Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m⁻³ for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m⁻³ to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.     

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing,and Implications for Late Blight Epidemiology

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.     

Genetic Analyses of Interspecific Hybrids between Phytophthora infestans and Phytophthora mirabilis

Goodwin, S. B., and Fry, W. E. 1994. Genetic analyses of interspecific hybrids between Phytophthora infestans and Phytophthora mirabilis. Experimental Mycology 18, 20-32. Four crosses were made between isolates of two host-specific Phytophthora species. Phytophthora infestans and Phytophthora mirabilis. In the two most successful crosses involving a common P. infestans A2 parent, allozyme analysis confirmed that 79 of 86 progeny were interspecific hybrids, 3 were presumed selfs, and 4 were either selfs or nonrecombinant parental types. Mating type, alleles at the allozyme locus glucose-6-phosphate isomerase, and the + alleles at a number of DNA fingerprinting loci segregated independently according to Mendelian expectation. Three DNA fingerprinting loci were tightly linked in P. mirabilis, but no other linkages were detected among these markers. Mitochondrial DNA was uniparentally inherited, mostly from the P. infestans parent. Growth rate segregated as a quantitative character. None of the 68 progeny tested infected Mirabilis jalapa (the host of P. mirabilis), 3 infected potato, and 4 were weakly pathogenic to tomato. Because most of the F₁ hybrids could not infect any of the hosts infected by the parents, host specialization could provide a postzygotic as well as a prezygotic reproductive isolating mechanism for P. infestans and P. mirabilis in central Mexico. These results indicate that P. mirabilis probably is capable of a regular outcrossing mating system.     

Genotypic diversity and migration patterns of Phytophthora infestans in the Nordic countries

In this study we investigated the genotypic diversity and the migration patterns of Phytophthora infestans in the Nordic countries. Isolates of P. infestans from outbreaks in 43 fields sampled in 2008 were collected using stratified sampling with country, field, and disease foci as the different strata. Microsatellites were used as markers to determine the genotypic variation in the sampled material. The results show a high genotypic variation of P. infestans in the Nordic countries with most of the genotypes found only once among the collected isolates. The major part of the genotypic variation was observed within the fields, with low differentiation between the fields. The observed low association of alleles among loci is consistent with frequent sexual reproduction of P. infestans in the Nordic countries. Coalescence analyses did not support a single common population for the four countries, thus indicating some degree of geographic differentiation. The analyses of migration patterns showed differing levels of gene flow among the Nordic countries. No correlation between migration rates and geographical distance could be seen. This could be explained by different degrees of genetic similarity between the pathogen populations in the different countries.     

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia + Tc Id, Tc Ia + Tc Ie and Tc Id + Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.     

Fine-scale genetic structure in populations of the Chagas’ disease vector Triatoma infestans (Hemiptera, Reduvidae)

Fine scale patterns of genetic structure and dispersal in Triatoma infestans populations from Argentina was analysed. A total of 314 insects from 22 domestic and peridomestic sites from the locality of San Martín (Capayán department, Catamarca province) were typed for 10 polymorphic microsatellite loci. The results confirm subdivision of T. infestans populations with restricted dispersal among sampling sites and suggest inbreeding and/or stratification within the different domestic and peridomestic structures. Spatial correlation analysis showed that the scale of structuring is approximately of 400 m, indicating that active dispersal would occur within this distance range. It was detected difference in scale of structuring among sexes, with females dispersing over greater distances than males. This study suggests that insecticide treatment and surveillance should be extended within a radius of 400 m around the infested area, which would help to reduce the probability of reinfestation by covering an area of active dispersal. The inferences made from fine-scale spatial genetic structure analyses of T. infestans populations has demonstrated to be important for community-wide control programs, providing a complementary approach to help improve vector control strategies.     

Development of microsatellite markers targeting (GATA) n motifs in sorghum [Sorghum bicolor (L.) Moench]

Microsatellite markers targeting (GATA) _n motifs are known to be highly polymorphic. Genome-wide development of such markers has not been reported in sorghum. The main objective of this study was to identify Class I microsatellites with (GATA) _n motifs in the sorghum genome through in silico analysis and assess their potential as molecular markers. The study identified a total of 128 such motifs, of which 14, 16 and 98 motifs were present in the genic, upstream and non-genic regions, respectively. The majority of the (GATA) _n motifs were found in the non-genic regions of the genome while 23.44 % of them were found within the genes and upstream of genes. About 110 PCR-based markers were developed targeting these microsatellites and 50 of them distributed across the genome were validated in 24 diverse sorghum genotypes representing different racial groups. Thirty-eight markers were polymorphic, with average polymorphism information content value of 0.69, and the sorghum genotypes could be grouped into two major clusters. These markers with robust amplification combined with good allelic diversity represent a new set of microsatellite markers in sorghum reported for the first time that will be highly useful for various genetics and molecular breeding applications.     

Partial resistance to late blight (Phytophthora infestans) in hybrid progenies of four South American Solanum species crossed with diploid S. tuberosum

Resistant genotypes of the diploid tuber-bearing South American species Solarium arnezii x hondelmannii, S. berthaultii, S. leptophyes and S. microdontum were crossed with three diploid genotypes of S. tuberosum that varied in resistance and maturity type. The progenies were field tested for 2 years for resistance to a complex race of Phytophthora infestans. A wealth of genetic variation for resistance was found in most of the progenies. At least two susceptibility groups could be distinguished in some progenies of S. microdontum. This could be explained by the presence of several major resistance genes in the wild parent and, unexpectedly, in the susceptible parent SH 82-44-111. In most of the wild parents and in the susceptible parent SH 77-114-2988 there appeared to be minor resistance genes. General combining ability effects were predominant; small specific combining ability effects were detected in some crosses of S. microdontum. Gene action appeared dominant in some crosses.     

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Microsatellite Typing of Clinical and Environmental Cryptococcus neoformans var. grubii Isolates from Cuba Shows Multiple Genetic Lineages

Background

Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods.

Methodology/Principal Findings

We selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68; from pigeon guano) C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5–51). A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70%) fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical). Clinical isolates were segregated over multiple microsatellite complexes.

Conclusions/Significance

A large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s) of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii.     

Hidden Sylvatic Foci of the Main Vector of Chagas Disease Triatoma infestans: Threats to the Vector Elimination Campaign?

Background

Establishing the sources of reinfestation after residual insecticide spraying is crucial for vector elimination programs. Triatoma infestans, traditionally considered to be limited to domestic or peridomestic (abbreviated as D/PD) habitats throughout most of its range, is the target of an elimination program that has achieved limited success in the Gran Chaco region in South America.

Methodology/Principal Findings

During a two-year period we conducted semi-annual searches for triatomine bugs in every D/PD site and surrounding sylvatic habitats after full-coverage spraying of pyrethroid insecticides of all houses in a well-defined rural area in northwestern Argentina. We found six low-density sylvatic foci with 24 T. infestans in fallen or standing trees located 110–2,300 m from the nearest house or infested D/PD site detected after insecticide spraying, when house infestations were rare. Analysis of two mitochondrial gene fragments of 20 sylvatic specimens confirmed their species identity as T. infestans and showed that their composite haplotypes were the same as or closely related to D/PD haplotypes. Population studies with 10 polymorphic microsatellite loci and wing geometric morphometry consistently indicated the occurrence of unrestricted gene flow between local D/PD and sylvatic populations. Mitochondrial DNA and microsatellite sibship analyses in the most abundant sylvatic colony revealed descendents from five different females. Spatial analysis showed a significant association between two sylvatic foci and the nearest D/PD bug population found before insecticide spraying.

Conclusions

Our study shows that, despite of its high degree of domesticity, T. infestans has sylvatic colonies with normal chromatic characters (not melanic morphs) highly connected to D/PD conspecifics in the Argentinean Chaco. Sylvatic habitats may provide a transient or permanent refuge after control interventions, and function as sources for D/PD reinfestation. The occurrence of sylvatic foci of T. infestans in the Gran Chaco may pose additional threats to ongoing vector elimination efforts.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

Extraordinary Trypanosoma cruzi diversity within single mammalian reservoir hosts implies a mechanism of diversifying selection

Trypanosoma cruzi is an evolutionarily ancient parasitic protozoan endemic to the Americas. Multiple genetic and phenotypic markers indicate that this parasite is highly diverse, with several divergent and discrete major genotypes reported. Infection multiclonality has been observed among numerous metazoan and unicellular endoparasitic species. However, few studies report the complexity of mixed infections within an individual host in any detail or consider their ecological and biological implications. Here we report extraordinary genetic diversity within single reservoir hosts of T. cruzi I using nine polymorphic microsatellite markers across 211 clones from eight mammals from three different sylvatic foci in South America. Forty-nine distinct multilocus genotypes were defined, with as many as 10 isolated from the same host. We discuss our data in the light of previous population genetic studies of this and related parasitic protozoa and contrast high levels of diversity within each host with the precarious nature of T. cruzi contaminative vectorial transmission. Finally, we propose that non-neutral processes could easily account for the diversity we observe and suggest a functional link with survival in the host.     

Chromosomal heteromorphism linked to the mating type locus of the oomycetePhytophthora infestans

The mating type locus of the oomycete,Phytophthora infestans, is embedded in a region of DNA that displays distorted and non-Mendelian segregation. By using DNA probes linked to the mating type locus to genetically and physically characterize that region, a large zone of chromosomal heteromorphism was detected. LocusS1 was shown to represent a tandemly repeated array of DNA that was typically present in a hemizygous state in A1 isolates while being absent from A2 isolates. The analysis of the parents and progeny of seven crosses indicated that the tandem array was linked in cis to the A1-determining allele of the mating type locus. A worldwide survey of genotypically diverse field isolates ofP. infestans indicated thatS1 was present in each of 48 isolates of the A1 mating type that were tested, but was absent in 46 of 47 A2 strains. Physical analysis ofS1 indicated that the tandemly repeated DNA sequence spanned about 300 kb and had evolved from a 1.35-kb monomer. Internal deletions occurred withinS1 during sexual propagation. This and other mutations apparently contributed to a high degree of polymorphism within theS1 array.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.     

Obtaining sexual genotypes for breeding in buffel grass

Buffel grass (Cenchrus ciliaris L. syn. Pennisetum ciliare (L.) Link) is a species that is highly tolerant to drought and is used primarily as forage in drier regions throughout the subtropics and tropics. It reproduces mainly by apomixis and the acquisition of obligate sexual genotypes or facultative apomicts with high levels of sexuality is required for performing crosses and plant improvement. The aim of this study was to obtain sexual genotypes from controlled crosses using obligate apomictic cultivars and a sexual line. Twelve putative hybrid F1 plants were selected morphologically and two of them were identified as sexual genotypes by PCR using specific primers for reproductive mechanism. Cytoembryological analysis showed 65.5 and 71.3% meiotic embryo sacs in these plants and their hybrid nature was corroborated by AFLP. Both highly sexual genotypes could be used as female parents in crosses for obtaining improved cultivars of buffel grass.