Transgenic fungus-resistant barley – effects on pathogenic and beneficial fungi

Topic

Two lines of genetically modified barley are to be investigated for their effects on parasitic and beneficial fungi (that live in symbiosis with the plant). One of the lines produces a chitinas e, a protein that breaks down the cell walls of pathogenic fungi. The other line produces a glucanase which breaks down glucans in the barley corn. This increases the feed value of the barley for poultry rearing and also improves its brewing properties. Since glucans are also a component of the hyphal wall of fungi, as a side effect this barley line could also display fungal resistance properties

The project is investigating the following questions:

• How and to what extent are transgenic plants infested with harmful fungi? Is the interaction between plant and beneficial fungi in the field affected? (Project partner: University of Giessen)

• How does the production of the recombinant proteins affect the expression of other genes and the content and quality of the corn? (Project partner: University of Erlangen)

The following description relates to the research area of the University of Giessen.

Experiment description

Plant material

Plant material is available from two transgenic barley lines, each with a pest-resistance gene, and their non-transgenic parent lines. The transgenic lines were developed and supplied by Washington State University in the US. The plants’ increased resistance potential to the important fungal pest Rhizoctonia solani has already been demonstrated in laboratory experiments over there. The pathogen Rhizoctonia solani is found in increasing numbers today, particularly in fields where ploughless soil tillage is used. Ploughless soil tillage is however recommended for ecological reasons (avoiding soil erosion, conserving the soil structure) as part of good farming practice.

Genes used to improve resistance to fungal pests

• Chitinase: A gene was inserted into the plants of the first barley strain which codes for the enzyme chitinase. It is produced in all parts of the plant. Chitinases cause the specific breakdown of chitin, the key component of fungal hyphal walls. The gene comes from a beneficial soil fungus found throughout the world (Trichoderma harzianum), which parasitizes a range of fungi which cause significant economic damage to crops.

• Glucanase: Plants in the second barley line produce the enzyme glucanase. The glucanase gene comes from a soil bacterium (Bacillus amyloliquefaciens) and was introduced into barley to improve its feed quality for poultry. Glucanases enhance the digestibility of cereals, which historically have had limited use in animal feed.
  The gene for glucanase is only active in the endosperm tissue of the grain (the tissue that feeds germinating seeds). Nonetheless, the possibility exists that glucanases could be released into the soil at germination. Because the enzyme can break down the cell walls of certain fungi, the enzyme could influence the ability of both beneficial and harmful fungi to colonise the plant.

Greenhouse and field

Experiments are being carried out in the greenhouse and in the field. The field trials are taking place within the grounds of the University of Giessen. From 2006 to 2008 each of the two transgenic barley lines and their non-transgenic parent lines are being sown and studied on a field measuring 9.6 square metres.

Field: For the infestation studies, half of the trial field is treated with a commercially available mycorrhizal fungus . This is applied to the field and incorporated in the soil. Both the two transgenic lines and the non-transgenic parent lines are sown on this plot. The same plant material is sown on the second half of the trial field but no mycorrhizal fungus is applied.

In the event of a negative effect on symbionts, it is suspected that the genetically modified plants react to oligotrophic soil with slower growth than the non-transgenic organisms. As well as growth measurements, root colonisation of the transgenic lines is analysed using modern microscopic techniques.

Microscopic studies of the transgenic barley lines

Both field and greenhouse plants are used for this. Assessing the fungal infestation of roots, leaves and ears should provide insight into the effect of the two genes on beneficial and parasitic fungi. The interaction between fungus and plant will be investigated under the microscope. Plant defence mechanisms against fungal pests will be made visible using special dyeing techniques.

Studies of non-target organisms

Chitin is also a component of the exoskeleton of insects. This means that the chitinases could also have an effect on insects. However, the chitin in insect shells differs from the chitin found in fungi. The chitinases are very unlikely to have a negative impact on insects because of their specificity to fungal chitin. Previous field experiments in the US confirm this assumption.

Results

Research into the effects on parasitic and beneficial fungi

Greenhouse: In the first and second year the team investigated whether the glucanase/chitinase produced stably in the transgenic barley lines has an impact on the plants’ colonisation by parasites (Rhizoctonia solani or Fusarium graminearum) or on beneficial fungi (Glomus mosseae) under defined environmental conditions in the greenhouse.

To assess the impact of the various fungal infections, the plants were examined in terms of height, number of side-shoots and ear-bearing stalks at the time of heading. 

Since the project partner (AG Sonnewald) also uses seed material from these trials, the plants were only slightly infected, so that only moderate symptoms were to be expected. However, no significant differences were found between transgenic and non-transgenic plants in the trials with regard to the parameters studied.

Field: Significant areas of the release site were destroyed before the ears ripened in 2006 and 2007. Because of this, the infestation analysis using mycorrhizal fungi was repeated in both years under greenhouse conditions on transgenic and non-transgenic barley lines (see above).

Research into the interaction between the transgenic lines and harmful fungi could not be carried out in either year because of the field being destroyed. Neither was there any quantification of the grain yield.

Microscopic studies and molecular analysis of the transgenic barley lines

In the second year the team carried out a detailed microscopic analysis of root colonisation by Glomus mosseae both on greenhouse plants and on the remaining plants from the field trials. It was found that hyphae formation (hyphae= fungal cell filaments), colonisation of the root cells and the formation of food cells (arbuscules) in the transgenic plants were no different from the results found in the non-transgenic line.

In addition to the microscopic investigation, molecular analysis using quantitative PCR was also carried out on this sample material to measure exactly the quantity of mycorrhizal fungi in the roots.
Transgenic lines and the control plants showed a successful colonisation with mycorrhizal fungi under field and greenhouse conditions.

Current experiments (autumn 2007): In 2005-2007, homozygous transgenic barley lines were produced by crossing in the USA. They have good agronomical properties (high resistance, good yield, low pesticide requirement) and release effective amounts of the enzyme chitinase from their roots.

Between now and April 2008 the team will investigate whether the enzyme chitinase can protect the plants against Rhizoctonia root rot under greenhouse conditions as expected, without harming beneficial fungi in the process.

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