Kerry B. Walsh

Sorting of Fruit Using near Infrared Spectroscopy: Application to a Range of Fruit and Vegetables for Soluble Solids and Dry Matter Content:

The performance of a single instrumentation platform, incorporating the use of a tungsten halogen light source, body transmittance optics and a silicon photodiode array detector, and a uniform chemometric approach is reported for the application of assessment of determination of soluble solids and dry matter content of a range of fruit. Spectra were acquired at integration times of 30 ms or less, with integration time varied between fruit types to achieve a similar signal level. Calibration performance was compared in terms of root mean standard error of cross validation (RMSECV), regression coefficient (R), and the SDR (SDR = SD / RMSECV (SD is standard deviation)]. The technology was well suited to sorting on soluble solids content (SSC) in apple (RMSECV 0.22%, SDR > 5; R 0.98), and useful, in decreasing order of accuracy, for sorting of stonefruit, mandarin, banana, melons, onions, tomato and papaya (RMSECV 1.1%, SDR 1.6, R 0.79). The technology also performed well in sorting on dry matter content in kiwifruit (RMSECV 0.38%, SDR > 3, R 0.95), and useful, in decreasing order of accuracy, for sorting of banana, mango, avocado, tomato and potato (RMSECV 1.0%, SDR 1.7, R 0.79). The limitations of the application of the technology to fruit sorting is discussed in terms of fruit type (“skin” thickness) and population range. For example, calibration RMSECV was only 0.20% on tomato SSC, but as population variation was low (SD 0.30%), a poor R (0.77) and SDR (1.5) was obtained.

Short-wavelength near-infrared spectra of sucrose, glucose, and fructose with respect to sugar concentration and temperature

Short-wavelength near-infrared (SW-NIR) (700–1100 nm) spectra of aqueous solutions of sucrose, d-glucose, and d-fructose were monitored with respect to change in temperature and sugar concentration. Sugar OH and CH related vibrations were identified by analysis of the spectra of sugar solutions in deuterium oxide (D2O), and of sucrose-d8 solutions in D2O. Absorption spectra were explained in terms of the second and third overtones of OH stretching vibrations and the third overtone of CH2 and CH stretchings. In deuterated solutions, CH and CH2 higher overtone vibration bands became apparent. The major spectral effect of decreased temperature or increased sugar concentration was a decrease in absorbance at 960 nm and an increase in absorbance at 984 nm, interpreted as an increase in the degree of H bonding. Partial least-squares (PLS) calibrations on sugar concentrations (with spectra collected at several sample temperatures) relied strongly on the 910 nm sugar CH related bands, whereas calibrations on temperature depended equally on all OH associated vibrations (750, 840, 960, and 985 nm).

Application of commercially available, low-cost, miniaturised NIR spectrometers to the assessment of the sugar content of intact fruit

Recent decreases in costs, and improvements in performance, of silicon array detectors open a range of potential applications of relevance to plant physiologists, associated with spectral analysis in the visible and short-wave near infra-red (far-red) spectrum. The performance characteristics of three commercially available ‘miniature’ spectrometers based on silicon array detectors operating in the 650–1050-nm spectral region (MMS1 from Zeiss, S2000 from Ocean Optics, and FICS from Oriel, operated with a Larry detector) were compared with respect to the application of non-invasive prediction of sugar content of fruit using near infra-red spectroscopy (NIRS). The FICS–Larry gave the best wavelength resolution; however, the narrow slit and small pixel size of the charge-coupled device detector resulted in a very low sensitivity, and this instrumentation was not considered further. Wavelength resolution was poor with the MMS1 relative to the S2000 (e.g. full width at half maximum of the 912 nm Hg peak, 13 and 2 nm for the MMS1 and S2000, respectively), but the large pixel height of the array used in the MMS1 gave it sensitivity comparable to the S2000. The signal-to-signal standard error ratio of spectra was greater by an order of magnitude with the MMS1, relative to the S2000, at both near saturation and low light levels. Calibrations were developed using reflectance spectra of filter paper soaked in range of concentrations (0–20% w/v) of sucrose, using a modified partial least squares procedure. Calibrations developed with the MMS1 were superior to those developed using the S2000 (e.g. coefficient of correlation of 0.90 and 0.62, and standard error of cross-validation of 1.9 and 5.4%, respectively), indicating the importance of high signal to noise ratio over wavelength resolution to calibration accuracy. The design of a bench top assembly using the MMS1 for the non-invasive assessment of mesocarp sugar content of (intact) melon fruit is reported in terms of light source and angle between detector and light source, and optimisation of math treatment (derivative condition and smoothing function).

Studies on spatial distribution of nickel in leaves and stems of the metal hyperaccumulator Stackhousia tryonii using nuclear microprobe (micro-PIXE)and EDXS techniques

Stackhousia tryonii Bailey is one of the three nickel hyperaccumulators reported from Australia. It is a rare, herbaceous plant that accumulates (Ni) both in leaf and stem tissues. Localisation of Ni in leaf and stem tissues of S. tryonii was studied using two micro-analytical techniques, energy dispersive X-ray spectrometry (EDXS) and micro-proton-induced X-ray emission spectrometry (micro-PIXE). Dimethylglyoxime complexation of Ni was also visualised by bright- and dark-field microscopy, but this technique was considered to create artefacts in the distribution of Ni. Energy dispersive X-ray spectrometric analysis indicated that guard cells possessed a lower Ni concentration than epidermal cells, and that epidermal cells and vascular tissue contained higher levels of Ni than mesophyll, as reported for other Ni hyperaccumulators. The highest Ni concentration was recorded (PIXE quantitative point analysis) in the epidermal cells and vascular tissue (5400 μg g-1 DW), approximately double that recorded in palisade cells (2500 μg g-1 DW). However, concentrations were variable within these tissues, explaining, in part, the similarity between average Ni concentrations of these tissues (as estimated by region selection mode). Stem tissues showed a similar distribution pattern as leaves, with relatively low Ni concentration in the pith (central) region. The majority of Ni (73-85% for leaves; 80-92% for stem) was extracted from freeze-dried sections by water extraction, suggesting that this metal is present in a highly soluble and mobile form in the leaf and stem tissues of S. tryonii.

Assessment of internal quality attributes of mandarin fruit. 1. NIR calibration model development

The utility of near infrared spectroscopy as a non-invasive technique for the assessment of internal eating quality parameters of mandarin fruit (Citrus reticulata cv. Imperial) was assessed. The calibration procedure for the attributes of TSS (total soluble solids) and DM (dry matter) was optimised with respect to a reference sampling technique, scan averaging, spectral window, data pre-treatment (in terms of derivative treatment and scatter correction routine) and regression procedure. The recommended procedure involved sampling of an equatorial position on the fruit with 1 scan per spectrum, and modified partial least squares model development on a 720–950-nm window, pre-treated as first derivative absorbance data (gap size of 4 data points) with standard normal variance and detrend scatter correction. Calibration model performance for the attributes of TSS and DM content was encouraging (typical Rc2 of >0.75 and 0.90, respectively; typical root mean squared standard error of calibration of <0.4 and 0.6%, respectively), whereas that for juiciness and total acidity was unacceptable. The robustness of the TSS and DM calibrations across new populations of fruit is documented in a companion study.

Robustness of NIR calibrations for soluble solids in intact melon and pineapple

The soluble solids content of intact fruit can be measured non-invasively by near infrared spectroscopy, allowing “sweetness” grading of individual fruit. However, little information is available in the literature with respect to the robustness of such calibrations. We developed calibrations based on a restricted wavelength range (700–1100 nm), suitable for use with low-cost silicon detector systems, using a stepwise multiple linear regression routine. Calibrations for total soluble solids (°Brix) in intact pineapple fruit were not transferable between summer and winter growing seasons. A combined calibration (data of three harvest dates) validated reasonably well against a population set drawn from all harvest dates (r2 = 0.72, SEP = 1.84 °Brix). Calibrations for Brix in melon were transferable between two of the three varieties examined. However, a lack of robustness of calibration was indicated by poor validation within populations of fruit harvested at different times. Further work is planned to investigate the robustness of calibration across varieties, growing districts and seasons.

The anatomy of the pathway of sucrose unloading within the sugarcane stalk

The physical path of sucrose unloading in the sugarcane stalk is described. About 50% of the vascular bundles in the internodes were located within 3 mm of the outside of the stalk. These bundles were inactive in long distance sucrose transport, as assessed by dye tracers of phloem flow. A sheath of fibres isolates the phloem apoplast from that of the storage parenchyma. In bundles associated with long distance transport (i.e. in the central region), the fibre sheath is narrowest to either side of the phloem fibre cap, and consists of living cells with plasmodesmata within pits in the secondary wall. Plasmodesmata were also arranged into pit fields between cells of the storage parenchyma. Since the vascular apoplast is isolated from the apoplast of the storage parenchyma, sucrose must move through the symplast of the fibre sheath. The calculated flux of sucrose through plasmodesmata of this cell layer was at the low end of reported values in the literature. Sucrose unloading within the storage parenchyma may also follow a symplastic route, with unloading into the apoplast of the storage parenchyma occurring as part of a turgor mechanism to increase sink strength.

Assessment of internal quality attributes of mandarin fruit. 2. NIR calibration model robustness

The robustness of multivariate calibration models, based on near infrared spectroscopy, for the assessment of total soluble solids (TSS) and dry matter (DM) of intact mandarin fruit (Citrus reticulata cv. Imperial) was assessed. TSS calibration model performance was validated in terms of prediction of populations of fruit not in the original population (different harvest days from a single tree, different harvest localities, different harvest seasons). Of these, calibration performance was most affected by validation across seasons (signal to noise statistic on root mean squared error of prediction of 3.8, compared with 20 and 13 for locality and harvest day, respectively). Procedures for sample selection from the validation population for addition to the calibration population (‘model updating’) were considered for both TSS and DM models. Random selection from the validation group worked as well as more sophisticated selection procedures, with approximately 20 samples required. Models that were developed using samples at a range of temperatures were robust in validation for TSS and DM.

Detection of phytoplasmas in dieback, yellow crinkle, and mosaic diseases of papaya using polymerase chain reaction techniques

Oligonucleotide primers complementary to regions specific to plant-pathogenic mycoplasma-like organisms (phytoplasmas) were used in polymerase chain reactions on tissue samples from dieback, yellow crinkle, and mosaic affected papaya plants. The primer pair P068/P069, which hybridise to internal regions of the 16s rRNA gene, amplified an approximately 560 bp product in dieback, yellow crinkle and mosaic affected papaya. The primer pair P3/P7, which hybridise to the spacer region between the 16s and 23s rRNA genes, amplified an approximately 300 bp fragment in yellow crinkle and mosaic affected papaya, with no product from dieback affected plants. No PCR product was obtained with either set of primers from healthy plants. An identical Alu I restriction enzyme profile was obtained with all three 560 bp products. This study provides the first evidence for the association of phytoplasmas with papaya mosaic and Australian papaya dieback.

Calibration transfer between miniature photodiode array-based spectrometers in the near infrared assessment of mandarin soluble solids content

The transfer of predictive models among photodiode array based, short wave near infrared spectrometers using the same illumination/detection optical geometry has been attempted using various chemometric techniques, including slope and bias correction (SBC), direct standardisation (DS), piecewise direct standardisation (PDS), double window PDS (DWPDS), orthogonal signal correction (OSC), finite impulse transform (FIR) and wavelet transform (WT). Additionally, an interpolation and photometric response correction method, a wavelength selection method and a model updating method were assessed. Calibration transfer was attempted across two populations of mandarin fruit. Model performance was compared in terms of root mean squared error of prediction (RMSEP), using Fearns significance testing, for calibration transfer (standardisation) between pairs of spectrometers from a group of four spectrometers. For example, when a calibration model (Root Mean Square Error of Cross-Validation [RMSECV = 0.26% soluble solid content (SSC)], developed on one spectrometer, was used with spectral data collected on another spectrometer, a poor prediction resulted (RMSEP = 2.5% SSC). A modified WT method performed significantly better (e.g. RMSEP = 0.25% SSC) than all other standardisation methods (10 of 12 cases), and almost on a par with model updating (MU) (nine cases with no significant difference, one case and two cases significantly better for WT and MU, respectively).