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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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants.

We have identified banana bract mosaic potyvirus (BBMV) in banana plants growing in the Coimbatore and Tiruchchirappalli regions of southern India based on symptomatology, particle morphology, sequence homology, and nucleic acid hybridization assays. Potyvirus-like particles typical of BBMV also were detected in sap dips from banana plants growing in Maharashtra State. Sequence comparisons of the C terminus of the coat protein-coding and 3′ untranslated regions revealed that the Indian isolates of BBMV had greater than 96.6 and 97.2% homology with a Philippines isolate at the nucleotide and amino acid levels, respectively. BBMV-infected banana cultivars from the Coimbatore region showed the characteristic mosaic on the bract of the banana inflorescence. In contrast, infected plants growing in the Tiruchchirappalli region and Maharashtra State displayed symptoms similar to those associated with cucumber mosaic cucumovirus and not the characteristic bract mosaic symptom. These results indicate that BBMV is more widespread than previously thought.

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro (Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNAmet-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near constitutive expression of either the green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter gene (uidA) in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters (Ubi-1). In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four and ten-fold lower than that of the double Cauliflower mosaic virus (CaMV) 35S (D35S) promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

Investigations were conducted into the transmission of Taro bacilliform badnavirus (TaBV) by seed and mealybugs. Seed transmission was investigated by artificially pollinating TaBV-infected taro. Seeds derived from four successfully pollinated plants tested positive for TaBV by PCR. Twenty seeds derived from each pollinated plant were also germinated; 2/80 seedlings showed TaBV-like symptoms and tested positive for TaBV by PCR. Pollen samples taken from TaBV-infected plants also tested positive for the virus. Mealybug transmission was investigated by exposing 51 healthy taro plants to Pseudococcus solomonensis that had been reared on TaBV-infected taro plants. Typical virus symptoms developed on 17 plants between 24 and 36 days after feeding; all these plants, in addition to 13 symptomless plants, tested positive for TaBV by PCR. This is the first report of TaBV transmission by P. solomonensis and the first report of P. solomonensis in Samoa.

Four viruses have been reported from taro; Dasheen mosaic virus (DsMV), Taro bacilliform virus (TaBV) and two putative rhabdoviruses, Colocasia bobone disease virus (CBDV) and Taro vein chlorosis virus (TaVCV). A fifth virus, tentatively named Taro reovirus (TaRV), has also been recently identified. The distribution of these viruses throughout the Pacific Islands, and the symptoms associated with their infection, are unknown in many cases due to a lack of sensitive diagnostic tests. We have used recently developed PCR-based diagnostic tests to survey taro growing in 11 Pacific Island countries for the presence of known viruses. DsMV and TaBV were widespread, whereas TaVCV and TaRV were more restricted in their distribution. CBDV was restricted to PNG and Solomon Islands and was always associated with the two most serious viral diseases of taro; alomae disease and bobone disease, but the causal agent of these two diseases remains unclear.

The movement protein coding region of six isolates of Tobacco yellow dwarf virus (TYDV), collected over a 30 year period from various regions throughout Australia, was analysed using PCR and DNA sequencing. Four isolates of Bean summer death virus, considered a synonym of TYDV, were also analysed. Phylogenetic analysis showed that all 10 isolates were greater than 95% homologous to the published TYDV sequence (GenBank accession number M81103). The weed Raphanus raphanistrum was identified as an alternative host of TYDV for the first time.

When the leaves of 4- to 6-weeks-old papaw seedlings were mechanically inoculated with sap extracts containing tobacco ringspot Nepovirus (TRSV), tobacco streak Ilarvirus (TSV), tomato spotted wilt Tospovirus (TSWV), lettuce necrotic yellows Rhabdovirus (LNYV) or solanum nodiflorum mottle Sobemovirus (SNMV), infection of some of the papaw seedlings resulted with TRSV,TSWV and TSV, but not with LNW and SNMV. However, LNW was transmitted to papaw seedlings by its aphid vectors, Hyperomyzus spp., without causing symptoms. Symptoms produced by TRSV and TSWV were similar, but not identical, to those shown by plants with naturally occurring Australian dieback disease. Infection by TSV caused a mild mottle. It is concluded that none of the five viruses is the likely cause of Australian papaw dieback disease.