James L. Dale

Banana Bunchy Top: An Economically Important Tropical Plant Virus Disease

Publisher Summary This chapter explores the possible reasons for the negligence of banana bunchy top virus (BBTV), the way in which it has been controlled in Australia, and the possible directions for future research that could reduce the often devastating effects of the disease. The economic effects of the disease were as dramatic as its spread. In New South Wales, practically no plantation was free of BBTV in the Tweed and Brunswick areas. Most attempts to control or eradicate banana bunchy top virus have been made in commercial banana-producing countries. In Australia, a control program has successfully contained the disease at a very low level in southeast Queensland and the north coast of New South Wales for about 50 years. However, the disease still requires continual monitoring. The basis for control came from the discovery that (1) banana bunchy top was caused by a virus, (2) this virus was transmitted by the aphid, P. nigronervosa, (3) the virus could be disseminated in infected vegetative planting material, and (4) there were no known non-musaceous alternate hosts. For the campaign to be successful, little more information about the virus was required and in the ensuing 60 years little more has been discovered. Most research has been confined to verification of the identity of the disease in particular areas, varietal reactions to infection, some epidemiology and more recently isolation of the dsRNAs associated with infection.

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) Embryogenic Cell Suspensions of Banana (Musa spp. Cavendish AAA and Lady finger AAB)

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (β-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile.

On the evolution and molecular epidemiology of the potyvirus Papaya ringspot virus

The potyvirus Papaya ringspot virus (PRSV) is found throughout the tropics and subtropics. Its P biotype is a devastating pathogen of papaya crops and its W biotype of cucurbits. PRSV-P is thought to arise by mutation from PRSV-W. However, the relative impact of mutation and movement on the structure of PRSV populations is not well characterized. To investigate this, we have determined the coat protein sequences of isolates of both biotypes of PRSV from Vietnam (50), Thailand (13), India (1) and the Philippines (1), and analysed them together with 28 PRSV sequences already published, so that we can better understand the molecular epidemiology and evolution of PRSV. In Thailand, variation was greater among PRSV-W isolates (mean nucleotide divergence 7.6%) than PRSV-P isolates (mean 2.6%), but in Vietnamese populations the P and W biotypes were more but similarly diverse. Phylogenetic analyses of PRSV also involving its closest known relative, Moroccan watermelon mosaic virus, indicate that PRSV may have originated in Asia, particularly in the Indian subcontinent, as PRSV populations there are most diverse and hence have probably been present longest. Our analyses show that mutation, together with local and long-distance movement, contributes to population variation, and also confirms an earlier conclusion that populations of the PRSV-P biotype have evolved on several occasions from PRSV-W populations.

Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA.

Virus-like particles were purified from banana plants with banana bunchy top disease. These particles were isometric with a diameter of 18 to 20 nm and a density of 1.28 to 1.30 g/ml in caesium sulphate. Associated with these particles were an ssDNA of about 1 kb and one major protein of Mr 20,100. DsDNA was synthesized from nucleic acid extracts from these particles and cloned. One clone, pBT338, hybridized specifically (i) with sap extracts from plants infected with banana bunchy top virus (BBTV) but not with sap extracts from healthy plants and (ii) with the small ssDNA in nucleic acid extracts from infected plants and virus-like particles. Banana bunchy top disease was transmitted from infected to healthy bananas by aphid inoculation and it was demonstrated that the small ssDNA was transmitted with the disease. It is probable that these particles represent the virions of BBTV containing small ssDNA and that the virus resembles subterranean clover stunt virus more than any other known virus.

Evidence for two groups of banana bunchy top virus isolates

Banana bunchy top virus (BBTV) DNA component 1 from isolates from 10 different countries was cloned and sequenced and the sequences were aligned and compared. This analysis indicated two groups: the South Pacific group (isolates from Australia, Burundi, Egypt, Fiji, India, Tonga and Western Samoa) and the Asian group (isolates from the Philippines, Taiwan and Vietnam). The mean sequence difference within each group was 1.9 to 3.0% and between isolates from the two groups was approximately 10%, but some parts of the sequences differed more than others. However, the protein encoded by the major open reading frame, which is probably a replicase, differed by approximately 5%. The region from the beginning of the stem-loop sequence to the potential TATA box was identical in all isolates except for a two nucleotide change in the Western Samoan isolate and a single change in that of the NSW isolate. These results, together with other evidence, suggest that BBTV has spread to bananas after the initial movement of bananas from the Asian Pacific regions to Africa and the Americas.

Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses.

SummaryTwo pairs of degenerate primers were designed from sequences within the potyviral CI (CIFor/CIRev) and HC-Pro-coding regions (HPFo/HPRev), and these were shown to be highly specific to members of the genus Potyvirus. Using the CIFor and CIRev primers, three novel potyviruses infecting crop and weed species from Vietnam were detected, namely telosma mosaic virus (TelMV) infecting telosma (Telosma cordata, Asclepiadaceae), peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii, Araceae) and wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum, Solanaceae). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these viruses and a banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to chilli veinal mottle virus (ChiVMV) and pepper veinal mottle virus (PVMV), while PeLMV, TelMV and BBrMV were related to different extents to members of the bean common mosaic virus (BCMV) subgroup.

PAPAYA RINGSPOT POTYVIRUS : ISOLATE VARIABILITY AND THE ORIGIN OF PRSV TYPE P (AUSTRALIA)

We have sequenced the coat protein gene of nine isolates of papaya ringspot virus (PRSV) including six Australian and three Asian isolates and compared these with four previously reported sequences of PRSV. There was up to 12% sequence variation between isolates at the nucleotide level. However, there was no significant difference between the sequences obtained from Australian isolates irrespective of whether they were PRSV type P (cucurbit or papaya infecting) or PRSV type W (cucurbit infecting) and these isolates were more closely related to one another than to any other isolate. These results imply that PRSV-P, first recorded in Australia in 1991, arose locally from PRSV-W (first recorded in Australia in 1978) rather than being introduced. Further, there was no consistent sequence difference between PRSV-P and PRSV-W isolates that would obviously account for their host range difference.

Sequence diversity of South Pacific isolates of Taro bacilliform virus and the development of a PCR-based diagnostic test

Summary. We have analysed the sequence variability in the putative reverse transcriptase (RT)/ribonuclease H (RNaseH) and the C-terminal coat protein (CP)-coding regions from Taro bacilliform virus (TaBV) isolates collected throughout the Pacific Islands. When the RT/RNaseH-coding region of 22 TaBV isolates from Fiji, French Polynesia, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands and Vanuatu was examined, maximum variability at the nucleotide and amino acid level was 22.9% and 13.6%, respectively. Within the CP-coding region of 13 TaBV isolates from Fiji, New Caledonia, PNG, Samoa and the Solomon Islands, maximum variability at the nucleotide and amino acid level was 30.7% and 19.5%, respectively. Phylogenetic analysis showed that TaBV isolates from the Solomon Islands showed greatest variability while those from New Caledonia and PNG showed least variability. Based on the sequences of the TaBV RT/RNaseH-coding region, we have developed a PCR-based diagnostic test that specifically detects all known TaBV isolates. Preliminary indexing has revealed that TaBV is widespread throughout Pacific Island countries. A sequence showing approximately 50% nucleotide identity to TaBV in the RT/RNaseH-coding region was also detected in all taro samples tested. The possibility that this may represent either an integrated sequence or the genome of an additional badnavirus infecting taro is discussed.

Banana bunchy top virus DNA-3 encodes the viral coat protein

SummaryBanana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense. A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein. The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6. This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV. The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit. The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.