RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

Purification of bromelain from pineapple wastes by ethanol precipitation

Bromelain is an aqueous extract of pineapple that contains a complex mixture of proteases and non-protease components. These enzymes perform an important role in proteolytic modulation of the cellular matrix in numerous physiologic processes, including anti-inflammatory, anti-thrombotic and fibrinolytic functions. Due to the scale of global production of pineapple (Ananas comosus L.), and the high percentage of waste generated in their cultivation and processing, several studies have been conducted on the recovery of bromelain. The aim of this study was to purify bromelain from pineapple wastes using an easy-to-scale-up process of precipitation by ethanol. The results showed that bromelain was recovered by using ethanol at concentrations of 30% and 70%, in which a purification factor of 2.28 fold was achieved, and yielded more than 98% of the total enzymatic activity. This enzyme proved to be susceptible to denaturation after the lyophilization process. However, by using 10% (w/v) glucose as a cryoprotector, it was possible to preserve 90% of the original enzymatic activity. The efficiency of the purification process was confirmed by SDS-PAGE, and native-PAGE electrophoresis, fluorimetry, circular dichroism and FTIR analyzes, showing that this method could be used to obtain highly purified and structurally stable bromelain. (C) 2012 Elsevier B.V. All rights reserved.

Effect of modified atmosphere packaging on the quality of minimally processed pineapple cv.'Smooth Cayenne' fruit

The effects of modified atmosphere (MA) conditions on the quality of minimally processed pineapple slices were determined. Commercial pineapple slice packs sealed with 40 pm thick polyester film were kept at 4.5 degrees C for 14 d. The oxygen transmission rate of the film was 23 ml m(-2) day(-1) atm(-1) (at 25 degrees C, 75% RH). In-built atmospheres and the quality of the products were determined. O-2 concentrations within the packs stabilised at 2%, while CO2 concentrations increased to 70% by day 14. The high CO2 level suggested an inappropriate lidding film permeability for the product, and hence affected its quality. Three batches of pineapple slices were packed in the laboratory using lidding films with oxygen transmission rate of 75, 2790 or 5000 ml m(-2) day(-1) atm(-1) (at 23 degrees C, 0% RH). Headspace atmospheres from laboratory-packed pineapple slices suggested an optimum equilibrium modified atmosphere of ca. 2% O-2 and 15% CO2. Respiration data from the laboratory-prepared packs were pooled together and used to develop a correlation model relating respiration rates to O-2 and CO2 concentrations. The model showed a decrease in respiration rate with decreasing O-2 and increasing CO2 concentrations. Respiration rate stabilised at 2% 02 and 10% CO2. The high concentrations of CO2 observed in the commercial packs did not fit the range in the respiration model. The model could aid in selection of MA conditions for minimally processed pineapple fruit.

Developing pineapple fruit has a small transcriptome dominated by metallothionein

In a first step toward understanding the molecular basis of pineapple fruit development, a sequencing project was initiated to survey a range of expressed sequences from green unripe and yellow ripe fruit tissue. A highly abundant metallothionein transcript was identified during library construction, and was estimated to account for up to 50% of all EST library clones. Library clones with metallothionein subtracted were sequenced, and 408 unripe green and 1140 ripe yellow edited EST clone sequences were retrieved. Clone redundancy was high, with the combined 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of the EST clone sequences clustered within 13.5% and 9.3% of contigs from green unripe and yellow ripe libraries, respectively, indicating that a small subset of genes dominate the majority of the transcriptome. Furthermore, sequence cluster analysis, northern analysis, and functional classification revealed major differences between genes expressed in the unripe green and ripe yellow fruit tissues. Abundant genes identified from the green fruit include a fruit bromelain and a bromelain inhibitor. Abundant genes identified in the yellow fruit library include a MADS box gene, and several genes normally associated with protein synthesis, including homologues of ribosomal L10 and the translation factors SUI1 and eIF5A. Both the green unripe and yellow ripe libraries contained high proportions of clones associated with oxidative stress responses and the detoxification of free radicals.

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.

Present and future potential of pineapple biotechnology

Pineapple is an important crop for many countries in Central and South America as well as the Asia-Pacific region. Even though the history of the crop dates to pre-Colombian times there is a remarkable lack of commercial varieties with a single cultivar ‘Smooth Cayenne’ dominating the whole industry. Variety improvement is a very difficult task for pineapple breeders and very little progress has been made in this respect when compared to other crops more suitable to classical breeding approaches. This special characteristic makes pineapple specially suited for genetic engineering approaches that can transfer specific traits from other species into pineapple. In this presentation past and present efforts to use biotechnological methods for the improvement of pineapple will be reviewed. On-going biotechnology projects include control of flowering and control of ‘blackheart’ disease. The development of pineapple biotechnology, as with any other crop, is dependent on the availability of a number of molecular tools, which will also be discussed. For pineapple, these tools can be roughly classified into three different categories: (1) availability of useful genes (2) availability of suitable promoters and (3) availability of an efficient transformation method.

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.

Measuring nitrous oxide emissions from conventional and controlled release fertilisers in south-east Queensland pineapple production

Nitrous oxide (N2O) is a potent greenhouse gas with a global warming potential 298 times higher than carbon dioxide. Soils are a natural source of N2O, contributing 65% of global emissions. This paper is the first in Australia to measure and compare N2O emissions from pre-plant controlled release (CR) and conventional granular (CV) fertilisers in pineapple production using static PVC chambers to capture N2O emissions. Farm 1 cumulative emissions from the CR fertiliser were 3.22 kg ha-1 compared to 6.09 kg ha-1 produced by the CV. At farm 2 the CV blend emitted 2.36 kg ha-1 in comparison to the CR blend of 2.92 kg ha-1. Daily N2O flux rates showed a relationship of direct response to rainfall and soil moisture availability. High emissions were observed for wheel tracks where increased N2O emissions may be linked to soil compaction and waterlogging that creates anaerobic conditions after rain events. Emission measurements over three months highlighted the inconsistencies found in other studies relative to reducing emissions through controlled release nitrogen. More investigations are required to verify the benefits associated with controlled release fertiliser use in pineapples, placement and seasonal timing to address N2O emissions in pineapples.

Leadership training and regular communication identified as key strategies for the development of the Australian pineapple industry

Changes in the circumstances of the Australian pineapple industry left growers with a leadership vacuum, limited technical support and no funds for conducting research and marketing. Inspirational leadership training together with regular district farm meetings were used to assist the Australian pineapple industry to successfully adapt to these challenges. All growers were assigned to one of a number of regional grower study groups and regular on-farm meetings commenced to facilitate communication between growers, transfer of technology, awareness of industry affairs and an opportunity to become involved in industry business. A leader was appointed within each study group and these leaders attended a leadership course consisting of three, three-day modules. These original course graduates formed the nucleus of a new grower representative group which subsequently instigated levies to fund research and marketing. Two more courses have since been conducted to provide the depth of leadership to satisfy the growers' desire to rotate industry leadership on a regular basis.

Effect of diazotrophic bacterium inoculation and organic fertilization on yield of Champaka pineapple intercropped with irrigated sapota.

The purpose of this study was to evaluate the response of the Champaka pineapple to inoculation with the diazotrophic bacterium Asaia bogorensis (strain 219) when grown with organic fertilizer in an irrigated sapota orchard. Plantlets were transplanted to tubes containing a mixture of worm compost and vermiculite and inoculated with 108 bacterial cells. After five and a half months of acclimatization the plantlets were transplanted in furrows in the sapota orchard. Fertilizer was placed at the bottom of the furrows and covered with three doses (2.5; 5.0 and 7.5 L linear m−1 row) of three organic composts. The successful association of the plantlets with the diazo-trophic bacterium was confirmed by most probable number analysis before transferring to the field. Plants inoculated with strain AB219 showed the greatest initial leaf growth and produced the heaviest fruits compared to uninoculated plants. Plant growth and fruit yield increased with increasing compost dosages. The results suggested that Champaka pineapple benefited from the association of A. bogorensis (strain 219) when grown under irrigation and with organic fertilizer.

Crowning glory at harvest time

Review of 'The Pineapple Queen', La Boite Theatre Company, published in The Australian, 31 July 2009.