meet this challenge. However, research is lacking on the role of antioxidant enzymes and their isoforms in the non-compatible pathogen and host plant interaction and resistance mechanisms in capsicum varieties. The present study was conducted to ascertain the defensive role of antioxidant enzymes and their isoforms in chilli varieties Hybrid, Desi, Serrano, Padron, and Shehzadi against bacterial spot disease-induced Xanthomonas sp. The seedlings were inoculated with bacterial pathogen @ 107 CFU/mL, and samples were harvested after regular intervals of 24 h for 4 days followed by inoculation. Total plant proteins were extracted in phosphate buffer and quantified through Bradford assay. The crude protein extracts were analyzed through quantitative enzymatic assays in order to document activity levels of various antioxidant enzymes, including peroxidase (POD), Catalase (CAT), Ascorbate peroxidase (APX), and Superoxide dismutase (SOD). Moreover, the profiles appearance of these enzymes and their isoforms were determined using native polyacrylamide gel electrophoresis (PAGE) analysis. These enzymes exhibited maximum activity in Hybrid (HiR) cultivar followed by Desi (R), Serrano (S), Padron, and Shehzadi (HS). Both the number of isoforms and expression levels were higher in highly resistant cultivars compared to susceptible and highly susceptible cultivars. The induction of POD, CAT, and SOD occurs at the early stages of growth in resistant Capsicum cultivars. At the same time, APX seems to make the second line of antioxidant defense mechanisms. We found that modulating antioxidant enzymes and isoforms activity at the seedling stage was an important mechanism for mitigating plant growth inhibition in the resistant ones.

Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

Prevalence of Xanthomonas euvesicatoria (formally X. perforans) associated with bacterial spot severity in Capsicum annuum crops in South Central Chihuahua, Mexico

Background Xanthomonas spp. causes bacterial spot disease, which reduces quality and yield of pepper crops in Mexico. Identification of phytopathogen species is necessary to implement more effective control strategies. Objective The aim of this study was to isolate and identify infecting Xanthomonas species in South Central Chihuahua pepper-producing areas. Methods Diseased plants were collected from 30 cultivation lots and bacteria were isolated from damaged tissues. Potential causative agents were isolated, identified, and characterized by biochemical and molecular analysis. Pathogenicity tests from each isolate were then performed on 30-d-old pepper seedlings, exposing five leaves to 10 µL of 1 × 108 CFU/mL bacterial suspensions of each isolate, using sterile distilled water as a control. Disease severity was determined after 10 d by calculating leaf damage percentage. Furthermore, we evaluated the susceptibility of the highest bacterial spot severity-causing isolates (13 isolates) to copper sulphate (CuS), copper gluconate (CuG), copper oxychloride + oxytetracycline hydrochloride (Cu + Ox), gentamicin + oxytetracycline hydrochloride (Gen + Ox), and gentamicin sulphate (GenS). Copper-resistance genes (copLAB) were detected by PCR analysis among isolates. Results Thirty-seven foliage isolates were identified as Xanthomonas euvesicatoria (14%), which were associated with bacterial spot disease in jalapeño pepper. Tested Xanthomonas isolates were resistant to Cu-based compounds, but susceptible to Cu + Ox. All isolates were susceptible to Gen + Ox and GenS. CopLAB genes were detected in all but one strain. Conclusions X. euvesicatoria (formally X. perforans) may be considered as an emerging pathogen of bacterial spot pepper in Mexico. Among disease management strategies, alternatives to copper should be taken into consideration.

Copper-fixed quat: a hybrid nanoparticle for application as a locally systemic pesticide (LSP) to manage bacterial spot disease of tomato

A hybrid core–shell silica nanoparticle system integrating Cu nanoclusters and Quat combats resistance development of Xanthomonas perforans responsible for bacterial spot disease of tomatoes.