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.

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.

Sensitivity of Clarireedia spp. to benzimidazoles and dimethyl inhibitors fungicides and efficacy of biofungicides on dollar spot of warm season turfgrass.

Dollar spot caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa) is an economically destructive fungal disease of turfgrass that can significantly compromise turf quality, playability, and aesthetic value. Fungicides are frequently used to manage the disease but are costly and potentially unfavorable to the environment. Repeated use of some active ingredients has resulted in reduced efficacy on C. jacksonii causing dollar spot in cool-season turfgrasses in the US. Experiments were conducted to study fungicide sensitivity of Clarireedia spp. as well as to develop alternatives to fungicides against dollar spot on warm-season turfgrass in Georgia. First, 79 isolates of Clarireedia spp. collected across the state were tested on fungicide-amended agar plates for their sensitivity to thiophanate-methyl (benzimidazole) and propiconazole (dimethyl inhibitor). Seventy-seven isolates (97.5%) were sensitive (0.001 to 0.654 µg/mL) and two isolates (2.5%) were found resistant (>1000 µg/mL) to thiophanate-methyl. However, in the case of propiconazole, 27 isolates (34.2%) were sensitive (0.005 to 0.098 µg/mL) while 52 isolates (65.8%) were resistant (0.101 to 3.820 µg/mL). Next, the efficacy of three bio- and six synthetic fungicides and ten different combinations were tested in vitro against C. monteithiana. Seven bio- and synthetic fungicide spray programs comprising Bacillus subtilis QST713 and propiconazole were further tested, either alone or in a tank mix in a reduced rate, on dollar spot infected bermudagrass 'TifTuf' in growth chamber and field environments. These fungicides were selected as they were found to significantly reduce pathogen growth up to 100% on in vitro assays. The most effective spray program in growth chamber assays was 100% B. subtilis QST713 in rotation with 75% B. subtilis QST713 + 25% propiconazole tank mix applied every 14 days. However, the stand-alone application of the biofungicide B. subtilis QST713 every seven days was an effective alternative and equally efficacious as propiconazole, suppressing dollar spot severity and AUDPC up to 75%, while resulting in acceptable turf quality (>7.0) in field experiments. Our study suggests that increased resistance of Clarireedia spp. to benzimidazoles and dimethyl inhibitors warrants continuous surveillance and that biofungicides hold promise to complement synthetic fungicides in an efficacious and environmentally friendly disease management program.

Genetics of Fusarium head blight resistance in soft red winter wheat using a genome-wide association study.

Host resistance is an effective and sustainable approach to manage the negative impact of Fusarium head blight (FHB) on wheat (Triticum aestivum L.) grain yield and quality. The objective of this study was to characterize the phenotypic responses and identify quantitative trait loci (QTL) conditioning different FHB resistance types using a panel of 236 elite soft red winter wheat (SRWW) lines in a genome-wide association study (GWAS). The panel was phenotyped for five FHB and three morphological traits under two field and two greenhouse environments in 2018-2019 and 2019-2020. We identified 160 significant marker-trait associations (MTAs) for FHB traits and 11 MTAs for plant height. Eleven QTL showed major effects and explained >10% phenotypic variation (PV) for FHB resistance. Among these major loci, three QTL were stable and five QTL exhibited a pleiotropic effect. The QTL QFhb-3BL, QFhb-5AS, QFhb-5BL, QFhb-7AS.1, QFhb-7AS.2, and QFhb-7BS are presumed to be novel. Pyramiding multiple resistance alleles from all the major-effect QTL resulted in a significant reduction in FHB incidence, severity, index, deoxynivalenol (DON), and Fusarium-damaged kernel (FDK) by 17, 43, 45, 55, and 25%, respectively. Further validation of these QTL could potentially facilitate successful introgression of these resistance loci in new cultivars for improved FHB resistance in breeding programs.

New Approaches to an Old Problem: Dollar Spot of Turfgrass.

Dollar spot, caused by fungal pathogens Clarireedia spp. (formerly Sclerotinia homoeocarpa), is the most common and widely distributed disease of turfgrass worldwide. It can drastically reduce the quality of turfgrass species and affect their aesthetic value and playability. Management of dollar spot typically includes a costly program of multiple application of fungicides within a growing season. Consequently, there have been reported cases of fungicide resistance in populations of Clarireedia spp. Host resistance could be an important component of dollar spot management; however, this approach has been hampered by the lack of sources of resistance because nearly all known warm- and cool-season turfgrass species are susceptible. With the recent advancement in genome sequencing technologies, studies on pathogen genomics and host-pathogen interactions are emerging with the hope of revealing candidate resistance genes in turfgrass and genes for virulence and pathogenicity in Clarireedia spp. Large-scale screening of turfgrass germplasm and quantitative trait locus (QTL) analysis for dollar spot resistance are important for resistance breeding, but only a handful of such studies have been conducted to date. This review summarizes currently available information on the dollar spot pathosystem, taxonomy, pathogen genomics, host-pathogen interaction, genetics of resistance, and QTL mapping and also provides some thoughts for future research prospects to better manage this disease.

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.

Turfgrass Disease Diagnosis: Past, Present, and Future.

Turfgrass is a multibillion-dollar industry severely affected by plant pathogens including fungi, bacteria, viruses, and nematodes. Many of the diseases in turfgrass have similar signs and symptoms, making it difficult to diagnose the specific problem pathogen. Incorrect diagnosis leads to the delay of treatment and excessive use of chemicals. To effectively control these diseases, it is important to have rapid and accurate detection systems in the early stages of infection that harbor relatively low pathogen populations. There are many methods for diagnosing pathogens on turfgrass. Traditional methods include symptoms, morphology, and microscopy identification. These have been followed by nucleic acid detection and onsite detection techniques. Many of these methods allow for rapid diagnosis, some even within the field without much expertise. There are several methods that have great potential, such as high-throughput sequencing and remote sensing. Utilization of these techniques for disease diagnosis allows for faster and accurate disease diagnosis and a reduction in damage and cost of control. Understanding of each of these techniques can allow researchers to select which method is best suited for their pathogen of interest. The objective of this article is to provide an overview of the turfgrass diagnostics efforts used and highlight prospects for disease detection.

Evaluating the impact of turf-care products on soil biological health.

Golf courses require extensive use of inputs to meet the needs of playability and aesthetics. The impact of these inputs on soil biological health is largely unknown. Two field trials were conducted at a golf course in Georgia to evaluate short-term effects of wetting agents (Cascade Plus and Duplex [C+D], Revolution [Rev]), plant growth regulators (PrimoMaxx [PM] and Cutless [CL]), and a product called PlantHelper (PH) on soil biological health by measuring microbial abundance and function. Quantitative polymerase chain reaction was used to measure microbial abundance, which included total bacteria, total fungi, and ammonia-oxidizing prokaryotes. Soil respiration and enzyme assays were used as additional indicators of soil health. In bentgrass putting green, total bacteria and ammonia-oxidizing bacteria decreased in abundance in response to the wetting agents and PH, indicating their sensitivity to the products. Whereas C+D stimulated urease activity, Rev and PH caused a short-lived but immediate increase in respiration, indicating that they acted as labile carbon sources. In a bermudagrass fairway, PM was the only product that caused an increase in total bacteria abundance. PrimoMaxx and CL caused a delayed increase in respiration, suggesting that they may have affected the microorganisms indirectly through their impact on root growth and exudate production later. Although CL caused a decrease in urease activity, none of the products significantly affected phosphatase activity. Overall, the products did not seem to have a lasting impact on soil biological health, although long-term studies are needed to confirm these observations.

First Report of Fusarium poae Causing Fusarium Head Blight of Wheat in Georgia, USA.

Fusarium head blight (FHB) is one of the most troublesome fungal diseases challenging US wheat (Triticum aestivum L.) production (Savary et al. 2019). Harmful mycotoxin contamination, primarily due to deoxynivalenol (DON) in the Fusarium-damaged kernels (FDK), can negatively impact human and livestock health (McMullen et al. 1997). Although Fusarium graminearum is the primary causal agent of FHB, several other species including F. poae could pose a risk by producing dangerous mycotoxins such as nivalenol, DON, HT-2, and T-2 (Stenglein 2009). Severe FHB epidemics on wheat have occurred in recent years along with increased corn acreage across the southeast US specifically in Georgia (Ghimire et al. 2020). Five symptomatic wheat heads displaying bleaching symptoms were randomly collected from 19 different fields across 13 counties of Georgia in late spring of 2018. Infected kernels were dipped in 6% sodium hypochlorite for 10 min and rinsed three times with sterilized water. Blot dried kernels were placed on potato dextrose agar (PDA) and incubated for 7 days at 25°C under 12-h photoperiod. Three isolates (GA18W-2.1.6, GA18W-6.1.4, and GA18W-10.2.3) from Terrell, Peach, and Sumter counties exhibited dense, whitish mycelium colony typical of F. poae (Leslie and Summerell 2006). When grown in carboxymethylcellulose broth, isolates produced globose to piriform microconidia (5.1 to 12.4 µm by 4.4 to 11.2 µm) that were aseptate or had a single septation. The morphological identification was further confirmed by DNA sequencing. Single hyphal tip isolates were grown on cellophane overlain on PDA for 10 days. Fungal DNA was extracted using a Qiagen DNeasy Plant Mini Kit. Genomic DNA was sequenced using TEF1 and TEF2 primer pairs that target the translation elongation factor 1-α (EF1-α) locus (O'Donnell et al. 1998). BLASTn query of the obtained sequences of GA18W-2.1.6 (accession no. MT856907) and GA18W-10.2.3 (accession no. MT856909) were identified as F. poae with a 99% sequence homology with GenBank reference accession MK629641, while GA18W-6.1.4 (accession no. MT856908) displayed 100% similarity with F. poae accession KJ947343. Koch's postulates were performed under greenhouse conditions. Three seeds of the FHB susceptible wheat cultivar 'SS8641' were planted in individual cone-tainers with three replications (two cone-tainers/replicate). Wheat plants were vernalized for six weeks and then moved back to the greenhouse. Each F. poae isolate was spray inoculated (50,000 spores/ml) at the flowering stage onto 18-24 wheat heads. A field isolate of F. graminearum was included as a positive control whereas heads mock-inoculated with water were used as a negative control. Inoculated wheat heads were incubated in black plastic bags for 48 hours. Disease severity and FDK were recorded three weeks post inoculation. Disease severities were 6.7% (GA18W-2.1.6), 8.3% (GA18W-10.2.3), and 15.2% (GA18W-6.1.4) compared to 90.0% in the positive control similar to Arrúa et al (2019). No symptoms were observed in the negative control. FDK was 18% (GA18W-2.1.6), 28% (GA18W-10.2.3) and 44% (GA18W-6.1.4). F. poae was re-isolated from the infected heads and found to be morphologically identical to the isolates used for inoculation. To our knowledge, this is the first report of F. poae associated with FHB of wheat in the state of Georgia, USA. F. poae isolates from Georgia might produce mycotoxins in addition to reducing grain yield which needs further study.

Fusarium Head Blight and Rust Diseases in Soft Red Winter Wheat in the Southeast United States: State of the Art, Challenges and Future Perspective for Breeding.

Among the biotic constraints to wheat (Triticum aestivum L.) production, fusarium head blight (FHB), caused by Fusarium graminearum, leaf rust (LR), caused by Puccinia triticina, and stripe rust (SR) caused by Puccinia striiformis are problematic fungal diseases worldwide. Each can significantly reduce grain yield while FHB causes additional food and feed safety concerns due to mycotoxin contamination of grain. Genetic resistance is the most effective and sustainable approach for managing wheat diseases. In the past 20 years, over 500 quantitative trait loci (QTLs) conferring small to moderate effects for the different FHB resistance types have been reported in wheat. Similarly, 79 Lr-genes and more than 200 QTLs and 82 Yr-genes and 140 QTLs have been reported for seedling and adult plant LR and SR resistance, respectively. Most QTLs conferring rust resistance are race-specific generally conforming to a classical gene-for-gene interaction while resistance to FHB exhibits complex polygenic inheritance with several genetic loci contributing to one resistance type. Identification and deployment of additional genes/QTLs associated with FHB and rust resistance can expedite wheat breeding through marker-assisted and/or genomic selection to combine small-effect QTL in the gene pool. LR disease has been present in the southeast United States for decades while SR and FHB have become increasingly problematic in the past 20 years, with FHB arguably due to increased corn acreage in the region. Currently, QTLs on chromosome 1B from Jamestown, 1A, 1B, 2A, 2B, 2D, 4A, 5A, and 6A from W14, Ning7840, Ernie, Bess, Massey, NC-Neuse, and Truman, and 3B (Fhb1) from Sumai 3 for FHB resistance, Lr9, Lr10, Lr18, Lr24, Lr37, LrA2K, and Lr2K38 genes for LR resistance, and Yr17 and YrR61 for SR resistance have been extensively deployed in southeast wheat breeding programs. This review aims to disclose the current status of FHB, LR, and SR diseases, summarize the genetics of resistance and breeding efforts for the deployment of FHB and rust resistance QTL on soft red winter wheat cultivars, and present breeding strategies to achieve sustainable management of these diseases in the southeast US.

First report of the stubby-root nematode Nanidorus minor infecting Paspalum vaginatum, seashore paspalum grass in Georgia, USA.

We found that Nanidorus spp. was pathogenic to seashore paspalum (Paspalum vaginatum) turfgrass as its population increased from 100 to 2,080 nematodes per pot 180 days after inoculation under greenhouse conditions. Morphological measurements of adult females were similar to those described for N. minor. Molecular analysis also confirmed the morphological identification by targeting three different regions of the genomic DNA. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) were used in singleplex PCR. Forward and reverse sequences of each individual primer set were then subjected to multiple alignment and the complimentary sequences were assembled into a consensus sequence. Upon nucleotide blast on the NCBI website, they were all confirmed to be N. minor. A one-step multiplex PCR method using specific primers and a fragment size of 190 bp also confirmed the identity of N. minor. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia.

Ubc2, an ortholog of the yeast Ste50p adaptor, possesses a basidiomycete-specific carboxy terminal extension essential for pathogenicity independent of pheromone response.

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile alpha motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.

MAP kinase and cAMP signaling pathways modulate the pH-induced yeast-to-mycelium dimorphic transition in the corn smut fungus Ustilago maydis.

Acid pH induces the yeast-to-mycelium transition in haploid cells of Ustilago maydis. We tested two signal transduction pathways known to be involved in dimorphism for roles in acid-induced filamentation. In wild-type cells intracellular cAMP levels were reduced under acid growth. A mutant defective in the regulatory subunit of PKA, ubc1, failed to respond to acid induction on solid medium, but in liquid medium showed a mycelial phenotype at acid pH. Mutants in the pheromone-responsive MAP kinase pathway lost the capacity to grow as mycelium at acid pH, while a mutant in the pheromone response-transcriptional regulator, prf1, behaved as wild-type. Filamentation by both ubc1 and prf1 mutants was inhibited by addition of cAMP. A putative MAP kinase cascade adaptor protein gene, ubc2, complemented a previously identified myc mutant strain defective in pH-induced myceliation. These results indicate that pH-dependent dimorphism is regulated by two known signaling pathways but that an effector for cAMP signaling alternative to Ubc1 is present in U. maydis and that Prf1 is not the sole downstream target of MAP kinase signaling.

Cloning and expression analysis of the ornithine decarboxylase gene (PbrODC) of the pathogenic fungus Paracoccidioides brasiliensis.

We describe the isolation and sequencing of PbrODC, the gene encoding ornithine decarboxylase (ODC) in Paracoccidioides brasiliensis. The gene contains a single open reading frame made of 1413 bp with a single intron (72 bp), and encodes a 447 amino acid polypeptide with a predicted molecular weight of 50.0 kDa, an isoelectric point of 4.9 and a high similarity to other fungal ornithine decarboxylases. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae odc null mutant. A phylogenetic tree generated with several fungal ODCs provided additional evidence to favour a taxonomic position for P. brasiliensis as an ascomycetous fungus, belonging to the order Onygenales. Expression of the PbrODC gene was determined by Northern analyses during growth of the mycelial and yeast forms, and through the temperature-regulated dimorphic transition between these two extreme phases. Expression of PbrODC remained constant at all stages of the fungal growth, and did not correlate with a previously observed increase in the activity of ornithine decarboxylase at the onset of the budding process in both yeast growth and mycelium-to-yeast transition. Accordingly, post-transcriptional regulation for the product of PbrODC is suggested.

Infection of alternative host plant species by Ustilago maydis.

• Here, the host specificity of the corn smut fungus Ustilago maydis was analyzed, with the long-term objective of understanding the different aspects of its pathogenic behavior. • Axenic plantlets obtained in vitro, including one gymnosperm, monocotyledons and dicotyledons, were inoculated with a diploid strain of U. maydis, incubated in a growth chamber, and observed periodically. • All plants were susceptible to infection. The most common symptoms were growth of fungal mycelium on stems and leaves, increase in root number in monocots, or development of adventitious roots in dicots. Other symptoms - chlorosis, increased anthocyanins, necrosis and stunting - varied among the different plant species. Ustilago penetrated and grew into the plant tissues in the form of pleomorphic mycelium, but no teliospores were formed. Noticeably, the fungus induced formation of lateral buds and tumors in papaya. • The results provide evidence that U. maydis is able to infect a variety of phylogenetically unrelated plants grown under axenic conditions. These results may be useful in the analysis of different phenomena associated with the complex pathogenic behavior of U. maydis.

Use of PCR to detect infection of differentially susceptible maize cultivars by Ustilago maydis strains of variable virulence / Utilización de la PCR para la detección de cultivars de maíz diferencialmente susceptibles por cepas de Ustilago maydis de virulencia variable

Ustilago maydis was specifically detected in infected maize plants by means of the polymerase chain reaction (PCR) using oligonucleotides corresponding to a specific region downstream of the homeodomain of the bE genes of the pathogen. The reaction gave rise to amplification of a ca. 500-bp product when tested with U. maydis DNA, but no amplification was detected with DNA from fungi not related to U. maydis. Using these primers, U. maydis was detected in infected maize plants from differentially susceptible cultivars as early as 4 days after inoculation with strains of variable degrees of virulence. Detection of U. maydis at early stages of infection, or in asymptomatic infected plants should assist in studies on plant-pathogen interactions (AU)

Mediante el empleo de PCR se detectó específicamente Ustilago maydis en plantas de maíz infectadas, usando oligonucleótidos correspondientes a una región específica situada pasado el homodominio de los genes bE del patógeno. La reacción amplifica una región de aproximadamente 500 pares de bases si se emplea sobre DNA de U. maydis, pero no muestra ninguna amplificación si se utiliza DNA de hongos no relacionados con U. maydis. Mediante estos cebadores, se detectó U. maydis en plantas de maíz de cultivars con distintas susceptibilidades, incluso cuando tan solo habían pasado cuatro días desde su inoculación con cepas con distintos grados de virulencia. La detección de U. maydis en los primeros estadios de la infección o en plantas asintomáticas infectadas es muy útil en los estudios de las relaciones entre plantas y patógenos. (AU)

Use of PCR to detect infection of differentially susceptible maize cultivars using Ustilago maydis strains of variable virulence.

Ustilago maydis was specifically detected in infected maize plants by means of the polymerase chain reaction (PCR) using oligonucleotides corresponding to a specific region downstream of the homeodomain of the bE genes of the pathogen. The reaction gave rise to amplification of a ca. 500-bp product when tested with U. maydis DNA, but no amplification was detected with DNA from fungi not related to U. maydis. Using these primers, U. maydis was detected in infected maize plants from differentially susceptible cultivars as early as 4 days after inoculation with strains of variable degrees of virulence. Detection of U. maydis at early stages of infection, or in asymptomatic infected plants should assist in studies on plant-pathogen interactions.

The Ustilaginales as plant pests and model systems.

The Ustilaginales are a vast and diverse group of fungi, which includes the plant pathogenic smuts that cause significant losses to crops worldwide. Members of the Ustilaginales are also valuable models for the unraveling of fundamental mechanisms controlling important biological processes. Ustilago maydis is an important fungal model system and has been well studied with regard to mating, morphogenesis, pathogenicity, signal transduction, mycoviruses, DNA recombination, and, recently, genomics. In this review we discuss the life cycles of members of the Ustilaginales and provide background on their economic impact as agricultural pests. We then focus on providing a summary of the literature with special attention to topics not well covered in recent reviews such as the use of U. maydis in mycovirus research and as a model for understanding the molecular mechanisms of fungicide resistance and DNA recombination and repair.