Preparation and In Vitro Release of Drug-Loaded Microparticles for Oral Delivery Using Wholegrain Sorghum Kafirin Protein

Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content, in vitro protein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released in in vitro conditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangifera indica) transcriptome for mapping and estimation of genetic diversity

The development of molecular markers for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here our identification of thousands of unambiguous molecular markers that can be easily assayed across genotypes of the species. With origin centered in Southeast Asia, mangos are grown throughout the tropics and subtropics as a nutritious fruit that exhibits remarkable intraspecific phenotypic diversity. With the goal of building a high density genetic map, we have undertaken discovery of sequence variation in expressed genes across a broad range of mango cultivars. A transcriptome sequence reference was built de novo from extensive sequencing and assembly of RNA from cultivar 'Tommy Atkins'. Single nucleotide polymorphisms (SNPs) in protein coding transcripts were determined from alignment of RNA reads from 24 mango cultivars of diverse origins: 'Amin Abrahimpur' (India), 'Aroemanis' (Indonesia), 'Burma' (Burma), 'CAC' (Hawaii), 'Duncan' (Florida), 'Edward' (Florida), 'Everbearing' (Florida), 'Gary' (Florida), 'Hodson' (Florida), 'Itamaraca' (Brazil), 'Jakarata' (Florida), 'Long' (Jamaica), 'M. Casturi Purple' (Borneo), 'Malindi' (Kenya), 'Mulgoba' (India), 'Neelum' (India), 'Peach' (unknown), 'Prieto' (Cuba), 'Sandersha' (India), 'Tete Nene' (Puerto Rico), 'Thai Everbearing' (Thailand), 'Toledo' (Cuba), 'Tommy Atkins' (Florida) and 'Turpentine' (West Indies). SNPs in a selected subset of protein coding transcripts are currently being converted into Fluidigm assays for genotyping of mapping populations and germplasm collections. Using an alternate approach, SNPs (144) discovered by sequencing of candidate genes in 'Kensington Pride' have already been converted and used for genotyping.

Protected cropping for specialty melons in North Queensland

Protective cropping could be an effective system for growing specialty melons in the dry tropics of North Queensland. The growing system could reduce outdoor risks for production loss, improve fruit quality, increase yield per m2, allow production offseason, and used for supplying niche markets in a segment of the larger melon market in Australia. First evaluations in Giru, Queensland, included seven cultivars of fruit types 'Galia', 'Hami', 'Charentais', small 'Canary', and 'Rockmelon', transplanted July 25, 2013 under a high polyethylene-covered tunnel. Plants were grown at a density of 2.8 plants m-2 in containers filled with volcanic rock and irrigated with a complete nutrient solution. Pruning and trellising was done to a single vertical stem, keeping lateral shoots on the main stem after the 7th leaf node. After bearing small fruit, lateral shoots were cut off after their second or third leaf node. To facilitate insect pollination, a screen window in the tunnel was left partially opened. On November 20 the cultivars had combined marketable yields that ranged from 2.8 to 8.2 fruits m-2 and 3.1 to 7.8 kg m-2. Total soluble solids levels in fruit ranged from 6 to 13 °Brix. Cultivars 'Tempo' ('Galia'), 'Tikal' ('Canary') and 'Sultan' ('Charentais') had fruit yields that were up to 2.6 times greater than yields commonly achieved with field-grown rockmelon crops. Sugar levels in fruits and marketable yields may be increased with changes in fertigation management. Promising results in this first evaluation justify examination of a greater number of genetic materials, in addition to the development of economic feasibility studies and further adaptive research to refine crop recommendations for growing melons in protective cropping systems.

Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera : Tenebrionidae) populations from Turkey

Fumigation with phosphine gas is the primary method of controlling stored grain pests. In Turkey, phosphine has been used extensively since the 1950's. Even though high levels of phosphine resistance have been detected in several key stored products pests across the world, it has never been studied in Turkey despite this long history of phosphine use. High-level phosphine resistance has been detected and genetically characterised previously in the rust red flour beetle, Tribolium castaneum in other countries. Since this pest is also a common problem in stored grain environment in Turkey, the current study was undertaken for the first time, to investigate the distribution and strength of phosphine resistance in T. castaneum. Four strains of T. castaneum were tested through bioassays for determining the weak and strong phosphine resistance phenotypes on the basis of the response of adults to discriminating phosphine concentrations of 0.03 mg/L and 0.25 mg/L, for 20 hour exposures respectively. Phenotype testing showed all strains exhibited some level of phosphine resistance with a maximum level of 196 fold. Sequencing and genetic testing of seven field-collected strains showed that all of them carried a strong resistance allele in at the rph2 locus similar to the one previously reported. Our results show that strong resistance to phosphine is common in Turkish strains of T. castaneum.

Random Mating Between Two Widely Divergent Mitochondrial Lineages of Cryptolestes ferrugineus (Coleoptera: Laemophloeidae): A Test of Species Limits in a Phosphine-Resistant Stored Product Pest

Effective pest management relies on accurate delimitation of species and, beyond this, on accurate species identification. Mitochondrial COI sequences are useful for providing initial indications in delimiting species but, despite acknowledged limitations in the method, many studies involving COI sequences and species problems remain unresolved. Here we illustrate how such impasses can be resolved with microsatellite and nuclear sequence data, to assess more directly the amount of gene flow between divergent lineages. We use a population genetics approach to test for random mating between two 8 ± 2% divergent COI lineages of the rusty grain beetle, Cryptolestes ferrugineus (Stephens). This species has become strongly resistant to phosphine, a fumigant used worldwide for disinfesting grain. The possibility of cryptic species would have significant consequences for resistance management, especially if resistance was confined to one mitochondrial lineage. We find no evidence of restricted gene flow or nonrandom mating across the two COI lineages of these beetles, rather we hypothesize that historic population structure associated with early Pleistocene climate changes likely contributed to divergent lineages within this species.

Seasonal and Species Variation of the Hepatotoxin Indospicine in Australian Indigofera Legumes as Measured by UPLC−MS/MS

Livestock industries have maintained a keen interest in pasture legumes because of the high protein content and nutritive value. Leguminous Indigofera plant species have been considered as having high feeding values to be utilized as pasture, but the occurrence of the toxic constituent indospicine in some species has restricted this utility. Indospicine has caused both primary and secondary hepatotoxicosis and also reproductive losses, but has only previously been determined in a small number of Indigofera species. This paper validates a high throughput ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method to determine indospicine content of various Indigofera species found in Australian pasture. Twelve species of Indigofera together with Indigastrum parviflorum plants were collected and analysed. Out of the 84 samples analyzed, *I. spicata contained the highest indospicine level (1003 ± 328 mg/kg DM, n = 4) followed by I. linnaei (755 ± 490 mg/kg DM, n = 51). Indospicine was not detected in 9 of the remaining 11 species, and at only low levels (<10 mg/kg DM) in 2 out of 8 I. colutea specimens and in 1 out of 5 I. linifolia specimens. Indospicine concentrations were below quantitation levels for other Indigofera spp. (I. adesmiifolia, I. georgei, I. hirsuta, I. leucotricha,* I. oblongifolia, I. australis and I. trita) and Indigastrum parviflorum. One of the more significant findings to emerge from this study is that the indospicine content of I. linnaei is highly variable (159 to 2128 mg/kg DM, n = 51), and differs across both regions and seasons. Its first re-growth after spring rain has a higher (p < 0.01) indospicine content than growth following more substantial summer rain. The species collected include the predominant Indigofera in Australia pasture, and of these, only *I. spicata and I. linnaei contain high enough levels of indospicine to pose a potential toxic threat to grazing herbivores.

Organic acids in Kakadu plum (Terminalia ferdinandiana): The good (ellagic), the bad (oxalic) and the uncertain (ascorbic)

The phenolic ellagic acid (EA) is receiving increasing attention for its nutritional and pharmacological potential as an antioxidant and antimicrobial agent. The Australian native Kakadu plum (Terminalia ferdinandiana) fruit is an abundant source of this phytochemical. The fruit also contains large amounts of vitamin C (mainly as ascorbic acid, AA) and possibly the undesirable oxalic acid (OA). Regular consumption of high oxalate foods poses a variety of health risks in humans including interference with calcium absorption and kidney stone formation. Oxalate is also the end-product of AA metabolism so that consumption of fruit with heightened AA content has the potential to elevate urinary oxalate levels. The aims of this study were to investigate the distribution of EA and the presence of other bioactives in other Kakadu plum tissues. Chemical analysis of Kakadu plum fruit and leaves for EA (free and total), OA (water-soluble and total), calcium (Ca) and AA indicated that EA and AA concentrations were high in the fruit while the leaves had significantly higher EA levels but little or no detectable AA. OA content in fruit and leaves was substantial with the fruit being placed in the high-Oxalate category. These findings suggest that there is potential to elevate oxalate levels in the urine of susceptible people and intake of fruit-derived products should be closely monitored. By measuring tissues collected from specific trees, high EA-producing or low OA-containing individuals were identified.

First report of Didymella americanaon baby lima bean (Phaseolus lunatus)

A new foliar disease was observed on baby lima bean (Phaseolus lunatus) in fields across western New York State, USA. The disease occurred in 10 fields with variable incidence and severity. Symptoms were initially necrotic, tan spots on leaves with red to reddish brown irregular margins that coalesced to encompass the entire leaf and cause abscission. Pycnidia were observed within the lesions. Isolations from diseased leaves yielded several pycnidial forming fungi, including a Didymella species. These isolates were characterized by morphology and sequencing of multiple reference genes (internal transcribed spacer (ITS), partial actin, β- tubulin (tub2), translation elongation factor 1-α (TEF), 28S rDNA large subunit (LSU), rpb2, and calmodulin). A four gene phylogeny (ITS, tub2, LSU, and rpb2) showed that the isolates from baby lima bean belonged to a well-supported clade that contained the type culture of Didymella americana. Pathogenicity of the isolates on three commonly grown cultivars of baby lima bean was confirmed. Symptoms that developed on inoculated plants were similar to those observed on diseased plants in the field. This is the first report of D. americana on baby lima bean.

Recent development and commercial adoption of legumes for heavy clay soils in Queensland

In previous chapters of this volume, various authors describe the development of herbaceous legumes for pastures on clay soils in Queensland until about the 1980s. Emphasis is on the collection and evaluation of the genus Desmanthus, given its relatively recent addition to agriculture and considerable potential for providing useful pasture legumes for clay soils, particularly in the seasonally dry areas of northern Australia. Other genera are also discussed, including early assessments of herbaceous legumes that were later developed for clay soils (Clitoria, Macroptilium and Stylosanthes). This chapter provides a summary of the development of herbaceous legumes for clay soils in Queensland from these earlier assessments until present. Beef cattle farming is the principal agricultural enterprise in seasonally dry areas of northern Australia, including large areas of clay soils in Queensland. Sown and naturally occurring grasses provide the key feed resource, and the inclusion of sown legumes can significantly improve live-weight gain and reproductive performance per unit area. Queensland has been the centre of development for legumes for clay soils in tropical and subtropical areas of Australia, mostly through assessing and developing plants held in the Australian Tropical Forages Genetic Resource Collection (ATFGRC) (now a component of the Australia Pastures Genebank (APG)). The systematic appraisal of genetic material for clay soils was a focus of well-resourced government research up to the early to mid-1990s, but declined thereafter as sown pasture research teams were dismantled and funding to maintain the ATFGRC declined. Cultivar development is now conducted by small government, private enterprise and university research teams that collaborate where possible. In recent studies the use of experienced researcher knowledge and old plant evaluation sites has been particularly valuable for identifying potentially useful material. Cultivars for long- and short-term pastures on clay soils have been developed to the level of commercial seed production for Desmanthus (five cultivars from four species with two cultivars (one composite) in current use), Clitoria ternatea (one cultivar), Macroptilium bracteatum (two) and Stylosanthes seabrana (two). Other potential cultivars of these species are currently in various stages of development. Each species has different production niches depending on climate, clay soil type and grazing strategy. Adoption of these cultivars is occurring but has variously been impeded by limited promotion, mismatch of seed supply and demand, and difficulty establishing legumes in pastures of some key grass species. Recent renewed investment by the Australian Beef Industry has seen revived government research into pasture legumes in Queensland and rejuvenation of the APG.

Performance of Sirex noctilio’s biocontrol agent Deladenus siricidicola, in known and predicted hosts

Survival of the free-living mycetophagous form of Deladenus siricidicola, the major biological control agent of Sirex woodwasp, Sirex noctilio, was tested in known (Pinus taeda) and predicted novel (P. elliottii subsp. elliottii × P. caribaea var. hondurensis) hybrid host taxa. Trials were established in the field to simulate nematode dispersal both naturally by infected wasps and following commercial inoculation, as well as in the laboratory under controlled conditions. Nematodes showed reduced survival in hybrid pine compared with P. taeda for all tree-associated treatments, but performed equivalently in petri-dish bioassays containing substrate of each taxon. Growth of Amylostereum areolatum, the food source of D. siricidicola was lower on plates containing ground hybrid substrate than on plates containing ground P. taeda. Some physical differences were found between taxa, including differences in bordered pit diameters, tracheid widths, and basic density, but these did not consistently explain reduced performance. More plant secondary compounds (predominantly oleoresins) were present in hybrid taxa than in P. taeda, and in standing trees compared with felled trees. Our results suggested that D. siricidicola may not be as effective in hybrid pine taxa for the biological control of S. noctilio as it is in its current known host taxa, possibly because of reduced growth of its food source, A. areolatum in hybrid pine.

A model of macadamia with application to pruning in orchards

A self-organising model of macadamia, expressed using L-Systems, was used to explore aspects of canopy management. A small set of parameters control the basic architecture of the model, with a high degree of self-organisation occurring to determine the fate and growth of buds. Light was sensed at the leaf level and used to represent vigour and accumulated basipetally. Buds also sensed light so as to provide demand in the subsequent redistribution of the vigour. Empirical relationships were derived from a set of 24 completely digitised trees after conversion to multiscale tree graphs (MTG) and analysis with the OpenAlea software library. The ability to write MTG files was embedded within the model so that various tree statistics could be exported for each run of the model. To explore the parameter space a series of runs was completed using a high-throughput computing platform. When combined with MTG generation and analysis with OpenAlea it provided a convenient way in which thousands of simulations could be explored. We allowed the model trees to develop using self-organisation and simulated cultural practices such as hedging, topping, removal of the leader and limb removal within a small representation of an orchard. The model provides insight into the impact of these practices on potential for growth and the light distribution within the canopy and to the orchard floor by coupling the model with a path-tracing program to simulate the light environment. The lessons learnt from this will be applied to other evergreen, tropical fruit and nut trees.

Comparison of firmness meters for measuring ‘Hass’ avocado fruit firmness

The temporal passage of fruit through the supply chain and the selection of consumable fruit by shoppers depend primarily upon fruit firmness. Traditionally, fruit firmness measuring methods, like Effegi and conical probes, are relatively inefficient and destructive. Simple, accurate and non-damaging methods of measuring fruit firmness are ideally required to help assure eating quality to the consumer without fruit wastage. The firmness of 'Hass' avocado fruit at a range of ripening stages was measured with the various different firmness measuring techniques of the Sinclair iQ Firmness Tester (SIQFT), the Electronic Firmometer (EF), the Analogue Firmness Meter (AFM) and hand squeezing. Measurements were made by each method at different points on the same fruit. Destructive bruise assessment was performed 48 h later, thereby allowing sufficient time for fruit to express any bruising resulting from the act of firmness measurements. Non-linear relationships were determined between fruit firmness values measured with the different techniques. The adjusted R2 for the relationship between the SIQFT and the EF was 91.6%. For the SIQFT and the AFM, the adjusted R2 was 73.7%. It was 77.7% for the SIQFT and hand squeezing. A significantly (P<0.05) high incidence of bruising was associated with firmness assessment by the EF as compared with either the SIQFT or the AFM. Among the methods compared, the SIQFT was non-damaging compared with the EF and relatively efficient for measuring the firmness. This instrument merits consideration as a quality control tool of choice in 'Hass' avocado supply chains.

Bioeconomic modelling of woody regrowth carbon offset options in productive grazing systems

Agricultural land has been identified as a potential source of greenhouse gas emissions offsets through biosequestration in vegetation and soil. In the extensive grazing land of Australia, landholders may participate in the Australian Government’s Emissions Reduction Fund and create offsets by reducing woody vegetation clearing and allowing native woody plant regrowth to grow. This study used bioeconomic modelling to evaluate the trade-offs between an existing central Queensland grazing operation, which has been using repeated tree clearing to maintain pasture growth, and an alternative carbon and grazing enterprise in which tree clearing is reduced and the additional carbon sequestered in trees is sold. The results showed that ceasing clearing in favour of producing offsets produces a higher net present value over 20 years than the existing cattle enterprise at carbon prices, which are close to current (2015) market levels (~$13 t–1 CO2-e). However, by modifying key variables, relative profitability did change. Sensitivity analysis evaluated key variables, which determine the relative profitability of carbon and cattle. In order of importance these were: the carbon price, the gross margin of cattle production, the severity of the tree–grass relationship, the area of regrowth retained, the age of regrowth at the start of the project, and to a lesser extent the cost of carbon project administration, compliance and monitoring. Based on the analysis, retaining regrowth to generate carbon income may be worthwhile for cattle producers in Australia, but careful consideration needs to be given to the opportunity cost of reduced cattle income.

Genetic diversity of stay-green sorghums and their derivatives revealed by microsatellites

The genetic variability of 28 sorghum genotypes of known senescence phenotype was investigated using 66 SSR markers well-distributed across the sorghum genome. The genotypes of a number of lines from breeding programmes for stay-green were also determined. This included lines selected phenotypically for stay-green and also RSG 03123, a marker-assisted backcross progeny of R16 (recurrent parent) and B35 (stay-green donor). A total of 419 alleles were detected with a mean of 6.2 per locus. The number of alleles ranged from one for Xtxp94 to 14 for Xtxp88. Chromosome SBI-10 had the highest mean number of alleles (8.33), while SBI-05 had the lowest (4.17). The PIC values obtained ranged from zero to 0.89 in Xtxp94 and Xtxp88, respectively, with a mean of 0.68. On a chromosome basis, mean PIC values were highest in SBI-10 (0.81) and lowest in SBI-05 (0.53). Most of the alleles from B35 in RSG 03123 were found on chromosomes SBI-01, SBI-02 and SBI-03, confirming the successful introgression of quantitative trait loci associated with stay-green from B35 into the senescent background R16. However, the alternative stay-green genetic sources were found to be distinct based on either all the SSRs employed or using only those associated with the stay-green trait in B35. Therefore, the physiological and biochemical basis of each stay-green source should be evaluated in order to enhance the understanding of the functioning of the trait in the various backgrounds. These genetic sources of stay-green could provide a valuable resource for improving this trait in sorghum breeding programmes.