M. P. Grisham

A Polymerase Chain Reaction Protocol for the Detection of Xanthomonas albilineans, the Causal Agent of Sugarcane Leaf Scald Disease

A polymerase chain reaction (PCR) protocol was developed that amplified a 360-bp DNA product unique to Xanthomonas albilineans (Xa), the causal agent of sugarcane leaf scald disease. The assay utilizes previously described PCR primers that target the intergenic transcribed spacer (ITS) region between the 16S and 23S rRNA genes. Primer pair Ala4/L1 allowed amplification of a 360-bp DNA fragment from 71 Xa strains including representatives of serovars I, II, and III. Fragments of different sizes were also amplified from three unidentified saprophytic bacteria from sugarcane. Xa could be detected at a lower bacterial concentration with the PCR protocol than with a serological dot blot assay. With PCR, as little as 1.25 pg of Xa genomic DNA (125 fg if followed by Southern blot hybridization), or as few as 0 to 5 CFU of Xa per reaction were detected from infected sugarcane sap and leaf diffusate. Five CFU of Xa per reaction were detected from suspension culture. The PCR protocol provides a rapid, reliable, and economical tool for routine detection and identification of Xa.

An assessment of the genetic diversity within a collection ofSaccharum spontaneum L. with RAPD-PCR

A local collection of 33Saccharum spontaneum L. clones and two sugarcane cultivars (LCP 82-89 and LCP 85-384) were assessed for genetic variability using random amplified polymorphic DNA (RAPD)-PCR. A total of 157 polymorphic RAPD-PCR bands were scored with 17 primers. The number of RAPD-PCR products per primer ranged from four to 16. The data were analyzed with two multivariate analysis software programs, NTSYSpc and DNAMAN®. Although these two programs yielded similar results, a bootstrapped phylogenetic tree could only be generated with the DNAMAN® software. A substantial degree of genetic diversity was found within the localS. spontaneum collection. Pairwise genetic homology coefficients ranged from 65% (SES, 196/Tainan 2n = 96) to 88.5% (IND 81-80/IND 81-144). LCP 82-89 and LCP 85-384 shared a greater similarity (82%) than either was to any clone ofS. spontaneum (ranging from 60.5 to 75.2%). The 33S. spontaneum clones were assigned to eight groups independent of their geographic origin or morphology, while the two sugarcane cultivars were assigned to the ninth group. All but two pairs ofS. spontaneum clones could be distinguished by a single RAPD primer OPBB-02. The use of a second primer, either OPBE-04 or Primer 262, separated allS. spontaneum clones. One amplification product from the RAPD primer OPA-11, OPA-11-336, proved to be cultivar-specific and has been adopted for use in our breeding program. Information from this study would help conserve the genetic diversity ofS. spontaneum.

Detecting Sugarcane yellow leaf virus infection in asymptomatic leaves with hyperspectral remote sensing and associated leaf pigment changes

Sugarcane infected with Sugarcane yellow leaf virus (SCYLV) rarely produces visual symptoms until late in the growing season. High-resolution, hyperspectral reflectance data from SCYLV-infected and non-infected leaves of two cultivars, LCP 85-384 and Ho 95-988, were measured and analyzed on 13 July, 12 October, and 4 November 2005. All plants were asymptomatic. Infection was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Results from discriminant analysis showed that leaf reflectance was effective at predicting SCYLV infection in 73% of the cases in both cultivars using resubstitution and 63% and 62% in LCP 85-384 and Ho 95-988, respectively, using cross-validation. Predictive equations were improved when data from sampling dates were analyzed individually. SCYLV infection influenced the concentration of several leaf pigments including violaxanthin, beta-carotene, neoxanthin, and chlorophyll a. Pigment data were effective at predicting SCYLV infection in 80% of the samples in the combined data set using the derived discriminant function with resubstitution, and 71% with cross-validation. Although further research is needed to improve the accuracy of the predictive equations, the results of this study demonstrate the potential application of hyperspectral remote sensing as a rapid, field-based method of identifying SCYLV-infected sugarcane plants prior to symptom expression.

Early detection of leifsonia xyli subsp. xyli in sugarcane leaves by real-time polymerase chain reaction

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

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.

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.

Molecular Variation of Sporisorium scitamineum in Mainland China Revealed by RAPD and SRAP Markers

Sugarcane smut caused by Sporisorium scitamineum occurs worldwide, causing serious losses in sugar yield and quality. To study the molecular variation of S. scitamineum, 23 S. scitamineum isolates collected from the six primary sugarcane production areas in mainland China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi provinces) were assessed by random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) markers. The results of RAPD, SRAP, and RAPD-SRAP combined analysis showed that, whereas the molecular variation of S. scitamineum was associated with geographic origin, there was no evidence of co-evolution between sugarcane and the pathogen. The results of RAPD, SRAP, or RAPD-SRAP combined analysis also did not provide any information about race differentiation of S. scitamineum. This suggests that the mixture of spores from sori collected from different areas should be used in artificial inoculations for resistance breeding and selection.

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.

Resistance of sugarcane relatives injected with Ustilago scitaminea

We evaluated the resistance of 102 clones of sugarcane (Saccharum spp.) relatives to Ustilago scitaminea, causal agent of sugarcane smut, in two greenhouse experiments. Relatives included Erianthus spp. section Ripidum, S. barberi/S. sinense, S. officinarum, S. robustum, S. spontaneum, and Saccharum interspecific hybrids (cultivars). Clones of Erianthus spp. section Ripidium were the most resistant and clones of S. officinarum and S. robustum were the most susceptible of the six taxonomic groups included in the first experiment

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.