Comparison of direct-RT-PCR and dot-blot hybridization for the detection of Potato spindle tuber viroid in natural host plant species

Potato spindle tuber viroid (PSTVd) is an EPPO A2-listed quarantine pathogen and its detection in large scale surveys requires complex decision schemes. In this study, a simple and rapid application of direct-RT-PCR was evaluated together with dot blot hybridization for the detection of PSTVd in dormant potato tubers harvested from primary infected plants, as well as in tomato and solanaceous ornamental plants. In all infected dormant potato tubers tested, both direct-RT-PCR and dot blot hybridization detected two different PSTVd isolates, with direct-RT-PCR being ten times more sensitive than dot blot. Similarly, in infected tomato and Brugmansia spp., PSTVd was detected by direct-RT-PCR with higher sensitivity compared to that of dot blot hybridization. However, in Brugmansia spp., a ten-fold decrease of the typical working concentration of the sap was required for an unequivocal detection of the viroid by direct-RT-PCR. The potential to use direct-RT-PCR for routine PSTVd examination is discussed.     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

An outbreak of Potato spindle tuber viroid in tomato is linked to imported seed

In 2011, an outbreak of the quarantine-regulated pathogen Potato spindle tuber viroid (PSTVd) occurred in a commercial glasshouse-grown tomato crop in Queensland, Australia. Phylogenetic studies showed that the genotype of this isolate grouped in a cluster of PSTVd genotypes from tomato and Physalis peruviana, and exhibited an interesting mutation (U₂₅₇→A) that has previously been linked to lethal symptom expression in tomato. Transmission studies showed that the viroid could be mechanically transmitted from crushed fruit sap, but not from undamaged fruits. A low rate of asymptomatic infection was determined for plants in the affected glasshouse, demonstrating the efficacy of using symptoms to detect PSTVd infections in tomato. No PSTVd infections were detected in solanaceous weeds located outside of the infected glasshouse, excluding them from playing a role in the viroid epidemiology. Monitoring and subsequent testing of new tomato crops grown in the facility demonstrated successful eradication of the pathogen. A trace-back analysis linked the outbreak of PSTVd to an infected imported tomato seed-lot, indicating that PSTVd is transmitted internationally through contaminated seed.     

Occurrence and molecular variability of Potato spindle tuber viroid and Tomato apical stunt viroid in ornamental plants in Croatia

Viroids of the genus Pospiviroid are able to induce diseases in a wide range of host plants including important crop species. Although occasional disease outbreaks of Potato spindle tuber viroid (PSTVd) and closely related pospiviroids have been reported in potato and tomato, recent studies found an increase in number of latent infections in ornamental solanaceous species. In order to verify the presence of PSTVd and other pospiviroids in Croatia, a survey was conducted between 2009 and 2012. A total of 182 samples belonging to five ornamental species and two solanaceous crops were analyzed. Eight plants belonging to two different species (Solanum jasminodes and Lycianthes rantonnetii) were found infected by PSTVd and, in addition, one S. jasminoides plant infected by Tomato apical stunt viroid (TASVd). Viroid infection was confirmed by mechanical inoculation on tomato plants to observe symptom expression. Molecular characterization of the isolates was done and complete viroid sequences were submitted to the GenBank. This is the first evidence of the presence of PSTVd and TASVd and their variability in Croatia.     

Mechanical transmission of <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> between plants of <Emphasis Type="Italic">Brugmansia suaveoles,Solanum jasminoides</Emphasis> and potatoes and tomatoes

Potato spindle tuber viroid (PSTVd) has been recently found in many solanaceous ornamental plant species. This study reports on the effectiveness of mechanical transmission between Brugmansia suaveolens, Solanum jasminoides, potato and tomato. Inoculation with ‘infected’ plant sap diluted in water, rubbing with contaminated finger tips and cutting with contaminated razor blades all resulted in transmission of PSTVd. Temperature, plant species and source of inoculum were found to be critical factors. An average temperature of 15°C only resulted in a few infections, whereas transmission at 20 and 25°C was more successful. Tomatoes were more susceptible to PSTVd than B. suaveolens, S. jasminoides and potatoes. Furthermore, S. jasminoides was a better source of inoculum than B. suaveolens. No transmission was obtained after repeated addition of inocula to tomato roots. These results indicate that PSTVd can be transmitted between plant species in practice by crop handling.     

Selection of virulent populations of Meloidogyne javanica by repeated cultivation of Mi resistance gene tomato rootstocks under field conditions

Field trials were conducted in a plastic house artificially infested with an avirulent population of Meloidogyne javanica to determine the durability of the resistance mediated by the Mi gene in tomato rootstocks after repeated cultivation for three consecutive years. Treatments included an experimental rootstock cv. PG76 ( Solanum lycopersicum  ×  Solanum sp.), a commercial rootstock cv. Brigeor ( S. lycopersicum  ×  S. habrochaites ), a resistant tomato cv. Monika ( S. lycopersicum ), and a susceptible cv. Durinta ( S. lycopersicum ). Based on the reproduction index (RI: number of eggs per g root on the resistant cultivar divided by number of eggs per g root on the susceptible cultivar × 100), rootstock cv. PG76 responded as highly resistant (RI = 7%) after the first cropping cycle (3·4 nematode generations), showed intermediated resistance (RI = 33%) after the second cropping cycle (3·3 generations), and was fully susceptible (RI = 94%) after the third cycle (3·3 generations). In contrast, rootstock cv. Brigeor and resistant cv. Monika retained intermediate resistance levels (RI = 41 and 25%, respectively) after the third cropping cycle. Virulent nematode populations were rapidly selected from an avirulent one after repeated cultivation of resistant tomatoes under field conditions. Bioassays conducted under controlled conditions confirmed that selection for virulence occurred more rapidly in plots with cv. PG76 followed by Brigeor and Monika. The nematode population in the field not exposed to Mi resistance remained avirulent to Mi genotypes. The genetic background of the resistant rootstocks and the frequency of cropping were critical factors for the appearance of virulent nematode populations. Irrespective of nematode infection, all resistant tomatoes yielded more than the susceptible cultivar.     

马铃薯纺锤块茎类病毒的检测和防治

马铃薯纺锤块茎类病毒病(potato spindle tuber viroid,PSTVd)是一种严重为害马铃薯生产的病害,降低产量20%—30%。防治的主要措施是应用无类病毒的种薯。由于目前还没有脱掉类病毒的有效措施,只能从未被饱和侵染的群体中鉴定筛选出未被侵染的个体,再脱掉其它病毒,作为核心繁殖材料。1987年以来,利用自制的电泳设备,以往复聚丙烯酰胺凝胶电泳法(return-polyacrylamide gel electrophoresis,R-PAGE)检测类病毒,筛选出未感病的个体,再用茎尖组织培养法脱掉其它病毒。经用马铃薯卷叶病毒等8种病毒酶标抗体鉴定筛选,获得既无类病毒也无主要马铃薯病毒的克新1、2、3和4号等主栽马铃薯品种的核心种。并已提供给省内外的良种场繁殖推广。1989和1990年抽样检测克山良种场繁殖的原种、一级和二级良种,未检测到类病毒。     

Potato germplasm poses the highest risk of introducing potato spindle tuber viroid in potatoes in the Netherlands: analysis and evaluation of an outbreak in potato breeding

In 2014, potato spindle tuber viroid (PSTVd) was identified in a potato clone originating from a breeding company in the Netherlands. This clone was submitted for micro propagation and therefore tested for PSTVd and a number of other pathogens. This finding of PSTVd initiated actions to track and eradicate the infections. In addition to the finding at the breeding company, PSTVd was also found at a research institute. At both locations the viroid was eradicated following extended testing and discarding of infected plants. Additional surveys including testing of each individual plant in all crossing glasshouses and random samples of pre-basic and basic seed potatoes, revealed no further infections in the Netherlands. This result concurred with the fact that mechanical spread of PSTVd in the field is not likely under climatic conditions in the Netherlands. Therefore, vegetative propagation seems the most important pathway for maintaining and spreading of PSTVd. Based on the evaluation of this outbreak, it was concluded that potato germplasm poses the highest risk of introducing this viroid in potatoes in the Netherlands.     

Genetic diversity and host differentiation among isolates of Phytophthora infestans from cultivated potato and wild solanaceous hosts in Peru

To test the hypothesis that isolates of Phytophthora infestans attacking wild Solanaceae exhibit specialization for particular host species, 115 isolates of P. infestans were collected from cultivated potatoes, nontuber-bearing Solanum spp. of the Basarthrum section and wild tomatoes from five departments in the northern and central highlands of Peru, and characterized using several neutral markers. All isolates belonged to one of four clonal lineages described previously in Peru: EC-1, US-1, PE-3 and PE-7. There was a strong association of three lineages with host species: PE-3 was only isolated from cultivated potato, while PE-7 and US-1 were only isolated from nontuber-bearing Solanum spp. ( Basarthrum section and wild tomatoes). EC-1 was isolated from all host groups sampled. A subset ( n  = 74) of the isolates was evaluated for metalaxyl resistance. High levels of resistance were found almost exclusively in EC-1 and PE-3, while US-1 and PE-7 isolates were generally sensitive. In a detached-leaf assay for lesion diameter using five EC-1 isolates from S. caripense and seven EC-1 isolates from cultivated potato, there was a significant interaction between isolate origin and inoculated host, caused by higher aggressiveness of EC-1 from cultivated potato on its host of origin. In a comparison of EC-1 (seven isolates from cultivated potato) and US-1 (three isolates from S. caripense ), each pathogen lineage was more aggressive on its original host species, causing a highly significant interaction between isolate origin and inoculated host. Wild tomatoes and nontuber-bearing Solanum spp. harbour several pathogen lineages in Peru and could serve as reservoirs of inoculum that might contribute to epidemics on potato or tomato. Potential risks associated with the use of wild Solanum hosts as sources of resistance to P. infestans are discussed .     

Stability of resistance to Phytophthora infestans in potato: an international evaluation

Ten institutions in nine countries joined together to test the stability of resistance of 14 potato genotypes to the oomycete pathogen Phytophthora infestans in three separate trials. Seven of the genotypes were tested in one trial involving seven locations, and all 14 were tested in two subsequent trials, each involving eight locations. Stability of resistance was tested with nonparametric tests and with an additive main effects and multiplicative interaction (AMMI) model. Overall, resistance to P. infestans was robust; resistant genotypes were consistently resistant in all locations and trials. The nonparametric analysis indicated that specific genotypes were basically stable across sites for resistance. In trial 3, the Z statistic for overall stability was significant at 0·05%, indicating a significant level of interaction across the trial, but there were no significant interactions for specific genotypes in this trial. The genotype by environment (G × E) effect of the AMMI model was highly significant in both trials, but the mean square of G × E was less than 10% of the genotype effect in each trial. The first two principal components (PCA1 and PCA2) of the AMMI analyses together explained 75 and 80% of the interaction effects in trials 2 and 3, respectively. Based on both nonparametric and AMMI analyses, Ecuador and Argentina were locations of relatively high interaction effects for both trials 2 and 3, although in Ecuador this interaction was not associated with any particular potato genotype. Other locations also had high interaction effects, but these occurred in only one trial. The genotypes Chata Blanca and, to a lesser extent, Torridon were relatively unstable in trials 2 and 3, but in the case of Torridon, resistant, this did not represent a significant loss of resistance.     

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. Its flexuous rod-shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.     

Aggressiveness and host specificity of Brazilian isolates of Phytophthora infestans

The population of Phytophthora infestans in Brazil consists of two clonal lineages, US-1 associated with tomatoes and BR-1 associated with potatoes. To assess whether host specificity in these lineages resulted from differences in aggressiveness to potato and tomato, six aggressiveness-related epidemiological components – infection frequency (IF), incubation period (IP), latent period (LP), lesion area (LA), lesion expansion rate (LER) and sporulation at several lesion ages (SSLA) – were measured on detached leaflets of late blight-susceptible potato and tomato plants. Infection frequency of US-1 was similar on potato and tomato leaflets, but IF of BR-1 was somewhat reduced on tomato. Incubation period was longer on both hosts with US-1, although this apparent lineage affect was not significant. Overall there was no host effect on IP. On potato, BR-1 had a shorter LP (110·3 h) and a larger LA (6·5 cm²) than US-1 (LP = 162·0 h; LA = 2·8 cm²). The highest LER resulted when isolates of BR-1 (0·121 cm² h⁻¹) and US-1 (0·053 cm² h⁻¹) were inoculated on potato and tomato leaflets, respectively. The highest values of the area under the sporulation capacity curve (AUSC) were obtained for isolates of US-1 inoculated on tomato leaflets (6146) and for isolates of BR-1 on potato leaflets (3775). In general, higher values of LA, LER, SSLA and AUSC, and shorter values of LP were measured when isolates of a clonal lineage were inoculated on their original host than with the opposite combinations. There is evidence that there are quantitative differences in aggressiveness components between isolates of US-1 and BR-1 clonal lineages that probably contribute to host specificity of P. infestans populations in Brazil.     

Selection for reproductive ability in Globodera pallida populations in relation to quantitative resistance from Solanum vernei and S. tuberosum ssp. andigena CPC2802

Four field populations of the nematode Globodera pallida were subjected to selection pressure for increased reproductive ability by rearing sub-populations continuously on four partially resistant potato genotypes for 12 generations. The resistance was derived from either Solanum vernei or from S. tuberosum spp. andigena CPC2802. After the 12th generation the original and sub-populations of nematodes were assessed for their reproductive ability on a susceptible genotype and on each of the partially resistant genotypes. Selection pressure was shown to have increased reproductive ability but the increases were specific to the source of resistance used. The average increase on the ex S. vernei clones was from 11% reproduction by the unselected populations to 35·5% reproduction after selection. On the clones derived from CPC2802, which had higher levels of resistance, the increases were larger with an average of 6·6% reproduction for the unselected but 47·4% reproduction after selection. The response to selection differed amongst the initial field populations with some rates of reproduction increased to as much as 79%. A RAPD based analysis of the original and sub-populations after selection indicated small but consistent changes in the genetic structure, which could have been a result of the selection pressure per se and/or the bottlenecks that the populations had gone though.     

Molecular and biological characterization of a severe isolate of <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis> containing a novel terminal right (T<Subscript>R</Subscript>) domain sequence

Tomato chlorotic dwarf viroid (TCDVd) manually inoculated to transgenic (cv.‘Desiree’) potato plants containing antimicrobial cationic peptides failed to develop symptoms in above ground plant parts, but infected tubers were symptomatic. Plants from the infected tubers (second generation plants) emerged as either severely stunted (bushy stunt isolate, BSI) or tall and symptomless. Molecular characterization of BSI isolates showed TCDVd sequence variants 95 to 98% identical to TCDVd sequences from the database, while a viroid variant identical to TCDVd type isolate (acc # AF162131) was cloned from symptomless plants. The TCDVd BSI variants had novel U165C, GU177-178AA, and UCAC181-184CUUU nucleotide substitutions in the terminal right (T_R) domain of the viroid molecule. The cloned viroid cDNAs of the BSI were infectious to experimental (cv. ‘Sheyenne’) tomato plants causing stunted plants with profuse auxiliary shoots. Visual evaluation of the susceptibility of the BSI to 18 potato and 21 tomato cultivars revealed severe symptoms in most cultivars of both species. The progeny variants accumulating in each potato and tomato cultivar exhibited the same novel T_R domain in most cultivars, with only a slight variation in a few. The severity of the stunting symptoms induced by TCDVd from BSI isolates in both potato and tomato cultivars has not been noted previously with other TCDVd isolates and, as such, it is proposed that this new isolate be recognized as a distinct genotype. Emergence of this type of sequence variant in commercial fields or commercial tomato greenhouses could potentially cause relevant losses in both crops.     

PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10² and 1 × 10³ colony forming units (CFU) mL⁻¹ and detection sensitivity was increased to as few as 2–4 CFU mL⁻¹ after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco

The survival and half-life of Clavibacter michiganensis ssp. michiganensis ( C. michiganensis ), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Aït Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Aït Melloul and Melk Zhar. The half-life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half-life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half-life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Aït Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half-life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.     

Characterization of Potato spindle tuber viroid (PSTVd) incidence and new variants from ornamentals

We analyzed the spreading and persistence of PSTVd variants in several ornamentals in the territory of the Czech Republic. The pool of PSTVd variants detected in Solanum jasminoides, S. muricatum, Datura sp. and Brugmansia sp. was biolistically transferred to Matricaria chamomilla, Argyranthemum frutescens and Diascia sp., species which we found as sensitive hosts for PSTVd from ornamentals. The PSTVd pool showed sequence changes and increased variation after its transfer to potato, suggesting a wide adaptation potential of PSTVd in this crop. Potato exhibited genotype-dependent leaf and spindle tuber symptoms, when inoculated with the sap from S. jasminoides infected with the predominant and sequence-stable PSTVd-S1.     

Quantitative relationships between inoculum of Melampsora larici-epitea and corresponding disease on Salix

Six Salix clones were inoculated with urediniospores of four isolates of Melampsora larici-epitea at five inoculum levels using a leaf-disc method. Disease reactions were recorded using a digital camera; the number and size of uredinia were examined using image analysis software; and spore yield per leaf disc was measured. In three Salix / Melampsora combinations, S.  ×  mollissima 'Q83'/Q1 (LET4); S. viminalis '78183'/V1 (LET1); and S.  ×  stipularis /V1, pustule numbers increased as inoculum density became higher. In the remainder, S. viminalis 'Mullatin'/V1; S.  ×  calodendron /DB (LET3); and S. burjatica 'Korso'/K (LR1), pustule numbers initially increased, then decreased as inoculum densities exceeded 140–360 spores per disc. Calculated infection efficiency ranged from 0·11 to 0·20 on the three willows inoculated with V1: 0·16–0·68 for S.  ×  calodendron /DB; 0·20–0·55 for 'Q83'/Q1; and 0·07–0·48 for Korso/K. In single-spore inoculations, up to 10% of spores produced single uredinia. Infection efficiency increased sharply between inoculum densities of 1–40 spores per leaf disc. Spore yield was more closely correlated to pustule area (accounting for 61·2% variance for the combined data) than to the number of pustules (42·7% variance). For spore yields in relation to pustule numbers, clone-specific individual lines having different intercepts and slopes fitted significantly better than either a single line for all the tested willows, or parallel lines fitted to each clone ( P  < 0·001). For spore yields in relation to pustule area, clone-specific individual parallel lines were significantly better than a single line ( P  < 0·001).     

A Novel Population of Phytophthora, Similar to P. infestans, Attacks Wild Solanum Species in Ecuador

ABSTRACT Twenty-six isolates of a Phytophthora population from two wild solanaceous species, Solanum tetrapetalum (n 11) and S. brevifolium (n = 15), were characterized morphologically, with genetic and phenotypic markers, and for pathogenicity on potato and tomato. Based on morphology, ribosomal internal transcribed spacer region 2 (ITS2) sequence, and pathogenicity, all isolates closely resembled P. infestans and were tentatively placed in that species. Nonetheless, this population of Phytophthora is novel. Its primary host is neither potato nor tomato, and all isolates had three restriction fragment length polymorphism (RFLP) bands (probe RG57) and a mitochondrial DNA haplotype that have not been reported for P. infestans. All the isolates were the A2 mating type when tested with a P. infestans A1 isolate. The A2 mating type has not been found among isolates of P. infestans from potato or tomato in Ecuador. Geographical substructing of the Ecuadorian A2 population was detected. The three isolates from the village of Nono, identical to the others in all other aspects, differed by three RFLP bands; those from Nono lacked bands 10 and 16, but possessed band 19. Most of the Ecuadorian A2 isolates were nonpathogenic on potato and tomato, but a few caused very small lesions with sparse sporulation on necrotic tissue. Cluster analysis of multilocus genotypes (RFLP, mating type, and two allozymes) dissociated this A2 population from genotypes representing clonally propagated populations of P. infestans worldwide. The current hypotheses for the historical global movements of P. infestans do not satisfactorily explain the origin or possible time of introduction into Ecuador of this A2 population. Assuming the population is P. infestans, its presence in Ecuador suggests either a hitherto unreported migration of the pathogen or an indigenous population that had not previously been detected.