L. M. Quesada-Ocampo

Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data

BackgroundRice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa (japonica group).ResultsThe Nipponbare genome assembly was updated by revising and validating the minimal tiling path of clones with the optical map for rice. Sequencing errors in the revised genome assembly were identified by re-sequencing the genome of two different Nipponbare individuals using the Illumina Genome Analyzer II/IIx platform. A total of 4,886 sequencing errors were identified in 321 Mb of the assembled genome indicating an error rate in the original IRGSP assembly of only 0.15 per 10,000 nucleotides. A small number (five) of insertions/deletions were identified using longer reads generated using the Roche 454 pyrosequencing platform. As the re-sequencing data were generated from two different individuals, we were able to identify a number of allelic differences between the original individual used in the IRGSP effort and the two individuals used in the re-sequencing effort. The revised assembly, termed Os-Nipponbare-Reference-IRGSP-1.0, is now being used in updated releases of the Rice Annotation Project and the Michigan State University Rice Genome Annotation Project, thereby providing a unified set of pseudomolecules for the rice community.ConclusionsA revised, error-corrected, and validated assembly of the Nipponbare cultivar of rice was generated using optical map data, re-sequencing data, and manual curation that will facilitate on-going and future research in rice. Detection of polymorphisms between three different Nipponbare individuals highlights that allelic differences between individuals should be considered in diversity studies.

The cucurbit downy mildew pathogen Pseudoperonospora cubensis.

UNLABELLED Pseudoperonospora cubensis[(Berkeley & M. A. Curtis) Rostovzev], the causal agent of cucurbit downy mildew, is responsible for devastating losses worldwide of cucumber, cantaloupe, pumpkin, watermelon and squash. Although downy mildew has been a major issue in Europe since the mid-1980s, in the USA, downy mildew on cucumber has been successfully controlled for many years through host resistance. However, since the 2004 growing season, host resistance has been effective no longer and, as a result, the control of downy mildew on cucurbits now requires an intensive fungicide programme. Chemical control is not always feasible because of the high costs associated with fungicides and their application. Moreover, the presence of pathogen populations resistant to commonly used fungicides limits the long-term viability of chemical control. This review summarizes the current knowledge of taxonomy, disease development, virulence, pathogenicity and control of Ps. cubensis. In addition, topics for future research that aim to develop both short- and long-term control measures of cucurbit downy mildew are discussed. TAXONOMY Kingdom Straminipila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Pseudoperonospora; Species Pseudoperonospora cubensis. DISEASE SYMPTOMS Angular chlorotic lesions bound by leaf veins on the foliage of cucumber. Symptoms vary on different cucurbit species and varieties, specifically in terms of lesion development, shape and size. Infection of cucurbits by Ps. cubensis impacts fruit yield and overall plant health. INFECTION PROCESS Sporulation on the underside of leaves results in the production of sporangia that are dispersed by wind. On arrival on a susceptible host, sporangia germinate in free water on the leaf surface, producing biflagellate zoospores that swim to and encyst on stomata, where they form germ tubes. An appressorium is produced and forms a penetration hypha, which enters the leaf tissue through the stomata. Hyphae grow through the mesophyll and establish haustoria, specialized structures for the transfer of nutrients and signals between host and pathogen. CONTROL Management of downy mildew in Europe requires the use of tolerant cucurbit cultivars in conjunction with fungicide applications. In the USA, an aggressive fungicide programme, with sprays every 5-7 days for cucumber and every 7-10 days for other cucurbits, has been necessary to control outbreaks and to prevent crop loss. USEFUL WEBSITES http://www.daylab.plp.msu.edu/pseudoperonospora-cubensis/ (Day Laboratory website with research advances in downy mildew); http://veggies.msu.edu/ (Hausbeck Laboratory website with downy mildew news for growers); http://cdm.ipmpipe.org/ (Cucurbit downy mildew forecasting homepage); http://ipm.msu.edu/downymildew.htm (Downy mildew information for Michigans vegetable growers).

Advances in Research on Phytophthora capsici on Vegetable Crops in The United States

Since L. H. Leonians first description of Phytophthora capsici as a pathogen of chile pepper in 1922, we have made many advances in our understanding of this pathogens biology, host range, dissemination, and management. P. capsici causes foliar blighting, damping-off, wilting, and root, stem, and fruit rot of susceptible hosts, and economic losses are experienced annually in vegetable crops including cucurbits and peppers. Symptoms of P. capsici infection may manifest as stunting, girdling, or cankers for some cultivars or crops that are less susceptible. P. capsici continues to be a constraint on production, and implementation of an aggressive integrated management scheme can still result in insufficient control when weather is favorable for disease. Management of diseases caused by P. capsici is currently limited by the long-term survival of the pathogen as oospores in the soil, a wide host range, long-distance movement of the pathogen in surface water used for irrigation, the presence of fungicide-resistant pathogen populations, and a lack of commercially acceptable resistant host varieties. P. capsici can infect a wide range of hosts under laboratory and greenhouse conditions including cultivated crops, ornamentals, and native plants belonging to diverse plant families. As our understanding of P. capsici continues to grow, future research should focus on developing novel and effective solutions to manage this pathogen and prevent economic losses due to the diseases it causes.

Resistance in tomato and wild relatives to crown and root rot caused by Phytophthora capsici.

Phytophthora capsici causes root, crown, and fruit rot of tomato, a major vegetable crop grown worldwide. The objective of this study was to screen tomato cultivars and wild relatives of tomato for resistance to P. capsici. Four P. capsici isolates were individually used to inoculate 6-week-old seedlings (1 g of P. capsici-infested millet seed per 10 g of soilless medium) of 42 tomato cultivars and wild relatives of tomato in a greenhouse. Plants were evaluated daily for wilting and death. All P. capsici isolates tested caused disease in seedlings but some isolates were more pathogenic than others. A wild relative of cultivated tomato, Solanum habrochaites accession LA407, was resistant to all P. capsici isolates tested. Moderate resistance to all isolates was identified in the host genotypes Ha7998, Fla7600, Jolly Elf, and Talladega. P. capsici was frequently recovered from root and crown tissue of symptomatic inoculated seedlings but not from leaf tissue or asymptomatic or control plants. The phenotype of the recovered isolate matched the phenotype of the inoculum. Pathogen presence was confirmed in resistant and moderately resistant tomato genotypes by species-specific polymerase chain reaction of DNA from infected crown and root tissue. Amplified fragment length polymorphisms of tomato genotypes showed a lack of correlation between genetic clusters and susceptibility to P. capsici, indicating that resistance is distributed in several tomato lineages. The results of this study create a baseline for future development of tomato cultivars resistant to P. capsici.

Investigating the Genetic Structure of Phytophthora capsici Populations

Phytophthora capsici Leonian is a destructive soilborne pathogen that infects economically important solanaceous, cucurbitaceous, fabaceous, and other crops in the United States and worldwide. The objective of this study was to investigate the genetic structure of 255 P. capsici isolates assigned to predefined host, geographical, mefenoxam-sensitivity, and mating-type categories. Isolates from six continents, 21 countries, 19 U.S. states, and 26 host species were genotyped for four mitochondrial and six nuclear loci. Bayesian clustering revealed some population structure by host, geographic origin, and mefenoxam sensitivity, with some clusters occurring more or less frequently in particular categories. Bayesian clustering, split networks, and statistical parsimony genealogies also detected the presence of non-P. capsici individuals in our sample corresponding to P. tropicalis (n = 9) and isolates of a distinct cluster closely related to P. capsici and P. tropicalis (n = 10). Our findings of genetic structuring in P. capsici populations highlight the importance of including isolates from all detected clusters that represent the genetic variation in P. capsici for development of diagnostic tools, fungicides, and host resistance. The population structure detected will also impact the design and interpretation of association studies in P. capsici. This study provides an initial map of global population structure of P. capsici but continued genotyping of isolates will be necessary to expand our knowledge of genetic variation in this important plant pathogen.

Susceptibility of Fraser Fir to Phytophthora capsici

Phytophthora cinnamomi, P. drechsleri, P. citricola, and P. cactorum limit Fraser fir production, whereas P. capsici affects Solanaceous, Cucurbitaceous, and Fabaceous crops. Some vegetable growers in Michigan plant conifers for the Christmas tree market in fields infested with P. capsici. To determine the susceptibility of Fraser fir to P. capsici, stems (no wound or 1- or 3-mm-diameter wound) or roots (2 or 4 g of infested millet seed or 2 or 5 × 103 zoospores/ml of a zoospore suspension) of seedlings were inoculated with each of four P. capsici isolates and incubated in growth chambers (20 or 25°C). In addition, Fraser fir seedlings were planted in two commercial fields naturally infested with P. capsici. All P. capsici isolates tested incited disease in the seedlings regardless of incubation temperature or inoculation method. Seedlings (72%) planted in P. capsici-infested fields developed disease symptoms and died. Most of the P. capsici isolates obtained from the Fraser fir seedlings infected while in the field were recovered from root tissue. Identification was confirmed by species-specific direct colony polymerase chain reaction. The pathogen was successfully recovered from stems of all stem-inoculated seedlings, and from roots and stems of all root-inoculated seedlings; the phenotype of the recovered isolate matched the phenotype of the inoculum. This study suggests that planting Fraser fir in fields infested with P. capsici could result in infection and that adjustments in current rotational schemes are needed.

The genetic structure of Pseudoperonospora cubensis populations

Pseudoperonospora cubensis is a destructive foliar pathogen of economically important cucurbitaceous crops in the United States and worldwide. In this study, we investigated the genetic structure of 465 P. cubensis isolates from three continents, 13 countries, 19 states of the United States, and five host species using five nuclear and two mitochondrial loci. Bayesian clustering resolved six genetic clusters and suggested some population structure by geographic origin and host, because some clusters occurred more or less frequently in particular categories. All of the genetic clusters were present in the sampling from North America and Europe. Differences in cluster occurrence were observed by country and state. Isolates from cucumber had different cluster composition and lower genetic diversity than isolates from other cucurbits. Because genetic structuring was detected, isolates that represent the genetic variation in P. cubensis should be used when developing diagnostic tools, fungicides, and resistant host varieties. Although this study provides an initial map of global population structure of P. cubensis, future genotyping of isolates could reveal population structure within specific geographic regions, across a wider range of hosts, or during different time points during the growing season.

Evidence for Positive Selection in Putative Virulence Factors within the Paracoccidioides brasiliensis Species Complex

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood–based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi.

The Genetic Structure of Pseudoperonospora cubensis Populations

Pseudoperonospora cubensis is a destructive foliar pathogen of economically important cucurbitaceous crops in the United States and worldwide. In this study, we investigated the genetic structure of 465 P. cubensis isolates from three continents, 13 countries, 19 states of the United States, and five host species using five nuclear and two mitochondrial loci. Bayesian clustering resolved six genetic clusters and suggested some population structure by geographic origin and host, because some clusters occurred more or less frequently in particular categories. All of the genetic clusters were present in the sampling from North America and Europe. Differences in cluster occurrence were observed by country and state. Isolates from cucumber had different cluster composition and lower genetic diversity than isolates from other cucurbits. Because genetic structuring was detected, isolates that represent the genetic variation in P. cubensis should be used when developing diagnostic tools, fungicides, and resistant host varieties. Although this study provides an initial map of global population structure of P. cubensis, future genotyping of isolates could reveal population structure within specific geographic regions, across a wider range of hosts, or during different time points during the growing season.

Resurgence of Pseudoperonospora cubensis: The Causal Agent of Cucurbit Downy Mildew

The downy mildew pathogen, Pseudoperonospora cubensis, which infects plant species in the family Cucurbitaceae, has undergone major changes during the last decade. Disease severity and epidemics are far more destructive than previously reported, and new genotypes, races, pathotypes, and mating types of the pathogen have been discovered in populations from around the globe as a result of the resurgence of the disease. Consequently, disease control through host plant resistance and fungicide applications has become more complex. This resurgence of P. cubensis offers challenges to scientists in many research areas including pathogen biology, epidemiology and dispersal, population structure and population genetics, host preference, host-pathogen interactions and gene expression, genetic host plant resistance, inheritance of host and fungicide resistance, and chemical disease control. This review serves to summarize the current status of this major pathogen and to guide future management and research efforts within this pathosystem.