Ring Rot Pathogen Research Articles

Latent Infestation of Potato Tubers with Soft Rot and Ring Rot Pathogens under Changing Weather Conditions in Ukraine

Potato is one of the most important food crops in the world. Cultivation of potatoes is an important segment of the agriculture and economy as a whole in Ukraine. Bacterial diseases such as ring rot caused by Clavibacter sepedonicus, as well as soft rot and blackleg caused by Pectobacterium atrosepticum evoke tremendous losses of the potato crop. Stored seed tubers are the main source of accumulating pathogens in latent form. In addition, potato crop yield and quality are significantly affected by weather conditions. Along with affecting potatoes directly, meteorological factors such as air temperature and humidity can also impact tuber infestation with bacterial pathogens. The aim of the study was to monitor the prevalence of bacterial ring rot pathogen C. sepedonicus and soft rot pathogen P. atrosepticum in potato tubers on the territory of Ukraine in 2020 and 2021 years and assess its association with different weather conditions. Methods. Potato seed lots from newly harvested crops in eight regions of Ukraine (Odesa, Kyiv, Donetsk, Kherson, Cherkasy, Mykolaiv, Dnipropetrovsk, and Zhytomyr regions) without visual symptoms of diseases were used for the experiments. The detection of C. sepedonicus and P. atrosepticum was conducted by the immunochemical method DAS ELISA. Basal meteorological data (the sum of precipitation (mm/month) and average air temperature (°C) were obtained from the local meteorological stations. The amplitude of air temperature and the sum of precipitation during potato planting, growing, and harvesting seasons, as well as Sielianinov’s hydrothermal coefficient (K) were additionally calculated. The Spearman correlation coefficient was used for the analysis of the correlation between pathogen prevalence and weather parameters. Results. Both ring rot pathogen C. sepedonicus and soft rot pathogen P. atrosepticum were detected in potato lots from all surveyed regions, but the prevalence of latent infestation varied between 2020 and 2021. In 2020, the highest soft rot pathogen prevalence values were registered for Mykolaiv, Kyiv, and Cherkasy regions (21.7, 10.5, and 10% respectively), where high rainfall levels in May and June were observed. In 2021, potato planting, growing, and harvesting seasons were marked by significantly higher precipitation levels, as compared to 2020, in all regions. Average occurrence values for P. atrosepticum in potato lots were 1.7 times higher than that in 2020. The highest prevalence (40%) was observed in the Donetsk region with extremely high air humidity during the potato growing period. Strong positive correlation (r = 0.721) was found between soft rot pathogen prevalence and K coefficients during the potato growing season, as well as between P. atrosepticum prevalence and the sum of precipitation during planting and early growing seasons. Prevalence of ring rot pathogen didn’t differ significantly in 2020 and 2021. The highest C. sepedonicus prevalence was observed in 2020 in the Kyiv region (18.9%), and in 2021 — in the Donetsk region (20%). A moderate positive correlation (r = 0.591) was found between pathogen prevalence and air temperature during planting and early growing season. Conclusions. Weather factors such as air temperature and humidity over the planting and early growing potato season can influence progeny tuber infestation with P. atrosepticum and C. sepedonicus. Knowledge of the effect of the changing weather conditions on the susceptibility of potato varieties with different genetic features to infestation with soft rot and ring rot pathogens warrants future investigation since it is crucially important for developing measures for disease control by potato producers.

PREVALENCE OF CAUSATIVE AGENTS OF RING ROT CLAVIBACTER SEPEDONICUS, BLACKLEG AND WET ROT PECTOBACTERIUM ATROCEPTICUM IN THE 2021 YEAR POTATO HARVEST IN THE TERRITORY OF UKRAINE

Aim. To investigate the prevalence of causative agents of ring rot and soft rot in the potato harvest by the DAS ELISA method. Materials and methods. Samples of the potato harvest from Ternopil, Zhytomyr, Kyiv, Cherkasy, Dnipropetrovsk, Donetsk, Mykolaiv, Odesa, and Kherson regions were used in the study. The detection of bacterial agents was conducted by enzyme-linked immunosorbent assay using commercial test systems LOEWE® Standard Complete Kit (Germany). Results. The highest percentage of affected potatoes was registered in the Donetsk region: 11.1% – by the Clavibacter seedonicus, 31.0% – by the Pectobacterium atrosepticum, and 8.9% – by both bacteria. The causative agent of soft rot was detected twice as often as a causative agent of ring rot in tuber samples from all regions, except the Ternopil region, where the predominance of Clavibacter sepedonicus was revealed. In addition to the Donetsk region, a high prevalence of the causative agent of soft rot was found in the potato harvest from Kyiv (10.2%) and Odesa (10.0%) regions, the highest abundance of the causative agent of ring rot was detected in the Ternopil region. The mixed bacterial infection of potato tubers was diagnosed in 6 areas, and the highest rate was detected in the Donetsk region. Conclusion. The analysis of the distribution of ring rot pathogen Clavibacter sepedonicus and soft rot pathogen Pectobacterium atrocepticum in the potato crop shows the presence of research pathogens in all studied regions. It was discovered the dominance of the representative of pectolytic bacteria in all regions except Ternopil, where the distribution of Clavibacter sepedonicus, the causative agent of ring rot, was dominated. High rates of spread of the studied pathogens were associated with increased local average annual precipitation, as well as with increased annual amplitude of this indicator.

Complete genome sequence resource for a recently isolated potato ring rot pathogen, Clavibacter sepedonicus K496.

We sequenced the genome of Clavibacter sepedonicus that was isolated from diseased potato tubers that we received in December 2020. These diseased potato tubers (Solanum tuberosum cv. Lady Liberty) exhibited external zones of pitting with necrosis of the subtending areas. Scattered brown or blackened internal symptoms were also observed, and in some cases, there was discoloration of the vascular cylinder or pith tissues. Spores of Fusarium sp. were observed in the necrotic areas, and overall, the symptoms were more consistent with a Fusarium dry rot than of bacterial ring rot caused by C. sepedonicus. To aid in isolation of pathogenic organisms without the confounding growth of non-pathogenic saprophytes, we grew plants so that we could sample at the margins of any symptomatic tissues. All tubers produced healthy sprouts, but after eight weeks the two plants grown from the symptomatic tubers showed wilting lower leaves. These symptoms progressed and within a week, water-soaking lesions developed on the affected leaflets followed by leaf desiccation. No symptoms were observed on plants grown from asymptomatic tubers. To isolate bacteria, we collected 1 cm of petiole tissue from leaves of symptomatic plants and the symptom-free control. The petioles pieces were surface sterilized, finely minced, and soaked in double-distilled water for 20 min. The solution was placed on yeast extract-dextrose CaCO3 (YDC) medium and incubated at room temperature (~21˚C). White-yellow mucoid colonies formed after four to five days from the samples taken from symptomatic plants and these were the only type of colonies recovered. In contrast, no colonies grew from the plant grown from the asymptomatic tuber piece. One colony from each symptomatic plant was purified by sub-culture on YDC agar at room temperature for two passages; these isolates were designated K496 and K497 and stored in 16% glycerol at -80 °C. Partial 16S rRNA gene sequences for both isolates were identical, and a BLAST search indicated the isolates were C. sepedonicus. We then sequenced the genome of C. sepedonicus K496 and produced a draft genome sequence using Nanopore R9 flowcell. The genome was then polished using short reads generated using NextSeq2000 platform with the 2x150 bp mode. The complete genome of C. sepedonicus K496 consists of a single circular chromosome and a large linear plasmid. The chromosome is 3,266,016 bp long with a GC content of 72.6%, and the linear plasmid is 135,489 bp with a GC content of 67.9%. This resource announcement is the first to report the complete sequence of a single 140 kb C. sepedonicus linear plasmid. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.5.3. This resource announcement is useful for comparative and diagnostic analysis of C. sepedonicus.

Antifungal Activity and Possible Mechanism of Bacillus amyloliquefaciens FX2 Against the Postharvest Apple Ring Rot Pathogen.

Botryosphaeria dothidea-induced apple ring rot is one of the most serious postharvest diseases in apple production. In our preliminary work, we isolated a bacterial strain (FX2) from an infested apple orchard. Here, we confirmed the strong antifungal activity of FX2 on B. dothidea. Through phylogenetic analysis and morphological observations, we identified FX2 as a Bacillus amyloliquefaciens strain. We also found that 10% cell-free supernatant (CFS) of FX2 significantly affected mycelial growth and morphology and almost completely inhibited spore germination and germ tube elongation in B. dothidea. Furthermore, 10% CFS damaged the cell ultrastructure, resulting in a remarkable increase in cellular leakage in B. dothidea mycelia. Thus, CFS has the potential to effectively reduce in vivo B. dothidea infection, reduced lesion diameters to 64.7% compared with the control group, and reduced disease incidence by 15%. Finally, ultrafiltration, desalting chromatography, and anion exchange chromatography showed that the antifungal constituents in CFS are composed mainly of antifungal proteins. We further characterized these potential antifungal proteins via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Herein, we provide novel insights into the antifungal mechanisms of B. amyloliquefaciens FX2, and we highlight its potential as a novel biocontrol agent for controlling postharvest apple ring rot.

Selenium Nanocomposites in Natural Matrices as Potato Recovery Agent.

The paper presents a study of the effect of chemically synthesized selenium nanocomposites (Se NCs) in natural polymer matrices arabinogalactan (AG) and starch (ST) on the viability of the potato ring rot pathogen Clavibacter sepedonicus (Cms), potato plants in vitro, and the soil bacterium Rhodococcus erythropolis. It was found that the studied Se NCs have an antibacterial effect against the phytopathogenic Cms, reducing its growth rate and ability to form biofilms. It was revealed that Se NC based on AG (Se/AG NC) stimulated the growth and development of potato plants in vitro as well as their root formation. At the same time, Se did not accumulate in potato tissues after the treatment of plants with Se NCs. The safety of the Se NCs was also confirmed by the absence of a negative effect on the growth and biofilm formation of the soil bacterium R. erythropolis. The obtained results indicate that Se NCs are promising environmentally safe agents for the protection and recovery of cultivated plants from phytopathogenic bacteria.

Starch-capped sulphur nanoparticles synthesised from bulk powder sulphur and their anti-phytopathogenic activity against Clavibacter sepedonicus.

Water‐soluble, stable nanoparticles of elemental sulphur with a size of 9‐52 nm have been synthesised using the stabilising potential of starch. Sulphide anions were used as sulphur precursors that were generated earlier from the bulk powder sulphur in the base‐reduction system NaOH‐N2H4·H2O followed by their oxidation with molecular oxygen to element sulphur atoms. Using a set of modern spectral and microscopic methods (XRD, optical spectroscopy, DLS, TEM), the phase state, elemental composition of the nanocomposites and their nanomorphological characteristics have been investigated. It was found that nanocomposites are formed as sulphur particles with the shape which is nearly spherical dispersed in the polysaccharide starch matrix with a pronounced tendency to cluster into ring formations. Water solubility and stability of the obtained nanoparticles is ensured by sorption of starch macromolecules on the surface of sulphur nanoparticles, with the thickness of the stabilising shell in a range of 10‐171 nm. In vitro experiments were carried out to study the anti‐microbial activity of the obtained sulphur nanocomposite (1.6% S) using the propidium iodide fluorescent dye staining method and the diffusion method. It showed that the water solution of the starch‐capped sulphur nanoparticles at the concentration of 6.25 µg/ml had a pronounced anti‐phytopathogenic activity against the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus.

Genome Assembly and Annotation of Botryosphaeria dothidea sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

Botryosphaeria dothidea is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by B. dothidea is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of B. dothidea strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

Detection of Potato ring rot Pathogen Clavibacter michiganensis subsp. sepedonicus by Loop-mediated isothermal amplification (LAMP) assay

Clavibacter michiganensis subsp. sepedonicus (CMS) is an important bacterial plant pathogen causing potato ring rot disease. Rapid diagnosis of CMS is crucial because of the economic losses caused by serious harvest losses. Although there are serological tests used in the rapid diagnosis of CMS, they are not widely used because of their low sensitivity. The DNA-based PCR methods, which are highly sensitive, do not have the possibility of on-site diagnosis, especially since they require serious laboratory infrastructure. In recent years, scientists have been working on alternative amplification methods to develop DNA-based point of care (POC) diagnostic methods. Accordingly, the loop-mediated isothermal amplification (LAMP) method, which was developed in the early 2000s, provides an important convenience for DNA-based tests to use in the field. Due to the unique design of primers, more amplification products could be create in a shorter time than conventional amplification methods without needing a temperature cycle, and it can be applied with the aid of a simple heater without requiring a laboratory environment. In this study, efficient LAMP method for the detection of CMS has optimized. For device-independent detection of LAMP products, colorimetric method and LFD has used.

Selenium Nanocomposites Having Polysaccharid Matrices Stimulate Growth of Potato Plants in Vitro Infected with Ring Rot Pathogen.

The influence of nanocomposites (NC) of selenium in matrices of arabinogalactan (Se/AG) and starch (Se/St) on in vitro vegetation of potato plants, peroxidase activity, and reactive oxygen species has been thoroughly studied. It has been shown that these nanocomposites of selenium have antimicrobial effect to the phytopathogenic bacterium Clavibacter michiganensis ssp. sepedonicus (Cms). In the present investigation, it has been shown that Se/AG NC (6.4% of Se) and Se/St NC (12.0% of Se) have no negative impact on the potato plants healthy and Cms infected, while stimulating their growth, number of leaves and weight of the vegetative part. Se/AG NC has shown a positive effect on potato plants by increasing its immune status by increasing the ROS content and increasing the peroxidase activity. With the use of the element analysis technique, it has been shown that scrutinized nanocomposites are not accumulated in potato plants after the bactericidal processing with the nanocomposites. Se/AG NC and Se/St NC as potential agents used for treatment of potato plants against pathogenic bacteria.

Calcium as a Modulator of the Adenylyl Cyclase Activity of Potato Cells in Bacterial Pathogenesis.

This is the first study to detect the effect of calcium ions on the activity of transmembrane adenylyl cyclase (tmAC), the key enzyme of the adenylyl cyclase signaling system, under normal conditions and after a short-term exposure to exopolysaccharides (EPS) of the bacterial ring rot pathogen Clavibacter michiganensis ssp. sepedonicus (Cms). After the treatment of the roots of plants with the Cms EPS, the response to Ca2+ changed: the activity of the tmAC of plants of the resistant cultivar significantly increased, whereas in the cells of the susceptible cultivar it remained unchanged.

Antimicrobial efficacy of some plant extracts on bacterial ring rot pathogen, clavibacter michiganensis ssp. sepedonicus

Abstract Control of plant bacterial diseases remains difficult due to the limited availability of efficient plant protection products with reduced negative effects either in the environment or with human and animal health. In order to reduce the usage of chemical pesticides alternative strategies for controlling plant pathogens and improve plant disease resistance are promoted. The aim of the study was to investigate the antibacterial activity of some natural compounds (plant extracts of Tamarix ramosissima, Rosmarinus officinalis, Chelidonium majus, Silybum marianum, Satureja hortensis essential oil and propolis) against bacterial ring rot pathogen, Clavibacter michiganensis ssp. sepedonicus (Cms). An agar diffusion method was used for the screening of the inhibitory effect of natural compounds on bacterial strains’ growth. Minimum inhibition concentrations (MICs) were determined by a twofold serial dilution method. The anti-pathogenic activity was investigated by the study of anti-biofilm activity of natural substances. The analyzed natural substances showed a good microbicidal activity and anti-biofilm activity. The results obtained from this study may contribute to the development of new bio-control agents as alternative strategies for prevention and control of ring rot pathogen.

The Relationship between rDNA-ITS Sequences and Biological Characteristics of the Apple Ring Rot Pathogen Botryosphaeria berengeriana de Not f. sp. piricola (Nose)

Ten fungal strains that cause apple ring rot disease (Physalospora piricola Nose) were used to study the role of ribosomal DNA-internal transcribed spacer (rDNA-ITS) in classifying and identifying the pathogen Botryosphaeria berengeriana de Not f. sp. piricola (Nose) Koganezawa et Sakuma, and the correlation between sequence mutations and biological characteristics, and pathogenicity. We used an effective method to screen for conidiospore induction in in vitro cultures of the pathologic pathogens. PCR amplification and clone sequencing were performed for the rDNA-ITS region and used to construct a phylogenetic tree. The growth status, sporulation ability, and phylogenetic effect of different strains were determined. The influence of culture medium types, pH, temperature and light, and the effect of mycelia scraping were investigated. Based on the 582-bp and 583-bp cloned fragments, the results show that there are two significant sequence variations in the ITS1 region, and there were variant bases found in the sequences from five strains. All ten strains were shown to be in theâ sub-variety of Botryosphaeria berengeriana f. sp. piricola (Nose) using homology comparisons. The strains with a higher degree of variation performed better in terms of biological characteristics and were more pathogenic than the strains with less variation. The variations in the ITS1 region might have more predictive power. The appropriate developmental conditions for ring rot colonies were determined to be 25-30°C on a potato-based medium at a pH 5.0-7.0. Alternating irradiation of 12 h/d black light and 12 h/d fluorescent light and scrape treatment of fungal mycelia were best able to induce conidiosporangia and sporulation.

First report of Clavibacter michiganensis subsp. sepedonicus causing bacterial ring rot of potato in Turkey

During the 2007 seed potato monitoring programme in Turkey, 336 tuber samples from Kayseri province were tested for the presence of ring rot and brown rot diseases according to the EC Directives 93/85/EEC (Anon., 1993) and 98/57/EC (Anon., 1998).An intensive survey in 1988-1989 had shown that the potato growing areas of Turkey were free of the ring rot pathogen (Benlioglu et al., 1991). This is the first report of Cms in Turkey. Strict control measures have been taken in contaminated and potentially contaminated fields since Cms is a regulated quarantine organism in Turkey.

Detection of extracellular proteinases in the potato ring rot pathogen

Detection of extracellular proteinases in the potato ring rot pathogen

The tomato pathogen Clavibacter michiganensis ssp. michiganensis: producer of several antimicrobial substances

To purify and analyse antimicrobial substances produced by the tomato pathogen Clavibacter michiganensis ssp. michiganensis (Cmm), with potential application in control of Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. After selection of a suitable producer and indicator strain, antimicrobial compounds were isolated using chromatographic techniques. The resulting preparations were analysed with respect to heat and protease sensitivity, amino acid composition, amino acid sequence and mass. Using this procedure we discovered one post-translationally modified 2145 Da peptide bacteriocin, one 14 kDa antimicrobial protein as well as low molecular weight (<1000 Da) antimicrobial compounds, putatively belonging to the tunicamycin family. Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. This study describes the first attempt to characterize antimicrobial substances from Cmm at the molecular level. This is an important step towards investigation of the possible use of these compounds to control the potato ring rot pathogen.

Proficiency Testing in a Laboratory Accreditation Program for the Bacterial Ring Rot Pathogen of Potato.

Variability of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tissue was analyzed to determine the magnitude of repeatability (within analyst variation) and reproducibility (among analyst variation) components. The analysis was based on data generated by analysts in eight laboratories testing proficiency panel samples distributed under a laboratory accreditation program. The standard deviation for repeatability of the ELISA test was small but increased at higher absorbance readings, while the standard deviation for reproducibility was larger and also increased at high absorbances. For immunofluorescence, the standard deviation for repeatability and reproducibility were similar to one another and increased with increasing bacterial concentration, as might be expected for count data and the inherent subjectivity of the test. The reproducibility standard deviation provided the basis for calculating "z-scores" by the Association of Official Analytical Chemists' procedure to evaluate proficiency of chemical analyses. More than 90 and 80% of the z-scores for samples tested in this study by ELISA and immunofluorescence, respectively, were in the acceptable range. The rescaled sums of z-scores for individual analysts were used as single combination scores to evaluate each analyst's results over all samples of a proficiency panel. This measure may be useful for tracking analyst performance on process control charts as part of a quality control system.

The Role of Extracellular pH‐homeostasis in Potato Resistance to Ring Rot Pathogen

Different cultivars of in vitro plants of potato Solanum tuberosum L. were cultivated with a bacterium Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh., causing wilt of the overground part and ring rot of tubers. Development of disease symptoms (chlorosis, growth suppression) closely correlated with strong acidification of the medium, caused by the bacterium producing extracellular polysaccharides. However, root cells of the host plant (potato) and other non‐host plants (seedlings of tomato, wheat and radish) were able to restore extracellular pH. The efficiency of such restoration correlated with the resistance of the potato cultivar to bacteriosis. Plasma membrane and vacuolar H+‐ATPases were not involved in the stabilization of extracellular pH‐homeostasis, but a significant role was played by the buffer capacity of the cells. The maintenance of pH‐homeostasis in the plants is inferred to be one of the main factors of nonspecific resistance.

Plant-to-plant transmission of the bacterial ring rot pathogenClavibacter michiganensis subsp.sepedonicus

Plant-to-plant transmission of the bacterial ring rot (BRR) pathogen,Clavibacter michiganensis subsp.sepedonicus (Spieck. et Kott.) Skapt. et Burkh. of potato (Solanum tuberosum L.), was studied in field trials over a three year period. Healthy and infected seed tubers were planted 35 cm apart. In one treatment, a subsurface barrier was placed between the healthy and infected seed tubers separating the root systems of neighbouring plants. In this treatment, none of 216 plants grown from healthy seed tubers was infected at harvest, as determined by indirect immunofluorescence antibody staining (IFAS) with monoclonal antibodies. In the other treatment, no subsurface barrier was used. In this treatment, two of 368 plants (0.5%) grown from healthy seed tubers were infected at harvest. It is concluded that plant-to-plant transmission may occur but at very low frequency, and is unlikely to play a significant role compared with the potential of transmission by shared potato handling equipment.

Extraction ofclaviba cter michiganensis subsp.sepedonicus from composite samples of potato tubers

The presence ofClavibacter michiganensis subsp.sepedonicus was detected by immunofluorescence and ELISA in diluent in which cores from 200 potato tubers had been incubated on a rotary shaker. By this procedure the ring rot pathogen was detected in composite samples from naturally infected tubers and tuber samples “spiked” with infected tissue or a pure culture of the pathogen. Populations of saprophytic bacteria increased about 100-fold in the diluent during 18 h of incubation but did not interfere with serological detection of C.m. sepedonicus. Some of the results suggest thatC. m. sepedonicus cells multiply during incubation of tuber samples. Extraction of ring rot bacteria from potato tuber tissue by incubation on a rotary shaker requires little work and minimizes the amount of plant tissue in the extract compared to the standard extraction procedure involving maceration and differential centrifugation.

Potato Flea Beetle (Coleoptera: Chrysomelidae) Evaluated as a Possible Vector of Ring Rot Bacterium in Potatoes

Commercial tolerances for the incidence of bacterial ring rot caused by Clavibacter michiganense subsp. sepedonicum (Spieck. & Kotth.), Davis, Gillaspie, Vidaver &Harris in potatoes, Solanum tuberosum L., are zero, requiring strict regulations to reduce field spread of the disease. Few insects have been shown to be directly associated with the dissemination of ring rot bacteria. Concentrations of bacteria within potato leaves as potential inoculum and the ability of the potato flea beetle, Epitrix cucumeris (Harris), to acquire and disseminate the ring rot bacterium were investigated. There was considerable variation in the concentrations of ring rot bacteria within potato leaves. The highest numbers of bacteria were found in the midveins. Fewest bacteria were present in the mesophyll of the laminae, but the basal sections of laminae contained more bacteria than did the midrib sections from leaflet tips. Potato flea beetles acquired the bacteria in 24 h or less. Beetles exposed to infected plant tissue were confirmed positive for the pathogen using indirect immunofluorescence antibody staining (IFAS). In addition, scanning electron photomicrographs of infected flea beetles showed their mouthparts to be contaminated with bacteria that were the same size and morphology as the ring rot pathogen. Positive IFAS reactions of potato test plants that had been fed upon by potato flea beetles previously exposed to ring rot-infected plants represented support for transmission of the bacteria by the beetles. Mode of transmission appears to be through feeding activity because IFAS analyses of regurgitants and fecal material from contaminated beetles were negative.