Detection of the phytotoxin coronatine by ELISA and localization in infected plant tissue

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR)in vitro, and the availability of purified toxin has facilitated development of immunological detection methods. A modified, indirect competitive ELISA using the COR-specific monoclonal antibody 11B8 was developed to detect COR in various host plants infected by P. syringae. The estimated detection limit for COR was 50 pg per well, and COR could be reliably quantified from 5 to 40 ng ml⁻¹. The subcellular localization of COR within infected tomato tissue was investigated using the COR-specific antibody MAb 8H3G2. Immunofluorescence microscopy and immunogold labelling showed that COR was present inside tomato cells and was associated with chloroplasts and particles of proteinase inhibitor I. Localization studies indicated that COR is mobile in infected plant tissue and can be detected in healthy tissue adjacent to the bacterial lesions.     

Elimination of common scab sensitive progeny from a potato breeding population using thaxtomin A as a selective agent

Common scab is one of the most important soil‐borne diseases of potato and is difficult to control. Selection of potato breeding lines for resistance to common scab is also cumbersome due to environmental factors influencing symptom development and an erratic spatial distribution of the scab pathogens (Streptomyces spp.) in the field. The bacterial phytotoxin thaxtomin A, which causes scab symptoms, can be used to screen large numbers of potato seedlings for tolerance in vitro, but few studies have investigated whether the results correspond to resistance to common scab observed in the field. In this study, 120 F1 potato progeny from a single cross were screened in vitro by exposing the seedlings to thaxtomin A added to the culture medium. Eighteen genotypes were selected based on high sensitivity or tolerance using shoot growth as the criterion, multiplied in vitro, and tested for resistance to common scab caused by S. turgidiscabies and S. scabies in a glasshouse and in three different fields. Evaluation of ca. 6500 tubers showed that the 18 potato genotypes differed in scab indices and disease severity (P < 0·0001). The relative shoot height in vitro (thaxtomin A used at 0·5 μg mL^?1) and the scab index in the field showed significant correlation (r_s = ?0·463, P = 0·0528, n = 18), also consistent with the results obtained under controlled conditions in the glasshouse. Hence, the in vitro bioassay may be used to discard scab‐susceptible genotypes and elevate the overall levels of common scab resistance in the potato breeding populations.     

Suppression of potato cyst nematode root penetration by the endoparasitic nematophagous fungusHirsutella rhossiliensis

The endoparasitic nematophagous fungusHirsutella rhossiliensis was tested for its ability to suppress root penetration and cyst formation by the potato cyst nematode speciesGlobodera pallida. Isolates ofH. rhossiliensis were obtained from infected potato cyst nematode juveniles from different starch potato fields in The Netherlands. The isolates showed no difference in spore adhesion to juveniles on agar plates (adhesion rate: ±90%). The most rapid growing isolate, CBS 108.94, was used for experiments. Vegetative mycelial colonies ofH. rhossiliensis CBS 108.94, grown in potato dextrose broth, were used as soil inoculum. During submerged cultivation the mycelial colonies produced phialides (spore-bearing cells) but no spores. Exposed to the air, however, spores were rapidly formed. The effect of different soil inoculum densities of mycelial colonies on root penetration byGlobodera pallida was examined in an experiment in 250-ml pots. Up to a mycelial colony concentration representing a potential spore density of 10⁴ g^–1 soil no suppression occurred. At approximated densities of 2.5×10⁴ and 10⁵ spores g^–1 soil the numbers of juveniles which penetrated roots were reduced by 30% and 34%, respectively. The distribution of the inoculum could be improved by fragmentation of the mycelial colonies before soil inoculation. Using mycelial fragments, again no suppression of root penetration was observed up to a potential spore density of 10⁴ g^–1 soil, but at densities of 10⁵ and 10⁶ g^–1 a suppression of 54% and 88%, respectively, was measured. In a greenhouse experiment, soil inoculation with mycelial colonies with a potential spore production of 2.5×10⁵ g^–1 soil resulted in a suppression of root penetration of 37% and 51% after 5 and 6 weeks, respectively, but the number of newly formed cysts after 18 weeks in soil was not different for control and inoculated pots. It is concluded thatH. rhossiliensis may be useful for the reduction of root damage caused by juveniles of potato cyst nematodes, but the usefulness for population control is doubtful.     

Isolation and partial characterization of polypeptides associated with phytotoxin in cultures of the cool-temperature biotype of Stemphylium botryosum pathogenic on alfalfa

Stemphylium botryosum pathogenic on alfalfa (Medicago sativa) produced a phytotoxin in a defined liquid medium which caused symptoms similar in sequence and appearance to stemphylium leafspot lesion development on attached alfalfa leaflets. The molecular weight of the phytotoxin was estimated to be 19500 by gel filtration chromatography. Treatment of partially purified phytotoxin with proteinase K or subtilisin resulted in loss of phytotoxic activity. Action of the proteases in inducing loss of phytotoxicity was inhibited by phenylmethylsulphonyl fluoride. Concentrated culture filtrates were fractionated by chromatofocusing, HPLC ion-exchange and gel filtration. Gel filtration also was followed by non-denaturing electrophoresis or isoelectric focusing (IEF). Analysis of sequential fractions from each procedure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that phytotoxic activity was consistently coincident with the presence of two major polypeptides (M_r = 26900 ± 2%; 19500 ± 4%) and a triplet of polypeptides (M_r = 15500). The constant association between the three polypeptides suggested an intermolecular interaction during fractionation. The phytotoxic activity eluted from the chromatofocusing column between pH 5·79 and 5·43 whereas IEF estimated the isoelectric point to be between 5·40 and 5·02.Immobilized concanavalin A and wheat germ lectin failed to bind phytotoxicity. Proteolytic activity in some phytotoxic gel filtration fractions was reduced by addition of phenyl-methylsulphonyl fluoride and separated from phytotoxicity by non-denaturing electrophoresis, indicating that phytotoxicity was not due to serine protease activity.Pathogenicity tests of the cool-temperature biotype on 20 plant species representing eight plant families resulted in a compatible interaction only on M. sativa and M. polymorpha. In contrast, sensitivity to the phytotoxin was observed on both pathogen-resistant and susceptible alfalfa clones along with all other plants tested with the exception of Triticum aestivum, Zea mays, Apium graveolens and Melilotus officinalis, indicating non-host-specificity of the phytotoxic polypeptides produced by the cool-temperature biotype.     

Interactions Between the Potato Cyst Nematode Globodera rostochiensis and Diseases Caused by Rhizoctonia solani AG3 in Potatoes Under Field Conditions

Findings from 2 years of field experiments investigating the relationship between Globodera rostochiensis and Rhizoctonia solani on unique field sites are reported. In 2000, a field experiment was positioned on land that had previously been used for experimental work investigating integrated potato cyst nematode (PCN) management methods. This study had produced an ‘untypical’ mosaic of PCN population densities ranging from 5 to 221 eggs g⁻¹ soil. In 2001, the field experiment was conducted on a different field site and overlaid on a focus of G. rostochiensis population densities ranging from 11 to 108 eggs g⁻¹ soil. In each experiment, potatoes (cv. Désirée) were grown in plots with similar population densities of G. rostochiensis that were either uninoculated or inoculated with R. solani. A series of potato plant harvests were undertaken to investigate the effects of nematode infestation on the incidence and severity of R. solani diseases and the associated development of plants. In both experiments, a clear relationship was found between the density of G. rostochiensis juveniles present in potato roots and the incidence of stolons infected by R. solani, 6 weeks after planting. For the first time this interaction has been determined under field conditions. The results of the study suggest that the interaction between nematode and fungus is indirect and possible mechanisms are discussed.     

Mycorrhiza induced resistance in potato plantlets challenged by Phytophthora infestans

Biological control of soil-borne pathogens by arbuscular mycorrhizal (AM) fungi has been repeatedly demonstrated. However, their role in the control of above-ground hemibiotrophic pathogens is less conclusive. Here, we investigated in vitro the impact of an AM fungus on Phytophthora infestans in potato plants. The leaf infection index was decreased in mycorrhizal potato plants. Real-Time Quantitative PCR revealed the induction of two pathogenesis related genes (PR1 and PR2) in the leaves of mycorrhizal plants shortly after infection with P. infestans. These results suggested a systemic resistance in mycorrhizal plants, related to the priming of the two PR genes in potato.     

Relationship Between Production of the Phytotoxin Prehelminthosporol and Virulence in Isolates of the Plant Pathogenic Fungus Bipolaris sorokiniana

A gas chromatographic method was developed to quantify the phytotoxin prehelminthosporol, which is a sesquiterpene metabolite of the plant pathogen Bipolaris sorokiniana. The toxin was extracted from mycelium or culture filtrates, pre-cleaned using solid phase extraction, and analyzed by gas chromatography as a trimethylsilyl-derivative. The detection limit of the method was 5ngl^–1 (signal to noise ratio 4:1) which corresponds to ca. 15ng prehelminthosporol per mg dry weight of mycelium or 15ng prehelminthosporol per ml culture filtrate. The total amount of prehelminthosporol (mycelium plus culture filtrate) increased with cultivation time when examined in six isolates of B. sorokiniana after 6, 9, 12 and 15 days of incubation. The screening experiment of 17 isolates for prehelminthosporol production after 8 days of incubation revealed significant differences in the toxin production between the isolates. The isolates with low toxin production had lower virulence towards barley roots compared to those with higher production of the toxin. However, the virulence did not increase with prehelminthosporol level among the high producing isolates. Prehelminthosporol was also analyzed in a number of related Bipolaris and Drechslera species. In addition to B. sorokiniana, three out of six Bipolaris species (B. setariae, B. zeicola, B. victoriae) produced prehelminthosporol, which indicates that ability to produce prehelminthosporol is conserved among closely-related Bipolaris species.     

Involvement of Iron and Ferritin in the Potato–Phytophthora infestans Interaction

This work shows that the infection of potato (Solanum tuberosum) detached leaves by the late blight pathogen Phytophthora infestans, was drastically reduced by adding deferoxamine, an exogenous iron chelator. Reactive oxygen species in leaves inoculated with P. infestans were also reduced after adding deferoxamine. A leaf ferritin cDNA fragment was obtained by PCR and used as probe for screening a tuber cDNA library. A cDNA (named StF1) encoding the iron-storing potato ferritin was cloned. StF1 is 915 bp in length and has an open reading frame of 230 amino acids that contains the information for the mature 28 kDa subunit of potato ferritin. StF1 was used as probe in northern blot hybridizations to analyze expression of the ferritin gene. In leaves, ferritin mRNA accumulated in response to pathogen attack. In tubers, ferritin mRNA increased upon treatment with the elicitor eicosapentaenoic acid. These results suggest that iron plays a role in the potato-P. infestans interaction.     

Variability of Cuban and International Populations of Alternaria solani from Different Hosts and Localities: AFLP Genetic Analysis

As causal agent of early blight disease in tomato and potato, Alternaria solani is an internationally important horticultural pathogen. Genetic variability was surveyed by amplified fragment length polymorphism analysis in a total of 112 isolates from potato and tomato, representing pathogen populations from different Cuban provinces together with isolates from the USA, Brazil, Turkey, Greece and China. Also included in the analysis were isolates from catenulated Alternaria spp. from Brazil, Canada, Greece and Russia, along with single isolates of Alternaria porri, Alternaria alternata and a Curvularia sp. UPGMA clustering revealed a differentiation between the isolates of A. solani and all other species with the exception of A. porri which could not be distinguished from A. solani. Among the isolates of A. solani, two distinct subclusters were formed, with high genetic significance revealed by bootstrapping, corresponding to a general subdivision based on the respective solanaceous host. The results are discussed with regard to potential host specificity of A. solani on tomato and potato, and in terms of the comparative contributions of regional and international genetic variability in populations of this ubiquitous plant pathogen.     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Effect of crop rotation on the incidence of soil-borne fungal diseases of potato

Effects of crop rotation on the incidence of soil-borne pathogens and on the performance of potato were investigated in five field experiments. Rotations differed in cropping frequency of potato and in crop sequence.Incidence of stem canker caused byRhizoctonia solani was strongly influenced by the cropping frequency of potato and not by crops with which the potato was alternated in the rotation. Cropping frequency of potato also affected the occurrence of black scurf, but less pronounced than for stem canker. The antagonistVerticillium bigutatum slightly reducedR. solani (black scurf) in plots on sandy soil continuously cropped with potato. Incidence of stem canker was also strongly affected by granular nematicides applied to the soil, nitrogen level and the cultivar grown.     

Evaluation of antagonistic bacteria for suppression of bacterial ring rot of potato

Bacterial strains with potential for biological control of bacterial ring rot of potato caused byClavibacter michiganensis subsp.sepedonicus were isolated from the surface of potato tubers. Eighty-eight potential biocontrol candidates, selected on the basis ofin vitro antibiosis toC. m. sepedonicus, produced inhibition zones with radii ranging from 0.5 to 16 mm on test plates. All antagonistic isolates were screened in the greenhouse for biocontrol activity on micropropagated potato plantlets root-inoculated withC. m. sepedonicus. Eight strains consistently prevented infection of plantlets but there was no significant correlation between the width of the inhibition zone in thein vitro assay and ring rot suppression in the plant bioassay. Three strains that showed a high level of biological control potential were identified as a saprophytic enteric bacterium (strain 7G), anArthrobacter sp. (strain 16C), and a soil coryneform bacterium (strain 18A). These were tested in a field plot by co-inoculating cut seed potato tubers withC. m. sepedonicus and antagonists. Strains 7G and 18A significantly increased plant stand whereas 16C decreased disease incidence. The relative number of ostensibly ring rot-free progeny tubers was generally greater when antagonists were present.     

Phosphite compounds reduce disease severity in potato seed tubers and foliage

Phosphites (Phi) are alkali metal salts of phosphorous acid, with the ability to protect plants against different pathogens. In this research, the effect of Phi applied to potato plants on severity of three important potato diseases in Argentina was assessed. Seed tubers and foliage of potato cvs Shepody and Kennebec were treated with Phi to assess effects on resistance against Phytophthora infestans, Fusarium solani and Rhizoctonia solani. Protection resulting from Phi treatment in seed tubers was high against P. infestans, intermediate against F. solani, and low against R. solani. In addition, seed tubers treated with calcium or potassium phosphites (CaPhi and KPhi, respectively) at 1% of commercial product emerged earlier than untreated ones. When Phi were foliarly applied two or four times at different doses, high levels of protection against P. infestans were achieved in both cultivars. Higher protection was observed in Kennebec when CaPhi was applied, while in Shepody this was true for KPhi. Expression of β-1,3-glucanases was induced at different times after treatment but no correlation between β-1,3-glucanases expression and foliar protection level was found. On the other hand, Phi positive protection effects did not produce negative effects in plant growth. Leaves from CaPhi-treated plants showed a darker green colour than leaves from control plants; also an increase in Rubisco protein and a delay in crop senescence was observed.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Occurrence of the A2 mating type and oospores ofPhytophthora Infestans in potato crops in Israel

Seventeen metalaxyl-sensitive and 21 metalaxyl-resistant isolates ofPhytophthora infestans collected from blighted potato fields during the years 1983–1988 were tested for mating type on rye seed agar medium. All isolates except one (MS3, collected in 1986 at Sufa, in the western Negev) were found to belong to the A2 mating type. A2 isolates produced oospores within 2 weeks when cultured together with isolate 163 (A₁ from the U.S.A.) or with the A₁ isolate MS3 from Israel. When cultured singly, A₂ isolates produced some oospores within 4–8 weeks. Blighted potato tubers harvested from potato crops artificially inoculated with a mixture of A1+A2 sporangia were found to contain some oospores. No oospores were detected in blighted tubers harvested from A₂ + A₂ inoculated crops. It was concluded that the A2 mating type ofP. infestans has occurred in Israel since 1983 or even earlier. The rare occurrence of the A1 mating type was unexpected and indicated that sexual reproduction of the fungus in the country might be limited.     

Specific inhibition of spore germination of <Emphasis Type="Italic">Alternaria brassicicola</Emphasis> by fistupyrone from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TP-A0569

Fistupyrone (FP), a metabolite from Streptomyces sp. TP-A0569, inhibited the in vivo infection of Chinese cabbage seedlings by Alternaria brassicicola. To detect the possible action sites of FP, the effect of FP on the infection behavior of A. brassicicola and A. alternata was investigated. When spores of A. brassicicola were suspended in FP solution and inoculated on host leaves, FP at 0.1ppm significantly inhibited spore germination, appressorial formation, and infection hypha formation of A. brassicicola. Host-specific AB-toxin production and lesion formation by A. brassicicola spores were also reduced significantly by treatment with FP 1ppm. The effect of FP seemed to be irreversible because significant washing of FP-treated spores with distilled water (DW) did not change the inhibitory effects. In contrast, A. alternata isolates such as Japanese pear pathotype, apple pathotype, and saprophyte behaved almost equally in both FP- and DW-treated spores. Mycelial dry weight in potato dextrose broth and mycelial diameters on potato dextrose agar, gelatin glucose agar, and Czapek solution agar of both A. brassicicola and A. alternata were not different with or without addition of FP. These results indicate that FP at low concentrations has a fungicidal effect on spores of A. brassicicola but not on spores of A. alternata; FP also does not affect the vegetative phase of these fungi.     

Density—yield relationships for Colorado potato beetle adults on potatoes

A model composed of two straight lines describes the relation between overwintering spring adults of Colorado potato beetle [Leptinotarsa decemlineata (Say)] and potato yield. Potato vines (Solarium tuberosum (L.), cv. ‘Superior’) had a damage boundary of around 5.8 spring adults per plant. However, under conditions favorable for reproduction, overwintering adults of 0.07 beetles per plant would produce a large population of first-generation larvae that would reduce considerably the potato yield if not controlled. There is no appreciable seasonal overlapping between adults and young larvae in the field. Monitoring activities should therefore be directed at the beginning of the season against overwintering adults and later against first-generation larvae. For summer adults, an action threshold of 10 per plant is proposed.     

Genetic Structure of the Population of Phytophthora infestans Attacking Solanum ochranthum in the Highlands of Ecuador

Thirty-nine isolates of Phytophthora infestans were collected from the wild host Solanum ochranthum in the highland tropics of Ecuador and characterized with a set of phenotypic and molecular markers (mating type, metalaxyl sensitivity, the allozyme loci Gpi, and Pep, mitochondrial DNA haplotype, RFLP, and SSR), as well as for pathogenicity on various hosts. Three groups of isolates (A, B, and C) were identified based on their multilocus genotypes and variable abilities to cause disease on different hosts. Group A had a combination of alleles for the Gpi (86/100), Pep (96/100) and mtDNA (Ia) loci, as well as an RFLP fingerprint, that have not been reported for P. infestans in Ecuador, or elsewhere. Group B shares many marker characteristics with the US-1 lineage described in Ecuador on tomato, pear melon (S. muricatum), and S. caripense, but has SSR alleles not present in typical US-1 isolates. Group C for all markers tested is identical to the EC-1 lineage described on cultivated and wild potatoes in Ecuador. All isolates from S. ochranthum were able to re-infect their host of origin in the detached leaf assay; however, we did not draw clear conclusions as to the relative aggressiveness of the three groups on this host. Isolates of group A were the most specialized and were generally non-pathogenic or weakly pathogenic on all hosts other than S. ochranthum. Groups B and C infected tuber-bearing hosts, including the cultivated potato but were generally non-pathogenic on other non-tuber bearing hosts. Solanum ochranthum was infected by isolates coming from tuber-bearing Solanum hosts (i.e., the EC-1 lineage of P. infestans) and some US-1 isolates from non-tuber bearing hosts. Thus, in nature this species might be a potential reservoir of inoculum of different pathogen populations able to infect the cultivated hosts potato, tomato and pear melon (S.␣muricatum). Unlike potato and tomato in Ecuador, each of which is primarily attacked by a highly specialized pathogen population, S. ochranthum appears to harbour at least three pathogen groups of␣different genetic make-up. The unresolved issue of potential host specificity in isolates found on S.␣ochranthum could complicate efforts to use this species in tomato improvement.     

An Aspartic Protease With Antimicrobial Activity is Induced after Infection and Wounding in Intercellular Fluids of Potato Tubers

Aspartic proteases (APs) one of the main proteinase classes, have different physiological functions in animals, fungi and viruses. In plants, knowledge of the biological roles of APs is less well developed. An AP has been purified from potato tuber and leaves (Guevara et al., 1999, 2001). In this paper, the changes in the level of AP in response to infection by Phytophthora infestans (P. infestans) and wounding were studied in intercellular washing fluids (IWFs) from tuber disks of two potato cultivars differing in their susceptibility to P. infestans. A differential induction was observed between both cultivars: in the resistant cultivar, induction was higher and faster in infected tissues than in wounded ones. In the susceptible cultivar, a lower and later accumulation was observed than in the resistant cultivar. In addition, AP had a direct inhibitory effect on the germination of cysts of P. infestans and conidia of Fusarium solani. The pattern of accumulation and in vitro activity of AP suggest that this enzyme may have a role in the defense response of potato.