Cavity spot of carrot (Daucus carota)

Cavity spot disease of carrot (Daucus carota) has been one of the intractable problems for both growers and scientists. Carrots are rejected at grading with one or two visible lesions, and when disease incidence passes a relatively low threshold it becomes uneconomic to harvest crops. For the scientist, there has been considerable pressure to produce both information on the cause of the disease and a cure. Many putative causes have been advanced over the years, but these were almost always contradicted by subsequent work. The first solid indication of involvement of a pathogen was when three different fungicides with activity against Oomycete fungi all reduced disease. Very quickly the causal agents Pythium violae and Pythium sulcatum were isolated from cavity spot lesions and Koch's postulates satisfied. The species are not typical of the more common pythia, having slow growth at normal temperatures, which means that in the context of isolation work, plates may be overgrown by other species before they are seen. Metalaxyl fungicide was identified as the most effective in controlling cavity spot caused by P. violae, but P. sulcatum is naturally tolerant of the fungicide. Recently, metalaxyl has been shown to be subject to enhanced microbial degradation. This phenomenon has been associated with failure to control cavity spot. No other fungicide has been shown to be consistently effective in the field, and none has been registered for disease control. For the future, this means that control of cavity spot can not be based solely on fungicidal control. Other, complementary strategies are necessary for reducing disease. Calcium carbonate is known to have significant effects on cavity spot, probably by inducing a soil microflora inhibitory to filamentous fungi. Management of agronomic aspects such as irrigation, soil cultivation and the length of time for which crops are grown may all be used, while carrot cultivars with some field resistance may be beneficial. However, one of the most significant factors is disease avoidance by not selecting fields with high inoculum levels. One serology‐based risk assessment test has been produced and commercialised, and molecular probes which could be the basis of more sensitive tests are available for both pathogens. The potential for disease reduction via a management strategy combining several key components is discussed.     

Production and Evaluation of Monoclonal Antibodies for the Detection of Pythium sulcatum in Soil

Monoclonal antibodies (MAbs) were raised against surface antigens from Pythium sulcatum. The immunogens were prepared from salt extractable cell wall protein to produce monoclonal antibodies. The MAbs showed high specificity to seven P. sulcatum isolates among 26 species of soil‐borne fungi. Weak cross‐reactivities were observed with Pythium aristosporum, Pythium myriotylum, and Pythium zingiberum in indirect enzyme‐linked immunosorbent assay (ELISA), but no reaction was obtained in Western blot analysis. The MAbs recognized glycoproteins in cell wall. Pythium sulcatum was detected in naturally infected carrot tissues and soil using indirect competition ELISA.     

Studies on the biology and control of cavity spot of carrots

Using a selective medium of corn meal agar with pimaricin and rifamycin, isolations from asymptomatic periderm of carrots grown on experimental plots or in commercial crops most frequently yielded the fast-growing species Pythium intermedium, P. sylvaticum or P. ultimum. In contrast isolations from cavity spot lesions mostly produced the slow-growing species P. violae and P. sulcatum. Following treatment of crops with metalaxyl + mancozeb, few isolations from asymptomatic periderm produced Pythium spp. and generally there was a reduction in the number of cavities. Treatment had little or no effect on size distribution of cavities. Cavity spot incidence was significantly less at higher pH values, fields of pH 8·0 and above producing carrots with little or no disease.     

Cavity spot of carrots: II. Cell-wall-degrading enzymes secreted by Pythium and pathogen-related proteins produced by the root cells

This study investigates the biochemical relationships between carrot roots and Pythium violae, the pathogen responsible for cavity spot (CS) disease. P. violae isolates obtained from CS lesions, cultured in Petri dishes on agar were used for inoculation of uninfected mature carrots. The fungus secreted a wide spectrum of enzymes that degraded the cellulose and pectic substances of the carrot cell walls. Cellulase and polygalacuronase (pg) showed the highest activity during the first day post-inoculation, subsequently declining. Pectin lyase (PnL), pectate lyase (PeL) and pectin methylesterase (PME) gradually increased to their highest levels of activity 14 to 30 days post-inoculation. This pattern of activity enables the penetration of the fungus through the walls of the host cells and the establishment of the hyphae. Several plant pathogen-related substances such as peroxidase, chitinase, glucanase and polyphenol oxidase were produced in the infected tissue. Peroxidase activity rose in the inoculated roots from day 1 post-inoculation. Chitinase, glucanase and polyphenol oxidase activities first appeared 3–4 days post-inoculation. At this time, two bands corresponding to chitinase at about 26 and 33 KDa and one band corresponding to glucanase at about 24 KDa could be resolved by SDS-PAGE.     

Pythium species associated with cavity spot on carrots in Norway

Carrot roots with cavity spot lesions from eight different counties in Norway were sampled and Pythium species were isolated on selective medium. Pythium spp. were characterised morphologically and by species-specific PCR. Laboratory experiments with inoculations of carrot roots were performed. A total of 130 isolates out of 230 Pythium -like isolates tested with PCR were identified as pathogenic species of Pythium. These were P. intermedium (29%), P. sulcatum (23%), P. sylvaticum (16%), P. violae (15%) and a possible new Pythium species designated P . ' vipa ' (18%). There were some differences between geographical regions and ages of cavities regarding the frequency of the different species isolated. When rating sunken lesions in the laboratory inoculation experiments, P. ' vipa ' was the most aggressive and P. violae the least aggressive species. P. intermedium and P. ' vipa ' caused more discolouration of the infected carrot tissue than the other species. The importance of the different Pythium spp. as agents of cavity spot in Norway is discussed.     

Biochemical characterisation of Pythium spp. involved in cavity spot of carrots in France

The pathogenicity and growth rate in vivo were assessed on 27 isolates of Pythium spp. recovered from cavity spot lesions on carrots grown in various parts of northwest France. Polyacrylamide gel electrophoresis of isoesterases was used to identify the Pythium spp. involved. Slow-growing isolates were more aggressive than fast-growing ones when inoculated on carrot tap roots. Isoesterase patterns identified the slow-growing isolates as P. violae and P. sulcatum; P. ultimum and P. intermedium were identified among the less aggressive fast-growing isolate group, in which some isolates were also classed as P. sylvaticum or P. irregulare, which have similar electrophoretic profiles. The incidence of Pythium spp. associated with the disease in France is discussed in regard to cavity spot in other countries.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

Microbiological differences between limed and unlimed soils and their relationship with cavity spot disease of carrots (Daucus carota L.) caused by Pythium coloratum in Western Australia

Application of lime (4000 kg ha^-1) to a soil used for commercial carrot production (pH 6.9) significantly (p<0.05) reduced the incidence of cavity spot disease of carrots compared to unlimed soil (pH 5.1). It significantly (p<0.01) increased soil microbial activity as measured by the hydrolysis of fluorescein diacetate and arginine ammonification. The application of lime resulted in a significant (p<0.01) increase in the total numbers of colony forming units (efu) of aerobic bacteria, fluorescent pseudomonads, Gram negative bacteria, actinomycetes and a significant (p<0.01) decrease in the cfu of filamentous fungi and yeasts compared to unlimed soil. Liming also increased the cfu of non-streptomycete actinomycetes rarely reported in similar studies. These non-streptomycete actinomycetes were estimated and isolated using polyvalent Streptomyces phages and the dry heat technique to reduce the dominance of streptomycetes on isolation plates. The non-streptomycete actinomycetes isolated included species of Actinoplanes, Micromonospora, Streptoverticillium, Nocardia, Rhodococcus, Microbispora, Actinomadura, Dactylosporangium and Streptosporangium. The numbers of actinomycetes antagonistic to Pythium coloratum, a causal agent of cavity spot disease of carrots increased in soil amended with lime. Application of lime also reduced the isolation frequency of P. coloratum from asymptomatic carrot roots grown in soil artificially infested with the pathogen, 3, 4 and 5 weeks after sowing.     

Interaction of the mycoparasite Pythium oligandrum with other Pythium species

Interactions of Pythium oligandrum and four plant‐pathogenic Pythium spp. (P. ultimum, P. vexans, P. graminicola and P. aphanidermatum,) were studied in vitro by (i) video microscopy of hyphal interactions on water agar films, (ii) counting of host and mycoparasite propagules in different regions of opposing colonies on sunflower‐seed extract agar films and (Hi) ability of P. oligandrum to overgrow plates of potato‐dextrose agar previously colonized by Pythium spp. Pythium oligandrum typically coiled round the hyphae of Pythium hosts and penetrated the host hyphae after approximately 50 min from the hyphal coils, causing disruption of host hyphal tips up to 1.2 mm ahead of contact points. The relative growth rates of mycoparasite and host hyphae, timing of penetration and distance (sub‐apical) at which penetration led to host tip disruption were used to assess the potential of mycoparasitism by P. oligandrum to prevent the growth of Pythium hosts. P. aphanidermatum was unique among the ‘host’ Pythium spp. in being largely unaffected by P. oligandrum and in antagonizing the mycoparasite by coiling and penetrating the mycoparasite hyphae. Other host Pythium spp. apparently differed in susceptibility, the most susceptible being P. vexans and P. ultimum, whereas P. graminicola was more resistant. The results are discussed in relation to the role of P. oligandrum as a biocontrol agent, especially for limiting the ability of other Pythium spp. to increase their propagule populations in crop residues.     

Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber

Two biological control agents, Pythium nunn and Trichoderma harzianum isolate T-95, were combined to reduce Pythium damping-off of cucumber in greenhouse experiments lasting 3–4 weeks. T. harzianum T-95, a rhizosphere competent mutant, was applied to seeds and P. nunn was applied to pasteurized and raw soils naturally and artificially infested with Pythium ultimum. Some treatments were also amended with bean leaves to enhance the activity of P. nunn. The biological control of Pythium damping-off was evaluated in a Colorado soil (Nunn sandy loam) and an Oregon soil mix, which were replanted twice after 2 and 3 months. Interactions between P. nunn and T-95 were detected in the Colorado but not the Oregon soil. No consistent evidence of antagonism between P. nunn and T. harzianum was seen, and significant interactions were detected in the Colorado, but not the Oregon soil. In the first planting of some treatments, the combination of P. nunn and T. harzianum gave greater control of damping-off than either applied alone. P. nunn was most effective in soils that were pasteurized or amended with bean leaves. T. harzianum controlled Pythium damping-off in the Colorado, but not the Oregon soil. In both soils, disease declined over time in treatments amended with bean leaves but without P. nunn or T. harzianum added. This suppression was greater in the Colorado soil, which contained an indigenous population of P. nunn. This work demonstrates that two compatible biological control agents can be combined to give additional control of a soil-borne plant pathogen.     

Studies on five species of Pythium parasitic on mustard and cabbage in India

Diseased seedlings and roots of cabbage and mustard of Khurpatal (Nainital), were surveyed for zoosporic plant pathogens. Pythium aphanidermatum and P. debaryanum were isolated from cabbage and P. middletonii, P. spinosum and P. undulatum were isolated from mustard. Pathogenicity tests conducted in the glasshouse indicated that P. debaryanum and P. spinosum are the most virulent pathogens of cabbage and mustard respectively.     

Pythium dimorphum from rhododendron

A pythiaceous phycomycete was isolated from diseased roots of rhododendron in New York. It was characterized by fast growth, preference for high temperature for growth, ovoid to elongate, non-papillate, internally proliferating sporangia and large spherical, pigmented chlamydospores, but failed to produce sex organs in single or dual cultures. It was identified as Pythium dimorphum Hendrix & Campbell. This appears to be the second report of its occurrence since its first isolation from lolly pine roots in Louisiana in 1971.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

Pythium root rot of crucifers

A destructive root disease of cabbage (Brassica oleracea L. var. capitata) and cauliflower (Brassica oleracea L. var. botrytis) incited by a species of Pythium Pringsheim is described as occurring in Varanasi, U.P. The pathogen was isolated on potato dextrose and corn meal agar. Pathogenicity and host range of the disease were studied. Cultural characters, morphology and developmental stages and life cycle of the fungus indicated its identity with Pythium middletonii Sparrow.     

First Report of Pythium indigoferae and P. irregulare Associated to Apple Trees Decline in Tunisia

Pythium indigoferae and Pythium irregulare, identified based on morphological and physiological characteristics, were isolated from necrotic roots, crown tissues and the rhizosphere of apple trees in Tunisia from 23 apple orchards in spring and autumn 2007–2009. The virulence assays on excised twigs, using different Pythium species isolated demonstrated that these oomycetes were pathogenic on the Anna, Lorka and Meski varieties and the MM106 rootstock. However, the biggest lesion area was noted on MM106 rootstock. Thus, it appeared that this rootstock is more susceptible to Pythium infections than Anna, Meski and Lorka apple varieties. Furthermore, it is important to note that in vitro tests showed that P. indigoferae seems to be more virulent than P. irregulare.     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pythium vexans Causing Stem Rot of Dendrobium in Yunnan Province,China

Since 2002 a severe root and stem disease of Dendrobium has occurred periodically each year in the plantations of Simao City, Yunnan Province, China. Symptoms included water‐soaked and brown lesions, and rot of tissues. Based on the morphological characteristics and the internal transcribed spacer‐1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2 and β‐tubulin gene sequences, the pathogen was identified as Pythium vexans de Bary. The pathogenicity of the fungus was confirmed by satisfying Koch’s postulates. This is the first world record of stem rot of Dendrobium caused by P. vexans.     

First records of Pythium aquatile and P. macrosporum isolated from soils in Japan

Pythium aquatile and P. macrosporum were isolated from the soil of a cultivated field in Gunma Prefecture and a forest in Nagano Prefecture for the first time in Japan. Their morphological characteristics are described, and their pathogenicity and taxonomy are discussed.     

Distribution and Expression of Elicitin‐like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin‐like proteins (POD‐1, POD‐2, POS‐1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D‐type isolate containing POD‐1 and POD‐2, and the S‐type isolate containing POS‐1. We identified the genes encoding these elicitin‐like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D‐type isolate MMR2 was constructed and genomic regions containing the elicitin‐like protein genes were identified. Southern blot analyses with probes derived from pod‐1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D‐type isolates carried pod‐1, pod‐2 and two oligandrin genes, termed oli‐d1 and oli‐d2, while the S‐type isolates carried pos‐1 and one oligandrin gene termed oli‐s1. Phylogenetic analysis of POD‐1, POD‐2, POS‐1, Oli‐D1, Oli‐D2 and Oli‐S1 with the previously defined elicitins and elicitin‐like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT‐PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D‐type and the S‐type isolates, physical maps of the flanking regions around pod‐1, pod‐2, pos‐1 and the oligandrin genes were constructed. The maps suggest that the D‐type isolates may be derived from the S‐type isolates due to gene duplication and deletion events.     

Cavity spot of carrots: Interactions between the host and pathogen, related to the cell wall

This study investigates the structural aspects of cavity spot pathogenesis. Different Pythium spp. isolated from infected carrots, apples and melons were cultured on agar in Petri dishes and used for inoculation of uninfected carrots. Only slow-growing Pythium spp. (< 15 mm day^-1), such as P. violae and P. sulcatum caused cavity spot lesions. It is suggested that slow-growing species are able to penetrate, albeit slowly, into the plant tissue for 3 to 4 days before a hypersensitive reaction develops. Fast-growing species, however, did not cause lesions. Based on ultrastructural observations, we suggest that the following sequence of events occurs between the plant and the pathogen: The fungus infects the walls and grows for several days, during which time small amounts of wall-degrading enzymes are secreted. Phenylalanine ammonia lyase (PAL) activity and phenols increase linearly immediately upon inoculation. There was a lag phase of about 5 days before lignin began to increase linearly for about a month. Dissolution of wall components decreases the solute potential and water potential in the apoplast. Thus, water moves from the symplast into the apoplast, the turgor pressure gradually dissipates, and the cells shrink and eventually die.