The enigma of pear stony pit

What causes pitted pears in South African orchards? Apple stem pitting virus or antestia stink bug? By Anna Mouton.

Pear stony pit is a disease of pear trees characterised by deformed fruit. Moderately affected fruit are dimpled or pitted. Severely impacted fruit become distorted and develop gritty flesh.

Pear stony pit has been reported internationally since 1939. A South African study published in 1965 found that the disease is graft transmissible. This suggests a viral cause, according to Dr Rachelle Bester of the Department of Genetics at Stellenbosch University.

“The study was repeated around the world, and apple stem pitting virus was associated with these disease symptoms,” said Bester, “but it was never shown conclusively that this virus causes pear stony pit. To complicate things a little more, similar symptoms are caused by stink-bug feeding damage.”

Bester is part of a team that has been delving into potential causes of pitted pears in South African orchards. Her co-workers are Prof. Hano Maree and MSc student Kayleigh Bougard.

Updated tests for virus variants

The researchers began by screening pear trees for apple stem pitting virus using an RT-PCR test they called the Menzel assay. There was a significant association between a positive test and signs of pear stony pit but roughly a third of trees without signs also tested positive.

Bester and colleagues used high-throughput sequencing to generate more data. “High-throughput sequencing is a technique that allows you to detect viruses and viroids without having any prior knowledge,” explained Bester. “So, you don’t need to know what you’re looking for. You can take a sample and bioinformatically identify all of the viruses present in that sample.”

Eighteen samples were examined using high-throughput sequencing — fourteen that had tested positive and four that had tested negative with the Menzel assay. High-throughput sequencing confirmed the presence of apple stem pitting virus in all the Menzel-positive samples as well as two of the Menzel-negative samples.

The results revealed the presence of a variant of apple stem pitting virus that is not detectable using the Menzel assay. This indicated that an updated test was needed. “Our MSc student Kayleigh Bougard designed a multiplex PCR assay that we think will be able to detect all variants of apple stem pitting virus currently known to us,” said Bester.

A total of 125 trees were tested with the updated PCR assay — 78% of symptomatic trees and 87% of asymptomatic trees tested positive for apple stem pitting virus. These results do not support a significant link between apple stem pitting virus and signs of pear stony pit. One reason could be that many infected trees appear healthy — it is common for fruit trees to show few or no signs when infected by viruses.

The various viruses of pear trees

“We wanted to get a better idea of how apple stem pitting virus is distributed in orchards,” said Bester. They surveyed a Beurre Bosc orchard in the Koue Bokkeveld using an ELISA assay. The ELISA assay is relatively inexpensive and suited to screening large numbers of samples. The disadvantage of ELISA is that it may not detect low virus concentrations or all variants of the virus.

A third of the trees tested positive with ELISA. “We found that the distribution of positive trees was completely random,” said Bester. “The virus didn’t seem to be spreading between trees.” These results were not a surprise as apple stem pitting virus is spread through infected plant material — there are no known vectors.

“What is interesting about this orchard is that we’ve been monitoring it for four years,” commented Bester. “Trees that displayed symptoms in year one had symptoms every year.” As she pointed out, stink-bug damage is unlikely to be limited to the same trees year after year. And stink bugs were not observed in this orchard.

In addition to apple stem pitting virus, the researchers found four other viruses in pear trees with the help of high-throughput sequencing — apple chlorotic leaf spot virus, apple stem grooving virus, apple rubbery wood virus 2, and citrus virus A. There were no obvious signs of disease associated with these viruses.

Roughly half of the surveyed trees tested positive for apple rubbery wood virus 2, and citrus virus A, neither of which have previously been reported from pear trees in South Africa.

Stink bugs under scrutiny

To investigate whether stink bugs are to blame for pitted pears, the researchers first had to go bug hunting in orchards showing signs of pear stony pit. They identified large populations of antestia bugs in the Elgin region but not in the Koue Bokkeveld. They then established a captive colony of stink bugs to supply insects for their trials.

Experiments were conducted on four Forelle trees that had tested positive and seven Rosemarie trees that had tested negative for apple stem pitting virus. Clusters of small fruit were enclosed in mesh bags early in the season. Stink bugs were placed in some of the bags whereas bags without stink bugs and unbagged fruit acted as controls.

“Unfortunately, there was a lot of antestia,” said Bester. “We suspect that it was possible for the antestia to feed through the bag.”

Nonetheless, fruit that were bagged with antestia or left open had significantly more damage and deformities than fruit that were enclosed with bags. Apple-stem-pitting-virus positive and negative trees had similar levels of damage.

This research suggests that antestia may well be answerable for pitted pears in the Elgin region. But the Koue Bokkeveld results point the finger at apple stem pitting virus. “The question still remains,” concluded Bester, “Is this virus involved in this disease?”

Image: Dr Rachelle Bester, Stellenbosch University. Supplied by Echo Media.