Seeing things at the electron scale

He sits in an unassuming, air-conditioned laboratory. The hum of a pump can be heard in the background. The pump works around the clock to maintain a constant, high vacuum in the interior of the microscope. Steffens feeds his meticulously-prepared samples into the device through a pressure lock – and his works of art immediately appear on the screen that sits next to the apparatus.

	What a picture! Laboratory Manager Dr. Andreas Goertz (left) and his assistant Ulf Steffens examine the image of an untreated, rust-infected leaf made using a scanning electron microscope (seen in the background).

Black-and-white displays show the surface structures of sampled plant parts in fine detail. The wave-like texture of the leaf surface is evident, as are the delicate stomata and the leaf hairs, which stick out from the surface like fine, pointed spines.

This “world at a minute scale” can present “a certain beauty,” as Steffens describes it, but one suspects that his passion for what he sees is actually more intense than his sober description suggests. In fact, the aesthetic side is only incidental to his work – he and his colleagues are really interested in something else when they peruse the images: the many small, spherical structures that are visible at various locations on the surface of the sample. Sometimes these occur individually, other times in large aggregations that remind you of oranges arranged on a market stall. Some of them are immaculately uniform spheres, without any protuberances; others have a filament emerging from them that extends for shorter or greater distances across the leaf surface before disappearing into the interior of the leaf through a stoma.

	The scanning electron microscope reveals details that are otherwise hidden to the naked eye: within the interior of the leaf, the brown rust pathogen has produced spores, which it is now thrusting through the leaf surface. With the naked eye, an advanced infection can be recognized as an accumulation of brown spots on the wheat leaf.

The spheres are fungal spores, and the filaments germ tubes (hyphae). In reality, the spores are only 20 microns (one fiftieth of a millimeter) in diameter. But on the monitor, they appear large and are clear to see – even at a mere thousand-fold magnification, which means that Steffens has hardly even started to use the electron microscope’s full power.

Threatening aesthetics
The state of the spores themselves, and the extent to which germ tubes have formed, give an indication of the stage a fungal infection has reached. And that is particularly interesting to Ulf Steffens – and to his laboratory director, Dr. Andreas Görtz. Goertz is working in Bayer CropScience’s Fungicide Research Department in the area of life-cycle management. His work involves close cooperation with product managers and product developers. Their common aim is to gain new insights into the properties of active substances. “The scanning electron microscope allows us to visualize the stage of fungal development at which a particular active substance starts to show its effects: for example, whether it already inhibits germination,” says Dr. Goertz.

To determine this, Steffens simulates what happens naturally in the field: he produces diseased cereal plants or those of other crops by spraying them with fungal spores suspended in water, a process he calls “inoculation”. After that, he maintains the plants in a specially-constructed incubation box. “Inside the box, the fungi are provided with the conditions they thrive under – 100 percent humidity,” he explains. He goes on to remove plant samples from the box at certain intervals, preparing them carefully in order to inspect them under the scanning electron microscope. Among the plants he samples are those that have also been treated with a certain active substance. “Comparison with the untreated samples will reveal exactly what the individual products do to the fungus,” says Steffens.

Take the active ingredient bixafen. Steffens did numerous investigations during the development phase of this new active substance. For example, he investigated how the substance can protect wheat against infection by brown rust, a fungus with the deceptively attractive name Puccinia recondita. The images he produced did indeed confirm that bixafen already starts to show an effect within five hours of inoculating the wheat. While the spherical spores on the untreated samples began to form germ-tubes after a short time, this hardly occurred at all on wheat leaves treated with bixafen. Which means that the first step in the life-cycle of the fungus has already been suppressed: without a germ tube, the fungus cannot enter the interior of the leaf. In contrast, this can occur uninhibited on untreated leaves, where the fungus continues to develop and eventually produces spores that are pushed towards the surface again so that they can be released and spread to new hosts. Even 28 hours after inoculation, virtually no germ-tubes are seen on the treated leaves.

On untreated leaves, the fungus rapidly penetrates into the interior of the leaf, where it uses the plant’s resources to propagate itself. This usually results in significant damage to plant tissues. Using the scanning electron microscope, Steffens can make this damage clearly visible. He takes quick-frozen leaves or stems, breaks them, and then investigates the interior of the tissues under the microscope wherever it is exposed at the point of breaking. In the bixafen study, investigation of untreated wheat samples ten days after inoculation with brown rust showed them to be covered with brown lesions easily visible to the naked eye.

The images show what lies below the spots. These are the so-called uredia – clusters of spores that the pathogen has produced within the leaf and is now thrusting towards the leaf surface, where the spores will be released in order to initiate new infections. From the image on the screen, Steffens can clearly discern how the spore clusters are oozing out of ruptures in the leaf surface. And where the leaf interior is exposed, it is clear to see how extensively the fungus has colonized the tissues. The places of the mycelium – the network of fungal hyphae – are differentiated from the plant cells.

The bixafen-treated wheat leaves look completely different. Here, the interior structures remain undisturbed, and there are no uredospores to be seen. The reproductive cycle of the brown rust pathogen has obviously been in its tracks.

The spherical brown rust spores on the untreated sample quickly start to develop germ tubes.

Bayer CropScience is happy to share the insights it gains from such scanning electron microscope investigations. “If we succeed in showing the effects of our compounds as clearly as this, then it is not just interesting for us,” explains Dr. Frank Göhlich, Head of Global Asset Management for fungicides at Bayer CropScience. “We are also pleased to show these images to our customers,” with whom the images go down very well – because a picture is worth a thousand words.

Valuable tips for improved product use
Beyond these impressive and beautiful pictures, the investigations often deliver concrete ideas for improving use, which are also valuable for farmers. In particular, the time-series images that Steffens produces from samples taken at intervals after inoculation and treatment are often a valuable source of information. “They help us to make clear statements about the optimal timing and application rate for a particular fungicide” says Thierry Gestate de Garambe, Product Manager for Fungicides, responsible for the active ingredients prothioconazole, tebuconazole, triadimenol and bitertanol.

	Successful control: treatment with the new active substance bixafen has suppressed germ-tube formation (colour contrast added to image). The image was made 28 hours after inoculation.

Here, the investigations carried out by Sascha Teitscheid can also be helpful. Like Steffens, Teitscheid is also regularly to be found at the scanning electron microscope, but working for a different business area – namely for the colleagues in Formulation Technology. When Teitscheid examines samples under the microscope, he is interested in the spray deposit that forms on the leaf after the spray mixture has dried out. How well does a particular formulation ensure that active substance molecules are distributed evenly over the leaf surface? Does the active substance crystallize-out in the spray deposit, which could delay penetration into the leaf interior and make the active substance vulnerable to washing off by rain? These are examples of questions that are important to the formulation technologists. The scanning electron microscope helps to answer them.

A comprehensive understanding of the behavior of a fungicide on the plant surface and of its effects on the different development stages of a fungal pathogen allows the protective and curative potential of the product to be assessed, thus predicting its flexibility during application in practice. This information ultimately provides farmers and gardeners with a basis for optimizing the application of a fungicide, and thus for fully exploiting its effectiveness.

Bayer CropScience’s continuous work to develop new, innovative products and to optimize already-approved products will provide a steady supply of questions that can be answered with the help of scanning electron microscopy into the future. So Ulf Steffens is likely to continue to produce his beautiful pictures on a regular basis. A biology technician, if you like, whose side-job is generating knowledge in a most artistic way. Karl Hübner