First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Ecuador.

Orange rust, Puccinia kuehnii (W. Krüger) E.J. Butler, is an important disease of sugarcane (complex hybrid of Saccharum L. species) that causes up to 53% yield loss (3), and can eliminate sugarcane clones in breeding programs. Initially confined to the Asia-Oceania region, P. kuehnii was reported in Florida in June 2007 (2) followed by confirmation in Central and South America. Orange rust pustules were observed on August 5, 2011, in commercial sugarcane fields located in the Ecuadorian Pacific coast of South America. Pustules were observed on cultivar SP79-2233 and sugarcane clones of the CINCAE breeding program (EC06-351, EC06-340, and EC01-744). Low levels of disease incidence and severity were observed in the sugarcane germplasm. Observation under a light microscope showed typical irregularly echinulate urediniospores that were pale in color with thickened apices and paraphyses inconspicuous to absent, such as those reported to be P. kuehnii (4). DNA of urediniospores were extracted and amplified using Pk1F and PK1R qPCR primers (5). Additionally, the 28s large ribosomal subunit DNA was sequenced (1), resulting in a qPCR and 100% sequence identity with a partial sequence of the P. kuehnii 28S ribosomal RNA gene, accession GU058010 (932/932 base pairs, GenBank Accession No. KF202306). Based on urediniospore morphology, DNA amplification, and sequence analysis, the causal agent of the rust observed in Ecuador was confirmed to be P. kuehnii. Commercial varieties have not yet shown symptoms of infections. However, a survey conducted in 2011 and 2012 showed an increase of disease severity from 3% to 28% in the susceptible cv. SP79-2233. Disease symptoms were evident from stalk growth to maturity (7 to 12 months), especially at the beginning of the harvesting season. To our knowledge, this is the first report of the presence, distribution, and disease spread by the sugarcane orange rust pathogen P. kuehnii in Ecuador. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) J. C. Comstock et al. ASSCT. 29:82, 2009. (4) L. Dixon and L. Castlebury. Orange Rust of Sugarcane - Puccinia kuehnii. Syst. Mycol. Microbiol. Lab. Retrieved from /sbmlweb/fungi/index.cfm, August 12, 2011. (5) N. C. Glynn et al. Plant Pathol. 59:703, 2010.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Ivory Coast and Cameroon.

Orange rust of sugarcane caused by Puccinia kuehnii was detected in Florida in 2007 (1). It was hypothesized that the pathogen originated from Africa because brown rust of sugarcane (synonym common rust) was introduced to the Western Hemisphere from Africa (3). Requests for rust-infected sugarcane samples were made to several western and central African countries to investigate if orange rust of sugarcane was present but as yet undetected. Orange rust had not previously been reported from western or central Africa. At Zuénoula, Ivory Coast in July 2009, symptoms of sugarcane rust were observed on cvs. SP 71-6180 and Co 997 and appeared distinct to those of brown rust of sugarcane. A year later (May 2010), rust-infected specimens of SP 71-6180 and Co 997 from the same location and also from Borotou in Ivory Coast were sent to the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Also in May 2010, sugarcane rust was observed at Mbandjock and Nkoteng in Cameroon on cvs. D 88172, FR 87482, and RB 72-454 and on breeding clones RCmr 08/319 and RCmr 08/1121. All specimens had orange uredinial lesions that ranged from 0.6 to 6.5 mm × 200 to 300 µm and were ellipsoidal to elongate. Urediniospores were consistent with P. kuehnii E.J. Butler observed on specimens from Florida (1). DNA isolated from all samples was successfully amplified with P. kuehnii specific primers targeting ITS1 of rDNA (2). The nuclear large subunit region of rDNA of the rust specimens from Ivory Coast (BPI 881015-881017, GenBank Accession No. HQ666888) and Cameroon (BPI 881010-881014, GenBank Accession Nos. HQ666889-HQ666891) were sequenced. DNA sequences for all were identical to sequences of P. kuehnii and distinct from known sequences of P. melanocephala available in GenBank. To our knowledge, this is the first confirmed report of orange rust of sugarcane in western and central Africa. There is evidence that brown rust of sugarcane was introduced to the Western Hemisphere from this region of Africa (3) making it also the likely source of introduction of orange rust. Further experimentation is required to confirm this hypothesis. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) N. C. Glynn et al. Plant Pathol. 59:703. 2010. (3) H. L. Purdy et al. Plant Dis. 69:689, 1985.

Evaluation of PCR assays for quantifying seed-borne infection by Fusarium and Microdochium seedling blight pathogens.

AIMS: To evaluate competitive PCR assays for quantifying seed-borne Microdochium and Fusarium seedling blight pathogen DNA and to determine test and year repeatability and sources of variability. METHODS AND RESULTS: Relationships between DNA and plate counts were significant for Fusarium and Microdochium seedling blight pathogens in 152 seed batches from 3 years. Coefficient of determinations, however, differed greatly (Fusarium; R(2) = 0.25, P = 0.029, Microdochium; R(2) = 0.73, P < 0.001). Significant differences between years were observed in the regression slopes for Microdochium. Pathogen DNA quantified in 16 extractions after sampling was highly correlated to results following storage for 1-2 years (R(2) > 0.90). Residual maximum likelihood analysis showed that the least and greatest variance components of the testing procedure were DNA extraction subsampling and PCR assay respectively. CONCLUSIONS: Amount of pathogen DNA is a useful estimator of seed batch contamination for Microdochium but not Fusarium seedling blight pathogens. Although reproducible over time, improvements to the testing procedure should focus on repeated PCR amplifications to reduce assay variability. SIGNIFICANCE AND IMPACT OF THE STUDY: Replacing plate counts with competitive PCR for determining the severity of seed batch contamination is feasible in areas where Microdochium seedling blight pathogens predominate.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Mexico, El Salvador, and Panama.

Symptoms of sugarcane orange rust were observed on July 17, 2008 on sugarcane cvs. Mex 57-1285, Mex 61-230, and Co 301 (a clone received in Mexico in 1953) at the Centro de Investigación y Desarrollo de la Caña de Azúcar en Tuxtla Chico, Chiapas, Mexico. In El Salvador, from August 2008 through January 2009, rust symptoms were observed on cv. CP 72-2086 (previously resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd.) in 117 dispersed sugarcane-production fields in various localities of El Salvador. Likewise, rust symptoms were first observed on sugarcane cv. SP 74-8355 (more than 25% severity and considered resistant to brown rust) at Natá, Coclé Province in Panama from January to February 2008. Dried herbarium leaf samples of sugarcane rust-infected leaves collected in El Salvador and Mexico were sent to the ARS, USDA Systematic Mycology and Microbiology Laboratory in Beltsville MD for identification. Panamanian samples were collected similarly and analyzed at the CALESA Biotechnology Laboratory. Morphological features of uredinial lesions and urediniospores were distinct from those of P. melanocephala and consistent with P. kuehnii E. J. Butler observed previously on specimens from Florida, Guatemala, Costa Rica, and Nicaragua (1-3). Analysis of the ITS1, 5.8S, and ITS2 and 28S large subunit rDNA sequences of the rust on infected cvs. Mex 57-1285, Mex 61-230, and Co 301 (BPI 878930, 879139, and 879140; GenBank Accession Nos. GO283006, GO283004, and GO283005, respectively) from Mexico and cv. CP 72-2086 from three locations in El Salvador (BPI 879135, 879136, and 879137; GenBank Accession Nos. GO283009, GO283007, and GO283008, respectively) all confirmed the identification of P. kuehnii. Similar analysis of the ITS1, 5.8S, and ITS2 rDNA sequence for the rust infecting cv. SP 74-8355 (GenBank Accession No. GO281584) confirmed the identification of P. kuehnii in Panama. To our knowledge, this is the first report of P. kuehnii causing orange rust disease of sugarcane in El Salvador, Mexico, and Panama. These findings also confirm the wider distribution of orange rust in the Western Hemisphere. References: (1) E. Chavarria et al. Plant Dis. 93:425, 2009. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) W. Ovalle et al. Plant Dis. 92:973, 2008.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Costa Rica and Nicaragua.

Symptoms and signs of orange rust on sugarcane (a complex hybrid of Saccharum L. species) were observed from July 2007 on cv. SP 71-5574 in Costa Rica at the Coopeagri Sugar Mill located in Pérez Zeledón, San José and on multiple cultivars (CP 72-2086, Pindar, Q 132, Q 138, SP 71-5574, and SP 79-2233) at the Providencia Sugar Mill near Muelle, San Carlos and Cutris Sugar Mill near Los Chiles during August 2007. The same symptoms and signs were observed on cv. CP 72-2086 during September 2007 in Nicaragua at Ingenio San Antonio, located near Chinandega, and Ingenio Monte Rosa near El Viejo, Nicaragua. Disease symptoms and uredinia appeared different from brown rust caused by Puccinia melanocephala, and brown rust usually does not occur on these cultivars. Uredinia and urediniospores were similar to those described for orange rust (1,2). Cvs. SP 71-5574 and SP 79-2233 are susceptible and cv. CP 72-2086 is moderately susceptible to orange rust in Costa Rica and cvs. ISACP 00-1075, ISA 00-1000, and CP 72-2086 are moderately susceptible in Nicaragua. Samples from both locations (Costa Rica BPI No. 878816 and Nicaragua BPI No. 878817) examined at the USDA-ARS Mycology and Microbiology Laboratory in Beltsville, MD showed morphological characteristics consistent with those of P. kuehnii. Analysis of ITS1, 5.8S, and ITS2 rDNA sequences of the rust infecting cv. CP 72-2086 (GenBank Accession No. FJ532477) from Costa Rica and cv. ISA 00-1000 from Nicaragua (GenBank Accession No. FJ532476) confirmed the identity as P. kuehnii, the causal agent of sugarcane orange rust. Beside the cultivars already mentioned, orange rust also was confirmed on cvs. RB 73-9735 and CPCL 02-2130 in Costa Rica. To our knowledge, this is the first report of orange rust of sugarcane in Costa Rica and Nicaragua and the third confirmation of the disease in the Western Hemisphere and Caribbean Basin. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) W. Ovalle et al. Plant Dis. 92:973, 2008.

First Report of Puccinia kuehnii, Causal Agent of Orange Rust of Sugarcane, in Guatemala.

In September 2007 at Masagua, Escuintla Department, Guatemala, uredial lesions that appeared different from those of brown rust were observed on a sugarcane (a complex hybrid of Saccharum L. species) cultivar (CP 72-2086) considered resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd. Samples were sent to the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Observed morphological features were consistent with P. kuehnii E.J. Butler and appeared similar to orange rust samples obtained from Florida in July (2). Uredinial lesions were hypophyllous, orange, and variable in size measuring 650 to 850 × 26 to 32 µm. Urediniospores were mostly obovoid to pyriform or broadly ellipsoidal, variable in size, 32 to 45 × 25 to 30 µm, and moderately echinulate with spines evenly distributed, 3 to 5 µm apart. Urediniospore walls were orange-to-light cinnamon brown, 1 to 2.5 µm thick with a pronounced apical wall and four to five equatorial pores. Telia and teliospores were not observed. The nuclear large subunit rDNA region of the rust infecting cv. CP 72-2086 (BPI 898289, GenBank Accession No. EU344904) and the ITS1, 5.8S, and ITS2 rDNA regions (GenBank Accession No. EU543434) were sequenced (1,3). DNA sequences matched sequences of P. kuehnii in GenBank and were distinct from known sequences of P. melanocephala available in GenBank (3). Thirteen cultivars were rated as to their relative resistance using severity of orange rust symptoms; CG 96-59, CG 96-135, CP 72-1312, CP 73-1547, and CP 88-1165 were resistant; CG 96-40, CG 98-121, CP 72-2086, CP 88-1508, and CP 89-2143 were intermediate; and CG 96-52, CG 98-0115, and SP 79-2233 were susceptible. Orange rust was previously reported in Florida (2), but to our knowledge, this is the second report of its occurrence in the Western Hemisphere. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

First Report of Puccinia kuehnii, Causal Agent of Orange Rust of Sugarcane, in the United States and Western Hemisphere.

In June 2007, approximately 8 km east of Belle Glade, FL, a rust disease was observed on a sugarcane (a complex hybrid of Saccharum L. species) cultivar (CP 80-1743) considered resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd. Approximately 10 km south of Canal Point, FL, another cultivar (CP 72-2086), also considered resistant to P. melanocephala, was found to be infected with a rust. Samples were sent to the USDA-APHIS National Mycologist and the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Observed morphological features were consistent with P. kuehnii E.J. Butler. Uredinial lesions were orange and variable in size, measuring 650 to 850 × 26 to 32 µm, hypophyllous, ellipsoidal to fusiform in shape, and distinctly lighter than pustules of P. melanocephala that were present in the area along with P. kuehnii. Urediniospores were mostly obovoid to pyriform or broadly ellipsoidal, variable in size, 32 to 45 × 25 to 30 µm, and moderately echinulate with mostly evenly distributed spines 2 to 4.5 µm apart. Walls were orange-to-light cinnamon brown, 1 to 2.5 µm thick with a pronounced apical wall thickening as much as 7 µm, and 4 to 5 equatorial pores. Similar orange uredinial lesions were subsequently observed on the same two cultivars and several other cultivars, including CPCL99-1777 and CPCL01-1055, at different locations in South Florida. Telia and teliospores were not observed. The nuclear large subunit rDNA region of the rust infecting cv. CP 80-1743 (BPI 878243, GenBank Accession No. EU164549) and the ITS1, 5.8S, and ITS2 rDNA regions of the rust infecting CP 80-1743 (GenBank Accession No. EU176009) and CP 72-2086 (GenBank Accession No. EU176008) were sequenced (1,4). All sequences were identical to sequences of P. kuehnii and distinct from known sequences of P. melanocephala (4). To our knowledge, this is the first confirmed record of P. kuehnii infecting sugarcane in the Western Hemisphere, and the disease appears to be distributed widely in the South Florida sugarcane-growing area. Although listed by P. Holliday (3) as occurring in Cuba, the Dominican Republic, and Mexico, CMI map no. 215 ed. 4 (2) does not include these three countries in the known distribution of P. kuehnii. P. kuehnii has also been reported in the literature as present in Hawaii (4). However, examination of the specimen label found that the specimen cited in those papers (BPI 079624) was actually collected in Tahiti. Therefore, the report from Hawaii is erroneous. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) CMI. Distribution Maps of Plant Diseases. No. 215, ed. 4. CAB International, Wallingford, UK, 1981. (3) P. Holliday. Fungus Diseases of Tropical Crops. Cambridge University Press, Cambridge, 1980. (4) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat.

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.