ofPhytophthora Infestans Research Articles

Large subclonal variation in Phytophthora infestans populations associated with Ecuadorian potato landraces

The population of Phytophthora infestans on potato landraces in three provinces (Carchi, Chimborazo and Loja) of Ecuador was analysed. All isolates (n = 66) were of the A1 mating type. Simple sequence repeats (SSR) were used to assess the genetic diversity of the isolates. The P. infestans isolates from the potato landraces grouped in a single clade together with reference isolates belonging to the clonal lineage EC‐1. In the 66 SSR profiles obtained, 31 multilocus genotypes were identified. The 66 isolates constituted 49 different races according to the Solanum demissum differential set ( R1 to R11). The P. infestans population was complex and virulent on 4 to 11 R genes. Analysis showed that the subclonal variation in the Ecuadorian EC‐1 clone is increasing over time and is much larger than clonal variation in lineages in the Netherlands and Nicaragua, suggesting high mutation rates and little or no selection in Ecuador.

An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

The 74-Kilodalton Immunodominant Antigen of the Pathogenic Oomycete Pythium insidiosum Is a Putative Exo-1,3-ß-Glucanase

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

Endophytic Colonization of Potato ( Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte, Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

A Phytophthora sojae G-Protein α Subunit Is Involved in Chemotaxis to Soybean Isoflavones

For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein alpha subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.

Post-harvest applications of zoxamide and phosphite for control of potato tuber rots caused by oomycetes at harvest

Potato storage tuber rots caused by the late blight and pink rot pathogens at harvest can cause severe economic losses, warranting the need for effective post-harvest fungicide applications. The purpose of this study was to evaluate the efficacy of select post-harvest fungicides in reducing tuber infections by the late blight and pink rot pathogens when applied at various post-inoculation time intervals. ‘Russet Burbank’ potatoes were inoculated by submersion in an aqueous suspension ofPhytophthora infestans orPhytophthora erythroseptica zoospores at 0, 1, 2, 4, and 6 h prior to receiving a post-harvest treatment. Products evaluated were zoxamide (various rates and formulation), phosphite (335 g a.i./MT), and a hydrogen peroxide/peroxyacetic acid mixture (HPPA, 9 g a.i./MT), all applied at 2.08 L/MT of tubers as a low pressure spray prior to storage. Zoxamide and phosphite significantly reduced late blight and pink rot incidence and severity when applied immediately after inoculation. HPPA was less effective at controlling disease development. Phosphite was effective at reducing late blight development at all time intervals up to 6 h post-inoculation (7% vs 80% in untreated). Zoxamide appeared to have good post-harvest disease control if applied soon after inoculation. The maximum time intervals between inoculation and treatment where significant reductions in pink rot incidence were observed was 0 h for HPPA (28%), 2 h for zoxamide (55%; 64 g a.i./MT) and 6 h for phosphite (13%) compared to the untreated (73%). Phosphite provided consistent disease control even when applied several h after inoculation and has potential to be a reliable post-harvest fungicide for the potato industry.

Foliar resistance toPhytophthora infestans (Mont.) de Bary (US-8) in 13 Mexican and South AmericanSolarium species having EBNs of 1, 2, and 4 and implications for breeding

The recent US-8 clonal lineage ofPhytophthora infestans, the pathogen causing late blight (LB) disease, is one of the most widespread and difficult to control. Sources of foliar resistance to US-8 were evaluated in 1927 seedling genotypes by sampling 49 plant introductions (PIs) representing 13 Mexican and South AmericanSolanum species having an Endosperm Balance Number (EBN) of 1, 2, or 4, and one species with an unknown EBN. Species evaluated were 2x(1EBN)S. bulbocastanum, S. cardiophyllum, S. commersonii, S. pinnatisectum, andS. trifidum; 2x(2EBN)S. berthaultii, S. megistacrolobum, S. microdontum, andS. verrucosum; 4x(2EBN)S. fendleri andS. stoloniferum; 6x(4EBN)S. guerreroense;2x unknown EBNS. polyadenium; as well as4x S. tuberosum families as controls. Foliar resistance in the field was monitored in 1998 and 1999. Resistance differed relative to species geographic origin, EBN, species themselves, PIs within a species, and genotypes within a PI. In 1998 Mexican germplasm had a lower area under the disease progress curve (AUDPC, 1140) and greater resistance than South American germplasm (1601), while the 1EBN (1119) and unknown EBN (1075) species had greater resistance than 2EBN (1470) and 4EBN (1888) species. AUDPC ranged among species from 555 (S. cardiophyllum) to 1932 (S. megistacrolobum) and among PIs within a species most widely forS. bulbocastanum (267–1007) and least widely forS.polyadenium (1019–1179). In 1999 LB symptoms were not observed on the species before an early freeze; however, all 1999S. tuberosum families showed LB symptoms and segregated for resistance, pointing to greater resistance in the species than in cultivated germplasm. Resistance found in diploid 2EBN germplasm is more accessible due to greater crossability with cultivated germplasm; however, though more difficult to introgress, resistance found in some of the more reproductively isolated 1EBN germplasm is worth pursuing.

Sites of entry ofphytophthora infestans (Mont.) de Bary into potato tubers in assessment of tuber blight resistance

During assessment of tuber blight resistance using three test methods, the sites of entry ofPhytophthora infestans into the tuber was estimated for 41 cultivars. The trials were conducted using a mix of three highly virulent isolates at Gros Lusewitz 1991–1993. The influence of date of harvest and intermediate storage before inoculation was examined with two cultivars in 1993. Two levels of soil moisture and five densities of inoculum were tested in 1994. Methods of assessment of tuber blight resistance, cultivars, years and all their interactions showed a significant effect on sites of entry. Eye infections identified the highest the level of resistance and correlated best with tuber blight resistance in net bag test, r=-0.58. Soil moisture and temperature before harvest varied the ontogenic development of resistance and the relation of sites of entry of the pathogen from July to September. Intermediate storage changed the resistance and the preferred site of cultivar-specific infection. Interactions cultivar x soil moisture and cultivar x density of inoculum were significant and should be considered in the resistance assessment of cultivars.

Oospores associated with tomato seed may lead to seedborne transmission ofPhytophthora infestans

Tomato fruits at the green mature stage were inoculated with a mixed sporangial suspension of A1 and A2 isolates ofPhytophthora infestans. Other fruits were inoculated with either A1 or A2 sporangia. Seeds were extracted from the blighted fruits and sown in soil or on agar media to test for the transmission of late blight to the emerging seedlings. Only 23 (0.09%) of approximately 25,000 seedlings developed symptoms. All blighted seedlings originated from fruits inoculated with mixed A1 + A2 sporangia. Isolates ofP. infestans recovered from the emerging blighted seedlings were seemingly of oosporic origin, as they differed phenotypically (mating type, virulence, sensitivity to metalaxyl) from the parent isolates used to inoculate the fruits. The results suggest that transmission ofP. infestans might occur by seeds extracted from fruits carrying oospores and less probably by seeds extracted from fruits having no oospores.

Thermal properties of overwintered piles of cull potatoes

Annual epidemics of late blight of potato have lead to debate as to the relative importance of overwintering sources of inoculum. Host availability is a key factor for overwinter survival ofPhytophthora infestans and the initiation of epidemics of potato late blight. Temperature within discarded piles of cull potatoes may influence tuber tissue temperature and therefore affect survival of meristematic tissue. Consequently the risk of initiation of an epidemic of late blight from cull piles is closely related to the thermal experience of overwintered potato culls. Temperature monitoring of cull piles over two years indicated that temperature was stable in the interior of the piles regardless of cull pile size (1–15 ton) or year. The possible use of ambient temperature information gathered during winter months in potato-growing regions may not help estimate the risk of an epidemic of late blight initiated from cull piles as, although highly correlated with the surface temperature of cull piles, ambient temperature was not correlated with the temperature within the cull piles. The presence of cull piles in excess of 1 ton may enhance the survival of the host and thus the development ofP. infestans from infected tubers.

Survey ofPhytophthora infestans populations in Serbia: Mating type, metalaxyl resistance and virulence properties

A total of 37 single-lesion isolates ofPhytophthora infestans were collected during 2000 from different potato growing regions in the central part of Serbia. They were paired in Al and A2 matings to determine their mating types. Of these 37 isolates, 28 were found to belong to the A2 mating type, eight to the Al type and one was found to be self-fertile (A1/A2). Using the in vitro mycelial radial growth method no metalaxyl resistantP. infestans isolate could be found. The rate of occurrence of intermediate isolates was not high, on the average 2.70%. All other isolates were sensitive to metalaxyl. There was no correlation between mating type and the intermediate metalaxyl resistance found.

Survival of detached sporangia ofPhytophthora infestans exposed to ambient, relatively dry atmospheric conditions

The effect of duration of exposure, daily weather conditions, and exposure to direct or indirect sunlight on survival of sporangia ofPhytophthora infestans under ambient, relatively dry atmospheric conditions was evaluated. Viability of sporangia was assessed by determining the proportion of potato tuber slices or leaflets that became infected after inoculation with exposed sporangia. The maximum survival time of sporangia was 24 h under moderately cool (mean of 15 C) and dry conditions (RH < 25%). Sky conditions were sunny during daylight hours during this exposure. Infection seldom occurred when sporangia were exposed directly to sunlight; only 25 of 566 (4.4%) groups of sporangia caused infection. Of these 25 groups, 23 (92%) had been exposed to mean temperatures below 33 C for 4 or fewer hours. Binary logistic regression analysis of the data showed that duration of exposure, direct sunlight, and type of day were all significant factors affecting survival of sporangia. Long exposure in direct sunlight limited survival. Survival of sporangia was more likely to occur on cool, cloudy days and cool, rainy days than on other day-types.

Characterization ofPhytophthora infestans from Maine during 1999 and 2000

A total of 77 isolates ofPhytophthora infestans was collected in 1999 and 93 isolates were collected in 2000 from growers’ fields, home gardens, and research plots in Maine. Genotype diversity based onglucose-6-phosphate isomerase (Gpi) allozyme analysis was greater in the collections from 2000, with six genotypes identified vs two genotypes identified in 1999. The US-8 (Gpi 100/111/122) genotype constituted 88% of the isolates collected in 1999 and 75% of those collected in 2000. The US-14 (Gpi 100/122) genotype constituted 12% and 7% of the isolates collected in 1999 and 2000, respectively. Three newGpi genotypes constituted 7% of the isolates collected from potato in northern Maine in 2000. A new genotype (A1 mating type,Gpi 100/100) that was aggressive on both tomato and potato was identified from home gardens in western Maine and constituted 11% of the isolates collected in 2000. A1, A2, and A1A2 mating types as well as self-fertile isolates were collected in both years. Self-fertility in artificial culture appeared to be relatively common in field isolatesof P. infestans. Potential origins of the new genotypes are discussed.

Comparative analysis of quantitative trait loci for foliage resistance toPhytophthora infestans in tuber-bearingSolanum species

Previously published data for potato foliage resistance to late blight were examined in a combined analysis to determine how the genetic backgrounds of the host and pathogen affect the occurrence of resistance QTLs. Data from 19 diploid populations and one tetraploid population that originated from at least 12 different tuber-bearingSolanum species were included. Comparative analysis across all populations revealed three highly active genomic regions on the distal parts of chromosomes 3,4, and 5. The region most consistently detected that conferred foliage resistance inSolanum was located on chromosome 5, near marker locus GP21. A previously identified cluster of three race-specific R-genes on chromosome 11 was not associated with polygenic resistance. Statistical examination of active QTLs indicates that congruence among QTLs is significantly affected by both genetic relatedness of mapping populations and race ofPhytophthora infestans used for resistance tests. The proportion of congruent QTLs for late blight resistance decreased from 0.52 detected in relatedSolanum populations tested with the same race ofPhytophthora infestans to 0.20 found in unrelated mapping populations tested with different races ofPhytophthora infestans. Analysis of resistance mapping tests provided statistical evidence for the occurrence of race-specific QTLs in tuber-bearingSolanum. The highly conserved genomic regions identified in the comparative analysis are likely to be good candidates for gene cloning or marker assisted selection in potato breeding programs.

Two decades of monitoring irish populations ofPhytophthora infestans for phenylamide resistance

Between 1981 and 1998,Phytophthora infestans populations in 878 potato crops in Ireland were monitored for phenylamide resistance. The incidence of resistance was very high in 1981 following intensive use of metalaxyl for blight control from 1977 onwards. It decreased rapidly in the absence of phenylamide use between 1981 and 1984. Following the reintroduction of phenylamide based fungicides in 1985, there was a rapid increase in phenylamide resistance following the widespread curative use of these fungicides. From 1990 onwards there was greater adherence among growers to the use of phenylamides in accordance with an antiresistance strategy and resistance distribution again decreased. During the 18-year period of the study, resistance was always lower at the beginning of the season and increased as the season progressed. Following the reintroduction of the phenylamide based fungicides in 1985 there was a steady increase in phenylamide use up to 1986 followed by a continuous decrease in use up to 1990. Since then there has been a steady increase in phenylamide use. They are currently used on some 60% of crops in Ireland and are still providing acceptable levels of disease control.

Phenotypes ofPhytophthora infestans in Nepal: mating types and metalaxyl sensitivity

Potato late blight has appeared in epidemic proportions in Nepal since the mid 1990s and fungicides have been reported to be decreasingly effective in managing the disease.Phytophthora infestans isolates were collected from potato crops during 1999–2000 and analysis of 371 isolates for mating types and 270 isolates for metalaxyl sensitivity revealed the presence of both new and old populations. This is the first report on the presence of metalaxyl-resistant isolates in Nepal. The frequencies of A1 and A2 were 83 and 17%, respectively. Metalaxyl-resistant, intermediate and sensitive isolates were recorded as 10%, 12% and 78% respectively. Metalaxyl resistance was distributed in both mating types. Sites with a history of metalaxyl use had a significantly higher number of resistant and intermediate isolates ({ie337-1}, P<0.01) than sites where metalaxyl had not been used. This study confirms the changes in population structure ofP. infestans in Nepal.

Jacqueline Lee: A late-blight-resistant tablestock variety

Jacqueline Lee has a bright golden skin, yellow flesh, attractive oval shape, and excellent cooking qualities that make it suitable for tablestock use. In addition, it has been determined to have a high level of foliar resistance to the US-8 genotype ofPhytophthora infestans (Mont.) de Bary under Michigan field and greenhouse conditions. It is a full-season variety with vine maturity similar to Snowden. Jacqueline Lee has high yield potential. It has a high set (14–16 tubers/hill) of medium sized tubers that leads to 65%–80% marketable yield. Tubers are low in internal and external defects. It has a scab reaction similar to Atlantic, and the tubers have been observed to have a long dormancy.

Race diversity and virulence complexity ofPhytophthora infestans in Nepal

A total of 251Phytophthora infestans isolates collected during 1999–2000 potato growing seasons from nine potato growing areas of Nepal were tested for virulence using a set of eleven potato differentials. The virulence test revealed the presence of 30 races with the most frequent being 4.11, 4 and 11. Twenty-nine percent of isolates belonged to simple races having none to one virulence factor, whereas 71% had complex virulence factors governed by 2 to 7 genes. Gleason and Shannon indices of 5.24 and 2.59, respectively, showed a moderate level of diversity in NepaleseP. infestans populations. Shannon indices comparison among regional populations revealed significant differences (t-test, P<0.001) in the diversity. The population from hills were the most diverse followed byterai and the high hill populations. The virulence complexities were minimal in Nepalese populations. The implications of these findings in association with the potato cultivation system in Nepal are discussed.

Relationships between pathotypes and RAPDs, Gpi-allozyme patterns, mating types, and resistance to metalaxyl ofPhytophthora infestans in Canada in 1997

Relationships between pathotypes and other markers such as mating types, metalaxyl resistance, Gpi-allozyme and random amplified polymorphic DNA (RAPD) patterns, were investigated among 33 isolates ofPhytophthora infestans obtained from both potato and tomato across Canada in 1997. Pathogenicity testings, based on virulence against potato differentials carrying single-resistance genes and the size of lesions on leaves, revealed higher pathogenic diversity in 1997 than in the few past years, with more pathotypes and more race-complex isolates. A higher pathogenic diversity was observed within A2 (US-8) than within A1 (US-11) isolates. There was no correlation between the level of resistance to metalaxyl of an isolate and its pathogenicity. Nevertheless, when grouped into metalaxyl-sensitive (MS), -intermediate (MI), and -resistant (MR) classes, both average pathotype complexity and average lesion size were lowest for MS group. RAPD data revealed a good distinction between A1 (US-11) and A2 (US-8) mating type groups, and between isolates from British Columbia and those from other provinces. The detection in the last few years of both A1 and A2 mating types in Canada and the occurrence in 1997 of some isolates sharing different characteristics of both of these two groups suggest that sexual reproduction may have occurred locally.

Production of phytoalexins, glycoalkaloids and phenolics in leaves and tubers of potato cultivars with different degrees of field resistance after infection withPhytophthora infestans

The kinetics of accumulation of phytoalexins, glycoalkaloids and phenolics was studied in two potato cultivars differing in their degrees of field resistance when infected withPhytophthora infestans. Tuber slices and leaves of cvs Pampeana INTA (high degree of field resistance, free of R genes) and Bintje (susceptible) were infected with race C (complex race 1, 3, 5, 7, 11) ofPhytophthora infestans. Phytoalexins and phenolics accumulated in tuber and leaf tissues which had been inoculated. The levels of these compounds in the susceptible cv. Bintje were relatively low and similar to those found before inoculation. Leaves of cv. Pampeana INTA had a very high glycoalkaloid content, suggesting that glycoalkaloids may play a role in protection of leaves against the fungus. However, we could find no correlation between resistance and glycoalkaloid content of tubers. Our results suggest a major role of phytoalexins, phenolics and glycoalkaloids in the complex mechanisms of field resistance.