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Efficacy of agricultural disinfectants on biofilms of the bacterial ring rot pathogen, Clavibacter michiganensis subsp. sepedonicus
Abstract
Susceptibility of biofilms of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, to three common agricultural disinfectants was tested. The MBECTM assay device was used to evaluate optimum parameters for growing artificial biofilms. These conditions were determined to be 7 days of growth at 23°C in a yeast extract-glucose-mineral salts medium. As expected, the bacteria in the biofilm state were more resistant to disinfection by chemical treatment with sodium hypochlorite, quaternary ammonium, and hydrogen peroxide when compared with planktonic cells. Artificial biofilms were also grown on five different surface materials typically found in commercial potato storage facilities (concrete, mild steel, rubber, polycarbonate and wood) using the BESTTM assay device to test the effect of surface type on biofilm susceptibility to disinfection. Sodium hypochlorite was the most effective disinfectant on the wood surface and hydrogen peroxide was best on the mild steel surface. Efficacies of the various disinfectants were not significantly different on concrete, rubber and polycarbonate surfaces. When artificially grown biofilms and those grown naturally in potato tissue were transferred to, and dried onto, coupons of the different surface materials, they were significantly more difficult to inactivate than in situ grown biofilms. The resistance of plant pathogenic bacteria in the biofilm state, particularly when spread and dried onto surfaces of agricultural machines and other equipment, to commonly used disinfectants, has important implications for disease control strategies that depend largely on strict sanitary and hygienic practices.
... As judged from their surface area, conveyer belts and the walls that shape the path of travel through the cutting process are the most likely candidates to catch Cs contamination. The type of material presented by these surfaces is one of the factors that may determine the chances of survival of the Cs bacteria attached (Nelson 1978(Nelson , 1980Howard et al. 2015). The experimental objects chosen for this study therefore were pieces cut from the original polyvinylchloride conveyer belt that carries the tubers inside the machine in the horizontal plane, pieces cut from the original conveyer belt that carries the tuber halves in vertical direction to the storage box and original pieces of lacquered steel that represent the machine walls and machine rims of the most commonly used potato cutter in the Netherlands. ...
... The Cs biofilms were subsequently suspended by rinsing the objects separately in 20 ml sterile neutralizer solution for 30 min using a laboratory shaker. The neutralizer solution, serving as scavenger for disinfectant remnants to protect the bacteria, consisted of 1 g/L L-histidine, 1 g/L L-cysteine and 2 g/L reduced glutathione in water (Tremaroli et al. 2008;Howard et al. 2015). The experiments were performed in 5-fold (experiments A-E). ...
... The smear of ring rotinfected tubers comprises Cs biofilms that are sticky and are able to become relatively firmly attached to various kinds of material. In addition, it should be noted that bacteria present in microbial biofilms generally exhibit a significantly higher tolerance towards antimicrobials than planktonic cells (Howard et al. 2015). In this study, we therefore investigated the efficacy of this conventional treatment using the ring rot bacterium present in Cs biofilms attached to relevant conveyer belt and lacquered steel materials. ...
A potential cause of the dissemination of the potato ring rot bacterium Clavibacter sepedonicus (Cs) is the use of automated seed potato cutters. The present study focuses on the question of whether disinfection practices are sufficient to prevent the transmission of Cs from contaminated machine parts to a new tuber lot. The disinfection efficacy was determined by establishing the culturability of Cs that remained after spray application of sodium-p-toluenesulfochloramide solution on clean and fouled specimens of machinery material that had been provided with an imprint of Cs biofilm.
Although conventional spraying, with the authorized concentration of sodium-p-toluenesulfochloramide, of inoculated rubber, PVC and lacquered steel led to a substantial decrease of colony forming units, the treatment was insufficient for complete eradication of Cs. The presence of dirt negatively affected the efficacy of the disinfectant.
... nebraskensis (Botti-Marino, 2017;Harding et al., 2011), bacterial wilt on many crops caused by Ralstonia solanacearum (Mori et al., 2016), ring rot on potato caused by Clavibacter michiganensis pv. sepedonicus (Howard et al., 2015), fire blight on fruit trees caused by Erwinia amylovora (Koczan et al., 2009), Stewart's wilt of corn caused by Pantoea stewartii (Koutsoudis et al., 2006), Pierce's disease of grape caused by Xylella fastidiosa (Marques et al., 2002), and crown gall caused by Agrobacterium tume-faciens (Tarbah and Goodman, 1987). In some instances, the ability to form biofilms is an essential virulence factor (Koczan et al., 2009;Mori et al., 2016). ...
... Using this multi-well plate-based method, one can easily determine the minimum concentration required to eradicated both biofilm and planktonic populations in the same reactor, making it especially useful for evaluation of chemical treatments and simultaneous comparison of treatment efficacy against both biofilm and planktonic cells (Harrison et al., 2010). For example, the MBEC Assay method has been used to characterize C. sepedonicus biofilms (Howard et al., 2015), C. michiganensis biofilms (Harding et al., 2011), and recently to identify novel, anti-biofilm chemicals such as oxidized silver compounds and optimal fungicide + metallic ion tank-mix partners (Harding et al., 2019). The availability of this assay was instrumental in developing the first American Society for Testing and Materials (ASTM) standard method for evaluation of disinfectant efficacy in a static reactor (ASTM Standard Method E2799-17). ...
Bacterial wilt is a re-emerging disease on dry bean and can affect many other crop species within the Fabaceae. The causal agent, Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), is a small, Gram-positive, rodshaped bacterium that is seed-transmitted. Infections in the host become systemic, leading to wilting and economic loss. Clean seed programs and bactericidal seed treatments are two critical management tools. This study characterizes the efficacies of five bactericidal chemicals against CFF. It was hypothesized that this bacterium was capable of forming biofilms, and that the cells within biofilms would be more tolerant to bactericidal treatments. The minimum biocide eradication concentration assay protocol was used to grow CFF biofilms, expose the biofilms to bactericides, and enumerate survivors compared to a non-treated control (water). Streptomycin and oxysilver bisulfate had EC95 values at the lowest concentrations and are likely the best candidates for seed treatment products for controlling seed-borne bacterial wilt of bean. The results showed that CFF formed biofilms during at least two phases of the bacterial wilt disease cycle, and the biofilms were much more difficult to eradicate than their planktonic counterparts. Overall, biofilm formation by CFF is an important part of the bacterial wilt disease cycle in dry edible bean and antibiofilm bactericides such as streptomycin and oxysilver bisulfate may be best suited for use in disease management.
... Infected packaging equipment and storage facilities, that is, potato crates, bags, vehicles, and machinery, support the survival of the pathogen in short and medium timeframes and are important in spreading the pathogen to healthy lots of seed potatoes (EFSA et al., 2019). Sodium hypochlorite is an effective disinfectant for decreasing the pathogen's survival on wooden surfaces, while hydrogen peroxide is common for treating metal surfaces of agricultural machines and other equipment (Howard et al., 2015). Prior to disinfection, surfaces should be washed because dirt negatively affects the efficacy of the disinfectant (Stevens et al., 2021b). ...
... In an in vitro assay, exposure to sodium monoiodoacetate as well as Lazurite preparation reduced biofilm formation (Perfileva et al., 2018b). It is notable that bacteria in a biofilm state are more resistant to chemical treatments than planktonic cells (Howard et al., 2015), and therefore disinfection of materials should be preceded by, or combined with, disruption of the biofilm matrix through washing (Stevens et al., 2017). A combination of moderate heat shock (45°C) and treatment with the glycolysis inhibitor monoiodoacetate negatively affected C. sepedonicus in vitro (Rymareva et al., 2008). ...
Background:
Bacterial ring rot of potato (Solanum tuberosum) caused by the gram-positive coryneform bacterium Clavibacter sepedonicus is an important quarantine disease threatening the potato industry around the globe. Since its original description in 1906 in Germany, management of ring rot has been a major problem due to the seedborne nature (via seed tubers not true seeds) of the pathogen allowing the bacterium to be transmitted long distances via infected tubers.
Disease symptoms:
On growing potato plants: interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. On infected tubers: vascular tissues become yellowish brown with a cheesy texture due to bacterial colonization and decay.
Host range:
Potato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet.
Taxonomic status of the pathogen:
Class: Actinobacteria; Order: Actinomycetales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter sepedonicus (Spieckermann and Kotthoff 1914) Li et al. 2018.
Synonyms (nonpreferred scientific names):
Aplanobacter sepedonicus; Bacterium sepedonicum; Corynebacterium sepedonicum; Corynebacterium michiganense pv. sepedonicum; Clavibacter michiganensis subsp. sepedonicus.
Microbiological properties:
Gram-positive, club-shaped cells with creamy to yellowish-cream colonies for which the optimal growth temperature is 20-23°C.
Distribution:
Asia (China, Japan, Kazakhstan, Nepal, North Korea, Pakistan, South Korea, Uzbekistan, the Asian part of Russia), Europe (Belarus, Bulgaria, Czech Republic, Estonia, Finland, Georgia, Germany, Greece, Hungary, Latvia, Lithuania, Norway, Poland, Romania, European part of Russia, Slovakia, Spain, Sweden, Turkey, Ukraine), and North America (Canada, Mexico, USA).
Phytosanitary categorization:
CORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2.
... Literature suggests that the efficacy of temperature treatments depends highly on the physical and chemical environment of the bacterium during temperature exposure; in particular, the presence of an organic matrix may provide significant protection to relatively high temperatures (Fatmi et al. 1991;Downer et al. 2008;Raviv et al. 2011;Steinmöller et al. 2013;Howard et al. 2015). The natural environment of Cs inside potato plants is constituted by their self-produced biofilm attached to the walls of xylem vessels (Marques et al. 2003). ...
... The extracellular polymeric matrix protects the bacterium against unfavourable conditions. In addition, bacterial cells immobilised inside biofilms are supposed to be more protected against adverse conditions than planktonic cells (Marques et al. 2003;Ramey et al. 2004;Howard et al. 2015). Eradication of Cs by means of pasteurisation or by controlled composting therefore requires knowledge about the effects of elevated temperature on Cs that is present in infected plant material. ...
The objective of the present study was to establish whether exposure to temperatures of 55-70 °C results in eradication of the pathogen Clavibacter sepedonicus (Cs) in colonised potato tissue, in order to evaluate the efficiency of composting for disinfection of Cs-infected potato waste. Pre-sprouted potato tubers were inoculated and planted to produce Cs-colonised stem and tuber material. After incubation in temperature-controlled water baths, the infected potato tissue was analysed for the presence of culturable and pathogenic Cs. Additional experiments were performed with Cs-colonised potato stem tissue crushed and deaerated, thus simulating macerated stem tissue in the compost heap. To enable a comparison with bacterial cells that are not enclosed by their natural organic matrix, temperature treatments were applied to non-infected stem tissue crushed and deaerated, and spiked with freshly prepared Cs-suspensions. Cs settled inside colonised potato tissue, as well as Cs present as Cs-suspensions supplemented to potato tissue, was eradicated by exposure to heat, even after a treatment of 1 h at 55 °C, with the exception of one case in which the pathogen present in intact stem material escaped a treatment of 6 h at 60 °C, indicating that incidentally stems may provide niches in which the pathogen is protected against heat.
... Disinfection of contaminated objects through the use of highly effective disinfectants remains the basis for the prevention of both infectious and invasive diseases (Maertens et al., 2020;Paliy et al., 2016;2020d). The scientifically based use of disinfectants makes it possible to control the epizootic situation for many animal diseases (Howard et al., 2015;Paliy et al., 2018a;. ...
The development of new import-substituting highly effective and environmentally friendly complex antiparasitic drugs and their implementation in a scientifically grounded system of measures to combat the causative agents of the main invasive diseases of farm animals and poultry remains an urgent task of veterinary medicine. Based on the results of studies of anthelmintic and antiprotozoal action of agents on exogenous forms of parasites, an experimental form of a complex antiparasitic agent "Tinidafen" was created and investigated. An experimental sample in the form of a powder was created on the basis of two active substances – tinidazole and fenbendazole with the addition of excipients (talc, starch). It has been established that the LD50 of "Tinidafen" for white rats with a single dose is 2564.36±349.13 mg/kg. The therapeutic dose of "Tinidafen" for rabbits is 7.5 g per 10 kg of live body weight. The extensefectevity of "Tinidafen" is 99.5% for helminths and 98.7% for eimeria. After administered orally of the agent, helminth eggs in the feces of infested animals were not registered from day 3, and the release of eimeria oocysts was observed until day 5 of the experiment.
... Biofilm matrix presents a diffusion barrier and a neutralising environment for some biocides. This protective effect varies with: substrate surface, age of biofilm, hydration, fluid shear conditions during biofilm formation, and the mix of microbe species in the biofilm (Akinbobola et al., 2017;Buckingham-Meyer et al., 2007;Das et al., 1998;Harding et al., 2014;Howard et al., 2015;Papaioannou et al., 2018). Biofilm-embedded organisms often show less biocide susceptibility than counterparts dried-on to surfaces (Buckingham-Meyer et al., 2007;Maris, 1992;Otter et al., 2015), and they are markedly less susceptible than suspended cells (Cabeça et al., 2012;Harding et al., 2014). ...
Disinfectants for veterinary and livestock use, plus skin antiseptics, are critical elements for the control of infectious agents, including zoonotic and antimicrobial‐resistant micro‐organisms, in managed animal species. Such agents impact animal welfare, economic performance and human health. Testing of disinfectants is needed for safety, efficacy and quality control. The present review examines the principal types of test (carrier, suspension, surface and field) that have been developed or attempted, plus the features inherent in the respective tests, particularly with respect to variability. Elements of testing that have to be controlled, or which can be manipulated, are discussed in the context of real‐world scenarios and anticipated applications. Current national and international testing regimes are considered, with an emphasis on the UK, continental Europe and North America, and with further detail provided in the Supporting Information. Challenges to disinfectant efficacy include: the nature of the biological targets (bacteria, fungi, yeasts, spores, viruses and prions), the need for economical and safe working concentrations, the physical and chemical nature of contaminated surfaces, constraints on contact times and temperatures, the presence of organic soil and other barrier or neutralising substances (including biofilms), and thoroughness of pre‐cleaning and disinfectant application. The principal challenges with veterinary disinfectant testing are the control of test variability, and relating test results to likely performance in variable field conditions. Despite some ambitions to develop standardised field tests for disinfectants, aside from skin antiseptic trials the myriad problems such tests pose with respect to cost, reproducibility and generalisability remain intractable.
... Much more important factors relating to the severity of the occurrence and spread of the disease are agrotechnical aspects. The use of clean equipment at every stage of production (Nelson 1980;Howard et al. 2015;Stevens et al. 2017), compliance with phytosanitary regulations (EC 1993), the use of healthy, certified seed potatoes play a fundamental role in preventing the disease. While the practice of cutting seed potatoes is common in North America, in Europe the practice of cutting seeds before planting is increasingly rare and unacceptable with the current state of knowledge Nolte et al. 2020). ...
The results of the studies carried out over the last 50 years have not provided a clear answer to questions concerning the interaction between Clavibacter sepedonicus (Cs), potato and the environment. The reason is insufficient knowledge of the biology of the causal agent of ring rot, problems of research methodology and the influence of many environmental and biotic factors on the degree of plant infestation. The research conducted in the years 2013–2016 in the Plant Breeding and Acclimatization Institute – National Research Institute concerned the determination of the influence of weather conditions, involved the influence of temperature and Sielianinov hydrothermal coefficient on the development of Cs bacteria in stems and progeny tubers, as well as the occurrence of disease symptoms on plants and in tubers of eight potato varieties. It was found that genotype had the greatest impact on leaf and tuber infestation, while weather conditions were less significant. The results of correlation analysis indicate that the weight of tubers of plants infected by Cs increased statistically significantly with increases in air and soil temperature. On the other hand, air and soil temperature in the initial vegetation period did not significantly affect the number of potato plant tubers infested by Cs. In a 4-year experiment, it was not shown how air and soil temperature and the Sielianinov hydrothermal coefficient affected the degree of potato leaf and tuber infestation. In most cases, potato infestation by Cs caused a decrease in yield.
... Sodium hypochlorite is the most effective disinfectant on the wood surface and hydrogen peroxide is best on the mild steel surface for eradication of the biofilms of C. sepedonicus (Howard et al. 2015). Further, the biofilm formation was reduced when exposed to sodium monoiodoacetate, as well as "Lazurite" preparation, while 2,4-D and "Ridomil Gold" stimulated the biofilm formation (Alla et al. 2018). ...
Clavibacter sepedonicus is one of the few major plant pathogens which is not widely distributed in the area where the main host crop (potato) evolved. Clavibacter sepedonicus has the propensity to exist asymptomatically as latent infections in potato; it is not known to naturally infect other plant species. Inadvertent dissemination of the bacterium to new places of production occurs with the movement of latently infected seed tubers used for planting. The bacterium also spreads from infected tubers through direct contact and by contamination of equipment used for potato production such as seed cutters, planters, harvesters, transport vehicles, and storages. Clavibacter sepedonicus survives for extended periods of many months to years in a dry and cool environment. Hence, its persistence on farm equipment, in storages, and on transport vehicles is an important means by which the bacterium is maintained and spread within farm units and disseminated to other production units. It persists in the field in unharvested potato tubers (i.e. volunteers or ground keepers) and in infected potato plant debris.
This datasheet on Clavibacter sepedonicus covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Environmental Requirements, Seedborne Aspects, Impacts, Uses, Prevention/Control, Further Information.
To improve and develop industrial products and processes, a standard is always needed. Nowadays, not only for academic activities, but also for industrial ones, various standards are developed beyond borders among nations. Therefore, the international standards are also needed. In light of that, we introduce various industrial standards relating to biofilms. Since the concept of biofilms is relatively new, there are not many existing standards for them. However, we already have some standards in advanced countries. In this chapter, we describe some related national standards for the United States, the European Union (EU), and Japan, as well as the International Organization for Standardization (ISO).
For many pathogenic microbes, biofilm formation is a critical component of the disease cycle and is required for pathogenicity or full virulence. Recent innovation in in vitro biofilm reactors has allowed high throughput evaluation and testing of chemical treatments against microbial biofilms. For example, the BESTTM Assay is a versatile and specialized biofilm reactor capable of culturing bacterial and fungal biofilms in a replicated, high throughput, multi-well format. The microorganism forms a biofilm on the surfaces of pegs extending into liquid media contained within the microtitre plate wells. Additionally, planktonic cells also grow within the media in each well which allows simultaneous growth of biofilms and planktonic cultures within the wells of each reactor. Comparisons and evaluations of microbial biofilms on different surface materials can also be performed. For example, the bacterial ring rot pathogen Clavibacter michiganensis subsp. sepedonicus was studied in the BESTTM Assay to determine the conditions that promote growth in the biofilm form, the sensitivities of biofilm and planktonic cells to biocides, and the effects of biocide concentrations, exposure times and substrate types on efficacy. Technologies such as the BESTTM Assay excel at efficacy testing of chemical and biological agents being screened or developed for control of plant diseases, including postharvest diseases. Plant pathology research, phenotype screening, evaluating and formulating biological control products and protocols often ignore the critical aspect of biofilm potential. As a result, biofilm biology remains uncharacterized or unconfirmed for many plant-pathogen interactions and for most biocontrol agents. Biofilm reactors, such as the BESTTM Assay, provide technology for characterizing and evaluating these important issues.
Clavibacter michiganensis subsp. michiganensis (Cmm) is an infectious, biofilm-forming bacterium that causes bacterial canker on tomato, a destructive disease of both field and greenhouse-grown tomatoes. Chemical disinfection of tools, equipment and structures is a commonly used method of disease management for bacterial canker. The potential of Cmm biofilm formation on hard surfaces in and around tomato production facilities has not been characterized. Furthermore, the ability of chemical biocides to disinfect or eradicate Cmm biofilms has not been characterized on contaminated surfaces. The BEST™ Assay, a versatile, high throughput, multiwell plate assay, was used to assess the ability of Cmm to form biofilms on six surface materials commonly found in tomato production, handling and storage systems. The BEST™ Assay was also used to compare the performances of eight chemical disinfectants in their abilities to eradicate or reduce Cmm biofilms on five surfaces. The results clearly demonstrated that Cmm forms biofilms on many surface materials, however some surfaces were more effectively colonized than others. For example, plywood surfaces were able to support the highest biofilm populations whereas metals and plastics supported the lowest populations. Biofilms, formed using the BEST™ Assay plate, were much more difficult to disinfect and eradicate than the planktonic cells free-floating in the wells of the plate. Disinfectant efficacy versus Cmm biofilms varied with surface material with porous surfaces being more difficult to disinfect than smooth. The data presented here highlight the importance of biofilm surface testing in the discovery, development and registration of chemical disinfectants and other disease management chemistries.
The impact of microbial biofilm formation on survival, tolerance and virulence is now well documented for many pathogenic bacteria and yeast. One of the challenges of biofilm research has been difficulty asssociated with culturing, characterizing and testing biofilms in traditional biofilm reactors. These traditional techniques can be cumbersome when many treatments or replicates are needed as they are not well-suited for rapid, high throughput screening experiments or factorial experiments with many replicates. The MBEC™ Assay is a technique that involves a patented, multi-well plate technology specifically designed for rapid, high throughput culturing, characterzing and testing of microbial biofilms. Furthermore, planktonic cell testing can be performed simultaneously in the same plate. Up to 96 biofilms can be cultured on pegs extending from the plate lid into the wells of the plate bottom. The lid, with biofilms attached, can then be conveniently transferred through a series of solutions or treatments such as rinses, challenges, neutralizations and etc. culminating with the recovery of planktonic cells, or biofilm cells which can be enumerated via standard plate count methods. This assay is now an ASTM standard method for disinfectant and biocide efficacy testing versus microbial biofilms (ASTM E2799-11). We report that this method can be used for many purposes including; simultaneous culture of planktonic cells and biofilms, characterization of biofilm formation and population growth curves, description of biofilm morphology, assessment of biocide efficacy breakpoints, and high througput screening for novel anti-biofilm compounds. This technique is highly versatile and can be used for virtually all culturable species of bacteria. As an example of this, we evaluated the biocidal effect of commercially available disinfectant formulations, approved by the Canadian Food Inspection Agency CFIA to be used in food and feed processing areas, based on MBC (Minimum Bactericidal Concentration) and MBEC (Minimum Biofilm Eradication Concentration) values of Escherichia coli O157:H7 (clinical isolate), and Listeria.
Xylella fastidiosa colonizes the xylem of various host plants, causing economically important diseases such as Pierce's disease in grapevine and citrus variegated chlorosis (CVC) in sweet oranges. The aggregative nature of this bacterium has been extensively documented in the plant xylem and the insect's foregut. Structured communities of microbial aggregates enclosed in a self-produced polymeric matrix and attached to a surface are defined as biofilms. In this study, we characterized biofilm formation by X. fastidiosa through the use of a novel in vitro assay for studying biofilm growth in a potential mimic system of what might occur in planta. We used wood, a xylem rich material, as a surface for bacterial attachment and biofilm formation, under shear force. We demonstrated that X. fastidiosa strains isolated from various hosts formed biofilm on wood in this in vitro assay. Different biofilm morphology was detected, which seems to vary according to the strain tested and microenvironmental conditions analyzed. We observed that strains from different hosts could be grouped according to three parameters: biofilm morphology, the ability to form clumps in liquid culture, and the ability to attach to glass surfaces. We hypothesize that biofilm formation is likely a major virulence factor in diseases related to X. fastidiosa, bringing a new perspective for disease treatment.
The term microbially influenced corrosion, or biocorrosion, refers to the accelerated deterioration of metals owing to the presence of biofilms on their surfaces. The detailed mechanisms of biocorrosion are still poorly understood. Recent investigations into biocorrosion have focused on the influence of biomineralization processes taking place on metallic surfaces and the impact of extracellular enzymes, active within the biofilm matrix, on electrochemical reactions at the biofilm–metal interface.
Detection of the bacterial ring pot pathogen (Clavibacter michiganensis subsp. spedonicus) in seed potato lots by laboratory indexing complements visual inspection. The probability of detecting symptomless infections is a function of sample size and incidence of infection. We determined the incidence of asymptomatic stem and tuber infections in four potato cultivars at three levels of inoculum. At the high inoculum level, 51–93% of stems were infected at 80 days after planting, and 10–59% of the tubers were infected at harvest. The effect of the different percentages of infected stems and tubers on the probability of detection for simple random sampling was calculated for a constant sample size. The actual detection levels for two cultivars planted in field plots with predetermined incidence levels of ring rot infected plants were reasonably close to predicted probabilities.