Population differentiation in the banana leaf spot pathogen Mycosphaerella musicola, examined at a global scale

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of the global population of Mycosphaerella musicola, the cause of Sigatoka (yellow Sigatoka) disease of banana. The isolates of M. musicola examined were grouped into four geographic populations representing Africa, Latin America and the Caribbean, Australia and Indonesia. Moderate levels of genetic diversity were observed for most of the populations (H = 0.22-0.44). The greatest genetic diversity was found in the Indonesian population (H = 0.44). Genotypic diversity was close to 50% in all populations. Population differentiation tests showed that the geographic populations of Africa, Latin America and the Caribbean, Australia and Indonesia were genetically different populations. Using F-ST tests, very high levels of genetic differentiation were detected between all the population pairs (F-ST > 0.40), with the exception of the Africa and Latin America-Caribbean population pair. These two populations differed by only 3% (F-ST = 0.03), and were significantly different (P < 0.05) from all other population pairs. The high level of genetic diversity detected in Indonesia in comparison to the other populations provides some support for the theory that M. musicola originated in South-east Asia and that M. musicola populations in other regions were founded by isolates from the South-east Asian region. The results also suggest the migration of M. musicola between Africa and the Latin America-Caribbean region.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction. (C) 2004 Elsevier B.V. All rights reserved.

The genetic structure of Australian populations of Mycosphaerella musicola suggests restricted gene flow at the continental scale

Mycosphaerello musicolo causes Sigatoka disease of banana and is endemic to Australia. The population genetic structure of M. musicola in Australia was examined by applying single-copy restriction fragment length polymorphism probes to hierarchically sampled populations collected along the Australian cast coast. The 363 isolates studied were from 16 plantations at 12 sites in four different regions, and comprised 11 populations. These populations displayed moderate levels of gene diversity (H = 0.142 to 0.369) and similar levels of genotypic richness and evenness. Populations were dominated by unique genotypes, but isolates sharing the same genotype (putative clones) were detected. Genotype distribution was highly localized within each population, and the majority of putative clones were detected for isolates sampled from different sporodochia in the same lesion or different lesions on a plant. Multilocus gametic disequilibrium tests provided further evidence of a degree of clonality within the populations at the plant scale. A complex pattern of population differentiation was detected for M. musicola in Australia. Populations sampled from plantations outside the two major production areas were genetically very different to all other populations. Differentiation was much lower between populations of the two major production areas, despite their geographic separation of over 1,000 km. These results suggest low gene flow at the continental scale due to limited spore dispersal and the movement of infected plant material.

Knowledge of birds and willingness to support their conservation: an Australian case study

This case study concentrates on the extent of knowledge among the Australian public of Australia's tropical bird species, and their willingness to support their conservation. In order to place this issue in context, we provide background information on the status of Australian bird species, focusing attention on species that occur in tropical Australia. Then, using questionnaire survey results, we consider the hypothesis that the public's support for the conservation of different bird species depends on their understanding of the species' existence and status. Based on results from a sample of residents in Brisbane, Queensland, we found that knowledge of bird species that occur exclusively in the Australian tropics (including tropical Queensland) was very poor compared with that of those occurring in the Brisbane area that are relatively common. Experimental results indicated that when respondents in the sample had an option to allocate A$1,000 between 10 bird species listed in the survey, they allocated more funds to the better-known and more common species, unless they were provided with balanced information about all the selected species. With balanced information, the average allocation to bird species confined mostly to the Australian tropics, particularly those threatened, increased. This demonstrates the conservation implications of information provision about bird species. The results showed that public education can play a crucial role in attempts to conserve bird species that are poorly known and threatened.

Sampling tissue for DNA analysis of trees: Trunk cambium as an alternative to canopy leaves

The number of studies of tropical tree species that use molecular tools is increasing, most of which collect leaf tissue for genomic DNA extraction. In tropical trees the canopy is not only frequently inaccessible, but also, once reached, the leaf tissue is often heavily defended against herbivory by high concentrations of anti-predation compounds, which may inhibit downstream applications, particularly PCR. Cambium tissue, accessed directly from the tree trunk at ground level, offers a readily accessible resource that is less hampered by the presence of defensive chemicals than leaf tissue. Here we describe a simple method for obtaining tissue from the cambial zone for DNA extraction and test the applicability of the method in a range of tropical tree species. The method was used successfully to extract DNA from 11 species in nine families. A subset of the DNA extracts was tested in more detail and proved to be highly suitable for AFLP analysis.

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed. (c) 2006 Published by Elsevier Ltd on behalf of The British Mycological Society.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.]

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

Delayed flowering in pineapples (Ananas comosus (L.) Merr.) caused by co-suppression of the ACACS2 gene

Natural flower induction is a major pineapple industry problem. It usually occurs when shortening days and low temperatures give raise to increased ethylene production in the leaf tissue and plant stem apex which in turn stimulates flowering. Natural flowering fruit matures 4 to 6 weeks ahead of the normal summer harvest resulting in the need for extra harvest passes and considerable yield losses. Ethylene is produced through the sequential action of ACC synthase and ACC oxidase. Our team has cloned an ACC synthase gene from pineapple (ACACS2), which is expressed in meristems and activated under the environmental conditions that induce flowering in nature. Genetic constructs have been produced containing ACACS2 in sense orienta¬tion to induce silencing of the host gene in the plant by co-suppression mechanisms. Two independent lines of transgenic plants have been produced and field trials have been conducted in Queensland for four years in order to study the characteristics of the transgenic lines. We have identified a group of transgenic plants demonstrating inherited flowering delay and confirmed co-suppression of the ACACS2 gene due to methylation.

The Pineapple EST sequencing and microarray project

We have initiated an EST sequencing project to survey a range of expressed sequences from green fruit, yellow fruit, roots, and root-knot nematode infected root/gall tissues. In total, 5681 edited EST sequences were retrieved. Clone redundancy was high in the fruit libraries, with the combined fruit 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of all fruit EST clone sequences clustered within approximately 14 and 9% of contigs from green unripe and yellow ripe libraries respectively, indicating that a small subset of genes dominates the majority of the transcriptome. The root and root/gall libraries had lower levels of redundancy than the fruit libraries. Half of the root/gall ESTs clustered within approximately 40% of all contigs, indicating the roots possess a more complex transcriptome. Contig assembly and cluster analysis revealed major differences in the abundant gene sequences expressed between the unripe green and the ripe yellow fruit tissues, or gene sequences expressed between the weeks 1-4 and weeks 5-10 nematode infected gall vascular cylinder libraries.