Potential agrifood application of seriguela (Spondias purpurea L.) residues extract and nanoZnO as antimicrobial, antipathogenic and antivirulence agents

The impact of phytopathogenic microorganisms in several crops causes significant losses in agrifood industry, spoilage throughout food chain and storage. Nanoparticles and plant extracts have been highlighted by their antimicrobial properties applied in food packaging, agriculture, drug delivery systems and other medical approaches. Over the past few years, this group have studied the application of ZnO nanoparticles and agroindustrial wastes in edible food coatings/films. This study aimed to evaluate active characteristics from the extract of seriguela processing wastes and nanoZnO regarding to their inhibitory activity against bacterial pathogenicity and virulence systems TTSS (Type Three Secretion System) and QS (Quorum Sensing) for Pseudomonas savastanoi. Also, antibacterial action (inhibition area) against species of Curtobacterium, Clavibacter, E. coli, Xanthomonas and Serratia, and antifungal against Botrytis cinerea (reduction in colony size). The 60% extract inhibited the activation of QS and TTSS system in 20.26% and 13.54%, respectively; while nanoZnO at 3% reduced 46.77% QS and increased 302.88% TTSS. Extract without dilution inhibited the growth of Clavibacter michiganensis pv michiganensis (Gram-positive) and Xanthomonas phaseoli (Gram-negative), inhibitory zone of 94.25 mm2 and 452.39 mm2 respectively. The latter also being inhibited by nanoZnO 1 and 2% (138.23 mm2) and 3% (275.67 mm2). Pure extract inhibited 17.38% growth of fungal colony and nanoZnO (1 and 3%) in 33.08%. Finally, the active agents studied showed to be promising in the prevention of phytopathogenic diseases and consequently economic losses, food films/coatings and the extract as a biopesticide, reducing the environmental impact.

Response of tomato (Solanum lycopersicum) varieties to Clavibacter michiganensis subsp. michiganensis infection

<p><em>Clavibacter michiganensis</em> subsp. <em>michiganensis</em> causes bacterial wilt and canker disease of tomato. The objectives of this research were i) to characterize and identify the most aggressive Cmm in Chignahuapan, Puebla, ii) to evaluate the tolerance in two phenological stages of 10 tomato varieties to infection by CP_Cmm1 in the greenhouse and the <em>in vitro</em> sensitivity of the bacteria to bactericides. 12 Cmm isolates were collected from tomato plants and a strain (CP_Cmm-1) with greater aggressiveness was selected according to the time of manifestation of symptoms in plants of var. Reserva. Afterward, the strain was inoculated by cutting with scissors embedded in a suspension with 3 x 108 UFC mL-1 in two phenological stages (at five and 10 true leaves). Symptoms were evaluated for 30 days after inoculation. Additionally, the sensitivity of CP_Cmm-1 to eight commercial bactericides <em>in vitro</em> was evaluated. The results showed that the CP_Cmm-1 strain was identified with 97% similarity with the API20 E system, positive with DAS-ELISA, and by PCR it showed 96.6% identity with <em>Clavibacter michiganensis</em> subsp. <em>michiganensis</em>. The Saher variety showed greater tolerance to infection (* = p?0.05) both in the stage of five and 10 leaves; the varieties Sv4401, Nápoles, and Súper óptimo showed greater susceptibility in the five-leaf stage. The Sv4401 variety was highly susceptible in both stages. Neither variety was resistant to infection by CP_Cmm-1. The use of the Sahel variety and kasugamycin could reduce the damage of this bacteria.</p>

Posibilidades del uso de Trichoderma harzianum en el biocontrol del marchitamiento y cancro bacteriano del tomate

El presente trabajo consistió en evaluar la posibilidad del uso de dos cepas de Trichoderma harzianum (Th118 y Th5cc) sobre la manifestación del marchitamiento y cancro bacteriano del tomate en plantas de tomate platense cultivadas en invernáculo. Ambas cepas se aplicaron al sustrato de la plantinera en forma sólida (S) y líquida (L) en el momento de la siembra. Clavibacter michiganensis subsp. michiganensis se inoculó en el momento del desbrote mediante una pequeña incisión en el tallo principal entre la quinta y sexta hoja. El ensayo se repitió durante dos años consecutivos. Para cada tratamiento se evaluaron los componentes del rendimiento (peso y número de frutos) y número de hojas afectadas. Si bien no se encontraron diferencias significativas entre tratamientos durante los dos años, el tratamiento Th118 L es el que presentó menor cantidad de hojas afectadas y valores promedio más altos de peso de frutos. De acuerdo con los resultados obtenidos, se puede afirmar que la cepa de Trichoderma harzianum Th 118 aplicada como riego a los plantines en el momento de la siembra, podría ser incorporada como una buena alternativa dentro de un plan de manejo integrado de enfermedades en el cultivo de tomate platense.

Synthesis of Adenine-based Fluorescent and Naked-eye Chemosensors: Specific DNA Probes for the Detection of Bacterial Pathogens

A rapid and confident tool to identify and diagnose bacterial pathogens with more accuracy using DNA as fingerprints is necessary. Herein, we report a smart chemosensor having a terminal adenine sticking to the thymine of single-stranded DNA (ssDNA) through supramolecular interactions and, which leaves ssDNA when the same ssDNA matches with the targeting desired DNA. We have synthesized a naked-eye coloured chemosensor with carbazole. As a model genetic material, DNA of Clavibacter michiganensis subsp. michiganensis was hybridized to ssDNA and immobilized over nitrocellulose membrane. The prepared adenine-chemosensor, by passing through the nitrocellulose-ssDNA membrane caused the formation of ssDNA nitrocellulose-ssDNA-adenine-chemosensor. FTIR results of the immobilized ssDNAs showed that the matching of same ssDNA releases the adenine-chemosensor from the surface of nitrocellulose-ssDNA that results in formation of the double stranded DNA. The selectivity of chemosensor was also confirmed with different bacterial DNA (Bacillus subtilis) as control. These data highlights accurate and reliable results of a new diagnostic kit prototype promising for further studies, which is able to diagnose DNA quickly and precisely.

A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Transcriptome analysis of Clavibacter michiganensis subsp. michiganensis-infected tomatoes: a role of salicylic acid in the host response

Bacterial canker of tomato (Solanum lycopersicon) caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is an economically important disease. To understand the host defense response to Cmm infection, transcriptome sequences in tomato cotyledons were analyzed by RNA-seq. Overall, 1788 and 540 genes were upregulated and downregulated upon infection, respectively. Gene Ontology enrichment analysis revealed that genes involved in the defense response, phosphorylation, and hormone signaling were over-represented by the infection. Induced expression of defense-associated genes suggested that the tomato response to Cmm showed similarities to common plant disease responses. After infection, many resistance gene analogs (RGAs) were transcriptionally upregulated, including the expressions of some receptor-like kinases (RLKs) involved in pattern-triggered immunity. The expressions of WRKYs, NACs, HSFs, and CBP60s encoding transcription factors (TFs) reported to regulate defense-associated genes were induced after infection with Cmm. Tomato genes orthologous to Arabidopsis EDS1, EDS5/SID1, and PAD4/EDS9, which are causal genes of salicylic acid (SA)-deficient mutants, were upregulated after infection with Cmm. Furthermore, Cmm infection drastically stimulated SA accumulation in tomato cotyledons. Genes involved in the phenylalanine ammonia lyase pathway were upregulated, whereas metabolic enzyme gene expression in the isochorismate synthase pathway remained unchanged. Exogenously applied SA suppressed bacterial growth and induced the expression of WRKYs, suggesting that some Cmm-responsive genes are regulated by SA signaling, and SA signaling activation should improve tomato immunity against Cmm.

Exogenous calcium modulates the activity of adenylate cyclases in potato plants under biotic stress

This article aims to study the influence of different concentrations of calcium ions on the activity of transmembrane (tmAC) and soluble forms of adenylyl cyclase (sAC) in the cells of roots and stems of the plants of two types of potatoes. It compares and contrasts their stability to the agent of the annular rot Clavibacter michiganensis ssp. Sepedonicus (Cms) when exposed to its exopolysaccharides. The experimental results have shown that the reaction of tmAC from the roots and stems to exogenous Ca2+ was almost opposite in the plants of both types. In the root cells of the plants of the resistant types, 1 and 10 mM of Ca2+ have activated tmAC in a very intensive way. In the stem, the average concentrations of Ca2+ inhibited the tmAC activity, while the highest, 1 and 10 mM, did not affect it. the activity of tmAC taken from the root cells of the receptive type of plants was not activated significantly by the increased concentrations of Ca2+, whereas, in the stems, all the concentrations of Ca2+, tmAC activity increased substantially starting with 1 μM. Thus, the unequal reaction of adenylate cyclases of the potato plants of both types to different concentrations of exogenous calcium, testifies, most likely, the presence of several isoform of this ferment that differ in the sensitivity to calcium ions. At the same time, it is possible that the plants of both types may also differ in the spectrum of such isoforms. Since the influence of Cms exopolysaccharides significantly changes the sensitivity to the calcium ions of both forms of adenylate cyclases in the cells of plants of both types, it can be assumed that this feature is one of the mechanisms of these plants’ resistance to the pathogen.

Antibacterial activity of Calendula officinalis and Echinacea purpurea extracts against the causal agent of tomatoes’ bacterial canker: Clavibacter michiganensis subsp. michiganensis

We aimed to investigate the effects of Calendula officinalis and Echinacea purpurea extracts in terms of growth parameters, antibacterial activity and phenolic profile in tomato infected by Clavibacter michiganensis subsp. michiganensis (CmmT7). A significant difference was observed in E. purpura extract, indicating the highest effects on plant height (27.25 cm), fresh plant weight (28.45 cm), root length (24.42 cm), and root weight (6.74 g) (p<0.05). Moreover, Calendula officinalis and Echinacea purpurea extracts showed significant inhibitory activity against CmmT7 (p<0.05). Among phenolic compounds, the only chlorogenic acid amounts were varied in the tomato seedlings leaves with C. officinalis extract (K3) + CmmT7, E. purpurea extract (E3) + CmmT7 and CmmT7 (p<0.01). Moreover, chlorogenic acid amount was approximately 9 times higher than in CmmT7-treated leaves when compared to control. The results showed that application of the extracts of these plants had a significant influence on bacterial canker and growth parameters.