Crown gall research continues

A Hortgro-funded project aims to isolate the responsible pathogen and investigate whether apple-associated bacteria can help protect trees against it. By Anna Mouton.

In the introduction to his 1910 monograph on crown gall in apple trees, plant pathologist George Hedgcock describes the urgent demand from nurserymen and horticulturists for more exact data on the spread and impact of this disease. At the time, Hedgcock was employed by the United States Department of Agriculture and had been studying crown gall for seven years.

More than a century later, crown gall outbreaks continue to plague nurserymen and horticulturists worldwide, affecting not only apples but many other crops, especially in the grape, walnut, and rose families. Although crown gall has long been present in South Africa, a resurgence of galls on apple trees in 2022 made it the focus of a renewed Hortgro-funded research effort.

Agrobacterium in isolation

Crown gall in apple trees is caused by bacteria called Agrobacterium tumefaciens (synonym Rhizobium radiobacter). The bacteria are attracted to wounded plant tissues, where they insert bacterial genes into the host’s genome, thereby transforming the infected cells. The transformed cells divide uncontrollably, giving rise to galls.

Galls provide a home for A. tumefaciens, and transformed cells produce combinations of amino acids and sugars on which the bacteria feed. Other microbes and insects often join the party.

Theoretically, crown gall can be diagnosed by isolating and identifying A. tumefaciens from galls. In practice, this isn’t straightforward because the bacteria may no longer be present by the time galls are noticed and sampled. Once transformed, cells continue proliferating whether or not the bacteria stick around.

“We’ve managed to isolate and purify pathogenic Agrobacterium once,” says Dr Yolanda Petersen, senior researcher at the ARC Infruitec-Nietvoorbij. Petersen is a plant pathologist specialising in bacterial diseases and has been leading the Hortgro-funded investigation into crown gall of apple trees.

The crown gall pathogen was isolated from a G.222 rootstock in July 2023, and its identity was confirmed by PCR. Although it is the project’s only successful isolation to date, having a South African A. tumefaciens in culture enabled further research.

“With those results, an MSc student could continue characterising the bacteria and inoculating rootstocks,” says Petersen. “We also brought on board a second MSc student to look at biocontrol.”

Agrobacterium in action

Not all strains of A. tumefaciens cause crown gall. To confirm that isolates are pathogenic, the bacteria are unleashed on living plant tissues, starting with carrots.

“Instead of using a whole plant, we use carrot discs,” explains Petersen. “We add the Agrobacterium to the discs, leave the discs in a Petri dish with moist paper for 2–3 weeks, and look for tissue proliferation. You expect to see that with pathogenic Agrobacterium.”

She has also tested the bacteria on green bean and tomato plants. The isolate from the G.222 rootstock caused galls on the stems of young green bean and tomato plants within 14 days of inoculation.

One of the MSc students on the project, Ashlee du Toit, inoculated A. tumefaciens into wounded stems and roots of 6–8 plants each of the following apple rootstocks: G.11, G.41, G.210, G.222, G.228, G.778, M.7, M.793, and MM.109. The aim was to see whether some rootstocks are more susceptible than others.

Unfortunately, only 13% of the stem-inoculated and 18% of the root-inoculated plants developed galls after five months. It was only possible to confirm crown gall using PCR in one of these.

“We redid these trials, but I don’t have the final results yet,” says Petersen. “Based on my observations, when the student was lifting the plants, not many had galls.” The second round of trials included G.41, G.202, G.210, G.213, G.214, G.222, G.228, G.757, G.778, G.890, G.969, M.7, M.9 T337, and MM.109.

Judging from the literature, many researchers struggle to induce galls in their trials. “When you read papers about apples or members of the rose family, the researchers report not getting consistent results, and the number of plants forming galls is low,” remarks Petersen.

Enlisting bacterial aid

Certain bacteria have been shown to control crown gall in some crops, so Petersen and MSc student Viwe Mbulawa are searching for apple-associated bacteria that could help protect South African apple trees.

“We’re specifically looking at bacteria that are present in the crown, galls, and roots,” says Petersen. “Those bacteria would be well-adapted to those niches, so an antagonist we find there would make a better candidate than just any old bacteria. Also, we wouldn’t be introducing totally new bacteria to the ecosystem.”

Preliminary investigation of the microbial community of apple galls revealed a diverse endophytic flora. Petersen and Mbulawa isolated bacteria from G.778 plants and screened 963 for activity against the South African Agrobacterium isolate and a reference strain.

“I think Viwe has seven candidates that showed antagonism on agar plates,” says Petersen. “From what we’ve done so far, we will move forward with three of those.”

The plan is first to test whether the biocontrol candidates can prevent A. tumefaciens from inducing cell proliferation in carrot discs. If successful, they will be tested in other susceptible hosts, such as tomato plants.

“Our last experiment will be in apple rootstocks,” says Petersen. Those results will likely be available toward the end of 2025.

Meanwhile, Petersen plans to continue with isolations. “I want to see whether I would be able to detect the Agrobacterium more consistently,” she says.

Read more about the biology and signs of crown gall in Fresh Quarterly issue 20.

What does industry say about this research?

“I think crown gall research is of paramount importance to the industry. Based on my experience, starting with my first observations of crown gall in 2018 in the EGVV, to the 2022 outbreak, it would be negligent not to do research because our current understanding of this disease is incomplete.

“I believe the extent of crown gall’s impact on the industry hasn’t yet fully manifested, and we need research to prepare ourselves and to become resilient.”

Technical adviser Angelique Pretorius. Kromco.