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An unknown sclerotium-forming fungus was observed and isolated in 2016 from sugarcane grown at Sugar Research Australia, Woodford Pathology farm, Woodford, Queensland. The fungus appeared to be causing germination failure of sugarcane setts. The fungus was isolated from infected tissues and mycelia from internal pithy sett tissues. White mycelia and abundant sclerotia were grown on PDA. Morphological observation and molecular analysis confirmed the identification of the fungus as Athelia rolfsii. Significant reductions in sett germination (>70%) were observed in the field when inoculated setts were planted. Reddish and patchy lesions were observed on the external surface of the setts and pale-red to red discolouration in internal tissues (associated with white mycelial growth in the pithy tissues and on the cut end of setts). Symptoms on young plants included water-soaked to light-brown lesions at their base, production of sclerotia / white mycelium in and around the lesions, and death of the young plants. This is the first report demonstrating that A. rolfsii can cause germination failure and young plant death in sugarcane.

Citrus black spot, caused by

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.

Assessing storage impacts on manure properties is relevant to research associated with nutrient-use efficiency and greenhouse gas (GHG) emissions. We examined the impact of cold storage on physicochemical properties, biochemical methane-emitting potential (BMP) and the composition of microbial communities of beef feedlot manure and poultry broiler litter. Manures were analysed within 2 days of collection and after 2 and 8 weeks in refrigerated (4 °C) or frozen (–20 °C) storage. Compared with fresh manure, stored manures had statistically significant (p 0.05) BMP in both manure types. We did not detect ammonium- or nitrite-oxidising bacterial taxa (AOB, NOB) using fluorescence in situ hybridisation (FISH). Importantly, the viability of microbes was unchanged by storage. We conclude that storage at –20 °C or 4 °C adequately preserves the investigated traits of the studied manures for research aimed at improving nutrient cycling and reducing GHG emissions.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.

Phosphorus (P) enters roots as inorganic phosphate (Pi) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into Pi by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to Pi-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with Pi only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of Pi supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.

Sugarcane is a crop with great potential for metabolic engineering, but progress has been limited by highly efficient transgene silencing. The potential exists to utilize efficient gene silencing in molecular improvement through down-regulation of sugarcane genes. However, sugarcane is highly polyploid and heterozygous, which might complicate efforts to employ transgene-mediated silencing of endogenous genes. To explore this issue, we tested in sugarcane a construct designed for hairpin-mediated silencing of the phytoene desaturase (PDS) gene in sugarcane. The hairpin construct was designed based on the sugarcane PDS EST collection containing multiple alleles, to down-regulate the suite of PDS alleles. Three out of four plants transformed using the PDS hairpin construct showed detectable hairpin transcripts when analyzed by northern analysis, and displayed the photo-bleaching phenotype characteristic of PDS knockout lines. There was near-complete reduction in the levels of endogenous PDS transcripts in leaf tissue, indicating efficient hairpin-mediated down-regulation despite the highly polyploid and heterozygous sugarcane genome. Hairpin-mediated gene silencing should therefore be a powerful tool for the molecular improvement of this important crop.

Sorghum grain provides an important calorific source for millions of people living in developing countries and is a principal animal feed and source of gluten-free flour for the livestock and food processing industries of developed nations. A versatile grain, sorghum is also widely utilized in the production of alcoholic beverages in countries such as China and several countries in sub-Saharan Africa, where the liquor baijiu and beer are a major end-use, respectively. Renowned as a hardy crop, sorghum is relatively drought tolerant and can be grown on marginal lands and is adaptable to a wide range of environmental conditions, giving this species particular advantages over other cereals. Despite its inherent benefits, sorghum has not proven to be a major alternative to the other notable cereals such as wheat and maize, due to significant problems concerning the low amount of specific essential amino acids, for example, lysine, lower protein content, lower starch digestibility, and smaller grain size, which has implications for the traits mentioned above as well as acting as an impediment to efficient grain handling in cereal-processing industries. The challenges in enhancing sorghum grain quality are not insurmountable and great strides have already been achieved in a relatively short time via scientific breeding to enhance grain yield and provide abiotic and biotic stress resistance. As the sorghum market has matured, demand for higher quality grain, whether for alcohol production or animal and human consumption, is increasing. Although yield and disease resistance are still the primary focus of breeders, advances in genomics, online bioinformatic data repositories, high-throughput phenotypic screening such as near-infrared reflectance (NIR), and the increasing affordability of next-generation sequencing, have allowed breeders to incorporate improved grain quality parameters into their programs. This chapter elaborates recent advances in genomics that have provided researchers with the tools to solve several of the issues surrounding grain quality in sorghum as well as future directions for experimentation.

The efficient use of transgenic technology for the improvement of phosphorus (P) nutrition in crop plants may require the expression of introduced genes only under conditions of phosphate deficiency and only in specific tissues. We have investigated the use of the promoter from the Arabidopsis Pht1;2 phosphate transporter to drive the expression of a secretable Aspergillus niger phytase gene only in the root epidermis of phosphate-deprived plants. Deletion analysis of this promoter indicated that, while the 933 bp upstream of the start codon is sufficient for root-specific and phosphate-responsive reporter gene expression, the 2000 bp upstream of the start codon are required for maximum expression levels. We show that transgenic Arabidopsis plants in which this 2000 bp promoter drives the Aspergillus phytase gene secrete phytase enzyme only from roots when grown on medium containing low phosphate concentrations, and that this transgene enables the plants to grow on medium containing phytate as a sole P source. The growth rates and shoot P concentrations of these plants were similar when grown on phytate or phosphate as the P source, and were similar to transgenic lines in which the phytase was driven by the constitutive CaMV 35S promoter. Our results demonstrate the potential usefulness of the promoter from the Pht1;2 phosphate transporter for driving genes that may improve crop P uptake and nutrition.

Transport of inorganic phosphate (Pi) through plant membranes is mediated by a number of families of transporter proteins. Studies on the topology, function, regulation and sites of expression of the genes that encode the members of these transporter families are enabling roles to be ascribed to each of them. The Pht1 family, of which there are nine members in the Arabidopsis genome, includes proteins involved in the uptake of Pi from the soil solution and the redistribution of Pi within the plant. Members of this family are H2PO4−/H+ symporters. Most of the genes of the Pht1 family that are expressed in roots are up-regulated in P-stressed plants. Two members of the Pht1 family have been isolated from the cluster roots of white lupin. These same genes are expressed in non-cluster roots. The evidence available to date suggests that there are no major differences between the types of transport systems that cluster roots and non-cluster roots use to acquire Pi. Differences in uptake rates between cluster and non-cluster roots can be ascribed to more high-affinity Pi transporters in the plasma membranes of cluster roots, rather than any difference in the characteristics of the transporters. The efficient acquisition of Pi by cluster roots arises primarily from their capacity to increase the availability of soil Pi immediately adjacent to the rootlets by excretion of carboxylates, protons and phosphatases within the cluster. This paper reviews Pi transport processes, concentrating on those mediated by the Pht1 family of transporters, and attempts to relate those processes involved in Pi acquisition to likely Pi transport processes in cluster roots.

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes beta-glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT-PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

This chapter contains sections titled: - Incidence and Practical Significance of Transgene Silencing - Factors Influencing Transgene Silencing - Box 12.1. An RNAi primer - Mechanisms of Transgene Silencing - Strategies to Avoid Transgene Silencing - Conclusions and Future Prospects - Box 12.2. Avoiding transgene silencing.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes beta-glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT-PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.

We have evaluated T-DNA mediated plant promoter tagging, with a left-border-linked promoterless firefly luciferase (luc) construct, as a strategy for the isolation of novel plant promoters. In a population of approximately 300 transformed tobacco plants, 10 lines showed LUC activity, including novel tissue-specific and developmental patterns of expression. One line, showing LUC activity only in the shoot and root apical meristems, was further characterised. Inverse PCR was used to amplify a 1.5 kb fragment of plant DNA flanking the single-copy T-DNA insertion in this line. With the exception of a 249 bp highly repetitive element, this sequence is present as a single copy in the tobacco genome, and is not homologous to any previously characterised DNA sequences. Sequence analysis revealed the presence of several motifs that may be involved in transcriptional regulation. Transgenic tobacco plants transformed with a transcriptional fusion of this putative promoter sequence to the β-glucuronidase (uidA) reporter gene, showed GUS activity confined to the shoot tip and mature pollen. This promoter may be useful to direct the expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

Citrus black spot, caused by Phyllosticta citricarpa, is characterized by fruit blemishes and premature fruit drop, resulting in significant economic losses in summer rainfall areas. The pathogen forms both conidia and ascospores during its life cycle. However, the occurrence of these spores and their contributions to infection of fruit in field conditions are not well understood. Our research using direct leaf litter monitoring and volumetric spore trapping in Queensland orchards revealed that pseudothecia and ascospores in leaf litter as well as trapped ascospores had low abundance, while pycnidia and conidia were highly abundant. Both P. citricarpa and endophytic Phyllosticta spp. were identified, with P. citricarpa being dominant. In replicated field trials, we determined that infection of Imperial mandarin fruit by P. citricarpa occurred from fruit set until week 20 of fruit development, with the key infection events taking place between weeks 4 and 16 in Queensland subtropical conditions. These results demonstrate that protecting fruit during weeks 4 to 16 significantly reduced P. citricarpa infection. We found no significant correlation between the disease incidence in fruit and P. citricarpa conidial abundance in leaf litter or ascospore abundance measured by volumetric spore trapping. Therefore, it is suggested that inoculum sources in the tree canopy other than those detected by spore trapping and direct leaf litter monitoring may play a major role in the epidemiology of citrus black spot. Improved knowledge regarding epidemiology of P. citricarpa and an understanding of propagules causing infection may aid in development of more effective disease management strategies.