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).

Influence of environmental and instrumental variables on the non-invasive prediction of Brix in pineapple using near infrared spectroscopy

The Brix content of pineapple fruit can be non-invasively predicted from the second derivative of near infrared reflectance spectra. Correlations obtained using a NIRSystems 6500 spectrophotometer through multiple linear regression and modified partial least squares analyses using a post-dispersive configuration were comparable with that from a pre-dispersive configuration in terms of accuracy (e.g. coefficient of determination, R2, 0.73; standard error of cross validation, SECV, 1.01°Brix). The effective depth of sample assessed was slightly greater using the post-dispersive technique (about 20 mm for pineapple fruit), as expected in relation to the higher incident light intensity, relative to the pre-dispersive configuration. The effect of such environmental variables as temperature, humidity and external light, and instrumental variables such as the number of scans averaged to form a spectrum, were considered with respect to the accuracy and precision of the measurement of absorbance at 876 nm, as a key term in the calibration for Brix, and predicted Brix. The application of post-dispersive near infrared technology to in-line assessment of intact fruit in a packing shed environment is discussed.

Non-invasive assessment of pineapple and mango fruit quality using near infra-red spectroscopy

The potential of near infra-red (NIR) spectroscopy for non-invasive measurement of fruit quality of pineapple (Ananas comosus var. Smooth Cayenne) and mango (Magnifera indica var. Kensington) fruit was assessed. A remote reflectance fibre optic probe, placed in contact with the fruit skin surface in a light-proof box, was used to deliver monochromatic light to the fruit, and to collect NIR reflectance spectra (760–2500 nm). The probe illuminated and collected reflected radiation from an area of about 16 cm2. The NIR spectral attributes were correlated with pineapple juice Brix and with mango flesh dry matter (DM) measured from fruit flesh directly underlying the scanned area. The highest correlations for both fruit were found using the second derivative of the spectra (d2 log 1/R) and an additive calibration equation. Multiple linear regression (MLR) on pineapple fruit spectra (n = 85) gave a calibration equation using d2 log 1/R at wavelengths of 866, 760, 1232 and 832 nm with a multiple coefficient of determination (R2) of 0.75, and a standard error of calibration (SEC) of 1.21 °Brix. Modified partial least squares (MPLS) regression analysis yielded a calibration equation with R2 = 0.91, SEC = 0.69, and a standard error of cross validation (SECV) of 1.09 oBrix. For mango, MLR gave a calibration equation using d2 log 1/R at 904, 872, 1660 and 1516 nm with R2 = 0.90, and SEC = 0.85% DM and a bias of 0.39. Using MPLS analysis, a calibration equation with R2 = 0.98, SEC = 0.54 and SECV = 1.19 was obtained. We conclude that NIR technology offers the potential to assess fruit sweetness in intact whole pineapple and DM in mango fruit, respectively, to within 1° Brix and 1% DM, and could be used for the grading of fruit in fruit packing sheds.

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 assessing fruit quality

Predictive models based on near infra-red spectroscopy for the assessment of fruit internal quality attributes must exhibit a degree of robustness across the parameters of variety, district and time to be of practical use in fruit grading. At the time this thesis was initiated, while there were a number of published reports on the development of near infra-red based calibration models for the assessment of internal quality attributes of intact fruit, there were no reports of the reliability ("robustness") of such models across time, cultivars or growing regions. As existing published reports varied in instrumentation employed, a re-analysis of existing data was not possible. An instrument platform, based on partial transmittance optics, a halogen light source and (Zeiss MMS 1) detector operating in the short wavelength near infra-red region was developed for use in the assessment of intact fruit. This platform was used to assess populations of macadamia kernels, melons and mandarin fruit for total soluble solids, dry matter and oil concentration. Calibration procedures were optimised and robustness assessed across growing areas, time of harvest, season and variety. In general, global modified partial least squares regression (MPLS) calibration models based on derivatised absorbance data were better than either multiple linear regression or `local' MPLS models in the prediction of independent validation populations . Robustness was most affected by growing season, relative to the growing district or variety . Various calibration updating procedures were evaluated in terms of calibration robustness. Random selection of samples from the validation population for addition to the calibration population was equivalent to or better than other methods of sample addition (methods based on the Mahalanobis distance of samples from either the centroid of the population or neighbourhood samples). In these exercises the global Mahalanobis distance (GH) was calculated using the scores and loadings from the calibration population on the independent validation population. In practice, it is recommended that model predictive performance be monitored in terms of predicted sample GH, with model updating using as few as 10 samples from the new population undertaken when the average GH value exceeds 1 .0 .

Near infrared reflectance as a rapid and inexpensive surrogate measure for fatty acid composition and oil content in peanuts (Arachis hypogaea L.)

The fatty acid composition of ground nuts (Arachis hypogaea L.) commonly known as peanuts, is an important consideration when a new variety is being released. The composition impacts on nutrition and, importantly, self-life of peanut products. To select for suitable breeding material, it was necessary to develop a rapid, non-derstructive and cost-efficient method. Near infrared spectroscopy was chosen as that methodology. Calibrations were developed for two major fatty-acid components, oleic and linoleic acids and two minor components, palmitic and stearic acids, as well as total oil content. Partial least squares models indicated a high level of precision with a squared multiple correlation coefficient of greater than 0.90 for each constitutent. Standard errors for prediction for oleic, linoleic, palmitic, stearic acids and total oil content were 6.4%, 4.5%, 0.8%, 0.9% and 1.3% respectively. The results demonstrated that reasonable calibrations could be developed to predict oil composition and content of peanuts for a breeding programme.

Quantitative assessment of total phenol contents of European oak (Quercus petraea and Quercus robur) by diffuse reflectance NIR spectroscopy on solid wood surfaces

Near infrared spectroscopy (NIRS) combined with multivariate analysis techniques was applied to assess phenol content of European oak. NIRS data were firstly collected directly from solid heartwood surfaces: in doing so, the spectra were recorded separately from the longitudinal radial and the transverse section surfaces by diffuse reflectance. The spectral data were then pretreated by several pre-processing procedures, such as multiplicative scatter correction, first derivative, second derivative and standard normal variate. The tannin contents of sawmill collected from the longitudinal radial and transverse section surfaces were determined by quantitative extraction with water/methanol (1:4, by vol). Then, total phenol contents in tannin extracts were measured by the Folin-Ciocalteu method. The NIR data were correlated against the Folin-Ciocalteu results. Calibration models built with partial least squares regression displayed strong correlation - as expressed by high determination correlation coefficient (r2) and high ratio of performance to deviation (RPD) - between measured and predicted total phenols content, and weak calibration and prediction errors (RMSEC, RMSEP). The best calibration was provided with second derivative spectra (r2 value of 0.93 for the longitudinal radial plane and of 0.91 for the transverse section plane). This study illustrates that the NIRS technique when used in conjunction with multivariate analysis could provide reliable, quick and non-destructive assessment of European oak heartwood extractives.

NIR model development and robustness in prediction of melon fruit total soluble solids

Near infrared spectroscopy (NIRS) can be used for the on-line, non-invasive assessment of fruit for eating quality attributes such as total soluble solids (TSS). The robustness of multivariate calibration models, based on NIRS in a partial transmittance optical geometry, for the assessment of TSS of intact rockmelons (Cucumis melo) was assessed. The mesocarp TSS was highest around the fruit equator and increased towards the seed cavity. Inner mesocarp TSS levels decreased towards both the proximal and distal ends of the fruit, but more so towards the proximal end. The equatorial region of the fruit was chosen as representative of the fruit for near infrared assessment of TSS. The spectral window for model development was optimised at 695-1045 nm, and the data pre-treatment procedure was optimised to second-derivative absorbance without scatter correction. The 'global' modified partial least squares (MPLS) regression modelling procedure of WINISI (ver. 1.04) was found to be superior with respect to root mean squared error of prediction (RMSEP) and bias for model predictions of TSS across seasons, compared with the 'local' MPLS regression procedure. Updating of the model with samples selected randomly from the independent validation population demonstrated improvement in both RMSEP and bias with addition of approximately 15 samples.

Detecting the attributes of a wheat crop using digital imagery acquired from a low-altitude platform

A low-altitude platform utilising a 1.8-m diameter tethered helium balloon was used to position a multispectral sensor, consisting of two digital cameras, above a fertiliser trial plot where wheat (Triticum spp.) was being grown. Located in Cecil Plains, Queensland, Australia, the plot was a long-term fertiliser trial being conducted by a fertiliser company to monitor the response of crops to various levels of nutrition. The different levels of nutrition were achieved by varying nitrogen application rates between 0 and 120 units of N at 40 unit increments. Each plot had received the same application rate for 10 years. Colour and near-infrared images were acquired that captured the whole 2 ha plot. These images were examined and relationships sought between the captured digital information and the crop parameters imaged at anthesis and the at-harvest quality and quantity parameters. The statistical analysis techniques used were correlation analysis, discriminant analysis and partial least squares regression. A high correlation was found between the image and yield (R2 = 0.91) and a moderate correlation between the image and grain protein content (R2 = 0.66). The utility of the system could be extended by choosing a more mobile platform. This would increase the potential for the system to be used to diagnose the causes of the variability and allow remediation, and/or to segregate the crop at harvest to meet certain quality parameters.

Deviant near-infrared spectra identifies Corymbia hybrids

The study examined the potential of Near Infrared Reflectance (NIR) spectroscopy for field diagnosis of hybrids between Corymbia (formerly Eucalyptus) species. NIR profiles were generated by scanning foliage from a total of 383 hybrid and 533 parental seedlings grown in a common garden and partial least squares discriminant analysis was used to test three-way model power to assign individuals to their appropriate taxon; either a parental or F1 hybrid class. Using the optimised conditions, fresh foliage from eight-month-old seedlings and a handheld NIR instrument (950–1800 nm), the mean assignment rates for the three hybrid groups ranged from 76% to 90%. Hybrid-parent contrast of NIR spectra deviated more so than parent–parent contrast. The F1 taxon assignment rates were usually higher than those for parents at 100% and 72%, respectively. Hybrid resolution was even greater for 2nd generation backcross hybrids. Similar to studies of morphology, taxon assignments tended to be more accurate for hybrid groups in which the parental taxa were more divergent. The practical application of this technique for hybrid diagnosis of seedlings in the nursery will require careful attention to control environmental factors because seedling age and storage effects influenced the ability of NIR to identify hybrids. The technique may also necessitate the generation of comparable reference populations, although exclusions approaches to analysis may circumvent the need for reference populations. The application of NIR in field diagnosis will be further complicated by the need to generate global models across environments but such models have been obtained for reliable prediction of chemistries in other situations.

Infrared spectroscopy as a rapid tool to detect methylglyoxal and antibacterial activity in Australian honeys

Methylglyoxal (2-oxopropanal) is a compound known to contribute to the non-peroxide antimicrobial activity of honeys. The feasibility of using infrared spectroscopy as a predictive tool for honey antibacterial activity and methylglyoxal content was assessed. A linear relationship was found between methylglyoxal content (279–1755 mg/kg) in Leptospermum polygalifolium honeys and bacterial inhibition for Escherichiacoli (R2 = 0.80) and Staphylococcusaureus (R2 = 0.64). A good prediction of methylglyoxal (R2 0.75) content in honey was achieved using spectroscopic data from the mid infrared (MIR) range in combination with partial least squares regression. These results indicate that robust predictive equations could be developed using MIR for commercial application where the prediction of bacterial inhibition is needed to ‘value’ honeys with methylglyoxal contents in excess of 200 mg/kg.

Phylogeography of the pharaoh cuttle Sepia pharaonis based on partial mitochondrial 16S sequence data

The pharaoh cuttle Sepia pharaonis Ehrenberg, 1831 (Mollusca: Cephalopoda: Sepiida) is a broadly distributed species of substantial fisheries importance found from east Africa to southern Japan. Little is known about S. pharaonis phylogeography, but evidence from morphology and reproductive biology suggests that Sepia pharaonis is actually a complex of at least three species. To evaluate this possibility, we collected tissue samples from Sepia pharaonis from throughout its range. Phylogenetic analyses of partial mitochondrial 16S sequences from these samples reveal five distinct clades: a Gulf of Aden/Red Sea clade, a northern Australia clade, a Persian Gulf/Arabian Sea clade, a western Pacific clade (Gulf of Thailand and Taiwan) and an India/Andaman Sea clade. Phylogenetic analyses including several Sepia species show that S. pharaonis sensu lato may not be monophyletic. We suggest that "S. pharaonis" may consist of up to five species, but additional data will be required to fully clarify relationships within the S. pharaonis complex.

Mapping Quantitative Trait Loci for Partial Resistance to Powdery Mildew in an Australian Barley Population

Genomic regions influencing resistance to powdery mildew [Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal] were detected in a doubled haploid (DH) barley (Hordeum vulgare L.) population derived from a cross between the breeding line ND24260 and cultivar Flagship when evaluated across four field environments in Australia and Uruguay. Significant quantitative trait loci (OIL) for resistance to B. graminis were detected on six of the seven chromosomes (1H, 2H, 3H, 4H, 5H, and 7H). A QTL with large effect donated by ND24260 mapped to the short arm of chromosome 1H (1 HS) conferring near immunity to B. graminis in Australia but was ineffective in Uruguay. Three OIL donated by Flagship contributed partial resistance to B. graminis and were detected in at least two environments. These OIL were mapped to chromosomes 3H, 4H, and 5H (5HS) accounting for up to 18.6, 3.4, and 8.8% phenotypic variation, respectively. The 5HS QTL contributed partial resistance to B. graminis in all field environments in both Australia and Uruguay and aligned with the genomic region of Rph20, a gene conferring adult plant resistance (APR) to leaf rust (Puccinia hordei Otth), which is found in some cultivars having Vada' or 'Emir' in their parentage. Selection for favorable marker haplotypes within the 3H, 4H, and 5H QTL regions can be performed even in the presence of single (major) gene resistance. Pyramiding such QTL may provide an effective and potentially durable form of resistance to B. graminis.

Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line '2-49'

We have tested the efficacy of putative microsatellite single sequence repeat (SSR) markers, previously identified in a 2-49 (Gluyas Early/Gala) × Janz doubled haploid wheat (Triticum aestivum) population, as being linked to partial seedling resistance to crown rot disease caused by Fusarium pseudograminearum. The quantitative trait loci (QTLs) delineated by these markers have been tested for linkage to resistance in an independent Gluyas Early × Janz doubled haploid population. The presence of a major QTL on chromosome 1DL (QCr.usq-1D1) and a minor QTL on chromosome 2BS (QCr.usq-2B1) was confirmed. However, a putative minor QTL on chromosome 2A was not confirmed. The QTL on 1D was inherited from Gluyas Early, a direct parent of 2-49, whereas the 2B QTL was inherited from Janz. Three other putative QTLs identified in 2-49 × Janz (on 1AL, 4BL, and 7BS) were inherited by 2-49 from Gala and were not able to be confirmed in this study. The screening of SSR markers on a small sample of elite wheat genotypes indicated that not all of the most tightly linked SSR markers flanking the major QTLs on 1D and 1A were polymorphic in all backgrounds, indicating the need for additional flanking markers when backcrossing into some elite pedigrees. Comparison of SSR haplotypes with those of other genotypes exhibiting partial crown rot resistance suggests that additional, novel sources of crown rot resistance are available.

Evaluation of partial replacement of live and fresh feeds with a formulated diet and the influence of weaning Panulirus ornatus phyllosomata onto a formulated diet during early ontogeny

We have evaluated the potential of a formulated diet as a replacement for live and fresh feeds for 7-day post-hatch Panulirus ornatus phyllosomata and also investigated the effect of conditioning phyllosomata for 14-21 days on live feeds prior to weaning onto a 100% formulated diet. In the first trial, the highest survival (>55%) was consistently shown by phyllosomata fed a diet consisting of a 50% combination of Artemia nauplii and 50% Greenshell mussel, followed by phyllosomata fed 50% Artemia nauplii and 50% formulated diet and, thirdly, by those receiving 100% Artemia nauplii. The second trial assessed the replacement of on-grown Artemia with proportions of formulated diet and Greenshell mussel that differed from those used in trial 1. Phyllosomata fed a 75% combination of formulated diet and 25% on-grown Artemia and 50% on-grown Artemia and 50% Greenshell mussel consistently showed the highest survival (>75%). Combinations of Greenshell mussel and formulated diet resulted in significantly (P < 0.05) reduced survival. In trial 3, phyllosomata were conditioned for 14, 18 or 21 days on Artemia nauplii prior to weaning onto a 100% formulated diet, which resulted in survival rates that were negatively related to the duration of feeding Artemia nauplii. In the final trial, phyllosomata were conditioned for 14 days on live on-grown Artemia prior to weaning onto one of three formulated diets (one diet with 44% CP and two diets with 50%). Phyllosomata fed a 44% CP diet consistently showed the highest survival (>35%) among all treatments, while those fed a 50%-squid CP diet showed a significant (P < 0.05) increase in mortality at day 24. The results of these trials demonstrate that hatcheries can potentially replace 75% of live on-grown Artemia with a formulated diet 7 days after hatch. The poor performance associated with feeding combinations of Greenshell mussel and formulated diet, and 100% formulated diet as well as conditioning phyllosomata for 14-21 days on live feeds prior to weaning onto a formulated diet highlights the importance of providing Artemia to stimulate feeding.