The effect of soil- and tuber-borne inoculum on the incidence of potato gangrene

Under optimum growing conditions neither tuber- nor soil-borne Phoma exigua var. foveata inoculum appreciably affected stand or yield of the subsequent potato crop. Seed tubers with gangrene rots caused high levels of stem and tuber symptoms when planted in var. foveata contaminated or uncontaminated land; contaminated seed tubers with no rots also produced progeny with a high gangrene potential. Sufficient soil-borne inoculum was carried over in land that produced a gangrene affected crop in the previous year to override the effect of tuber disinfection. Effective gangrene control was achieved by a combination of tuber disinfection shortly after harvest over successive years with a 1 in 5 yr potato crop rotation. Gangrene rots usually developed through injuries to the tuber periderm, rots in other tubers being associated with pustules of powdery scab (Spon-gospora subterranea).     

The infection of potatoes by Phoma exigua

The pathogenicities of Phoma exigua var. foveata and P. exigua var. exigua were compared by determining the relationship between incidence of infection of whole tubers or tuber pieces and various inoculum levels in soil. In natural soil var. foveata was more pathogenic than var. exigua, but in sterilized soil there were no clear differences between the two. Both varieties were isolated from some lesions derived from inoculum to which only one variety had been added indicating either natural contamination of soil or latent infection in tubers; the proportion of such lesions increased at the lower inoculum concentrations. In studies where potato plants were grown in infested soils, the incidence of stem disease and tuber disease increased at increased inoculum concentrations of var. foveata; var. exigua was not added to the inoculum but was isolated from some lesions, the proportions of such lesions again increasing at lower inoculum concentrations. The infectivity of the infested soil, assessed by a tuber-baiting method, declined soon after planting but increased prior to harvest. Laboratory studies indicated that in natural soil the infectivity was highest after 2–3 months and lowest after 10 months incubation, but in sterilized soil infectivity was highest after 5 months and lowest after 7 months incubation.     

The effect on gangrene incidence of dusting seed potatoes with benomyl at time of planting

In 1 out of 2 years' field trials benomyl applied as a dust treatment at time of planting seed potatoes resulted in an increase in potato gangrene in the progeny of both gangrene-free tubers planted in land contaminated with Phoma exigua var. foveata and gangrene-diseased tubers planted in clean land. Gangrene-infected seed tubers treated with benomyl also produced more stems infected with var. foveata than untreated tubers. Two hypotheses are presented to account for this increase in gangrene which does not occur in seed potatoes treated shortly after lifting. In all cultivars tested an organo-mercury dip-treatment increased total numbers of tubers in the seed and chat-size grades without increasing total weight whereas benomyl dust increased the numbers in these grades in Majestic only. The treatment of gangrene-diseased seed with benomyl dust affected neither total weight nor total number of tubers.     

The role of seed-tuber and stem inoculum in the development of gangrene in potatoes

To determine the relative importance of seed tubers and stems as sources of inoculum for potato gangrene in progeny tubers, different levels of inoculum of Phoma exigua var. foveata were established in field experiments by planting rotting or contaminated seed tubers and by inoculating stems shortly before haulm desiccation. The pathogen was only occasionally detected by isolation from inside green stems in June and July on plants growing from contaminated but unrotted seed. The incidence of pycnidia on desiccated stems increased with increasing concentration of inoculum contaminating seed tubers and with increasing time interval between desiccation and harvest. Stem infection was probably derived from inoculum on seed tubers spreading via the soil to the stem bases. Stem inoculation of plants growing from uninoculated or uncontaminated seed greatly increased both the gangrene potential of progeny tubers (defined as % wounds with gangrene after uniformly damaging tubers and storing them at 5°C for 12 wk), and tuber contamination (defined as % wounds with gangrene after spreading tuber-borne soil onto test tuber slices and storing them at 5°C for 8 wk). However, when stems of plants growing from rotting or contaminated seed were cut at ground level and removed before desiccation, gangrene potential of progeny tubers was only slightly less than that of tubers from untreated plots. There was no evidence that soil inoculum or aerial spread played a significant role in disease development. Gangrene potential and contamination of progeny tubers were related to Contamination levels on seed tubers. Some transmission also occurred from rotted seed tubers to progeny. Inoculum levels around progeny tubers increased rapidly after desiccation even in plots where stems had previously been cut at soil level and removed to eliminate pycnidial development above ground as a source of inoculum. Both gangrene potential and contamination of progeny tubers were greater on cv. Ulster Sceptre than on cv. Pentland Crown. The results showed that the inoculum on seed tubers, whether from rots or surface contamination, contributed more to the contamination of progeny tubers at harvest than did the inoculum from pycnidia on stems following desiccation of the haulm.     

Aerial dispersal of the potato gangrene pathogen, Phoma exigua var. foveata

A semi-selective medium for isolating the potato gangrene pathogen, Phoma exigua var. foveata, from air is described. Airborne propagules of P. exigua var. foveata were generated for at least 57 min after the start of simulated raindrop impaction on senescent potato stems bearing pycnidia from a natural infection. The number of propagules generated as aerosols was 0·17% of that available for aerosolisation. The aerodynamic particle size of at least 92% of the aerosols was in the range 2·1–4·7 μm in diameter. Using a Casella High Volume Bacterial Sampler airborne propagules of P. exigua var. foveata were detected in the open air during rainfall from September to November at three sites in Southern Scotland. They were recovered at least 800 m down wind from the nearest potato crop. P. exigua var. exigua and P. eupyrena were also recovered from the air during rainfall. Very low numbers of P. exigua var. foveata were occasionally detected in the absence of rain. The role of airborne inoculum of P. exigua var. foveata is discussed in relation to reinfection of virus-tested stocks of seed potatoes derived from stem cuttings.     

Development and Validation of Conventional and Quantitative Polymerase Chain Reaction Assays for the Detection of Storage Rot Potato Pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata

The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular‐based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real‐time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real‐time PCR methods produced similar results. In terms of sensitivity, the detection limits for real‐time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real‐time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative C_t method (ΔΔCt) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene‐causing potato pathogens.     

Pathogenesis of Potato Gangrene Caused by Phoma exigua var. foveata. I. Location of Phosphatase Activity in Diseased Tuber Tissue

The nitrocellulose blotting method was adapted for locating phosphatase activities in gangrene‐diseased tuber tissue. Adenine and glucose phosphates were used as enzyme substrates. A strong dephosphorylation of high‐energy phosphates took place in the UV‐fluorescent tissue adjacent to dry rots caused by Phoma exigua var. foveata, but the enzyme remained at a low activity in the other areas of the diseased tuber. Phosphatases were probably induced by P. exigua var. foveata, and intense dephosphorylation of high‐energy compounds leads to the death of potato tuber tissue.     

Effects of delaying fungicide treatment on the incidence of gangrene in stored potato tubers

Potato titbers infested with Phoma exigua var. foveata were uniformly wounded and sprayed or dipped in fungicide suspensions either immediately or after periods of up to 21 days' storage at 5, 10, 15 or 20 °C. Tubers were then stored at 5 °C and gangrene assessed after 12 wk. Incidence of gangrene on untreated tubers was progressively decreased by increasing the length of storage at 15 or 20° (curing) but was not affected by 3 days' storage at any temperature. Fungicide treatment immediately after wounding gave best control of the disease; treatment after 3 days' delay was less effective and after 14 or 21 days was usually ineffective. Gangrene was decreased by fungicides more on tubers stored for 3 or 7 days at 5 °C than at higher temperatures. Control of gangrene by curing or fungicides diminished when the amount of inoculum on tubers was increased. Increasing the amount of fungicide applied improved control and fungicides were more effective in decreasing gangrene on cut and crush wounds than on cut wounds. At the arbitrary concentrations used in these experiments imazalil gave better disease control than thiabendazole, prochloraz, carbendazim plus quinolin 8-ol or triadimefon.     

The spread of Phoma exigua within the potato crop

The incidence of gangrene that developed in tuber samples harvested in a systematic way from potato crops planted with surface sterilized gangrene-free seed surrounding a small plot planted with gangrene-affected seed showed that in two out of the three years marked outward spread of Phoma exigua var.foveata occurred in an uneven manner to about 4 ft (1–2 m) from the central plot. The occurrence of var. foveata was restricted to the immediate area in and around the gangrene infection locus whilst P. exigua var. exigua was more widely and evenly distributed throughout the experimental area. The incidence of infection in and around the central plot varied from year to year and it is suggested that the main method of spread of var. foveata inoculum is by rain splash. Infection by both fungi appeared to be influenced in a similar manner in all three seasons.     

Factors affecting the occurrence of gangrene (Phoma exigua) in potatoes

The presence in soil from Scotland and England of Phoma exigua f. sp. exigua and P. exigua f.sp. foveata, which cause gangrene, is confirmed by isolation, and it is established that infection of tubers occurs before lifting, and after lifting from soil adhering to tubers. The distribution of the disease is related to soil moisture, gangrene being most prevalent in tubers from the north-eastern counties of Scotland where the moisture content of arable soils remains high throughout the growing season. The incidence of the disease may be affected by the haulm, either as a source of infection—though not an important source—or by its effect on the maturity of the tubers. The incidence of gangrene is less where haulm destruction is rapid. The incidence of gangrene in a crop is not related to its incidence in the seed tubers planted and, unless infection is severe, the effect on yield is not serious. Symptoms of skin necrosis were associated with tubers from acid soils, infected with P. exigua f.sp. foveata and stored at low temperature.     

The significance of tuber damage and inoculum concentration of Phoma exigua var. foveata in the development of gangrene in stored potato tubers

The susceptibility to gangrene infection of wounds of various shapes and depths on potato tubers was studied by inflicting wounds using differently-shaped brass teeth and rods of different diameters. Inoculating wounds with spore suspensions or damaging tubers which had been previously contaminated with Phoma exigua var. foveata or which had been recently lifted from plots of field experiments showed that wounds in which tissue was crushed were most susceptible to infection. Over a wide range of inoculum concentrations and in experiments using several different cultivars the incidence of infection of any wound type was compared to that of the standard severe cut and crush wound. Using a probit transformation a linear relationship was established, the slope of the line indicating the relative susceptibility of the wound. In 1977 and 1978, crops of cv. Pentland Dell were surveyed for damage incidence, inoculum and inoculum potential on arrival at a commercial bulk store. Nets of tubers buried among the tuber bulk were recovered after storage and gangrene incidence compared with damage and inoculum assessments. Inoculum potential and incidence of severe damage both influenced disease development but damage incidence was of greater importance, showing that priority should be given to decreasing damage and to curing to promote rapid wound healing in endeavours to control the disease.     

The effects of various chemical and physical haulm treatments on the incidence of potato gangrene

In studies of the influence of haulm defoliants on gangrene incidence in storage the use of diquat dibromide was consistently found to exacerbate levels of Phoma exigua vzr.foveata infection particularly on tubers harvested 3–4 wk after burning down. Intermediate levels occurred where the haulm was chemically defoliated with dinoseb or was physically cut and removed and least where the defoliants were sodium chlorate or sulphuric acid. Pycnidia of P. exigua var. foveata developed within 10 days on stems desiccated with diquat dibromide, sulphuric acid or dinoseb and most prolifically on those treated with diquat dibromide but tuber infection was not always related to their abundance. Speed of kill was not considered to be important in determining effect on gangrene incidence.     

Seasonal occurrence of the potato gangrene pathogen, Phoma exigua var. foveata, in the open air

Using a Casella High Volume Bacterial Sampler air samples were collected down wind of potato fields in central Scotland when it was raining and when it was dry between December 1978 and October 1982. Airborne propagules of Phoma exigua var. foveata were recovered from the open air during rainfall in most months of the year but the frequency of recovery was greatest in September, October and November. The numbers of colonies of P. exigua var. foveata detected during rainfall were much greater in September and October than during the rest of the year. Low numbers of colonies of P. exigua var. foveata were recovered in the absence of rainfall and their occurrence appeared to be associated with strong winds and dry soil conditions. These latter airborne propagules were detected during all the months except November to February when few samples were made. Sampling simultaneously up wind and down wind of potato fields in September and October demonstrated that the potato crop was the main source of airborne propagules caught down wind but small numbers of P. exigua var. foveata were recovered up wind in four out of the seven experiments.     

Immunoenzymatic techniques for the detection of Phoma exigua in infected potato tissues

Antisera to Phoma exigua var. foveata and var. exigua were prepared by injecting rabbits and mice with protein solutions from mycelium. Specific antibodies were isolated and immunoenzymatic techniques (double antibody sandwich ELISA and indirect ELISA) were used to test for the fungus in inoculated tubers and sprouts and in stems grown from these tubers. The fungus was detected in these different tissues, with var. foveata being more aggressive, demonstrating the applicability and sensitivity of the techniques. The antibodies produced to the two varieties of the fungus were not specific to their own varieties. They also reacted with Phoma tracheiphila but did not react with several other common potato pathogens. Preliminary results obtained with antibodies from mouse ascite liquid suggest the possibility of producing specific monoclonal antibodies.     

Pathogenesis of Potato Gangrene Caused by Phoma exigua var. foveata: II. Activities of some Hydrolases and Dehydrogenases

The location of enzyme activity in gangrene‐diseased tubers was determined using the nitrocellulose blotting method. The activity of aminopeptidase and esterase was located in tissues adjacent to dry rot caused by Phoma exigua var. foveata and in other apparently healthy tissues. The activity of glucuronidase, succinic and glucose‐6‐phosphate dehydrogenases (G‐6‐PDH), however, was confined to tissues adjacent to the rotted tissue. The pathogen produces very active α‐ and β‐glycosidases, so their highest activity occurred in rotten tissue that was filled with fungal mycelium. Results suggest that all these enzymes are involved in alteration of cell metabolism and the destruction of diseased tuber tissue.     

The effect of harvest date and duration of wound healing conditions on the susceptibility of damaged potato tubers to infection by Phoma exigua (gangrene)

In experiments extending over 3 yr, King Edward tubers harvested on various dates from early September until early November were uniformly wounded and kept at 15 ^oC and c. 95% r.h. for periods of up to 21 days for wounds to cure. When tubers were subsequently inoculated with Phoma exigua var.foveata and incubated at 5 ^oC for 12 wk, curing for 3 and 7 days decreased gangrene incidence to respectively c. 13 and 4% of the non-cured controls. When tubers were inoculated before curing and immediately after wounding, curing for 7 days decreased gangrene incidence to only c. 68% of the non-cured controls. Curing was also progressively less effective the later the date of harvesting, suggesting that there would be advantages in harvesting potato crops early in the autumn.     

The influence of cultivar and isolate on the development of gangrene (Phoma exigua var. foveata) in potato tubers

In studies on the infection of potato cultivars with different field isolates of Phoma exigua var. foveata, cultivar × isolate interactions, although sufficiently large to influence the relative pathogenicities of isolates on different cultivars, were not substantial enough to affect cultivar ranking order. Cultivar rank was markedly influenced by pathogen isolate only when both field and culture collection isolates were compared. This suggests that the complications of cultivar × isolate interactions can be avoided in cultivar screening tests by the use of recent field isolates of high pathogenicity. It was found that cultivar was considerably more important than isolate in determining gangrene lesion size. The need to consider both lesion diameter and depth when estimating rot size following point inoculation of different cultivars was confirmed.     

Variation in pathogenicity among field isolates of Phoma exigua var. foveata

Experiments carried out using a point inoculation method to infect potato tubers with Phoma exigua var. foveata demonstrated considerable variation in pathogenicity among field isolates. This variation was unlikely to be due to differential reductions in isolate pathogenicities during axenic culture. However, fresh field isolates generally produced larger lesions than stored isolates. An investigation of the distribution of pathogenicity variation in the fungus revealed that differences among isolates from different lesions taken from the same potato stock were greater than those between stocks, but pathogenicity variation within each lesion isolate was small relative to that between isolates. The importance of using isolates with a high level of pathogenicity, hence recent field isolates, in studies of this pathogen is stressed.     

Factors affecting the assessment of resistance of potatoes to gangrene (Phoma exigua var. foveata)

Three experiments designed to elucidate the effects and interactions of different inoculation methods, inoculation positions, strains of Phoma exigua var. foveata and clones of potato tubers inoculated were analysed using as data dimensions of the lesions and a more general surface score. The assessment of susceptibility was affected by all these factors. The ranking of varieties for resistance altered considerably depending upon whether the surface area or the depth of lesions was considered. Derivation of more complex scores combining these measurements reduced the relative genetic component and hence would be of no value to potato breeders.     

Fungus diseases on potato seed tubers planted in England and Wales, 1963-76

During 1963-76 samples of potato tubers from commercial seed stocks of cvs King Edward (14 yr), Pentland Crown (9 yr), Majestic (7 yr), Pentland Dell (3 yr), Record and Arran Pilot (2 yr) were received from farms in England and Wales. Fifty tubers from each sample were examined macroscopically for fungus diseases and eyes were excised from a 20-tuber sub-sample, incubated and examined for pathogenic fungi; 50 tubers were stored on trays to sprout and examined for diseases and sprouting in May and in most years samples of 50 tubers were wounded by dropping onto expanded metal, stored at 5° C and examined for gangrene and dry rot after 12 wk. Amounts of disease varied between years and during 14 yr black scurf and powdery scab on King Edward tended to increase and skin spot and late blight decrease. On average 44% of King Edward tubers were affected with skin spot, 25% with black scurf and 16% with powdery scab. Gangrene affected 5% of tubers and 97% of the isolates from rots were identified as Phoma exigua var. foveata. Wounding tubers increased the incidence of gangrene three-fold. During 1963-69 late blight affected 2% of King Edward tubers but fewer in later years and in other cultivars. Majestic had most common scab (44% tubers) and Arran Pilot most dry rot (9% tubers) and this disease was increased by wounding tubers. Conidiophores of Helminthosporium solani (silver scurf) were more common on excised eyes of Pentland Crown, Record and Arran Pilot than of other cultivars, and isolations from verticillate conidiophores that developed on the side of incubated eye plugs of King Edward and Majestic stocks gave pure cultures of Verticillium tricorpus (78%), V. nigrescens (9%) and V. nubilum (3%). Proportions of tubers with different diseases were affected by their country of origin; Scottish seed had most skin spot and gangrene, Irish seed most powdery scab and English seed most common scab, late blight and H. solani. There was also evidence of differing disease incidence in seed from different geographical areas in Scotland and England. Up to half the King Edward and Pentland Crown stocks examined in 1975 and 1976 were derived from stem cuttings and average amounts of diseased tubers were similar to those in stocks not derived from stem cuttings. Annual and cultivar differences in disease incidence and effects of date of receipt of seed on farms are discussed.