Guirong Zhang

First Report of the Soybean Frogeye Leaf Spot Fungus (Cercospora sojina) Resistant to Quinone Outside Inhibitor Fungicides in North America

Quinone outside inhibitor (QoI; also known as strobilurin) fungicides sometimes are applied to soybean (Glycine max) fields to help manage frogeye leaf spot of soybean (caused by Cercospora sojina) in the United States. In August 2010, soybean leaflets exhibiting severe frogeye leaf spot symptoms were collected from a field in Lauderdale County, TN that had been treated twice with pyraclostrobin during that growing season. The field had been planted into soybean annually since at least 2008, and a QoI fungicide had been applied to the field in each of those years. Fifteen single-spore isolates of C. sojina were recovered from the affected soybean leaflets. These isolates were identified as C. sojina based on the observed symptoms on the soybean leaflets and the morphology and size of conidiophores and conidia (3). In addition, DNA was extracted from the cultures, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region was conducted (2), and the resulting PCR product was sequenced at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequences were compared with sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of C. sojina. The isolates were tested for their sensitivity to technical-grade formulations of the QoI fungicides azoxystrobin, pyraclostrobin, and trifloxystrobin with an in vitro conidial germination assay with fungicide + salicylhydroxamic acid (SHAM)-amended potato dextrose agar as described by Bradley and Pedersen (1). The effective concentration at which 50% conidial germination was inhibited (EC50) was determined for all 15 C. sojina isolates, with mean values of 3.1644 (2.7826 to 4.5409), 0.3297 (0.2818 to 0.6404), and 0.8573 (0.3665 to 2.5119) μg/ml for azoxystrobin, pyraclostrobin, and trifloxystrobin, respectively. When compared with previously established mean EC50 values of C. sojina baseline isolates (4), EC50 values of the C. sojina isolates collected from the Lauderdale County, TN soybean field were approximately 249- to 7,144-fold greater than the EC50 values of the baseline isolates. These results indicate that all isolates recovered from the Lauderdale County, TN soybean field were highly resistant to QoI fungicides. To our knowledge, this is the first report of QoI fungicide resistance occurring in C. sojina, and surveys for additional QoI fungicide-resistant C. sojina isolates are needed to determine their prevalence and geographic distribution. In light of these findings, soybean growers in Tennessee and adjacent states should consider utilizing alternative frogeye leaf spot management practices such as planting resistant cultivars, rotating to nonhost crops, and tilling affected soybean residue (3). References: (1) C. A. Bradley and D. K. Pedersen. Plant Dis. 95:189, 2011. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) D. V. Phillips. Page 20 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999. (4) G. Zhang et al. Phytopathology (Abstr.) 100(suppl.):S145, 2010.

Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Genetic diversity of Cercospora sojina revealed by amplified fragment length polymorphism markers

Abstract Cercospora sojina Hara, the causal agent of frogeye leaf spot of soybean (Glycine max (L.) Merr.), causes yield reductions worldwide. Although the phenotypic diversity (physiological races) of this pathogen has been assessed through its ability to affect soybean lines with different resistant genes (Rcs genes), little is known about the pathogens genetic diversity. In order to better understand the genetic diversity that exists with C. sojina, a historical collection of 62 C. sojina isolates from Brazil (10 isolates), China (7 isolates), Nigeria (1 isolate), and United States (44 isolates) was used for genetic diversity analysis with amplified fragment length polymorphism (AFLP) markers. The average genetic similarity of the isolates was 0.56 on a scale between 0 and 1, indicating a high degree of genetic diversity within the species. Cluster analysis resulted in two major clusters and seven sub-clusters. Two isolates collected from Georgia were clustered together, and two isolates from China were clustered together. Besides these four isolates, no clear separation of isolates based on origin was found. Our results provide evidence that substantial genetic diversity exists within the species of C. sojina and that selection for broad-spectrum host-resistance should be targeted in soybean breeding programmes.

Draft genome sequence of Cercospora sojina isolate S9, a fungus causing frogeye leaf spot (FLS) disease of soybean

Fungi are the causal agents of many of the worlds most serious plant diseases causing disastrous consequences for large-scale agricultural production. Pathogenicity genomic basis is complex in fungi as multicellular eukaryotic pathogens. The fungus Cercospora sojina is a plant pathogen that threatens global soybean supplies. Here, we report the genome sequence of C. sojina strain S9 and detect genome features and predicted genomic elements. The genome sequence of C. sojina is a valuable resource with potential in studying the fungal pathogenicity and soybean host resistance to frogeye leaf spot (FLS), which is caused by C. sojina. The C. sojina genome sequence has been deposited and available at DDBJ/EMBL/GenBank under the project accession number AHPQ00000000.

A comparative genome analysis of Cercospora sojina with other members of the pathogen genus Mycosphaerella on different plant hosts

Fungi are the causal agents of many of the worlds most serious plant diseases causing disastrous consequences for large-scale agricultural production. Pathogenicity genomic basis is complex in fungi as multicellular eukaryotic pathogens. Here, we report the genome sequence of C. sojina, and comparative genome analysis with plant pathogen members of the genus Mycosphaerella (Zymoseptoria. tritici (synonyms M. graminicola), M. pini, M. populorum and M. fijiensis - pathogens of wheat, pine, poplar and banana, respectively). Synteny or collinearity was limited between genomes of major Mycosphaerella pathogens. Comparative analysis with these related pathogen genomes indicated distinct genome-wide repeat organization features. It suggests repetitive elements might be responsible for considerable evolutionary genomic changes. These results reveal the background of genomic differences and similarities between Dothideomycete species. Wide diversity as well as conservation on genome features forms the potential genomic basis of the pathogen specialization, such as pathogenicity to woody vs. herbaceous hosts. Through comparative genome analysis among five Dothideomycete species, our results have shed light on the genome features of these related fungi species. It provides insight for understanding the genomic basis of fungal pathogenicity and disease resistance in the crop hosts.

First report of phomopsis stem canker of sunflower in Illinois caused by phomopsis helianthi

Stem cankers were observed on confection sunflower (Helianthus annuus) plants growing in a field in Champaign County, Illinois in August 2008. Lesions were brown to reddish brown, elongated (approximately 10 to 15 cm long), and centered over the area where leaf petioles connected to the stems. Stem tissues underneath the lesions were degraded. Lesions from diseased stems were cut into 5- to 7-mm pieces and immersed in a 0.5% NaOCl solution for 1 min, rinsed with sterilized distilled water, and placed into petri dishes containing acidified potato dextrose agar (APDA; 4 ml of 25% lactic acid per liter). Fungal colonies that grew from the stem lesion pieces on APDA were white, floccose, and dense with dark colored substrate mycelia. On the basis of the symptoms on sunflower plants and the growth characteristics on APDA, the fungus was tentatively identified as Phomopsis helianthi (1). To confirm the identity of the fungus, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was done (2). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of Diaporthe helianthi, teleomorph of P. helianthi. To confirm pathogenicity of the fungus, sunflower plants (cv. Cargill 270) were grown in the greenhouse and inoculated with the isolated fungus. The stems of sunflower plants between the V2 and V4 growth stages (3) were excised just below the uppermost node. Mycelia plugs of the fungus were placed into the large end of disposable micropipette tips (200 μl). The micropipette tip containing the fungus was subsequently placed over a cut sunflower stem. The fungal isolate was used to inoculate five stems. To serve as controls, five cut sunflower stems were inoculated with micropipette tips containing plugs of noninfested PDA and five cut stems were not inoculated. Mean lesion length on the stem was measured from the inoculated tip toward the soil line 7 days after inoculation. The experiment was replicated over time. Mean lesion length over both replications averaged 24 mm on the fungus-inoculated plants, 2 mm on the noninfested PDA-inoculated control plants, and no lesions were present on the noninoculated control plants. The fungus was reisolated on PDA from the inoculated plants in the greenhouse. To our knowledge, this is the first report of P. helianthi causing a stem canker of sunflower in Illinois. Although commercial sunflower production in Illinois is currently limited, it is being evaluated as a potential crop to follow winter wheat in portions of the state. If sunflower production were to increase in the state, growers may have to monitor for and manage Phomopsis stem canker. References: (1) T. Gulya et al. Sunflower diseases. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) A. A. Schneiter and J. F. Miller, Crop Sci. 21:901, 1981.

First report of charcoal rot caused by Macrophomina phaseolina on sunflower in Illinois.

In September 2009, sunflower (Helianthus annuus L.) plants (cv. Mycogen 8C451) from a University of Illinois field research trial in Fayette County, Illinois exhibited silvery gray girdling lesions on the lower stems and premature death. When lower stems and roots were split open, the pith tissue was compressed into layers. Black microsclerotia (90 to 180 μm) were present on the outside of the lower stem tissue and in the stem vascular tissue. Five pieces (approximately 1 cm long) of symptomatic stem tissue from five different affected plants (25 pieces total) were soaked in a 0.5% solution of NaOCl for 30 s, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA; Becton, Dickinson, and Company, Franklin Lakes, NJ). Gray hyphae grew from all of the stem pieces, which subsequently turned black and formed black microsclerotia (75 to 175 μm). On the basis of plant symptoms and size and color of the microsclerotia, the disease was diagnosed as charcoal rot caused by Macrophomina phaseolina (Tassi) Goid (2). To confirm that the isolated fungus was M. phaseolina, DNA was extracted from the pure culture, and PCR amplification of a subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was performed (3). The Keck Biotechnology Center at the University of Illinois, Urbana sequenced the PCR product. The resulting nucleotide sequence shared the highest homology (99%) with sequences of M. phaseolina when compared with the subunit rDNA and ITS sequences in the nucleotide database ( http://www.ncbi.nlm.nih.gov ). A greenhouse experiment was conducted to confirm pathogenicity; the greenhouse temperature was approximately 27°C and sunflower plants (cv. Cargill 270) were grown in pots and watered daily to maintain adequate soil moisture for growth. Sterile toothpicks were infested with M. phaseolina and placed through the stems (10 cm above the soil surface) of five 40-day-old sunflower plants that were approximately at growth stage R4 (1,4). Five sterile, noninfested toothpicks were similarly placed through sunflower plants to act as controls. Parafilm was used to hold the toothpick in the stem and seal the stem injury. Thirty-five days after inoculation, the mean lesion length on stems inoculated with M. phaseolina was 595 mm and no lesions developed on the control plants. M. phaseolina-inoculated plants also began to wilt and die. Cultures identical to the original M. phaseolina isolate were reisolated from stem lesions of the M. phaseolina-inoculated plants. This is the first report of charcoal rot on sunflower in Illinois to our knowledge. Sunflower is currently not a major crop grown in Illinois, but on-going research is focused on evaluating sunflower as a potential late-planted crop to follow winter wheat. If sunflower production increases in Illinois, growers may need to take precautions to manage charcoal rot. References: (1) L. K. Edmunds. Phytopathology 54:514, 1964. (2) T. Gulya et al. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (3) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (4) A. A. Schneiter and J. F. Miller. Crop Sci. 21:901, 1981.