Mellein: Production in culture by Macrophomina phaseolina isolates from soybean plants exhibiting symptoms of charcoal rot and its role in pathology.

Macrophomina phaseolina (Mp) is a fungal pathogen proposed to enter host roots by releasing toxins that induce local necrosis in roots allowing entry of hyphae. Mp is reported to produce several potent phytotoxins, including (-)-botryodiplodin and phaseolinone, but isolates that do not produce these phytotoxins retain virulence. One hypothesis explaining these observations is that some Mp isolates may produce other unidentified phytotoxin(s) responsible for virulence. A previous study of Mp isolates from soybean found 14 previously unreported secondary metabolites using LC-MS/MS, including mellein, which has various reported biological activities. This study was conducted to investigate the frequency and amounts of mellein produced in culture by Mp isolates from soybean plants exhibiting symptoms of charcoal rot and to investigate the role of mellein in any observed phytotoxicity. LC-MS/MS analysis of cell-free culture filtrates (CCFs) from 89 Mp isolates revealed that 28.1% produced mellein (49-2,203 µg/L). In soybean seedlings in hydroponic culture, Mp CCFs diluted to 25% (vol/vol) in hydroponic growth medium induced phytotoxic symptoms with frequencies of 73% chlorosis, 78% necrosis, 7% wilting, and 16% death, and at 50% (vol/vol) induced phytotoxicity with frequencies of 61% chlorosis, 82% necrosis, 9% wilting, and 26% death. Commercially-available mellein (40-100 µg/mL) in hydroponic culture medium induced wilting. However, mellein concentrations in CCFs exhibited only weak, negative, insignificant correlations with phytotoxicity measures in soybean seedlings, suggesting that mellein does not contribute substantially to observed phytotoxic effects. Further investigation is needed to determine if mellein plays any role in root infection.

Defining Fungicide Resistance Mechanisms in the Corynespora cassiicola Population from Mississippi Soybean.

Target spot, caused by Corynespora cassiicola, is a common lower canopy soybean disease in the southern United States. Recently, target spot has resurged in importance especially following the identification of resistance to the quinone outside inhibitor (QoI) fungicides. As a result, a survey of C. cassiicola from soybean throughout Mississippi began in 2018. A total of 819 C. cassiicola monoconidial isolates were obtained from 228 fields in 75 counties. The molecular mechanism of QoI resistance was determined, which resulted from an amino acid substitution from glycine (G) to alanine (A) at position 143 using a PCR-RFLP method and comparing nucleotide sequences of the cytochrome b gene. Five previously defined geographic regions were used to present the distribution of the G143A substitution and included the Capital, Coast, Delta, Hills, and Pines. The Capital had the greatest proportion of G143A-containing isolates (95.0%), followed by the Coast (92.9%), Delta (89.8%), Pines (78.8%), and Hills (69.4%). In all, 85.8% of the C. cassiicola isolates carried the G143A substitution. In addition, the effective fungicide concentration (EC50) of randomly selected C. cassiicola isolates to azoxystrobin was used to characterize isolates as resistant (n = 14) (based on the presence of the G143A substitution and EC50 values >52 µg/ml) or sensitive (n = 11) (based on the absence of the G143A substitution and EC50 values <46 µg/ml). The EC50 values varied among isolates (P < 0.0001), with QoI-sensitive isolates exhibiting lower EC50 values than QoI-resistant isolates. The current study revealed that a reduction in sensitivity to QoI fungicides has likely resulted based on the percentage of C. cassiicola isolates containing the G143A substitution identified in Mississippi.

Evaluating In Vitro Fitness Parameters of G143A-Containing and Wild-Type Corynespora cassiicola Isolates from Mississippi Soybean.

Quinone outside inhibitor (QoI) fungicides have been widely used to manage diseases of soybean including target spot caused by Corynespora cassiicola. However, resistance to QoI fungicides has recently been reported within the C. cassiicola population from Alabama, Arkansas, Mississippi, and Tennessee as a result of isolates in the population containing the G143A amino acid substitution. Therefore, the relative fitness and stability of isolates containing the G143A substitution compared with wild-type C. cassiicola isolates from Mississippi soybean were investigated by analyzing several fitness parameters in vitro. In addition, in vivo virulence assays were conducted in the greenhouse on a target spot-susceptible cultivar. The evaluations of fitness considered the difference between isolates from the wild-type and G143A-containing genotypes by evaluating colony growth parameters following the first and the 10th subcultures on microbiological media. When considered as an average of all G143A-containing isolates, the G143A-containing isolates following the 10th subculture produced 6.2% greater colony diameter growth but produced 2.3% less conidia. Conversely, over the same period, wild-type isolates produced 6.7% less colony growth but produced 10.9% more conidia. Based on our results, the C. cassiicola isolates that contained the G143A substitution appear stable since successive subculturing did not significantly affect the measured fitness parameters. The lack of fitness cost accompanying the genotypic shift to the G143A amino acid substitution which confers fungicide resistance in C. cassiicola indicates that these isolates may have fitness advantages and may remain stable in the population as well as displace wild-type isolates with repeated fungicide applications of QoI-containing products.

Metagenomic Analyses of the Soybean Root Mycobiome and Microbiome Reveal Signatures of the Healthy and Diseased Plants Affected by Taproot Decline.

Invading pathogens interact with plant-associated microbial communities, which can be altered under the pressure of pathogen infection. Limited information exists on plant-microbe interactions occurring during natural outbreaks in agricultural fields. Taproot decline (TRD) of soybean is an emerging disease caused by Xylaria necrophora. TRD disease occurrence and yield loss associated with TRD are outstanding issues in soybean production. We applied nuclear ribosomal DNA Internal Transcribed Spacers and 16S rRNA gene taxonomic marker sequencing to define the composition of the fungal and bacterial communities associated with healthy and diseased soybean roots collected from the Mississippi Delta. The plant compartment was a significant factor regulating taxonomic diversity, followed by the disease status of the plant. TRD impacted the root endophytes, causing imbalances; at the intermediate and advanced stages of TRD, X. necrophora decreased mycobiome diversity, whereas it increased microbiome richness. Networks of significant co-occurrence and co-exclusion relationships revealed direct and indirect associations among taxa and identified hubs with potential roles in assembling healthy and TRD-affected soybean biomes. These studies advance the understanding of host-microbe interactions in TRD and the part of biomes in plant health and disease.

Gaeumannomyces nanograminis, sp. nov., a hyphopodiate fungus identified from diseased roots of ultradwarf bermudagrass in the United States.

The genus Gaeumannomyces (Magnaporthaceae, Magnaporthales, Sordariomycetes, Ascomycota) includes root-infecting pathogens, saprobes, and endophytes. Morphological, biological, and phylogenetic analyses were employed to identify fungal isolates derived from turfgrass roots colonized with ectotrophic, dark runner hyphae. Phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes were obtained via maximum likelihood and Bayesian methods. Our isolates consistently formed a distinct and highly supported clade within Gaeumannomyces. Common and distinctive biological and morphological characters reinforced these findings. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davey), its native host, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Gaeumannomyces nanograminis, associated with rotted roots of ultradwarf bermudagrass.

Xylaria necrophora, sp. nov., is an emerging root-associated pathogen responsible for taproot decline of soybean in the southern United States.

Taproot decline (TRD) is a disease of soybean that has been reported recently from the southern United States (U.S.). Symptoms of TRD include foliar interveinal chlorosis followed by necrosis. Darkened, charcoal-colored areas of thin stromatic tissue are evident on the taproot and lateral roots along with areas of necrosis within the root and white mycelia within the pith. Upright stromata typical of Xylaria can be observed on crop debris and emerging from infested roots in fields where taproot decline is present, but these have not been determined to contain fertile perithecia. Symptomatic plant material was collected across the known range of the disease in the southern U.S., and the causal agent was isolated from roots. Four loci, ⍺-actin (ACT), ß-tubulin (TUB2), the nuclear rDNA internal transcribed spacers (nrITS), and the RNA polymerase subunit II (RPB2), were sequenced from representative isolates. Both maximum likelihood and Bayesian phylogenetic analyses showed consistent clustering of representative TRD isolates in a highly supported clade within the Xylaria arbuscula species complex in the "HY" clade of the family Xylariaceae, distinct from any previously described taxa. In order to understand the origin of this pathogen, we sequenced herbarium specimens previously determined to be "Xylaria arbuscula" based on morphology and xylariaceous endophytes collected in the southern U.S. Some historical specimens from U.S. herbaria collected in the southern region as saprophytes as well as a single specimen from Martinique clustered within the "TRD" clade in phylogenetic analyses, suggesting a possible shift in lifestyle. The remaining specimens that clustered within the family Xylariaceae, but outside of the "TRD" clade, are reported. Both morphological evidence and molecular evidence indicate that the TRD pathogen is a novel species, which is described as Xylaria necrophora.

First Report of the Production of Mycotoxins and Other Secondary Metabolites by Macrophomina phaseolina (Tassi) Goid. Isolates from Soybeans (Glycine max L.) Symptomatic with Charcoal Rot Disease.

Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57-79.9 µg/L), moniliformin (61.8% of cultures at 0.011-12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71-1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012-0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants.

Magnaporthiopsis cynodontis, a novel turfgrass pathogen with widespread distribution in the United States.

The genus Magnaporthiopsis of Magnaporthaceae (Magnaporthales, Sordariomycetes, Ascomycota) contains species that are predominantly necrotrophic pathogens, often producing simple hyphopodia and dark, ectotrophic runner hyphae on plant roots and stems during colonization. Fungal isolates from turfgrass roots with dark and ectotrophic runner hyphae were examined and identified based on morphological, biological, and phylogenetic analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions, and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes. Our isolates consistently formed a distinct and highly supported clade within Magnaporthiopsis. These findings were reinforced by common and distinctive biological and morphological characters. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass, one of its native hosts, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Magnaporthiopsis cynodontis, which has widespread distribution in the United States.

Draft genome sequence of Xylaria sp., the causal agent of taproot decline of soybean in the southern United States.

The draft genome of Xylaria sp. isolate MSU_SB201401, causal agent of taproot decline of soybean in the southern U.S., is presented here. The genome assembly was 56.7 Mb in size with an L50 of 246. A total of 10,880 putative protein-encoding genes were predicted, including 647 genes encoding carbohydrate-active enzymes and 1053 genes encoding secreted proteins. This is the first draft genome of a plant-pathogenic Xylaria sp. associated with soybean. The draft genome of Xylaria sp. isolate MSU_SB201401 will provide an important resource for future experiments to determine the molecular basis of pathogenesis.

Curvularia malina sp. nov. incites a new disease of warm-season turfgrasses in the southeastern United States.

A novel species of Curvularia was identified as a foliar pathogen of Cynodon dactylon (bermudagrass) and Zoysia matrella (zoysiagrass), two important warm-season turfgrasses in the southeastern United States. Field symptoms were conspicuous chocolate brown to black spots in turf of both species on golf course putting greens and fairways. Leaves of plants within these spots exhibited prominent, black eyespot lesions from which a darkly pigmented fungus was consistently isolated. The fungus produced gray- to black-olivaceous mycelium within 10 d on potato dextrose agar at 25 C but never produced conidia despite numerous attempts to induce them. Field symptoms were reproduced in inoculated plants of both grasses, and re-isolation of the pathogen from symptomatic tissues confirmed its pathogenicity in fulfillment of Koch's postulates. A phylogenetic analysis was performed using sequence markers of internal nuclear ribosomal transcribed spacer region (ITS), glyceralde-hyde-3-phosphate dehydrogenase (GPD1) and translation elongation factor 1-α (TEF 1). The concatenated phylogenetic tree showed strong support for a new species within Curvularia that is distinctly divergent from other Curvularia spp. Therefore, the darkly pigmented pathogen of warm-season turfgrasses is described and illustrated as a new species, Curvularia malina.

Anthracnose disease of centipedegrass turf caused by Colletotrichum eremochloae, a new fungal species closely related to Colletotrichum sublineola.

Colletotrichum is a cosmopolitan, anamorphic fungal genus responsible for anthracnose disease in hundreds of plant species worldwide, including members of the Poaceae. Anthracnose disease of the widely planted, non-native, warm-season lawn grass, Eremochloae ophiuroides (centipedegrass), is commonly encountered in the southern United States, but the causal agent has never been identified. We use DNA sequence data from modern cultures and archival fungarium specimens in this study to determine the identity of the fungus responsible for centipedegrass anthracnose disease and provide experimental confirmation of pathogenicity. C. eremochloae sp. nov., a pathogen of centipedegrass, is proposed based on phylogenetic evidence from four sequence markers (Apn2, Apn2/ Mat1, Sod2, ITS). C. eremochloae isolates from centipedegrass shared common morphology and phenotype with C. sublineola, a destructive pathogen of cultivated sorghum and Johnsongrass weeds (Sorghum halepense, S. vulgaris). Molecular phylogenetic analysis identified C. eremochloae and C. sublineola as closely related sister taxa, but genealogical concordance supported their distinction as unique phylogenetic species. Fixed nucleotide differences between C. eremochloae and C. sublineola were observed from collections of these fungi spanning 105 y, including the 1904 lectotype specimen of C. sublineola. C. eremochloae was identified from a fungarium specimen of centipedegrass intercepted at a USA port from a 1923 Chinese shipment; the multilocus sequence from this specimen was identical to modern samples of the fungus. Thus, it appears that the fungus might have migrated to the USA around the same time that centipedegrass first was introduced to the USA in 1916 from China, where the grass is indigenous. The new species C. eremochloae is described and illustrated, along with a description and discussion of C. sublineola based on the lectotype and newly designated epitype.

Seasonal Variation in Frequency of Isolation of Ophiosphaerella korrae from Bermudagrass Roots in Mississippi and Pathogenicity and Optimal Growth of Selected Isolates.

Isolation frequency of Ophiosphaerella korrae (spring dead spot pathogen) from Cynodon dactylon (bermudagrass) roots at a golf course near West Point, Mississippi, was monitored over a 3-year investigation. Laboratory and greenhouse experiments were conducted to determine optimal temperatures for the growth of selected O. korrae isolates collected from the field study and to evaluate those isolates for pathogenicity potential. Isolation frequencies of the pathogen from naturally infested root samples were significantly higher in the winter and spring and lowest in the fall regardless of cultural, nutrient, and chemical treatments. Annual soil temperatures ranged between 8 and 29 degrees C, and no correlation was observed between temperature and percent isolation of O. korrae. Optimal in vitro growth of selected O. korrae isolates ranged from 21 to 25 degrees C. Root discoloration was significantly greater in the presence of O. korrae compared to non-inoculated roots in greenhouse studies. Results of this study confirm and are the first to document that O. korrae naturally infests roots throughout the bermudagrass growth cycle, but factors other than temperature and management practices may influence O. korrae in situ.

Two Mutations in ß-Tubulin 2 Gene Associated with Thiophanate-Methyl Resistance in Colletotrichum cereale Isolates from Creeping Bentgrass in Mississippi and Alabama.

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass putting greens during the summer in Mississippi and Alabama over the last 15 years. Thiophanate-methyl is a single-site mode-of-action fungicide applied to control C. cereale. In vitro bioassays were performed to evaluate the sensitivity of 103 isolates to thiophanate-methyl concentrations ranging from 0.039 to 10 µg/ml. Eighty-three isolates were collected from creeping bentgrass in Mississippi and Alabama that had been exposed to thiophanate-methyl. An additional 20 isolates were included from nonexposed turfgrasses. Radial colony growth in amended media was relative to nonamended media for all in vitro bioassays. With thiophanate-methyl at 10 µg/ml, relative growth of exposed isolates ranged from 77.5 to 130.7% with a mean of 99.3% compared with nonexposed, baseline isolates that ranged from 0.0 to 48.7% with a mean of 20.4%. A representative sample of thiophanate-methyl-exposed and nonexposed isolates was used to determine the mechanism of resistance by comparing amino acid sequences of the ß-tubulin 2 protein. All of the thiophanate-methyl-exposed isolates that were sequenced had a point mutation resulting in substitutions from glutamic acid to alanine at position 198 or from phenylalanine to tyrosine at position 200 of the ß-tubulin 2 protein. These amino acid substitutions in C. cereale isolates from Mississippi and Alabama appear to confer resistance to thiophanate-methyl and differ from those reported previously for this pathogen.

Occurrence and Molecular Identification of Azoxystrobin-Resistant Colletotrichum cereale Isolates from Golf Course Putting Greens in the Southern United States.

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass and annual bluegrass putting greens throughout the southern United States. Strobilurin (QoI) fungicides such as azoxystrobin are single-site mode-of-action fungicides applied to control C. cereale. In vitro bioassays with azoxystrobin at 0.031 and 8 µg/ml incorporated into agar were performed to evaluate the sensitivity of 175 isolates collected from symptomatic turfgrasses in Alabama, Mississippi, North Carolina, Tennessee, and Virginia. Three sensitivity levels were identified among C. cereale isolates. Resistant, intermediately resistant, and sensitive isolates were characterized by percent relative growth based on the controls with means of 81, 23, and 4%, respectively, on media containing azoxystrobin at 8 µg/ml. The molecular mechanism of resistance was determined by comparing amino acid sequences of the cytochrome b protein. Compared with sensitive isolates, C. cereale isolates exhibiting QoI resistance had a G143A substitution, whereas isolates expressing intermediate resistance had a F129L substitution. C. cereale isolates displaying azoxystrobin resistance in vitro were not controlled by QoI fungicides in a field evaluation. The dominance of QoI-resistant C. cereale isolates identified in this study indicates a shift to resistant populations on highly managed golf course putting greens.

Biologically active tetranorditerpenoids from the fungus Sclerotinia homoeocarpa causal agent of dollar spot in turfgrass.

Nine new tetranorditerpenoid dilactones (2-10), together with two previously reported norditerpenoids dilactones (1, 11), and two known putative biosynthetic intermediates, oidiolactone-E (12) and 13, were isolated from an ethyl acetate extract of a culture medium of Sclerotinia homoeocarpa. Structures and absolute configurations of these compounds were determined by spectroscopic methods and confirmed by X-ray crystallographic analysis of representative compounds. Compounds were evaluated for herbicidal, antiplasmodial, and cytotoxic activities. Compounds 1, 2, 6, 7, and 11 were more active as growth inhibitors in a duckweed bioassay (I(50) values of 0.39-0.95 microM) than more than half of 26 commercial herbicides previously evaluated using the same bioassay. Some of these compounds exhibited strong antiplasmodial activities as well, but they also had cytotoxic activity, thus precluding them as potential antimalarial agents.