Early detection of tomato bacterial canker (Cmm) using quantitative PCR (qPCR)

Abstract

Background: Tomato (Solanum lycopersicum L.) is not only one of the key vegetable crops worldwide but also an important source of bioactive compounds, including lycopene, vitamin C, and polyphenols, which are associated with antioxidant and health-promoting properties. In the Republic of Armenia, the intensive development of greenhouse vegetable production contributes to increased crop productivity; however, it simultaneously increases the risk of bacterial diseases. One of the most problematic diseases is Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), which can lead to massive plant losses and significant yield reduction. Early diagnosis using quantitative polymerase chain reaction (qPCR) is crucial for effective disease control and the prevention of epiphytotics in greenhouses. 

Objective: To study the manifestation of bacterial canker of tomato under commercial greenhouse conditions and to evaluate the effectiveness of a qPCR method for the early and latent detection of the Cmm pathogen.

Methods: The studies were conducted in commercial hydroponic greenhouses of the Kotayk and Ararat regions of Armenia. DNA was extracted from infected tomato stems and leaves using the CTAB method with Genetic Analysis Strategies S.L. kits and was spectrophotometrically analysed (A260/280 =1.78-1.92). Identification of Cmm was performed by qPCR using species-specific primers, and quantitative assessment of the pathogen was carried out using a standard curve based on serial tenfold dilutions. The reliability of the method was confirmed by analysis of sensitivity, linearity, repeatability, and reproducibility (Efficiency 98.1%, R² =1.0, LOD 3.1±0.4 × 10¹ copies/µl).

Results: In commercial greenhouses, the first manifestations of bacterial canker in tomato include unilateral leaf wilting, darkening and necrosis of the stem vascular system, as well as alterations in fruit structure. The qPCR method enabled the detection of Cmm in all 12 examined samples with high amplification efficiency (97.5%). Samples with low Ct values (12.5-17.5) corresponded to a high number of pathogen DNA copies and exhibited pronounced visual disease symptoms. In contrast, samples with higher Ct values (26.7-36.8) contained low pathogen concentrations and, in most cases, showed no symptoms, indicating a latent stage of infection.

Application of qPCR for early diagnosis reduced the proportion of infected plants from 13.2-14.9% to 4.8-5.1%, and yield losses from 9.4-10.1% to 3.5-3.9%. In addition to yield reduction, bacterial infection may affect the physiological processes of fruit development. In particular, the present study revealed a decrease in average fruit weight from 165.4 to 134.2 g, corresponding to a reduction of 18.9%. At the same time, accelerated fruit ripening was observed. The obtained results show that qPCR ensures effective early detection of infection and limits its spread under protected cultivation conditions, leading to reduced production losses and contributing to the production of healthier and high-quality plant products.

Novelty: This study presents, for the first time, a comprehensive diagnosis of Cmm in commercial greenhouses in Armenia using qPCR to detect both symptomatic and latent tomato infections. The data demonstrates a direct correlation between bacterial load and symptom severity and confirms that early molecular detection of the pathogen can significantly reduce yield losses. The results highlight the practical value of implementing qPCR in greenhouse monitoring systems and provide a basis for developing a rapid phytosanitary control strategy, which has not previously been documented for the study region.

Conclusion: The qPCR method demonstrated high efficiency for the early diagnosis of bacterial canker in tomato, enabling the detection of the pathogen at latent stages and preventing widespread infection. Early detection of Cmm allows timely implementation of protective measures, minimizes yield losses, and enhances the resilience of greenhouse tomato production. Thus, early molecular detection of Cmm is important not only for plant disease management but also for preserving the bioactive compounds and functional food value of tomato.

Keywords: tomato, Clavibacter michiganensis subsp. michiganensis, bacterial canker, qPCR, greenhouse, early detection