Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

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.

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.

Genetic segregation of microsatellite markers in Saccharum officinarum and S. spontaneum.

Genetic mapping techniques can be used to study the interaction between two different genomes after hybridization. This study investigated a Saccharum officinarum (Green German or GG, 2n approximately 11x approximately 110) x S. spontaneum (IND 81-146 or IND, 2n approximately 7x approximately 56) interspecific cross. Segregation of 193 microsatellite (SSR) loci was evaluated in the F(1) progeny of 169 full-sibs of the cross. Following the two-way pseudo-testcross strategy and 'cross pollination' population type, linkage groups (LG) and phases were established for each parent map, using the criteria of LOD score > or = 3.0 and a maximum recombination frequency of 0.35. Of the 193 markers analyzed, 61 were IND-specific, 106 were GG-specific, and 26 were heterozygous in both parents. About 78% of the markers segregated in a Mendelian fashion and 22% were distorted (as evaluated by chi(2)-tests, P < or = 0.01). The GG map included 91 marker loci arranged into 25 LG covering 1180 cM of the officinarum genome. The IND map consisted of 46 marker loci assembled into 10 LG, which spanned 614 cM of the spontaneum genome. A specific chromosome associated with segregation distortion was detected in the female (GG) genome only, probably as a result of double reduction. The segregation patterns of the marker loci indicated a centromere-driven distortion process with the shared allelic markers (as putative centromeres) regulating the placement and association of markers with opposite phase (coupling vs repulsion) and dosage on either side. Although incomplete, the framework maps were informative with respect to segregation distortion, chromosome fusion, rearrangements, and translocations, observed in both parental genomes as a result of their merger.

Report of Sugarcane yellow leaf virus in Ecuador, Guatemala, and Nicaragua.

In 1998, sugarcane plants with symptoms similar to yellow leaf syndrome were observed in Ecuador, Guatemala, and Nicaragua. These plants showed yellowing of the central portion of the third to sixth leaves on the abaxial surface from the youngest expanding spindle leaf. Intense yellowing and necrosis of the leaf tip and the central portion of the leaf blade near the midrib occurred in severe cases. A tissue blot immunoassay was used to detect Sugarcane yellow leaf virus (SCYLV) in the midrib of the top visible dewlap leaf (2) using an antiserum specific to a Florida isolate of SCYLV (1). Since the virus can be detected in asymptomatic plants, leaf samples were collected from both symptomatic and asymptomatic plants. Symptom expression was most intense in plants at maturity that were under stress. Cut ends of leaf samples were imprinted on nitrocellulose membranes in the country of origin, and control samples of healthy and SCYLV-infected leaves were imprinted in Florida on each membrane prior to serological processing. The results from the following locations and cultivars, and the ratio of SCYLV-positive samples over the total samples is indicated: Milagro, Ecuador, PR 70-2085 (11/24) and PR 76-3385 (48/63) in 1999; Escuintla, Guatemala, CP 57-603 (1/10), CP 73-1547 (0/10), CP 72-2086 (120/308), PR 75-2002 (8/11), PR 78-294 (10/10), and PR 87-2080 (13/13) in both 2000 and 2001; Tipitapa, Nicaragua, L 68-40 (21/70) in 1998; and Chinandega, Nicaragua, CP 72-2086 (30/30) and CP 74-2005 (13/45) in 2000. CP 72-2086 is a major commercial cultivar in Central American countries and was infected in both Guatemala and Nicaragua. SCYLV was detected in 9 of 10 cultivars sampled. An exception was noticed in CP 73-1547 in Guatemala where none of the 10 plants tested were infected; however this cultivar has a high incidence of SCYLV in Florida. Only 1 of 10 samples of CP 57-603 was SCYLV positive in Guatemala; however, this cultivar has a low incidence of infection in Florida and is considered more resistant than the other CP cultivars sampled. To our knowledge, this is the first report SCYLV in Ecuador, Guatemala, and Nicaragua. References: (1) S. M. Scagliusi and B. E. L. Lockhart. Phytopathology 90:120, 2000. (2) S. Schenck et al. Sugar Cane 4:5, 1997.

Outbreak of Sugarcane Mosaic in Commercial Sugarcane Cultivar CP 72-2086, in Florida.

In May 1996, an outbreak of sugarcane mosaic virus (SCMV) was detected in Florida affecting a major commercial sugarcane (Saccharum spp. hybrid) cultivar, CP 72-2086. Identification of SCMV was confirmed by enzyme-linked immunosorbent assay (ELISA) with SCMV antiserum (ATCC no. PVAS 115), and by reverse transcription(RT)-PCR (1,2). The strain of SCMV was identified as E with an RT-PCR-based restriction fragment length polymorphism analysis (1). Although SCMV strain E has been detected in Florida for many decades, it had been confined primarily to S. officinarum clones, to occasional susceptible sugarcane clones in breeding programs, and to various grasses throughout the sugarcane production area. CP 72-2086 plus seven other sugarcane cultivars and 10 sorghum cultivars reacted similarly to inoculation with the isolate of SCMV found recently on CP 72-2086 and an isolate of SCMV collected in 1986. CP 72-2086 was released in 1982 and currently is the second most widely grown cultivar, constituting 18.0% of the commercial sugarcane area in Florida. At the epicenter of infection, located 11 km southeast of Canal Point, over 50% of CP 72-2086 plants had SCMV symptoms. The incidence of mosaic decreased rapidly away from the epicenter. No SCMV was observed in the western area of the sugarcane production area, west of South Bay, FL. References: (1) T. E. Mirkov and J. E. Irvine. Sugar y Azucar 9:23, 1996. (2) G. R. Smith et al. Plant Dis. 78:557, 1994.