Probe-based loop-mediated isothermal amplification assay for rapid detection of two Clarireedia spp., the causal agent of dollar spot of turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental US. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount purified DNA needed for detection was 0.05 ng µL-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple and reliable detection in 1.25 hours. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Dose-dependent physiological effects of UV-C radiation on seashore paspalum.

Positive effects of ultraviolet-C (UV-C) radiation on plants have been documented in previous literature with a focus on extending shelf life and reducing disease development. However, its effect on plant growth habits has been scarcely explored, especially in turfgrass where a compact shoot growth is a desirable trait. Seashore paspalum (Paspalum vaginatum) is a warm-season perennial turfgrass requiring low fertilizer and pesticide inputs. This project aimed to test the effects of different doses of UV-C radiation on growth and performance of seashore paspalum cv. Seastar. Here, we provide evidence of dose-dependent effects. Lower UV-C doses (6 s and 1 min daily) improved the performance of seashore paspalum, as manifested by higher tiller density, reduced clipping yields, increased chlorophyll level on selected dates as well as enhanced photosynthetic efficiency compared to control. Contrastingly, higher doses (6 min and 30 min daily) resulted in severe damage with 30-min treatment being lethal to seashore paspalum, causing marked declines in all measured parameters. This is the first time that UV-C-induced growth response was reported in turf. Conclusions drawn from this study would shed light into the effects of UV-C radiation on the growth and performance of seashore paspalum and offer exciting potential for the utilization of UV-C at non-lethal dosage in turfgrass management.

Genetic Diversity, Mycotoxin Profiles, and Population Structure of Fusarium spp. Associated with Fusarium Head Blight in Georgia, U.S.A.

Fusarium head blight (FHB) has become a limiting factor in soft red winter wheat production in the southeast US. Recent epidemics have occurred in Georgia, however genetic information on the Fusarium species responsible for FHB is unknown. This study aimed to assess pathogen population structure and genetic diversity, trichothecene profiles, and representative pathogenicity of 196 Fusarium isolates collected from 44 wheat (n = 85) and 53 corn (n = 111) fields in Georgia. Phylogenetic analysis using the translation elongation factor 1-alpha (635 bp) and RNA polymerase second largest subunit (930 bp) sequence data resolved isolates into 185 haplotypes, representing 12 Fusarium species grouped under five species complexes. F. graminearum with 15-acetyl-deoxynivalenol (15ADON) chemotype (75.6%) and F. incarnatum (57.7%) predominated in wheat and corn, respectively, with a surprisingly higher frequency of NIV F. graminearum (21.8%). Using nine variable number of tandem repeat markers, 82 multilocus genotypes out of 86 F. graminearum isolates were identified and grouped into two genetic clusters, pop1fg (n = 29) and pop2fg (n = 32), as part of the North American populations (NA1 and NA2), but with no chemotype differentiation. F. graminearum populations in Georgia are mostly clonal and might have evolved through at least two introductions from the northeast US and Canada and local adaptation to maintain high genetic diversity. Pathogenicity of F. graminearum isolates from wheat and corn had high FHB severity (>60%) in wheat, depicting the risk they can pose towards future FHB outbreaks. Overall, this baseline study provided important information on Fusarium species diversity including F. graminearum associated with FHB in Georgia that will be useful to formulate integrated disease management incorporating improved host resistance and fungicide spray program.

Detection of Maternal and Cytoplasmic Effects on Resistance to Zymoseptoria tritici in Durum Wheat.

Septoria tritici blotch (STB) is a major disease problem of wheat worldwide. To optimize the introgression of resistance genes in elite genotypes throughout traditional or molecular breeding programs, a full understanding of the quantitative inheritance of resistance to Zymoseptoria tritici, plant height (PH), and thousand kernel weight (TKW) is needed. In this study, maternal and cytoplasmic effects of resistance to STB were investigated using P1 (susceptible, high-yielding line) and P2 (resistant, low-yielding line) durum wheat lines and their F1, RF1, F2, RF2, BC1, RBC1, BC2, and RBC2 progeny, assessed for resistance to STB during three growing seasons. Duncan mean's analysis revealed significant differences between generation means for STB, PH, and TKW. The two parents had an extreme pattern. The F1 and RF1 segregated close to their respective parents, suggesting the presence of cytoplasmic and maternal genetic effects for Z. tritici resistance, PH, and TKW. Separate generation mean's analysis confirmed the results of the Duncan test. A three-parameter model was found to be not adequate for all traits in all three growing years; while a digenic epistatic model with cytoplasmic or/and maternal effect was adequate for all cases. Narrow-sense heritability was in the range of 50-60%, 30-69%, and 28-31% for STB, PH, and TKW, respectively. For STB, high heritability and the presence of fixable epistatic effect is encouraging and could lead to creating varieties with the right female parent to exploit cytoplasmic and maternal effects in order to improve resistance to Z. tritici in durum wheat.

Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass.

Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, Clarireedia monteithiana and C. jacksonii (formerly Sclerotinia homoeocarpa) have been reported so far in the United States To study the Clarireedia genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced via Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as C. aff. paspali, a novel species in the United States Using short and long reads, C. aff. paspali H3 contained 193 contigs with 48.6 Mbp and presented the most completed assembly and annotation among Clarireedia species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher Clarireedia pathogenicity, C. aff. paspali genomes (H2 and H3), as well as available C. jacksonii (LWC-10 and HRI11), C. monteithiana (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in S. sclerotiorum. Seventeen orthologs of pathogenicity genes have been identified in Clarireedia species involved in oxalic acid production (pac1, nox1), mitogen-activated protein kinase cascade (pka1, smk3, ste12), appressorium formation (caf1, pks13, ams2, rgb1, rhs1) and glycolytic pathway (gpd). Within these genes, 366 species-specific SNPs were recorded between Clarireedia species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among Clarireedia spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among Clarireedia species.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 µm (average 8.24 µm) wide by 15.96 to 29.26 µm (average 25.40 µm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

First report of stem rust, caused by Puccinia graminis, and crown rust, caused by Puccinia coronata var. avenae f. sp. avenae, on tall fescue (Festuca arundinacea) in Georgia, USA.

Stem rust, caused by Puccinia graminis, and crown rust, caused by P. coronata, are common rust diseases on cool-season grasses (Karakkat et al. 2018), for which long-distance spore dispersal was recorded in northern US (Harder and Haber 1992). During the summers of 2019 and 2020, severe infection of stem rust and crown rust was observed on > 60% of tall fescue (Festuca arundinacea) germplasm plants in a breeding nursery located at the University of Georgia, Griffin GA. Rust-infected leaves first presented uredinia pustules, then black telia towards the end of the season. The uredinia pustules of stem rust and crown rust were brick-red and, yellow and arranged along the host veins, respectively. The urediniospores were one-celled, round to ovoid and measured from 20.75±2.44 µm (crown rust) to 27±3.60 µm long (stem rust). The teliospores were two-celled and measured from 45.75±10.14 µm (stem rust) to 51.60±4.0 µm long (crown rust) (Leonard et al. 2005; Cummins 1971). Urediniospores of both rusts were collected from infected plants in the field in April of 2020 using a Piston vacuum pump (Welch by Gardner Denver Ltd.) and stored at -80 °C in 1.5 ml Eppendorf tubes. Genomic DNA was extracted by grinding the urediniospores in liquid nitrogen using mortar and pestle, followed by the cetyltrimethylammonium bromide method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using the ITS5-ITS4 primers (White et al. 1990). BLASTn and phylogenetic analyses revealed that the sequence of stem rust (GenBank acc. no. MW430963) and crown rust (GenBank acc. no. MW431324) pathogens had >99% similarity with P. graminis (GenBank acc. no. HQ317538) and P. coronata var. avenae f. sp. avenae (clade V; Liu and Hambleton 2013) (GenBank acc. no. EU014044), respectively. Pathogenicity tests were conducted on the tall fescue cultivar 'Bandit'. For each rust, 12 pots (10 cm × 10 cm) were planted, each containing 13 seeds in a Sungro professional growing mix soil (Sun Gro Horticulture Distribution Inc.). The plant materials were kept in the greenhouse at 20°C/ 25°C (night/day),15-hrs of light, and watered twice a week for 4-weeks. Urediniospores were recovered from -80°C and allowed to acclimate at room temperature for 1 h. For each rust, 20 ml of suspension containing 1×105 urediniospores ml-1 and 5 µl of Tween-twenty (Agdia Inc. Elkhart, IN) were used to inoculate 6 pots; while 6 control pots were sprayed with sterile water. After inoculation, plants were allowed to dry for 1 h and then transferred to a dark chamber at 20°C and 90% of humidity for 12-15 h. At 10-days post inoculation, all inoculated plants developed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. Stem and crown rust pathogens were re-isolated from the artificially inoculated tall fescue plants. Based on form, size, color and numbers of cells forming the spores, a 1947 Festuca elatior specimen from Georgia mentioning Puccinia coronata (Hanlin 1966), held at the Julian H. Miller Mycological Herbarium (Catalog No. GAM00013162), was discarded as an earlier record of P. coronata var. avenae and could have been misdiagnosed. Due to the fragile integrity of the original infected plant sample as well as the incipient infection, DNA identification was unsuccessful. To our knowledge, this is the first morphological, genetic and taxonomic report of P. graminis and P. coronata var. avenae f. sp. avenae on tall fescue in Georgia, USA.

Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm (Phoenix dactylifera) and related species.

Date palm (Phoenix dactylifera), one of the most ancient crops, is grown commercially in >30 countries. Using whole plastome assemblies, phylogenetic analyses revealed that cultivated date palm accessions share the same clade with P hoenix sylvestris, P hoenix pusilla and P hoenix acaulis, which are native to the Indian subcontinent, and Phoenix caespitosa that is native to the Arabian Peninsula and the deserts of Somalia. Analysis of genetic diversity and genetic relationships among date palm accessions from 13 producing countries involved 195 date palm accessions that were genotyped at 19 microsatellite loci. Extensive genetic diversity was observed, with many accessions heterozygous for most markers in this clonally propagated crop. The average number of alleles per locus (42.1), expected heterozygosity (0.8), observed heterozygosity (0.47) and fixation indices (FST = 0.42) demonstrated substantial genetic diversity and population structure. Iraqi accessions were found to have the richest allelic diversity, and the most private alleles. The model-based Bayesian method indicated that these accessions could be broadly divided into two structure groups, one group with predominantly African accessions and another predominantly Asian. Some germplasm, especially from Tunisia and Iraq, deviated from this generalization. Many accessions in the STRUCTURE-derived groups were found to be genetic admixtures, with gene flow between Asian and African groups. Indian and Pakistani date palms were found to be most closely related to North African germplasm.

Genetic diversity and population structure of six species of Capparis in Tunisia using AFLP markers.

In order to study the genetic diversity, the phylogeographic pattern and hybridization between six Tunisian Capparis species, 213 accessions of Caper were genotyped with three primer combinations of amplified fragment length polymorphism (AFLP) markers. Out of 750 fragments generated, 636 were polymorphic and 407 of them were restricted to a single species. STRUCTURE and PCoA analyses clearly separated morphologically different populations into six distinct genetic ones. The UPGMA analysis grouped the species into three main clusters: G1 grouped C. spinosa subsp. spinosa var. spinosa and C. sicula subsp. sicula; G2 grouped C. ovata subsp. ovata and C. orientalis and G3 clustered C. zoharyi and C. aegyptia. Populations from G1, G2 and G3 were mainly distributed in arid, subhumid, and semi-arid bioclimates, respectively. Additional genetic studies on Capparis could help to identify genes underlying speciation events and local adaptation to geographic areas leading to the development of breeding programs.