J. E. Thomas

Banana and Plantain

Banana and plantain (Musa spp.) are grown in more than 125 countries (Anon., 2001) and are a major source of carbohydrate for more than 400 million people (Swennen et al, 1995). The progenitors of almost all domesticated Musa are two wild species, Musa acuminata and M. balbisiana, whose genomes are represented using the letter codes A and B, respectively. The most commonly cultivated cultivars have the triploid genotypes AAA, AAB or ABB.

Genetic Diversity Among Banana streak virus Isolates from Australia

ABSTRACT Banana streak virus (BSV) is an important pathogen of bananas and plantains (Musa spp.) throughout the world. We have cloned and sequenced part of the genomes of four isolates of BSV from Australia, designated BSV-RD, BSV-Cav, BSV-Mys, and BSV-GF. These isolates originated from banana cvs. Red Dacca, Williams, Mysore, and Goldfinger, respectively. All clones contained a sequence covering part of open reading frame III and the intergenic region of the badnavirus genome. The sequences were compared with those of other badnaviruses, including BSV-Onne, a previously characterized isolate from Nigeria. The BSV-RD sequence was virtually identical to that of BSV-Onne, differing by only two nucleotides over 1,292 bp. However, BSV-Cav, -Mys, and -GF were divergent in nucleotide sequence. Phylogenetic analyses using conserved sequences in the ribonuclease H domain revealed that all BSV isolates were more closely related to each other than to any other badnavirus. BSV-Cav was most closely related to BSV-Onne, and there was 95.1% identity between the two amino acid sequences. Other relationships between the BSV isolates were less similar, with sequence identities ranging from 66.4 to 78.2%, which is a magnitude comparable to the distance between some of the recognized badnavirus species. Immunocapture-polymerase chain reaction assays have been developed, allowing specific detection and differentiation of the four isolates of BSV.

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

The nucleotide sequence of the infectious cloned dna component of tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants

An infectious clone of the Australian geminivirus tobacco yellow dwarf virus (TobYDV) was constructed from virus-specific double-stranded DNA isolated from infected tobacco and used to demonstrate a single-component genome. The nucleotide sequence of TobYDV DNA comprises 2580 nucleotides. TobYDV DNA has three coding regions, two in the virion sense and one in the complementary sense, homologous to those identified for other geminiviruses, particularly those infecting monocotyledonous (monocot) plants. The complementary sense coding region is comprised of two overlapping reading frames, with an intron of 86 nucleotides. Efficient splicing of the mRNA for this coding region was observed in the infected dicotyledonous (dicot) hosts bean and tobacco despite the intron having an A + U content (57%) more typical of geminiviruses of monocot plants. TobYDV encapsidates a small oligonucleotide able to prime synthesis of the complementary DNA strand in vitro. The TobYDV genome organization, low A + U intron, and encapsidated oligonucleotide primer resemble those of the monocot-infecting geminiviruses. These results strongly suggest that TobYDV is a monocot geminivirus which has become adapted to dicot hosts.

Purification, Characterization and Serological Detection of Virus-Like Particles Associated with Banana Bunchy Top Disease in Australia

Isometric virus-like particles, 18 nm in diameter, have been isolated from banana (Musa spp.) affected by bunchy top disease in Australia. Banana bunchy top disease-associated virus-like particles (BBTV) banded as a single component with buoyant density of 1.28 to 1.29 g/ml in Cs2SO4 and sedimented at about 46S in isokinetic sucrose density gradients. The A260/A280 of purified preparations was about 1.33. A single coat protein of Mr 20,500 was identified with antibodies to BBTV particles from Australia. Single-stranded DNA of about 1 kb as well as ssRNA smaller than 0.45 kb was also associated with the particles. A polyclonal antiserum to BBTV, suitable for use in ELISA, was prepared. Stability and antigenicity of purified BBTV was impaired by storage at pH greater than or equal to 8.5 and freezing at -20 degrees C without protectants. BBTV was detected by double antibody sandwich-ELISA with monoclonal and polyclonal antibodies, in field-infected banana plants, single aphids from an infective colony, and in experimentally aphid-inoculated banana plants. After transmission of BBTV particles by aphids from a banana bunchy top disease-affected to an uninfected banana plant, the disease was induced and BBTV was detected by ELISA in symptomatic leaves only. BBTV isolates from Australia, Taiwan, Peoples Republic of China, Tonga, Western Samoa and Hawaii were found to be serologically related, which suggests a common aetiology for the disease.

The diversity of Banana streak virus isolates in Uganda

Summary.In a study of the variation among isolates of Banana streak virus (BSV) in Uganda, 140 sequences were obtained from 49 samples by PCR across the conserved reverse transcriptase/RNaseH region of the genome. Pairwise comparison of these sequences suggested that they represented 15 different species and phylogenetic analyses showed that all species fell into three major clades based on 28% sequence difference. In addition to the Ugandan sequences, clade I also contained BSV species that are known as both integrated sequences and episomal viruses; clade II also contained integrated BSV sequences but which have not previously been identified as episomal viruses. Clade III comprised of Sugarcane bacilliform virus isolates and Ugandan BSV sequences and for which there is no evidence of integration. The possible reasons for the extraordinary levels of virus sequence variation and the potential origins and epidemiology of these viruses causing banana streak disease are discussed.

A new tospovirus serogroup IV species infecting capsicum and tomato in Queensland, Australia.

A previously undescribed tospovirus infecting tomato, capsicum and chilli in Queensland was characterised. The virus reacted with antibodies to serogroup IV tospoviruses, and the complete nucleotide sequence of its nucleocapsid protein gene indicated that it was a new tospovirus species. Sequence identities at the nucleotide and amino acid levels were <85% with recognised members of serogroup IV The virus infected test plants in five families and, importantly, overcame the Sw-5 resistance gene in tomato and TSWV resistance in Capsicum chinense accessions PI 152225, PI 159236 and AVRDC 00943. The name Capsicum chlorosis virus (CaCV) is proposed for this virus.

Tospoviruses—an Australian perspective

The detection, distribution, molecular and biological properties, vector relations and control of tospoviruses present in Australia, including Tomato spotted wilt virus (TSWV), Capsicum chlorosis virus (CaCV) and Iris yellow spot virus (IYSV), are reviewed. TSWV occurs throughout Australia where it has caused serious sporadic epidemics since itwas first described in the 1920s. The frequency and distribution of outbreaks has increased in the 1990s, with the arrival and dispersal of the western flower thrips (Frankliniella occidentalis) being one factor favouring this situation. The crops most frequently and severely affected are capsicum, lettuce, tomato, potato and several species of ornamentals. Minimal differences were found between the nucleocapsid (N) gene amino acid sequences of Australian isolates and these were most closely related to a clade of northern European isolates. CaCV was first detected in Australia in 1999 and is most closely related to Watermelon silver mottle virus, a serogroup IV tospovirus. The natural hosts include capsicum, tomato, peanut and Hoya spp. The virus also occurs in Thailand and Taiwan. IYSV was first found in Australia in 2003, infecting onion and leek, with the distribution in three States suggesting that the virus has been present for some time.

Characterisation of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa

Summary.We have sequenced the complete genome of an isolate of Banana streak virus from banana cv. ‘Mysore’ and show that it is sufficiently different from a previously characterised isolate from cv. ‘Obino l’Ewai’ to warrant recognition as a distinct species, for which the name Banana streak Mysore virus (BSMysV) is proposed. The structure of the BSMysV genome was typical of badnaviruses in general, although ORF I had a non-conventional start codon. Evidence that at least part of the BSMysV genome is integrated in the B genome of cultivated Musa is presented and transmissibility by the mealybug Planococcus citri also demonstrated.

High global diversity of cycloviruses amongst dragonflies

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra- and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution.