Assessment of the response of carrot somaclones to Pythium violae, causal agent of cavity spot

Cavity spot is a major disease of carrots, causing cavities on the surface of the root. Available commercial varieties show a range of susceptibility but no significant resistance. Seed progeny from 46 tissue culture-derived carrot ( Daucus carota ) somaclones were screened for viability, then 19 selected somaclone families were sown under glasshouse conditions, along with commercial cultivars (Bertan, Nandor, Bolero and Vita Longa) as controls. Mature roots were exposed to Pythium violae in a cavity spot bioassay to determine their response as measured by disease incidence and severity. Some somaclones formed fewer lesions than the least susceptible control cultivar, Vita Longa. Seven somaclonal families that showed a range of susceptibility were sown under field conditions and the assessment was repeated. Although there was little relationship between glasshouse and field-trial results, under field conditions one of the somaclones had a mean incidence of disease, as estimated by transformed data, of 1·9 compared with 37·9 for the most susceptible somaclone and 3·5 for Bolero, the most resistant commercial cultivar. The results indicated that significant genetic variation in susceptibility to cavity spot disease was present in the somaclones.     

Pythium violae isolated from cavity spot lesions on carrots in Israel

Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on carrots in Israel and the resulting isolates were compared with isolates from the UK, France and Spain. The isolates from all locations responded similarly to temperature and had similar colony morphology, oogonial diameters and soluble protein patterns.     

Aggressiveness and production of cell-wall degrading enzymes by Pythium violae, Pythium sulcatum and Pythium ultimum, responsible for cavity spot on carrots

This study investigated the relationships between pathogenesis, types of symptoms and in vitro production of cell-wall degrading enzymes by P. violae, P. sulcatum and P. ultimum. The three pathogens, considered as the three Pythium species principally responsible for cavity spot on carrot roots, secreted only low levels of fatty acid esterases activity, suggesting they have limited ability to degrade suberin in the walls of the outer cell layers of carrot tissues. Among the enzymes that degrade cell-wall polysaccharides, only pectate lyases and cellulases were produced by P. violae, and these were produced late and in small amounts: the symptoms caused by P. violae were limited and typical of cavity spot. Conversely, P. ultimum caused maceration of tissues, and secreted polygalacturonases and -1,4-glucanases earlier and in larger amounts than P. violae. P. ultimum also produced a large diversity of proteins and cellulase isoenzymes. Although secreting all the monitored enzymes in higher quantity than the two previous species, P. sulcatum was responsible for only typical limited symptoms of cavity spot, with a brown colouring. The role of plant reactions induced in response to early pectinolytic enzyme production by P. sulcatum may account for this apparent inconsistency.Died on April 11, 1997     

Modulation of primary and secondary infections in epidemics of carrot cavity spot through agronomic management practices

The relative importance of primary and secondary infections (auto- and alloinfections) in the development of a carrot cavity spot (CCS) epidemic caused by Pythium spp. were investigated. Three cropping factors: fungicide application, soil moisture and planting density, were selected as the key variables affecting the disease tetrahedron. Their effects on: (i) disease measurements at a specific time, (ii) the areas under the disease progress curves (AUDPCs) and (iii) a time-dependent parameter in a pathometric incidence-severity relationship, were studied. Mefenoxam applications 5 and 9 weeks after sowing reduced the intensity of a field CCS epidemic that involved both primary and secondary infections. In microcosm experiments, mefenoxam reduced secondary infections by Pythium violae obtained by transplanting infected carrot roots and slowed disease progress (1·6 lesions per root in treated versus 5·8 lesions in non-treated microcosms). A deficit of soil moisture limited the movement of Pythium propagules to host tissue, and thus reduced primary infections in the field; it also promoted the healing of lesions, limiting lesion expansion and the potential for alloinfections (6·8–7·5 lesions per root in irrigated plots compared with 2·4 lesions in non-irrigated plots). A negative relationship between the mean root-to-root distance and the rate of alloinfections was established in microcosms; a reduction in mean planting density was also effective in limiting CCS development (0·5, 1·6 and 2·0 lesions per root in microcosms containing 8, 16 and 31 roots, respectively). An integrated disease management system based on a combination of cultural methods, such as optimized fungicide application, date of harvest versus soil moisture content, and host density versus planting pattern, may make a useful contribute to the control of CCS.     

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic -(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

Lateral roots of carrot have a low impact on alloinfections in cavity spot epidemic caused by <Emphasis Type="Italic">Pythium violae</Emphasis>

Carrot cavity spot, caused by a complex of Pythium species, is characterized by sunken elliptical lesions on the taproot. Recent epidemiological studies of P. violae have demonstrated the occurrence of both primary and secondary infections, with two types of secondary infection, autoinfection and alloinfection. Investigating the mechanisms underlying alloinfection and the role of carrot lateral roots, we asked whether direct physical root contact plays a role in alloinfection and whether root exudates enhance mycelial growth in soil alone. A rhizobox system was designed to differentiate the effects of each mechanism: a buffer zone created by nylon mesh was used to test the first mechanism, and young carrots with a root system similar to lateral roots were used to test the second. Alloinfections were generated in rhizoboxes via diseased taproots transplanted close to healthy, mature carrots. The nylon mesh had no significant effect on disease intensity (reflecting alloinfection), providing evidence that mycelial growth in soil contributed more to disease spread than did physical contact among roots. Nor did young carrots significantly affect alloinfection; thus root exudates had little effect on mycelial growth.     

Reduced persistence of metalaxyl in soil associated with its failure to control cavity spot of carrots

Metalaxyl was used to control Pythium diseases of carrots in experiments on farms with a history of cavity spot. The first experiment compared the method of application (sprayed, banded or broadcast) and rate (0, 1.5, 3 or 6 kg a.i. ha⁻¹) one week after sowing. Three additional experiments compared the rate (0, 0.75, 1.5 or 3 kg a.i. ha⁻¹) and time (sowing, 1- to 2- or 4- to 5-true-leaf stage) of application. In expt 1, the application of metalaxyl, but not the method by which it was applied, increased yield by 20% and significantly reduced the incidence of cavity spot, forking and misshapen carrots. In expts 2, 3 and 4, neither the rate nor time of application affected yield or reduced the incidence of Pythium diseases. Comparison of the sites showed that they differed in past metalaxyl usage. Metalaxyl had not been used on the site of expt 1, but had been used previously at sites 2, 3 and 4. Laboratory experiments were conducted to determine whether these differences in efficacy resulted from reduced sensitivity of Pythium isolates to metalaxyl, or reduced persistence of metalaxyl in soil. ED₅₀ values showed that there was no reduction in metalaxyl sensitivity. The half-life of metalaxyl was 82 days in soil from expt 1, but was 10 days or fewer in soils from expts 2, 3 and 4. Thus the failure of metalaxyl to control Pythium diseases was associated with reduced persistence in soil, not reduced sensitivity of the target fungi.