Corn Pathogen Research Articles

Development of a qPCR Assay for Species-Specific Detection of the Tar Spot Pathogen Phyllachora maydis

Phyllachora maydis is a fungal plant pathogen that causes tar spot of corn ( Zea mays) in North and South America, causing devastating yield losses under favorable conditions. Although the causal agent is relatively easy to diagnose via macroscopic and microscopic observations, other diseases and conditions, such as insect frass, have been mistaken for tar spot of corn. Furthermore, conidia and ascospores in isolation can be difficult to visually distinguish from other fungi, and the development of signs and symptoms of the disease may not be observed until 12 to 20 days after infection. Therefore, we developed a TaqMan quantitative polymerase chain reaction (qPCR) assay for the detection and quantification of this pathogen to be used for diagnostics and airborne spore quantification. The assay was designed for the internal transcribed spacer region of P. maydis. The specificity of the assay was confirmed and tested against various nontarget Phyllachora species, corn pathogens, endophytes, and P. maydis samples from several states in the Midwest and from Mexico. The detection limit of this assay was determined to be 100 fg of genomic P. maydis DNA. To demonstrate the transferability of this technology, the assay was tested in different labs using various qPCR thermal cyclers. This assay can be used in downstream research involving latency period, disease prediction, and diagnostics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides

Cutinases are serine esterases that belong to the α/β hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp (Cannabis sativa) in Missouri.

Charcoal rot, caused by Macrophomina phaseolina, is abundantly present in the soil and has been reported as pathogenic to both soybean and corn, as well as numerous other hosts, including hemp grown for fiber, grain, and cannabinoids (Casano et al. 2018; Su et al. 2001). Hemp (Cannabis sativa) production in Missouri was a relatively new addition to the 2021 growing season. Charcoal rot was reported in Reynolds, Knox, and Boone counties in Missouri from commercial and experimental fields. One of the fields in question experienced heavy disease pressure and had an uneven stand loss, but the total loss was estimated at approximately 60% of the field and was attributed to charcoal rot. Charcoal rot signs and symptoms, microsclerotia on the lower stem and root tissue, wilting and stem discoloration, were observed on a majority of the hemp plants received at the University of Missouri Plant Diagnostic Clinic in July and late Fall of 2021, including samples from Bradford Research Farm in Boone County and Greenley Research Center in Knox County. Root and crown tissue from the hemp plants from the Greenley Research Center were cultured onto acidified potato dextrose agar (APDA). Macrophomina phaseolina and other fungi grew from the plated tissue after about three days of incubation at room temperature. Macrophomina phaseolina was confirmed based on the presence of melanized hyphae and microsclerotia (Siddique et al. 2021). The microsclerotia were black, round to ovoid shaped and ranged from about 34-87 µm (average 64 µm) in length and 32-134 µm (average 65 µm) in width (n = 44). A single-hyphae isolation from a putative M. phaseolina isolate was conducted to obtain a pure culture. The M. phaseolina culture from the Greenley Research Center was used to complete Koch's postulates of charcoal rot on four hemp cultivars. Sterilized toothpicks were added to pure cultures of M. phaseolina on APDA and incubated at room temperature for one week to allow for colonization and for use in greenhouse inoculation. Four hemp cultivars (Katani, Grandi, CFX-2, and CRS-1) were grown in a sterilized silt loam for three weeks in a greenhouse. About four plants per cultivar were grown for inoculation and one plant per cultivar was used as a control. The plants were inoculated with the M. phaseolina colonized toothpicks that were gently rubbed onto stem tissue and subsequently inserted into the soil at the stem. For six weeks, the plants were kept in greenhouse conditions of 25°C with a 12-hour light and dark cycle and were watered when soil appeared dry. Plants were kept in a loosely sealed container constructed from wood and vinyl sheeting to minimize cross contamination with other plants grown in the same greenhouse. Plants were monitored weekly for charcoal rot symptoms. Symptoms that resembled charcoal rot, wilting and microsclerotia on the lower stem, were present on inoculated plants after about four weeks and symptoms were not present on the control plants. Isolates resembling M. phaseolina in culture were recovered from symptomatic plants; therefore, Koch's postulates were successfully fulfilled and the fungus was recovered from the inoculated plants. DNA was extracted from the pure cultures of both the initial isolate and the isolate obtained from Koch's postulates using GeneJet Plant Genomic DNA Purification Kit (Thermo Scientific, California, USA) and the internal transcribed spacer (ITS) region of ribosomal DNA including ITS1, 5.8S, and ITS4 regions were amplified using universal primers ITS1 and ITS4 (White et al. 1990). The ITS region was sequenced and compared to reference sequences in GenBank by BLAST analysis. Recovered isolates (GenBank accession no. OQ455934.1) showed closest sequence similarity (100%) to M. phaseolina accession number GU046909.1. Little is known about the life cycle, growth conditions, and possible inoculum buildup in the soil in hemp in Missouri. In addition, M. phaseolina is a known pathogen of corn and soybean and effective management strategies are challenging for these crops as well due to the broad host range of the pathogen. Cultural management practices, such as crop rotations to reduce inoculum in the soil and closely monitoring for symptoms, may help reduce the severity of this disease.

Elucidating the obligate nature and biological capacity of an invasive fungal corn pathogen.

Tar spot is a devasting corn disease caused by the obligate fungal pathogen Phyllachora maydis. Since its initial identification in the United States in 2015, P. maydis has become an increasing threat to corn production. Despite this, P. maydis has remained largely understudied at the molecular level due to difficulties surrounding its obligate lifestyle. Here, we generated a significantly improved P. maydis nuclear and mitochondrial genome using a combination of long- and short-read technologies and also provide the first transcriptomic analysis of primary tar spot lesions. Our results show that P. maydis is deficient in inorganic nitrogen utilization, is likely heterothallic, and encodes for significantly more protein coding genes, including secreted enzymes and effectors, than previous determined. Furthermore, our expression analysis suggests that following primary tar spot lesion formation, P. maydis might reroute carbon flux away from DNA replication and cell division pathways and towards pathways previously implicated in having significant roles in pathogenicity, such as autophagy and secretion. Together, our results identified several highly expressed unique secreted factors that likely contribute to host recognition and subsequent infection, greatly increasing our knowledge of the biological capacity of P. maydis, which have much broader implications for mitigating tar spot of corn.

First Report of Fusarium verticillioides Causing Fusarium Ear Rot of Corn in Jordan.

Sweet corn (Zea mays L.) is one of the most popular crops grown in Jordan. Fusarium verticillioides (Sacc.) is a major pathogen of corn and a producer of mycotoxin fumonisins (Blacutt et al. 2018). During September and October 2019, ear rot symptoms were observed on ̴30% of the sweet corn variety Mis Dolce grown in the Jordan Valley. The disease caused substantial losses, including damage to greater than 50% of the kernels within 15-20 days after harvesting. A total of 350-corn kernels were randomly taken from 70 plants distributed in five fields with a total area of 2 ha. About 35% of the samples showed typical symptoms of the disease. Discolored corn kernels were surface sterilized with 5% NaOCl solution for 1 min, then rinsed three times with sterilized distilled water (SDW), plated on potato dextrose agar (PDA) at 25°C, and incubated in the dark for 7 days. Twelve putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar (SNA) with a ca. 1 × 2-cm strip of sterile filter paper on the agar surface (Nirenberg 1976). Cultures were incubated for 10 to 14 days at 20°C in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined under the microscope at 400x. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Macroconidia were abundant, falcate to straight, three- to five-septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 μm. Microconidia produced on polyphialides and aggregating in heads, were unicellular, ovoidal, or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 μm (Fig. 1A, B, C, D, E, and F). Based on morphological characteristics, isolates were tentatively identified as F. verticillioides (Al-Hatmi et al. 2016; Guarro 2013). Two representative isolates were DNA extracted and the translation elongation factor 1-α gene (TEF1) was amplified (O'Donnell et al. 1998), and sequenced from both directions at Macrogen Inc, South Korea. The consensus sequences of the two isolates Fvcorn2021JO-03 (OK040159) and Fvcorn2021JO-04 (OK040160) were used as BLASTn query on the NCBI website and were 100% and 99% similar with F. verticilloides JF740717 and JF740737 accessions, respectively. Similarly, the two isolates were 100% and 99.85% similar with F. verticilloides reference sequences MH582332 and MH582327 on the Fusarium MLST database, respectively. The pathogenicity of the two isolates was tested on 15 cobs by injecting 2 ml of a 2.5 × 105 conidia/ml suspension into the silk channel and into kernel wounds of the primary ear (three replicates) for each treatment (Reid and Hamilton 1996). Inoculated kernels were incubated at 25°C for 2 weeks in plastic boxes. The healthy kernels were injected with 2 ml of SDW as a negative control. Grains started to rot after 2 weeks, in the form of a thick, cottony, crimson-looking growth between the ear and its covers, with only some grains or a group of a adjacent grains rotting, and then white lines appear on the outer skin of the grain, yielding symptoms similar to those in the field (Fig. 2A, B, C, and D). The fungus was re-isolated from the inoculated kernels and was morphologically identified as F. verticilloides thus fulfilling Koch's postulates. The fumonisins-producing potential of the isolated F. verticillioides was confirmed using the AgroQuant Total Fumonisins Assay (Romer Labs, Singapore). To our knowledge, this is the first report of F. verticillioides causing Fusarium ear rot on corn in Jordan. Further investigation is needed to gain a better understanding of the spatio-temporal dynamics of this novel pathogen.

First Report of Aspergillus flavus Causing Fruit Rot on Kiwifruit in China.

In October 2020, fruit rot symptoms were detected on kiwifruit (Actinidia chinensis var. deliciosa 'Xuxiang') in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Mature kiwifruit, during the harvest period, exhibited soft rot and brown lesions. The symptoms were similar than those reported for Alternaria alternata, Colletotrichum spp., Fusarium avenaceum and Rhizopus oryzae causing fruit rot on kiwifruit (Feng et al. 2019; Li et al. 2017; Kim et al. 2018; Zhao et al. 2020). The symptoms were observed in approximately 15% of the fruit in 6 kiwifruit orchards (31 ha in total). Ten samples of symptomatic tissue, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 30 seconds and washed twice with sterilized water. The pathogen was isolated from all collected samples via culturing on PDA medium, containing 50 µg/mL chloramphenicol, at 28 ºC. Green powdery-like colonies were detected after 5 days (Figure 1). A total of 12 isolates were obtained via single spore isolation. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and RNA polymerase II subunit (RPB2) genes were amplified using ITS5/ITS4, A_EF1_F/A_EF1_R and RPB2-5F/RPB2-7cR (NJC03), or RPB2-7cF/RPB2-11aR (NJC04), primers, respectively. Eleven isolates shared the same sequences (NJC03), MZ801787 (ITS), MZ701709 (EF1-α) and MZ701707 (RPB2), while one of the isolates provided different sequences (NJC04), OK618459 (ITS), OK634020 (EF1-α) and OL331017 (RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from A. flavus KU20018.4 (MT487825), the EF1-α sequences shared 100% homology to the EF1-α gene from A. flavus clinical2342 (KP054370) and the RPB2 sequences shared >99% homology to the RPB2 genes from A. flavus PW3170 (LC000581) and A. flavus NRRL3357 (XM_041293948). Molecular phylogenetic tree was constructed using MEGA7 with reference Aspergillus strains (Figure 2). Microscope observations of all isolates showed the presence of septate mycelium, circular unicellular conidia (2-4 µm diameter) and conidiophores, and agree with the morphology of A. flavus (Horn 2005). The pathogenicity of all isolates was screened using intact and wounded 'Xuxiang' kiwifruits (ten kiwifruits were used for each combination with 3 replicates), which were purchased from a local market. A 1 × 106 spores/mL (10 µL) solution of the isolates was used for the inoculation. Sterilized water was used in the control experiment. Inoculated kiwifruits were storage at 26 °C and 60% relative humidity for 10 days. Rot lesions in the wounded kiwifruits were totally covered by green mycelia, while the lesions on the intact kiwifruits were similar to the symptoms observed in the field. The pathogen was recovered and its identity was confirmed by sequence analysis of ITS, EF1-α and RPB2, fulfilling Koch's postulates. A. flavus is known to be an important fungal pathogen of corn, cotton and peanuts (Zhang et al. 2020). During recent years, A. flavus was reported to cause fruit rot on grapes (Ghuffar et al. 2020), and was identified on almond, fig, organic spelt and pistachio (Krulj et al. 2017; Ortega-Beltran et al. 2019). The presence of A. flavus in food products is an issue of global concern due to A. flavus is able to produce carcinogenic aflatoxin (Maxwell et al. 2021). As far as we know, this is the first report of A. flavus causing fruit rot on kiwifruit. This report will help to understand the distribution of A. flavus in crops and the food safety hazards that are present in China.

My Personal Journey from the Fascination for Phages to a Tumor-Inducing Fungal Pathogen of Corn.

My path in science began with a fascination for microbiology and phages and later involved a switch of subjects to the fungus Ustilago maydis and how it causes disease in maize. I will not provide a review of my work but rather focus on decisive findings, serendipitous, lucky moments when major advances made the U. maydis-maize system what it is now-a well-established model for biotrophic fungi. I also want to share with you the joy of finding the needle in a haystack at the very end of my scientific career, a fungal structure likely used for effector delivery, and how we were able to translate this into a potential application in agriculture.

Mono-/Bis-Alkenoic Acid Derivatives From an Endophytic Fungus Scopulariopsis candelabrum and Their Antifungal Activity

During a screening for antifungal secondary metabolites, six new mono-/bis-alkenoic acid derivatives (2–7) and one known alkenoic acid derivative (1) were isolated from an endophytic fungi Scopulariopsis candelabrum. Their chemical structures were identified by 1H-NMR, 13C-NMR, 2D NMR, and high-resolution mass spectrometry, as well as comparisons with previously reported literatures. Among them, fusariumesters C‒F (2–5) are bis-alkenoic acid derivatives dimerized by an ester bond, while acetylfusaridioic acid A (6) and fusaridioic acid D (7) are alkenoic acid monomers. All the isolates were submitted to an antifungal assay against Candida albicans and the corn pathogen Exserohilum turcicum using the filter paper agar diffusion method. As a result, only compound 1 decorating with β-lactone ring turned out to be active against these two tested fungi. The broth microdilution assay against Candida albicans showed the minimum inhibitory concentration (MIC) value of 1 to be 20 μg/ml, while the minimum inhibitory concentration value of the positive control (naystatin) was 10 μg/ml. And the half maximal inhibitory concentration (IC50) value (21.23 μg/ml) of 1 against Exserohilum turcicum was determined by analyzing its inhibition effect on the mycelial growth, using cycloheximide (IC50 = 46.70 μg/ml) as the positive control.

Completion of Maize Stripe Virus Genome Sequence and Analysis of Diverse Isolates.

Maize stripe virus is a pathogen of corn and sorghum in subtropical and tropical regions worldwide. We used high-throughput sequencing to obtain the complete nucleotide sequence for the reference genome of maize stripe virus and to sequence the genomes of ten additional isolates collected from the United States or Papua New Guinea. Genetically, maize stripe virus is most closely related to rice stripe virus. We completed and characterized the RNA1 sequence for maize stripe virus, which revealed a large open reading frame encoding a putative protein with ovarian tumor-like cysteine protease, endonuclease, and RNA-dependent RNA polymerase domains. Phylogenetic and amino acid identity analyses among geographically diverse isolates revealed evidence for reassortment in RNA3 that was correlated with the absence of RNA5. This study yielded a complete and updated genetic description of the tenuivirus maize stripe virus and provided insight into potential mechanisms underpinning its diversity.

Long term crop rotation effect on subsequent soybean yield explained by soil and root-associated microbiomes and soil health indicators

Crop rotation is an important management tactic that farmers use to manage crop production and reduce pests and diseases. Long-term crop rotations may select groups of microbes that form beneficial or pathogenic associations with the following crops, which could explain observed crop yield differences with different crop sequences. To test this hypothesis, we used two locations each with four long-term (12–14-year), replicated, rotation treatments: continuous corn (CCC), corn/corn/soybean (SCC), corn/soybean (CSC), and soybean/corn (SCS). Afterwards, soybean was planted, and yield and soil health indicators, bulk soil microbiome, and soybean root-associated microbiome were assessed. Soybean yields, as well as soil protein, and POXC as soil health indicators were higher following CCC than in the other three treatments at both locations. A bacterial taxon in family JG30-KF-AS9 was enriched in CCC, whereas Microvirga, Rhodomicrobium, and Micromonosporaceae were enriched in SCS. Several ascomycetes explain lowered yield as soybean pathogens in SCS. Surprisingly, Tumularia, Pyrenochaetopsis and Schizothecium were enriched in soybean roots after CCC, suggesting corn pathogens colonizing soybean roots as nonpathogens. Our finding of associations between soil health indicators related to microbiomes and soybean yield has wide-ranging implications, opening the possibility of manipulating microbiomes to improve crop yield potential.

Involvement of the Mitochondrial Protein Tyrosine Phosphatase PTPM1 in the Promotion of Conidiation, Development, and Pathogenicity in Colletotrichum graminicola.

The phosphorylation status of proteins, which is determined by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), governs many cellular actions. In fungal pathogens, phosphorylation-mediated signal transduction has been considered to be one of the most important mechanisms in pathogenicity. Colletotrichum graminicola is an economically important corn pathogen. However, whether phosphorylation is involved in its pathogenicity is unknown. A mitochondrial protein tyrosine phosphatase gene, designated CgPTPM1, was deduced in C. graminicola through the use of bioinformatics and confirmed by enzyme activity assays and observation of its subcellular localization. We then created a CgPTPM1 deletion mutant (ΔCgPTPM1) to analyze its biological function. The results indicated that the loss of CgPTPM1 dramatically affected the formation of conidia and the development and differentiation into appressoria. However, the colony growth and conidial morphology of the ΔCgPTPM1 strains were unaffected. Importantly, the ΔCgPTPM1 mutant strains exhibited an obvious reduction of virulence, and the delayed infected hyphae failed to expand in the host cells. In comparison with the wild-type, ΔCgPTPM1 accumulated a larger amount of H2O2 and was sensitive to exogenous H2O2. Interestingly, the host cells infected by the mutant also exhibited an increased accumulation of H2O2 around the infection sites. Since the expression of the CgHYR1, CgGST1, CgGLR1, CgGSH1 and CgPAP1 genes was upregulated with the H2O2 treatment, our results suggest that the mitochondrial protein tyrosine phosphatase PTPM1 plays an essential role in promoting the pathogenicity of C. graminicola by regulating the excessive in vivo and in vitro production of H2O2.

Review of Pythium Species Causing Damping-Off in Corn

Seedling blights and root rots caused by Pythium species account for almost US$25 million in annual losses to corn (Zea mays) production in the United States and Ontario. Variations in annual rainfall and increasing use of no-till agriculture can favor soilborne pathogens like Pythium. To date, 44 species have been reported as pathogenic to corn in the United States. The average annual corn planting date in the United States has shifted approximately 1 week earlier in the past decade, exposing young corn plants to longer germination periods of generally cooler temperatures, favoring attack by Pythium. Optimal temperatures, aggressiveness, and response to chemical and biological treatment options vary by species. This review consolidates the species of Pythium reported as corn pathogens in literature to date. It also provides an insight into management strategies and discusses variations in fungicide sensitivity observed in corn-related Pythium species.

Prevention and Detection of Fungicide Resistance Development in Rhizoctonia zeae from Soybean and Corn in Nebraska

The goal of this research was to advance the foundational knowledge required to quantify and mitigate fungicide resistance in Rhizoctonia zeae, the seedling disease pathogen of soybean and corn. In vitro sensitivity to azoxystrobin, fludioxonil, sedaxane, and/or prothioconazole was determined for 91 R. zeae isolates obtained mostly from soybean and corn fields in Nebraska. Isolates were sensitive to fludioxonil, sedaxane, and prothioconazole (EC50 < 3 µg/ml) and had a positively skewed EC50 distribution. Isolates were not sensitive to azoxystrobin in vitro (EC50 > 100 µg/ml) or in planta. Application of azoxystrobin did not significantly decrease disease severity or improve total dry weight of the soybean plants (P > 0.05). The risk of resistance development in R. zeae was estimated by characterizing its population structure. Eighty-one R. zeae isolates were genotyped using six microsatellite markers. Results showed that the population has a mixed mode of reproduction and is structured according to geographic region, suggesting limited dispersal. These population characteristics suggest that R. zeae has an intermediate risk of resistance development. Overall, this research established the current status of fungicide sensitivity in R. zeae in Nebraska and estimated its risk of resistance development, which can inform fungicide resistance management for R. zeae.

Kilbournase, a protease-associated domain subtilase secreted by the fungal corn pathogen Stenocarpella maydis

Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.

A Rab GTPase protein FvSec4 is necessary for fumonisin B1 biosynthesis and virulence in Fusarium verticillioides.

Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. But the role of Rab GTPases in Fusarium verticillioides, one of the key corn pathogens worldwide, remains elusive. These Small GTPases in fungi, particularly those homologous to Saccharomyces cerevisiae Sec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. In this study, our aim was to investigate the molecular functions of FvSec4 in F. verticillioides associated with physiology and virulence. Interestingly, the FvSec4 null mutation did not impair the expression of key conidiation-related genes. Also, the mutant did not show any defect in sexual development, including perithecia production. Meanwhile, GFP-FvSec4 localized to growing hyphal tips and raised the possibility that FvSec4 is involved in protein trafficking and endocytosis. The mutant exhibited defect in corn stalk rot virulence and also significant alteration of fumonisin B1 production. The mutation led to higher sensitivity to oxidative and cell wall stress agents, and defects in carbon utilization. Gene complementation fully restored the defects in the mutant demonstrating that FvSec4 plays important roles in these functions. Taken together, our data indicate that FvSec4 is critical in F. verticillioides hyphal development, virulence, mycotoxin production and stress responses.

Identification of Fungal Communities Within the Tar Spot Complex of Corn in Michigan via Next-Generation Sequencing.

Tar spot is a fungal disease complex of corn that has been destructive and yield limiting in Central and South America for nearly 50 years. Phyllachora maydis, the causal agent of tar spot, is an emerging corn pathogen in the United States, first reported in 2015 from major corn producing regions of the country. The tar spot disease complex putatively includes Monographella maydis (syn. Microdochium maydis), which increases disease damage through the development of necrotic halos surrounding tar spot lesions. These necrotic halos, termed "fish-eye" symptoms, have been identified in the United States, though Monographella maydis has not yet been confirmed. A recent surge in disease severity and loss of yield attributed to tar spot in the United States has led to increased attention and expanded efforts to understand the disease complex and how to manage it. In this study, next-generation sequencing of the internal transcribed spacer-1 (ITS1) ribosomal DNA was used to identify fungal taxa that distinguish tar spot infections with or without fish-eye symptoms. Fungal communities within tar spot only lesions were significantly different from communities having fish-eye symptoms. Two low abundance OTUs were identified as Microdochium sp., however, neither were associated with fish-eye symptom development. Interestingly, a single OTU was found to be significantly more abundant in fish-eye lesions compared to tar spot lesions and had a 91% ITS1 identity to Neottiosporina paspali. In addition, the occurrence of this OTU was positively associated with Phyllachora maydis fish-eye symptom networks, but not in tar spot symptom networks. Neottiosporina paspali has been reported to cause necrotic lesions on various monocot grasses. Whether the related fungus we detected is part of the tar-spot complex of corn and responsible for fish-eye lesions remains to be tested. Alternatively, many OTUs identified as Phyllachora maydis, suggesting that different isolate genotypes may be capable of causing both tar spot and fish-eye symptoms, independent of other fungi. We conclude that Monographella maydis is not required for fish-eye symptoms in tar spot of corn.

Effect of Tebuconazole Enantiomers and Environmental Factors on Fumonisin Accumulation and FUM Gene Expression in Fusarium verticillioides.

Fusarium verticillioides is an important corn pathogen that can produce fumonisins (FBs) under certain environmental conditions. In this study, we evaluated the enantioselective impact of tebuconazole enantiomers on the growth and FB production of F. verticillioides on maize-based media at different abiotic factors. The expression of FB biosynthetic genes ( FUM1 and FUM6) was quantified by real-time reverse transcription polymerase chain reaction. The results showed that water activity ( aw), temperature, and types of tebuconazole significantly affected the growth of F. verticillioides. The order of fungicidal activity was (-)-tebuconazole > rac-tebuconazole > (+)-tebuconazole. (-)-tebuconazole exhibited the maximal selective fungicidal activity (242-fold) against F. verticillioides at 0.95 aw and 35 °C. Production of fumonisin B1 (FB1) and fumonisin B2 (FB2) by F. verticillioides was influenced by aw, temperature, types of tebuconazole, and dose. Under most conditions, (-)-tebuconazole showed stronger inhibition for FB1 and FB2 production than (+)-tebuconazole (1.87-2.85-fold reduction in FBs) and rac-tebuconazole. The optimal environmental condition for FB production was at 0.99 aw and 25 °C. Tebuconazole enantiomers differently affected FB biosynthetic gene ( FUM1 and FUM6) expression, but the effects on FB production and gene expression showed no positive correlation. The present study provides a better understanding on ways to minimize FB production in corn treated with fungicides.

Identification and Characterization of Fusarium proliferatum, a New Species of Fungi that Cause Fungal Keratitis

Fusarium proliferatum (F. proliferatum) is known as a pathogen of corn and other crops, but its role in fungal keratitis has not been well investigated. Among 877 Fusarium isolates, we identified 155 (17.7%) stains as F. proliferatum according to their morphological features and partial DNA sequencing of translation elongation factor-1alpha (EF-1alpha ) in this study. In vitro antifungal susceptibility tests showed that the F. proliferatum strains were sensitive to natamycin and vorionazole but resistant to amphotericin B, fluconazol, ketoconazole and itaconazole. Most of the F. proliferatum-positive keratitis patients (44/155,28.4%) were aged 51–60 years old. The main cause of infection was injury by a plant (51/155, 32.9%). A combination of 1% amphotericin B and 3% ketoconazole cured 45.2% (14/31) and a combination of 0.5% natamycin and 0.5% voriconazole cured 59.1% (13/22) of F. proliferatum-positive patients. The date suggests that F. proliferatum identified through EF-1ɑ DNA sequencing is an important new species that causes fungal keratitis. Based on antifungal susceptibility, treatment with a combination of 0.5% natamycin and 0.5% voriconazole improves the therapeutic efficacy in F. prolifertum-positive patients.

Detection of Fusarium verticillioides by PCR-ELISA based on FUM21 gene

Fusarium verticillioides is a primary corn pathogen and fumonisin producer which is associated with toxic effects in humans and animals. The traditional methods for detection of fungal contamination based on morphological characteristics are time-consuming and show low sensitivity and specificity. Therefore, the objective of this study was to develop a PCR-ELISA based on the FUM21 gene for F. verticillioides detection. The DNA of the F. verticillioides, Fusarium sp., Aspergillus sp. and Penicillium sp. isolates was analyzed by conventional PCR and PCR-ELISA to determine the specificity. The PCR-ELISA was specific to F. verticillioides isolates, showed a 2.5 pg detection limit and was 100-fold more sensitive than conventional PCR. In corn samples inoculated with F. verticillioides conidia, the detection limit of the PCR-ELISA was 1 × 104 conidia/g and was also 100-fold more sensitive than conventional PCR. Naturally contaminated corn samples were analyzed by PCR-ELISA based on the FUM21 gene and PCR-ELISA absorbance values correlated positively (p < 0.05) with Fusarium sp. counts (CFU/g). These results suggest that the PCR-ELISA developed in this study can be useful for F. verticillioides detection in corn samples.

A high proportion of NX-2 genotype strains are found among Fusarium graminearum isolates from northeastern New York State

Fusarium graminearum, a fungal pathogen of wheat, barley, and corn, produces a variety of trichothecene mycotoxins that are important as virulence factors and as seed contaminants reducing grain quality. A previous survey of the pathogen in New York State identified variation in genes indicative of trichothecene diversity. Recently F. graminearum strains that produce a newly characterized trichothecene mycotoxin called NX-2 have been identified in North America. Using a large collection of F. graminearum strains from Willsboro NY, we found that the frequency of NX-2 genotype strains was 7–14 times higher than at other locations where it was reported previously. NX-2 genotypes were not only found in wheat heads but also found in high frequency from air samples and on maize ears and stubble. Because NX-2 genotypes may represent as much as 20% of the total F. graminearum population, this regional fungal population provides an opportunity to assess the effects of the novel NX-2 trichothecene on fungal virulence, toxin loading, and patterns of host specificity that could inform future disease management and plant breeding.