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Sweet potato collusive virus (SPCV) is a member of the genus Cavemovirus, family Caulimoviridae, for which only one full-length genome sequence has been reported. SPCV was first detected in Australia in 2007 in two sweet potato accessions using the nitrocellulose membrane ELISA kit developed by the International Potato Centre (CIP). Infected plants were also shown to contain isometric virions of ~ 50 nm, typical of members of the genera Cavemovirus, Caulimovirus, Petuvirus and Soymovirus. We have now sequenced and characterised the complete genomes of the two SPCV isolates (designated SPCV-Aus1 and -Aus2) using a combination of next-generation sequencing and PCR/Sanger sequencing. The sequences of both isolates encode three major ORFs with a genome organisation typical of cavemoviruses. However, isolate SPCV-Aus2 possesses a considerably shorter genome length of 7275 bp compared to SPCV-Aus1 (7712 bp) and the only available full-length sequence from a Portuguese isolate (7723 bp; GenBank accession number NC_015328). Further, ORF 1 of SPCV-Aus2 is considerably shorter than the ORF 1 length of both SPCV-Aus1 and SPCV-Mad1. Phylogenetic and PASC analysis showed that SPCV-Aus1 is closely related to SPCV isolates from North and Central America, whereas SPCV-Aus2 clustered together with isolates from Portugal and Africa.

Yam is an important food staple in many Pacific Island countries, but its production is limited by several factors including virus infection. The presence of viruses also hinders the exploitation of the crop by restricting the international dissemination of yam germplasm due to quarantine concerns. In this study, a total of 224 yam accessions present in the Pacific germplasm collection at SPC-CePaCT, Fiji, were tested for the presence of badnaviruses using a combination of rolling circle amplification (RCA) and sequencing. Of these, 35 tested positive for either Dioscorea bacilliform AL virus (DBALV) or Dioscorea bacilliform AL virus 2 (DBALV2). DBALV was only detected in samples from Vanuatu and Tonga, while DBALV2 was restricted to Papua New Guinean samples. Ten complete genome sequences of DBALV and DBALV2 were obtained and pairwise comparison of the partial reverse transcriptase/ribonuclease H (RT/RNase H)-coding sequence revealed identities ranging from 89% to 100% and 92% to 100%, respectively. The results suggest that DBALV and DBALV2 are the prevalent badnavirus species infecting yams in the Pacific region.

Bananas and plantains (Musa spp.) are an important staple and food security crop in sub-Saharan Africa. In Uganda, where the consumption of East African highland banana (EAHB) is the highest in the world, the population suffers from a high incidence of vitamin A deficiency (VAD). Since the consumption of pro-vitamin A carotenoids (pVAC) made available through the food staple can help alleviate these ailments, we set out to identify the most suitable banana variety to use in future biofortification strategies through genetic engineering. The study focussed on eight popular Musa cultivars grown in the heart of banana farming communities and across the three major agricultural zones of Uganda. The fruit pVAC concentration varied considerably within and across the cultivars tested. These variations could not be explained by the altitude nor the geographical location where these fruits were grown. More than 50% of the total carotenoids present in EAHB cultivars was found to comprise of α- and β-carotene, while the retention of these compounds following traditional processing methods was at least 70%. Storage up to 14 days post-harvest improved carotenoid accumulation up to 2.4-fold in the cultivar Nakitembe. The technical challenge for a successful biofortification approach in Uganda using genetically modified EAHB lies in guaranteeing that the fruit pVAC content will invariably provide at least 50% of the estimated average requirement for vitamin A regardless of the growing conditions.

Three tannia (Xanthosoma sp.) plants showing mosaic, mottling and vein chlorosis symptoms were observed in Buikwe district, Uganda. Next generation sequencing (NGS) of RNA extracted from the three plants revealed the presence of Cucumber mosaic virus (CMV). The complete genome sequence of this isolate, designated CMV-Xa, was assembled and sequence analyses based on the three genomic RNAs revealed that CMV–Xa belongs to subgroup IB. This is the first report of CMV infecting Xanthosoma sp. and also the first CMV isolate from subgroup IB detected from sub-Saharan Africa.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9–1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0–1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Although cowpea is an important food security crop in Africa, its production is constrained by insect pests such as the legume pod borer (Maruca vitrata) (Lepidoptera). Potential control strategies have focussed on using insecticidal toxins such as the crystal Cry proteins and vegetative insecticidal proteins (Vips) encoded by the cry and vip genes, respectively, of the bacterium Bacillus thuringiensis (Bt). This study sought to identify vip genes encoding toxins active against Maruca Pod Borer (MPB), from Australian Bt isolates. A collection of 224 Bt isolates was screened with gene-specific primers to identify those containing target vip genes namely vip3Aa35, vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1. The coding sequences of the vip3 genes were cloned and over-expressed in Escherichia coli to produce Vip3 protein. The proteins were incorporated into Maruca artificial diets for use in insect bioassays with MPB larvae to screen for toxicity. Of these, Vip3Ba1 protein was found to strongly inhibit larval growth and was selected as the candidate gene for cowpea transformation. A vip3Ba gene reconstructed for plant expression was used in transforming cowpea via Agrobacterium tumefaciens. Transgenic lines expressing Vip3Ba protein were used in insect feeding trials to assess protection against Maruca and were found to be completely protected from this pest. We propose that the vip-cowpea lines could be combined with existing cry-transgenic cowpea to introgress this additional resistance trait and thus avoid or greatly delay the development of resistance in Maruca.

Summary Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum‐free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol‐inducible AlcR:alcA gene switch. hVN expression was maximal 4–5 days postactivation of the INPACT platform with a dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of 643 ± 78 mg/kg fresh weight. A three‐stage purification protocol was developed using heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant‐made hVN product of >90% purity. Storage conditions for plant‐made hVN were identified that maximized the capacity of the recombinant protein to promote cell adhesion. Critically, plant‐made hVN was shown to be functionally equivalent to commercial, plasma‐derived hVN at promoting one‐half maximal attachment of murine fibroblast cells (BALB‐C/3T3) in serum‐free medium at

The complete genome sequences of three new badnaviruses associated with yam (Dioscorea spp.) originating from Fiji, Papua New Guinea and Samoa were determined following rolling circle amplification of the virus genomes. The full-length genomes consisted of a single molecule of circular double-stranded DNA of 8106 bp for isolate FJ14, 7871 bp for isolate PNG10 and 7426 bp for isolate SAM01. FJ14 and PNG10 contained three open reading frames while SAM01 had an additional open reading frame which partially overlapped the 3′ end of ORF 3. Amino acid sequence analysis of ORF 3 from the three isolates confirmed the presence of conserved motifs typical of other badnaviruses. Phylogenetic analysis revealed the sequences to be closely related to other Dioscorea–infecting badnaviruses. FJ14 and PNG10 appear to be new species, which we have tentatively named dioscorea bacilliform ES virus (DBESV) and dioscorea bacilliform AL virus 2 (DBALV2), respectively, while SAM01 represents a Pacific isolate of the recently published dioscorea bacilliform RT virus 2 and is described as dioscorea bacilliform RT virus 2-[4RT] (DBRTV2-[4RT]).

Summary Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β-carotene equivalent (β-CE) in the fruit. Expression of a Fe'i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop “Golden Rice 2”, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic pathway, as well as extended fruit maturation time, are essential factors to achieve optimal PVA concentrations in banana fruit.

Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with “clean” single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.

An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into the banana genome.

The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMV-AU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.

Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the β-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism.

The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.

Taro (

Background Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis. Results Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1–2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform. Conclusions In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

Uganda is a tropical country with a population in excess of 30 million, >80% of whom live in rural areas. Bananas (Musa spp.) are the staple food of Uganda with the East African Highland banana, a cooking banana, the primary starch source. Unfortunately, these bananas are low in pro-vitamin A (PVA) and iron and, as a result, banana-based diets are low in these micronutrients which results in very high levels of inadequate nutrition. This inadequate nutrition manifests as high levels of vitamin A deficiency, iron deficiency anemia, and stunting in children. A project known as Banana21 commenced in 2005 to alleviate micronutrient deficiencies in Uganda and surrounding countries through the generation of farmer- and consumer-acceptable edible bananas with significantly increased fruit levels of PVA and iron. A genetic modification approach was adopted since bananas are recalcitrant to conventional breeding. In this review, we focus on the PVA-biofortification component of the Banana21 project and describe the proof-of-concept studies conducted in Australia, the transfer of the technology to our Ugandan collaborators, and the successful implementation of the strategy into the field in Uganda. The many challenges encountered and the potential future obstacles to the practical exploitation of PVA-enhanced bananas in Uganda are discussed.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

International audience; In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance., The newly recognized Fusarium wilt pathogen tropical race 4 is threatening worldwide banana production. Here, the authors transform Cavendish bananas with a resistance gene, RGA2, from diploid banana or a nematode-derived gene, Ced9, and confer resistance to natural infection under field conditions.

Many protocols have been used for extraction of DNA from Thraustochytrids. These generally involve the use of CTAB, phenol/chloroform and ethanol. They also feature mechanical grinding, sonication, N2 freezing or bead beating. However, the resulting chemical and physical damage to extracted DNA reduces its quality. The methods are also unsuitable for large numbers of samples. Commercially-available DNA extraction kits give better quality and yields but are expensive. Therefore, an optimized DNA extraction protocol was developed which is suitable for Thraustochytrids to both minimise expensive and time-consuming steps prior to DNA extraction and also to improve the yield. The most effective method is a combination of single bead in TissueLyser (Qiagen) and Proteinase K. Results were conclusive: both the quality and the yield of extracted DNA were higher than with any other method giving an average yield of 8.5 µg/100 mg biomass.

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

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. Banana is a major commercial crop and serves 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 demonstrates 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.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweetpotato, Ipomoea batatas, inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field-derived and pathogen-tested (PT) clones of 14 sweetpotato cultivars and the yield benefits of using healthy planting materials. The field-derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem-tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane-enzyme-linked immunosorbent assay and graft- inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study. Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus-susceptible commercial cultivars.

Banana plants 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 among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization 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 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.

There is a paucity of data on the distribution of Cicadellidae (leafhoppers) in Australia. This study quantifies the relative abundance, seasonal activity and diversity of leafhoppers in the Ovens Valley region of north-east Victoria, Australia. Species diversity and abundance was assessed at four field sites in and around the field borders of commercially grown tobacco crops using three sampling techniques (pan trap, sticky trap and sweep net). Over 51 000 leafhopper samples were collected, with 57 species from 11 subfamilies and 19 tribes identified. Greater numbers and diversity of leafhoppers were collected in yellow pan traps. The predominant leafhopper collected was Orosius orientalis (Matsumura). Twenty-three leafhopper species were recorded for the first time in Victoria and eight economically important pest species were recorded. Seasonal activity of selected leafhopper species, covering two sampling seasons, is presented.

Factors that determine the epidemiology of Tobacco yellow dwarf virus (TbYDV), including alternative host plants and insect vector(s), were assessed over three consecutive growing seasons at four field sites in Northeastern Victoria in commercial tobacco growing properties. In addition, these factors were assessed for one growing season at three bean growing properties. Overall, 23 leafhopper species were identified at the 7 sites, with Orosius orientalis as the predominant leafhopper. Of the leafhoppers collected, only O. orientalis and Anzygina zealandica tested positive for TbYDV by polymerase chain reaction (PCR). The population dynamics of O. orientalis was assessed using sweep net sampling over three growing seasons and a trimodal distribution was observed. Despite large numbers of O. orientalis occurring early in the growing season (September–October), TbYDV was only detected in these leafhoppers between late November and end of January. The peaks in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and were associated with warmer temperatures and lower rainfall. Spatial and temporal distribution of vegetation at selected sites was determined using quadrat sampling. Of the 40 plant species identified, TbYDV was detected only in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. The proportion of host and non-host availability for leafhoppers was associated with climatic conditions.

Orosius orientalis is a leafhopper vector of several viruses and phytoplasmas affecting a broad range of agricultural crops. Sweep net, yellow pan trap and yellow sticky trap collection techniques were evaluated. Seasonal distribution of O. orientalis was surveyed over two successive growing seasons around the borders of commercially grown tobacco crops. Orosius orientalis seasonal activity as assessed using pan and sticky traps was characterised by a trimodal peak and relative abundance as assessed using sweep nets differed between field sites with peak activity occurring in spring and summer months. Yellow pan traps consistently trapped a higher number of O. orientalis than yellow sticky traps.

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4-D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settled cell volume while approximately 60% of the embryos regenerated into plants.

Callus was initiated in three different “esculenta” taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and ~72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.

The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls, 1956), tomato big bud (Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Harding & Teakle, 1985), and Australian lucerne yellows (Pilkington et al., 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne, 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill, 1937, 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne, 1968; Thomas, 1979; Moran & Rodoni, 1999). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr E bicki, unpubl.).

The fungus Fusarium oxysporum is responsible for causing a destructive wilting disease in numerous crops including banana. To date, only two non-TIR-NBS-LRR genes that confer resistance to F. oxysporum, the I2 and Fom-2 genes from tomato and melon, respectively, have been isolated. We previously identified two partial non-TIR-NBS sequences, designated RGC2 and RGC5, from FOC race 4 resistant banana, Musa acuminata ssp. malaccensis. In the present study, we have isolated and characterized the entire cDNA sequences of RGC2 and RGC5 from the root transcriptome of M. acuminata ssp. malaccensis. The open reading frames (ORFs) of both genes were predicted to encode the typical structure of non-TIR-NBS-LRR proteins. Similarity searches using the entire ORF of RGC2 and RGC5 revealed considerable sequence similarity to the I2 gene. Further, phylogenetic analyses showed that the banana RGC2 and RGC5 clustered within the ancient non-TIR clade N1.1. Interestingly, although phylogenetically distantly related, the known Fusarium R genes I2 and Fom-2 also grouped within the clade N1.1. The fact that the I2 and Fom-2 genes cluster in the same phylogenetic clade N1.1 opens the possibility that other unknown Fusarium R genes from different plant families may share the same clade. These findings may assist future attempts to identify Fusarium R genes in banana and other plants.

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

The death of plant cells in culture following exposure to Agrobacterium tumefaciens remains a major obstacle in developing Agrobacterium-mediated transformation into a highly efficient genotype-independent technology. Here, we present evidence that A. tumefaciens exposure induces cell death in banana cell suspensions. More than 90% of embryogenic banana cells died after exposure to A. tumefaciens and cell death was accompanied by a subset of features associated with apoptosis in mammalian cells, including DNA laddering, fragmentation, and formation of apoptoticlike bodies. Importantly, these cellular responses were inhibited in cells expressing the animal antiapoptosis genes Bcl-xL, Bcl-2 3′ untranslated region, and CED-9. Inhibition of cell death resulted in up to 90% of cell clumps transformed with Bcl-xL, a 100-fold enhancement over vector controls, approaching the transformation and regeneration of every “transformable” cell. Similar results using sugarcane, a crop plant known for recalcitrance to Agrobacterium transformation, suggest that antiapoptosis genes may inhibit these phenomena and increase the transformation frequency of many recalcitrant plant species, including the major monocot cereal crop plants. Evidence of inhibition of plant cell death by cross-kingdom antiapoptotic genes also contributes to the growing evidence that genes for control of programmed cell death are conserved across wide evolutionary distances, even though these mechanisms are not well understood in plants. Additional keywords: necrosis.

We report the first genome sequence of a Colocasia bobone disease-associated virus (CBDaV) derived from bobone-affected taro [Colocasia esculenta L. Schott] from Solomon Islands. The negative-strand RNA genome is 12,193 nt long, with six major open reading frames (ORFs) with the arrangement 3′-N-P-P3-M-G-L-5′. Typical of all rhabdoviruses, the 3′ leader and 5′ trailer sequences show complementarity to each other. Phylogenetic analysis indicated that CBDaV is a member of the genus Cytorhabdovirus, supporting previous reports of virus particles within the cytoplasm of bobone-infected taro cells. The availability of the CBDaV genome sequence now makes it possible to assess the role of this virus in bobone, and possibly alomae disease of taro and confirm that this sequence is that of Colocasia bobone disease virus (CBDV).

Banana bunchy top virus (BBTV), a multi-component circular ssDNA virus, replicates via a dsDNA intermediate that also serves as a template for virion sense transcription. Seven virus-derived transcripts have been previously identified and analysed in BBTV-infected bananas by northern analysis and 3' rapid amplification of cDNA ends (3' RACE). In this study, we have used RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-RACE) to complete the mapping of the BBTV gene transcripts and have now fully mapped the transcribed regions of each BBTV component and effectively defined the upstream regulatory region.

Banana bunchy top virus(BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV ‘master’ Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.

The complete nucleotide sequences of Fiji disease virus (FDV) genome segments S2, S4 and S7 were determined. This now completes the sequencing of all ten dsRNA genome segments of the Fijivirus type member, FDV, which comprises a total of 29339 nt. FDV S2, S4 and S7 comprised 3820, 3568 and 2194 nt, respectively. S2 and S4 each contained a single open reading frame (ORF), which encoded putative proteins of 137 and 133 kDa, respectively, while S7 contained two ORFs, which encoded putative proteins of 42 and 37 kDa. The putative amino acid sequences of FDV S2 and S4 showed most similarity to the gene products of Rice black-streaked dwarf virus (RBSDV) S2 and RBSDV S3, respectively. The putative amino acid sequences of FDV S7 ORF I and II showed most similarity to Maize rough dwarf virus (MRDV) S6 ORF I and RBSDV S7 ORF II, respectively. Phylogenetic analyses showed that FDV was most closely related to the group 2 fijiviruses.

Sequencing of the monopartite RNA genome of a Fijian isolate of Taro vein chlorosis virus (TaVCV) confirmed that it is a definitive rhabdovirus with most similarity to members of the genus Nucleorhabdovirus. The TaVCV 12 020 nt negative-sense RNA genome contained six ORFs in the antigenomic sequence, equivalent to the N, P, 3, M, G and L genes that have been identified in other rhabdoviruses. The putative gene products had highest similarity to those of the nucleorhabdovirus Maize mosaic virus. A characteristic 3′-AAUUCUUUUUGGGUUGU/A-5′ sequence was identified in each of the intergenic regions and the TaVCV leader and trailer sequences comprised 140 and 61 nt, respectively. Assignment of TaVCV to the genus Nucleorhabdovirus was supported by thin-section electron microscopy of TaVCV-infected taro leaves, which identified virions budding from nuclear membranes into the perinuclear space. Variability studies identified high levels of TaVCV sequence diversity. Within the L gene of 20 TaVCV isolates from Fiji, the Federated States of Micronesia, New Caledonia, Papua New Guinea, Solomon Islands and Vanuatu, maximum variability at the nucleotide level was 27·4 %. Within the N gene, maximum variability among 15 isolates at the nucleotide level was 19·3 %. The high level of TaVCV variability observed suggested that the introduction of TaVCV to the Pacific Islands was not a recent occurrence.

We report the first genome sequence of a Colocasia bobone disease-associated virus (CBDaV) derived from bobone-affected taro [Colocasia esculenta L. Schott] from Solomon Islands. The negative-strand RNA genome is 12,193 nt long, with six major open reading frames (ORFs) with the arrangement 3′-N-P-P3-M-G-L-5′. Typical of all rhabdoviruses, the 3′ leader and 5′ trailer sequences show complementarity to each other. Phylogenetic analysis indicated that CBDaV is a member of the genus Cytorhabdovirus, supporting previous reports of virus particles within the cytoplasm of bobone-infected taro cells. The availability of the CBDaV genome sequence now makes it possible to assess the role of this virus in bobone, and possibly alomae disease of taro and confirm that this sequence is that of Colocasia bobone disease virus (CBDV).

The genome of an Australian isolate of Sugarcane bacilliform virus (SCBV-IM) was cloned, sequenced and analysed. The genome consisted of 7687 nucleotides and contained three open reading frames which were similar in size and organisation to those of other badnaviruses. SCBV-IM was found to be most similar to the SCBV-Morocco isolate with amino acid sequence similarity of 91.4 %, 83.8 % and 85.3 % in the ORF I, II and III coding regions, respectively. Phylogenetic analysis of the SCBV-IM ORF III deduced amino acid sequence showed that SCBV isolates were more closely related to each other than to other badnaviruses. Amplification of SCBV sequences from three different sugarcane varieties revealed considerable variability in the viral populations, both within single infected plants as well as between infected plants, suggesting that the SCBV isolates sequenced to date may not be representative of the range of virus variability.

Bananas are one of the top ten world food crops. Unlike most other major food crops, bananas are difficult to genetically improve. The challenge is that nearly all banana cultivars and landraces are triploids, with high levels of male and female infertility. There are a number of international conventional breeding programs and many of these are developing new cultivars. However, it is virtually impossible to backcross bananas, thus excluding the possibility of introgressing new traits into a current cultivar. The alternative strategy is to “modify” the cultivar itself. We have been developing the capacity to modify Cavendish bananas and other cultivars for both disease resistance and enhanced fruit quality. Initially, we were using transgenes; genes that were derived from species outside of the Musa or banana genus. However, we have recently incorporated two banana genes (cisgenes) into Cavendish; one to enhance the level of pro-vitamin A and the other to increase the resistance to Panama disease. Modified Cavendish with these cisgenes have been employed in a field trial. Almost certainly, the next advance will be to edit the Cavendish genome, to generate the desired traits. As these banana cultivars are essentially sterile, transgene flow and the outcrossing of modified genes into wild Musa species. are highly unlikely and virtually impossible in other triploid cultivars. Therefore, genetic changes in bananas may be compatible with organic farming.

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.

We have isolated a banana actin (ACT1) gene and characterised its expression levels, tissue-specificity and upstream regulatory sequences in banana. The ACT1 promoter (2.2 kbp) was isolated by a combination of ligation-mediated polymerase chain reaction and a method for amplifying flanking sequences. A single transcriptional start site was mapped to within 215 bp of the ATG, within which was located a large intron of 840 bp with almost consensus monocot 5′ and 3′ splice recognition sites. The ACT1 gene was amplified from genomic DNA, and the deduced amino acid sequence showed the highest levels of similarity to vegetatively expressed plant actins. Northern analysis indicated that the ACT1 transcript was present in all tissues examined (petals/sepals, stigma/stamens, leaves and roots). Expression studies in transgenic banana plants showed the ACT1 promoter drove strong reporter gene expression in both leaves and roots. Truncations of the ACT1 promoter suggested that all of the necessary regulatory elements required for strong (twofold greater than CaMV 35S) near-constitutive expression were located within 1.2 kbp of the ACT1 ATG.

Intron-containing fragments derived from the 5′ untranslated regions of the maize ubi1, maize adh1, rice act1 and sugarcane rbcS genes were tested for their enhancing effects on the banana bunchy top virus DNA-6 promoter (BT6.1) in banana (Musa spp. cv. Bluggoe) embryogenic cells. The rice act1 and maize ubi1 introns provided the highest levels of intron-mediated enhancement of GUS expression, increasing native BT6.1 promoter activity by about 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about tenfold, whereas the adh1 intron had no significant effect. In regenerated transgenic banana plants, the ubi1 intron significantly enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35 S promoter and did not appear to affect the tissue specificity of the promoter.