Molecular characterisation of six badnavirus species associated with leaf streak disease of banana in East Africa

Banana leaf streak disease, caused by several species of Banana streak virus (BSV), is widespread in East Africa. We surveyed for this disease in Uganda and Kenya, and used rolling-circle amplification (RCA) to detect the presence of BSV in banana. Six distinct badnavirus sequences, three from Uganda and three from Kenya, were amplified for which only partial sequences were previously available. The complete genomes were sequenced and characterised. The size and organisation of all six sequences was characteristic of other badnaviruses, including conserved functional domains present in the putative polyprotein encoded by open reading frame (ORF) 3. Based on nucleotide sequence analysis within the reverse transcriptase/ribonuclease H-coding region of open reading frame 3, we propose that these sequences be recognised as six new species and be designated as Banana streak UA virus, Banana streak UI virus, Banana streak UL virus, Banana streak UM virus, Banana streak CA virus and Banana streak IM virus. Using PCR and species-specific primers to test for the presence of integrated sequences, we demonstrated that sequences with high similarity to BSIMV only were present in several banana cultivars which had tested negative for episomal BSV sequences.

Development of a novel rolling-circle amplification technique to detect Banana streak virus which also discriminates between integrated and episomal virus sequences

Bananas are hosts to a large number of banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity amongst BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence non-specific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterisation of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal and/or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV) and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV) and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.

Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

Frequency response function based structural damage detection using artificial neural networks

Damage detection in structures has become increasingly important in recent years. While a number of damage detection and localization methods have been proposed, few attempts have been made to explore the structure damage with frequency response functions (FRFs). This paper illustrates the damage identification and condition assessment of a beam structure using a new frequency response functions (FRFs) based damage index and Artificial Neural Networks (ANNs). In practice, usage of all available FRF data as an input to artificial neural networks makes the training and convergence impossible. Therefore one of the data reduction techniques Principal Component Analysis (PCA) is introduced in the algorithm. In the proposed procedure, a large set of FRFs are divided into sub-sets in order to find the damage indices for different frequency points of different damage scenarios. The basic idea of this method is to establish features of damaged structure using FRFs from different measurement points of different sub-sets of intact structure. Then using these features, damage indices of different damage cases of the structure are identified after reconstructing of available FRF data using PCA. The obtained damage indices corresponding to different damage locations and severities are introduced as input variable to developed artificial neural networks. Finally, the effectiveness of the proposed method is illustrated and validated by using the finite element modal of a beam structure. The illustrated results show that the PCA based damage index is suitable and effective for structural damage detection and condition assessment of building structures.

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy-transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Bananas are a major commercial crop and serve as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrate that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

The acquisition of an artificial logographic script and bilingual working memory : Evidence for L1-specific orthographic processing skills transfer in Chinese-English bilinguals

Studies of orthographic skills transfer between languages focus mostly on working memory (WM) ability in alphabetic first language (L1) speakers when learning another, often alphabetically congruent, language. We report two studies that, instead, explored the transferability of L1 orthographic processing skills in WM in logographic-L1 and alphabetic-L1 speakers. English-French bilingual and English monolingual (alphabetic-L1) speakers, and Chinese-English (logographic-L1) speakers, learned a set of artificial logographs and associated meanings (Study 1). The logographs were used in WM tasks with and without concurrent articulatory or visuo-spatial suppression. The logographic-L1 bilinguals were markedly less affected by articulatory suppression than alphabetic-L1 monolinguals (who did not differ from their bilingual peers). Bilinguals overall were less affected by spatial interference, reflecting superior phonological processing skills or, conceivably, greater executive control. A comparison of span sizes for meaningful and meaningless logographs (Study 2) replicated these findings. However, the logographic-L1 bilinguals’ spans in L1 were measurably greater than those of their alphabetic-L1 (bilingual and monolingual) peers; a finding unaccounted for by faster articulation rates or differences in general intelligence. The overall pattern of results suggests an advantage (possibly perceptual) for logographic-L1 speakers, over and above the bilingual advantage also seen elsewhere in third language (L3) acquisition.