Neil C. Glynn

Improving sugarcane for biofuel: engineering for an even better feedstock

Sugarcane is a proven biofuel feedstock and accounts for about 40% of the biofuel production worldwide. It has a more favorable energy input/output ratio than that of corn, the other major biofuel feedstock. The rich resource of genetic diversity and the plasticity of autopolyploid genomes offer a wealth of opportunities for the application of genomics and technologies to address fundamental questions in sugarcane towards maximizing biomass production. In a workshop on sugarcane engineering held at Rutgers University, we identified research areas and emerging technologies that could have significant impact on sugarcane improvement. Traditional plant physiological studies and standardized phenotypic characterization of sugarcane are essential for dissecting the developmental processes and patterns of gene expression in this complex polyploid species. Breeder friendly DNA markers associated with target traits will enhance selection efficiency and shorten the long breeding cycles. Integration of cold tolerance from Saccharum spontaneum and Miscanthus has the potential to expand the geographical range of sugarcane production from tropical and subtropical regions to temperate zones. The Flex‐stock and mix‐stock concepts could be solutions for sustaining local biorefineries where no single biofuel feedstock could provide consistent year‐round supplies. The ever increasing capacities of genomics and biotechnologies pave the way for fully exploring these potentials to optimize sugarcane for biofuel production.

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.

Phylogenetic analysis of EF-1 alpha gene sequences from isolates of Microdochium nivale leads to elevation of varieties majus and nivale to species status

Degenerate PCR primers were designed based on EF-1 alpha (EF-1alpha) gene sequences of several filamentous fungi retrieved from sequence databases. These primers were used to isolate a partial sequence, approximately 830 bp in length of the EF-1alpha from isolates of Microdochium nivale obtained from various geographic locations across the world. Two distinct groups of isolates were evident among those isolates examined. Sequence homology for comparisons within group was 99.7% for group A and 99.8% for group B. Primers specific to either group A or group B sequences were designed and tested on isolates from around the world. Comparisons were made with primers previously reported for the two varieties of M. nivale and revealed that Group A type isolates correlated with M. nivale var. majus and group B isolates with M. nivale var. nivale. The primers from this study and those previously reported were in agreement for all isolates with the exception of one isolate (NRRL 3289) which failed to amplify with previously published M. nivale primers. Sequence analysis of NRRL 3289 suggested that it was an isolate of M. nivale var. nivale as indicated by the EF-1alpha based primers developed in this study. This study provides sequence based phylogenetic evidence of two species and when taken together with biological differences reported, leads to the recognition of M. majus comb. nov. (syn. Fusarium nivale var. majus). Descriptions of the two species are provided.

Orange Rust Effects on Leaf Photosynthesis and Related Characters of Sugarcane

Orange rust of sugarcane (Saccharum spp. hybrids), caused by Puccinia kuehnii, is a relatively new disease in the Western Hemisphere that substantially reduces yields in susceptible sugarcane genotypes. The objective of this study was to determine the physiological mechanisms of orange rust-induced reductions in sugarcane growth and yield by quantifying effects of the disease on leaf SPAD index (an indication of leaf chlorophyll content), net photosynthetic rate, dark respiration, maximum quantum yield of CO2 assimilation, carbon fixation efficiency, and the relationships between these leaf photosynthetic components and rust disease ratings. Plants growing in pots were inoculated with the orange rust pathogen using a leaf whorl inoculation method. A disease rating was assigned using a scale from 0 to 4 with intervals of 0.5. At disease ratings ≥2, the rust-infected leaf portion of inoculated plants showed significant reductions in SPAD index, maximum quantum yield, carbon fixation efficiency, stomatal conductance, leaf transpiration rate, and net photosynthetic rate; but the rusted portion of the infected leaves had increased intercellular CO2 concentration and leaf dark respiration rate. Although leaf SPAD index, photosynthetic rate, stomatal conductance, and transpiration rate at the rust-infected portion decreased linearly with increased rust rating, the effect of orange rust on photosynthetic rate was much greater than that on stomatal conductance and transpiration. Unlike earlier reports on other crops, reduction in leaf photosynthesis by orange rust under low light was greater than that under high light conditions. These results help improve the understanding of orange rust etiology and physiological bases of sugarcane yield loss caused by orange rust.

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.

Phylogenetic relationships of sugarcane rust fungi

The phylogenetic positions of Puccinia spp. infecting sugarcane (a complex hybrid of Saccharum spp.) were determined using 38 newly generated rust sequences and 26 sequences from GenBank. Rust specimens on sugarcane were collected from 164 locations in 23 countries and identified based on light microscopy. The morphology for all samples matched that of Puccinia kuehnii or P. melanocephala, the orange and brown rust pathogens of sugarcane, respectively. Nuclear ribosomal DNA sequences (rDNA) including portions of the 5.8S rDNA, the complete internal transcribed spacer 2 (ITS2) and 5′ region of the large subunit (nLSU) rDNA were obtained for each species along with 36 additional rust taxa. Despite a shared host, the two Puccinia spp. on sugarcane are not closely related within the Pucciniales. Phylogenetic analyses place P. melanocephala most closely to P. miscanthi, P. nakanishikii, and P. rufipes infecting Miscanthus sinensis, Cymbopogon citratus, and Imperata cylindrica, respectively. Puccinia kuehnii is basal to a clade of Poaceae-infecting rusts including P. agrophila, P. polysora, P. substriata, and Uromyces setariae-italicae infecting Schizachyrium spp., Zea mays, Digitaria spp., and Urochloa mosambicensis, respectively. Light and scanning electron microscopy images highlight morphological differences distinguishing the two sugarcane-infecting species. This study confirms the separation of rust species infecting Poaceae from Cyperaceae- and Juncaceae-infecting rusts and also provides support for the presence of an additional group that includes P. kuehnii and other grass-infecting relatives.

Leaf Whorl Inoculation Method for Screening Sugarcane Rust Resistance

Brown rust, caused by Puccinia melanocephala, and orange rust, caused by P. kuehnii, are agronomically important diseases of sugarcane in Florida. Cultivar resistance is the best means of controlling these diseases. Natural infection has been the primary means of assessing resistance in sugarcane cultivars against rusts; unfortunately, natural infection is not always efficient in identifying resistant cultivars due to variable environmental conditions. Therefore, a more reliable screening method is needed to effectively select resistant genotypes. An inoculation technique was evaluated for identification of brown and orange rust resistance in sugarcane cultivars. Inoculations were performed in the field by placing a 0.5-ml urediniospore suspension in the leaf whorl of three individual sugarcane stalks per plant using a pipette. Symptoms developed on leaves of all the susceptible cultivars after 4 weeks, and appeared as a band of pustules. Plants were rated for their reaction to rust 4 weeks after inoculation. The optimum concentrations of inoculum for expression of brown and orange rust symptoms were determined. The most severe brown rust and orange rust symptoms were observed using inoculum containing 105 and 104 urediniospores/ml, respectively. Clones in several stages of the Canal Point breeding program were screened for their rust reaction by leaf whorl inoculation. The technique enabled rapid screening of a large number of cultivars in field plantings using a small amount of inoculum and limited man hours.

Optimized construction of microsatellite-enriched libraries

The construction of microsatellite‐enriched libraries is an indispensable tool to search for molecular markers as complete genome sequences are still not available for the majority of species of interest. Numerous protocols are available in the literature for the construction of these libraries; however, sometimes their low efficiency or lack of optimization in the protocols can restrict their efficacy. We have designed and tested various adapters and ligation methods; we also tested oligo‐repeat combinations and hybridization temperatures, and created libraries with this new protocol for four organisms: Ipomoea batatas (L.) Lam, Chionanthus retusus Lindley & Paxton, Rotylenchulus reniformis Linford & Olivera and Puccinia kuehnii W. Krüger. The number of microsatellites detected for these species ranged from 2494 to 3919 per Mb of nonredundant sequence, that was 0.86 and 1.53 microsatellites per contig, with 37–66% of di‐nucleotide motifs and 21–49% of tri‐ to octa‐nucleotide repeats combined. A simplified protocol is provided for the successful generation of SSR‐enriched libraries.

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.

Promoting Utilization of Saccharum spp. Genetic Resources through Genetic Diversity Analysis and Core Collection Construction

Sugarcane (Saccharum spp.) and other members of Saccharum spp. are attractive biofuel feedstocks. One of the two World Collections of Sugarcane and Related Grasses (WCSRG) is in Miami, FL. This WCSRG has 1002 accessions, presumably with valuable alleles for biomass, other important agronomic traits, and stress resistance. However, the WCSRG has not been fully exploited by breeders due to its lack of characterization and unmanageable population. In order to optimize the use of this genetic resource, we aim to 1) genotypically evaluate all the 1002 accessions to understand its genetic diversity and population structure and 2) form a core collection, which captures most of the genetic diversity in the WCSRG. We screened 36 microsatellite markers on 1002 genotypes and recorded 209 alleles. Genetic diversity of the WCSRG ranged from 0 to 0.5 with an average of 0.304. The population structure analysis and principal coordinate analysis revealed three clusters with all S. spontaneum in one cluster, S. officinarum and S. hybrids in the second cluster and mostly non-Saccharum spp. in the third cluster. A core collection of 300 accessions was identified which captured the maximum genetic diversity of the entire WCSRG which can be further exploited for sugarcane and energy cane breeding. Sugarcane and energy cane breeders can effectively utilize this core collection for cultivar improvement. Further, the core collection can provide resources for forming an association panel to evaluate the traits of agronomic and commercial importance.