Oospore Production of Phytophthora infestans in Potato and Tomato Leaves

ABSTRACT Fungal, host, and environmental factors affecting sexual reproduction of Phytophthora infestans in planta were studied. Intact and detached leaves were coinoculated with sporangia of various combinations of A(1) and A(2) mating-type isolates; leaves were incubated under various conditions, and oospore production was estimated microscopically within whole, clarified leaflets. Some A(1) + A(2) isolate combinations were more reproductive than others, whereas some potato genotypes better supported oospore formation than others. Tomato usually supported more oospore formation than potato. To induce oospore formation, A(1) and A(2) sporangia were usually mixed at a 1:1 ratio. Ratios of 1:19 to 19:1, however, also allowed abundant production of oospores. Optimal temperatures for sexual sporulation ranged from 8 to 15 degrees C, but oospores also were produced at 23 degrees C. Oogonia developed 5 to 6 days after sporangial coinoculation, and oospores developed after 8 to 10 days. Light had little effect on oospore formation in both tomato and potato leaves provided that initial lesions were established under photoperiodic conditions. Although A1 and A(2) sporangia usually were mixed before inoculation on leaves to obtain oospores, we found that discrete A(1) and A(2) lesions produced on opposite sides of the midvein of tomato leaves also induced oospore formation in the midvein and adjacent tissues. Oospores also formed when the two halves of the leaves were cut and separated at 3 days after sporangial coinoculation, which corresponded with the appearance of late blight lesions. The continuous supply of moisture to infected leaves was essential to oospore production. No oospores or oogonia formed in severely diseased plants kept at 50 to 80% relative humidity. Such plants did allow some oospore formation when kept continuously wet for 2 weeks in plastic boxes or tents. Detached leaves floated on water supported the highest sexual sporulation. Under optimal conditions of wetness and temperature, as many as 100 oospores per mm(2) of tissue were observed.     

Population Genetic Structure of Phytophthora infestans in Ecuador

ABSTRACT The population genetic structure of Phytophthora infestans in Ecuador was assessed from 101 isolates collected from 1990 to 1992 and 111 isolates collected in 1993. All isolates were analyzed for mating type and allozyme genotype. Both samples were dominated (>95%) by a clonal lineage (EC-1) defined from neutral markers: 90/100 genotype for glucose-6-phosphate isomerase, 96/100 genotype for peptidase, A1 mating type, and a previously unreported nuclear DNA fingerprint. The remaining isolates belonged to the US-1 clonal lineage, which has a worldwide distribution. Isolates in the 1993 sample were analyzed for virulence and metalaxyl sensitivity. All representatives of EC-1 had complex patho-types, with three pathotypes representing >60% of the collection. There was variation for metalaxyl sensitivity. There was no evidence for geographical substructuring on the basis of neutral markers, but there was evidence for limited substructuring based on metalaxyl sensitivity and specific virulence. We hypothesize that EC-1 has been recently introduced to Ecuador.     

Aggressiveness and Competitive Fitness of Phytophthora infestans Isolates Collected from Potato and Tomato in France

ABSTRACT To test the hypothesis that host-related differences in the genotypic composition of populations of the late blight pathogen Phytophthora infestans can be explained by differential pathogenicity, the aggressiveness of isolates of the pathogen collected in France from potato and tomato was measured on detached leaflets of potato (cv. Bintje) and tomato (cv. Marmande). A preliminary trial with four isolates (two each from potato and tomato) showed that lesion appearance and development were similar for each isolate in detached leaflets and in whole plant tests in growth cabinets. Isolates collected from tomato were more pathogenic to tomato than isolates collected from potato. This was particularly the case for isolates belonging to the A2 mating type. Isolates originating from potato had a higher infection efficiency and a higher sporulation capacity on this host, but they induced lesions that generally spread more slowly than those caused by isolates from tomato. Extensive variation for components of aggressiveness on potato, and to a lesser extent on tomato, was observed in collections of isolates from each of the two hosts. Competition experiments between one potato isolate and one tomato isolate in field plots of the susceptible potato cv. Bintje clearly demonstrated the higher competitive fitness of the potato isolate on its host of origin. Therefore, differential pathogenicity to potato and tomato certainly contributes to the differentiation between P. infestans populations present on potato and tomato in France; however, additional factors, possibly related to survival ability or random genetic drift, are probably also involved and may explain the persistence of weakly pathogenic isolates in these populations.     

Phytophthora infestans Populations from Tomato and Potato in North Carolina Differ in Genetic Diversity and Structure

ABSTRACT Phytophthora infestans causes a destructive disease on tomato and potato. In North Carolina (NC) potatoes are mostly grown in the east, whereas tomatoes are grown in the mountainous areas in the western part of the state. Five genotypes of P. infestans were identified from 93 and 157 isolates collected from tomato and potato over a 5 year period between 1993 and 1998. All isolates collected from potato in eastern NC were the US-8 genotype, whereas only a single isolate was the US-1 genotype. Tuber blight was found on immature daughter tubers in a single field in 1997, however infection on mature tubers was not observed. Within potato fields, a range of sensitivity to metalaxyl was observed among isolates but all were either intermediate or highly resistant to the fungicide. In contrast, isolates from tomatoes included previously reported US-7 and US-8 genotypes and two new genotypes called US-18 and US-19 (A2 mating type, allozyme genotype Gpi 100/100 and Pep 92/100). These genotypes had unique restriction fragment length polymorphism banding patterns, were sensitive to metalaxyl, and have not been reported elsewhere. All genotypes, with the exception of the US-1, were the Ia mitochondrial haplotype. Thus, isolates of P. infestans from tomato were more genetically diverse over time in NC than those from potato and include two new genotypes that are sensitive to metalaxyl.     

Oospore Formation by Phytophthora infestans in Potato Tubers

ABSTRACT The ability of Phytophthora infestans, the causal agent of potato and tomato late blight, to produce oospores in potato tuber tissue was studied in the field and under laboratory conditions. In 1998 and 2000 field experiments, the canopy of potato cvs. Alpha and Mondial, respectively, were coinoculated with A1 + A2 sporangia of the fungus, and the infected tubers collected at harvest were examined for the presence of oospores. In 1998, only 2 of 90 infected tubers had oospores, whereas none of the 90 tubers examined in 2000 had any oospores. In the latter experiment, infected tubers kept in storage up to 12 weeks after harvest had no oospores. Artificial co-inoculations of whole tubers with A1 + A2 sporangia resulted only rarely in the formation of oospores inside the tubers. Co-inoculations of potato tuber discs taken from dormant tubers 0 to 16 weeks after harvest failed to support any oospore production, whereas discs taken from sprouting tubers of >/=18 weeks after harvest allowed oospores to form. Tuber discs showed enhanced oospore formation when treated before inoculation with either sugars, amino acids, casein hydrolysate, beta-sitosterol, or chloroethylphosphonic acid. In contrast, reducing airflow into the petri dishes where potato tuber discs were incubated reduced the number of oospores produced. The number of oospores produced in tuber tissue was lower compared with that in leaf tissue regardless of the origin of isolates used. The data show that the ability of Phytophthora infestans to produce oospores in potato tuber tissue is very limited and increases with tuber aging.     

Phytophthora infestans Produces Oospores in Fruits and Seeds of Tomato

ABSTRACT Tomato fruits at the mature green stage coinoculated with A1 + A2 sporangia of Phytophthora infestans, the late blight causal fungus, showed abundant oospores in the vascular tissues, pericarp, columella, and placenta. Oospores were also formed on the surface of fruits kept in moisture-saturated atmosphere. Occasionally, oospores were enclosed between the epidermal hairs of the seed coat. In a few seeds, oospores were detected inside the embryo. The data suggest that blighted tomato fruits may carry a large number of oospores, thus making them a threatening source of blight inoculum. Such fruits may also release airborne oosporic inoculum that may introduce recombinant genotypes within a growing season. Although Phytophthora infestans is seedborne in tomato, to our knowledge, this is the first report on the occurrence of oospores in tomato seeds. Whether such tomato seeds produce blighted seedlings remains to be shown.     

Tomato late blight (Phytophthora infestans) in Uganda

A survey on the tomato late blight situation and current practices for disease management was carried out in Uganda using an informal structured questionnaire approach. Ten districts from different agroclimatic zones were selected for the survey. Phytophthora infestans isolates from tomatoes were obtained from the zones and only the A1 mating type was recovered. Tomato cultivation is practised year-round. The major commercial varieties grown, Moneymaker, Marglobe, Heinz and Roma, were susceptible to late blight. Late blight and bacterial wilt ( Pseudomonas solanacearum ) were the major diseases, with bollworm ( Helicoverpa armigera ) and thrips ( Thrips spp .) the major insect pests. Fungicides, mostly mancozeb, were the predominant control method used by farmers. Sanitation is practised by some farmers, but only in association with fungicides, to control late blight. Plant nutrition is not perceived as playing a major role in late blight management. Problems associated with pesticides and poor marketability were identified as major constraints to tomato production in Uganda.     

Temporal and Spatial Patterns of Genetic Structure of Phytophthora infestans from Tomato and Potato in the Del Fuerte Valley

ABSTRACT The temporal and spatial patterns of Phytophthora infestans population genetic structure were analyzed in the Del Fuerte Valley, Sinaloa, Mexico, during the crop seasons of 1994 to 1995, 1995 to 1996, and 1996 to 1997 by geographical information systems. Isolates of P. infestans were obtained from infected tissue of tomato and potato collected from two areas: (i) where both potatoes and tomatoes are grown, and (ii) where only tomatoes are grown. The isolates were characterized by mating type, allozymes at the glucose-6-phosphate isomerase and peptidase loci, restriction fragment length polymorphism (RFLP) fingerprint with probe RG57, metalaxyl sensitivity, and aggressiveness to tomato and potato. The results suggest presence of an asexual population with frequent immigrations from outside the valley. There was a shift of mating type in the population from predominantly A2 to completely A1 in this period. The co-occurrence of mating types was restricted to very few fields in the area around Los Mochis where tomato and potato crops are grown. Genotype variation based on allozyme analysis and mating type was low with only one genotype affecting both crops each year. The genotypes affecting both crops were the only genotypes highly aggressive to both tomato and potato in laboratory aggressiveness tests and the only genotypes widespread on both the tomato and potato crops in the valley each year. These predominant genotypes were highly resistant to the fungicide metalaxyl. Data on metalaxyl sensitivity indicate that allozyme analysis can discriminate between sensitive and resistant isolates in the Del Fuerte Valley. RFLP analysis with the probe RG57 gives further discrimination of genotypes within an allozyme genotype. In the 1995 to 1996 season, four different RFLP genotypes were found within an allozyme genotype. However, there were five other dilocus allozyme genotypes that could not be further split by RFLP analysis in 1995 to 1996 and 1996 to 1997 seasons. Spatial analysis of genotypes suggests that each season individual fields near Los Mochis became infected with one or more genotypes, but only a single genotype, aggressive on both potato and tomato, occurred south and east to the Guasave area.     

Phytophthora infestans Secretes Extracellular Proteases with Necrosis Inducing Activity on Potato

The proteolytic activity present in the extracellular preparation (ECP) from suspension media of infective structures of the late blight pathogen Phytophthora infestans was partially characterized. The proteolytic activity was analyzed in gelatin-containing SDS-PAGE. A discrete band of digested gelatin was visualized at approximately 45 kDa in ECPs from zoospores and germinating cysts media. Treatment of ECP with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) or incubation at 100°C for 5 min completely abolished the proteolytic activity in the zymograph assay. When microinjected in potato leaves, ECP induce localized necrosis within 24 h post inoculation. This necrosis appeared in potato and was not visible in two non-host plants. Moreover, the necrosis seems to be dependent on active host metabolism. Treatments of ECP with Proteinase K, PMSF and boiling inhibited their ability to induce the necrotic response. These results suggest a correlation between, proteolytic and necrosis-inducing activities in ECP. A preliminary characterization with protease inhibitors suggests that the ECPs contain serine protease(s).     

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.     

Aggressiveness of Phytophthora infestans and phenotypic analysis of resistance in wild Petota accessions in Ecuador

The aggressiveness of four Phytophthora infestans isolates collected from wild and cultivated potato species (sect. Petota ) and the level of resistance of nine Petota species were assessed in the highland tropics of Ecuador. For this, isolates of P. infestans were inoculated on whole plants of Petota species in the field and net house and six epidemiological components – infection frequency (IF), incubation period (IP), latent period (LP), lesion size (LS), lesion growth rate (LGR), and relative area under the lesion expansion curve (RAULEC) – were measured during a single infection cycle. Additionally, host specificity was determined by testing for a significant host by pathogen interaction using the same components. The results showed significant differences among isolates of the EC-1 clonal lineage for IP, IF, and RAULEC. Significant differences among isolates were not found for the other components measured. There were significant differences in resistance among the accessions of Petota hosts tested. RAULEC, LGR, LP, and LS were in general more adequate in differentiating among the more resistant and more susceptible accessions but the importance of each component varied with host species. There was slight and inconsistent evidence for the existence of host specificity in some isolates of Petota hosts. IP was the only component for which a significant host by isolate interaction was observed and in most cases the isolates had the greatest aggressiveness on their hosts of origin.     

Potential of sexual reproduction among host-adapted populations of Phytophthora infestans sensu lato in Ecuador

To determine the potential of sexual reproduction among host-adapted populations of Phytophthora infestans sensu lato in Ecuador, 13 A1 isolates belonging to clonal lineages US-1, EC-1 and EC-3, and 11 A2 isolates belonging to the clonal lineage EC-2, were paired on agar plates to induce crossing. In the first experiment, six A1 isolates (three US-1, two EC-1 and one EC-3) were each crossed with three A2 isolates (total = 18 crosses). Matings involving isolates of the EC-1 lineage produced more oospores of healthy appearance than did matings with isolates of US-1 or EC-3. In the second experiment, the oospores of 35 crosses (21 EC-1 × EC-2; 10 US-1 × EC-2; four EC-3 × EC-2) were dispersed on water agar to assess oospore germination. Overall, germination percentages were low. Only one cross produced enough progeny for evaluation. Twenty-three single-oospore offspring were isolated and evaluated for mating type; electrophoretic patterns of glucose-6-phosphate isomerase ( Gpi ) and peptidase ( Pep ) alloenzyme loci; mitochondrial DNA haplotype; and genomic DNA fingerprint. Multilocus genotype data indicated that all 23 isolates resulted from meiotic recombination. Four progeny with homothallic phenotype appeared to be unstable heterokaryons. Markers at several loci segregated according to simple Mendelian expectations for a diploid organism, but the ratios of three RFLP loci and the Pep locus were not consistent with Mendelian expectations. All progeny were nonpathogenic on hosts of the parental genotypes. Reduced mating success and reduced pathogenic fitness of progeny appear to be postmating mechanisms of reproductive isolation in populations of P. infestans sensu lato in Ecuador.     

Spatial Analysis of Phytophthora infestans Genotypes and Late Blight Severity on Tomato and Potato in the Del Fuerte Valley Using Geostatistics and Geographic Information Systems

ABSTRACT Genetic structure of Phytophthora infestans, the causal agent of potato and tomato late blight, was analyzed spatially in a mixed potato and tomato production area in the Del Fuerte Valley, Sinaloa, Mexico. Isolates of P. infestans were characterized by mating type, allozyme analysis at the glucose-6-phosphate isomerase and peptidase loci, restriction fragment length polymorphism with probe RG57, metalaxyl sensitivity, and aggressiveness to tomato and potato. Spatial patterns of P. infestans genotypes were analyzed by geographical information systems and geo-statistics during the seasons of 1994-95, 1995-96, and 1996-97. Spatial analysis of the genetic structure of P. infestans indicates that geographic substructuring of this pathogen occurs in this area. Maps displaying the probabilities of occurrence of mating types and genotypes of P. infestans, and of disease severity at a regional scale, were presented. Some genotypes that exhibited differences in epidemiologically important features such as metalaxyl sensitivity and aggressiveness to tomato and potato had a restricted spread and were localized in isolated areas. Analysis of late blight severity showed recurring patterns, such as the earliest onset of the disease in the area where both potato and tomato were growing, strengthening the hypothesis that infected potato tubers are the main source of primary inoculum. The information that geostatistical analysis provides might help improve management programs for late blight in the Del Fuerte Valley.     

Foliar Aggressiveness of Phytophthora infestans in Three Potato Growing Regions in the Netherlands

Thirty-six isolates of Phytophthora infestans originating from three different potato growing regions in the Netherlands were tested for their aggressiveness to leaves of potato cultivar Bintje under controlled conditions. Measurements of latent period, maximal growth rate, infection efficiency and sporulation intensity were made and a composite aggressiveness index was calculated. Large variation in aggressiveness was present among isolates for each regional P. infestans population studied. The three populations differed significantly in latent period, but not for maximal growth rate and infection efficiency. Phenotypic variation existed for all components of aggressiveness and the aggressiveness index in P. infestans from each regional source. No association was found between mating type and aggressiveness. It is concluded that high levels of variation for aggressiveness are being generated and maintained through sexual reproduction in P. infestans strains from regional potato growing practices.     

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.     

Quantifying the Rate of Release and Escape of Phytophthora infestans Sporangia from a Potato Canopy

ABSTRACT A means for determining the rate of release, Q (spores per square meter per second), of spores from a source of inoculum is paramount for quantifying their further dispersal and the potential spread of disease. Values of Q were obtained for Phytophthora infestans sporangia released from an area source of diseased plants in a potato canopy by comparing the concentrations of airborne sporangia measured at several heights above the source, with the concentrations predicted by a Lagrangian Stochastic simulation model. An independent estimate of Q was obtained by quantifying the number of sporangia per unit area of source at the beginning of each sampling day by harvesting diseased plant tissue and enumerating sporangia from these samples. This standing spore crop was the potential number of sporangia released per area of source during the day. The standing spore crop was apportioned into time segments corresponding to sporangia concentration measurement periods using the time trace of sporangia sampled above the source by a Burkard continuous suction spore sampler. This apportionment of the standing spore crop yielded potential release rates that were compared with modeled release rates. The two independent estimates of Q were highly correlated (P = 0.003), indicating that the model has utility for predicting release rates for P. infestans sporangia and the spread of disease between fields.     

Adaptation of Phytophthora infestans to Partial Resistance in Potato: Evidence from French and Moroccan Populations

ABSTRACT The use of partially resistant cultivars should become an essential component of a sustainable management strategy of potato late blight, caused by Phytophthora infestans. It is therefore important to determine to what extent P. infestans populations can be selected for increased aggressiveness by potato cultivars with different levels of partial resistance. To this end, we sampled P. infestans populations from France and Morocco, chosen as locations where late blight occurs regularly but which differ in the distribution of potato cultivars. Cross-inoculation experiments were used to determine the aggressiveness of all populations to potato cvs. Bintje (prevalent in France but not grown in Morocco) and Désirée (popular in Morocco but cultivated to a very small extent in France). French populations were more aggressive on cv. Bintje than on cv. Désirée, irrespective of the site they were sampled from. Their aggressiveness increased between early and late samplings, suggesting that both cultivars selected for increased aggressiveness during epidemics. By contrast, Moroccan populations were more aggressive on Désirée, regarded as partially resistant in Europe, than on Bintje, highly susceptible under European conditions. These data indicate that P. infestans populations adapt to locally dominant cultivars, irrespective of their resistance levels, and can therefore overcome polygenic, partial resistance. This adaptive pattern may render partial resistance nondurable if not properly managed.