Validation and accreditation of a duplex real‐time PCR test for reliable in planta detection of Chalara fraxinea1

This work reports the optimization and provides statistically supported validation data for a real‐time detection tool targeting the emerging fungus Chalara fraxinea. The test proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as little as 20 fg of C. fraxinea DNA. The duplex real‐time polymerase chain reaction assay proved to be significantly more efficient and rapid than isolation on agar plates. To monitor the performance of the whole analysis process and to ensure reliability of the results, a set of controls was used systematically during the assays. A diagnosis flow diagram is proposed. The Laboratoire National de la Protection des végétaux was recently accredited by the French accreditation body for this protocol.     

Biocontrol potential of Alternaria sp. PMK2, against a devastating weed: Parthenium hysterophorus L

Parthenium hysterophorus (Asteraceae) is known as one of the most aggressive invasive weeds, causing severe economic, environmental, human and animal health problems in India and around the world. During a series of extensive surveys for natural enemies of P. hysterophorus, a leaf spot pathogen was isolated from the affected parts of the parthenium following the standard isolation techniques using potato dextrose agar (PDA) and parthenium dextrose agar (PeDA) media. Koch's postulates were performed and found satisfactory for the isolate and proved to be pathogenic to this weed. On the basis of cultural, morphological and molecular characteristics, the pathogen was identified as Alternaria sp. PMK2. The growth of the pathogen was studied on eight selected media and it exhibited varying degrees of growth on different media. Phytotoxicity of fungal cultural filtrates was also confirmed on parthenium leaves in laboratory conditions. Due to the virulent nature of the isolated pathogen, it may be selected for further studies to develop mycoherbicide for control of this devastating weed.     

Hymenoscyphus species associated with European ash1

The causative agent of dieback on European ash (Fraxinus excelsior) was first described as Chalara fraxinea based on cultural morphology because no sexual stage of the fungus was known. Later, based on culturing of ascospores of a candidate teleomorph, morphological comparison and nuclear ribosomal internal transcribed spacer sequencing, the sexual stage of C. fraxinea was assigned as Hymenoscyphus albidus, a native and widespread species in Europe. Recently, the morphological species concept of H. albidus was shown to cover two species that cannot be separated from each other based on teleomorph characters, but which can be distinguished by several DNA markers. As a result, the strains causing ash dieback were reassigned as H. pseudoalbidus. The closely related H. albidus is presumably a non‐pathogenic endophyte, but pathogenicity tests to confirm this hypothesis have not yet been performed. Genotyping of herbarium specimens has shown that H. pseudoalbidus was present in Switzerland for at least a decade prior to the epidemic outbreak in Europe. The origin of the ash dieback pathogen, and the general importance of correct pathogen identification to development of effective disease control, are discussed.     

Quantitative detection of <Emphasis Type="Italic">Colletotrichum godetiae</Emphasis> and <Emphasis Type="Italic">C. acutatum sensu stricto</Emphasis> in the phyllosphere and carposphere of olive during four phenological phases

A duplex qPCR detection method was developed to detect and quantify Colletotrichum godetiae and C. acutatum sensu stricto (s.s.) in olive tissues. The method proved highly specific and sensitive with a detection limit of 10 pg for each pathogen. The analysis of green and senescent leaves, fertilized fruitlets with floral residues, green fruit and symptomatic and asymptomatic fruit collected in May, June, October and December revealed a high incidence of both C. godetiae and C. acutatum s.s. in Calabria, southern Italy. In comparison with previous reports, these results highlighted an ongoing population shift from C. godetiae to C. acutatum s.s. Interestingly, C. godetiae was slightly more abundant in terms of number of infected samples, yet the quantity of C. acutatum in infected samples was always higher, suggesting greater aggressiveness and/or sporulation ability of the latter pathogen. The populations of both C. godetiae and C. acutatum s.s. increased sharply in December even though both pathogens were detected widely in asymptomatic samples in May, June and October, confirming an important role of latent infections in the disease cycle. A large quantity of both C. godetiae (1.7 × 10⁸ cells/mg of tissue) and C. acutatum s.s. (7.5 × 10⁸ cells/mg of tissue) was estimated in symptomatic fruit, presenting an enormous inoculum potential for secondary infections. Two other important observations were a high incidence and quantity of both pathogens in senescent leaves and in fertilized fruitlets with floral residues as compared to green leaves.     

Cultural characteristics of Sporisorium sorghi and detection of the pathogen in plant tissue by microscopy and polymerase chain reaction

Despite the economic importance of covered kernel smut of sorghum (Sporisorium sorghi) in many African states and other parts of the world, only limited information is available on laboratory cultivation methods for this fungus and techniques for its diagnosis in plant tissue. The current paper describes laboratory and greenhouse experiments performed with field material of S. sorghi. When intact sori were kept at 5°C, 80% of the spores germinated even after 24?months of storage. Spore germination on agar medium and production of mycelial dry weight in still culture were highest between 20° and 35°C, with a peak at 30°C. Both showed a steady increase from pH 4.5 to pH 7.5, followed by a decline at pH 8.5 and 9.5. In shake culture in different broth media the addition of 0.3% peptone from soybean caused an increase in fungal growth compared with the media alone. Of the media tested, mycelial production was highest in malt dextrose broth supplemented with peptone. When cultivated on different agar media, the morphology of single spore isolates differed both among isolates and depending on the agar medium. In greenhouse experiments, five short, early maturing sorghum breeding accessions proved to be partially or fully resistant to covered kernel smut. Among the plant materials tested, cv. ??Dorado?? appeared to be the one best suited for greenhouse experiments with covered kernel smut. By microscopy of hand-cut sections stained with trypan-blue, hyphae of S. sorghi were seen in apical buds and in nodes of young sorghum plants. Diagnostic PCR amplified a 903?bp element comprising the internal region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene and enabled the detection of S. sorghi in both nodes and apical buds of infected sorghum seedlings. Both techniques, i.e., microscopy and diagnostic PCR, have the potential to be used in studies for the identification of effective sorghum seed treatments already at the seedling stage.     

Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

Newly emerging or re‐emerging diseases are a constant and significant threat to agricultural production, so prompt and accurate identification of the causative agents is required for rapid and appropriate disease management. Classical methods of pathogen detection can be successfully supplemented by next‐generation sequencing (NGS), whereby sequence analysis can help in the discovery of new or emerging diseases. In 2007, hop growers in Slovenia reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; therefore, single step high‐throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was employed to identify a novel pathogen. The sequences were assembled de novo and also mapped to reference genomes, resulting in identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants. Furthermore, the presence of this novel pathogen on hop was confirmed by RT‐PCR analysis of 59 plants with symptoms from 15 hop gardens, representing the main outbreak locations identified by systematic disease monitoring, and small RNA Illumina sequencing of the bulked RNA sample. The high infectivity of the newly identified CBCVd was also confirmed by biolistic inoculation of two hop cultivars, which developed aggressive symptoms in controlled conditions. This study shows the feasibility of deep sequencing for the identification of causative agents of new diseases in hop and other plants.     

Nonhost resistance: Self-inflicted DNA damage by fungal DNase accumulation is a major factor in terminating fungal growth in the pea-Fusarium solani f.sp. phaseoli interaction

Pea endocarp tissue generates a total nonhost resistance response against inappropriate pathogens such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph) within 6 h. An array of plant components induced include: Pisatin (a phytoalexin), defensins, PR genes and hydrolytic enzymes in the non-host resistance response. This nonhost resistance response is similar but swifter than the responses induced by the compatible true pathogen, F. solani f. sp. pisi (Fspi). It was previously noted that a DNase released by both fungi is involved in induction of these resistance responses within pea endocarp tissue. This report demonstrates the cytological damage that occurs within nuclear DNA of both compatible and incompatible fungi when in contact with pea endocarp tissue and in the presence of DNase activity. The severity of damage to the bean pathogen exceeds that of the pea pathogen and requires only 2 h of contact with the pea tissue to develop. This accumulation of DNA damage is proposed to be the ultimate termination factor in this and other non-host resistance reactions. An updated DNase signaling scheme of the nonhost resistance of pea is presented.     

Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR

Rhizoctonia cerealis E.P. van der Hoeven (anamorph of Ceratobasidium cereale D.I. Murray and Burpee), which causes sharp eyespot in wheat, is a major soilborne fungal pathogen that severely impairs yield and quality of winter wheat in China. Because the pathogen is difficult to identify and quantify in soil using conventional methods, a rapid and reliable method is needed to detect and quantify the fungus. In this study, we developed an SYBR Green-based quantitative real-time polymerase chain reaction assay for specific, sensitive detection and quantification of R. cerealis in soil samples. Using a specific primer pair based on the β-tubulin gene of the fungal DNA sequence, we could specifically detect R. cerealis at quantities as low as 100?fg of purified pathogen DNA. Using the real-time PCR assays, we were able to quantify R. cerealis in artificially and naturally infested soil samples. This new technique to quantify R. cerealis is rapid and accurate and will be a useful tool for future studies of pathogenic R. cerealis.     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.     

A sensitive real-time PCR assay for the detection of the two Melampsora medusae formae speciales on infected poplar leaves

Melampsora medusae is a quarantine fungus in the European Union (EU) that causes a damaging leaf rust disease on poplars. Two formae speciales of the pathogen can be distinguished, M. medusae f. sp. deltoidae and M. medusae f. sp. tremuloidae, but the EU plant health directive 2000/29/EC currently in force does not make the distinction between them. EU countries must have the ability to detect and identify rapidly the introduction of these quarantine fungi and to conduct extensive surveys in case of outbreaks. Efficient detection tools are thus needed. In this study, a sensitive real-time PCR assay was developed to detect the presence of M. medusae in poplar leaf samples. A unique primer/hydrolysis probe combination targeting both formae speciales was designed using species-specific polymorphisms observed within the internal transcribed spacer region. An additional primer/hydrolysis probe combination was designed from a region of the 28S rDNA that is highly conserved in the genus Melampsora and used in a separate real-time PCR assay in order to check the quality of the DNA extracted from Melampsora urediniospores. The test developed demonstrated a high sensitivity since it enables the reproducible detection of two M. medusae urediniospore in a mixture of 2 mg of urediniospores (ca 800 000 urediniospores) of other Melampsora species. This new real-time PCR tool should be useful for laboratories in charge of official analyses since it has many advantages over the techniques currently used to monitor this quarantine pathogen in Europe.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Rhabdocline needle cast—investigations on various Douglas fir tissue types

Douglas fir (Pseudotsuga menziesii) is one of the most important non-indigenous tree species in Germany. The species is characterized by a wide amplitude of growing conditions, and is of increasing interest, in particular from the perspective of climate change. Douglas fir is nevertheless particularly susceptible to fungal pathogens, such as Rhabdocline pseudotsugae . The aim of the present study was therefore to develop an early detection method for R. pseudotsugae based on the polymerase chain reaction (PCR). Existing molecular techniques were adapted and optimized to detect the pathogen in small sample volumes. Both healthy and infected Douglas firs were examined, with various tissue types (buds, cambial tissue, needles) and seeds tested for the presence of R. pseudotsugae. Non-infected Douglas firs did not give positive responses in the molecular analyses, but the pathogen was clearly detected in buds, cambial tissues, needles and seeds of infected trees. To date, the fungus has been considered an obligate biotrophic needle parasite. The present results provide clear evidence, however, for the existence of an endophytic stage in the life cycle of R. pseudotsugae. In contrast with previous studies, this paper investigates the dispersal of the fungus via seeds.     

Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Fusarium oxysporum f. sp. cubense race 4

Fusarium wilt (Panama disease), caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (Foc race 4), is one of the most destructive diseases affecting banana (Musa). Early and accurate detection of Foc race 4 is essential to protect the banana industry. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the detection of Foc race 4 based on a SCAR marker sequence. The detection limit for this assay was 10 fg per 25 μl reaction in pure culture and DNA amplification was completed within 60 min. The assays detected 69 different isolates of Foc race 4 from geographically distinct counties in China, and no cross-reaction was observed with other fungal pathogens. When 26 infected and eight healthy looking but infested banana samples naturally from different fields were examined, the detection rate of LAMP was 100 %. The LAMP assay developed in this study was simple, fast, sensitive, and specific, and can be used in the field to detect Foc race 4 in infected banana plant tissue in resource-poor settings.     

Detection and identification of Xanthomonas arboricola pv. pruni from symptomless plant material: results of an Italian test performance study

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruits, is a regulated quarantine pathogen in the European Union, listed as an A2 pest by the European and Mediterranean Plant Protection Organization (EPPO). Because detection and identification of this pathogen is key for its management and to ensure the production of pest free propagation material, it should be based on reliable tests, in particular when dealing with symptomless material. The current EPPO diagnostic Standard (PM 7/64) does not provide specific molecular methods for detection of this pest. The present paper summarizes the results of a test‐performance study (TPS) to validate, at a national level, a detection procedure for this bacterium. A working group was established in order to evaluate the performance criteria for tests included in the current EPPO Standard, and for a conventional PCR. On the basis of the obtained performance criteria, a diagnostic procedure was elaborated and then applied to perform an inter‐laboratory comparison. Screening tests for the detection of the bacterium on symptomless plant material based on IF and/or PCR were proposed, in parallel with isolation on agar media. For identification two methods were suggested: a molecular test or IF. This paper reports on the results of the TPS and proposes a flow diagram for the detection and identification of X. arboricola pv. pruni.     

Pyruvate-amended modified SMSA medium: improved sensitivity for detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

The viable but nonculturable (VBNC) state is induced in the bacterial wilt pathogen Ralstonia solanacearum under prolonged environmental stress. These VBNC cells lose their ability to grow on standard media such as CPG agar, but some of the cells can recover this ability on media supplemented with sodium pyruvate (SP), that degrades hydrogen peroxide. Recently, we suggested that some of the cells in the low-temperature-induced SP-recoverable VBNC state regained their ability to grow on CPG agar after exposure to moderate temperature. These revived cells also retained their virulence on tomato. Although R. solanacearum is detectable on semiselective media, VBNC cells are not detectable on any known semiselective media for the pathogen. To create a suitable medium to detect VBNC cells, we therefore added various compounds that can either degrade hydrogen peroxide or serve an antioxidant function in a semiselective medium, modified SMSA. SP at 5 g/l most improved the sensitivity of R. solanacearum detection. Furthermore, counts on modified SMSA plates for R. solanacearum that had been added to field soil also increased after the addition of 5 g/l SP. SP thus improved the medium’s sensitivity for the detection of R. solanacearum by rescuing a portion of the VBNC cells.     

Development of specific markers for identification of Indian isolates of Fusarium oxysporum f.sp. ricini

Fusarium oxysporum f. sp. ricini (F. o. ricini) is a ubiquitous soil borne pathogen which causes wilt disease on castor (Ricinus communis L). Rapid and reliable detection of the pathogen is essential for undertaking appropriate and timely disease management measures. Identification based on cultural, morphological characteristics and pathogenicity tests are time-consuming and laborious. Traditional methods are now being increasingly replaced by molecular detection techniques, which are much faster and more specific. In this study we have identified two RAPD markers of 1100?bp and 1350?bp in size which can be amplified by OPJ-14 and OPK-12 primers respectively for detection of F. o. ricini. These two fragments were fully sequenced and two pairs of SCAR primers (For-J14 Fwd/Rev and For-K12 Fwd/Rev) were designed. The specific primer pairs amplified a single band from all F. o. ricini isolates and there was no amplification from another thirteen Fusarium species / subspecies tested. These results clearly demonstrate that the designed SCAR primer pairs can be used consistently to detect F. o. ricini isolates, isolated from the diseased samples or soil samples. To our knowledge this is the first report on generation of SCAR markers for identification of Indian F. o. ricini isolates.     

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis

Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.     

Chalara fraxinea is an invasive pathogen in France

Decline induced by Chalara fraxinea is an emerging disease that severely affects ash stands in Europe. The disease appears to have an invasive spread from East to West of Europe in the last decade. The teleomorphic stage, Hymenoscyphus pseudoalbidus, that occurs as apothecia on ash rachis in the litter was recently described. The origin of ash decline remains unclear as a cryptic species, H. albidus, a long-established fungus in Europe, could be present in the same niche, and as in Switzerland, H. pseudoalbidus was shown to have been present long before the recent epidemic outbreak. In France, the emerging disease is very recent and clearly restrained to Northeastern France. We thus collected isolates from infected hosts and from apothecia/ash rachis both inside and outside the infected area in France in order to compare them on the basis of pathogenicity towards ash seedlings and sequences of the ITS regions and of three single-copy genes. We showed that two population types exhibiting about 2% base pair polymorphism in the sequences analysed were present in Northern France. The first type, corresponding to H. pseudoalbidus, was present on rachis and infected hosts only in Northeastern France and showed strong pathogenicity towards ash seedlings in inoculation tests. By contrast, the second type, which corresponds to H. albidus, was present throughout Northern France and showed no pathogenicity towards ash seedlings. Our study confirms the results of Queloz et al. (2010) who presented molecular evidences for the existence of two cryptic species, H. albidus and H. pseudoalbidus. The results strongly suggest that Chalara fraxinea/H. pseudoalbidus is a recent invader in France.     

Exclusion of viroids from potato resources and the modified use of a cDNA probe1

Threats from potato spindle tuber viroid (PSTV) to potato breeding and centralized elite seed-tuber production have been identified in world potato genetic resources. In the UK effective diagnostic testing has proved essential in preventing acquisition. Inoculation of potato nucleic acids to tomato and subsequent viroid detection by polyacrylamide gel electrophoresis (PAGE) has proved a sensitive, but cumbersome, test over 8 years. Additionally, over 2 years, ³²P-labelled PSTV cDNA was used to probe denatured sap and nucleic acid extracts: 10^-4 of peak viroid concentrations in tissue could be detected. Spurious positives were seen in particular circumstances, but could be avoided. Probing of non-denatured samples was not as sensitive. Tubers became infected and PSTV was readily detected by PAGE in leaves of potato experimentally inoculated and maintained below 20°C, but the cDNA probe could not detect infection in tuber sprouts growing at 8–10°C in darkness. Otherwise similar green-leaved sprouts were faintly positive. Detection for all sprouts was unproblematic after movement to 25°C and light for 10 days.