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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.

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.

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.

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.

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.

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.

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.