Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

Laboratory testing of potato tubers for multigenic resistance to late blight

Resistance in the tubers of potato clones with various levels of foliar multigenic resistance toPhytophthora infestans was measured to detect possible correlations between tuber and foliar resistance. A highly significant correlation was found using wound-healed tuber tissues. High levels of tuber resistance were detected in most foliar-resistant clones when inoculations with race 1,2,3,4 were made after 24–48 hours of wound periderm formation at 20 C. The resistance of wound-healed tissues increased as the time between wounding and inoculation increased. This increase in resistance was significantly greater for clones with foliar resistance than for clones with susceptible foliage.     

Methods of assessing potato tubers for resistance to bacterial soft rot

Summary The susceptibility to soft rot caused byErwinia carotovora subsp.atroseptica of five potato cultivars and potato clones from the potato breeding programme at J?geva Plant Breeding Institute was assessed in a laboratory test. Three test methods were used: a single site inoculation, vacuum infiltration and slice inoculation. The severity of the disease varied widely with the different methods, which gave different cultivar rankings.     

Resistance of tubers from different potato cultivars to soft rot caused byErwinia carotovora subsp.atroseptica

Mechanically harvested tubers of 14 potato cultivars grown on both loamy sand and silt loam soils were evaluated for resistance to bacterial soft rot caused byErwinia carotovora subsp.atroseptica. Cultivars were also assayed for calcium and dry matter content to determine possible correlations with soft rot resistance. Resistance of potato tubers to bacterial soft rot was assayed after harvest by inoculating tubers and determining percent surface area decayed after four days in a mist chamber. Tubers of the cultivars differed widely in resistance to bacterial soft rot. The ranking of the resistance of tubers from different cultivars to bacterial soft rot was similar for both soils, although disease severity in tubers from a particular soil type varied with the season. Of the cultivars tested, tubers of Butte, Russet Burbank, and Nor-gold Russet were ranked as the most resistant, whereas tubers of Redsen, Norchip, Norland and Belchip were rated as the most susceptible. Tubers of Rhinered, Red La Soda, Superior, Pontiac, Atlantic, and Nooksack were intermediate in resistance. Neither calcium concentration in medullar tissues nor dry matter of potato tubers alone could be correlated with ranking of the cultivars with respect to severity of bacterial soft rot. However, a multiple linear regression model incorporating both variables indicated that tubers with a high content of both calcium and dry matter were likely to be less susceptible to bacterial soft rot than were tubers with lower percentages of these variables.     

Studies on the initiation of bacterial soft rot in potato tubers

Summary Non-inoculated whole tubers rot readily under anaerobic conditions when their surface is maintained moist in a water-saturated atmosphere. Rotting is reduced under otherwise similar but aerobic conditions, but there is some rotting under anaerobic conditions even if their surface is dry. The evidence obtained suggests the following sequence of events under wet anaerobic conditions. Water is absorbed causing the lenticels to open. Oxygen deficiency affects cell membrane integrity and there is leakage of water and solutes from turgid cells which establishes a continuous liquid phase between the cortex and the lenticels. Organisms in the lenticels then penetrate deeper into the tuber in this liquid phase. The leaked cell contents, together with the apparent reduction of tuber resistance to infection under anaerobic conditions, allow rapid growth of bacteria. If pectolytic bacteria are present a soft rot lesion is then established.

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Zusammenfassung Nicht inokulierte Knollen der Sorte Majestic faulten nicht, wenn sie in N₂ bei 93, 95 oder 98% relativer Feuchtigkeit und bei Raumtemperatur w?hrend 10 Tagen gelagert wurden (Tabelle 1). Unter anaeroben Bedingungen faulten sie jedoch sofort, wenn ihre Oberfl?che in einer wasserges?ttigten Atmosph?re feucht gehalten wurde (Tabelle 2). Unter gleichen, jedoch aeroben Bedingungen wurde das Verfaulen gehemmt, aber unter anaeroben Bedingungen und bei trockenen Oberfl?chen trat keine F?ulnis auf (Tabelle 2). Wenn einzelne Lentizellen inokuliert wurden mitE. carotovora var.atroseptica (Isolat 27), entwickelte sich eine L?sion weder unter aeroben noch unter anaeroben Bedingungen, sofern die Lentizellen geschlossen und die Knollenoberfl?che trocken waren (Tabelle 3). Bei offenen und wuchernden Lentizellen trat jedoch sofort F?ulnis ein, aber nur wenn anaerobe Bedingungen vorherrschten (Tabelle 3). Knollen mit teilweise nassen Oberfl?chen, gelagert in N₂, nahmen an Gewicht zu und wurden turgeszenter (Tabelle 4). Gleichzeitig mit diesen Aenderungen ?ffneten sich die Lentizellen zunehmends, und ihr Gewebe begann zu wuchern (Abb. 1). Die Abnahme an Elektrolyten und der Wasserverlust der Knollengewebescheiben waren unter anaeroben Bedingungen gr?sser als unter aeroben (Abb. 2). Die Abnahme an Elektrolyten und der Verlust im Frischgewicht waren unter anaeroben Bedingungen bei Scheiben mit einem hohen (turgeszenten) Wasserstatus gr?sser als bei solchen mit einem niedrigen (nicht turgeszenten) Wasserstatus (Abb. 3). Die Unterschiede im ‘Apparent Free Space’ der turgeszenten und nicht turgeszenten Scheiben, die unter aeroben und anaeroben Bedingungen gehalten wurden, zeigen ferner an, dass die Durchl?ssigkeit der Zellen unter anaeroben Bedingungen und besonders in turgeszenten Gewebe zunimmt (Tabelle 5). Diese Unterschiede werden wiederspiegelt in der Anf?lligkeit der Scheiben für das Entstehen der F?ulnis, das in erster Linie von der An- oder Abwesenheit von O₂ und in geringerem Umfang vom Wasserstatus in den Scheiben abh?ngt. Wenn wir diese Ergebnisse zusammenfassen, k?nnnte die Ausl?sung des F?ulnisprozesses wie folgt beschrieben werden: Unter nassen anaeroben Bedingungen wird das Wasser aufgenommen und veranlasst die Lentizellen, sich zu ?ffnen. Sauerstoffmangel beeinflusst die Integrit?t der Zellmembranen, und es tritt ein Verlust an Wasser und gel?sten Bestandteilen der turgeszenten Zellen ein, was zu einer st?ndigen flüssigen Phase zwischen der Rinde und den Lentizellen führt. Die Organismen in den Lentizellen dringen dann tiefer durch diese flüssige Zone in die Knolle ein. Der abgeflossene Zellinhalt, zusammen mit der offensichtlichen Verringerung der Resistenz der Knolle gegen die Infektion unter anaeroben Bedingungen, erlauben das rasche Wachstum der Bakterien. Wenn pectolytische Bakterien vorhanden sind, bildet sich eine Nassf?ule-L?sion.

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Résumé Des tubercules non inoculés de la variété Majestic ne pourissent pas quand ils sont conservés dans N₂ à 93, 95 ou 98% d'humidité relative (RH) à température ambiante pendant 10 jours (tableau 1). Cependant, quand on maintient une certaine humidité à la surface des tubercules dans un milieu anaérobe la pourriture se developpe rapidement (tableau 2). La pourriture est réduite dans des conditions semblables à d'autres égards mais aérobies; toutefois il n'y a aucune pourriture en condition anaérobie si la surface des tubercules est sèche (tableau 2). Lorsqu'on inocule des lenticelles individuellement avecErwinia carotovora var.atroseptica (isolat 27), aucune lésion ne se produit en conditions aérobies et anaérobies lorsque les lenticelles sont fermées et la surface des tubercules sèche (tableau 3). Toutefois la pourriture apparait rapidement si les lenticelles s'ouvrent et prolifèrent mais seulement lorsque des conditions anaérobies sont prédominantes (tableau 3). Les tubercules présentant une surface partiellement humide et conservés dans N₂ augmentent en poids et deviennent plus turgescents (tableau 4). Simultanement il y a une ouverture progressive des lenticelles et proliférations de leur tissu (fig. 1). Les pertes d'électrolytes et d'eau provenant de disques de tissu de tubercule sont plus importantes en milieu anaérobie qu'aérobie (fig. 2). La perte d'electrolytes et la perte de poids frais en condition anaérobie sont également plus grandes pour les disques ayant une forte teneur en eau (turgescents) que les disques à faible teneur d'eau (non turgescents) (fig. 3). Les différences en espace libre apparent entre disques turgescents et non turgescents tenus dans des conditions aérobie et anaérobie indiquent ultérieurement que la perméabilité cellulaire augmente en condition anaérobie et notablement dans les tissus turgescents (tableau 5). Ces différences se reflètent dans la susceptibilité des disques à pourrir, susceptibilité qui depend en premier lieu de la présence ou de l'absence de O₂ et, dans une moindre mesure, de la quantité d'eau dans les disques. Prenant en considérations ces results, la pourriture dans les tubercules pourrait débuter comme suit. Dans des conditions humides anaérobies, l'eau est absorbée causant l'ouverture des lenticelles. Le manque d'oxygène porte atteinte à l'intégrité de la membrane cellulaire et il y a une fuite d'eau et de produits solubles à partir de cellules turgescentes, ce qui réalise une phase liquide continue entre le cortex et les lenticelles. Dans cette phase liquide, les organismes se trouvant dans les lenticelles pénètrent alors plus profondément dans le tubercule. La perte de matières cellulaires, en meme temps que la réduction apparante de la résistance du tubercule à l'infection en condition anaérobie, permettent un développement rapide des bactéries. Si des bactéries pectolytiques sont présentes, des lésions de pourriture molle s'etablissent dès lors.

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The substance of this paper was presented at a session of the Pathology Section at the 5th Triennial Conference of the EAPR (Norwich, September 1972).     

A Study of the relationship of late blight resistance to glycoalkaloid content in fifteen potato clones

Tuber glycoalkaloid (TGA) content, leaf glycoalkaloid (LGA) content, and the level of multigenic resistance to late blight were determined for 15 potato clones. There was no association between the level of blight resistance and the TGA or LGA contents in these clones. However, TGA and LGA contents were highly correlated. The genotypic correlation coefficient between TGA and LGA contents for the 15 clones was 0.82 (SE = 0.115). TGA contents from blight-infected plants were not significantly higher than TGA contents from healthy (fungicide-protected) plants. The results indicate that breeders should be able to select for multigenic late blight resistance withohut increasing TGA contents of clones in their breeding programs.     

Resistance to powdery dry rot (Fusarium sulphureum) in potato tubers

Summary Fourteen cultivars were tested for resistance to penetration and colonization byF. sulphureum by a point inoculation technique. Differences in resistance to penetration were small when compared with those reported forF. solani var.coeruleum, and were more apparent in one year than another. Tubers grown at different sites gave similar results. The medullary tissue was generally more susceptible than the cortex. There were large differences between cultivars in resistance to colonization and these were consistent between years and sites. A selection derived from a cross between established cultivars was more resistant than its parents.

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Zusammenfassung Um die Resistenz von 14 Kartoffelsorten gegenFusarium sulphureum zu prüfen, wurde eine Punktinokulationsmethode verwendet, die der von Pietkiewicz & Jellis (1975) für Phomaf?ule beschriebenen Methode ?hnelt, das Inokulum bestand aber aus 100 Konidien in 0,01 ml sterilem Wasser und die Inkubation betrug 4 Wochen. Die zu prüfenden Knollen stammten von zwei Herkünften und sieben der Sorten werden in zwei Jahren getestet. Die Eindringungsresistenz wurde durch Bonitur der F?ulen, die mehr als 2 mm über die Verletzungen hinausgingen, erfasst und die Ausbreitungsresistenz in der Rinde durch eine Gesamtbonitur jeder Wiederholung, bei der eine 1–3 Skala verwendet wurde: 1=2–20 mm Durchmesser, 2=20–40 mm, 3=>40 mm. Nicht verwertet wurden F?ulen unter 2 mm im Durchmesser. Insgesamt waren King Edward, Record und Pentland Squire signifikant weniger anf?llig gegenüber der Eindringung als die anderen geprüften Sorten (Tabelle 1) aber keine Sorte besass eine hohe Resistenz. Die Ergebnisse waren zwischen den Herkünften, dem Gewebe und den Jahren vergleichbar, aber die Unterschiede zwischen den Sorten waren in einem Jahr deutlicher als im anderen. Das Markgewebe war im allgemeinen gegen die Eindringung anf?lliger als die Rinde. Die Rangfolge für die Ausbreitungsresistenz war zwischen den Herkünften konstant und wenn auch geringer, zwischen den Jahren (Tabelle 2). Sorten mit Eindringungsresistenz hatten geringe Boniturwerte für die Ausbreitung, aber das umgekehrte stimmte nicht immer. Erste Prüfungen von selektiertem Zuchtmaterial zeigten, dass in der Familie B31, die aus (Pentland Crown x Maris Piper) x Pentland Squire gezüchtet wurde (Tabelle 3a), Resistenz vorhanden sein k?nnte. Ein Wiederholungstest im folgenden Jahr best?tigte, dass der Klon B31/46 eine deutlich h?here Eindringungsresistenz in der Rinde aufwies als King Edward und erfolgreiche Inokulationen ergaben nur kleine Faulzonen (Tabelle 3b). Daraus wurde geschlossen, dass es erfolgversprechend ist, auf Resistenz gegenFusarium sulphureum zu züchten unter Verwendung der in den britischen Sorten vorhandenen genetischen Variabilit?t.

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Résumé Une technique d'inoculation par pointe a été appliquée sur 14 variétés de pommes de terre, afin d'étudier leur résistance àFusarium sulphureum. Cette technique est semblable à celle décrite par Pietkiewicz & Jellis (1975) pour la gangrène, mais la concentration d'inoculum est de 100 conidies par 0,01 ml d'eau stérile et l'incubation est de 4 semaines. Les tubercules testés proviennent de 2 régions et 7 variétés ont été étudiées pendant 2 années. La pénétration du parasite est évaluée en notant la proportion de pourritures se développant sur plus de 2 mm au-delà de la blessure, la surface du sympt?me est notée pour chaque répétition à partir d'une échelle de 1 à 3∶1= diamètre de 2 à 20 mm; 2=20 à 40 mm et 3= supérieur à 40 mm. Les pourritures d'un diamètre inférieur à 2 mm ne sont pas notées. Dans tous les cas, King Edward, Record et Pentland Squire sont significativement moins sensibles à la pénétration que les autres variétés testées (tableau 1), bien qu'aucune variété ne soit hautement résistante. Les résultats sont sensiblement comparables suivant les endroits, les tissus et les années, mais les différences entre les variétés sont plus nettes d'une année sur l'autre. Le tissu médullaire est en général plus sensible à la pénétration que le cortex. L'ordre de résistance au parasite est conforme suivant les endroits et sensiblement le même suivant les années (tableau 2). Les variétés présentant une certaine résistance à la pénétration ont des notes faibles au développement du sympt?me, mais l'inverse n'est pas toujours vrai. Des tests initiaux sous-abri à partir des sélections de plants ont montré une bonne résistance pour la famille B31, croisement de (Pentland Crown x Maris-Piper) x Pentland Squire (tableau 3a). Un test répété l'année suivante a confirmé la forte résistance à la pénétration dans le cortex du cl?ne B31/46 par rapport à King Edward et des inoculations réussies n'ont donné que de petites pourritures (tableau 3b). En conclusion, il est possible de sélectionner, en vue d'une résistance àF. sulphureum, à partir des variabilités génétiques disponibles au sein de variétés anglaises.

     

Inheritance of resistance to late blight in foliage and tubers of progenies from parents differing in resistance

Summary TenSolanum tuberosum genotypes differing in resistance to late blight (Phytophthora infestans (Mont.) de Bary) in foliage and tubers were intercrossed to determine the inheritance of disease resistance in their progenies. Plots of 10–15 clones per progeny were established in each of 2 years and resistance assessed by field or laboratory tests. The parental genotypes were similarly tested each year. The parents differed in general combining ability (GCA) for both foliage blight (FB) and tuber blight (TB). The parental and GCA scores were significantly correlated for both aspects of the disease, but the correlations between foliage and tuber scores for parents and for GCAs were not significant. Three parental genotypes were highly resistant in both foliage and tubers, and the genotype with the highest GCA for resistance to both FB and TB (cv. stirling) is recommended as the best parent. There was no evidence of strong genetic correlation between both aspects of resistance, and it is suggested that both be selected for in a breeding programme.     

Suppression of bacterial soft rot in potato tubers by application of kasugamycin

An immersion in aqueous solutions of Kasumin®, containing kasugamycin at 10 to 320 mg/L (ppm) for 1 to 320 sec delayed or prevented the development of soft rot on inoculated (Erwinia carotovora subsp.carotovora) tuber disks incubated over wet filter paper at 24C for 24 to 72 hr. Treatment efficacy decreased as inoculum level and incubation period increased. Short immersion periods were not as effective as longer ones. When stored tubers were inoculated withE. c. carotovora and incubated up to 5 days in fog chamber at 20 C, immersion treatments in 20 to 400 ppm kasugamycin either had no effect or increased soft rot development. By contrast, when the stored tubers were cut into sections before inoculation and treatment, immersion in 300 ppm reduced soft rot development on the cut surfaces from 83% (inoculated controls) to 3.2%. With non-inoculated sections, treatment with 25 ppm reduced the SAD from 13.3 to 0.6%. Wash or rinse treatment of freshly harvested tubers in 40 to 160 ppm produced a significant reduction in the severity of bacterial soft rot among tubers incubated for 72 to 96-hr. The 160-ppm treatment delayed disease onset by at least 24 hr. Pre-washing freshly harvested tubers with clean or chlorinated water prior to antibiotic treatment did not consistently improve the efficacy of the kasugamycin treatment.     

Assessing progenies of potato for resistance to early blight

Summary Progenies from crosses between cultivars varying widely in resistance to early blight (Alternaria solani Sor.), were assessed for resistance as true seedlings in a glasshouse in Scotland. The resistance of a representative sample of surviving genotypes from each progeny was compared with samples of the same progenies not previously exposed to the fungus, both in the glasshouse in Scotland and in the field in Israel. The exposed population was more resistant. Resistance was identified more effectively in adult plants from tubers in the glasshouse than in true seedlings and agreement between glasshouse and field assessment was better when progenies were compared rather than individual genotypes. The mid parent and progeny mean scores of the unexposed population were correlated at both sites, thus confirming that the resistance is heritable. Selecting resistant individuals at the seedling stage is suggested as a useful tool for resistance breeding, having first chosen the best parents for crossing.     

A method for estimating the decrease in marketable tubers caused by potato late blight

A regression analysis was used to develop a method to estimate the decrease in marketable tubers caused by late blight of potato (Phytophthora infestans), based on estimates of percentage loss in total tuber yield computed from a published method. The economic loss due to late blight in table stocks is the relative percentage marketable yield loss, calculated from the expression: {fx19-01} when total yield losses (in percentage) did not exceed 40%. A graphical method is presented as an alternative. Yield results from seven field experiments conducted in the Maritime Provinces of Canada during the period 1953 to 1971 were used in the study, where marketable tubers were defined as 2 1/4 inch or more in diameter.     

Reducing the potential for bacterial soft rot in potato tubers by chemical treatments and drying

The potential for bacterial soft rot caused byErwinia carotovora in freshly inoculated potato tubers was reduced up to 99% by immersion for 5 min in solutions of sodium hypochlorite (chlorine bleach) containing up to 10,000 ppm chlorine. Reductions up to 93% were achieved using a combination treatment of immersion in 1% citric acid for 5 min followed by airdrying. Immersion treatment alone in 1% aqueous solutions of citric, acetic, ascorbic, or malonic acid also significantly reduced the soft rot potential. In contrast, no reductions accompanied treatment with solutions of potassium or calcium acetate. All treatments including the combination immersion/airdrying treatment were much less effective if tubers had been infiltrated initially with soft rotErwinia or had numerous mechanical injuries. If tubers had not been infiltrated with the causal organism, immersing them in 1% citric acid reduced the potential nearly as much as a similar treatment with 1000 ppm chlorine. A 30-sec immersion in the latter was less effective than a similar treatment with 500 ppm a.i. CGA 78039, an experimental bactericide. However, air-drying, coupled with provisions for keeping tuber surfaces free from moisture, remains the most effective means of reducing losses to bacterial soft rot.     

Bacterial soft rot potential in washed potato tubers in relation to temperatures of tubers and water during simulated commercial handling practices

The bacterial soft rot potential in potato tubers was affected by differences in temperature between tubers and suspensions ofErwinia carotovora at the time of inoculation by immersion. In general, disease severity after incubation was higher when tuber temperatures were higher as opposed to equal to or lower than that of water containing cells of the bacterium. Disease severity was also greater when tubers were stored at 20° to 23°C in contrast to 4° or 26° to 30°C for 16–18 h prior to immersion. Tubers with a pulp temperature of 8°C were more prone to bruise injury as compared with those at 18°C when struck with a pendulum type bruising instrument. When the injured tubers were inoculated and incubated, most bruises became infected with soft rot bacteria.     

Correlation between resistance to late blight in foliage and tubers in potato clones from parents of contrasting resistance

Summary The relationship between resistance to late blight in foliage and tuber was examined on 50 clones of each of five progenies from crosses where one parent was resistant in both foliage and tuber and the other susceptible. Foliage resistance was assessed in a field trial and tuber resistance in a laboratory test on glasshouse-grown tubers. The genetical and environmental components of variance and the phenotypic and genetical correlations between foliage and tuber blight scores were determined for each progeny. Foliage and tuber resistance were correlated, indicating either that both are determined by the same genes or by different linked genes. The environmental component of variation was greater for tuber blight than foliage blight. We suggest that the most effective way of selecting for resistance to both aspects of the disease in a breeding programme is to select those resistant in the foliage and then screen them for tuber resistance.     

Effect of calcium soil amendments on phenolic compounds and soft rot resistance in potato tubers

Nutrients such as boron, nitrogen and calcium stimulate the production of phenols. This research focuses on the role of calcium in increasing phenol metabolism in potato peels and the ensuing tuber resistance to soft rot pathogens was investigated. Two field experiments were conducted at the University of Zimbabwe campus plots in 2008 and 2009 summer seasons. Sprouted tubers of cv. BP1 were planted in plots treated with different fertilizer combinations. The treatments were: 1) compound S (7N: 21P: 8K) + ammonium nitrate (34 %N) 2) compound D (7N: 14P: 7K) + calcium nitrate (19 Ca: 15.5N) 3) compound S + calcium nitrate and 4) compound D + ammonium nitrate. The harvested progeny tubers were inoculated with Pectobacterium carotovorum subsp. brasiliense. Calcium amendment increased the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidases) involved in the metabolism of phenolics and total soluble phenols. Calcium amendment significantly reduced (P < 0.05) the maceration effect of P. carotovorum subsp. brasiliense in tuber tissues. Chlorogenic acid, caffeic acid and calcium, but not ferulic acid content were significantly higher (P < 0.05) in plants grown in calcium treated plots. Calcium positively and significantly correlated with polyphenol oxidase (PPO), phenylalanine (PAL) and peroxidase (POD), while chlorogenic and caffeic acid showed a positive relationship with POD and PPO. Calcium amendment significantly reduced maceration symptom caused by the bacteria (P < 0.05), resulting in smaller decayed zone diameters on inoculated tubers from calcium-treated plots. This study shows that soil amendments of calcium increase concentration of calcium, caffeic and chlorogenic acid in tuber peels and also reduces maceration effect of pectinolytic pathogens. Reduced maceration could be due to increased levels of caffeic and chlorogenic acid which have antimicrobial properties.