Search

Cercospora leaf blight (CLB) causes extensive losses in soybean production in worldwide, including major soybean-producing countries such as Argentina. Cercospora kikuchii, C. cf. sigesbeckiae, C. cf. flagellaris, and C. cf. nicotianae are identified as pathogens of CLB. Soybean resistance against CLB is still unknown. Also, chemical control for CLB is losing effectiveness because of fungicide resistance of pathogens such as C. kikuchii. We urgently need to breed a CLB-resistant cultivar. Unfortunately, efficient methods for the screening of a resistant soybean genotype have not yet been established. In this study, we designed a new, high-throughput inoculation method for identifying resistance against one of the CLB pathogens, C. kikuchii. We used liquid-cultured mycelia of the pathogen C. kikuchii on detached soybean leaves. Lesions on soybean leaflets appeared 9 days postinoculation by this method. We used this method to select four C. kikuchii-resistant genotypes from 80 genotypes in the World Soybean Core Collection. The high-throughput screening method developed in this study can contribute to the research about C. kikuchii resistance by facilitating identification of resistant varieties. EEA Pergamino Fil: Kashiwa, Takeshi. Japan International Research Center for Agricultural Sciences (JIRCAS). Biological Resources and Post-harvest Division; Japón Fil: Lavilla, Miguel Ángel. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Diaz Paleo, Antonio Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Sección Laboratorio Biotecnología; Argentina Fil: Diaz Paleo, Antonio Horacio. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Ivancovich, Antonio Juan Gerardo. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Yamanaka, Naoki. Japan International Research Center for Agricultural Sciences (JIRCAS). Biological Resources and Post-harvest Division; Japón

In many parts of the world including the Great Lakes region of North America, Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is a major foliar disease of sugar beet (Beta vulgaris). Management of CLS involves an integrated approach which includes the application of fungicides. To guide fungicide application timings, disease prediction models are widely used by sugar beet growers in North America. While these models have generally worked well, they have not included information about pathogen presence. Thus, incorporating spore production and dispersal could make them more effective. The current study used sentinel beets to assess the presence of C. beticola spores in the environment early in the 2017 and 2018 growing seasons. Weather variables including air temperature, relative humidity, rainfall, leaf wetness, wind speed, and solar radiation were collected. These data were used to identify environmental variables that correlated with spore levels during a time when CLS is not generally observed in commercial fields. C. beticola spores were detected during mid-April both years, which is much earlier than previously reported. A correlation was found between spore data and all the weather variables examined during at least one of the two years, except for air temperature. In both years, spore presence was significantly correlated with rainfall (P < 0.0001) as well as relative humidity (P < 0.0090). Rainfall was particularly intriguing, with an adjusted R2 of 0.3135 in 2017 and 0.1652 in 2018. Efforts are ongoing to investigate information on spore presence to improve prediction models and CLS management.

Cercospora leaf blight (CLB), primarily caused by Cercospora cf. flagellaris, is one of the most important diseases of soybean (Glycine max) in Louisiana. The pathogen produces cercosporin, a nonspecific toxin and an important virulence factor. There are no commercial cultivars with CLB resistance, and the pathogen has developed substantial resistance to the frequently used fungicides. Consequently, alternative methods are needed to manage CLB. One possibility is the RNA interference-based topical application of double-stranded (ds)RNA. The present study addressed the two most critical steps for this novel approach to be practical: inexpensively producing large quantities of dsRNA and identifying the right target genes for silencing. A screening method was developed to compare the effectiveness of Escherichia coli-produced dsRNAs targeting five fungal genes involved in cercosporin production for silencing in liquid culture. As much as 151.6 mg of dsRNA-containing total nucleic acids (TNAs) was produced from 1 liter of E. coli Luria broth culture using the L4440 vector. All tested dsRNAs reduced cercosporin production. However, significant target gene suppression was only detected in the cultures treated with dsRNAs from Avr4 and CTB8. The most potent dsRNA was from Avr4, which reduced 50% of cercosporin production at an estimated TNA concentration of 10.4 µg/ml (half maximal effective concentration [EC50]), and the least potent dsRNA was from HN-2, with an estimated EC50 of 46.7 µg/ml TNA. The present study paves the road for managing CLB under field conditions using dsRNA. Additionally, this approach could be adapted to identify the best dsRNAs to manage other fungal diseases.

Chinese mesona (Platostoma palustre) plays an important role as special crop in Southeast Asia and Taiwan for the production of herbal tea, grass jelly, and further processed food. In order to assess the potential threat of fungi to Chinese mesona, we surveyed isolates from symptomless plants in the area of mesona production, as well as from leaf spots of potted plants in a garden shop and a plantation in a botanical garden in Taiwan. From leaves, stems, and roots of 15 symptomless plants sampled at five collection events over two years, 154 isolates from 810 surface-sterilized plant fragments were obtained and identified based on DNA sequence data of the internal transcribed spacer region, and partially of the β-tubulin and histone H3 genes. The most common species belonged to the genera Cercospora, Colletotrichum, and Fusarium and were considered to be potential plant pathogens. Latent pathogenicity was confirmed by an infection experiment with an endophytic strain of Corynespora cassiicola. Observation of leaf spot disease associated with Cercospora kikuchii suggested pathogenicity of this fungus, which was also isolated as an endophyte from symptomless leaves. We hypothesize that the most common endophytic fungi are latent pathogens in the host and may cause plant disease when the host becomes weakened by senescence or changed cultivation condition. Leaf spots of plants in the botanical garden were associated with a species of Pseudocercospora, which was not found among the endophytic isolates and is newly described based on morphology and analysis of translation elongation factor 1 alpha gene sequences.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection “toolbox” for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in β-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the dominant foliar disease affecting table-beet production in New York. CLS epidemics occur annually and, if uncontrolled, will rapidly lead to defoliation. In broad-acre production, season-long maintenance of healthy leaves is important to facilitate harvest by top-pulling. Fungicides are the dominant means of CLS control and applications are initiated at an action threshold of 1 CLS lesion/leaf. Regular fungicide application occurs thereafter without regard for scheduling based on weather-based risk. The current action threshold was evaluated with selected fungicides in two replicated field trials. Copper oxychloride + copper hydroxide and propiconazole significantly improved CLS control if initiated prior to infection. Pydiflumetofen + difenoconazole significantly reduced area under the disease progress stairs compared with other fungicides tested and was most efficacious when applications began at 1 CLS lesion/leaf. Six replicated field trials also evaluated the utility of scheduling fungicides on weather-based risk rather than a calendar approach. Two risk thresholds (moderate and high) integrating the accumulation of daily infection values based on temperature and relative humidity from a forecaster for CLS in sugar beet were evaluated. Applications of pydiflumetofen + difenoconazole were reduced from three to two by using the forecaster at either risk threshold compared with calendar applications without affecting CLS control. For propiconazole, the moderate risk threshold provided CLS control equivalent to calendar applications and saved one spray per season. Thus, there was substantial scope to reduce spray frequency by scheduling based on weather-based risk rather than calendar applications. The optimal risk thresholds for pydiflumetofen + difenoconazole and propiconazole were high and moderate, respectively. In these trials, periods of high risk occurred less frequently than moderate risk, increasing the reapplication intervals and, hence, represented a less conservative approach to disease management.

Cercospora beticola and Phoma betae are important pathogens of table beet, sugar beet, and Swiss chard (Beta vulgaris subsp. vulgaris), causing Cercospora leaf spot (CLS) and Phoma leaf spot, root rot, and damping-off, respectively. Both pathogens may be seedborne; however, limited evidence is available for seed infestation by C. beticola. Due to the limitations of culture-based seed assessment methods, detection of these pathogens was investigated using PCR. A P. betae-specific quantitative PCR assay was developed and used in conjunction with a C. beticola-specific assay to assess the presence of pathogen DNA in 12 table beet seed lots. DNA of C. beticola and P. betae was detected in four and eight seed lots, respectively. Plate tests and BIO-PCR confirmed the viability of each pathogen; however, competitive growth of other microbes and low incidence limited the frequency and sensitivity of detection in some seed lots. The results for P. betae support previously described infestation of seed. Further investigation of C. beticola-infested seed lots indicated the ability of seedborne C. beticola to cause CLS on plants grown from infested seed. Detection of viable C. beticola on table beet seed demonstrates the potential for pathogen dispersal and disease initiation via infested seed, and provides valuable insight into the epidemiology of CLS. Surveys of commercial table beet seed are required to determine the frequency and source of C. beticola seed infestation and its role as primary inoculum for epidemics, and to evaluate the effectiveness of seed treatments.

Gray leaf spot (Cercospora zeae-maydis) is a foliar disease of corn (Zea mays) that consistently reduces yields across the United States and is an annual concern in Indiana corn production. Field trials were conducted in West Lafayette, IN, over 3 years (2016 to 2018) to evaluate the effectiveness of 12-leaf collar stage (V12) foliar fungicide applications compared with tasseling (VT) applications for gray leaf spot management and yield. Results indicated that during years in which foliar disease severity was less than 4%, there was no effect of application timing on gray leaf spot severity. In 2018, when gray leaf spot levels exceeded 5%, significantly less disease was observed in treatments receiving VT applications compared with V12 applications. Application timing did not affect yield in any year of the experiment. In 2016, benzovindiflupyr + azoxystrobin + propiconazole resulted in greater yields compared with the nontreated control, and in 2018, pyraclostrobin + metconazole and benzovindiflupyr + azoxystrobin + propiconazole resulted in greater yields compared with the nontreated control. This research indicates that in high disease pressure environments and years, Indiana farmers may want to continue to apply fungicides at VT rather than apply prior to tassel.

Cercospora beticola, the cause of Cercospora leaf spot (CLS) of sugar beet and table beet, has a broad range of potential alternative hosts. The role of these hosts as inoculum sources in the field is unclear and has had limited investigation since the advent of DNA-based pathogen identification. The presence of C. beticola on alternative hosts associated with table beet fields of New York was assessed in field surveys during 2016. Lesions were collected, and 71 cercosporoid conidia were isolated for phylogenetic comparison. C. beticola was identified from Solanum ptycanthum (n = 4), Chenopodium album (n = 2), and Spinacia oleracea (n = 1), whereas C. chenopodii was identified on Chenopodium album (n = 51). Artificial inoculation of 21 plants species demonstrated that C. beticola was pathogenic to Brassica kaber, Chenopodium album, Carthamus tinctorius, Rumex obtusifolius, and Spinacia oleracea. These results indicate that although C. beticola may be pathogenic to a range of plant species, the role of symptomatic tissue for inoculum production on alternative hosts in the field appears limited. Observations of C. beticola on necrotic and naturally senescent tissue suggest saprophytic survival on plant debris of a range of species, which has implications for CLS epidemics and disease management.

Cercospora leaf spot, caused by Cercospora beticola, is a highly destructive disease of Beta vulgaris subsp. vulgaris worldwide. C. beticola populations are usually characterized by high genetic diversity, but little is known of the relationships among populations from different production regions around the world. This information would be informative of population origin and potential pathways for pathogen movement. For the current study, the genetic diversity, differentiation, and relationships among 948 C. beticola isolates in 28 populations across eight geographic regions were investigated using 12 microsatellite markers. Genotypic diversity, as measured by Simpson’s complement index, ranged from 0.18 to 1.00, while pairwise index of differentiation values ranged from 0.02 to 0.42, with the greatest differentiation detected between two New York populations. In these populations, evidence for recent expansion was detected. Assessment of population structure identified two major clusters: the first associated with New York, and the second with Canada, Chile, Eurasia, Hawaii, Michigan, North Dakota, and one population from New York. Inferences of gene flow among these regions suggested that the source for one cluster likely is Eurasia, whereas the source for the other cluster is not known. These results suggest a shared origin of C. beticola populations across regions, except for part of New York, where population divergence has occurred. These findings support the hypothesis that dispersal of C. beticola occurs over long distances.

Annual epidemics of Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, can result in substantial defoliation in table beet fields in New York. High allelic and genotypic diversity have been described within C. beticola populations; however, information on the temporal stability of populations is lacking. C. beticola isolates were obtained from symptomatic leaves in three table beet fields in successive years. Two of the fields were organic mixed-cropping farms and the third was managed conventionally in a broad-acre cropping system. C. beticola isolates (n = 304) were genotyped using 12 microsatellite markers. Genotypic diversity (Simpson’s complement index = 0.178 to 0.990), allele frequencies, and indices of differentiation between years varied. Pairwise index of differentiation values ranged from 0.02 to 0.25 for clone-corrected data, and indicated significant genetic differentiation at Farm 2. No multilocus genotype was shared between years. The shift in multilocus genotypes between years questions the role of clonally reproducing primary inoculum. Collectively, these results suggest that a dominant inoculum source for initiating annual CLS epidemics is external to the field of interest. These findings have implications for CLS disease management in conventional and organic table beet production.

Although previous research showed that the purple symptom of Cercospora leaf blight (CLB) is associated with lower biomass of Cercospora cf. flagellaris and lower concentrations of cercosporin, a reactive oxygen species producer, as compared with blighted leaves, the hypothesis that the purple symptom is a plant reaction to the pathogen has never been tested. In this study, we demonstrated that high levels of coumestrol (COU) were associated with purple symptoms of CLB and that COU has strong antioxidant activity. Additionally, we found that COU is restricted to the pigmented areas of purple leaves, and the pigmentation is restricted to the adaxial surfaces, suggesting that COU may be acting as a sunscreen. Even though COU is associated with the purple leaf symptom, this coumestan is not the direct cause of discoloration in that COU is colorless. Quantification of chlorophyll a and b and total carotenoids suggested that blighted but not purple or asymptomatic leaves were undergoing photooxidative stress. Because the purple symptom is associated with high COU concentrations, lower biomass of C. cf. flagellaris, and lower cercosporin concentrations, we conclude that the purple symptom is a disease resistance reaction, mediated in part by COU, which provides a high level of antioxidant activity and, hence, partial resistance.

Nicotiana benthamiana is a valuable model organism in plant biology research. This report describes its extended applicability in the field of molecular plant pathology by introducing a nonbiotrophic fungal pathogen Cercospora nicotianae that can be conveniently used under laboratory conditions, consistently induces a necrotic leaf spot disease on Nicotiana benthamiana, and is specialized on solanaceous plants. Our inoculation studies showed that C. nicotianae more effectively colonizes N. benthamiana than its conventional host, N. tabacum. The functions of two critical regulators of host immunity, coronatine-insensitive 1 (COI1) and ethylene-insensitive 2 (EIN2), were studied in N. benthamiana using Tobacco rattle virus-based virus-induced gene silencing (VIGS). Perturbation of jasmonic acid or ethylene signaling by VIGS of either COI1 or EIN2, respectively, resulted in markedly increased Cercospora leaf spot symptoms on N. benthamiana plants. These results suggest that the N. benthamiana–C. nicotianae host–pathogen interaction is a prospective but hitherto unutilized pathosystem for studying gene functions in diseased plants.

Okra [Abelmoschus esculentus (L.) Moench], which belongs to the family Malvaceae, is widely grown in the tropics, sub-tropics and warmer areas of the temperate zones for its immature seed pods which are consumed as a vegetable. In China, okra pods are consumed as not only vegetables but also as a traditional medicine to cure dental diseases and gastric ulcers. During September 2018 to June 2019, extensive spots on okra leaves were observed in several commercial fields (approximately 2.0 hectares), with disease incidence of approximately 25%~50% in the Yanqing District (115°98'E, 40°46'N) of Beijing, China. Symptoms of the disease initially appeared as small pale brown spots with yellow haloes. As the disease progressed, some spots gradually coalesced, forming larger irregular dark brown lesions. The centers of the lesions became grayish white. A total of 13 small fragments (3 to 5 mm) excised from the lesion margins were sterilized in 1% sodium hypochlorite (NaClO) for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. In total, 21 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 286/span> to 711 μm (avg. = 578 μm, n = 50). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3-17-septate, and measuring 52 to 231 μm (avg. = 182 μm, n = 50). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). Pathogenicity tests were conducted on potted okra plants cv. 'Jiayuan'. Twenty four healthy okra plants at the true leaf stage were sprayed with conidial suspensions (1 × 106 conidia/mL), incubated at a glass cabinet maintained at 25°C and 90% relative humidity (RH). To each leaf approximately 10 mL of conidial suspension was applied. Plants sprayed with water were used as controls. Seven days later, dark brown spot, which were identical to those observed in the fields, were observed on inoculated leaves, whereas the control plants remained healthy. C. cf. flagellaris was reisolated from symptomatic leaves, confirming Koch's Postulates. Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). The nuclear ribosomal internal transcribed spacer region (ITS), and portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone & Kohn 1999). The resulting 542 bp ITS, 226 bp ACT, 410 bp HIS3 and 306 bp TEF1 sequences of isolate QK14091813 were deposited in GeneBank (Accession nos. MT949700, MT949701, MT949702 and MT949703, respectively). The ITS, ACT, HIS3 and TEF1 sequences shared 99.42% to 100% identities to previously published sequences of C. cf. flagellaris (Accession nos. MN633275 for ITS, MF680960 for ACT, MK991295 for HIS3, and MK991292.1 for TEF1, respectively). Multi-locus phylogenetic analyses (ITS, ACT, HIS3, and TEF1) were performed by neighbor-joining method using MEGA 7.0. The resulting trees showed that C. cf. flagellaris isolate QK14091813 (this study) nested within the clade that includes other isolates of C. cf. flagellaris with a 99% confidence level. To our knowledge, this is the first report of C. cf. flagellaris causing leaf spot on okra (Farr and Rossman 2020). The pathogen has a worldwide distribution and an unusually broad host range, which can be of great significance, and the plant protection policy of priority to prevention and synthetical prevention should be followed.

Cercospora leaf spot (CLS), caused by Cercospora beticola, is one of the major diseases affecting productivity and profitability of beet production worldwide. Fungicides are critical for the control of this disease and one of the most commonly used products is the quinone outside inhibitor (QOI) azoxystrobin. In total, 150 C. beticola isolates were collected from two commercial processing table beet fields in Batavia, NY in 2014. The mating types of the entire population were determined, and genetic diversity of a subset of samples (n = 48) was assessed using five microsatellite loci. Sensitivity to azoxystrobin was tested using a spore germination assay. The cytochrome b gene was sequenced to check for the presence of point mutations known to confer QOI resistance in fungi. High allelic diversity (He = 0.50) and genotypic diversity (D* = 0.96), gametic equilibrium of the microsatellite loci, and equal ratios of mating types were suggestive of a mixed mode of reproduction for C. beticola. Resistance to azoxystrobin was prevalent because 41% of the isolates had values for effective concentrations reducing spore germination by 50% (EC50) > 0.2 μg/ml. The G143A mutation, known to cause QOI resistance in C. beticola, was found in isolates with EC50 values between 0.207 and 19.397 μg/ml. A single isolate with an EC50 of 0.272 μg/ml carried the F129L mutation, known to be associated with low levels of QOI resistance in fungi. This is the first report of the F129L mutation in C. beticola. The implications of these findings for the epidemiology and control of CLS in table beet fields in New York are discussed.

In November 2017, leaf spots were observed in a fresh market spinach crop of cultivar SV2157VB in New Jersey at ∼20% incidence but not in the adjacent crop of Unipack-151. Pale tan lesions (3 to 8 mm in diameter) had gray sporulation at the centers. Symptomatic leaf pieces were surface-sterilized in 0.6% NaOCl for 60 s, triple-rinsed in sterilized water, dried, and plated onto PDA and water agar (each with 100 µg of chloramphenicol/ml). Seven isolates all produced hyaline, straight to slightly curved conidia, mostly two- to three-septate, 20.0 to 82.0 µm (45.2 ± 11.3 µm, mean ± standard deviation) × 6.0 to 11.0 µm (8.1 ± 1.0 µm) (n = 312), with no significant differences among isolates. Spore characteristics matched those of Cercospora spp. (Świderska-Burek 2015). The consensus sequence of the calmodulin gene (cmdA) amplified from all seven isolates using primers CALDF1 and CALDR (Lawrence et al. 2013) (GenBank accession no. MN422444) was 99.78% identical to that of Cercospora cf. chenopodii (JX142839.1), which is not differentiated from C. chenopodii by this gene (Groenewald et al. 2013). Actin (actA) primers ACT-512F and ACT-783R generated an identical sequence for the isolates (MN422442) that was 99.55% identical to that of C. chenopodii (JX143081.1) (Carbone and Kohn 1999). The translation elongation factor 1-α (TEF1-α) sequence of the isolates (MN422445) had 99.45% identity to the sequence of a voucher specimen of C. chenopodii (JX143327.1). C. beticola-specific primers CbCALF and CbCALR did not amplify DNA of these isolates, only DNA of control isolates of C. beticola (Knight et al. 2019). Based on a phylogenetic tree inferred from concatenated actA, cmdA, ITS rDNA, and TEF1-α sequences, the isolates clustered with the voucher strain C. chenopodii CBS 132620. Six isolates were each tested for pathogenicity on four 33-day-old plants of each of spinach cultivars Mandolin, SV2157VB, Unipack-151, and Viroflay; sugar beet cultivar KDH4-9 (USDA National Plant Germplasm System PI 683513, homozygous susceptible to C. beticola), red beet cultivars Red Ace and Ruby Queen, and Swiss chard cultivar Silverado. Plants were enclosed in plastic bags for 24 h, atomized with a spore suspension (1 × 10⁴ conidia/ml) prepared from clarified V8 agar plates of each isolate, and returned to the bags for 24 h. Four non-inoculated control plants of each cultivar were atomized with water as control treatments. Beet and chard plants were placed in a greenhouse and spinach plants in a growth chamber with 10 h lighting/day to avoid bolting under 16-h summer days in Washington. Symptoms had not developed after 7 days, at which time spinach plants were moved to the greenhouse. By 9 days, leaf spots resembling those on the original leaves were observed on Mandolin and SV2157VB. By 14 days, there were leaf spots on all four spinach cultivars. Symptoms were most severe on SV2157VB (18.3 ± 2.0% mean ± standard error of leaf area), followed by Mandolin and Viroflay (8.8 ± 1.6%), and then Unipack-151 (2 ± 0.5%). Symptoms did not develop on non-inoculated spinach or inoculated and non-inoculated sugar beet, table beet, and Swiss chard. Cercospora could only be reisolated from symptomatic spinach plants, with the isolates confirmed as C. chenopodii based on actA sequences. Although C. beticola has been reported to cause Cercospora leaf spot of spinach (Knight et al. 2018), this is the first report of C. chenopodii causing a leaf spot of spinach in the United States. Research is needed to evaluate impacts of this species on additional spinach cultivars and management options.

During the summer of 2017, 38 Cercospora spp. isolates were collected from soybean leaves displaying symptoms of Cercospora leaf blight (CLB) from commercial soybean fields at three locations in Santa Cruz, Bolivia. Portions of cytochrome b (N = 38) and calmodulin (N = 37) were amplified, sequenced, edited, and assembled. Two representative isolates from each lineage (with distinct calmodulin haplotypes, if present) were selected for sequencing of two additional loci, histone 3 and elongation factor 1-alpha, for species identification. Based on the corresponding multilocus phylogenetic analysis, the 37 isolates belong to three species: C. nicotianae (N = 14), C. sp. “P” (N = 16), and C. kikuchii (N = 7). All 38 isolates (100%) possessed the G143A mutation in cytochrome b, and none carried the F129L mutation. Results of phenotypic assays on a subset of 15 isolates (five from each species) supported a high level of resistance to azoxystrobin. This is the first report of the G143A mutation in Cercospora species associated with CLB in South America and the first report of C. nicotianae associated with CLB. Future monitoring for G143A mutants of Cercospora spp. in South America will be necessary to assess whether strobilurin resistance is widespread.

Foliar fungicide use in the U.S. Corn Belt increased in the last decade; however, questions persist pertaining to its value and sustainability. Multistate field trials were established from 2010 to 2012 in Illinois, Iowa, Ohio, and Wisconsin to examine how hybrid and foliar fungicide influenced disease intensity and yield. The experimental design was in a split-split plot with main plots consisting of hybrids varying in resistance to gray leaf spot (caused by Cercospora zeae-maydis) and northern corn leaf blight (caused by Setosphaera turcica), subplots corresponding to four application timings of the fungicide pyraclostrobin, and sub-subplots represented by inoculations with either C. zeae-maydis, S. turcica, or both at two vegetative growth stages. Fungicide application (VT/R1) significantly reduced total disease severity relative to the control in five of eight site-years (P < 0.05). Disease was reduced by approximately 30% at Wisconsin in 2011, 20% at Illinois in 2010, 29% at Iowa in 2010, and 32 and 30% at Ohio in 2010 and 2012, respectively. These disease severities ranged from 0.2 to 0.3% in Wisconsin in 2011 to 16.7 to 22.1% in Illinois in 2010. The untreated control had significantly lower yield (P < 0.05) than the fungicide-treated in three site-years. Fungicide application increased the yield by approximately 6% at Ohio in 2010, 5% at Wisconsin in 2010 and 6% in 2011. Yield differences ranged from 8,403 to 8,890 kg/ha in Wisconsin 2011 to 11,362 to 11,919 kg/ha in Wisconsin 2010. Results suggest susceptibility to disease and prevailing environment are important drivers of observed differences. Yield increases as a result of the physiological benefits of plant health benefits under low disease were not consistent. National Institute of Food and Agriculture/[2009-51101-05820]/NIFA/Estados Unidos Risk Avoidance and Mitigation Program/[2009-51101-05820]/RAMP/Estados Unidos United States Department of Agriculture/[P526P-08-00769]/USDA/Estados Unidos United States Department of Agriculture/[P526P-08-01569]/USDA/Estados Unidos United States Department of Agriculture/[P526P-10-01323]/USDA/Estados Unidos UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomía

Purple seed stain (PSS), caused by Cercospora kikuchii, is a prevalent soybean disease that causes latent seed infection, seed decay, purple seed discoloration, and overall quality deterioration. The objective of this research was to screen soybean accessions from the United States Department of Agriculture germplasm collection for resistance to PSS. In total, 123 plant introductions (PI) from 28 different countries, representing maturity groups (MG) III, IV, and V, were screened. Incidence of Cercospora leaf blight (% CLB), visual PSS (% PSS), and seed infected by C. kikuchii (% C. kikuchii) in harvested seed were determined. In 2007, % C. kikuchii was 2 to 51% for MG III, 2 to 35% for MG IV, and 0 to 33% for MG V. In 2008, % C. kikuchii was 0 to 45% for MG III, 1 to 71% for MG IV, and 0 to 15% for MG V. In total, four and ten PI from MG III and IV, respectively, were identified as resistant to PSS in both years. Highly positive correlations were found for inoculated versus noninoculated treatments and for % PSS versus % C. kikuchii infection. The PSS-resistant PI identified in this study will be valuable to breeders in developing resistant cultivars.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.

de Paiva Custodio, A. A., Pozza, E. A., de Paiva Custodio, A. A., de Souza, P. E., Lima, L. A., and da Silva, A. M. 2014. Effect of center-pivot irrigation in the rust and brown eye spot of coffee. Plant Dis. 98:943-947. Incidence of rust (caused by Hemileia vastatrix) and brown eye spot (caused by Cercospora coffeicola) of coffee was evaluated for 2 years in southern Minas Gerais, Brazil, in a center-pivot-irrigated coffee crop with different water application levels. Treatments consisted of six water levels of 0 (nonirrigated), 60, 80, 100, 120, and 140% applied according to class A evaporation pan (CAE). Progress of disease was influenced significantly by different water levels; the highest incidence of rust occurred with lower water levels—0 (nonirrigated), 60, and 80% CAE—whereas the lowest incidence was detected with larger water levels: 120 and 140% CAE. There was one peak of rust incidence in August. The highest incidence of brown eye spot occurred with water levels of 100 and 140% CAE and the lowest incidence was found in the nonirrigated treatment. Two incidence peaks occurred in March and June. This study identified that irrigation methods and water levels can decisively affect the epidemiological behavior of diseases and, therefore, their management. Brazil is the world’s largest producer of coffee (Coffea arabica L.) and the second largest consumer market. In addition, coffee is largely consumed worldwide. In this country, the state of Minas Gerais is responsible for approximately 50% of the national output, with the largest planted area in the state’s southern region (8). In all, 25% of Brazilian coffee production comes from irrigated areas, although irrigated crops only cover 240,000 ha or 10% of the total area (8). Rainfall in southern Minas Gerais is favorable for coffee production but adverse weather causing droughts and a growing need for increased crop yield and quality of coffee due to global demand have encouraged producers to use irrigation in the region (4,15). Despite the benefits, irrigated coffee production can intensify diseases such as rust (caused by Hemileia vastatrix Berk. & Broome) and brown eye spot (caused by Cercospora coffeicola Berk. & Cooke), causing significant losses (19,20,23).

Spinach (Spinacia oleracea L.) is one of the most important leafy vegetables, cultivated throughout Pakistan. During September to November 2016, five spinach (cultivar ‘Kandiari’) fields (31°36′17″N, 74°34′23″E) were inspected near Lahore city, Pakistan. Leaf spot symptoms on plants were observed in 10 to 30% of inspected fields. Symptoms began as small circular to subcircular light-brown spots with a distinct dark brown margin. In mature more or less sunken lesions, the center was whitish gray to black, owing to sporulation. Microscopic study of the whitish-gray lesions revealed the presence of small and black stromata within the leaf substomatal cavities. The stromata produced loose fascicles of conidiophores, up to 15 conidiophores in a fascicle. Conidiophores were simple, one to four septate, golden brown but subhyaline at the apex, straight, 4.5 to 5 × 20 to 100 µm (n = 50), geniculate, having distinctive spore scars, and unbranched. Conidia were solitary, hyaline, filiform, straight to slightly curved, with obtuse to subacute at the apex and subtruncate bases, multiseptate (one to 18), nonconstricted at the septa, 28 to 230 × 2.5 to 4 µm (n = 50). On the basis of morphological characteristics, the causal organism was identified as Cercospora beticola Sacc. (Chupp 1954; Crous and Braun 2003). To obtain single-spore isolates from samples, conidial suspension from one lesion per sample was inoculated on separate 2% water agar plates and incubated at 24°C. After 24 h, individual germinating conidia were planted on separate potato dextrose agar (PDA) plates and incubated at 24°C for 10 days. Pure cultures of six C. beticola isolates were used for DNA extraction. The partial internal transcribed spacer (ITS) regions of the fungal rDNA and calmodulin (CAL) gene were amplified using the primers ITS1/ITS4 and CAL228F/CAL737R and sequenced. Obtained DNA sequences of the six isolates were identical; therefore, ITS (MH318663) and CAL (MK333232) sequences of a representative isolate (IM084) were submitted to GenBank. The resulting ITS and CAL sequences of IM084 showed 99 to 100% similarity with C. beticola accessions (MH42448, MF681169, JX142819, AY752227, and FJ473438). A pathogenicity test was accomplished by placing PDA discs (0.5 mm²) from a 10-day-old representative fungal culture onto leaves of six 3-week-old potted spinach (cv. Kandiari) and sugar beet (cv. ‘9597’) (three plants of each). For noninoculated controls, three plants of each type were inoculated with noncolonized PDA plugs. After inoculation, plants were kept 48 h in a dew chamber at 25°C and then transferred to the greenhouse under 12/12-h light/dark conditions at 25°C. Lesions developed on fungal inoculated leaves of spinach and sugar beet plants with similar characteristic symptoms as those observed on naturally infected spinach leaves within 10 days after inoculation. Control spinach and sugar beet plants remained asymptomatic. Sporulation of C. beticola was observed in lesions similar to initial symptoms observed in field samples. The pathogen was reisolated from infected leaf lesions and confirmed as C. beticola. Previously, C. beticola causing Cercospora leaf spot has been reported on sugar beet (Khan et al. 2007); however, to our knowledge this is a new record of C. beticola on spinach in Pakistan. Spinach is one of the high demand leafy vegetables, and incidence of this new pathogen can be a significant threat for spinach production.

Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beet (Beta vulgaris) worldwide. Despite the great agronomical importance of this disease, little is known about its underlying molecular processes. Technical resources are scarce for analyzing this important crop species. We developed a sugar beet microarray with 44,000 oligonucleotides that represent 17,277 cDNAs. During the four stages of C. beticola–B. vulgaris interactions, we profiled the transcriptional responses of three genotypes: susceptible, polygenic partial resistance, and monogenic resistant. Similar genes were induced in all three genotypes during infection but with striking differences in timing. The monogenic resistant genotype displayed strong defense responses at 1 day postinoculation (dpi). The other genotypes displayed defense responses in a later phase (15 dpi) of the infection cycle. The partially resistant genotype displayed a strong defense response in the late phase of the infection cycle. Furthermore, the partially resistant genotype expressed pathogen-related transcripts that the susceptible genotype lacked. These results indicate that resistance was achieved by the ability to mount an early defense response, and partial resistance was determined by additional defense and signaling transcripts that allowed effective defense in the late phase of the infection cycle.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

Quinone outside inhibitor (QoI) foliar fungicides can be effective at reducing foliar diseases in corn (Zea mays), and they have been shown to provide physiological benefits experimentally in other crops in the absence of disease. A new supplemental label for pyraclostrobin that was approved in January 2009 by the United States Environmental Protection Agency (EPA) states that corn plants applied with pyraclostrobin may have better tolerance to damage caused by hail. To determine the effects of QoI foliar fungicides on hail-damaged corn, field research trials were conducted near Champaign, IL in 2007 and 2008. Hail damage was simulated with a gasoline-powered string-mower at the V12 growth stage, which caused injury to leaves and defoliation. At VT, the foliar fungicides azoxystrobin and pyraclostrobin were applied to corn. Control treatments included a nontreated control and a nondamaged control. The simulated hail damage significantly (P ≤ 0.05) increased gray leaf spot severity (caused by Cercospora zeae-maydis) in 2007 but not in 2008. Simulated hail damage also significantly reduced yield compared with the nondamaged control in both 2007 and 2008. Foliar fungicides significantly reduced disease severity compared with the nontreated control in 2007 but not in 2008; however, foliar fungicides did not significantly improve yield in either the damaged or nondamaged plots compared with the nontreated controls. Results from our research trials indicated that foliar fungicides provided very little benefit to corn injured by simulated hail; thus, growers should consider factors other than hail damage when making fungicide application decisions for corn.

In October 2016, disease was observed on stems and branches of asparagus (Asparagus officinalis) in an organic crop at a fresh market farm in Ontario Co., NY. Lesions were small (

Cercospora leaf spot, caused by Cercospora beticola, is the most damaging foliar disease of sugar beet in Minnesota (MN) and North Dakota (ND). Research was conducted to characterize the temporal progression of aerial concentration of C. beticola conidia in association with the environment and disease severity in sugar beet. In 2003 and 2004, volumetric spore traps were placed within inoculated sugar beet plots to determine daily dispersal of conidia at Breckenridge, MN, and St. Thomas, ND. Plots were rated weekly for disease severity. At both locations, conidia were first collected in early July 2003 and late June in 2004. Peaks of conidia per cubic meter of air were observed with maxima in late August 2003 and in early September 2004 at both locations. Peaks of airborne conidium concentration were significantly correlated with the average temperature of daily hours when relative humidity was greater than 87%. Weekly mean hourly conidia per cubic meter of air was significantly (P < 0.01) associated with disease severity during both years and across locations. This study showed that C. beticola conidial numbers may be used to estimate potential disease severity that, with further research, could be incorporated in a disease forecasting model to rationalize Cercospora leaf spot management.

Cercospora leaf spot is a destructive fungal disease that has become a threat to the coffee industry in Brazil. Nevertheless, little is known about populations of its causal agent, Cercospora coffeicola. We evaluated the potential of using nitrogen-nonutilizing (nit) mutants and vegetative compatibility groups (VCGs) to characterize the genetic variability of the C. coffeicola population associated with coffee plantings in Minas Gerais state (MG), Brazil. A total of 90 monosporic isolates were obtained from samples collected according to a hierarchical sampling scheme: (i) state geographical regions (Sul, Mata, and Triângulo), and (ii) production systems (conventional and organic). Nit mutants were obtained and 28 VCGs were identified. The 10 largest VCGs included 72.31% of all isolates, whereas each of the remaining 18 VCGs included 1.54% of the isolates. Isolates of the largest VCGs were found in the three regions sampled. Based on the frequencies of VCGs at each sampled level, we estimated the Shannon diversity index, as well as its richness and evenness components. Genetic variability was high at all hierarchical levels, and a high number of VCGs was found in populations of C. coffeicola associated with both conventional and organic coffee plantings.

Random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction (MP-PCR) were used to characterize 164 isolates of Cercospora kikuchii, most of which were collected from Louisiana. Plant tissue (seeds versus leaves), but not host cultivar, had a significant impact on pathogen population differentiation. Cluster analysis showed that the Louisiana population was dominated by a primary lineage (group I) with only a few Louisiana isolates belonging to the minor lineage that also included the non-Louisiana isolates (group II). A previous study showed that isolates could be differentiated according to vegetative compatibility groups (VCGs). However, RAPD and MP-PCR data demonstrated that isolates of C. kikuchii were not generally clustered according to these VCGs. Furthermore, genetic relationships within and between VCGs were examined using sequences of the intergenic spacer region of rDNA. These analyses showed that VCG is not an indicator of evolutionary lineage in this fungus. Our results suggest the likely existence of a cryptically functioning sexual stage in some portion of the C. kikuchii population.

Cercospora beticola survives as stromata in infected crop residue. Spores produced on these survival structures serve as primary inoculum during the next cropping season. This study was conducted to determine how long C. beticola can survive at different soil depths, the mechanism of inoculum dispersal, and the primary infection site in sugar beet. Longevity of C. beticola was studied over a 3-year period under field conditions at Fargo, ND. C. beticola-infected leaves were placed at depths of 0, 10, and 20 cm and retrieved after 10, 22, and 34 months. Survival of C. beticola inoculum declined with time and soil depth. Inoculum left on the soil surface, 0 cm in depth, survived the longest (22 months) compared with that buried at 10 cm (10 months) and 20 cm (10 months). C. beticola dispersal from the primary source of inoculum was studied in the field for three growing seasons. Sugar beet plants were surrounded with plastic cages with and without ground cover, or exposed with and without ground cover. Significantly higher disease severity was observed on exposed plants than caged plants with or without ground cover, suggesting that wind was the major dispersal factor for C. beticola inoculum. The primary infection site by C. beticola was determined in a greenhouse study. Leaves, roots, and stems of healthy sugar beet plants were inoculated with C. beticola. Cercospora leaf spot symptoms were observed only on plants that were leaf inoculated, suggesting that the leaf was the primary infection site for C. beticola.

Disease dynamics of Cercospora leaf spot (CLS) of sugar beet was analyzed at two hierarchical scales: as vertical profiles within individual plants and in relation to disease on neighboring plants. The relative contribution of different leaf layers to increase in CLS was analyzed using a simple continuous-time model. The model was fitted to data from two field trials in the Netherlands: one in an area with a long history of CLS, the other in an area where CLS has only recently established; in each case these were unsprayed and twice-sprayed treatments. There were differences in the relative contribution of different leaf layers to disease increase on the target leaf layer according to the CLS history and whether the plants were sprayed or unsprayed. In both field trials, parameter estimates giving the relative contribution of the target leaf layer to disease increase at that leaf layer were higher than those for the lower leaf layer. On only a few occasions the contribution of an upper leaf layer to disease increase at the target leaf layer was significant. Thus, CLS increase at the target leaf layer was determined mainly by disease severity at that leaf layer and to a lesser extent by disease at the lower leaf layer. Our continuous-time model was also used to analyze CLS increase on an individual sugar beet plant in relation to its own and its neighbor's level of disease in field trials at five locations in the two CLS areas over two years. In all field trials, the contribution of the target plant itself to disease increase (auto-infection) was larger than that of its neighboring plants (allo-infection). The overall analysis in the two CLS areas also indicated a larger contribution of the target plant to its disease increase than of neighboring plants, and this pattern was also apparent in a pooled analysis across all sites. Thus, CLS increase on a sugar beet plant was mainly determined by the disease severity on that plant and to a lesser extent by its within-row neighboring plants.

Management of Cercospora leaf spot, caused by Cercospora beticola, is necessary for the economic production of sugar beet (Beta vulgaris). The objectives of this study were to evaluate the impact of two relative humidity thresholds (87 and 90%) on the daily infection values (DIVs) used to determine when fungicide applications were required, to determine whether current Cercospora management recommendations for northern areas of Minnesota and North Dakota could be used by growers in the southern areas of these states, and to compare the utility of calendar-based fungicide applications with the Cercospora management model. Research was conducted in Breckenridge, MN and St. Thomas, ND in 2003 and 2004. Fungicide applications significantly (P = 0.05) reduced maximum disease severity (ymax) and area under the disease progress curve (AUDPC) when compared with the nontreated control at both locations during 2003 and 2004. Fungicides applied according to DIVs calculated at RH ≥ 87% or RH > 90% gave similar results. The mandatory second fungicide application 14 days after the first application for southern areas did not significantly decrease disease severity or AUDPC, or improve root yield or recoverable sucrose compared with treatments without the mandatory application. This research illustrates that a DIV calculated at RH ≥ 87% would result in similar timing of fungicide applications compared with DIVs calculated at RH > 90%. The results further show that the recommendation of fungicide applications at initial symptom and subsequent applications based on DIV and disease severity should be used for both northern and southern growers. Finally, this research showed that fungicide applications based on the Cercospora management model provided similar, effective disease control with fewer fungicide applications compared with calendar-based applications.

Fungal leaf blight diseases caused by Cercospora carotae and Alternaria dauci occur annually on processing carrot in New York, with growers applying up to eight fungicide sprays to manage these diseases. An integrated pest management (IPM) program involving the use of a 25% disease incidence threshold to prompt the first fungicide application and timing subsequent sprays by monitoring for increases in disease severity and weather forecasts in conjunction with a 10- to 14-day spray interval was evaluated in grower fields in 1997 and 1998. The IPM plots, compared with the grower plots, required two to six fewer fungicide applications but showed no yield reduction. From 1999 to 2004, the IPM program was validated and the effect of crop rotation and carrot cultivar susceptibility also were assessed. Carrot plants growing in fields with 2-year or longer crop rotation intervals reached the 25% disease incidence threshold later in the season and required fewer fungicide applications. The less-susceptible carrot cultivars also reached the 25% disease incidence threshold later, required fewer fungicide applications, and were less severely diseased than more susceptible cultivars. Validation of the IPM program in New York showed that both fungal leaf blights can be managed effectively using a 25% incidence threshold to prompt the first fungicide spray and making the subsequent fungicide applications based on increases in disease severity, weather forecasts, and a 10- to 14-day spray interval.

Safflower is an oilseed crop adapted to the small-grain production areas of the western Great Plains, including the Northern Plains Area (NPA). In the NPA, safflower production is being evaluated for potential rotation with sugar beet. Safflower is susceptible to Cercospora carthami, whereas sugar beet is susceptible to C. beticola C. carthami has not been observed on safflower in the NPA but C. beticola is ubiquitous on sugar beet. Observation of unusual leaf spots on irrigated safflower cv. Centennial at Sidney, MT prompted this investigation of safflower as a potential alternate host of C. beticola. Safflower plants were inoculated with four isolates of C. beticola (C1, C2, Sid1, and Sid2) and incubated in growth chambers; leaf spot symptoms appeared between 3 and 4 weeks later. Polymerase chain reaction (PCR) amplification of extracts from lesion leaf tissue with C. beticola-specific primers produced fragments comparable with amplified fragments from purified cultures of control C. beticola. PCR assay of cultures of single spores from diseased safflower leaf lesions also produced fragments comparable with fragments from C. beticola cultures. Antibody that was raised from isolate C2 also bound to antigens from the single-spore cultures of the four C. beticola isolates. Inoculum from single-spore cultures from infected safflower also infected sugar beet and produced typical Cercospora leaf spot symptoms. Assay of these leaf lesions by PCR resulted in amplification of target fragments with the C. beticola-specific primers. Our results demonstrate that safflower is a new host of C. beticola.

Regression and artificial neural network (ANN) modeling approaches were combined to develop models to predict the severity of gray leaf spot of maize, caused by Cercospora zeae-maydis. In all, 329 cases consisting of environmental, cultural, and location-specific variables were collected for field plots in Iowa between 1998 and 2002. Disease severity on the ear leaf at the dough to dent plant growth stage was used as the response variable. Correlation and regression analyses were performed to select potentially useful predictor variables. Predictors from the best 9 of 80 regression models were used to develop ANN models. A random sample of 60% of the cases was used to train the networks, and 20% each for testing and validation. Model performance was evaluated based on coefficient of determination (R2) and mean square error (MSE) for the validation data set. The best models had R2 ranging from 0.70 to 0.75 and MSE ranging from 174.7 to 202.8. The most useful predictor variables were hours of daily temperatures between 22 and 30°C (85.50 to 230.50 h) and hours of nightly relative humidity ≥90% (122 to 330 h) for the period between growth stages V4 and V12, mean nightly temperature (65.26 to 76.56°C) for the period between growth stages V12 and R2, longitude (90.08 to 95.14°W), maize residue on the soil surface (0 to 100%), planting date (in day of the year; 112 to 182), and gray leaf spot resistance rating (2 to 7; based on a 1-to-9 scale, where 1 = most susceptible to 9 = most resistant).

Wolf, P. F. J., and Verreet, J. A. 2005. Factors affecting the onset of Cercospora leaf spot epidemics in sugar beet and establishment of disease-monitoring thresholds. Phytopathology 95:269-274. Severe Cercospora leaf spots epidemics in sugar beet during the late 1980s and early 1990s in southern Germany prompted us to initiate investigations on the epidemiology of the causal agent, Cercospora beticola. The data set involved 69 field trials (1993 to 2003) focusing on factors affecting the epidemic onset of this disease. Observations were made at weekly intervals, recording the calendar week when canopy closure occurred (growth stage according to BBCH scale = 39) and symptom development by assessing the percentage of infected leaf area on a single-leaf basis (n = 40 plants). These monitoring trials revealed that epidemic onset varied between early July and mid-September. Hence, the target was to identify the reasons for this variation in order to deduct the most suitable approach for predicting epidemic onset. Differences in cultivar resistance explained part of epidemic onset variability, as did different timings of canopy closure, presumably due to associated microclimate changes. Moreover, meteorological variables were considered as potential reasons for variation in epidemic onset. The weather-dependent infection probability was assessed by daily infection values (DIV) in the range from 0 to 1 using hourly weather data. For calculating DIVs, the temperature effect was quantified by the proportions of the latent period (LP) relative to the optimum at 20 to 25°C, established by artificial inoculation of sugar beet plants in growth cabinets. Artificial infection experiments further established that air relative humidity (RH) >95% or leaf wetness was required for infection and subsequent lesion development. Under field conditions, the probability of leaf wetness was 75% at RH >90%. Therefore, DIVs were set to 0 for RH ≤90% in the absence of precipitation (moisture index I). Alternatively, the effect of moisture was modeled with a sigmoidal function describing the occurrence of leaf wetness in dependence of RH in the field (moisture index II). Using this approach, DIV values were cumulated (c-DIV) for each of the 69 trials beginning either at fixed starting dates (1 May, 16 May, or 1 June) or the dates of canopy closure. Accumulation of DIV ended at the time of epidemic onset. The two different moisture index definitions had no significant influence on c-DIV; whereas, for starting time of summation of DIV, the date of canopy closure was more suitable. Values of c-DIV ranged from 7 to 19 in highly susceptible cultivars and 12 to 25 in cultivars with lower susceptibility. Given this variation, c-DIV values were insufficient to explain differences in the date of epidemic onset and thus were not considered suitable for making accurate and precise management decisions. However, a negative prognosis assessing the most likely periods of disease absence was possible by determining the minimum c-DIV as a threshold. This threshold was 7 c-DIV for highly susceptible cultivars and 12 c-DIV for cultivars with low susceptibility. Crop monitoring is recommended as soon as these threshold values are exceeded so that the exact epidemic onset time can be observed.

Nitrogen nonutilizing (Nit) mutants were used to assess vegetative compatibility of 58 isolates of Cercospora kikuchii, 55 of which were isolated from soybean plants in Louisiana. Two isolates were vegetatively self-incompatible. Of 56 self-compatible isolates, 16 were assigned to six multimember vegetative compatibility groups (VCGs), 01 to 06, with 2 or 3 isolates in each VCG. The other 40 isolates each belonged to a distinct VCG. All six multimember VCGs contained isolates from different soy bean cultivars, and three included isolates from different locations. Only one of six multimember VCGs included isolates both from soybean leaves and seed, while the other five included isolates from only leaves or seed. The likelihood of tissue specificity or preference was discussed. All isolates and tested Nit mutants produced cercosporin on potato dextrose agar under light. Significantly different amounts of cercosporin were produced among wild-type isolates, and two Nit mutants produced significantly more cercosporin than their wild-type counterparts. All isolates produced typical Cercospora leaf blight symptoms on soybean plants in greenhouse pathogenicity tests.

Magarey, R. D., Sutton, T. B., and Thayer, C. L. 2005. A simple generic infection model for foliar fungal plant pathogens. Phytopathology 95:92-100. In this study, a simple generic infection model was developed for predicting infection periods by fungal foliar pathogens. The model is designed primarily for use in forecasting pathogens that do not have extensive epidemiological data. Most existing infection models require a background epidemiological data set, usually including laboratory estimates of infection at multiple temperature and wetness combinations. The model developed in this study can use inputs based on subjective estimates of the cardinal temperatures and the wetness duration requirement. These inputs are available for many pathogens or may be estimated from related pathogens. The model uses a temperature response function which is scaled to the minimum and optimum values of the surface wetness duration requirement. The minimum wetness duration requirement (Wmin) is the number of hours required to produce 20% disease incidence or 5% disease severity on inoculated plant parts at a given temperature. The model was validated with published data from 53 controlled laboratory studies, each with at least four combinations of temperature and wetness. Validation yielded an average correlation coefficient of 0.83 and a root mean square error of 4.9 h, but there was uncertainty about the value of the input parameters for some pathogens. The value of Wmin varied from 1 to 48 h and was relatively uniform for species in the genera Cercospora, Alternaria, and Puccinia but less so for species of Phytophthora, Venturia, and Colletotrichum. Operationally, infection models may use hourly or daily weather inputs. In the case of the former, information also is required to estimate the critical dry-period interruption value, defined as the duration of a dry period at relative humidities

Risk assessment models for gray leaf spot of maize, caused by Cercospora zeae-maydis, were developed using preplanting site and maize genotype data as predictors. Disease severity at the dough/dent plant growth stage was categorized into classes and used as the response variable. Logistic regression and classification and regression tree (CART) modeling approaches were used to predict severity classes as a function of planting date (PD), amount of maize soil surface residue (SR), cropping sequence, genotype maturity and gray leaf spot resistance (GLSR) ratings, and longitude (LON). Models were development using 332 cases collected between 1998 and 2001. Thirty cases collected in 2002 were used to validate the models. Preplanting data showed a strong relationship with late-season gray leaf spot severity classes. The most important predictors were SR, PD, GLSR, and LON. Logistic regression models correctly classified 60 to 70% of the validation cases, whereas the CART models correctly classified 57 to 77% of these cases. Cases misclassified by the CART models were mostly due to overestimation, whereas the logistic regression models tended to misclassify cases by underestimation. Both the CART and logistic regression models have potential as management decision-making tools. Early quantitative assessment of gray leaf spot risk would allow for more sound management decisions being made when warranted.

Genetic variability and population structure of Cercospora sorghi from wild and cultivated sorghum were investigated to gain insight into their potential impact on epidemics of gray leaf spot of sorghum in Africa. Population structure was examined using data derived from amplified fragment length polymorphism (AFLP) of C. sorghi by Nei's test for population differentiation, GST, and analysis of molecular variation (AMOVA). Two ecological populations of C. sorghi in Uganda were devoid of population structure (GST = 0.03, фFST = 0.01, P = 0.291). AMOVA revealed that genetic variability was due mainly to variations within (99%) rather than between (0.35%) populations, and Nei's genetic distance between the two populations was 0.014. Phenetic analysis based on AFLP data and polymerase chain reaction-restriction fragment length polymorphism analyses of the internal transcribed spacer regions of rDNA and mitochondrial small subunit rDNA separated Cercospora cereal pathogens from dicot pathogens but did not differentiate among C. sorghi isolates from wild and cultivated sorghum. Our results indicate that Ugandan populations of C. sorghi compose one epidemiological unit and suggest that wild sorghum, while not affecting genetic variability of the pathogen population, provides an alternative host for generating additional inoculum.

Conidia of Cercospora zeae-maydis are the primary inoculum causing gray leaf spot of maize. On nutrient-deficient substrates, but not on water on the leaf surface, conidia germinate and develop secondary conidia on conidiophores produced from germ tubes or conidial cells. A population of conidia increases its numbers more than twofold by 2 days on the surface of a water droplet and by fourfold on trichomes. This microcycle conidiation is suppressed by hydrogen peroxide and ammonium compounds but not by nitrate compounds, amino acids, or simple sugars. Microcycle conidiation is sensitive to α-amanitin and cycloheximide, suggesting that new RNA and proteins must be synthesized. Upon transfer from a humid to a dry atmosphere, secondary conidia and conidiophores dehydrate and collapse. Mature, dehydrated, secondary conidia are liberated by wind speeds approximately one-third those required to liberate hydrated conidia. The dispersed secondary conidia can rehydrate and germinate normally. Because this microcycle conidiation occurs at the expense of endogenous reserves, the ability to produce secondary conidia is lost after four successive cycles without a period of growth on nutrient media. This alternative method of maintaining inoculum potential during periods of fluctuating relative humidity may have epidemiological consequences when primary conidia fail to infect.

The use of genetically resistant maize hybrids is the preferred means of control of gray leaf spot, caused by Cercospora zeae-maydis. One problem faced by maize breeders attempting to breed for resistance to gray leaf spot is the high degree of genotype-environment interactions observed in disease trials. In North Carolina gray leaf spot trials conducted at four locations in the western part of the state, we found consistent hybrid-location interactions over the 1995 and 1996 growing seasons. Isolates of C. zeae-maydis from those test locations were evaluated on the same hybrids used in the multilocation testing at a location in central North Carolina that does not have a history of gray leaf spot. The hybrid-isolate interactions observed in the isolate trial mirrored the hybrid-location effects seen in the multilocation testing. Most of the interactions arose from changes in the magnitude of differences between hybrids when inoculated with the isolates rather than from any change in hybrid ranking. Analysis of internal transcribed spacer-restriction fragment length polymorphisms (RFLPs) and mitochondrial rDNA RFLPs of those isolates and others revealed that both type I and type II sibling species of C. zeae-maydis, as well as C. sorghi var. maydis, are isolated from typical gray leaf spot lesions. Breeders should use the most aggressive isolates of C. zeae-maydis to maximize discrimination between genotypes in gray leaf spot trials.

The effects of the application of 0, 60, and 120 kg N ha-1 and of 0, 25, 50, and 150 kg K ha-1 on final disease severity, standardized area under disease progress curve, and grain yield were investigated at Cedara, South Africa, on a maize (Zea mays) hybrid susceptible to gray leaf spot (GLS), caused by Cercospora zeae-maydis. The trial was a randomized 3 × 4 factor design, split for fungicide treatments, and replicated three times. With increased N and K levels, final percent leaf blighting and the standardized area under disease progress curve were higher. In fungicide-treated maize, grain yields increased with increasing levels of N and K, as expected. In non-fungicide-treated maize, grain yield increased significantly with increased levels of N, despite increased disease severity. This was in contrast to small increases in grain yields from non-fungicide-treated maize with increased K levels, which were not significant. This was probably because grain yield response, which should have occurred at higher K applications, was reduced by increased disease severity. The effect of N, P, and K on GLS wasinvestigated at Ahrens. Maize was grown in a 4 × 4 × 4 N-P-K factorial, in a randomized complete block design. Fertilizer was applied at 0, 60, 120, and 180 kg N ha-1, 0, 30, 60, and 120 kg P ha-1, and 0, 50, 100, and 150 kg K ha-1. No fungicides were applied. A single disease assessment at physiological maturity showed that final disease severity increased with increasing levels of N, P, and K. These results have implications for small-scale farmers who are encouraged to fertilize for increased grain yields but may not have the resources to apply fungicide sprays to control fungal diseases.

Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.

Gray leaf spot, caused by the fungus Cercospora zeae-maydis, causes considerable yield losses in hybrid maize grown in the north-central United States and elsewhere. Nonchemical management tactics have not adequately prevented these losses. The probability of profitably using fungicide application as a management tool for gray leaf spot was evaluated in 10 field experiments under conditions of natural inoculum in Iowa. Gray leaf spot severity in untreated control plots ranged from 2.6 to 72.8% for the ear leaf and from 3.0 to 7.7 (1 to 9 scale) for whole-plot ratings. In each experiment, fungicide applications with propiconazole or mancozeb significantly reduced gray leaf spot severity. Fungicide treatment significantly (P ≤ 0.05) increased yield by as much as 1.65 t/ha with a single propiconazole application. There were significant (P < 0.05) correlations between gray leaf spot severity and yield. We used a Bayesian inference method to calculate for each experiment the probability of achieving a positive net return with one or two propiconazole applications, based on the mean yields and standard deviations for treated and untreated plots, the price of grain, and the costs of the fungicide applications. For one application, the probability ranged from approximately 0.06 to more than 0.99, and exceeded 0.50 in six of nine scenarios (specific experiment/hybrid). The highest probabilities occurred in the 1995 experiments with the most susceptible hybrid. Probabilities were almost always higher for a single application of propiconazole than for two applications. These results indicate that a single application of propiconazole frequently can be profitable for gray leaf spot management in Iowa, but the probability of a profitable application is strongly influenced by hybrid susceptibility. The calculation of probabilities for positive net returns was more informative than mean separation in terms of assessing the economic success of the fungicide applications.

Two taxonomically identical but genetically distinct sibling species, designated groups I and II, of Cercospora zeae-maydis cause gray leaf spot of maize in the United States. Isolates of the gray leaf spot pathogen from Africa were compared with isolates from the United States by amplified fragment length polymorphism (AFLP) analysis and restriction digests of internal transcribed spacer (ITS) regions and 5.8S ribosomal DNA (rDNA), as well as by morphological and cultural characteristics. The isolates from Africa were morphologically indistinguishable from the U.S. isolates in both groups, but like isolates of group II, they grew more slowly and failed to produce detectable amounts of cercosporin in culture. Analysis of restriction fragments from the ITS and rDNA regions digested with five endonucleases indicated that all of the African isolates shared the profile of the C. zeae-maydis group II population from the eastern United States and, thus, are distinct from the group I population, which is more prevalent in the United States and other parts of the world. Cluster analysis of 85 AFLP loci confirmed that the African and U.S. group II populations were conspecific (greater than 97% average similarity) with limited variability. Among all group II isolates, only 8 of 57 AFLP loci were polymorphic, and none was specific to either population. Thus, although gray leaf spot was reported in the United States several decades prior to the first record in Africa, the relative age of the two populations on their respective continents could not be ascertained with confidence. The absence of C. zeae-maydis group I in our samples from four countries in the major maize-producing region of Africa as well as the greater AFLP haplotype diversity found in the African group II population, however, suggest that Africa was the source of C. zeae-maydis group II in the United States. The overall paucity of AFLP variation in this sibling species further suggests that its origin is recent or that the ancestral population experienced a severe bottleneck prior to secondary migration.

Symptoms associated with fungal damage, viral diseases, and immature soybean (Glycine max) seeds were characterized using image processing techniques. A Red, Green, Blue (RGB) color feature-based multivariate decision model discriminated between asymptomatic and symptomatic seeds for inspection and grading. The color analysis showed distinct color differences between the asymptomatic and symptomatic seeds. A model comprising six color features including averages, minimums, and variances for RGB pixel values was developed for describing the seed symptoms. The color analysis showed that color alone did not adequately describe some of the differences among symptoms. Overall classification accuracy of 88% was achieved using a linear discriminant function with unequal priors for asymptomatic and symptomatic seeds with highest probability of occurrence. Individual classification accuracies were asymptomatic 97%, Alternaria spp. 30%, Cercospora spp. 83%, Fusarium spp. 62%, green immature seeds 91%, Phomopsis spp. 45%, soybean mosaic potyvirus (black) 81%, and soybean mosaic potyvirus (brown) 87%. The classifier performance was independent of the year the seed was sampled. The study was successful in developing a color classifier and a knowledge domain based on color for future development of intelligent automated grain grading systems.

Monoconidial isolates of the fungus causing gray leaf spot of maize were obtained from diseased leaves collected throughout the United States and analyzed for genetic variability at 111 amplified fragment length polymorphism (AFLP) loci. Cluster analysis revealed two very distinct groups of Cercospora zeae-maydis isolates. Both groups were found to be relatively uniform internally with an average genetic similarity among isolates of approximately 93 and 94%, respectively. The groups were separated from each other by a genetic distance of approximately 80%, a distance greater than that separating each group from the sorghum pathogen, C. sorghi (67 to 70%). Characteristics and dimensions of conidia and conid-iophores produced on infected plants or nutrient media were unreliable criteria for taxonomic differentiation of isolates composing the two groups of C. zeae-maydis. Nucleotide sequences of 5.8S ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) regions were identical within each group but different between the two groups and different from C. sorghi. Restriction fragment length polymorphisms generated by digestion of the 5.8S rDNA and ITS regions with TaqI readily distinguished each group and C. sorghi. Isolates in one group were generally distributed throughout maize-producing regions of the United States; isolates in the other group were localized in the eastern third of the country. Both types were present in the same fields at some locations. The genetic distance based on AFLP profiles and different ITS nucleotide sequences between the two morphologically indistinguishable groups indicate that they are sibling species. Although it is unlikely that breeding for resistance to gray leaf spot will be confounded by local or regional variation in the pathogen, a vigilant approach is warranted, because two pathogenic species exist with unknown abilities to evolve new pathotypes.