First report of areolate mildew of cotton, caused by Ramulariopsis pseudoglycines in Mississippi.

In August of 2022, cotton (Gossypium hirsutum L.) growing in several north central Mississippi counties was observed to exhibit yellowish lesions on the adaxial leaf surface with white powdery fungal growth on the corresponding abaxial surface. By the end of the 2022 growing season, 19 Mississippi counties were observed to have infected cotton. Symptomatic leaves were collected from affected plants, sealed in plastic freezer bags, stored on ice in a cooler, and transported to the laboratory. Prior to isolation, the pathogen was microscopically examined and determied to be morphologically similar to the description of Ramulariopsis spp. (Ehrlich and Wolf 1932). Using a sterile needle, conidia were transferred to V8 medium amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated in the dark at 25°C. After 14 days, the colony diameter was measured, and morphological characteristics were consistent with previous descriptions (Videira et al. 2016; Volponi et al. 2014). On V8 medium, the 7 mm diameter colonies grew raised, lumpy, and lobed, with iron-grey coloration. The mycelia were hyaline, septate, branched, and 1 to 3 µm in diameter. Conidia ranged from 2.8 to 25.6 µm in length and 1.0 to 4.9 µm in width (x = 12.8 × 3.1 µm; n = 20). Pure cultures were obtained on V8 medium, and DNA was extracted from a 14-day-old-culture. TW098-22, a representative isolate, was amplified, and sequenced targeting the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF 1-α), and actin (ACT) genes as described by Videira et al. (2016). The consensus sequences were deposited in GenBank (accession no. OQ653427, OR157986, OR157987). BLASTn query of the NCBI GenBank showed 100% identity of the 483-bp (ITS) and 706-bp TEF 1-α sequences from TW098-22 with Ramulariopsis pseudoglycines CPC 18242 (type culture; Videira et al. 2016). Koch's postulates were performed after multiplying individual colonies by streaking on V8 medium as above. Culture plates were subsequently incubated at 25°C for 14 days in the dark. Colonies were aseptically transferred to 50 ml centrifuge tubes containing 50 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%). The resulting inoculum suspension was adjusted to 13.5 × 105 conidia/ml using a hemocytometer. The foliage of five, 25-day-old cotton plants were sprayed with 10 ml of the suspension and a plastic bag was placed over each plant to maintain humidity for 30 days. Five plants were sprayed with sterilized RO water to serve as controls. Plants were incubated in a growth chamber at 25°C and ~70% relative humidity with 16:8 h of light:dark. Thirty days post-inoculation, foliar symptoms and signs were observed on all inoculated plants including small necrotic lesions and white powdery growth. Control plants remained asymptomatic. The trial was repeated. When reisolated, the colony and conidia morphology and DNA sequence (ITS) were consistent with the description of the original field isolate. Areolate mildew of cotton can be caused by two species of Ramulariopsis: R. gossypii and R. pseudoglycines (Videira et al. 2016). The two species have been reported in Brazil (Mathioni et al. 2021); however, this is the first report of R. pseudoglycines in the United States. In addition, even though areolate mildew has previously been reported from much of the southeastern U.S. (Anonymous 1960), the report herein serves as the first description of R. pseudoglycines in Mississippi and U.S. cotton.

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.

First Report of Neocosmospora Stem Rot of Soybean Caused by Fusarium neocosmosporiellum in Mississippi.

In September 2021, diseased soybean [Glycine max (L.) Merr.] plants were observed in a commercial field in Lamar County, Mississippi (MS). Foliar symptoms included mild interveinal chlorosis and necrosis in the affected plants (Fig. 1a). Stems and roots exhibited orange to black lesions and pith discoloration (Fig. 1b). Signs of the fungus included orange-to-red spherical perithecia, observed in clusters on crowns and roots. Perithecia were surface-sterilized with 70% ethanol for 1 min. Subsequently they were gently pressed in a droplet of 5 µl of sterilized reverse osmosis (RO) water and the suspension was streaked onto potato dextrose agar (PDA) amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter). Plates were incubated in the dark at 25°C. White to light pink and floccose mycelia developed after four days of incubation and hyaline, one celled, cylindrical to oblong-ellipsoidal microconidia were observed (Fig. 2b; ×400). Conidia measured 8.8 to 19 × 2.8 to 5 µm (n=15). Abundant orange to brown perithecia developed on PDA after three weeks of incubation (Fig. 2a). The asci within the perithecia were cylindrical, eight-spored, thin walled and measured 94.6 to 123.6 µm × 10 to 14 µm (n=10). Ascospores were uniseriate, globose to ellipsoidal, hyaline to brown and measured 11.6 to 16 µm × 7 to 10 µm (n=10; Fig. 2c). The morphological characteristics were consistent with those of Fusarium neocosmosporiellum O'Donnell & Geiser (≅ Neocosmospora vasinfecta E. F. Sm.; Geiser et al. 2013). Genomic DNA was extracted from isolate TW068-21 from a 3-week-old culture plate. The internal transcribed spacer region (ITS), elongation factor 1-alpha (EF1-alpha) and calmodulin (cmdA) gene were amplified, and consensus sequences deposited in GenBank (OM640625, OM681343, OM681344). ITS and EF1-alpha sequence comparison using NCBI BLAST, showed > 99.2% similarity with N. vasinfecta JL2210 while cmdA sequence was 99.8% similar to strain CBS 517.71. A pathogenicity test was performed on 2-week-old Asgrow 46X6 soybean seedlings grown in 10.2 cm pots in a growth chamber. Isolate TW068-21 was grown on antibiotic-amended PDA for 4-weeks and inoculum suspension was prepared with sterilized RO water and adjusted to 2 × 105 ascospores/ml. Soybean seedlings (n=8) were removed from pots and roots were dipped into the inoculum suspension for 20 min. Four control plants were dipped in autoclaved RO water. Plants were re-planted in potting mix, and the inoculated plants were immediately drenched with 20 ml of the inoculum and placed in a growth chamber (25°C; 14 h light). The experiment was repeated once. Inoculated plants presented dark brown discoloration at the base of the stem after 3 weeks of incubation, but no foliar lesions were observed. Control plants remained asymptomatic. Symptomatic stems were placed in a moist chamber (≈23°C; 12 h:h light:dark) and light brown perithecia developed after 1 week. Fusarium neocosmosporiellum was re-isolated from perithecia and stems and colony and spore morphology were similar as described above. To our knowledge, this is the first report of F. neocosmosporiellum in MS. Signs and symptoms of this disease resemble red crown rot. Consequently, careful morphological and molecular assessments should be used for confirmation. Neocosmospora stem rot has been previously reported in Alabama (Gray et al. 1980), Arkansas (Greer et al. 2015), and Georgia (Phillips 1972). Yield losses due to this pathogen in MS are currently unknown.

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.

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.