A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed-tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution-plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet-replicated onto fresh plates of CVP with or without 35 μg ml^-1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft-rot erwinias typically form deep cup-like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.     

Serological recognition of Streptomyces species causing scab on potato tubers

Antisera were prepared against extracts of two tyrosinase-positiveStreptomyces spp., one of which caused a “deep” and the other a “russet” scab. Tyrosinase-positiveStreptomyces isolates not reacting with either of these antisera proved to be nonpathogenic to potato tubers, with few exceptions only. Not all isolates reacting with one or both antisera, however, were pathogenic and so all the serological positive ones had to be tested for pathogenicity to potato tubers. To obtain this relative specificity the antisera had to be absorbed with an extract of a non-pathogenic tyrosinase-positive isolate.     

Cross-reactions of Corynebacterium sepedonicum antisera with soil bacteria associated with potato tubers

The occurrence of low numbers of fluorescing coryneform bacteria located by immunofluorescence microscopy (IF) in heel-end extracts of healthy potatoes was demonstrated to be a normal phenomenon. The number of bacteria found was highly dependent on the antiserum dilution. Bacteria isolated from other healthy potato material also cross-reacted, mostly at low antiserum dilutions compared with homologous isolates or bacteria of the same species, which reacted at a dilution of 11280. Only relatively small differences were found between the antisera tested. Failure to recognize the occurrence of cross-reacting unknown soil bacteria, indicated by the data presented in this paper, can only increase the dangers of incorrect interpretation of IF results.Samenvatting Het optreden van lage aantallen fluorescerende, coryneforme bacteriën gevonden met behulp van immuno fluorescentiemicroscopie in extracten van naveleinden van genzonde aardappelen, bleek een normaal verschijnsel te zijn. Het aantal gevonden bacteriën was sterk afhankelijk van de verdunning van het antiserum. Bacteriën die geïsoleerd werden van ander gezond aardappelmateriaal vertoonden ook kruisreacties, meestal bij lage verdunningen van het antiserum in vergelijking met homologe isolaten of bacteriën van dezelfde soort, die beide nog reageerden bij een verdunning van 11280. Tussen de getoetste antisera bleken slechts geringe onderlinge verschillen te bestaan. De gegevens in dit artikel onderstrepen het gevaar van een onjuiste interpretatie van IF-resultaten, wanneer het voorkomen van kruisreagerende, onbekende bodembacteriën over het hoofd wordt gezien.     

A sampling protocol to detect latent infections in potato tubers

A sampling protocol for detecting latent infections in potato tubers stored in bins, based on the preparation of standard samples of 200 tubers, was tested for reliability. Bins of tubers were manually distributed in windrows and 1% of tubers were replaced with painted tubers, simulating tubers with latent infections. The painted tubers were distributed in windrows with clustered, regular and random distribution. Tubers were harvested and transferred to bins. Each bin was considered to be divided into layers 15 cm in depth, and 20 tubers were collected along an X-shaped sampling path of each bin layer. A simulated sampling approach was applied to obtain 10 bulk samples (200 tubers) for each kind of tuber distribution. The number of marked tubers, the mean percentage and the standard deviation of the bulk samples of each group was computed. The results confirmed that the sampling design adopted in this study ensured preparation of representative bulk samples. In fact, marked tubers were effectively detected; the observed mean percentages of marked tubers were acceptable in value, even though an overestimate of the true percentage was generally obtained. The percentage of marked tubers was underestimated in 50% of cases when considering a regular distribution of marked tubers. However, it was never underestimated when considering the clustered distribution, which is most representative of the situation for tubers with latent infections.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Mixed rots of potato tubers in Ukraine

The pathogens involved in rotting potato tubers at harvest or in store have been identified in the forest and forest-steppe zones of Ukraine. Fusarium spp., Phytophthora infestons, Erwinia carotovora and Ditylenchus destructor are the most important.     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.     

Pathogen resistance in aerial tubers of potato cultivars

Increases in resistance of aerial tubers (cultivars Désirée, Epicure and King Edward) against nine pathogenic potato fungi were determined in vitro using freeze-dried powder derived from subterranean and aerial tubers as a culture medium, and in vivo by inoculating both tuber types with the storage pathogens Fusarium sulphureum, F . solani var. coeruleum, Phoma exigua var. foveata and Polyscytalum pustulans . Significantly reduced growth rates and spore and conidiophore numbers were recorded when pathogens were cultured on freeze-dried powder derived from aerial tubers. No effects on spore size of F . solani var. coeruleum were recorded but spore and conidiophore lengths of remaining pathogens were reduced and width increased, except for Colletotrichum coccodes , where the diameter of acervuli increased, and P . exigua var. foveata , where spore length and width were reduced. Reduced disease incidence, severity and spore numbers were recorded on aerial compared with subterranean tubers inoculated in vivo with the four storage pathogens. Spore length of P . pustulans was reduced and spore width increased, while spore length and width of P . exigua var. foveata were reduced on aerial compared with subterranean tubers inoculated with these pathogens. No effects on spore size of Fusarium spp. were recorded. Concentrations of glycoalkaloids, chlorogenic acid, calcium and magnesium were higher in aerial than in subterranean tuber tissue, which may have contributed to increased resistance and effects on size and morphology of fungal spores. The implications of using aerial tubers as an alternative technique for the production of seed potatoes and their importance to the potato industry are discussed.     

Necrotic diseases caused by viruses in Swedish potato tubers

Potato tuber necrosis in the form of spraing symptoms is caused by infection with Potato mop‐top virus (PMTV) or Tobacco rattle virus (TRV); spraing has become more important in the Swedish potato crop production. In this study, the presence in Sweden of three potato‐infecting viruses associated with necrotic symptoms in tubers was demonstrated: PMTV, TRV and Tobacco necrosis virus (TNV). This study shows that both PMTV and TRV are frequent in Swedish potato fields. PMTV was found in all Swedish counties, except the two most northern ones. TRV was present at several locations in southern and up to the central parts of Sweden. Both viruses were found further north than observed in earlier surveys. PMTV and TRV were analysed in tubers with spraing symptoms and it was not possible to decide visually if the symptoms were caused by either PMTV or TRV. Furthermore, a high occurrence of symptomless tuber infections was observed for several potato cultivars. A unique observation from this study was a mixed infection of both PMTV and TRV in tubers of cv. Berber, where no visual differences on symptom development were detectable for these tubers compared to single infections. During the survey, tubers of cv. Melody were found to display necrotic symptoms in the skin that are characteristic for the ABC disease. This suggested infection by TNV, which also could be confirmed.     

Detection of the root lesion nematode Pratylenchus penetrans in potato tubers

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species-specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species.     

Differences in host range, pathogenicity to potato cultivars and response to soil temperature among Streptomyces species causing common and netted scab in France

The pathogenicity and ecology of some isolates representative of the four main Streptomyces species ( S. scabies , S. europaeiscabiei , S. stelliscabiei and S. reticuliscabiei ) identified as pathogenic to potato tubers were investigated. Three pathogenicity groups could be distinguished. Group 1 included all isolates of S. scabies , S. europaeiscabiei and S. stelliscabiei from common scab lesions of potato and other susceptible root crops. All these produced similar symptoms and were pathogenic to potato, carrot and radish. Group 2 included all isolates from S. reticuliscabiei netted scab lesions; they were pathogenic to both tubers and roots of only a few potato cultivars, and did not infect carrot or radish. Group 3 included three isolates of S. europaeiscabiei from netted scab lesions on cv. Bintje, which produced either common or netted scab symptoms depending on the potato cultivar or plant species. In an experiment on a few isolates from each of the three groups, held at various soil temperature regimes, the three from group 1 were most pathogenic at higher temperatures (20°C or 20/30°C), the two from group 2 were most pathogenic at a lower temperature (17°C). The group 3 isolate caused netted scab symptoms on susceptible cultivars at low temperatures (≤ 20°C) and deep-pitted lesions at higher temperatures. Since the groups identified differ in ecological requirements, it is important to adapt the control methods to the pathogenic species present in the soil.     

Phosphite compounds reduce disease severity in potato seed tubers and foliage

Phosphites (Phi) are alkali metal salts of phosphorous acid, with the ability to protect plants against different pathogens. In this research, the effect of Phi applied to potato plants on severity of three important potato diseases in Argentina was assessed. Seed tubers and foliage of potato cvs Shepody and Kennebec were treated with Phi to assess effects on resistance against Phytophthora infestans, Fusarium solani and Rhizoctonia solani. Protection resulting from Phi treatment in seed tubers was high against P. infestans, intermediate against F. solani, and low against R. solani. In addition, seed tubers treated with calcium or potassium phosphites (CaPhi and KPhi, respectively) at 1% of commercial product emerged earlier than untreated ones. When Phi were foliarly applied two or four times at different doses, high levels of protection against P. infestans were achieved in both cultivars. Higher protection was observed in Kennebec when CaPhi was applied, while in Shepody this was true for KPhi. Expression of β-1,3-glucanases was induced at different times after treatment but no correlation between β-1,3-glucanases expression and foliar protection level was found. On the other hand, Phi positive protection effects did not produce negative effects in plant growth. Leaves from CaPhi-treated plants showed a darker green colour than leaves from control plants; also an increase in Rubisco protein and a delay in crop senescence was observed.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses S and M in potato tubers

Enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase successfully detected potato virus S (PVS) and potato virus M (PVM) in secondarily infected tubers of some Dutch potato cultivars. Extinction was higher for PVS than for PVM but values for both declined slightly within 8 weeks of lifting and it is suggested that testing be carried out within this period. Values (A405) of ELISA reactions between healthy and infected tubers were statistically significant and storage at 4° or 20°C had no effect on detectability of the viruses.Samenvatting Aardappelvirus S kon zowel in knollen van oogst 1978, die gedurende 49 weken bij 4°C waren bewaard en waarvan de kiemrust reeds was verbroken, als in knollen van oogst 1979, die bij 4° of 20°C waren bewaard en nog in de kiemrust verkeerden, tot 8 weken na rooien betrouwbaar met ELISA worden aangetoond. Aardappelvirus M kon eveneens met ELISA betrouwbaar worden aangetoond in knollen van oogst 1979, bewaard bij 4° of 20°C, tot 8 weken na het rooien.De extinctiewaarden voor aardappelvirus S waren hoger dan die voor aardappelvirus M. De waarden voor beide virussen vertoonden een daling gedurende de onderzoekperiode (tot 8 weken na rooien). Er kon geen effect van de bewaartemperatuur (4° en 20°C) op de aantoonbaarheid van de virussen worden aangetoond. Geen verschillen werden waargenomen tussen de extinctiewaarden van het sap uit navel- en krooneinden van de knollen, die nog in de kiemrust verkeerden.Guest worker from April–September 1979 as a fellow of the International Agricultural Centre, Wageningen, the Netherlands, from the Agricultural Research Centre, Yanco, NSW 2703, Australia.     

Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens

To evaluate whether the location of bacterial endophyte communities contributes to disease resistance in potato tubers ( Solanum tuberosum ), the population density, biodiversity and antibiotic activity of endophytic bacteria was examined from the tuber peel (periderm plus top 3 mm of tissue) of four cultivars (Russet Burbank, Kennebec, Butte and Shepody). There were no significant differences for population density of bacteria among the layers of peel examined and no cultivar × peel layer interaction. Endophytic bacteria from several layers of peel were challenged in in vitro bioassays to the soil-borne plant pathogens Fusarium sambucinum , Fusarium avenaceum , Fusarium oxysporum and Phytophthora infestans (mating types A1 and A2). In general, antibiosis of bacterial endophytes against these pathogens was significantly higher ( P  = 0.01) in isolates recovered from the outermost layer of tuber peel and decreased progressively toward the centre of the tuber. Antibiosis against P. infestans was variable, with a progressive decrease in antibiotic activity from outer to inner layers of peel occurring in cvs Russet Burbank and Kennebec only. For antibiosis there were significant cultivar × peel, and cultivar × pathogen interactions ( P  = 0.01). In all cases the inhibitory activity of endophytic bacteria was significantly greater ( P  = 0.01) against the A1 than the A2 mating type of P. infestans . In four of seven cases, where the same species of bacteria were recovered from all three peel layers, antibiosis to pathogens decreased significantly ( P  = 0.01) with depth of recovery (from the periderm to inside the tuber), indicating that in certain communities of endophytic bacteria, defence against pathogens may be related to bacterial adaptation to location within a host and may be tissue-type and tissue-site specific.     

Factors affecting the development and control of black dot on potato tubers

Field trials were carried out over a 4 year period (2004–2007) to determine the effect of agronomic factors, specifically cultivar resistance, irrigation, crop duration and chemical control (in‐furrow application of azoxystrobin), on black dot development on potato tubers grown in fields where soilborne inoculum of Colletotrichum coccodes was present. In 2004, 2005 and 2006, two field trials were performed each year and in 2007, 19 mini‐field trials were carried out across Scotland and England. Cultivar resistance was clearly demonstrated to be an effective method of reducing black dot disease severity on tubers (described here as the percentage of unmarketable tubers, i.e. those with symptoms covering a surface area of >10%). In the four field trials carried out in 2004 and 2005, in irrigated and fungicide‐untreated plots, 43·8% of tubers of cv. Maris Piper were unmarketable, compared with 17·0% of tubers of cv. Sante. Assessments of disease development on underground plant parts (stems, stolons and roots) revealed that cultivar resistance acted only at the tuber level, as disease symptoms on other parts were often high irrespective of published disease resistance ratings. Irrigation increased the severity of disease on tubers in two trials (England 2004 and 2006), but its effect was less significant when rainfall was high. Delaying harvest by 2 weeks increased disease severity in all six trials, whilst application of azoxystrobin consistently reduced black dot severity. There were significant interactions between factors. The results clearly show how black dot disease severity can be reduced through an integrated approach to disease management.     

Resistance to thiabendazole in Fusarium species isolated from potato tubers affected by dry rot

Fusarium species obtained from stored potato tubers affected with dry rot were grown on agar containing thiabendazole. All 40 isolates of F. coeruleum and 60 isolates of F. avenaceum tested were sensitive to the fungicide, but 68% of the 85 isolates of F. sulphureum and one isolate of F. culmorum were classified as resistant. When isolates were made from dry rots on tubers that had been treated with thiabendazole during loading into store, all 81 isolates of F. sulphureum were resistant, whereas all the isolates of F. coeruleum (25), F. avenaceum (4) and Phoma foveata (10) were sensitive. Resistance was not found in five isolates of Cylindrocarpon destructans. All the Fusarium spp. were sensitive to imazalil and were pathogenic when inoculated into potato tubers. Resistant and sensitive isolates of F. sulphureum caused rots of similar size.     

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to low infection rates of plants inoculated at the postflowering stage and a decrease in the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insectproof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to postflowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibility to PVY infection, in return, early and late PVY inoculations lead to similar rates of infected tubers at the plant scale and equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.