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INVASIVE INSECTS – POTENTIAL VECTORS OF PLANT PATHOGENS

Press Releases   •   Nov 14, 2011 10:01 GMT

 

 

Besides the direct damage that insects cause to plants by their feeding, some insects also a pose a serious indirect threat to the plant’s health by acting as vectors of plant pathogens. In fact, numerous plant pathogens require insects for their dispersal. This includes the well known example of Dutch Elm Disease caused by Ophiostoma ulmi, and spread by Elm Bark Beetles.

Various insects have been recorded to transmit plant pathogens in nurseries. Of these, fungus gnats (flies in the family Sciaridae) are often prevalent in nurseries of various crops and have been recorded to transmit Botrytis cinerea, Verticillium albo-atrum and Fusarium oxysporum f. sp. radicis-lycopersici. The most serious pathogen in pine nurseries of South Africa is the pitch canker fungus, Fusarium circinatum. Insects are known to be associated with the spread of this fungus on pine trees in forests, but no association between insects and this fungus has been recorded on pine seedlings in nurseries.

Our study was initiated to examine the presence, species composition and genetic diversity of fungus gnats present in South African pine nurseries and to examine their possible association with F. circinatum. Only one fungus gnat species, Bradysia difformis, was detected, and this species was detected in all the main pine nurseries. Examination of a 395bp portion of the COI gene from nursery populations in South Africa and Europe indicated a historical connection between the two regions. South African populations showed a high genetic diversity and low genetic differentiation, reflecting multiple and / or relatively large introductions of B. difformis into South Africa, with frequent movement between nurseries (probably with the movement of plants). These results indicate the ease at which invasive insects can enter into and spread within the country. Fortunately, investigations using standard isolation techniques and sensitive DNA markers have shown that adult fungus gnats do not have a major role in the transmission of F. circinatum. The role of the larvae requires further examination.


 

Key reference:

Hurley BP, Slippers B, Wingfield BD, Govender P, Smith JE, Wingfield MJ. 2009. Genetic diversity of Bradysia difformis (Sciaridae: Diptera) populations reflects movement of an invasive insect between forestry nurseries. Biological Invasions DOI 10.1007/s10530-009-9509-1.

Other references:


Hurley BP, Govender P, Coutinho TA, Wingfield BD, Wingfield MJ. 2007. Fungus gnats and other Diptera and their possible association with the pitch canker fungus. South African Journal of Science 103:43-46.

Hurley BP, Slippers B, Coutinho TA, Wingfield BD, Govender P, Wingfield MJ. 2007. Molecular detection of fungi carried by Bradysia diffomris (Sciaridae: Diptera) in forestry nurseries of South Africa. Southern Hemisphere Forestry Journal 69:103-109.

FABI was established on the foundation of the highly successful and internationally acclaimed Tree Protection Co-operative Programme (TPCP). 

invasive insects can enter into and spread within the country with relative ease. Fortunately, investigations using standard isolation techniques and sensitive DNA markers have shown that adult fungus gnats do not have a major role in the transmission of F. circinatum. The role of the larvae requires further examination.

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“Gene discovery” in cowpea, a drought tolerant African plant

Press Releases   •   Nov 14, 2011 08:24 GMT

Authors: Inge Gazendam (PhD project) and Nanette Coetzer (MSc project)

 

Drought is a serious problem affecting crop production throughout the world, especially in areas that are dependent on natural rainfall such as large parts of sub-Saharan Africa. Climate change forecasts an even larger impact on food security.

Since plants are stationary, they cannot move away from conditions that do not suit them. They have developed various mechanisms to adapt to adverse conditions such as drought, salt, heat, cold and various other conditions that are termed “abiotic” stresses.

A large amount of research is going into figuring out the mechanisms that enable plants to survive such unfavourable abiotic conditions. These adaptations include “macro” scale changes to plant morphology such as leaf area and stomata behaviour, and “micro scale” changes like switching on the production of specific protective proteins and other molecules, ion channels and changing membrane characteristics inside cells.

Drought tolerance is governed by multiple genes, and if the most important genes can be identified they can be used to engineer crop plants with improved drought tolerance. In a collaborative project between the Agricultural Research Council Vegetable and Ornamental Plant Institute and the Department of Plant Science, FABI at the University of Pretoria, we chose cowpea (Vigna unguiculata) for drought gene discovery. Some varieties of this important African crop are known to be quite drought tolerant.

Gene discovery can be divided into two main steps:

Step 1: Lab experiments with treated/control plants and isolate the genes; in our case drought-treated cowpea,

Step 2: Use bioinformatics to help you identify the genes responding to the treatment; in our case we developed two bioinformatics software packages.

 

We grew drought hardy cowpea plants under simulated drought conditions in a greenhouse (Figure 1). Plants of a less hardy variety were well-watered and used to “subtract” the background of genes that are normally expressed. A molecular technique called “suppression subtraction hybridisation” (SSH) was employed which picks out the genes that are present in only the drought-stressed plant.

Because so many genes were potentially involved, the high-throughput technique called microarrays was used to “fish-out” drought tolerance genes. Microarrays are glass slides spotted with small droplets containing DNA of your genes of interest. We used the Microarray robot and scanner at the ACGT Microarray Facility at FABI, UP (http://microarray.up.ac.za).

Microarray experiments produce thousands of data points, so this is where we needed to develop the first software package “SSHscreen”, using the statistical programming language called ‘R’. The input to SSHscreen was a large dataset of intensity values from the DNA spots on the microarray slides. The output from SSHscreen - after various data analysis steps - was a list of potential drought tolerance genes.

The SSHscreen software helped us to choose a selection of genes for DNA sequencing. To help interpret and manage the sequence information we developed the second software, a database called SSHdb (Figure 2). SSHdb allowed us to compare the unknown sequences against all known sequences in the international internet database called “Genbank”, and find out which proteins they are most likely to code for. Our results indicated that cowpea plants protect themselves against drought by detoxification of unwanted compounds, stabilization of useful proteins, and down-regulation of photosynthesis. The next step of the PhD project is to test one of the most promising candidate genes in transgenic plants.

Our work has not only shed light on the drought response in plants but the software pipeline can be used for gene discovery in any biological system, not just plants!

To make it easy for scientists to apply this approach, we have written up the experimental protocol in a book chapter (Berger et al., 2007), and the cowpea work and SSHscreen/SSHdb are described in (Coetzer et al., 2010). The software is freely available from http://microarray.up.ac.za/SSHscreen/. Users can start their own sequence database at SSHdb and share the data with collaborators of their choice by registering at http://sshdb.bi.up.ac.za/.

 

Further reading

Berger DK, Crampton BG, Hein I, Vos W (2007) Screeing cDNA libraries on glass slide microarrays. In J Brampal, ed, Microarrays, Second Edition, Volume II, Applications and Data Analysis, Ed 2nd. Humana press, Totowa, New Jersey, USA, pp 177-203

Coetzer N, Gazendam I, Oelofse D, Berger D (2010) SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea. Plant Methods 6: 10

 

 

 

FABI was established on the foundation of the highly successful and internationally acclaimed Tree Protection Co-operative Programme (TPCP). 

Our work has not only shed light on the drought response in plants but the software pipeline can be used for gene discovery in any biological system, not just plants!

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Genome sequence of Pantoea ananatis – a Eucalyptus pathogen

Press Releases   •   Nov 14, 2011 08:22 GMT

Pantoea ananatis is a Gram-negative bacterium belonging to the family Enterobacteriaceae which causes diseases on a broad range of agronomically important crops including maize, rice, onion and pineapple fruits. It has also been associated with human disease. In South Africa, P. ananatis poses a serious threat to the forestry industry, causing blight and dieback of commercial hybrids and clones of Eucalyptus grandis (Fig. 1). There are no effective means of control for this pathogen and infected material needs to be removed. Little is known about the mechanisms by which Pantoea ananatis causes disease on its plant hosts. Figure 1: P. ananatis blight on Eucalyptus   In this paper, the whole genome of the Eucalyptus-pathogenic Pantoea ananatis strain LMG20103 was sequenced using the newly available 454 pyrosequencing technology. Subsequently, the genome was assembled and annotated (Fig. 2). This constitutes the first genome of a phytopathogenic bacterium to be sequenced in Africa. The genome consists of a single chromosome, 4.69 million nucleotides in size and with a G+C content of 53.69%. A total of 4,237 protein coding genes are encoded on the chromosome. This genome sequence will provide an extensive resource which can be used to analyse various aspects of P. ananatis biology, including how it causes disease. Further analysis has revealed the presence of 433 protein coding genes on the genome which have been experimentally shown to play a role in disease in other bacteria. Interestingly, two secretion systems, the Type II and III systems, are absent from P. ananatis LMG20103. These two systems play a major role in disease in both plant- and animal-pathogenic bacteria, raising questions on how P. ananatis can cause disease in the absence of these factors. However, three copies of the Type VI secretion, a recently described pathogenicity factor, are present. Interestingly, the putative effector proteins which are secreted via these secretion systems appear to be recently acquired through horizontal gene transfer. These effector proteins may have a role in P. ananatis infection of both plant and animal hosts.     Figure 2: The genome sequence of P. ananatis LMG20103.

 

 

Another putative pathogenicity determinant identified from the genome, the exopolysaccharide ananatan, was experimentally demonstrated to play a role in disease on both onion seedlings and pineapple fruits. This was done through the production of a library of mutants which encompasses all the genes on the P. ananatis genome. This library can be further used to elucidate the functions of other putative pathogenicity factors on the genome. The information gained from the genome and from the mutant library will allow a better understanding of how P. ananatis causes disease on its plant hosts and can be utilised to develop and implement directed and effective control measures against this important plant pathogen.   Related article: Pieter De Maayer, Wai Yin Chan, Stephanus N. Venter, Ian K. Toth, Paul R. J. Birch, Fourie Joubert, Teresa A. Coutinho. 2010. Genome sequence of Pantoea ananatis LMG20103, the causative agent of Eucalyptus blight and dieback. Journal of Bacteriology 192: 2936-2937

FABI was established on the foundation of the highly successful and internationally acclaimed Tree Protection Co-operative Programme (TPCP). 

Another putative pathogenicity determinant identified from the genome, the exopolysaccharide ananatan, was experimentally demonstrated to play a role in disease on both onion seedlings and pineapple fruits. This was done through the production of a library of mutants which encompasses all the genes on the P. ananatis genome.

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UP scientists unravel hidden plant pathogenic fungi

Press Releases   •   Nov 14, 2011 08:20 GMT

Species of fungi can usually be distinguished based on what they look like. This method is known as the morphological species concept (study of the form, structure and configuration of organisms). But not all fungi can be studied in this way. Some fungi that occupy the same functions in ecosystems look identical, even though they are different species. Such species are cryptic, because they cannot be distinguished from each other with morphology. One way of dealing with such species is to apply the biological species concept, which states that if two individuals can mate with each other and produce fertile offspring, they should be the same species. This concept is useful if the sexes exist in different individuals, but some fungi are self-fertile – an individual can mate with itself or with another individual, and it is very difficult to distinguish these two events in nature.


One such fungal organism is known as Chrysoporthe austroafricana (the Latin for “orange destroyer from southern Africa”). This fungus is closely related to Chrysoporthe cubensis (orange destroyer from Cuba, although it occurs throughout South America). Both can self and both of them cause a serious stem disease of plantation Eucalyptus (blue gum) trees, which can weaken the stem and cause the tree to die or fall down during high wind. Chrysoporthe cubensis is also present in Southeast Asia, where it causes an identical disease. Scientists thought for a long
time that the fungus from South America and the one from Southeast Asia might be different species, simply because of the vast distances between them, but evidence for this separation was not forthcoming.

An example of the devastation that Chrysoporthe
austroafricana
can cause in gum tree plantations in South
Africa. This plantation of susceptible Eucalyptus clones are
approximately 40% infected with the fungus, and some trees
such as the one in the center have already fallen down after high
wind conditions. Albe van der Merwe (right) is busy sampling
C. austroafricana from the base of a dead young tree, and he is
assisted by Daleen van Dyk.

 

In a paper by Van der Merwe and coworkers (Department of Genetics and Forestry and Agricultural Biotechnology Institute, University of Pretoria), this problem was finally resolved. They used population genetics to show that populations of C. cubensis from South America and Southeast Asia are genetically different, and do not exchange genetic material. Armed with this knowledge, they searched for a quicker and easier way of distinguishing the species, because it is impractical to perform population genetic analyses in order to identify a single individual.

 

Sexual fruiting structures of Chrysoporthe cubensis
from Colombia, with spore drops oozing from the necks of
perithecia.

By sequencing the beta-tubulin gene, which is a gene that codes for proteins that form part of the cytoskeleton of a cell, two signature DNA sequences were uncovered that could aid to differentiate the three species of Chrysoporthe. When specific enzymes are added to amplifications of this gene from individuals of the fungi, they differentially cut the DNA at these sites, forming different sizes of fragments. These fragments could then be separated based on size, and a profile was obtained. This represented a breakthrough in developing a quick identification technique, because most laboratories have the equipment and expertise needed to perform the technique.

 

A “map” of the beta-tubulin gene of C. austroafricana, C. cubensis and C. deuterocubensis can aid to distinguish these species
quickly and easily. A and B show the profiles obtained using two different restriction enzymes (HindIII and AvaI). 1-2,
Chrysoporthe austroafricana; 3-6, Chrysoporthe cubensis; 7-10, Chrysoporthe deuterocubensis. C shows a map of the beta-
tubulin gene region, the sites where the enzymes cut the DNA, and the sizes of the DNA fragments that are produced.

 

Now that the species could be distinguished from each other, the final task was to describe the fungus from Southeast Asia as a new species. But there was a problem, because it looks exactly the same as C. cubensis from South America and causes the same disease, and it can self fertilize. It was thus impossible to apply either the morphological or the biological species concept. Fortunately, scientists have recently started to use DNA identification techniques to describe cryptic species in other fungi, although only a few such fungi have been described with new names. The UP scientists followed this trend and used information from the quick identification technique to convince the scientific community that these three species really are different. The fungus was thus described as Chrysoporthe deuterocubensis (the “other” cubensis).

 

Albe van der Merwe beside a 30-year-old
Eucalyptus grandis tree with a stem infection by
Chrysoporthe austroafricana clearly visible. Although such
old trees are not easily toppled by wind, the fungus girdles
the stem and causes the tree to die.

 

The new information gathered by UP scientists has important implications for quarantine and the continued prosperity of forestry in South Africa. The unholy trinity of gum tree pathogens is represented by three species of Chrysoporthe, each of which occurs in a different part of the world. Trade in wood and wood products could introduce one of these species to another part of the world, where it could intensify disease on forest trees. In fact, both C. cubensis and C. deuterocubensis have already been introduced onto the African continent, although those populations are far north of South Africa. Nobody knows what the effect will be when these fungi reach South Africa, and that is why it is important to never transport wood between different countries without a permit.

FABI was established on the foundation of the highly successful and internationally acclaimed Tree Protection Co-operative Programme (TPCP). 

New information gathered by UP scientists has important implications for quarantine and the continued prosperity of forestry in South Africa.

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A new species of Phytophthora associated with dying Pine Needles in Chile

Press Releases   •   Oct 31, 2011 10:48 GMT

During the course of the past three years, a new needle disease has appeared in Pinus radiata plantations, chiefly in the Arauco province of Chile . The disease, locally referred to as Daño Foliar del Pino (DFP), is typified by the relatively rapid death of ...

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UP scientists unravel hidden plant pathogenic fungi

News   •   Oct 19, 2011 11:23 GMT

Species of fungi can usually be distinguished based on what they look like. This method is known as the morphological species concept (study of the form, structure and configuration of organisms). But not all fungi can be studied in this way.

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Genome sequence of Pantoea ananatis – a Eucalyptus pathogen

News   •   Oct 19, 2011 11:15 GMT

Pantoea ananatis is a Gram-negative bacterium belonging to the family Enterobacteriaceae which causes diseases on a broad range of agronomically important crops including maize, rice, onion and pineapple fruits...

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About FORESTRY AND AGRICULTURAL BIOTECHNOLOGY INSTITUTE

Future Forests and Foods

FABI was established on the foundation of the highly successful and internationally acclaimed Tree Protection Co-operative Programme (TPCP). This programme, initiated in 1990, has supported South African Forestry for more than a decade and has become an Institution of that industry. The TPCP provides the entire forestry industry of South Africa with support in the field of tree health, and more particularly Forest Pathology and Forest Entomology. The group includes projects not only in South Africa, but in other parts of Africa, South America, South East Asia, Europe, Australia and North America.