A Multilocus Phylogeny Places Hymenula cerealis (syn. Cephalosporium gramineum) in the Helotiales, Leotiomycetes.

The soilborne fungus Hymenula cerealis causes Cephalosporium stripe, a vascular wilt disease of wheat and other grasses in the United States and other wheat-producing countries where winter wheat is subjected to snow cover and frozen soil. No sexual stage is known for H. cerealis, and consequently, its phylogenetic position relative to other fungi has been difficult to establish. The purpose of this study was to conduct a multilocus sequence analysis to determine the phylogenetic position of H. cerealis. Sequence data for five genes, the internal transcribed spacer (ITS), partial large subunit nrDNA (LSU), partial RNA polymerase II second largest subunit region (RPB2), ß-tubulin gene, and translation elongation factor 1-α (TEF1-α), from a diverse set of C. gramineum isolates and other related fungi was obtained from GenBank or directly from isolates in the Murray lab and used to construct maximum-likelihood and Bayesian trees. Based on phylogenetic analysis of the single LSU and ß-tubulin genes, Cephalosporium gramineum is closely related to the Drepanopezizaceae and Ploettnerulaceae of Helotiales. Based on analyses of the DNA sequence of the ITS, RPB2, and TEF1-α genes, as well as the combined five-gene data set, C. gramineum belongs to the family Drepanopezizaceae, which is a sister taxon to the Ploettnerulaceae, and formed a well-supported clade (MLBP/BIPP = 95%/100%). In conclusion, H. cerealis belongs to the Helotiales, Leotiomycetes.

Seed Infection Rate, but Not Pathogen Titer, Positively Correlates with Disease Index of Cephalosporium Stripe in Winter Wheat.

Cephalosporium gramineum survives primarily in colonized plant residue but is also transmitted by seed at a low frequency. The purpose of this study was to correlate disease intensity in the field with percentage of infected seed and amount of pathogen DNA using a high-throughput PCR method. Field-grown seed of three wheat cultivars was collected over 4 years from plots with a known disease index. The culture-based seed infection rate was determined by isolation of C. gramineum from 2,016 seeds per seed lot. DNA of 380 seeds from each seed lot was extracted individually, and a PCR assay with a fluorescent-labeled forward primer for detecting C. gramineum was performed on each seed. C. gramineum was isolated from 0.12% of the seed on average (range 0 to 0.74%), whereas it was detected in 3.7% on average (range 1.3 to 7.6%) using PCR detection. The single-seed PCR assay was more sensitive than either the culture-based method or conventional PCR. DNA of 674 seeds that tested positive by this PCR was quantified using a real-time PCR with newly designed primers for the amount of pathogen per seed. Seed contained 0.017 to 77.1 pg/seed of C. gramineum DNA (mean 3.0 pg/seed). Disease index was positively correlated with seed infection rate but not with pathogen titer in seed. This fluorescent-labeled PCR, along with quantitative PCR, improved our understanding of seed transmission of C. gramineum in wheat.

Carbohydrate Accumulation and Differential Transcript Expression in Winter Wheat Lines with Different Levels of Snow Mold and Freezing Tolerance after Cold Treatment.

Winter wheat (Triticum aestivum L.) undergoes a period of cold acclimation in order to survive the ensuing winter, which can bring freezing temperatures and snow mold infection. Tolerance of these stresses is conferred in part by accumulation of carbohydrates in the crown region. This study investigates the contributions of carbohydrate accumulation during a cold treatment among wheat lines that differ in their snow mold tolerance (SMT) or susceptibility (SMS) and freezing tolerance (FrT) or susceptibility (FrS). Two parent varieties and eight recombinant inbred lines (RILs) were analyzed. The selected RILs represent four combinations of tolerance: SMT/FrT, SMT/FrS, SMS/FrT, and SMS/FrS. It is hypothesized that carbohydrate accumulation and transcript expression will differ between sets of RILs. Liquid chromatography with a refractive index detector was used to quantify carbohydrate content at eight time points over the cold treatment period. Polysaccharide and sucrose content differed between SMT and SMS RILs at various time points, although there were no significant differences in glucose or fructose content. Glucose and fructose content differed between FrT and FrS RILs in this study, but no significant differences in polysaccharide or sucrose content. RNAseq was used to investigate differential transcript expression, followed by modular enrichment analysis, to reveal potential candidates for other mechanisms of tolerance, which included expected pathways such as oxidative stress, chitinase activity, and unexpected transcriptional pathways. These differences in carbohydrate accumulation and differential transcript expression begin to give insight into the differences of wheat lines when exposed to cold temperatures.

Genetic Dissection of Snow Mold Tolerance in US Pacific Northwest Winter Wheat Through Genome-Wide Association Study and Genomic Selection.

Snow mold is a yield-limiting disease of wheat in the Pacific Northwest (PNW) region of the US, where there is prolonged snow cover. The objectives of this study were to identify genomic regions associated with snow mold tolerance in a diverse panel of PNW winter wheat lines in a genome-wide association study (GWAS) and to evaluate the usefulness of genomic selection (GS) for snow mold tolerance. An association mapping panel (AMP; N = 458 lines) was planted in Mansfield and Waterville, WA in 2017 and 2018 and genotyped using the Illumina® 90K single nucleotide polymorphism (SNP) array. GWAS identified 100 significant markers across 17 chromosomes, where SNPs on chromosomes 5A and 5B coincided with major freezing tolerance and vernalization loci. Increased number of favorable alleles was related to improved snow mold tolerance. Independent predictions using the AMP as a training population (TP) to predict snow mold tolerance of breeding lines evaluated between 2015 and 2018 resulted in a mean accuracy of 0.36 across models and marker sets. Modeling nonadditive effects improved accuracy even in the absence of a close genetic relatedness between the TP and selection candidates. Selecting lines based on genomic estimated breeding values and tolerance scores resulted in a 24% increase in tolerance. The identified genomic regions associated with snow mold tolerance demonstrated the genetic complexity of this trait and the difficulty in selecting tolerant lines using markers. GS was validated and showed potential for use in PNW winter wheat for selecting on complex traits such tolerance to snow mold.

The Identification and Conservation of Tunicaminyluracil-Related Biosynthetic Gene Clusters in Several Rathayibacter Species Collected From Australia, Africa, Eurasia, and North America.

Tunicaminyluracil antibiotics are a novel class of toxigenic glycolipids that are synthesized by several soil-associated Actinomycetes. The acquisition of a tunicaminyluracil biosynthetic gene cluster (TGC) in Rathayibacter toxicus has led to the emergence of the only described, naturally occurring tunicaminyluracil-associated mammalian disease, annual ryegrass toxicity of livestock. Endemic to Australia, R. toxicus is obligately vectored by Anguinid seed gall nematodes to the developing seedheads of forage grasses, in which the bacteria synthesize tunicaminyluracils that may subsequently be consumed by livestock and result in high rates of mortality and morbidity. The potential impact of R. toxicus on U.S. agriculture has led the U.S. Department of Agriculture - Animal and Plant Health Inspection Service to list R. toxicus as a Plant Pathogen Select Agent. R. toxicus is the only characterized phytopathogenic bacterium to produce tunicaminyluracils, but numerous R. toxicus-like livestock poisonings outside Australia suggest additional bacterial sources of tunicaminyluracils may exist. To investigate the conservation of the TGC in R. toxicus and whether the TGC is present in other Rathayibacter species, we analyzed genome sequences of members of the Rathayibacter genus. Putative TGCs were identified in genome sequences of R. toxicus, R. iranicus, R. agropyri, and an undescribed South African Rathayibacter species. In the latter three species, the putative TGCs have homologs of tunicaminyluracil-related genes essential for toxin production, but the TGCs differ in gene number and order. The TGCs appear at least partially functional because in contrast to atoxigenic species, TGC-containing Rathayibacter species were each able to tolerate exogenous applications of tunicamycin from Streptomyces chartreusis. The North American R. agropyri TGC shows extensive diversity among the sequenced isolates, with presense/absense polymorphisms in multiple genes or even the whole TGC. R. agropyri TGC structure does not appear to correlate with date or location of isolate collection. The conservation and identification of tunicaminyluracil-related gene clusters in three additional Rathayibacter species isolated from South Africa, the Middle East, and the United States, suggests a wider global distribution of potentially neurotoxigenic plant-associated bacteria. This potential for additional endemic and exotic toxigenic Rathayibacter species could have widespread and severe implications for agriculture.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Genome-wide association mapping for eyespot disease in US Pacific Northwest winter wheat.

Eyespot, caused by the soil-borne necrotrophic fungi Oculimacula yallundae and O. acuformis, is a disease of major economic significance for wheat, barley and rye. Pacific Northwest (PNW) winter wheat (Triticum aestivum L.) grown in areas of high rainfall and moderate winters is most vulnerable to infection. The objective of this research was to identify novel genomic regions associated with eyespot resistance in winter wheat adapted to the PNW. Two winter wheat panels of 469 and 399 lines were compiled for one of the first genome-wide association studies (GWAS) of eyespot resistance in US winter wheat germplasm. These panels were genotyped with the Infinium 9K and 90K iSelect SNP arrays. Both panels were phenotyped for disease resistance in a two-year field study and in replicated growth chamber trials. Growth chamber trials were used to evaluate the genetic resistance of O. acuformis and O. yallundae species separately. Best linear unbiased predictors (BLUPs) were calculated across all field and growth chamber environments. A total of 73 marker-trait associations (MTAs) were detected on nine different chromosomes (1A, 2A, 2B, 4A, 5A, 5B, 7A, 7B and 7D) that were significantly associated (p-value <0.001) with eyespot resistance in Panel A, and 19 MTAs on nine different chromosomes (1A, 1B, 2A, 2D, 3B, 5A, 5B, 7A, and 7B) in Panel B. The most significant SNPs were associated with Pch1 and Pch2 resistance genes on the long arms of chromosome 7D and 7A. Most of the novel MTAs appeared to have a minor effect on reducing eyespot disease. Nevertheless, eyespot disease scores decreased as the number of resistance alleles increased. Seven SNP markers, significantly associated with reducing eyespot disease across environments and in the absence and presence of Pch1 were identified. These markers were located on chromosomes 2A (IWB8331), 5A (IWB73709), 5B (IWB47298), 7AS (IWB47160), 7B (IWB45005) and two SNPs (Ex_c44379_2509 and IAAV4340) had unknown map positions. The additive effect of the MTAs explained most of the remaining phenotypic variation not accounted for by Pch1 or Pch2. This study provides breeders with adapted germplasm and novel sources of eyespot resistance to be used in the development of superior cultivars with increased eyespot resistance.

Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii).

Aplanobacter agropyri was first described in 1915 by O'Gara and later transferred to the genus Corynebacterium by Burkholder in 1948 but it was not included in the Approved Lists of Bacterial Names in 1980 and, consequently, is not recognized as a validly published species. In the 1980s, bacteria resembling Corynebacterium agropyri were isolated from plant samples stored at the Washington State Mycological Herbarium and from a diseased wheatgrass plant collected in Cardwell, Montana, USA. In the framework of this study, eight additional isolates were recovered from the same herbarium plant samples in 2011. The isolates are slow-growing, yellow-pigmented, Gram-stain-positive and coryneform. The peptidoglycan is type B2γ containing diaminobutyric acid, alanine, glycine and glutamic acid, the cell-wall sugars are rhamnose and mannose, the major respiratory quinone is MK-10, and the major fatty acids are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0, all of which are typical of the genus Rathayibacter. Phylogenetic analysis of 16S rRNA gene sequences placed the strains in the genus Rathayibacter and distinguished them from the six other described species of Rathayibacter. DNA-DNA hybridization confirmed that the strains were members of a novel species. Based on phenotypic, chemotaxonomic and phylogenetic characterization, it appears that strains CA-1 to CA-12 represent a novel species, and the name Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev. is proposed; the type strain is CA-4T (=DSM 104101T;=ATCC TSD-78T).

Afrina sporoboliae sp. n. (Nematoda: Anguinidae) Associated with Sporobolus cryptandrus from Idaho, United States: Phylogenetic Relationships and Population Structure.

The dropseed gall-forming nematode, Afrina sporoboliae sp. n., is described from seed galls of Sporobolus cryptandrus (Poaceae: Chloridoideae: Sporobolinae) collected in Idaho, USA. This is the third report of an Afrina species in North America and the first report of this genus in a natural plant population on this continent. Morphological, morphometric, and molecular analyses placed this nematode in genus Afrina and demonstrated that it differs from Afrina hyparrheniae and Afrina spermophaga by having longer body and stylet lengths for females and males, and from Afrina wevelli by the absence of tip irregularities on the tails of female and presence of lips noticeably protruding beyond the body contour. The new species has several characters that overlap with Afrina tumefaciens, but differs from this species by inducing seed galls, whereas Afrina tumefaciens induces ovoid galls on stems, leaves, and in flower heads. Evolutionary relationships of Afrina sporoboliae sp. n. with other representatives of the family Anguinidae are presented based on analysis of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA and the D2-D3 regions of the rRNA genes. Analysis of 270 sequences of the cox1 gene from 25 populations of Afrina sporoboliae sp. n. revealed seven haplotypes with sequence divergence up to 5%. This study did not demonstrate a significant positive relationship between genetic difference and geographic distance. Seed gall nematodes are important quarantine pests in many countries. The association of this and other seed gall nematodes with Rathayibacter species and their ability to serve as vectors, especially of R. toxicus, is of concern for U.S. agriculture.

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

Rathayibacter toxicus, Other Rathayibacter Species Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings.

Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of Australia. The Rathayibacter species are unusual among phytopathogenic bacteria in that they are transmitted by anguinid seed gall nematodes and produce extracellular polysaccharides in infected plants resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing animals feeding on infected plants to develop convulsions and abnormal gate, which is referred to as "staggers," and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. "agropyri" was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall nematode vectors present in the United States.

Genomic Regions Associated with Tolerance to Freezing Stress and Snow Mold in Winter Wheat.

Plants grown through the winter are subject to selective pressures that vary with each year's unique conditions, necessitating tolerance of numerous abiotic and biotic stress factors. The objective of this study was to identify molecular markers in winter wheat (Triticum aestivum L.) associated with tolerance of two of these stresses, freezing temperatures and snow mold-a fungal disease complex active under snow cover. A population of 155 F2:5 recombinant inbred lines from a cross between soft white wheat cultivars "Finch" and "Eltan" was evaluated for snow mold tolerance in the field, and for freezing tolerance under controlled conditions. A total of 663 molecular markers was used to construct a genetic linkage map and identify marker-trait associations. One quantitative trait locus (QTL) associated with both freezing and snow mold tolerance was identified on chromosome 5A. A second, distinct, QTL associated with freezing tolerance also was found on 5A, and a third on 4B. A second QTL associated with snow mold tolerance was identified on chromosome 6B. The QTL on 5A associated with both traits was closely linked with the Fr-A2 (Frost-Resistance A2) locus; its significant association with both traits may have resulted from pleiotropic effects, or from greater low temperature tolerance enabling the plants to better defend against snow mold pathogens. The QTL on 4B associated with freezing tolerance, and the QTL on 6B associated with snow mold tolerance have not been reported previously, and may be useful in the identification of sources of tolerance for these traits.

Mapping resistance genes for Oculimacula acuformis in Aegilops longissima.

KEY MESSAGE: This study identified three QTL conferring resistance to Oculimacula acuformis in Aegilops longissima and their associated markers, which can be useful in marker-assisted selection breeding for eyespot resistance. Oculimacula acuformis is one of two species of soilborne fungi that cause eyespot of wheat, the other being Oculimacula yallundae. Both pathogens can coexist in the same field and produce elliptical lesions on stem bases of wheat that are indistinguishable. Pch1 and Pch2 are the only two eyespot resistance genes readily available to wheat breeders, but neither provides complete control. A new source of eyespot resistance was identified from Aegilops longissima (2n = 14, S(l)S(l)), a wild relative of wheat. Three QTL for resistance to O. acuformis were mapped in chromosomes 1S(l), 3S(l), and 5S(l) using a recombinant inbred line population developed from the cross Ae. longissima accessions PI 542196 (R) × PI 330486 (S). The three QTL explained 66 % of phenotypic variation by ß-glucuronidase score (GUS) and 84 % by visual rating. These QTL had LOD values of 10.6, 8.8, and 6.0 for GUS score, and 16.0, 10.0, and 13.0 for visual rating. QTL associated with resistance to O. acuformis have similar chromosomal locations as some for resistance to O. yallundae, except that a QTL for resistance to O. yallundae was found in chromosome 7S(l) but not for O. acuformis. Thus, it appears that some genes at the same locus in Ae. longissima may control resistance to both eyespot pathogens. QTL effective against both pathogens will be most useful for breeding programs and have potential to improve the effectiveness and genetic diversity of eyespot resistance.

Genetic variation of wheat streak mosaic virus in the United States Pacific Northwest.

Wheat streak mosaic virus (WSMV), the cause of wheat streak mosaic, is a widespread and damaging pathogen of wheat. WSMV is not a chronic problem of annual wheat in the United States Pacific Northwest but could negatively affect the establishment of perennial wheat, which is being developed as an alternative to annual wheat to prevent soil erosion. Fifty local isolates of WSMV were collected from 2008 to 2010 near Lewiston, ID, Pullman, WA, and the United States Department of Agriculture Central Ferry Research Station, near Pomeroy, WA to determine the amount of genetic variation present in the region. The coat protein gene from each isolate was sequenced and the data subjected to four different methods of phylogenetic analyses. Two well-supported clades of WSMV were identified. Isolates in clade I share sequence similarity with isolates from Central Europe; this is the first report of isolates from Central Europe being reported in the United States. Isolates in clade II are similar to isolates originating from Australia, Argentina, and the American Pacific Northwest. Nine isolates showed evidence of recombination and the same two well-supported clades were observed when recombinant isolates were omitted from the analysis. More polymorphic sites, parsimony informative sites, and increased diversity were observed in clade II than clade I, suggesting more recent establishment of the virus in the latter. The observed diversity within both clades could make breeding for durable disease resistance in perennial wheat difficult if there is a differential response of WSMV resistance genes to isolates from different clades.

Mapping QTL for resistance to eyespot of wheat in Aegilops longissima.

Eyespot is an economically important disease of wheat caused by the soilborne fungi Oculimacula yallundae and O. acuformis. These pathogens infect and colonize the stem base, which results in lodging of diseased plants and reduced grain yield. Disease resistant cultivars are the most desirable control method, but resistance genes are limited in the wheat gene pool. Some accessions of the wheat wild relative Aegilops longissima are resistant to eyespot, but nothing is known about the genetic control of resistance. A recombinant inbred line population was developed from the cross PI 542196 (R) × PI 330486 (S) to map the resistance genes and better understand resistance in Ae. longissima. A genetic linkage map of the S(l) genome was constructed with 169 wheat microsatellite markers covering 1261.3 cM in 7 groups. F(5) lines (189) were tested for reaction to O. yallundae and four QTL were detected in chromosomes 1S(l), 3S(l), 5S(l), and 7S(l). These QTL explained 44 % of the total phenotypic variation in reaction to eyespot based on GUS scores and 63 % for visual disease ratings. These results demonstrate that genetic control of O. yallundae resistance in Ae. longissima is polygenic. This is the first report of multiple QTL conferring resistance to eyespot in Ae. longissima. Markers cfd6, wmc597, wmc415, and cfd2 are tightly linked to Q.Pch.wsu-1S ( l ), Q.Pch.wsu-3S ( l ), Q.Pch.wsu-5S ( l ), and Q.Pch.wsu-7S ( l ), respectively. These markers may be useful in marker-assisted selection for transferring resistance genes to wheat to increase the effectiveness of resistance and broaden the genetic diversity of eyespot resistance.

Influence of Cold-Hardening and Soil Matric Potential on Resistance to Speckled Snow Mold in Wheat.

The influence of soil matric potential, cold-hardening temperature, and duration on resistance to speckled snow mold caused by Typhula ishikariensis in wheat was investigated. Six winter wheat lines were subjected to cold-hardening temperatures of 2 or 4°C for 1, 2, 3, or 4 weeks with soil matric potential of -0.1 or -0.01 MPa. Plants were inoculated with T. ishikariensis after cold-hardening, incubated at 10°C for 25 days in the dark, and then evaluated for regrowth. Overall recovery from snow mold was least when plants were hardened at 2°C for 1 week at -0.01 MPa and greatest when hardened at 4°C for 4 weeks at -0.1 MPa. Survival of plants following snow mold was greater when plants were cold-hardened at 4 than at 2°C and at -0.1 than -0.01 MPa soil matric potential. The greatest difference in survival among lines and correlation with field observations occurred when plants were hardened at 4°C at -0.1 MPa matric potential for 3 weeks. Understanding the influence of temperature and soil matric potential during cold-hardening on speckled snow mold resistance will be useful to breeding programs developing snow-mold-resistant cultivars under controlled environment conditions.

Seed Transmission of Cephalosporium gramineum in Winter Wheat.

Although isolation of Cephalosporium gramineum from wheat (Triticum aestivum) seed has been reported, development of Cephalosporium stripe in plants from infected seed has not been demonstrated experimentally. Winter wheat seed was collected from three experimental field plots where Cephalosporium stripe was present, and C. gramineum was isolated from the seed following surface-disinfection and incubation on a semi-selective medium. C. gramineum was isolated from 0.10 to 0.88% of seed from 11 of 12 cultivars in a field experiment at Pullman, WA, and from 0.10 to 0.30% of seed from 3 of 4 genotypes in a field experiment at Fort Hall, ID; differences among cultivars were not significant in either experiment. C. gramineum was isolated from 0.35 and 0.55% of cv. Stephens plants with no symptoms and severe symptoms, respectively, from a uniform seeding in Pullman. Seed of the four genotypes from Fort Hall and Stephens from Pullman were grown under controlled environment in a soilless potting mix with no added inoculum and in which C. gramineum was not detected. Symptoms of Cephalosporium stripe developed in 0.08 and 0.17% of Stephens and breeding line 87-00314A plants, respectively, from Fort Hall, and from 0.18 and 0.55% of Stephens plants with no symptoms and severe symptoms, respectively. Although development of Cephalosporium stripe in plants grown from seed lots harvested from diseased plants was low, infected seed can provide an important source of inoculum for introducing the pathogen and initiating epidemics in areas where the pathogen did not occur previously.

Influence of pH and Matric Potential on Germination of Cephalosporium gramineum Conidia.

Germination of Cephalosporium gramineum conidia in soil was up to twofold greater at -0.064 MPa than at -0.037 and -0.007 MPa when incubated at 5°C for 2 days. Soil pH from 4.7 to 7.5 did not have a significant influence on germination of conidia and the interaction between soil pH and matric potential on germination was not significant. Soil fungistasis, which was previously observed for conidia of C. gramineum, was not observed in these studies. Germination of conidia on mineral salts agar containing phosphate buffer was significantly less at pH 4.5 than at 5.5, 6.5, or 7.5 at 5°C in one of two experiments; however, pH had no influence on germination at 10 or 20°C in two experiments. Although Cephalosporium stripe is more severe under conditions of high soil moisture and low soil pH, increased germination of conidia in response to these factors does not explain the observed increase in disease.