Pathogens and other factors contributing to dark staining on pistachio shells (PS16002)
What’s it all about?
Established in late 2017, this project is delving into dark staining of pistachio shells and nuts – an issue that leaves produce unmarketable and that has been tentatively linked to fungal pathogens Fusarium and Alternaria in preliminary studies.
Its work involves field and laboratory studies to evaluate the occurrence of dark staining and to provide information on causal agents associated with the problem. It is also looking at factors that could influence fungal infection, such as insect damage or environmental conditions.
The project will ultimately develop a monitoring tool for growers, to use in evaluating the risk of fungal disease, and assist in the future development of management strategies.
The research team continued to gather samples from the processing plant for investigation.
They have found that the incidence of dark staining may be seasonally dependent, with environmental parameters such as high relative humidity at the time of hull slip necessary for fungal infection to occur. Weather data from past seasons has been compiled to assess if there is a correlation between dark staining with temperature, rainfall and relative humidity. The 2017 season was not considered wet, however relative humidity at hull slip may have been an influencing factor in the development of dark staining.
With the onset of dark staining at hull slip, oxygen and moisture are exposed to the nutshell. To assess if other environmental factors contributed to dark staining, pistachios shells were analysed for polyphenol oxidase (PPO) activity. Nuts were categorised into varying degrees of dark staining and compared to unstained shells. The enzyme PPO was detected in shells regardless of the level of staining and with no staining at all, suggesting PPO activity occurred with the breakdown of the cell tissue. There was no evidence to suggest dark stained shells had a higher level of PPOs.
To gain a greater understanding of dark staining and associated possible fungal pathogens, planned histological studies for the coming season will find out if fungi invade the pistachio shell or cause breakdown of cell tissue. Pistachio clusters will be inoculated with fungi isolated from dark stained shells under conditions conducive for disease development. This line of study will find out if fungi cause dark staining and assist future management strategies.
To find out more about the cause of the problem in pistachios, fruit was collected at various times at trial sites in Victoria and NSW that had previously had high levels of dark staining. Collections every 5-7 days from ripening to two weeks post-harvest showed dark staining occurred predominantly at hull slip and feathering when the hull no longer adhered to the shell. Dark staining was first detected 8-10 days prior to optimum harvest and the incidence increased after the first harvest shake.
Fungal pathogens were isolated from pistachio hull and shell but the same organisms were found on clean shells. Researchers tried to reproduce the dark staining by inoculating fruit with the fungal infections that they found, but the effects were quite different.
The team has found some clues though. They found that staining was more likely to occur from the inside of the hull and was more predominant at maturity and over-ripening on the tree. More staining was found when the shell was exposed to moisture and air at feathered and broken hull stages, suggesting that the same oxidation that causes browning of fruit might be a factor.
The team is continuing investigations with samples gathered daily from the processing plant to study them further.
As noted in the last project update, in its first year the project had two trials sites where monitoring and sampling during the season and harvest took place, to help determine the development and timing of dark staining. Where dark staining was detected on developing fruits, Alternaria was found as well – however because stained fruit deteriorated prior to ripeness, come harvest and hulling the project team were unable to directly align fruit necrosis with dark staining of shells.
After harvest, fungal pathogens were isolated from shells, including Alternaria, Fusarium and Rhizopus. However these fungi were found in ‘clean’ shells as well as those with dark staining, so the project team were unable to make a strong correlation between the presence of fungi and dark staining.
Meanwhile, the project team conducted experiments where they infected fruit with adhered gulls with fungi, to try to reproduce dark staining of the shells. They note that dark staining occurred up to 28 days after infection, but under the microscope the staining was not like the typical staining caused by fungal infection. They suggest that the staining was more likely associated with phenological oxidation.
Their second year of research will now focus on further pathology work, environmental factors, and biochemical interactions relating to dark staining.
The project has field trial sites at Robinvale in Victoria and Kyalite in New South Wales to monitor the occurrence and development of dark staining. The trials are in orchard blocks that have previously shown high levels of the staining.
During the latest growing season, the crops were monitored for signs of fungal disease on leaves and on developing fruit, with samples collected and fungal pathogens isolated from clusters showing black spots and/or dieback. As noted in the last edition of Hortlink, fruit samples taken during the season showed little evidence of dark staining, and in fruit showing characteristic disease symptoms, Alternaria was detected. Testing is currently underway to assess whether Alternaria is the causal agent of dark staining, or a secondary pathogen – however the project team report that “the infection of pistachio fruits and bunch dieback observed during the growing season is unlikely to contribute to dark staining.”
In 2017, nut processing information showed the incidence of dark staining increased through the harvest season. For this reason, this season a comprehensive sampling of nuts was undertaken during the harvest period at five-day intervals, beginning at 15 days prior to the optimum harvest date and 25 days after. At each timing, fruit were examined for the development and presence of dark staining of the shell in relation to external and internal damage/blackening of the hull.
Nuts were graded according to existing industry standards, including light staining and dark staining. The researchers report that dark staining occurred when hull slip was possible – that is, when the hull no longer adhered to the shell.
Culturing of hull and shell material from symptomatic and non-symptomatic nuts is currently being conducted from each sampling period to look for fungal pathogens. So far a number of detections have been made from infected nuts, including Alternaria, Fusarium and Rhizopus. Work is ongoing to assess their roles as causal agents of dark staining.
This project is a strategic levy investment in the Hort Innovation Pistachio Fund