Raman Spectroscopy and Machine-Learning for Early Detection of Bacterial Canker of Tomato: The Asymptomatic Disease Condition

Bacterial canker of tomato is caused by Clavibacter michiganensis subsp. michiganensis (Cmm). The disease is highly destructive, because it produces latent asymptomatic infections that favor contagion rates. The present research aims consisted on the implementation of Raman spectroscopy (RS) and machine-learning spectral analysis as a method for the early disease detection. Raman spectra were obtained from infected asymptomatic tomato plants (BCTo) and healthy controls (HTo) with 785 nm excitation laser micro-Raman spectrometer. Spectral data were normalized and processed by principal component analysis (PCA), then the classifiers algorithms multilayer perceptron (PCA + MLP) and linear discriminant analysis (PCA + LDA) were implemented. Bacterial isolation and identification (16S rRNA gene sequencing) were realized of each plant studied. The Raman spectra obtained from tomato leaf samples of HTo and BCTo exhibited peaks associated to cellular components, and the most prominent vibrational bands were assigned to carbohydrates, carotenoids, chlorophyll, and phenolic compounds. Biochemical changes were also detectable in the Raman spectral patterns. Raman bands associated with triterpenoids and flavonoids compounds can be considered as indicators of Cmm infection during the asymptomatic stage. RS is an efficient, fast and reliable technology to differentiate the tomato health condition (BCTo or HTo). The analytical method showed high performance values of sensitivity, specificity and accuracy, among others.

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.

Qtl Mapping of Clavibacter Michiganensis Subsp. Michiganensis (cmm) Resistance Originating from Solanum Pimpinellifolium g1.1554

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), is considered the most serious bacterial threat in tomato and causes large damages in production areas. Worldwide, Cmm is subjected to quarantine regulations. There is no cultivar on the market containing Cmm resistance. A mapping study was done in order to identify Quantitative Trait Loci (QTL) for resistance in a cross between Solanum lycopersicum and S. pimpinellifolium GI.1554, a wild relative of tomato. Besides wilting, symptoms like stem discoloration and bacterial titer were considered. Using single trait and multi-trait approaches, we have identified five QTL regions that are associated with wilting, stem discoloration and bacterial titer in three different environments. These QTLs can be used in breeding programs to develop cultivars with higher levels of resistance.

Detection and identification of bacterial canker of tomato Clavibacter michiganensis subsp. michiganensis

The study is devoted to the diagnosis of bacterial canker of tomato (Cmm). The method of sampling and plant sample preparation has been adapted. PCR recommended by the international diagnostic protocol and other sources have been tested. The use of methods made it possible to identify Cmm in plant material

Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen

The Gram-positive actinobacterium Clavibacter michiganensis is the causal agent of bacterial canker of tomato, an economically impactful disease with a worldwide distribution. This seedborne pathogen systemically colonizes tomato xylem leading to unilateral leaflet wilt, marginal leaf necrosis, stem and petiole cankers, and plant death. Additionally, splash dispersal of the bacterium onto fruit exteriors causes bird’s-eye lesions, which are characterized as necrotic centers surrounded by white halos. The pathogen can colonize developing seeds systemically through xylem and through penetration of fruit tissues from the exterior. There are currently no commercially available resistant cultivars, and bactericidal sprays have limited efficacy for managing the disease once the pathogen is in the vascular system. In this review we summarize research on epidemiology, host colonization, the bacterial genetics underlying virulence, and management of bacterial canker. Finally, we highlight important areas of research into this pathosystem that have the potential to generate new strategies for prevention and mitigation of bacterial canker.

Biogenic AgNPs—A Nano Weapon against Bacterial Canker of Tomato (BCT)

Bacterial canker of tomato caused by the bacterial pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) is a major limiting factor for tomato production worldwide. Currently there exists no resistant variety of tomato to bacterial canker; only cultural and chemical controls are available. This study synthesized AgNPs (silver nanoparticles) via a green chemistry route and investigated their bactericidal potential against bacterial canker of tomato (BCT). AgNPs were prepared using mycellial aqueous extract of agriculturally beneficial fungi Pythium oligandrum. The formation of AgNPs was confirmed by using UV–Vis spectroscopy for the absorbance pattern while their morphology was investigated by the transmission electron microscopy (TEM). The X-ray diffraction profile for the biogenic AgNPs confirmed a crystalline structure with an average particle size of 12 nm. AgNPs treated seeds showed a normal germination rate with normal seedling growth. An in-vitro study found that the prepared AgNPs caused the maximum inhibition of the bacterial pathogen. In the greenhouse the introduction of AgNPs significantly prevents and inhibits the bacterial pathogen Cmm on tomato plants. These results suggest that this process is a strong candidate for industrial scale production of AgNPs. These particles act as an inhibitor and broad spectrum antibacterial agent against cmm, and hence offer a new and eco-friendly alternative in BCT control.

Variation in Streptomycin Resistance Mechanisms in Clavibacter michiganensis

Clavibacter michiganensis is the causal agent of bacterial canker of tomato, which causes significant economic losses because of the lack of resistant tomato varieties. Chemical control with streptomycin or cupric bactericides is the last defensive line in canker disease management. Streptomycin is an aminoglycoside antibiotic that inhibits protein synthesis and targets the 30S ribosomal protein RpsL. Streptomycin has been used to control multiple plant bacterial diseases. However, identification and characterization of streptomycin resistance in C. michiganensis have remained unexplored. In this study, a naturally occurring C. michiganensis strain TX-0702 exhibiting spontaneous streptomycin resistance was identified, with a minimum inhibitory concentration of 128 μg/ml. Additionally, an induced streptomycin-resistant strain BT-0505-R was generated by experimental evolution of the sensitive C. michiganensis strain BT-0505. Genome sequencing and functional analyses were used to identify the genes conferring resistance. A point mutation at the 128th nucleotide in the rpsL gene of strain BT-0505-R is responsible for conferring streptomycin resistance. However, in TX-0702, resistance is not attributed to mutation of rpsL, streptomycin inactivation enzymes, or multidrug efflux pumps. The mechanism of resistance in TX-0702 is independent of previously reported bacterial loci. Taken together, these data highlight diverse mechanisms used by a Gram-positive plant pathogenic bacterium to confer antibiotic resistance.