Clavibacter Research Articles

An HLD Model for Tomato Bacterial Canker Focusing on Epidemics of the Pathogen Due to Cutting by Infected Scissors

A healthy, latently infected, diseased (HLD) plant model for botanical epidemics was defined for tomato bacterial canker (TBC) caused by the pathogenic plant bacteria, Clavibacter michiganensis subsp. michiganensis (Cmm). To estimate the infection probability parameter, inoculation experiments were conducted in which it was assumed that infection is transferred to healthy plants through contaminated scissors used to cut symptomless infected plants. The approximate concentration of Cmm in symptomless infected plants was 1 × 106 cells/mL, and the probability of infection of healthy tomato plants was approximately 0.75 due to cutting with scissors soaked in a cell suspension of Cmm at 1 × 106 cells/mL. Three different HLD models were developed by changing some parameters, and the D curve calculated by the developed HLD model A was quite similar to the curve of the proportion of diseased plants observed in fields that had a severe disease incidence. Under a simulation of disease incidence using this model, the basic reproduction number (R0) was 2.6. However, if the infected scissors were disinfected using ethanol, R0 was estimated as 0.3. The HLD model for TBC can be used to simulate the increasing number of diseased plants and the term of disease incidence.

Anti-phytopathogenic-bacterial fatty acids from the mycelia of the edible mushroom Agaricus blazei.

Five compounds including a new compound (1) were isolated from mycelia of a mushroom-forming fungus Agaricus blazei. Compound 2 was isolated from nature for the first time. Their structures were determined by the interpretation of spectroscopic data. In the bioassay examining growth inhibitory activity against phytopathogenic bacteria Clavibacter michiganensis, Burkholderia glumae, and Peptobacterium carotovorum, all the compounds showed inhibition effects on C. michiganensis. Compounds 3 and 4 also showed weak inhibitory activity against growth of B. glumae.

RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis

The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO4 in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO4 induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO4. These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.

Epiphytic and endophytic microorganisms associated to different cultivar of tomato fruits in greenhouse environment and characterization of beneficial bacterial strains for the control of post-harvest tomato pathogens

Several bacterial and fungal diseases affect greenhouse-grown tomato crops, causing severe annual yield losses worldwide. The need to reduce chemical compound applications has encouraged the search of alternative approach for the control of tomato diseases, including the use of biological control agents. The presence of total and beneficial microbial populations was investigated on the surface and in the pulp of seven cultivars of tomato fruit coming from eleven greenhouses in the Pachino district (south-east of Sicily), recognized by the European Community with the “Protected Geographical Indication” label. Principal component analysis (PCA) showed that epiphytic and endophytic microbial populations clustered into groups according to the areas of origin. Approximately 240 tomato fruit–associated bacterial isolates were selected and a high percentage of them showed antagonistic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria pv. perforans, Botrytis cinerea and Alternaria alternata. Analysis of the 16S rRNA gene sequences revealed a predominance of bacteria in Bacillus and Pseudomonas genera, followed by Citrobacter and Enterobacter. The presence of these genera differed according to the geographical areas of tomato samples, whereas their antagonistic capabilities varied according to the five tomato pathogens. The in vitro effectiveness of eight representative bacterial strains belonging to Pseudomonas, Bacillus and Enterobacter genera was confirmed in in vivo assays, carried out on tomato fruit artificially inoculated with A. alternata and B. cinerea. Altogether, these results revealed differences in population density of native microbiota of “Pomodoro di Pachino” fruits and allowed the selection of antagonistic bacterial strains that can be applied in field and in postharvest conditions as alternatives to chemical compounds.

Influence of the high infectious load of the causal agent of bacterial ring rot of potatoes on variations in hydrogen peroxide and cAMP levels in transgenic potato plants <i>in vitro</i> with increased contents of endogenous hydrogen peroxide

Effects of the infectious load of the causal agent Clavibacter michiganensis sps. Sepedonicus (Cms) of ring rot of potatoes on hydrogen peroxide and cAMP levels in potato plants in vitro were studied on three transgenic lines of the Scarb cultivar: line L17.2 – plants transformed with the native gene of glucose oxidase gox from the highly active fungal strain Penicillium funiculosum 46.1; line M7.3 – plants transformed with the modified gene gox-mod; and line Pb14.10 – plants transformed with a vector without a target gene. In addition, non-transgenic plants of the same cultivar (medium resistant against Cms), Lugovskaya (resistant against Cms) and Lukyanovsky (susceptible to Cms) were examined. In the plants infected with Cms (0.2х10 8 cells/ml), the dynamics of cAMP and Н2О2 indicated a balanced system signal “root–stem”. The Cms titre (2х10 8 cells/ml) caused a systemic increase in the concentration of Н 2 О 2 in the organs of almost all plant cultivars; however, a systemic increase in the level of cAMP occurred only in the Lugovskaya cultivar. In all the studied transgenic lines, cAMP levels remained at the control level, accompanied by both local and extensive necrosis. In transgenic potato lines, the observed decrease in protective responses (both local and extensive necrosis) is likely to be associated with an extremely high baseline level of endogenous hydrogen peroxide and an imbalance in cAMP levels. The introduction of the gox gene into potato plants was found to increase their resistance against high Cms titres.

Corchorus olitorius L. (Jute) leaf and seed extracts exerted high antibacterial activity against food and plant pathogenic bacteria

Abstract Aim of this study was to comparatively evaluate antibacterial activities of methanol (MetOH), acetone (Ace), petroleum ether (PE) and aqueous (dw) leaf (L), root (R), and seed (S) extracts of Corchorus olitorius L. on both food- and plant-borne pathogens, with DPPH radical scavenging activities (DRSA), and quantitative and qualitative constituent analysis. Leaf PE has the highest strain susceptibility on both food- and plant-borne pathogens. Clavibacter michiganensis, Pseudomonas tomato, and Erwinia caratovora were susceptible to nearly all the leaf and seed extracts. Very low minimum inhibitory concentration (8-128 μg mL−1) and minimum bactericidal concentration (32-2048 μg mL−1) were determined for both leaf and seed extracts against C. michiganensis. Total phenolic contents were correlated to DRSA. The phenolic compounds tested were higher in the leaf MetOH, cholorogenic acid being the most abundant one. Palmitic acid was determined in leaf PE and seed PE extracts. Results presented here demonstrate high antibacterial activity of C. olitorius leaf seed extracts against phytopathogens for the first time, and provide the most comprehensive data on the antibacterial activity screening against food-borne pathogens. Considering limitations in plant disease control, antibacterial activities of these extracts would be important in plant disease control.

Determination of Resistance Levels to Clavibacter michiganensis subsp. michiganensis in Some Solanum Species

Clavibacter michiganensis subsp. michiganensis (Cmm), is a devastating bacterial disease agent causing bacterial wilt and canker in tomatoes. There is no definitive solution to prevent yield losses by Cmm in tomatoes. Moreover, there is currently no commercially successful Cmm resistant tomato cultivar on the market. Therefore, we aimed to determine the tolerance level of some tomato accessions to Cmm in the present study. For this purpose, we screened seven tomato accessions representing four species (Solanum arcanum, S. habrochaites, S. pennellii, and S. peruvianum) from Peru, Ecuador, and Mexico against the highly virulent isolates Cmm-244 and Cmm-9. A root immersion method was used to identify new sources of resistance to this important disease. Two accessions, S. habrochaites LA1777, and S. arcanum LA2157 were found to be moderate and highly tolerant, respectively, and could serve as tolerance resources for tomato breeding in Türkiye. These materials can also be investigated more extensively to determine their intrinsic Cmm tolerance mechanism.

Chiral analysis of glycerol phosphates - can bacteria biosynthesize heterochiral phospholipid membranes?

Phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in bacteria. However, the chirality of the carbon atom on glycerol phosphate is different between the three kingdoms, Archaea, Bacteria, and Eukarya. Archaea membranes consist of phospholipids with glycerol-1-phosphate (G1P) in the S configuration, whereas phospholipids of the other two kingdoms contain glycerol-3-phosphate (G3P) having R stereochemistry. In the present study, GC/MS and LC/MS methods sensitively detected G3P and G1P from four bacterial strains (Bacillus amyloliquefaciens, B. subtilis, Clavibacter michiganensis, and Geobacillus stearothermophilus). Strain selection was carried out based on a GenBank search that revealed bacterial sequences associated with both enzymes involved in glycerol-phosphate synthesis, i.e., glycerol-3-phosphate dehydrogenase and glycerol-1-phosphate dehydrogenase. The detection of G1P and G3P was made by comparing the retention times of synthetic standards with those of analyzed samples. The structures of both glycerol phosphates were confirmed by selected ion monitoring (SIM) at m/z 171.006. The total concentration of G3P and G1P was around 30 µM, with a ratio of G3P to G1P of 4:1. We showed that PG was the most abundant phospholipid in all four bacteria by using the following analytical techniques and chromatographic modes: hydrophilic interaction liquid chromatography (HILIC), reversed-phase high-performance liquid chromatography high-resolution electrospray ionization tandem mass spectrometry (RP-HPLC/HR-ESI tandem MS) in negative and positive ionization modes, and an enzymatic cleavage by phospholipase C. By using chiral chromatography, the presence of both enantiomers in the glycerol backbone of some molecular species of PG was revealed. These results allow us to conclude that the bacteria examined here produce both enantiomer glycerol phosphates.

Efficacy of Biorational Products for Managing Diseases of Tomato in Greenhouse Production

Gray mold (Botrytis cinerea), late blight (Phytophthora infestans), powdery mildew (Leveillula taurica), pith necrosis (Pseudomonas corrugata), and bacterial canker (Clavibacter michiganensis) are major diseases that affect tomato (Solanum lycopersicum L.) in greenhouse production in Mexico. Management of these diseases depends heavily on chemical control, with up to 24 fungicide applications required in a single season to control fungal diseases, thus ensuring a harvestable crop. While disease chemical control is a mainstay practice in the region, its frequent use increases the production costs, likelihood of pathogen-resistance development, and negative environmental impact. Due to this, there is a need for alternative practices that minimize such effects and increase profits for tomato growers. The aim of this study is to evaluate the effect of biorational products in the control of these diseases in greenhouse production. Four different treatments, including soil application of Bacillus spp. or B. subtilis and foliar application of Reynoutria sachalinensis, Melaleuca alternifolia, harpin αβ proteins, or bee honey were evaluated and compared to a conventional foliar management program (control) in a commercial production greenhouse in Central Mexico in 2016 and 2017. Disease incidence was measured at periodic intervals for six months and used to calculate the area under the disease progress curve (AUDPC). Overall, the analysis of the AUDPC showed that all treatments were more effective than the conventional program in controlling most of the examined diseases. The tested products were effective in reducing the intensity of powdery mildew and gray mold, but not that of bacterial canker, late blight, and pith necrosis. Application of these products constitutes a disease management alternative that represents cost-saving to tomato growers of about 2500 U.S. dollars per production cycle ha−1, in addition to having less negative impact on the environment. The products tested in this study have the potential to be incorporated in an integrated program for management of the examined diseases in tomato in this region.

Nicotiana benthamiana, a Surrogate Host to Study Novel Virulence Mechanisms of Gram-Positive Bacteria, Clavibacter michiganensis, and C. capsici in Plants.

Clavibacter michiganensis is a Gram-positive bacterium that causes bacterial canker and wilting in host plants like tomato. Two major virulence genes encoding a cellulase (celA) and a putative serine protease (pat-1) have been reported. Here we show that Nicotiana benthamiana, a commonly used model plant for studying molecular plant–pathogen interactions, is a surrogate host of C. michiganensis and C. capsici. When a low concentration of two Clavibacter species, C. michiganensis and C. capsici, were infiltrated into N. benthamiana leaves, they caused blister-like lesions closely associated with cell death and the generation of reactive oxygen species and proliferated significantly like a pathogenic bacterium. By contrast, they did not cause any disease symptoms in N. tabacum leaves. The celA and pat-1 mutants of C. michiganensis still caused blister-like lesions and cankers like the wild-type strain. When a high concentration of two Clavibacter species and two mutant strains were infiltrated into N. benthamiana leaves, all of them caused strong and rapid necrosis. However, only C. michiganensis strains, including the celA and pat-1 mutants, caused wilting symptoms when it was injected into stems. When two Clavibacter species and two mutants were infiltrated into N. tabacum leaves at the high concentration, they (except for the pat-1 mutant) caused a strong hypersensitive response. These results indicate that C. michiganensis causes blister-like lesions, canker, and wilting in N. benthamiana, and celA and pat-1 genes are not necessary for the development of these symptoms. Overall, N. benthamiana is a surrogate host of Clavibacter species, and their novel virulence factors are responsible for disease development in this plant.

Isolation of Potato Endophytes and Screening of Chaetomium globosum Antimicrobial Genes.

Antimicrobial peptides (AMPs) have natural antibacterial activities that pathogens find difficult to overcome. As a result of this occurrence, AMPs can act as an important substitute against the microbial resistance. In this study, we used plate confrontation tests to screen out 20 potential endophytes from potato tubers. Among them, endophyte F5 was found to significantly inhibit the growth of five different pathogenic fungi. Following that, phylogenetic analysis revealed that the internal transcribed spacer (ITS) sequences were 99% identical to Chaetomium globosum corresponding sequences. Thereafter, the Bacillus subtilis expression system was used to create a C. globosum cDNA library in order to isolate the resistance genes. Using this approach, the resistance gene screening technology in the indicator bacteria built-in library was used to identify two antimicrobial peptides, CgR2150 and CgR3101, with broad-spectrum antibacterial activities. Furthermore, the results showed that CgR2150 and CgR3101 have excellent UV, thermal, and enzyme stabilities. Also, these two peptides can significantly inhibit the growth of various bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Clavibacter michiganensis, and Clavibacter fangii) and fungi (Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea). Scanning electron microscopy (SEM) observations revealed that CgR2150 and CgR3101 peptides act against bacteria by disrupting bacterial cell membranes. Moreover, hemolytic activity assay showed that neither of the two peptides exhibited significant hemolytic activity. To conclude, the antimicrobial peptides CgR2150 and CgR3101 are promising in the development of a new antibacterial agent and for application in plant production.

Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties.

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato‐growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid‐cured strains found that Cm‐mediated hypersensitive response (HR) in the Cm‐resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI‐encoded serine proteases into Cm∆PI identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (CmΩchpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm‐susceptible tomato. Virulence assays found that while wild‐type Cm and the CmΩchpG complemented strain were nonpathogenic on eggplant, CmΩchpG caused wilt and canker symptoms. Additionally, bacterial populations in CmΩchpG‐inoculated eggplant stem tissues were c.1000‐fold higher than wild‐type and CmΩchpG‐complemented Cm strains. Pathogenicity tests conducted in multiple Cm‐resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG‐recognition is conserved in eggplant. ChpG‐mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.

BIOCIDAL EFFECTS OF METABOLITES OF STREPTOMYCES GLOBISPORUS 3-1 AND STREPTOMYCES CYANOGENUS S136 STRAINS

Research of the biocide action of streptomycete-antagonist metabolites on the causative agent of bacterial canker of tomato and on the host-plant fertility is the actual question. Aim. To determine the action of metabolites of Streptomyces globisporus 3-1 and Streptomyces cyanogenus S136 on the phytopathogen Clavibacter michiganensis 102 in vitro and on seed germination, growth and yield of tomatoes in vivo. Methods. Microbiological methods in laboratory conditions and descriptive-comparative analysis of growth and development of tomatoes in hothouse conditions were used. Results. S. globisporus 3-1 and S. cyanogenus S136 metabolites inhibited the growth of the phytopathogen C. michiganensis 102 with the formation of clean zones 45 and 35 mm in vitro, respectively. Pre-sowing treatment with metabolites of S. globisporus 3-1 and S. cyanogenus S136 of tomatoes Lana seeds reduced germination by 27% for both and led to defects in leaf shape and plant sterility, respectively. Treatment by metabolites of both Streptomyces strains of phytopathogen infected seeds did not improve germination, growth, and yield compared to grown plants from infected seeds, though it were visually not defective, sterile and with signs of bacterial canker of tomato. Conclusion. It was found that metabolites of S. globisporus 3-1 and S. cyanogenus S136 had a biocide action on the phytopathogen C. michiganensis 102 in vitro and on Lana tomatoes in hothouse conditions.

Morphological and molecular characterization of Clavibacter michiganensis subsp. michiganensis causing bacterial canker in tomatoes

Bacterial canker is one of the emerging diseases of tomatoes caused by Clavibacter michiganensis subsp michiganensis (Cmm) in the Northern hill region of India. The present investigation was conducted to estimate the presence and spread of Cmm in the newer areas, to isolate and identify the cause of bacterial canker through conventional, serological and molecular diagnostic tools. 25 tomato plant samples along with the infected fruits collected from different tomato growing regions of the Uttarakhand and Himachal Pradesh were analyzed, and the bacterium was isolated from the seed of the infected tomato fruits. The present study revealed overall mean Cmm incidence of 19.57% and overall disease severity of 16.11% in the Northern hill region of India. The isolated bacterial strains were characterized culturally in semi-selective media (D2ANX and SCM), morphologically (SEM) and molecularly (16S rRNA and pat-1 genes). A total of 10 samples produced yellow colored colonies on D2ANX medium and 9 samples produced grey colored colonies on the SCM medium suggesting them to be of Clavibacter michiganensis subsp michiganensis (Cmm). Amongst the 10 isolates collected three isolates were serologically identified as Cmm through the DAS – ELISA technique. However with PCR reactions we found that only 2 strains out of 10 were of Cmm and the sequence of the most virulent Cmm isolate was submitted at NCBI with the accession number MH321815. These findings suggest that Cmm is present in the tomato seed, which can serve as a potential means of spread of a quarantine pest in the relatively newer areas of Northern hill regions of India.

A new lanostane triterpenoid from the mushroom Hypholoma fasciculare.

A novel compound (1) and 3 known compounds (2-4) were isolated from the fruiting bodies of Hypholoma fasciculare. The structure of 1 was determined by the interpretation of spectroscopic data. Compounds 2-4 were identified by comparing the spectra data of known compounds. In the bioassay examining growth inhibitory activity against phytopathogenic bacteria Clavibacter michiganensis, Burkholderia glumae, and Peptobacterium carotovorum, compounds 1, 2, and 4 showed inhibition effects on C. michiganensis only.

Comparative Analysis of the NDVI and NGBVI as Indicators of the Protective Effect of Beneficial Bacteria in Conditions of Biotic Stress.

Precision agriculture has the objective of improving agricultural yields and minimizing costs by assisting management with the use of sensors, remote sensing, and information technologies. There are several approaches to improving crop yields where remote sensing has proven to be an important methodology to determine agricultural maps to show surface differences which may be associated with many phenomena. Remote sensing utilizes a wide variety of image sensors that range from common RGB cameras to sophisticated, hyper-spectral image cameras which acquire images from outside the visible electromagnetic spectrum. The NDVI and NGBVI are computer vision vegetation index algorithms that perform operations from color masks such as red, green, and blue from RGB cameras and hyper-spectral masks such as near-infrared (NIR) to highlight surface differences in the image to detect crop anomalies. The aim of the present study was to determine the relationship of NDVI and NGBVI as plant health indicators in tomato plants (Solanum lycopersicum) treated with the beneficial bacteria Bacillus cereus-Amazcala (B. c-A) as a protective agent to cope with Clavibacter michiganensis subsp. michiganensis (Cmm) infections. The results showed that in the presence of B. c-A after infection with Cmm, NDVI and NGBVI can be used as markers of plant weight and the activation of the enzymatic activities related to plant defense induction.

An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants.

Clavibacter michiganensis, a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1Cm (a putative serine protease) in pathogenicity and plant immunity. Pat-1Cm was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1Cm mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1Cm, only chp-7Cs from Clavibacter sepedonicus, a potato pathogen, successfully complemented pat-1Cm function in pathogenicity in tomato, whereas all failed to complement pat-1Cm function in HR induction in N. tabacum. Based on the structural prediction, Pat-1Cm carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1Cm in C. michiganensis. Ectopic expression of pat-1Cm with an SP from tobacco secreted protein triggered HR in N. tabacum, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1Cm mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1Cm is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1Cm in plants.

Determination of biocontrol capacity of endophytic bacteria to the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato

Bitki gelişimini teşvik eden bakterilerin (PGPB) alt grubu olan Endofit bakteriler (EB) bitki dokuları içinde de bulunabilmeleri nedeniyle bitki hastalıkları ile savaşımda önemli bir potansiyele sahiptir. Bu çalışmada dört farklı endofit bakteri izolatının domateste Clavibacter michiganensis subsp. michiganensis (Cmm) patojeninin neden olduğu bakteriyel kanser ve solgunluk hastalığının kontrolü ve bitki gelişimi üzerine olan etkilerinin belirlenmesi amaçlanmıştır. Torf/perlit karışımında yetiştirilen domates fidelerine 108 CFU/mL yoğunluğundaki EB süspansiyonları kotiledon yaprak aşaması ve dördüncü bileşik yaprak oluşum aşamasında olmak üzere iki defa içirme metodu ile uygulanmıştır. Cmm’nin 108 CFU/mL yoğunluğundaki süspansiyonu ise ikinci EB uygulamasından 48 saat sonra fidelerin üçüncü bileşik yaprağının alt ve üstüne inokule edilmiştir. Çalışmalar 24±2 oC derecede, %40-60 nem ve 14 saat ışık/10 saat karanlık koşullarına sahip iklim odasında yürütülmüştür. Patojen inokulasyonundan 7 hafta sonra 0-4 skalası ile hastalık şiddeti, bitki gelişim parametreleri ve klorofil içeriği tespit edilmiştir. In vitro çalışmalarda dört endofit bakteri izolatından üçünün patojen gelişimini sınırlandırdığı gözlenmiştir. Bu izolatların aynı zamanda in vivo saksı çalışmalarında da Cmm’nin neden olduğu hastalık gelişimini sınırlandırdığı belirlenmiştir. EB T2K2-1 izolatı %40 etki ile en başarılı izolat olurken, bunu %17,5 ve 15,5 etki ile V30G2 ve T14K1-1 izolatları takip etmiştir. İzolatlar arasında farklılık olsa da bitki gelişim parametrelerine pozitif katkı sağladığı gözlenmiştir. Yaprak sayısında, T2K2-1 ve V35Y1 izolatlarının uygulandığı bitkilerde artış gözlemlenirken hastalık baskısı altında bu etki gözlenmemiştir. Bitki boyu ve klorofil içeriği bakımından uygulamalar arasında önemli bir fark belirlenmemiştir. Kullanılan EB, farklı biyolojik savaş mekanizması ile Cmm’nin hastalık gelişimini sınırlandırabilmesi ve bitki gelişimine pozitif katkı ortaya koymaları,bitkisel üretimde pestisit ve sentetik gübre kullanımının azaltılmasına yardımcı olabilecekleri hipotezimizi desteklemiştir.

Advances in the Characterization of the Mechanism Underlying Bacterial Canker Development and Tomato Plant Resistance

Bacterial canker caused by the Gram-positive actinobacterium Clavibacter michiganensis is one of the most serious bacterial diseases of tomatoes, responsible for 10–100% yield losses worldwide. The pathogen can systemically colonize tomato vascular bundles, leading to wilting, cankers, bird’s eye lesions, and plant death. Bactericidal agents are insufficient for managing this disease, because the pathogen can rapidly migrate through the vascular system of plants and induce systemic symptoms. Therefore, the use of resistant cultivars is necessary for controlling this disease. We herein summarize the pathogenicity of C. michiganensis in tomato plants and the molecular basis of bacterial canker pathogenesis. Moreover, advances in the characterization of resistance to this pathogen in tomatoes are introduced, and the status of genetics-based research is described. Finally, we propose potential future research on tomato canker resistance. More specifically, there is a need for a thorough analysis of the host–pathogen interaction, the accelerated identification and annotation of resistance genes and molecular mechanisms, the diversification of resistance resources or exhibiting broad-spectrum disease resistance, and the production of novel and effective agents for control or prevention. This review provides researchers with the relevant information for breeding tomato cultivars resistant to bacterial cankers.

Abstract P5-06-04: A multi-omics approach to study the host-microbiota interaction in breast cancer tissue

Abstract The microbiota is considered a component of the tumor microenvironment (TME) but its functional role is still not completely clear. In this study, we use both meta-proteomic/transcriptomic and metabarcoding approaches to investigate the composition and the role of microbiota-derived proteins in breast cancer (BC) and microbial diversity, respectively. Three tumoral regions (peripheral tumor, tumor core, in situ carcinoma) and 2 healthy regions (intra-tumoral fibrosis, healthy tissue) were selected on 12 formalin-fixed, paraffin-embedded (FFPE) BC samples. Ten µm tissue slides were used for DNA/RNA extraction and proteomics. The proteomic analysis was performed by nanoLC-MS/MS analysis and data analysis by Perseus and Maxquant, transcriptomic by NEBNext Ultra II Library Prep kit for Illumina and analyzed by Kraken2. Metabarcoding was performed by Ion 16S metagenomics kit for Ion s5 and analyzed by extracting the reads covering the V4 hypervariable region, denoising them with DADA2 and finally taxonomically annotating the obtained ASVs in BioMaS. The proteomic analysis of the 5 areas identified 29 bacterial proteins: 13 chaperonins, 7 ATP synthase subunits, 7 metabolic enzymes, 2 elongation factors. Most of the proteins (16) were located in the intra-tumoral fibrosis. A Glyceraldehyde-3-phosphate dehydrogenase (gapA) and three Chaperone proteins DnaK were overexpressed in the tumor compared to the healthy tissue. Only one of the 4 proteins (DnaK ID:B8IHL3) was associated with a single species: Methylobacterium nodulans. In order to narrow the number of possible species to which the other 3 proteins belong, we have compared the proteomic results with the taxonomic results obtained by metatranscriptomic. The remaining two DnaKs possibly belong to Methylobacterium sp4-46 and Clavibacter michiganensis, while the gapA to Escherichia coli. Overall the investigation based on the V4 hypervariable region of the 16S rRNA gene allowed to highlight a higher alpha diversity in tumor samples than in the healthy ones. In conclusion, the meta-proteomic investigation highlighted the overexpression in tumor tissues of DnaKs, bacterial chaperones able to interfere with important pathways related to DNA damage control/repair and cell-cycle/apoptosis and supposed to promote cancer, and gapA, which plays an important role in multiple cellular functions including metabolism and gene transcription. The most recurrent genera is Methylobacterium which had previously been found as more abundant in BC tissues than healthy tissues. We hypothesize that bacteria supply the tumor cells with proteins that potentially promote cancer progression, and that Methylobacterium could have a key role in breast cancer, but more studies are needed to confirm the hypothesis. Citation Format: Francesca Pirini, Tania Rossi, Soulaimane Aboulouard, Bruno Fosso, Maurizio Puccetti, Sara Ravaioli, Maria Maddalena Tumedei, Michela Cortesi, Michele Zanoni, Luca Magnani, Giovanni Martinelli, Michel Salzet, Sara Bravaccini. A multi-omics approach to study the host-microbiota interaction in breast cancer tissue [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-04.