Best Practices for Population Genetic Analyses.

Population genetic analysis is a powerful tool to understand how pathogens emerge and adapt. However, determining the genetic structure of populations requires complex knowledge on a range of subtle skills that are often not explicitly stated in book chapters or review articles on population genetics. What is a good sampling strategy? How many isolates should I sample? How do I include positive and negative controls in my molecular assays? What marker system should I use? This review will attempt to address many of these practical questions that are often not readily answered from reading books or reviews on the topic, but emerge from discussions with colleagues and from practical experience. A further complication for microbial or pathogen populations is the frequent observation of clonality or partial clonality. Clonality invariably makes analyses of population data difficult because many assumptions underlying the theory from which analysis methods were derived are often violated. This review provides practical guidance on how to navigate through the complex web of data analyses of pathogens that may violate typical population genetics assumptions. We also provide resources and examples for analysis in the R programming environment.

Genomic Analyses of Dominant U.S. Clonal Lineages of Phytophthora infestans Reveals a Shared Common Ancestry for Clonal Lineages US11 and US18 and a Lack of Recently Shared Ancestry Among All Other U.S. Lineages.

Populations of the potato and tomato late-blight pathogen Phytophthora infestans are well known for emerging as novel clonal lineages. These successions of dominant clones have historically been named US1 through US24, in order of appearance, since their first characterization using molecular markers. Hypothetically, these lineages can emerge through divergence from other U.S. lineages, recombination among lineages, or as novel, independent lineages originating outside the United States. We tested for the presence of phylogenetic relationships among U.S. lineages using a population of 31 whole-genome sequences, including dominant U.S. clonal lineages as well as available samples from global populations. We analyzed ancestry of the whole mitochondrial genome and samples of nuclear loci, including supercontigs 1.1 and 1.5 as well as several previously characterized coding regions. We found support for a shared ancestry among lineages US11 and US18 from the mitochondrial genome as well as from one nuclear haplotype on each supercontig analyzed. The other nuclear haplotype from each sample assorted independently, indicating an independent ancestry. We found no support for emergence of any other of the U.S. lineages from a common ancestor shared with the other U.S. lineages. Each of the U.S. clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Spatial and Temporal Analysis of Populations of the Sudden Oak Death Pathogen in Oregon Forests.

Sudden oak death caused by the oomycete Phytophthora ramorum was first discovered in California toward the end of the 20th century and subsequently emerged on tanoak forests in Oregon before its first detection in 2001 by aerial surveys. The Oregon Department of Forestry has since monitored the epidemic and sampled symptomatic tanoak trees from 2001 to the present. Populations sampled over this period were genotyped using microsatellites and studied to infer the population genetic history. To date, only the NA1 clonal lineage is established in this region, although three lineages exist on the North American west coast. The original introduction into the Joe Hall area eventually spread to several regions: mostly north but also east and southwest. A new introduction into Hunter Creek appears to correspond to a second introduction not clustering with the early introduction. Our data are best explained by both introductions originating from nursery populations in California or Oregon and resulting from two distinct introduction events. Continued vigilance and eradication of nursery populations of P. ramorum are important to avoid further emergence and potential introduction of other clonal lineages.

Five Reasons to Consider Phytophthora infestans a Reemerging Pathogen.

Phytophthora infestans has been a named pathogen for well over 150 years and yet it continues to "emerge", with thousands of articles published each year on it and the late blight disease that it causes. This review explores five attributes of this oomycete pathogen that maintain this constant attention. First, the historical tragedy associated with this disease (Irish potato famine) causes many people to be fascinated with the pathogen. Current technology now enables investigators to answer some questions of historical significance. Second, the devastation caused by the pathogen continues to appear in surprising new locations or with surprising new intensity. Third, populations of P. infestans worldwide are in flux, with changes that have major implications to disease management. Fourth, the genomics revolution has enabled investigators to make tremendous progress in terms of understanding the molecular biology (especially the pathogenicity) of P. infestans. Fifth, there remain many compelling unanswered questions.

Diversity of Foliar Phytophthora Species on Rhododendron in Oregon Nurseries.

The genus Phytophthora contains some of the most notorious plant pathogens affecting nursery crops. Given the recent emergence of the sudden oak death pathogen Phytophthora ramorum, particularly in association with Rhododendron spp., characterization of Phytophthora communities associated with this host in nursery environments is prudent. Many taxa may present symptoms similar to P. ramorum but we do not necessarily know their identity, frequency, and importance. Here, we present a survey of Phytophthora taxa observed from seven nurseries in the U.S. state of Oregon. Incidence and diversity of Phytophthora communities differed significantly among nurseries and among seasons within nursery. The taxa P. syringae and P. plurivora were widespread and detected at most of the nurseries sampled. Nine other taxa were also detected but were found either in a single nursery or were shared among only a few nurseries. Characterization of the Phytophthora communities present in nurseries is an important step toward understanding the ecology of these organisms as well as an aid to nursery managers in determining what risks may be present when symptomatic plants are observed. This study builds on an increasing literature, which characterizes Phytophthora community structure in nurseries.

Lineage, Temperature, and Host Species have Interacting Effects on Lesion Development in Phytophthora ramorum.

There are four recognized clonal lineages of the pathogen Phytophthora ramorum. The two major lineages present in North America are NA1 and NA2. With a few exceptions, NA1 is found in natural forest ecosystems and nurseries, and NA2 is generally restricted to nurseries. Isolates from the NA1 and NA2 lineages were used to infect rhododendron, camellia, and California bay laurel in detached leaf assays to study the effects of lineage, temperature, and host on pathogenicity and host susceptibility. Isolates within both lineages were highly variable in their ability to form lesions on each host. There was also a tendency toward reduced lesion size in successive trials, suggesting degeneration of isolates over time. Temperature had a significant effect on lesion size, with a response that varied depending on the host and isolate. Phenotypic differences between lineages appear to be heavily influenced by the representation of isolates used, host, and temperature. The importance of temperature, host, and lineage are discussed with respect to disease management, as well as future range expansions and migrations of the pathogen.

Evidence for multiple introductions and clonality in Spanish populations of Fusarium circinatum.

Fusarium circinatum is thought to have been moved around the world with pine planting stock consisting, most probably, of infected seed. In this effort, we investigate the genetic structure of F. circinatum in Spain and globally. In total, 223 isolates were studied from five regions in northern Spain and eight countries. Eight microsatellite markers revealed 66 multilocus genotypes (MLGs). Minimum spanning network analysis of MLGs by region within Spain as well as globally, discriminant analysis of principal components, and analysis of molecular variance revealed that Spanish populations are significantly differentiated and structured into two distinct groups, each one including one of the dominant genotypes observed. This result suggests that two independent introductions occurred into Spain that subsequently underwent clonal divergence and admixture. This result is further supported by the linkage disequilibrium and clonality observed for F. circinatum populations in northern Spain. The maintenance of differentiation between the clusters could result from the lack of or rare sexual reproduction in Spain. Possible introduction pathways from other countries and subsequent routes of dispersion of F. circinatum in Spain are discussed.

First Report of Camphor Tree (Cinnamomum camphora) as a Host of Phytophthora ramorum.

Cinnamomum camphora (Lauraceae) is an evergreen shade tree grown in many parts of the United States, including California. From 2007 to 2011, an arborist working in a residential neighborhood in Mill Valley (Marin Co.) noticed several camphor trees with branch dieback and decline. Affected trees had patchy, irregular cankers on the branches and shoot blight. Cankers were black and most had horizontal fissures. Cankers were most abundant in the inside and lower portions of the canopies. In 2011, samples sent to Bartlett Tree Laboratory tested positive for Phytophthora sp. using the Agdia ELISA Phytophthora kit (Agdia, Elkhart, IN). In February 2009 and April 2011, camphor leaf samples were collected by Sacramento Co. inspectors during an annual nursery inspection for Phytophthora ramorum and submitted to CDFA. The normally bright green leaves were reddish with small necrotic spots surrounded by green halos. Camphor samples from Marin Co. were also collected and sent to CDFA in September 2011. An organism with coralloid coenocytic hyphae, chlamydospores, and ellipsoidal semi-papillate sporangia grew on CMA-PARP (4) from both Marin and Sacramento Co. samples. Morphologically, it matched the description of P. ramorum (3). rDNA sequences of the internal transcribed spacer (ITS) region of the Marin (GenBank KC473521) and Sacramento (KC473522) isolates, amplified using primers ITS1 and ITS4 (4), were 100% identical to P. ramorum by a BLAST query (AY038058). Microsatellite loci placed the Marin isolate in the NA1 clonal lineage, while the Sacramento isolate belonged to the NA2 lineage (2). Pathogenicity of both isolates was tested on 5 trees grown in 18.93-liter pots. Three leaves on each tree were inoculated with 6-mm agar plugs taken from the margin of 7-day-old cultures grown on V8 juice agar (V8). Leaves were wounded with a sterile pushpin and two colonized plugs of each isolate were covered with a freezer tube cap filled with sterile dH2O and attached to the leaves with a pin-curl clip (4). Three branches of the same plants were wounded and inoculated with a 3-mm colonized agar plug for each isolate and secured with Parafilm. An equal number of leaves and stems were treated with uncolonized V8 plugs as controls. Plants were sprayed with dH2O, covered in large plastic bags, and placed in a growth chamber at 18°C. After 4 days, the bags, caps, and plugs were removed from the leaves. Black lesions were seen 7 days after inoculation on most leaves and 10 to 14 days on inoculated branches. After 32 days, P. ramorum was isolated from leaf lesions and canker margins onto CMA-PARP. No Phytophthora spp. grew from the controls. The experiment was repeated once with similar results. Overall, leaf and stem lesions were larger with the NA2 lineage isolate than the NA1 lineage isolate, which is consistent with previous research (1). Leaf abscission was seen in 30% of the leaves inoculated with the NA2 lineage isolate but none of the NA1 or control leaves. To our knowledge, this is the first report of P. ramorum on camphor in nursery and landscape settings. Mill Valley is known for its mild temperatures and abundant summer fog. Optimal weather conditions likely led to the spread of P. ramorum from infected neighboring forest hosts to camphor in Mill Valley, rather than from an introduction of infected nursery plants. References: (1) E. Elliott et al. For. Pathol. 41:7, 2011. (2) E. M. Goss et al. Phytopathology 101:166, 2011. (3) S. Werres et al. Mycol. Res. 105:1155, 2001. (4) L. E. Yakabe et al. Plant Dis. 93:883, 2009.

The 2009 Late Blight Pandemic in the Eastern United States - Causes and Results.

The tomato late blight pandemic of 2009 made late blight into a household term in much of the eastern United States. Many home gardeners and many organic producers lost most if not all of their tomato crop, and their experiences were reported in the mainstream press. Some CSAs (Community Supported Agriculture) could not provide tomatoes to their members. In response, many questions emerged: How did it happen? What was unusual about this event compared to previous late blight epidemics? What is the current situation in 2012 and what can be done? It's easiest to answer these questions, and to understand the recent epidemics of late blight, if one knows a bit of the history of the disease and the biology of the causal agent, Phytophthora infestans.

Fungal Planet description sheets: 154-213.

Novel species of microfungi described in the present study include the following from South Africa: Camarosporium aloes, Phaeococcomyces aloes and Phoma aloes from Aloe, C. psoraleae, Diaporthe psoraleae and D. psoraleae-pinnatae from Psoralea, Colletotrichum euphorbiae from Euphorbia, Coniothyrium prosopidis and Peyronellaea prosopidis from Prosopis, Diaporthe cassines from Cassine, D. diospyricola from Diospyros, Diaporthe maytenicola from Maytenus, Harknessia proteae from Protea, Neofusicoccum ursorum and N. cryptoaustrale from Eucalyptus, Ochrocladosporium adansoniae from Adansonia, Pilidium pseudoconcavum from Greyia radlkoferi, Stagonospora pseudopaludosa from Phragmites and Toxicocladosporium ficiniae from Ficinia. Several species were also described from Thailand, namely: Chaetopsina pini and C. pinicola from Pinus spp., Myrmecridium thailandicum from reed litter, Passalora pseudotithoniae from Tithonia, Pallidocercospora ventilago from Ventilago, Pyricularia bothriochloae from Bothriochloa and Sphaerulina rhododendricola from Rhododendron. Novelties from Spain include Cladophialophora multiseptata, Knufia tsunedae and Pleuroascus rectipilus from soil and Cyphellophora catalaunica from river sediments. Species from the USA include Bipolaris drechsleri from Microstegium, Calonectria blephiliae from Blephilia, Kellermania macrospora (epitype) and K. pseudoyuccigena from Yucca. Three new species are described from Mexico, namely Neophaeosphaeria agaves and K. agaves from Agave and Phytophthora ipomoeae from Ipomoea. Other African species include Calonectria mossambicensis from Eucalyptus (Mozambique), Harzia cameroonensis from an unknown creeper (Cameroon), Mastigosporella anisophylleae from Anisophyllea (Zambia) and Teratosphaeria terminaliae from Terminalia (Zimbabwe). Species from Europe include Auxarthron longisporum from forest soil (Portugal), Discosia pseudoartocreas from Tilia (Austria), Paraconiothyrium polonense and P. lycopodinum from Lycopodium (Poland) and Stachybotrys oleronensis from Iris (France). Two species of Chrysosporium are described from Antarctica, namely C. magnasporum and C. oceanitesii. Finally, Licea xanthospora is described from Australia, Hypochnicium huinayensis from Chile and Custingophora blanchettei from Uruguay. Novel genera of Ascomycetes include Neomycosphaerella from Pseudopentameris macrantha (South Africa), and Paramycosphaerella from Brachystegia sp. (Zimbabwe). Novel hyphomycete genera include Pseudocatenomycopsis from Rothmannia (Zambia), Neopseudocercospora from Terminalia (Zambia) and Neodeightoniella from Phragmites (South Africa), while Dimorphiopsis from Brachystegia (Zambia) represents a novel coelomycetous genus. Furthermore, Alanphillipsia is introduced as a new genus in the Botryosphaeriaceae with four species, A. aloes, A. aloeigena and A. aloetica from Aloe spp. and A. euphorbiae from Euphorbia sp. (South Africa). A new combination is also proposed for Brachysporium torulosum (Deightoniella black tip of banana) as Corynespora torulosa. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Clonal expansion of the Belgian Phytophthora ramorum populations based on new microsatellite markers.

Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey. Our main objectives were genetic characterization of this population to test for sexual reproduction, determination of population structure, evolution and spread, and evaluation of the effectiveness and impact of control measures. Novel, polymorphic simple sequence repeat (SSR) markers were developed after screening 149 candidate loci. Eighty isolates of P. ramorum, broadly representing the Belgian population, were analyzed using four previously described and three newly identified polymorphic microsatellite loci as well as amplified fragment length polymorphisms. SSR analysis was most informative and was used to screen the entire Belgian population. Thirty multilocus genotypes were identified, but 68% of the isolates belonged to the main genotype EU1MG1. Although accumulated mutation events were detected, the overall level of genetic diversity within the Belgian isolates of P. ramorum appears to be limited, indicating a relatively recent clonal expansion. Based on our SSR analysis there is no evidence of sexual recombination in the Belgian population of P. ramorum. Metalaxyl use decreased the genetic diversity of P. ramorum until 2005, when the majority of the isolates had become resistant. Most genotypes were site-specific and despite systematic removal of symptomatic and neighbouring plants, some genotypes were detected over a period of several years at a single site, sometimes discontinuously, indicating (latent) survival of the pathogen at those sites.

Migration patterns of the emerging plant pathogen Phytophthora ramorum on the West Coast of the United States of America.

Phytophthora ramorum (oomycetes) is the causal agent of sudden oak death and ramorum blight on trees, shrubs, and woody ornamentals in the forests of coastal California and southwestern Oregon and in nurseries of California, Oregon, and Washington. In this study, we investigated the genetic structure of P. ramorum on the West Coast of the United States, focusing particularly on population differentiation potentially indicative of gene flow. In total, 576 isolates recovered from 2001 to 2005 were genotyped at 10 microsatellite loci. Our analyses of genetic diversity and inferences of reproductive mode confirm previous results for the Oregon and California populations, with the strong majority of the genotypes belonging to the NA1 clonal lineage and showing no evidence for sexual reproduction. The high incidence of genotypes shared among populations and the lack of genetic structure among populations show that important large-scale, interpopulation genetic exchanges have occurred. This emphasizes the importance of human activity in shaping the current structure of the P. ramorum population on the West Coast of the United States.

Ancient isolation and independent evolution of the three clonal lineages of the exotic sudden oak death pathogen Phytophthora ramorum.

The genus Phytophthora includes some of the most destructive plant pathogens affecting agricultural and native ecosystems and is responsible for a number of recent emerging and re-emerging infectious diseases of plants. Sudden oak death, caused by the exotic pathogen P. ramorum, has caused extensive mortality of oaks and tanoaks in Northern California, and has brought economic losses to US and European nurseries as well due to its infection of common ornamental plants. In its known range, P. ramorum occurs as three distinct clonal lineages. We inferred the evolutionary history of P. ramorum from nuclear sequence data using coalescent-based approaches. We found that the three lineages have been diverging for at least 11% of their history, an evolutionarily significant amount of time estimated to be on the order of 165,000 to 500,000 years. There was also strong evidence for historical recombination between the lineages, indicating that the ancestors of the P. ramorum lineages were members of a sexually reproducing population. Due to this recombination, the ages of the lineages varied within and between loci, but coalescent analyses suggested that the European lineage may be older than the North American lineages. The divergence of the three clonal lineages of P. ramorum supports a scenario in which the three lineages originated from different geographic locations that were sufficiently isolated from each other to allow independent evolution prior to introduction to North America and Europe. It is thus probable that the emergence of P. ramorum in North America and Europe was the result of three independent migration events.

First Report of the European Lineage of Phytophthora ramorum on Viburnum and Osmanthus spp. in a California Nursery.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004.

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out-of-state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.

First Report of Haplotype I-b of Phytophthora infestans in Central Mexico.

Central Mexico is considered a center of genetic diversity for Phytophthora infestans on the basis of a range of genotypic and phenotypic characteristics (3). Surprisingly, while mitochondrial DNA (mtDNA) haplotypes I-a, II-a, and II-b have been reported from central Mexico, haplotype I-b has not been found in central Mexico (1). Therefore, a more extensive search for haplotypes was conducted in areas where sexual reproduction occurs. During the summer of 2003, leaflets of cvs. Rosita and Tollocan with a single lesion of late blight were collected in the area of Villarreal, located in Terrenate County in Tlaxcala, Mexico (170 km northeast of Mexico City). Fourteen P. infestans isolates were characterized for mtDNA haplotype, isozyme genotype (glucose 6- phosphate isomerase [Gpi] and peptidase [Pep]), and mating type. Isolation, mating type, and isozyme genotype were characterized following reported protocols (1,4). MtDNA haplotype was determined by amplifying and digesting the P2 and P4 regions and comparing amplicons to those of reference strains of known haplotype (1,2). Twelve isolates were mtDNA haplotype I-a and two were I-b. While the mtDNA I-b has been associated with the US-1 lineage (mating type: A1, Gpi: 86/100, Pep: 92/100), the genotypes for the Mexican isolates were A2, 86/100 Gpi, 100/100 Pep from cv. Rosita and A2, 86/100 Gpi, 92/100 Pep from cv. Tollocan. To our knowledge, this is the first report of the I-b mtDNA haplotype of P. infestans from central Mexico and it is now clear that all four haplotypes exist in Mexico. This finding therefore, stresses the importance of including a representative regional sampling of Mexican and Andean isolates in studies inferring the origin of this species. References: (1) W. G. Flier et al. Phytopathology 93:382, 2003. (2) G. W. Griffith and D. S. Shaw. Appl. Environ. Microbiol. 64:4007, 1998. (3) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (4) N. J. Grünwald et al. Phytopathology 91:882, 2001.

First Report of Blight on Ipomoea purpurea Caused by Phytophthora ipomoeae.

Several wild species of Ipomoea grow in the central highlands of Mexico. During the summer of 1999, in Metepec, Mexico, blighted leaves and petioles of Ipomoea purpurea were collected from diseased plants and placed in a moist chamber to induce sporulation. Sporangia that formed on the lesions were transferred with a piece of agar to selective rye agar medium (2). Phytophthora ipomoeae was consistently isolated. Species identification was based on sporangial and gametangial characteristics of five cultures grown on rye agar. Sporangia were mainly ellipsoid but occasionally ovoid, semipapillated, and deciduous with a short pedicel. All isolates were homothallic with smooth-walled and aplerotic oospores. Genotypic analysis for the allozymes Peptidase (Pep) and Glucose-6-phosphate isomerase (Gpi) indicated that all five isolates belonged to one genotype with alleles 78/78 (Pep) and 108/108 (Gpi). Morphological characteristics and the allozyme genotype correspond to the new, recently described species P. ipomoeae Flier & Grünwald (1) isolated from I. orizabensis (Pelletan) Ledeb. ex Steud. (I. tyrianthina) Lindl. and I. longepedunculata (Mart. & Gal.) Hemsl. Pathogenicity tests were carried out with leaves from greenhouse-grown I. purpurea plants. Detached leaves were inoculated with a suspension of 103 sporangia per ml and kept in a moist chamber at room temperature (17 ± 3°C). Lesions were observed between 7 and 15 days after inoculation and were characteristic of those observed in the field. The pathogen was reisolated from inoculated symptomatic tissue. To our knowledge, this is the first report of blight on I. purpurea caused by P. ipomoeae. References: (1) W. Flier et al. Mycol. Res. 106:848, 2002. (2) N. J. Grünwald et al. Phytopathology 91:882, 2001.

First Report of Pythium irregulare on Lentils in the United States.

In late June and early July 2002, stunted, chlorotic, and partially defoliated lentils (Lens culinaris Medik.) were observed throughout the lentil-growing areas of eastern Washington. These symptoms were investigated in two fields near Garfield, WA and one field near Genesee, ID. Cv. Mason was more affected than cv. Brewer. Roots were dry and brittle with black discoloration in some cases. Isolates of Fusarium oxysporum and F. solani were obtained from washed roots plated on water agar, but they were nonpathogenic in greenhouse testing in pasteurized field soil and peat-based growing mixes. On 21 April 2003, volunteer lentils growing in the same fields showed symptoms of root rot, and Pythium oospores were observed in the roots. Pythium spp. were isolated by using a selective medium (2). Oospores were aplerotic, intercalary, 12.6 to 21 µm long × 11.2 to 18.2 µm wide, mostly smooth, and often formed in chains. Isolates resembled P. paroecandrum Drechs. and P. irregulare Buisman on the basis of morphological characters (3), but DNA sequences of the internal transcribed spacer region were closer to P. irregulare on the basis of a comparison with a worldwide database of Pythium sequences (C. A. Lévesque, personal communication). Isolates were deposited with the USDA-ARS Western Regional Plant Introduction Station, Pullman, WA. Four hyphal-tip isolates were tested in the greenhouse with inoculum grown in autoclaved sandy loam amended with 1% ground rolled oats. Experiments were performed twice in Thatuna silt loam, first in pasteurized and then in nonpasteurized soil. Inoculum was added to the soil at 500 CFU/g, and seeds were planted on the same day. Each isolate was tested on cvs. Brewer and Mason, with five replicates per treatment. Plants were grown in 4- × 20.5-cm plastic tubes (two plants per tube) for 1 month at 16 to 22°C and supplemented with 14 h of light per day. P. irregulare was reisolated from infected roots in both experiments. Damping-off, stunting, chlorosis, and root rot were observed in the Pythium-inoculated treatments, which corresponded to symptoms observed in the field in 2002. In pasteurized soil, only one isolate reduced the whole, dry, plant weight of Brewer, but the other three isolates reduced the dry weight of Mason. All isolates reduced the root dry weight of Mason in natural soil, but only two isolates reduced the root dry weight of Brewer. To our knowledge, Pythium spp., but not P. irregulare, have been reported previously from lentils (1). P. irregulare also causes root rot on winter wheat, which is rotated with lentils, and this pathogen likely causes yield reduction in both crops. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) S. M. Mircetich and J. M. Kraft. Mycopathol. Mycol. Appl. 50:151, 1973. (3) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.