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.

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.

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.

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.

Integration of the gene for carboxin resistance does not impact the Ustilago maydis-maize interaction.

A previous report indicated that insertion of the carboxin resistance (cbxR) gene into the Ustilago maydis genome impaired the pathogenic ability of the fungus towards Zea mays, the corn host. Because we had anecdotal evidence from work in our laboratory that this was not necessarily the case we decided to determine how general was the observation of reduced pathogenicity associated with cbxR. To accomplish this we tested the pathogenicity of several strains that had been transformed with the cbxR gene and compared them with non-transformed strains or strains transformed with the gene conferring hygromycin resistance which is a commonly used selectable marker in this fungus. Our results indicate that carboxin resistance does not significantly alter pathogenicity and is therefore a suitable marker for use in genetic analysis of U. maydis.

Double chitin synthetase mutants from the corn smut fungus Ustilago maydis.

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize (Zea mays) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3/chs4, and chs4/chs5. Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO2+ , and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.