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.     

The influence of potato harvesting and grading machinery on contact spread of Phoma exigua var. foveata on tubers

The incidence of wounds infected by Phoma exigua var. foveata was increased if freshly damaged tubers (recipients) were shaken in a bag with diseased tubers (donors) to simulate the tuber-to-tuber contact that occurs during potato handling. An increase in the number of gangrene rots on damage points also occurred if the recipient tubers were wounded after contact with the diseased tubers, rather than before, and when the donor tubers were heavily infested with P. exigua var. foveata but were free of gangrene lesions. Increasing the proportion of donor to recipient tubers increased the percentage of infected wounds on recipients. Increased incidences of infection in recipient tubers also occurred after they had been passed over an elevator digger when it was lifting stocks of tubers heavily infested with P. exigua var. foveata. When spores of an E +ve isolate of P. exigua var. foveata were sprayed onto the webs of manned potato harvesters, tubers harvested immediately after developed gangrene rots from many of which the E +ve isolate was cultured. An E +ve isolate was also used to demonstrate the transfer of P. exigua var. foveata inoculum from tubers onto soil on riddles of a potato grader and from these soil-coated surfaces onto other tubers during grading.     

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 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.     

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.     

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.     

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).     

Evaluation of selective media for the isolation of Fusarium solani var. coeruleum and Fusarium sulphureum from soil and potato tuber tissue

A new selective medium containing pentachloronitrobenzene and 2-aminobutane (the PAB medium) was developed for soil-dilution plate enumeration of fungal propagules of Fusarium solani var. coeruleum and F. sulphureum from field soil. Growth of ‘weed-fungi’ was less on the PAB medium than on the previously developed F. solani var. coeruleum isolation medium (the PM70 medium) and significantly more propagules of F. solani var. coeruleum were detected. Propagule counts (x) of F. solani var. coeruleum and F. sulphureum from the PAB medium, after log₁₀ (x/10 + 1) transformation, were linearly related to the angular transformation of measurements of soil infectivity from the tuber bait method using the susceptible cv. Catriona. Slopes from disease-inoculum regressions for F. solani var. coeruleum and F. sulphureum in November and May were similar and this suggests that the mode of pathogenic action of F. solani var. coeruleum and F. sulphureum was similar. Recently harvested tubers, inoculated in November, however, were more resistant to infection by both pathogens than stored tubers inoculated in May. Storage of air dry soil at 4°C for 6 months reduced the population of F. sulphureum but not the population of F. solani var. coeruleum. Whereas the PAB medium is recommended primarily for use in the isolation of F. solani var. coeruleum and F. sulphureum from field soil, the PM70 medium appears to be more suitable for recovery of these pathogens and others, including Phoma exigua var. foveata from diseased tuber tissue.     

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.     

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 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.     

Two phytoalexin glycosides from potato tubers infected with Phoma

Four coumarins and seven isoprenoid compounds have been identified in potato tubers infected with Phoma exigua var. foveata. Among these were the 7-O-β-D-glucopyranoside of 7-hydroxy-6,8-dimethoxycoumarin (isofraxidin) and the sesquiterpene 2-(11,12-dihydroxy-11-methylethyl)-6,10-dimethyl-spiro[4,5]dec-6-en-8-one and its 12-O-β-D-glucopyranoside, which apparently have not been previously identified in potato tubers. At least two diastereoisomers of the latter glucoside were present. Analysis of eight fluorescent compounds in different parts of infected potatoes was performed by an improved HPLC technique.     

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.     

Two new isoprenoid spiro compounds from potato tubers infected with Phoma exigua

Two new isoprenoid compounds, 6, 10-dimethylspiro [4,5] dec-6-ene-2, 8-dione and 2-(1′, 2′-dihydroxy-1′-methylethyl)-6, 10- dimethyl-9-hydroxyspiro [4,5] dec-6-en-8-one, have been isolated from potato tubers infected with Phoma exigua var. foveata.     

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.     

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.     

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.     

Behaviour of Phytophthora citrophthora and P. nicotianae var. in soil, and Differences in Their Tolerance to Antimicrobial Components of Selective Media Used for Isolation of Phytophthora spp.

Phytophthora citrophthora was inhibited to a greater extent than P. nicotianac var. parasitica by chloramphenicol, hymexazol, PCNB and pimaricin at concentrations used in selective media for the isolation of Phytophthora spp. Reduced concentrations of the antimicrobial components of the selective media to tolerant levels for P. citrophthora markedly increased the recovery of the two brown rot pathogens from soil. Mycelium of both Phytophthora spp. survived in air-dried soil for at least 5 months while mycelium of most Phytophthora spp. do not survive in dry soil. In moist soil, P. nicotianae var. parasitica produced hyphal swellings, sporangia and chlamydospores. P. citrophthora produced hyphal swellings and sporangia, but no chlamydospores. No oospores were produced, even in pairing cultures on agar plates with isolates obtained from several locations of citrus groves andfruits by both species. Sporania were obtained in both species in citrus groves on mycelium mats, in the rhizosphere, in infected leaves and fruits buried at soil depths of 5–35 cm. Numbers of propagules declined during the incubation period, but conside, rable numbers survived throughout the experimental period (6 months). Although P. nicotianae var. parasitica produced chlamydospores while P. citrophthora did not, numbers of surviving propagules recovered from soil after 6 months were comparable with both species. The brown rot pathogens survived in soil both as colonizers of detached leaves and fruits and as parasites in living root tissues.     

Survival and recovery of Phytophthora capsici and oomycetes in tailwater and soil from vegetable fields in Florida

A current trend in Florida agriculture to conserve water is to irrigate with surface runoff water (tailwater) recovered in retention ponds and canals. Water filtration and lemon leaf baiting recovered Phytophthora capsici and other plant pathogenic Oomycetes in runoff water from ponds and canals. A total of 196 isolates of Phytophthora spp. and 471 isolates of Pythium spp. were recovered. Phytophthora spp. included P. capsici, P. cinnamomi, P. lateralis, P. nicotianae, P. citricola, P. cryptogea and P. erythroseptica. Species of Pythium were P. aphanidermatum, P. catenulatum, P. helicoides, P. irregulare, P. myriotylum, and Pythium‘group F’. Isolates of P. aphanidermatum, P. irregulare, P. myriotylum, and Pythium‘group F’ were pathogenic on pepper and tomato. Recovery of P. capsici propagules was related to soil moisture‐holding capacity and time interval but not temperature. Recovery of P. capsici propagules at 100% soil moisture‐holding capacity and 30° C was 57 days. In tailwater, recovery of propagules of P. capsici was 63 days at 24°C to 25°C. The potential exists to reintroduce and disseminate species of Phytophthora and Pythium when using tailwater for irrigation or other practices.