Michael G. O'Shea

Near-Infrared Spectroscopy for the Prediction of Disease Ratings for Fiji Leaf Gall in Sugarcane Clones

This paper demonstrates how inferential measurements or indirect methods using near-infrared (NIR) methodology and chemometrics can be used to predict sugarcane clonal performance. Fiji leaf gall resistance is used in this study as an example. Fiji leaf gall is one of Australias most serious sugarcane diseases, representing a significant problem in almost half of the total area under production. Traditional rating of sugarcane clones for resistance/susceptibility is difficult and expensive because of the nature of field-based methods and variable infection levels of the trials. Thus, the aim of this work was to investigate the potential of NIR spectroscopy as an alternative means to rate clones from direct measurement of sugarcane leaf spectra and to examine its ability to successfully predict traditional resistance ratings using a calibration model based on a chemometrics method such as partial least squares (PLS). A scanning electron microscopy (SEM) study of the leaf substrate was undertaken to elucidate the nature of the NIR sample site. In addition, an NIR study of freeze-dried sugarcane leaf samples resolved the heavily overlapping O–H bands present in the NIR spectrum due to water/cellulose interaction. A significant decrease in the spectral intensity between 5205 and 5393 cm−1 was observed and a similar decrease was noted in the OH stretching overtone (7114 cm−1) with an accompanying shift to lower wavenumbers. PLS modeling based on traditional ratings as the dependent variable and the corresponding NIR spectra showed satisfactory results with standard error of validation (SEV) and standard error of prediction (SEP) values being 0.98 (R2 = 0.97) and 1.20 (R2 = 0.88), respectively. This methodology has now been recommended for more extensive field trials.

Design, development, and use of molecular primers and probes for the detection of Gluconacetobacter species in the pink sugarcane mealybug.

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of “squashed” whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.

Complex biopolymeric systems at stalk/epicuticular wax plant interfaces: a near infrared spectroscopy study of the sugarcane example.

Naturally occurring macromolecules present at the epicuticular wax/stalk tissue interface of sugarcane were investigated using near infrared spectroscopy (NIRS). Investigations of water, cellulose, and wax‐cellulose interrelationships were possible using NIRS methods, where in the past many different techniques have been required. The sugarcane complex interface was used as an example of typical phenomena found at plant leaf/stalk interfaces. This detailed study showed that sugarcane cultivars exhibit spectral differences in the CHn, water OH, and cellulose OH regions, reflecting the presence of epicuticular wax, epidermis, and ground tissue. Spectrally complex water bands (5276 cm−1 and 7500–6000 cm−1) were investigated via freeze‐drying experiments which revealed sequentially a complex band substructure (7500–6000 cm−1), a developing weak H‐bonding system (∼7301 cm−1), and strong H‐bonding (∼7062 cm−1) assigned to water—cellulose interactions. Principal component analysis techniques clarified complex band trends that developed during the desorption experiment. Bands from wax‐free stalk were minimized in the 4327–4080 cm−1 region (Cuf8ffHn vibrational modes associated with long chain fatty compounds), while bands from the stalk tissue (particularly lignin and moisture) became more pronounced. This work is a comprehensive guide to similar studies by scientists involved in a variety of plant and fiber research fields.

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchariCockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarum L.). In this study, bothGlac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated withGlac. sacchari.Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.

Rapid Determination of Carbon, Nitrogen, Silicon, Phosphorus, and Potassium in Sugar Mill By-products, Mill Mud, and Ash using Near Infrared Spectroscopy

This article describes a proof-of-concept exercise to examine the ability of near infrared spectroscopy (NIRS)–based methods to predict the major nutrient properties of sugar mill by-products, particularly mill mud, ash, and mixtures of mud and ash. Sixty mill mud, mixed mud/ash, and ash samples were subsampled three times and analyzed using traditional analytical techniques for carbon (C), nitrogen (N), silicon (Si), phosphorus (P), and potassium (K), and the NIR spectra were recorded. Two different partial least squares (PLS) regression models were constructed, one using all samples and the other without the ash samples included in the model development. Three mud, one mixed mud/ash, and two ash samples were retained for predictive purposes and were not included in the model development process. R2 values in the range of 0.77 to 0.98 were obtained for all constituents across both sets of PLS models. The standard errors of prediction (SEP) were similar for both models for N (0.10 and 0.08), P (0.17 and 0.16), and K (0.05 and 0.05). However, the SEP obtained for Si (3.53 and 1.04) and C (1.92 and 1.00) varied between the two models. These preliminary results are very encouraging. Future research will extend to robust NIRS calibrations for these nutrients and develop applications for their use within laboratory or field situations to permit nutrient monitoring in various sugar mill by-products.

Advancing Energy Cane Cell Wall Digestibility Screening by Near-Infrared Spectroscopy

Breeding energy cane for cellulosic biofuel production involves manipulating various traits. An important trait to optimize is cell wall degradability as defined by enzymatic hydrolysis. We investigated the feasibility of using near-infrared spectroscopy (NIRS) combined with multivariate calibration to predict energy cane cell wall digestibility based upon fiber samples from a range of sugarcane genotypes and related species. These samples produced digestibility values ranging between 6 and 31%. To preserve the practicality of the technique, spectra obtained from crudely prepared samples were used. Various spectral preprocessing methods were tested, with the best NIRS calibration obtained from second derivative, orthogonal signal–corrected spectra. Model performance was evaluated by cross-validation and independent validation. Large differences between the performance results from the two validation approaches indicated that the model was sensitive to the choice of test data. This may be remedied by using a larger calibration training set containing diverse sample types. The best result was obtained through independent validation which produced a R 2 value of 0.86, a root mean squared error of prediction (RMSEP) of 1.59, and a ratio of prediction to deviation (RPD) of 2.7. This study has demonstrated that it is feasible and practical to use NIRS to predict energy cane cell wall digestibility.

Development of near Infrared Spectroscopic Methods for Monitoring Major Nutrient Elements in Sugar Mill Byproducts

Introduction T he Australian sugarcane industry is geographically situated in close proximity to environmentally sensitive and World Heritage listed areas of the Great Barrier Reef Marine Park and the Wet Tropics. As a result, the Australian sugar industry is subjected to a high level of scrutiny with respect to nutrient application rates and associated nutrient runoff. The Queensland State Government passed legislation at the beginning of 2010 enabling it to regulate certain farming activities. In particular, the legislation was designed to minimise the impact of fertilisers and pesticides on the quality of run-off water entering the Great Barrier Reef lagoon by controlling maximum application rates on nearby land. Sugar mill byproducts are a valuable source of mineral nutrients and organic matter and their application to surrounding cane farms is a mutually beneficial practice for sugarcane growers and millers. It is estimated that the value of available nutrient inputs from mill mud and ash application across the industry is approximately

A chemometrics investigation of sugarcane plant properties based on the molecular composition of epicuticular wax

36M per year. Mill mud (Figure 1) is produced as a byproduct of a juice purification process known as clarification. During clarification, predominantly high molecular weight and/ or insoluble impurities present in the juice such as gums, waxes and residual fibre particles are entrapped in a flocculated calcium phosphate system. A scum or mud forms during the process and is subsequently removed. This waste product is referred to as “mill mud” and contains significant amounts of calcium (approximately 878 kg Ca per 150 wet tonnes) and phosphorous (341 kg P per 150 wet tonnes) and is often combined with boiler ash (Figure 1) prior to field application. Boiler ash is the residue remaining after burning bagasse (sugarcane fibre which has had sucrose removed) in the boilers to produce steam and contains significant amounts of potassium (296 kg K per 150 wet tonnes) and silicon (3600 kg per 150 wet tonnes) (internal communication). The application rate of the byproducts back to the cane field can vary between 120 and 250 wet tonnes/ha depending on the region. Transportation of the byproduct from the mill to the cane field is usually by truck; Figure 2 shows a truck waiting to be loaded via a hopper at the sugar mill. A specifically designed mechanised applicator spreader is mounted on the tailgate of the truck in order to deposit the byproduct evenly between rows in the field (Figure 3). At present, the nutrient content of these byproducts is not included in the determination of nitrogen and phosphorus levels applied to sugarcane farms under existing legislation. The research reported in this article has been designed to produce rapid, easy to use NIR spectroscopic methods for the prediction of nutrient elements: carbon (C), nitrogen (N), silicon (Si), phosphorus (P) and potassium (K) levels in mill mud and boiler ash. This information will likely be required in the future in order to ensure that sugarcane growers comply with all aspects of the Great Barrier Reef Protection Legislation. While there is considerable literature precedent for carbon and nitrogen calibrations, there are theoretical issues regarding the potential to develop NIR calibrations for inorganic materials such as silicon, phosphorus Figure 1. Typical sample of mill mud (left) and boiler ash (right).