First Report of Pantoea ananatis causing leaf streak disease on wheat (Triticum aestivum) in the United States of America.

Wheat (Triticum aestivum) loses 21.5% yield to pests and diseases annually (Savary et al. 2019). Among the wheat diseases, bacterial leaf streak (BLS) is a growing problem, costing $78.5 million in losses (https://cropprotectionnetwork.org/). In July 2022, we sampled winter wheat leaf samples at Volga (44.30, -96.92), South Dakota, USA with an estimated disease incidence of 40% (n=100). The typical symptoms were water-soaking with large necrotic and chlorotic streaks extending the length of the leaves and were strikingly similar to BLS. To isolate the pathogen, leaves were cut lengthwise into 1 cm pieces and surface-sterilized using a 10% NaOCl solution for 3 min, followed by 70% ethanol for 3 min, and then rinsed with sterile distilled water and placed in 500 ul of sterile distilled water for 5 min and using a sterile loop the water was streaked over a plate of Nutrient Agar (NA). Following Duveiller et al. (1997), the streaked plate was incubated in the dark at 28℃ for 48 h. Observed single colonies were sub-cultured thrice onto fresh NA plates to obtain a pure culture. We named the culture SD101. Bacteria were found to be gram-negative with a colony morphology initially raised, smooth, and white that later turned yellow. DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's protocol, and sequenced using Nanopore MinION R9.4 (Oxford Nanopore Technology). We used the Rapid Annotation Using Subsystems Technology approach (Aziz eal. 2008) to extract the 16S rDNA, DNA gyrase subunit B (gyrB), and translation initiation factor IF-2 (infB) gene sequences that were deposited in GenBank under accession numbers PP329908.1 for 16S rDNA, PP496481 for infB, and PP328920.1 for gyrB. Homology analysis using CLC Genomics Workbench 22.0.2 (QIAGEN) and BLASTn against the GenBank nucleotide database resulted in a 99.74% match (1543/ 1547 bp) of the 16S sequence, 99.59% match (2674/ 2685 bp) of the infB sequence, and 99.42% match (2396/ 2410 bp) of the gyrB sequence with Pantoea ananatis strain AJ13355 (AP012032). To test pathogenicity, seeds of spring wheat breeding line SD4892 were planted in 30 cm × 30 cm pots in a greenhouse under a 16 h light photoperiod. The inoculum was prepared from 48-h-old NA plates of SD101 rinsed with 1X Phosphate Buffer Saline (PBS buffer), adjusted to an OD600 = 1.0, and amended with two drops of Tween 20 (polyoxyethylene sorbitol ester, Millipore Sigma). PBS with Tween 20 was used as a negative control. The inoculum was sprayed on 15 replicates of 15-day-old seedlings, kept at 95% relative humidity for 48 h, then moved to the greenhouse at 23 to 25°C. The symptoms appeared as water soaking that later turned to necrotic streaks with surrounding chlorosis on all 15 inoculated plants while control plants remained healthy. The pathogen was re-isolated from the leaves as described above. The 16S rDNA, infB, and gyrB sequences amplified and sequenced were identical to the gene sequences from the whole genome sequencing. The experiment was repeated with the same results, completing Koch's postulates. Although P. ananatis is pathogenic on corn, rice, and other plant species in the United States (Coutinho et al. 2009), and was reported pathogenic on wheat in Poland (Krawczyk et al. 2020), this is the first report of its pathogenicity on wheat in the United States. The prevalence, and incidence of BLS disease caused by P. ananatis in wheat is needed to estimate its threat to wheat production and to develop management strategies.

Histopathological Investigation of Varietal Responses to Cercospora beticola Infection Process on Sugar Beet Leaves.

Cercospora leaf spot (CLS) is the most destructive foliar disease in sugar beet (Beta vulgaris). It is caused by Cercospora beticola Sacc., a fungal pathogen that produces toxins and enzymes which affect membrane permeability and cause cell death during infection. In spite of its importance, little is known about the initial stages of leaf infection by C. beticola. Therefore, we investigated the progression of C. beticola on leaf tissues of susceptible and resistant sugar beet varieties at 12-h intervals during the first 5 days after inoculation using confocal microscopy. Inoculated leaf samples were collected and stored in DAB (3,3'-diaminobenzidine) solution until processed. Samples were stained with Alexa Fluor-488-WGA dye to visualize fungal structures. Fungal biomass accumulation, reactive oxygen species (ROS) production, and the area under the disease progress curve were evaluated and compared. ROS production was not detected on any variety before 36 h postinoculation (hpi). C. beticola biomass accumulation, percentage leaf cell death, and disease severity were all significantly greater in the susceptible variety compared with the resistant variety (P < 0.05). Conidia penetrated directly through stomata between 48 to 60 hpi and produced appressoria on stomatal guard cells at 60 to 72 hpi in susceptible and resistant varieties, respectively. Penetration of hyphae inside the parenchymatous tissues varied in accordance with time postinoculation and varietal genotypes. Overall, this study provides a detailed account to date of events leading to CLS disease development in two contrasting varieties.

First Report of Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 µm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and ß-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (ß-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (ß-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

Expansion of Internal Hyphal Growth in Fusarium Head Blight-Infected Grains Contributes to the Elevated Mycotoxin Production During the Malting Process.

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Worldwide surveys of commercial beers have reported that the trichothecene mycotoxin deoxynivalenol (DON) is the most frequent contaminant in beer. Although the DON content of grain generally declines during steeping due to its solubilization, Fusarium spp. can continue to grow and produce DON from steeping through the early kilning stage of malting. DON present on malt is largely extracted into beer. The objective of the current study was to localize the growth of Fusarium spp. within FHB-infected kernels by developing an improved method and to associate fungal growth with the production of DON during malting. FHB-infected barley, wheat, rye, and triticale grains that exhibited large increases in the amount of Fusarium Tri5 DNA and trichothecene mycotoxins following malting were screened for hyphal localization. The growth of fungal hyphae associated with grain and malt was imaged by scanning electron microscopy and confocal laser-scanning microscopy assisted with WGA-Alexa Fluor 488 staining, respectively. In barley, hyphae were present on or within the husk, vascular bundle, and pericarp cavities. Following malting, vast hyphal growth was observed not only in these regions but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. However, these grains already had an extensive internal presence of Fusarium hyphae in the unmalted grain, thus representing an enhanced chance of fungal expansion during the malting.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization.

BACKGROUND: In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis. RESULTS: We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. CONCLUSIONS: RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

The Barley HvWRKY6 Transcription Factor Is Required for Resistance Against Pyrenophora teres f. teres.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt) has emerged as a global threat to barley production and diverse populations of Ptt have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse Ptt isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised Ptt resistance were identified from an M2 population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F2 populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F1 individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F2 individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the HvWRKY6 transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of HvWRKY6 in barley line CI5791 resulted in Ptt susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of HvWRKY6 from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that HvWRKY6 functions as a conserved signaling component of defense mechanisms that restricts Ptt growth in barley.

Characterization of genes required for both Rpg1 and rpg4-mediated wheat stem rust resistance in barley.

BACKGROUND: Puccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an "integrated sensory domain" resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9). RESULTS: The rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F6:7 recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein. CONCLUSION: Genetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.

Shedding Light on Penetration of Cereal Host Stomata by Wheat Stem Rust Using Improved Methodology.

Asexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.

Molecular manipulation of the mating-type system and development of a new approach for characterizing pathogen virulence in Pyrenophora tritici-repentis.

The ascomycete Pyrenophora tritici-repentis (Ptr) is an important fungal pathogen worldwide that causes tan spot of wheat. The fungus is self-fertile because each isolate contains both mating type (MAT) idiomorphs. In this work, we developed knockouts of the MAT genes in Ptr and tested fertility of the knockout strains and outcrossing between the knockout strains carrying the opposite mating type. The fungal strains with deletions of either MAT1-1-1 or MAT1-2-1 did not form mature pseudothecia making them functionally heterothallic. The cross between the heterothallic strains of the same isolate (86-124) was fully fertile with the only difference compared to the homothallic wild type strain being the slightly lower percentage of pseudothecium formation. However, the cross between 86-124 (race 2, ToxA-containing isolate) and DW5 (race 5, ToxB-containing isolate) was partially fertile and had fewer mature pseudothecia. Furthermore, most mature asci produced only two or four instead of eight functional ascospores. A collection of ascospores from this cross was obtained and genotyped for the presence of the ToxA, ToxB and MAT genes as well as simple sequence repeat markers. The segregation of these genes and markers and recombination of different allele types at these loci was observed. This work clearly demonstrates that the fungus requires both MAT genes for sexual production and can undergo outcrossing and sexual recombination. It also establishes a new and practical way for further characterizing fungal virulence in Ptr through the development of segregating fungal populations and subsequent genetic analysis.