WGCNA Identifies a Comprehensive and Dynamic Gene Co-Expression Network That Associates with Smut Resistance in Sugarcane.

Sugarcane smut is a major fungal disease caused by Sporisorium scitamineum, which seriously reduces the yield and quality of sugarcane. In this study, 36 transcriptome data were collected from two sugarcane genotypes, YT93-159 (resistant) and ROC22 (susceptible) upon S. scitamineum infection. Data analysis revealed 20,273 (12,659 up-regulated and 7614 down-regulated) and 11,897 (7806 up-regulated and 4091 down-regulated) differentially expressed genes (DEGs) in YT93-159 and ROC22, respectively. A co-expression network was then constructed by weighted gene co-expression network analysis (WGCNA), which identified 5010 DEGs in 15 co-expressed gene modules. Four of the 15 modules, namely, Skyblue, Salmon, Darkorange, and Grey60, were significantly associated with smut resistance. The GO and KEGG enrichment analyses indicated that the DEGs involving in these four modules could be enriched in stress-related metabolic pathways, such as MAPK and hormone signal transduction, plant-pathogen interaction, amino acid metabolism, glutathione metabolism, and flavonoid, and phenylpropanoid biosynthesis. In total, 38 hub genes, including six from the Skyblue module, four from the Salmon module, 12 from the Darkorange module, and 16 from the Grey60 module, were screened as candidate hub genes by calculating gene connectivity in the corresponding network. Only 30 hub genes were amplifiable with RT-qPCR, of which 27 were up-regulated upon S. scitamineum infection. The results were consistent with the trend of gene expression in RNA-Seq, suggesting their positive roles in smut resistance. Interestingly, the expression levels of AOX, Cyb5, and LAC were higher in ROC22 than in YT93-159, indicating these three genes may act as negative regulators in response to S. scitamineum infection. This study revealed the transcriptome dynamics in sugarcane challenged by S. scitamineum infection and provided gene targets for smut resistance breeding in sugarcane.

Eight Species of Poaceae Are Hosting Different Genetic and Pathogenic Strains of Sugarcane Mosaic Virus in the Everglades Agricultural Area.

Sugarcane mosaic virus (SCMV) was detected by reverse transcription polymerase chain reaction in eight different species of the Poaceae family in the Everglades Agricultural Area (EAA) of south Florida: broadleaf signalgrass (Urochloa platyphylla), Columbus grass (Sorghum almum), goosegrass (Eleusine indica), maize (Zea mays), sorghum (Sorghum bicolor), St. Augustine grass (Stenotaphrum secundatum), southern crabgrass (Digitaria ciliaris), and sugarcane (Saccharum interspecific hybrids). Based on their coat protein (CP) gene sequence, 62 isolates of SCMV from Florida and 29 worldwide isolates representing the known genetic diversity of this virus were distributed into eight major phylogenetic groups. SCMV isolates infecting Columbus grass, maize, and sorghum in Florida formed a unique group, whereas virus isolates infecting sugarcane in the United States (Florida and Louisiana) clustered with isolates from other countries. Based on the entire genome coding region, SCMV isolates infecting sugarcane in Florida were closest to virus isolates infecting sorghum species or St. Augustine grass. Virus isolates from Columbus grass, St. Augustine grass, and sugarcane showed different virulence patterns after mechanical inoculation of Columbus grass, St. Augustine grass, and sugarcane plants, thus proving that these isolates were different pathogenic strains. Sugarcane was symptomless and tested negative for SCMV by tissue blot immunoassay after inoculation with crude sap from SCMV-infected Columbus grass, indicating that Columbus grass was not a reservoir for SCMV infecting sugarcane in the EAA. Close CP sequence identity between isolates of SCMV from Columbus grass, maize, and sorghum suggested that the same virus strain was naturally spreading between these three plants in south Florida.

Relationship of Soil Properties and Sugarcane Yields to Red Stripe in Louisiana.

Symptoms of red stripe disease caused by Acidovorax avenae subsp. avenae in Louisiana between 1985 and 2010 were limited to the leaf stripe form, which caused no apparent yield loss. During 2010, the more severe top rot form was observed, and a study was initiated to investigate the distribution of red stripe in the field and determine its effects on cane and sugar yields. Soil properties data, red stripe incidence, and sugarcane yields were all highly variable and were not randomly distributed in the field. Combined harvest data showed a negative correlation between yield components and red stripe incidence, with the strongest relationship between sucrose per metric ton and disease incidence. Red stripe incidence was positively correlated with several soil properties, including phosphorus, potassium, zinc, and calcium. Red stripe incidence also was found to increase with increasing nitrogen rate, with the greatest effects in heavy soils. Results also indicated that using red-stripe-infected cane as a seed source can significantly decrease shoot emergence, stalk population, and subsequent cane and sugar yields. These combined data suggest that red stripe disease can exhibit a highly variable rate of infection in commercial sugarcane fields and may also significantly decrease sugar yields.

Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane.

Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials (setts) are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of C. falcatum. C. falcatum genomic DNA was isolated from pure mycelium culture and infected tissues. Four sets of primers corresponding to a unique DNA sequence specific to C. falcatum were designed. Specificity of the LAMP test was checked with DNA of another fungal pathogen of sugarcane, Puccinia melanocephala, as well as two closely-related species, Colletotrichum fructivorum and Colletotrichum acutatum. No reaction was found with the three pathogens. When C. falcatum DNA from pure culture was used in a detection limit analysis, sensitivity of the LAMP method was observed to be ten times higher than that of conventional PCR; however, sensitivity was only 5 times higher when DNA from C. falcatum-infected tissues was used. Using the LAMP assay, C. falcatum DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions. Moreover, visual judgment of color change in <1 h without further post-amplification processing makes the LAMP method convenient, economical, and useful in diagnostic laboratories and the field.

Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane.

Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneupolyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible through capillary electrophoresis (CE) using fluorescence-labeled SSR markers. Twenty-four sugarcane cultivars, 12 each from India and the USA, were genetically assessed using 21 fluorescence-labeled polymorphic SSR markers. These markers primed the amplification of 213 alleles. Of these alleles, 161 were common to both Indian and US cultivars, 25 were specific to the Indian cultivars, and 27 were observed only in the US cultivars. Only 10 alleles were monomorphic. A high level of heterozygosity was observed in both Indian (82.4%) and US (91.1%) cultivars resulting in average polymorphism information content (PIC) values of 0.66 and 0.77 and marker index (MI) values of 5.07 and 5.58, respectively. Pearson correlation between PIC and MI was significant in both sets of cultivars (r = 0.58 and 0.69). UPGMA clustering separated cultivars into three distinct clusters at 59% homology level. These results propose the potential utility of six Indian cultivar-specific SSR alleles (mSSCIR3_182, SMC486CG_229, SMC36BUQ_125, mSSCIR74_216, SMC334BS_154, and mSSCIR43_238) in sugarcane breeding, vis a vis transporting CE-based evaluation in clone or variety identity testing, cross fidelity assessments, and genetic relatedness among species of the genus Saccharum and related genera.

Biogeographical variation and population genetic structure of Sporisorium scitamineum in Mainland China: insights from ISSR and SP-SRAP markers.

A total of 100 Sporisorium scitamineum isolates were investigated by inter simple sequence repeat (ISSR) and single primer-sequence related amplified polymorphism (SP-SRAP) markers. These isolates were clearly assorted into three distinct clusters regardless of method used: either cluster analysis or by principal component analysis (PCA) of the ISSR, SP-SRAP, or ISSR + SP-SRAP data set. The total gene diversity (H t) and gene diversity between subpopulations (H s) were estimated to be 0.34 to 0.38 and 0.22 to 0.29, respectively, by analyzing separately the ISSR and SP-SRAP data sets, and to be 0.26-0.36 by analyzing ISSR + SP-SRAP data set. The gene diversity attributable to differentiation among populations (G st) was estimated to be 0.35 and 0.22, and the gene flow (Nm) was 0.94 and 1.78, respectively, when analyzing separately ISSR and SP-SRAP data set, and was 0.27 and 1.33, respectively, when analyzing ISSR + SP-SRAP data set. Our study showed that there is considerable genetic variation in the analyzed 100 isolates, and the environmental heterogeneity has played an important role for this observed high degree of variation. The genetic differentiation of sugarcane smut fungus depends to a large extent on the heterogeneity of their habitats and is the result of long-term adaptations of pathogens to their ecological environments.

Genome-wide characterization of sugarcane catalase gene family identifies a ScCAT1 gene associated disease resistance.

In plants, catalase (CAT) mainly scavenges H2O2 from reactive oxygen species (ROS) and regulates the growth and development. So far, genome-wide identification of CAT gene family in Saccharum has not yet been reported. Here, 16 SsCAT genes were identified based on a Saccharum spontaneum genome. They were clustered into three subfamilies, with closer genes sharing similar structures. Most SsCAT proteins contained three conserved amino acids, one active catalytic site, one heme-ligand signature, and three peroxisomal targeting signal 1 (PTS1) sequences. The cis-regulatory element prediction revealed that SsCAT genes were involved in growth and development, and in response to various hormones and stresses. RNA-Seq databases showed that SsCAT genes were differentially expressed in Saccharum tissues and under cold, drought, and Sporisorium scitamineum stresses. The ScCAT1 gene transcript (GenBank accession number KF664183) and relevant CAT activity were up-regulated under S. scitamineum stress. Overexpression of ScCAT1 gene in Nicotiana benthamiana could enhance its resistance to pathogen infection through scavenging of excessive toxic ROS and up-regulated expressions of genes related to hypersensitive response (HR), ROS and salicylic acid (SA) pathways. This study provides comprehensive information for the CAT gene family and sets up a basis for its function identification in sugarcane.

Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq.

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca2+ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.

Sugarcane Mosaic Disease: Characteristics, Identification and Control.

Mosaic is one of the most important sugarcane diseases, caused by single or compound infection of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and/or Sugarcane streak mosaic virus (SCSMV). The compound infection of mosaic has become increasingly serious in the last few years. The disease directly affects the photosynthesis and growth of sugarcane, leading to a significant decrease in cane yield and sucrose content, and thus serious economic losses. This review covers four aspects of sugarcane mosaic disease management: first, the current situation of sugarcane mosaic disease and its epidemic characteristics; second, the pathogenicity and genetic diversity of the three viruses; third, the identification methods of mosaic and its pathogen species; and fourth, the prevention and control measures for sugarcane mosaic disease and potential future research focus. The review is expected to provide scientific literature and guidance for the effective prevention and control of mosaic through resistance breeding in sugarcane.

Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation.

In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

Heterologous expression of a Glyoxalase I gene from sugarcane confers tolerance to several environmental stresses in bacteria.

Glyoxalase I belongs to the glyoxalase system that detoxifies methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates. The concentration of MG increases rapidly under stress conditions. In this study, a novel glyoxalase I gene, designated as SoGloI was identified from sugarcane. SoGloI had a size of 1,091 bp with one open reading frame (ORF) of 885 bp encoding a protein of 294 amino acids. SoGloI was predicted as a Ni2+-dependent GLOI protein with two typical glyoxalase domains at positions 28-149 and 159-283, respectively. SoGloI was cloned into an expression plasmid vector, and the Trx-His-S-tag SoGloI protein produced in Escherichia coli was about 51 kDa. The recombinant E. coli cells expressing SoGloI compared to the control grew faster and tolerated higher concentrations of NaCl, CuCl2, CdCl2, or ZnSO4. SoGloI ubiquitously expressed in various sugarcane tissues. The expression was up-regulated under the treatments of NaCl, CuCl2, CdCl2, ZnSO4 and abscisic acid (ABA), or under simulated biotic stress conditions upon exposure to salicylic acid (SA) and methyl jasmonate (MeJA). SoGloI activity steadily increased when sugarcane was subjected to NaCl, CuCl2, CdCl2, or ZnSO4 treatments. Sub-cellular observations indicated that the SoGloI protein was located in both cytosol and nucleus. These results suggest that the SoGloI gene may play an important role in sugarcane's response to various biotic and abiotic stresses.

A Comparative Study of Three Detection Techniques for Leifsonia xyli Subsp. xyli, the Causal Pathogen of Sugarcane Ratoon Stunting Disease.

The ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically devastating diseases impacting sugarcane. RSD causes significant yield losses and variety degradation. Diagnosis of RSD is challenging because it does not exhibit any discernible internal and external symptoms. Moreover, the Lxx bacteria are very small and difficult to isolate, cultivate, and detect. In this study, conventional polymerase chain reaction (PCR), real-time quantitative PCR (RT-qPCR), and Lxx-loop-mediated isothermal amplification (Lxx-LAMP) were utilized to specifically detect the presence of Lxx pathogens in the juice from Lxx-infected sugarcane stalks and an Lxx-pMD18-T recombinant plasmid. The results showed that Lxx was a highly specific causal pathogen for RSD. All three techniques provided great reproducibility, while Lxx-LAMP had the highest sensitivity. When the DNA extract from Lxx-infected sugarcane juice was used as a template, Lxx-LAMP was 10 and 100 times more sensitive than RT-qPCR and conventional PCR, respectively. When the Lxx-pMD18-T recombinant plasmid was used as a template, Lxx-LAMP was as sensitive as RT-qPCR but was 10 times more sensitive than conventional PCR. Based on the Lxx-LAMP detection system established, adding 0.4 µM loop primers (LF/LP) can accelerate the reaction and reduce the total time required. In addition, the optimal amount of Bst DNA polymerase for Lxx-LAMP reactions was determined to be 6.0 U. The results provide technical support for the detection of RSD Lxx pathogen that will help manage sugarcane RSD.

Relationship between sugarcane rust severity and soil properties in louisiana.

ABSTRACT The extent of spatial and temporal variability of sugarcane rust (Puccinia melanocephala) infestation was related to variation in soil properties in five commercial fields of sugarcane (interspecific hybrids of Saccharum spp., cv. LCP 85-384) in southern Louisiana. Sugarcane fields were grid-soil sampled at several intensities and rust ratings were collected at each point over 6 to 7 weeks. Soil properties exhibited significant variability (coefficients of variation = 9 to 70.1%) and were spatially correlated in 39 of 40 cases with a range of spatial correlation varying from 39 to 201 m. Rust ratings were spatially correlated in 32 of 33 cases, with a range varying from 29 to 241 m. Rust ratings were correlated with several soil properties, most notably soil phosphorus (r = 0.40 to 0.81) and soil sulfur (r = 0.36 to 0.68). Multiple linear regression analysis resulted in coefficients of determination that ranged from 0.22 to 0.73, and discriminant analysis further improved the overall predictive ability of rust models. Finally, contour plots of soil properties and rust levels clearly suggested a link between these two parameters. These combined data suggest that sugarcane growers that apply fertilizer in excess of plant requirements will increase the incidence and severity of rust infestations in their fields.

Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection.

Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed and tested for each virus. Three primer sets designed for detecting SCMV and four for detecting SrMV were successful in the RT-LAMP assay. The effective primer sets were not only specific for their target virus, but also able to detect multiple virus strains. The magnesium sulfate concentration of the reaction solution was optimized, with both viruses requiring a minimum of 5mM for detection. The sensitivity of this RT-LAMP assay was less than that of conventional and real-time RT-PCR.

Biplot evaluation of test environments and identification of mega-environment for sugarcane cultivars in China.

Test environments and classification of regional ecological zones into mega environments are the two key components in regional testing of sugarcane cultivars. This study aims to provide the theoretical basis for test environment evaluation and ecological zone division for sugarcane cultivars. In the present study, sugarcane yield data from a three-year nationwide field trial involving 21 cultivars and 14 pilot test locations were analysed using both analysis of variance (ANOVA) and heritability adjusted-genotype main effect plus genotype-environment interaction (HA-GGE) biplot. The results showed that among the interactive factors, the GE interaction had the greatest impact, while the genotype and year interaction showed the lowest impact. Kaiyuan, Lincang and Baoshan of Yunnan, Zhangzhou and Fuzhou of Fujian, and Hechi, Liuzhou and Chongzuo of Guangxi, and Lingao of Hainan were ideal test environments with a demonstrated high efficiency in selecting new cultivars with a wide adaptability, whereas Baise of Guangxi was not. Based on HA-GGE biplot analysis, there are three ecological sugarcane production zones in China, the Southern China Inland Zone, the Southwestern Plateau Zone, and the Southern Coastal Zone. The HA-GGE biplot analysis here presents the ideal test environments and also identifies the mega-environment for sugarcane cultivars in China.

Rational regional distribution of sugarcane cultivars in China.

Knowing yield potential and yield stability of sugarcane cultivars is of significance in guiding sugarcane breeding and rationalising regional distribution of sugarcane cultivars. In the present study, a heritability-adjusted genotype main effect plus genotype × environment (HA-GGE) biplot program was used to analyze the cane and sucrose yields of 44 newly released sugarcane cultivars at eight pilot test sites. The cane and sucrose yields of nine cultivars were higher than those of the control cultivar ROC22. From the perspective of cane yield, cultivars FN 40 and YZ 06-407 were well adapted to a wider range of conditions and produced relatively high cane yields in several pilot sites. From the perspective of sucrose yield, cultivars LC 03-1137, FN 38, FN 41, MT 01-77 and LC 05-136 were well adapted to a wide range of conditions and produced relatively high sucrose yields. Based on these results, three high yielding and widely adapted cultivars, namely, FN 39, LC 05-136, and YZ 05-51 were recommended for production in three major Chinese sugarcane planting areas. The results will provide a theoretical basis for recommending the effective use and rational regional distribution of sugarcane cultivars in China.

Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum.

Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugars, and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. We have studied the nature of DNA damage induced directly by the pulsed 46.9-nm (26.5 eV) radiation provided by an extreme ultraviolet (XUV) capillary-discharge Ne-like Ar laser (CDL). Doses up to 45 kGy were delivered with a repetition rate of 3 Hz. We studied the dependence of the yield of SSBs and DSBs of a simple model of DNA molecule (pBR322) on the CDL pulse fluence. Agarose gel electrophoresis method was used for determination of both SSB and DSB yields. The action cross sections of the single- and double-strand breaks of pBR322 plasmid DNA in solid state were determined. We observed an increase in the efficiency of strand-break induction in the supercoiled DNA as a function of laser pulse fluence. Results are compared to those acquired at synchrotron radiation facilities and other sources of extreme-ultraviolet and soft x-ray radiation.

Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane.

Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane.

Clarification properties of trash and stalk tissues from sugar cane.

The effect of the U.S. and worldwide change from burnt to unburnt (green) sugar cane harvesting on processing and the use of sugar cane leaves and tops as a biomass source has not been fully characterized. Sugar cane whole-stalks were harvested from the first ratoon (repeat) crop of five commercial, Louisiana sugar cane varieties (LCP 85-384, HoCP 96-540, L 97-128, L 99-226, and L 99-233). Replicated sample tissues of brown, dry leaves (BL), green leaves (GL), growing point region (GPR), and stalk (S) were separated. Composite juice from each tissue type was clarified following a hot lime clarification process operated by most U.S. factories. Only GPR and GL juices foamed on heating and followed the normal settling behavior of factory sugar cane juice, although GL was markedly slower than GPR. GPR juice aided settling. S juice tended to thin out rather than follow normal settling and exhibited the most unwanted upward motion of flocs. Most varietal variation in settling, mud, and clarified juice (CJ) characteristics occurred for GL. The quality rather than the quantity of impurities in the different tissues mostly affected the volume of mud produced: After 30 min of settling, mud volume per unit tissue juice degrees Brix (% dissolved solids) varied markedly among the tissues (S 1.09, BL 11.3, GPR 3.0, and GL 3.1 mL/degrees Brix). Heat transfer properties of tissue juices and CJs are described. Clarification was unable to remove all BL cellulosic particles. GL and BL increased color, turbidity, and suspended particles in CJs with BL worse than GL. This will make the future attainment of very high pol (VHP) raw sugar in the U.S. more difficult. Although optimization of factory unit processes will alleviate extra trash problems, economical strategies to reduce the amount of green and brown leaves processed need to be identified and implemented.