Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens

P. aeruginosa strain FG106 was isolated from the rhizosphere of tomato plants and identified through morphological analysis, 16S rRNA gene sequencing, and whole-genome sequencing. In vitro and in vivo experiments demonstrated that this strain could control several pathogens on tomato, potato, taro, and strawberry. Volatile and non-volatile metabolites produced by the strain are known to adversely affect the tested pathogens. FG106 showed clear antagonism against Alternaria alternata, Botrytis cinerea, Clavibacter michiganensis subsp. michiganensis, Phytophthora colocasiae, P. infestans, Rhizoctonia solani, and Xanthomonas euvesicatoria pv. perforans. FG106 produced proteases and lipases while also inducing high phosphate solubilization, producing siderophores, ammonia, indole acetic acid (IAA), and hydrogen cyanide (HCN) and forming biofilms that promote plant growth and facilitate biocontrol. Genome mining approaches showed that this strain harbors genes related to biocontrol and growth promotion. These results suggest that this bacterial strain provides good protection against pathogens of several agriculturally important plants via direct and indirect modes of action and could thus be a valuable bio-control agent.

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.

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.

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.

Efecto de los ácidos fenólicos en el sistema antioxidante de plantas de tomate (Solanum lycopersicum Mill.)

Introducción. Los ácidos fenólicos pertenecen al grupo de los compuestos fenólicos, su síntesis y concentración en las plantas aumenta cuando estas se encuentran bajo condiciones de estrés biótico o abiótico. Objetivo. Evaluar el efecto de los ácidos fenólicos sobre el sistema de defensa antioxidante enzimático y no enzimático en plantas de tomate sometidas a estrés biótico. Materiales y métodos. El experimento se realizó de marzo a diciembre de 2016, en Saltillo, México. Se estableció un cultivo de tomate tipo Saladette de la variedad Río Fuego (Solanum lycopersicum Mill.). A plantas de tomate inoculadas con Clavibacter michiganensis subsp. michiganensis (1X105 UFC ml-1) se les realizaron aspersiones foliares de ácidos fenólicos a una dosis de 1 kg ha-1 con el producto Defens Gr® (IA: ácidos fenólicos 10 000 ppm). Se muestrearon hojas a los 15, 31 y 92 días después del trasplante (ddt) y frutos a los 90 ddt. Se trabajó con seis tratamientos: 1) testigo absoluto (T0), 2) aplicación de ácidos fenólicos antes de inocular Clavibacter (AFA), 3) aplicación de ácidos fenólicos después de inocular Clavibacter (AFD), 4) aplicación de ácidos fenólicos antes y después de inocular Clavibacter (AFAD), 5) solo aplicación de ácidos fenólicos (AF) y 6) solo inoculación con Clavibacter (Cmm). Resultados. La aplicación de ácidos fenólicos intervino en la actividad de antioxidantes enzimáticos y no enzimáticos. Se encontró mayor capacidad antioxidante en hoja que en fruto, la cual se determinó por ABTS [2,2’-azino-bis (3-etilbenzotiazolin-6-ácido sulfónico)] y DPPH (1,1-difenil-2-picrilhidrazil). La inoculación de las plantas de tomate aumentó la actividad de las enzimas catalasa y fenilalanina amonio liasa en hoja; además, hubo reducción de la actividad enzimática del superóxido dismutasa y el contenido de fenoles totales. Conclusión. Los ácidos fenólicos intervinieron en los mecanismos de defensa enzimáticos de la planta y redujeron los niveles de estrés ocasionados por la inoculación.

Adsorption of Recombinant Human β-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect against Clavibacter michiganensis subsp. michiganensis

Solanum lycopersicum L. is affected among other pests and diseases, by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), causing important economic losses worldwide. Antimicrobial peptides (AMPs) are amphipathic cationic oligopeptides with which the development of pathogenic microorganisms has been inhibited. Therefore, in this study, we evaluate antimicrobial activity of mesoporous silica nanoparticles (MSN5.4) loaded with human β-defensin-2 (hβD2) and two mutants (TRX-hβD2-M and hβD2-M) against Cmm. hβD2, TRX-hβD2-M and hβD2-M presented a half-maximum inhibitory concentration (IC50) of 3.64, 1.56 and 6.17 μg/mL, respectively. MSNs had average particle sizes of 140 nm (SEM) and a tunable pore diameter of 4.8 up to 5.4 nm (BJH). AMPs were adsorbed more than 99% into MSN and a first release after 24 h was observed. The MSN loaded with the AMPs inhibited the growth of Cmm in solid and liquid media. It was also determined that MSNs protect AMPs from enzymatic degradation when the MSN/AMPs complexes were exposed to a pepsin treatment. An improved AMP performance was registered when it was adsorbed in the mesoporous matrix. The present study could expand the applications of MSNs loaded with AMPs as a biological control and provide new tools for the management of phytopathogenic microorganisms.

Analyses of Virulence Genes of Clavibacter michiganensis subsp. michiganensis Strains Reveal Heterogeneity and Deletions That Correlate with Pathogenicity

Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato. Differences in virulence between Cmm strains have been reported. The aim of this study was the characterization of nine Cmm strains isolated in Chile to reveal the causes of their differences in virulence. The virulence assays in tomato seedlings revealed different levels of severity associated with the strains, with two highly virulent strains and one causing only mild symptoms. The two most virulent showed increased cellulase activity, and no cellulase activity was observed in the strain causing mild symptoms. In three strains, including the two most virulent strains, PCR amplification of the 10 virulence genes analyzed was observed. In the strain causing mild symptoms, no amplification was observed for five genes, including celA. Sequence and cluster analyses of six virulence genes grouped the strains, as has been previously reported, except for gene pelA1. Gene sequence analysis from the genomes of five Chilean strains revealed the presence of deletions in the virulence genes, celB, xysA, pat-1, and phpA. The results of this study allow us to establish correlations between the differences observed in disease severity and the presence/absence of genes and deletions not previously reported.