You are reading: Flicking the switch for a ‘high-vis’ look inside disease-affected lentils

Flicking the switch for a ‘high-vis’ look inside disease-affected lentils

Green Fluorescent Protein (GFP) is an imaging tool used by modern scientists to more easily observe organisms of interest. The GFP gene is introduced to the species of interest – in this case the fungus – causing it to fluoresce and making it as visible through a microscope as a worker on a construction site wearing a vest of the high-vis variety. Author: Amanda Iannuzzi Nov 11, 2019 Read Time: 4 minutes

Lights off. Lights on. Just as the simple flicking of a switch can help what was unseen to be seen, our researchers have adopted a handy technique to shine new light on their study of the host-pathogen interactions behind the progression of crop diseases.

It’s called Green Fluorescent Protein (GFP) – a protein composed of 238 amino acid residues that exhibits bright green fluorescence in response to blue light and occurs naturally within organisms including some species of jellyfish.

GFP is an imaging tool used by modern scientists to more easily observe organisms of interest. The GFP gene is introduced to the species of interest – in this case the fungus – causing it to fluoresce and making it as visible through a microscope as a worker on a construction site wearing a vest of the high-vis variety.

Centre for Crop and Disease Management’s pulse researchers, eager to try every tool in their disease-busting toolkit in their efforts to rid us of the destructive ascochyta blight disease, are now putting the GPF technique to very productive use. The disease is a huge problem for pulse growers around the world, affecting lentil, chickpea, field pea and faba bean, among other crops, so the more our team can do to combat it the better the outcomes for our Australian growers.

The team’s latest published research, made possible with investment from the GRDC, is focused on Ascochyta lentis (A. lentis) the pathogen that causes the disease in lentil crops and a major threat to production of the crop worldwide.

Why GFP is useful for studying resistance to pathogens

CCDM’s Dr Bernadette Henares says the team’s use of a combination of fluorescent tagging with GFP and advanced microscopy is opening up a world of new possibilities in their studies of the disease.

“Fluorescence microscopy and the more sensitive confocal laser scanning microscopy enable us to non-invasively and efficiently observe fluorescent fungal structures, both on the surface and inside infected living cells and tissues of whole plants, under natural conditions and at all stages of infection,” said Dr Henares.

“This strategy is particularly valuable for studying interaction mechanisms between pathogen and host plant systems and will be useful in our search for disease-resistant lines in lentil germplasm collections and in future breeding programs.”

This news is sure to light up the faces of lentil growers looking to limit yield losses to ascochtya blight, which produces lesions that cause stem damage, loss of leaves and pod discolouration. If uncontrolled it can result in reduced yield and poor seed quality.

The Australian lentil industry produces an average of 292,000 tonnes annually. Average annual yield and grain quality losses cost the industry $0.9 million, and control costs a further $15.3 million, a year (Murray and Brennan 2012).

CCDM’s Dr Robert Lee says current management strategies for ascochyta blight include cultural practices, seed and foliar applications of fungicide treatment and the use of resistant cultivars. However, the disease remains difficult to control.

“Populations of A. lentis persist in lentil cropping areas by growing on plant residues and stubble at the start of the growing season,” said Dr Lee.

“The pathogen also persists through adaptation via the exchange of genetic material in the mating process and is distributed through the production of windborne spores.

“The pathogen’s ability to evolve new virulence forms or levels of aggressiveness present a particular challenge for researchers and the industry, so exploring new techniques such as the use of fluorescently-labelled fungal strains to learn everything we can about the disease is critical to finding new and more effective ways to overcome it.”

What has our researchers so excited?

A well-orchestrated set of events occurs between a host plant and a pathogen that ultimately determine the progression and outcome of disease.

Using a fluorescently-labelled strain of A. lentis, our researchers were able to get a good look at the progression of the pathogen during the full infection cycle – from spore germination on the plant surface to full colonisation. Differences in disease progression in resistant and susceptible lentil varieties were also examined.

Valuable insights were gained into the fungal development of A. lentis, including the germination of spores; penetration of the plant; colonisation of leaf tissues; and spore production. The host plant’s response to the pathogen was also observed. Key findings include:

  • Germination of the spores occurs early, within 24 hours of them making contact with the leaf surface.
  • Proliferation of the fungus was evident in the early stages of the disease infection cycle – spore germination and development of infection structures on the leaf surface occurred over the first 3 days following inoculation in both the resistant and susceptible lentil varieties.
  • Host recognition of, and response to, the invading pathogen was evident in both resistant and susceptible lentil varieties during the early stages of infection. Resistant lentil varieties deployed defence mechanisms earlier than susceptible types.
  • During the early stage of infection, both resistant and susceptible lentil varieties appeared healthy, with no leaf damage or lesions observed in the first six days. In susceptible lentil varieties, the onset of symptoms usually occurred 6 to 8 days after inoculation.
  • Most apparent to our researchers was that colonisation of leaf tissues did not occur in resistant lentil varieties, whereas the susceptible varieties were completely overwhelmed by fungal growth.

Let’s hope this and future studies using GFP continue to boost our researchers’ efforts to keep Australia’s lentil cultivars as green and disease-free as the protein they are using to study them!

The full paper, “Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil”, was recently published in the prestigious science journal, PLOS ONE.

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