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

(2005 – 2008) University of Giessen, Institute of Phytopathology and Applied Zoology

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 chitinase, 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. Both barley lines showed an increased resistance against pathogenic fungi. The glucanase-barley also demonstrated an improved feed value for poultry farming.

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 expressionof 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.

Summary

In the greenhouse and outdoors studies, no significant differences were seen between the transgenic and non-transgenic barley plants in the colonisation by the beneficial mycorrhizal fungi.

In addition, the functionality of the mycorrhizal fungi, i.e. its supportive effect on plant growth, was not influenced through the genetic modification. This was shown though the formation of intact arbuscle cells (nutrient cells).

The investigations of the interaction of transgenic lines with harmful fungi could not be carried through in either year of the test because of vandalism of the trial fields. For the same reason the yield of corn could not be quantitated.

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 of the second strain of barley (parent plant: Baronesse) form the enzyme β-1,3-1,4-glucanase. The glucanase gene originates from a soil bacterium (Bacillus amyloliquefaciens) was introduced into barley to improve the feed value of the barley for poultry farming. It increases the digestibility of barley, the use of which in animal feed has until now been only limited. From the sustainability point of view, the utilisation of barley would be welcome.

Greenhouse and field

Greenhouse and field trials were undertaken. Approximately 5000 plants per year were sown outdoors, but because of vandalism only a fraction of these could be investigated further.

Trial field with genetically modified barley

Barley in the field trial under a safety tent

Young barley plants

Young barley plants

Comparison with mycorrhiza (right) and without (left) Colonisation by mycorrhizal fungi protects plants against diseases. Here: infestation with a barley pathogen.

Plants are protected against diseases by colonising them with beneficial mycorrhizal fungi: right with mycorrhiza, left without

Research into effects on parasitic and beneficial fungi

The greenhouse and field trials are designed to record and assess the spread of fungal diseases on genetically modified barley plants.

Since the introduced resistances could have undesirable effects on beneficial fungi, a particular focus of the research will be on fungi living in symbiosis with the plant. Symbiosis, a community of two organisms, is of mutual benefit for both partners (fungus and plant) and is of great importance, both ecologically and agronomically.

Greenhouse: For the greenhouse experiments, the transgenic barley lines are infected with two parasites (Rhizoctonia solani or Fusarium graminearum) and a beneficial mycorrhizal fungus (Glomus mosseae). Plants colonised by G. mosseae show a number of positive effects, such as stronger growth, increased resistance to pathogens and tolerance to heat stress. In order to determine the possible effect of various enzyme concentrations, barley lines are used that produce chitinase in three different quantities. In addition, the project is testing a line that produces glucanase and the parent strains.

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

Studies of the effects of the transgene on parasitic and beneficial fungi

Greenhouse: The question here was whether the glucanase that was stably expressed in the transgenic barley line or the chitinase had an influence on the colonisation through parasites (Rhizoctonia solani or Fusarium graminearum) or on beneficial fungi (Glomus mosseae) under the defined environmental conditions in the greenhouse.

The height of the plants, and the number of tiller and ear-carrying stems at the time of ear emergence were used to assess the influence of different fungal infections. In the long-term studies no significant difference was seen between the transgenic and non-transgenic plants with respect to colonisation with beneficial mycorrhizal fungi. However, the transgenic chitinase plants showed resistance against root Rhizoctonia attack.

Outdoors: Large areas of the outdoor trial areas were vandalised in 2006 and 2007 before the ears had ripened. In 2008 the field was occupied by gene technology opponents. In 2009 the trials were repeated in Groß Lüsewitz under otherwise similar conditions. There too attempts were made to destroy the plants. Because of these intrusions the mycorrhiza colonisation studies were repeated in each year under greenhouse conditions on transgenic and non-transgenic barley lines.

The studies on the interaction of the transgenic lines with harmful fungi could not be carried out in either year because of the destruction of the fields. It was also not possible to quantitate the yield of corn.

Because of the vandalism, the running time of the research project was prolonged until March 2010. AgroBioTechnikums Groß Lüsewitz in Mecklenburg-West Pomerania also participated in the field trials in 2009. The hyphal network of the mycorrhizal fungi in the plant cells was examined microscopically after staining.

Microscopic study and molecular analysis of the transgenic barley lines

In the second and third years of the project a detailed microscopic analysis of the root colonisation by Glomus mosseae was undertaken on both greenhouse plants and those that were still standing from the field trials. This showed that the formation of hyphae, the colonisation of the root cells and the formation of nutrient cells (arbuscles) in the transgenic compared to non-transgenic plants were not affected.

In addition to the microscopic examination, samples were also analysed by quantitative PCR, through which the amount of mycorrhizal fungi in the roots could be exactly quantified.

Transgenic lines and control plants grown under both field and greenhouse conditions showed a successful colonisation with mycorrhizal fungi. No quantitative or qualitative differences in colonisation were detected between transgenic plants and their parent plants. The functionality of the mycorrhizal fungi was demonstrated through their forming intact arbuscles.