Functional Variation of Plant-Pathogen Interactions: New Concept and Methods for Virulence Data Analyses.

Classical virulence analysis is based on discovering virulence phenotypes of isolates with regard to a composition of resistance genes in a differential set of host genotypes. With such a vision, virulence phenotypes are usually treated in a genetic manner as one of two possible alleles, either virulence or avirulence in a binary locus. Therefore, population genetics metrics and methods have become prevailing tools for analyzing virulence data at multiple loci. However, a basis for resolving binary virulence phenotypes is infection type (IT) data of host-pathogen interaction that express functional traits of each specific isolate in a given situation (particular host, environmental conditions, cultivation practice, and so on). IT is determined by symptoms and signs observed (e.g., lesion type, lesion size, coverage of leaf or leaf segments by mycelium, spore production and so on), and assessed by IT scores at a generally accepted scale for each plant-pathogen system. Thus, multiple IT profiles of isolates are obtained and can be subjected to analysis of functional variation within and among operational units of a pathogen. Such an approach may allow better utilization of the information available in the raw data, and reveal a functional (e.g., environmental) component of pathogen variation in addition to the genetic one. New methods for measuring functional variation of plant-pathogen interaction with IT data were developed. The methods need an appropriate assessment scale and expert estimations of dissimilarity between IT scores for each plant-pathogen system (an example is presented). Analyses of a few data sets at different hierarchical levels demonstrated discrepancies in results obtained with IT phenotypes versus binary virulence phenotypes. The ability to measure functional IT-based variation offers promise as an effective tool in the study of epidemics caused by plant pathogens.

Sr36- and Sr5-Mediated Resistance Response to Puccinia graminis f. sp. tritici Is Associated with Callose Deposition in Wheat Guard Cells.

Race-specific resistance of wheat to Puccinia graminis f. sp. tritici is primarily posthaustorial and often involves the induction of a hypersensitive response (HR). The aim of this study was to investigate host defense responses induced in interactions between P. graminis f. sp. tritici races and wheat lines carrying different race-specific stem rust resistance (Sr) genes. In incompatible interactions between wheat lines carrying Sr36 in three genetic backgrounds (LMPG, Prelude, or W2691) and avirulent P. graminis f. sp. tritici races MCCFC or RCCDM, callose accumulated within 24 h in wheat guard cells contacted by a P. graminis f. sp. tritici appressorium, and P. graminis f. sp. tritici ingress was inhibited following appressorium formation. Accordingly, the expression of transcripts encoding a callose synthase increased in the incompatible interaction between LMPG-Sr36 and avirulent P. graminis f. sp. tritici race MCCFC. Furthermore, the inhibition of callose synthesis through the infiltration of 2-deoxy-D-glucose (DDG) increased the ability of P. graminis f. sp. tritici race MCCFC to infect LMPG-Sr36. A similar induction of callose deposition in wheat guard cells was also observed within 24 h after inoculation (hai) with avirulent P. graminis f. sp. tritici race HKCJC on LMPG-Sr5 plants. In contrast, this defense response was not induced in incompatible interactions involving Sr6, Sr24, or Sr30. Instead, the induction of an HR and cellular lignification were noted. The manifestation of the HR and cellular lignification was induced earlier (24 hai) and was more extensive in the resistance response mediated by Sr6 compared with those mediated by Sr24 or Sr30. These results indicate that the resistance mediated by Sr36 is similar to that mediated by Sr5 but different from those triggered by Sr6, Sr24, or Sr30. Resistance responses mediated by Sr5 and Sr36 are prehaustorial, and are a result of very rapid recognition of molecules derived from avirulent isolates of P. graminis f. sp. tritici, in contrast to the responses triggered in lines with Sr6, Sr24, and Sr30.

Molecular cytological characterization of two novel durum--Thinopyrum intermedium partial amphiploids with resistance to leaf rust, stem rust and Fusarium head blight.

Thinopyrum intermedium, a wild relative of wheat, is an excellent source of disease resistance. Two novel partial amphiploids, 08-47-50 and 08-53-55 (2n = 6x = 42), were developed from wide crosses between durum wheat and Th. intermedium. Meiotic analysis showed that pollen mother cells of the two partial amphiploids formed an average 20.49 bivalents for 08-47-50 and 20.67 bivalents for 08-53-55, indicating that they are basically cytologically stable. GISH analysis revealed that the two partial amphiploids carried different chromosome compositions. 08-47-50 had fourteen chromosomes from Th. intermedium and its alien chromosomes included six St-, four E(e) - and four E(e)-St translocated chromosomes, whereas 08-53-55 had four St- and ten E(e)-St translocated chromosomes. Fungal disease evaluation indicated that both partial amphiploids had a high level of resistance to FHB, leaf rust and stem rust race Ug99. These two novel partial amphiploids with multiple disease resistance could be used as a new source of multiple disease resistance in bread wheat and durum wheat breeding programs.

Identification and mapping of leaf, stem and stripe rust resistance quantitative trait loci and their interactions in durum wheat.

Leaf rust (Puccinia triticina Eriks.), stripe rust (Puccinia striiformis f. tritici Eriks.) and stem rust (Puccinia graminis f. sp. tritici) cause major production losses in durum wheat (Triticum turgidum L. var. durum). The objective of this research was to identify and map leaf, stripe and stem rust resistance loci from the French cultivar Sachem and Canadian cultivar Strongfield. A doubled haploid population from Sachem/Strongfield and parents were phenotyped for seedling reaction to leaf rust races BBG/BN and BBG/BP and adult plant response was determined in three field rust nurseries near El Batan, Obregon and Toluca, Mexico. Stripe rust response was recorded in 2009 and 2011 nurseries near Toluca and near Njoro, Kenya in 2010. Response to stem rust was recorded in field nurseries near Njoro, Kenya, in 2010 and 2011. Sachem was resistant to leaf, stripe and stem rust. A major leaf rust quantitative trait locus (QTL) was identified on chromosome 7B at Xgwm146 in Sachem. In the same region on 7B, a stripe rust QTL was identified in Strongfield. Leaf and stripe rust QTL around DArT marker wPt3451 were identified on chromosome 1B. On chromosome 2B, a significant leaf rust QTL was detected conferred by Strongfield, and at the same QTL, a Yr gene derived from Sachem conferred resistance. Significant stem rust resistance QTL were detected on chromosome 4B. Consistent interactions among loci for resistance to each rust type across nurseries were detected, especially for leaf rust QTL on 7B. Sachem and Strongfield offer useful sources of rust resistance genes for durum rust breeding.

Genetic Diversity of Gibberella zeae Isolates from Manitoba.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight, one of the most important diseases of cereals in the Canadian prairies for the last decade. In 2002, 60 isolates of G. zeae were collected and single spored from naturally infected spikes of wheat from Carman and Winnipeg in Manitoba. These isolates were compared using vegetative compatibility analysis and polymerase chain reaction (PCR)-based sequence related amplified polymorphisms (SRAP). Sixteen vegetative compatibility groups (VCG) were found among the 50 isolates tested. Five VCGs were found in the two locations, five in Carman and six in Winnipeg. Eight SRAP primer pairs amplified 90 polymorphic DNA fragments from 60 isolates and identified 59 distinct haplotypes. Among seven pairs of isolates, each pair from a distinct spike, four had isolates with different VCGs and six comprised different SRAP haplotypes. Principal component analysis and UPGMA separated the dataset into two main groups, each with isolates from both locations. The analysis of molecular variance also revealed that 75 and 20% of the variance was associated with differences among individual isolates and varieties sampled, respectively. Geographic location was not a significant source of variation at P = 0.05 and accounted for only 4% of total variance. A low correlation between VCG and SRAP marker data was detected. This study showed that, although genetic diversity is high among G. zeae isolates, Carman and Winnipeg collections have a similar genetic makeup and are likely part of the same population. The significant proportion of variance accounted by the variety compared with the geographic origin of isolates suggests that seedborne inoculum might have contributed to the genetic diversity within the G. zeae collection under study.

Inheritance and QTL analysis of durable resistance to stripe and leaf rusts in an Australian cultivar, Triticum aestivum 'Cook'.

An F4-derived F6 recombinant inbred line population (n = 148) of a cross between the durable stripe (yellow) rust (caused by Puccinia striiformis) and leaf (brown) rust (caused by Puccinia triticina) resistant cultivar, Triticum aestivum 'Cook', and susceptible genotype Avocet-YrA was phenotyped at several locations in Canada and Mexico under artificial epidemics of leaf or stripe rusts and genotyped using amplified fragment length polymorphism (AFLP) and microsatellite markers. Durable adult plant resistance to stripe and leaf rusts in 'Cook' is inherited quantitatively and was based on the additive interaction of linked and (or) pleiotropic slow-rusting genes Lr34 and Yr18 and the temperature-sensitive stripe rust resistance gene, YrCK, with additional genetic factors. Identified QTLs accounted for 18% to 31% of the phenotypic variation in leaf and stripe rust reactions, respectively. In accordance with the high phenotypic associations between leaf and stripe rust resistance, some of the identified QTLs appeared to be linked and (or) pleiotropic for both rusts across tests. Although a QTL was identified on chromosome 7D with significant effects on both rusts at some testing locations, it was not possible to refine the location of Lr34 or Yr18 because of the scarcity of markers in this region. The temperature-sensitive stripe rust resistance response, conditioned by the YrCK gene, significantly contributed to overall resistance to both rusts, indicating that this gene also had pleiotropic effects.

Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL4452x'AC Domain'.

Relatively little is known about the genetic control of agronomic traits in common wheat (Triticum aestivum L.) compared with traits that follow Mendelian segregation patterns. A doubled-haploid population was generated from the cross RL4452x'AC Domain' to study the inheritance of the agronomic traits: plant height, time to maturity, lodging, grain yield, test weight, and 1000-grain weight. This cross includes the genetics of 2 western Canadian wheat marketing classes. Composite interval mapping was conducted with a microsatellite linkage map, incorporating 369 loci, and phenotypic data from multiple Manitoba environments. The plant height quantitative trait loci (QTLs), QHt.crc-4B and QHt.crc-4D, mapped to the expected locations of Rht-B1 and Rht-D1. These QTLs were responsible for most of the variation in plant height and were associated with other agronomic traits. An additional 25 agronomic QTLs were detected in the RL4452x'AC Domain' population beyond those associated with QHt.crc-4B and QHt.crc-4D. 'AC Domain' contributed 4 alleles for early maturity, including a major time to maturity QTL on 7D. RL4452 contributed 2 major alleles for increased grain yield at QYld.crc-2B and QYld.crc-4A, which are potential targets for marker-assisted selection. A key test weight QTL was detected on 3B and prominent 1000-grain weight QTLs were identified on 3D and 4A.

Reaction of a Diverse Collection of Barley Lines to Fusarium Head Blight.

Fusarium head blight (FHB) is currently the primary disease of concern in barley in Canada and sources of FHB resistance need to be identified. A diverse collection of 77 two-rowed and 81 six-rowed barley lines was screened for resistance to FHB in inoculated, irrigated nurseries from 1995 to 1998. Barley spikes were spray inoculated with conidia of Fusarium graminearum and visual symptoms of FHB were scored to determine an FHB index. Deoxynivalenol (DON) content was determined from harvested seed samples during 1997 and 1998. Although there was variation in the average FHB index and DON content among the different years of testing, the rankings of the lines generally were correlated among the years of testing and the two measures of FHB. Based on the FHB index from 1995 and 1996, a subset of 18 lines with low FHB and 12 lines with high FHB were compared with 20 FHB resistance sources and 7 Canadian lines or cultivars during 1997 and 1998 in both inoculated and noninoculated nurseries. Lines Nepolegajuscij, Krasnojarskij, Nordic, Murakaki-mochi, Golozernyj 1, Maris Mink, Symko, Ussurijskij 8, Suvenir, and Canadian cultivars AC Sterling and Morrison were similar to the best resistance sources (Zhedar1, Seijo II, and Chevron) and had consistently low DON content.

Barrage Zone Formation Between Vegetatively Incompatible Fusarium graminearum (Gibberella zeae) Isolates.

ABSTRACT Vegetative compatibility has been used to assess the population biology of many fungal plant pathogens. However, for many species, including Fusarium graminearum, this has meant making auxotrophic mutants to force heterokaryon formation. A method was developed to observe barrage zones of thick, raised mycelium at the junctions of vegetatively incompatible F. graminearum isolates. The appearance of the barrage zones was influenced by the growth medium and the light. Barrage zones on V8 agar were thicker and better defined than those on potato dextrose agar, Spezieller Nahrstoffarmer agar, and water agar. The addition of ground wheat kernels to V8 agar enhanced barrage zone formation. Incubating the cultures under constant light at 2,150 lx produced more distinct barrage zones than constant light at 3,400 lx, constant darkness, or ambient room light. Forty-three F. graminearum isolates from 34 vegetative compatibility groups, determined previously using nit auxotrophic mutants, were paired in all combinations using these optimized conditions. Isolates in different vegetative compatibility groups typically formed distinct, thick barrage zones at their junctions. Pairs of isolates in the same vegetative compatibility group had a very slight or no visible reaction, or rarely, a distinct "line gap" of sparse mycelium. Subcultures from the same isolate typically had no visible reaction at their colony junctions; however, subcultures from some isolates had thin, slight barrage zones. This method was used to identify the proportion of each of four F. graminearum isolates from infected barley spikes in the field, inoculated previously with a mixture of conidia from these four isolates. Barrage zone formation represents a rapid method to screen vegetative compatibility groups in F. graminearum and may be useful for other Fusarium species.

Moniliformin in barley inoculated with Fusarium avenaceum.

To assess production of the mycotoxin moniliformin, inoculation trials with four barley cultivars and local isolates of Fusarium avenaceum were performed at two different experimental farms in southern Manitoba, Canada, during 1997-99. In the 1997 study, moniliformin was detected in 11 of 16 barley rows at levels between 0.19 and 1.62 mg g(-1), and F. avenaceum infection ranged from 10 to 57% in rows where the toxin was detected. In the 1998 study, moniliformin was detected in only three of 16 barley rows, with levels between 0.06 and 0.43 micro g g(-1), and F. avenaceum infection between 16 and 39%. In the 1999 study, moniliformin was detected in 11 of 16 barley rows with levels between 0.09 and 0.42 micro g g(-1), and F. avenaceum infection between 37 and 76%. In the 1997 trial, moniliformin was found in a single water-inoculated control row at 0.15 micro g g(-1). The data suggest that in years of high rainfall and F. avenaceum infection, moniliformin is likely to be found in Manitoba barley.

Comparison of Canadian Fusarium graminearum isolates for aggressiveness, vegetative compatibility, and production of ergosterol and mycotoxins.

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada.

Effect of exposure to 900 MHz radiofrequency radiation on intrachromosomal recombination in pKZ1 mice.

Radiofrequency (RF) radiation emitted from mobile phones is not considered to be directly genotoxic, but it may have downstream effects on cellular DNA. We studied the effect of 4 W/kg pulsed 900 MHz RF radiation on somatic intrachromosomal recombination in the spleen in the pKZ1 recombination mutagenesis model. Somatic intrachromosomal recombination inversion events were detected in spleen tissue of pKZ1 mice by histochemical staining for E. coli beta-galactosidase protein in cells in which the lacZ transgene has undergone an inversion event. pKZ1 mice were exposed daily for 30 min to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms for 1, 5 or 25 days. Three days after the last exposure, spleen sections were screened for DNA inversion events. There was no significant difference between the control and treated groups in the 1- and 5-day exposure groups, but there was a significant reduction in inversions below the spontaneous frequency in the 25-day exposure group. This observation suggests that exposure to RF radiation can lead to a perturbation in recombination frequency which may have implications for recombination repair of DNA. The biological significance of a reduction below the spontaneous frequency is not known. The number of mice in each treatment group in this study was small (n = 10 or n = 20). Therefore, repetition of this study with a larger number of animals is required to confirm these observations.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Characterization and screening for mutations of the growth arrest-specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer.

Loss of heterozygosity involving the long arm of chromosome 16 is a frequent event seen in a number of human carcinomas, including breast, prostate, hepatocellular, and ovarian cancers. A region found to be commonly deleted in breast and prostate carcinomas is located at 16q24.3, which suggests the presence of a tumor suppressor gene that may be altered in these two malignancies. A detailed physical and transcription map of this region that includes the loci defining the smallest region of deletion has been constructed. This report describes the characterization of a transcript located in this region, the growth arrest-specific 11 (GAS11) gene, which was viewed as a potential tumor suppressor gene due to the expression of its mouse homolog specifically during growth arrest. The gene consists of 11 exons spanning approximately 25 kb. Northern blot analysis identified two ubiquitously expressed mRNAs of 3.4 and 1.8 kb produced by the use of alternative polyadenylation sites. Another gene, C16orf3 (chromosome 16 open reading frame 3), was found to lie within intron 2 of GAS11. This gene appears intronless, is transcribed in the orientation opposite to that of GAS11, and is expressed at low levels. These genes were examined for mutations in breast tumor DNA, and both were excluded as tumor suppressor genes involved in breast cancer.

Comparison of Virulence and Isozyme Phenotypes of Pgt-QCCJ and Great Plains Races of Puccinia graminis f. sp. tritici.

ABSTRACT Stem rust race Pgt-QCCJ was first found in the Great Plains of the United States in 1989, collected primarily from barley. This race became a major part of the Puccinia graminis f. sp. tritici population, even though it is virulent to only a few hard red winter wheat cultivars in the central Great Plains and to barley in the northern Great Plains. It threatens barley production in the northern Great Plains of the United States and Canada due to virulence to Rpg-1. Six differences in virulence and two in isozyme banding patterns from the most similar stem rust races make it unlikely that QCCJ arose as a mutant. Thus, QCCJ likely arose through sexual or parasexual recombination. Sexual recombination in the Great Plains is unlikely, as it has not been detected in many years. Avirulence to 'McNair 70l' is only known from the Pacific Northwest of the United States and adjacent Canada. The rust population in this area is of sexual origin, and the pattern of virulence/avirulence and isozyme banding for QCCJ occurs there. Pgt-QCCJ likely originated in the Pacific Northwest during or before 1989 and was wind-transported into the Great Plains.

Muscarinic and ganglionic blocking properties of atropine compounds in vivo and in vitro: time dependence and heart rate effects.

High doses (3 mg/kg) of methylatropine nitrate have been used in vivo to produce long-lasting muscarinic blockade during physiologic experiments. At these levels, the possibility exists that ganglionic blockade may also be responsible for some heart rate effects. Therefore, the effects of methylatropine nitrate (0.0012-2.4 mg.kg(-1)) and atropine sulfate (0.0036 - 0.060 mg.kg(1)) were evaluated in vivo using conscious dogs and in vitro using canine right atria and isolated stellate ganglia. The lowest doses of either agent given in vivo caused bradycardia, while intermediate doses induced excess tachycardia. High doses of methylatropine nitrate transiently decreased the heart rate, followed by slow recovery. In vitro using the canine right atria, neither drug caused pacemaker shifts nor directly altered the atrial rate, but postvagal tachycardia occurred with acetylcholine challenge and was prevented by metoprolol or 6-hydroxydopamine. In vitro studies using the canine stellate ganglia indicate that both agents depressed postganglionic compound action potentials at high doses. In conclusion, with high-dose methylatropine nitrate, ganglionic blockade yields the mechanism for a reduction of excess tachycardia as well as a likely explanation for opposing chronotropic effects in conscious and anesthetized dogs. In experimental studies where high doses of atropine compounds are used for long-term muscarinic blockade, it is possible that ganglionic blocking effects may also be produced.

Fourier transform magnitudes are unique pattern recognition templates.

Fourier transform magnitudes are commonly used in the generation of templates in pattern recognition applications. We report on recent advances in Fourier phase retrieval which are relevant to pattern recognition. We emphasise in particular that the intrinsic form of a finite, positive image is, in general, uniquely related to the magnitude of its Fourier transform. We state conditions under which the Fourier phase can be reconstructed from samples of the Fourier magnitude, and describe a method of achieving this. Computational examples of restoration of Fourier phase (and hence, by Fourier transformation, the intrinsic form of the image) from samples of the Fourier magnitude are also presented.