The science behind the control strategy. By Anna Mouton.
Figuring out plum marbling is like building a jigsaw. The discovery of plum viroid I was that essential first corner-piece needed to start solving the puzzle. Researchers have since expanded the picture to include new information on testing and transmission — but many areas still have to be filled in. Fresh Quarterly spoke to Dr Rachelle Bester and Prof. Hano Maree of the Department of Genetics at Stellenbosch University about their progress in understanding plum marbling.
“There are different pathogens that will cause exactly the same or very similar symptoms,” explains Maree. “When people first saw marbling, it looked like plum pseudo-pox which is caused by apple chlorotic leaf spot virus. So they developed tests for apple chlorotic leaf spot virus.”
Testing confirmed the presence of apple chlorotic leaf spot virus but over time a problem became apparent. Not all trees with marbling tested positive for the virus.
“There was a suspicion that the test was ineffective,” recalls Maree, “so a new test was developed.” But the improved test did not reduce the incidence of marbling. We now know the reason — it targeted the wrong pathogen.
The breakthrough came when Maree and Bester were approached to relook at marbling. They formed Team Marbling and co-opted key people in the stone-fruit industry to assist. “We’d had success in the past in sorting out the cause of diseases using high-throughput sequencing technology,” says Maree.
High-throughput sequencing enabled Maree and Bester to compare the genetic material present in the cells of trees with and without marbling. Trees with marbling contained genetic material that was absent in trees without marbling. This genetic material belonged to the pathogen — plum viroid I —responsible for marbling.
Bester and Maree used these results to develop an accurate test for plum viroid I. The test is based on PCR — short for polymerase chain reaction — technology, and can reliably detect a single positive sample in a pool of ten samples. The implication is that samples from ten trees can be pooled to save costs without the risk of missing any positives due to dilution.
The researchers evaluated the performance of the test on different sample types and at different times of year. They were able to detect plum viroid I in leaves, stems, flowers, and the peel of fruit. They also showed that the viroid could be found in leaves throughout the season from September to March. The best time to test is early in the season when leaves are growing actively — older leaves sometimes fail to yield a conclusive test result.
“Most of the effort with this project has been to develop tests,” says Bester, “so that we can ensure that the plant material that is distributed within the industry is clean.”
The PCR test developed by Bester and Maree is accurate and sensitive — but also expensive and resource-intensive. This limits its application to parent plant material such as trees in foundation units or mother blocks. Large-scale testing of trees in nurseries and orchards is not feasible due to financial and capacity constraints.
Bester and Maree are currently collaborating with a group in Spain to develop a new test that would be more cost-effective than the existing PCR. The new test is based on a technique known as hybridisation. Hybridisation involves a molecule — called a probe — that binds specifically to a target — in this case plum viroid I — and thereby effectively sticks a label on it.
The Spanish scientists previously developed a hybridisation test for viruses and viroids in stone-fruit trees. Their test could simultaneously detect eight different viruses and two viroids.
Unfortunately progress on the new test for plum viroid I has been delayed due to — you guessed it — COVID-19. The South African research team has been unable to meet directly with their Spanish counterparts. Virtual collaboration is a poor substitute for in-person contact when developing a practical laboratory-based test.
Routes of transmission
Understanding how plum marbling spreads is essential to controlling the disease. Accounts from growers suggested that infected plant material is the most likely source of plum viroid I. The marbling team therefore started by looking at graft transmission.
“We established an experimental orchard at Welgevallen,” recounts Bester. “We have ten different cultivars with cross-pollinators. In each group we have ten healthy trees and ten trees which we deliberately tried to infect by grafting them with an infected bud.”
The researchers tested all their experimental trees after nine months. Nearly sixty percent of the trees grafted with infected buds tested positive. The healthy controls remained negative. Bester and Maree plan to retest all the trees after a second season. They also intend to repeat some of the bud grafting.
“There were some problems with some of the grafts,” says Maree. “We grafted in December, which was a little late, so some of the buds didn’t take.”
The trial so far has conclusively shown that plum marbling can be transmitted by grafting. “We had symptomatic fruit on those little trees after the first year,” emphasises Bester.
Seed transmission was another important area of investigation. More than two hundred seedlings were raised from plums with symptoms of marbling. The seedlings were tested at five months. All were negative for plum viroid I. The researchers also plan to germinate a second batch of seed and test those. “But the viroid appears not to be seed transmissible,” says Maree.
Team Marbling is conducting trials to test whether marbling can spread within an orchard by mechanical means such as pruning. They tested two groups of healthy trees to ensure that they were free of plum viroid I. The healthy trees were then cut multiple times with a blade contaminated with sap from a known positive tree.
“We’ll be testing those trees at the end of this year,” says Bester, “and if they test positive, we’ll know this is mechanically transmissible. At the moment we don’t know so the best recommendation is to sanitise your pruning equipment.”
The recommended current best practice is to remove trees that show symptoms of marbling. But some growers have — understandably — been reluctant to do this. Trees represent a considerable investment. Is there not a way to salvage some of their value?
Bester and Maree have responded to requests from industry by initiating a trial on top-working infected trees. They identified twenty infected trees from two different orchards and cut them back to below the graft union. Five of the rootstocks have since regrown and been tested for plum viroid I. All tested positive.
The next step is to graft scions from uninfected trees onto the rootstocks of the infected trees. This will take place later this year once all the rootstocks have regrown. These scions will be tested to see whether they become infected.
Bester thinks top-working infected trees is unlikely to be a solution. “There’s no way to revert a diseased tree to a healthy tree. If it has the viroid, then it has it for life.”
“Until you chop it out,” agrees Maree.
Surveying the scene
Team Marbling has tested samples from nine different cultivars in 34 different orchards on seventeen farms in the Western Cape. Close to one hundred percent of the samples from the symptomatic trees tested positive for plum viroid I.
“Wherever we went, we found the viroid,” recalls Maree. “We didn’t test a complete list of cultivars because we relied on self-reporting by growers. We spread the word to get samples.”
They also tested the trees for apple chlorotic leaf spot virus — you may recall that this was originally thought to cause plum marbling. All the trees tested negative. “This is a feather in the cap of the Scheme,” says Maree. Testing for apple chlorotic leaf spot virus forms part of the Deciduous Fruit Plant Improvement Scheme, and they ensure that plant material which tests positive is removed from the propagation pipeline.
The researchers will continue monitoring trees in one infected orchard to see whether plum marbling spreads. Thus far their own observations have been similar to those of growers. “We have trees that we’ve been sampling since 2017 and we don’t see spread. When we return to those orchards, it’s exactly the same trees that are infected every time,” confirms Bester.
The distribution of infected trees in an orchard appears to be random. This supports the theory that the disease is introduced by planting infected material. There is no evidence to suggest that plum viroid I is transmitted by insects or other vectors.
These research projects on plum marbling are set to continue through the current season. The work is funded by Hortgro.
Image: Dr Rachelle Bester and Prof. Hano Maree in their experimental orchard at Welgevallen.