Epidemiology of the aster yellows phytoplasma: the influence of non-crop hosts on geographic distribution and movement of the pathogen

Epidemiology of the aster yellows phytoplasma: the influence of non-crop hosts on geographic distribution and movement of the pathogen

Summary

A method for quantifying aster yellows phytoplasma (AYp) titer in the aster leafhopper (ALH) was developed and used to examine AYp growth pattern and titer variation among ALHs. Our study demonstrated quantitative real-time PCR (Polymerase Chain Reaction) as a reliable and accurate method for measuring AYp titer in ALHs and detecting differences of AYp titers among insects. An examination of 8 years of ALH scouting data revealed that the period of time associated with above average aster yellows risk is early-June through Mid-July. Thus, the number of pesticide applications could be reduced if plant protection were targeted toward periods of elevated AYp risk.

Objectives/Performance Targets

The original proposal contained two primary objectives:

Objective 1: Accurately identify of the primary reservoir hosts of AYP in habitats surrounding carrot fields and determine which have the greatest epidemiological importance as potential inoculum sources.

Objective 2: I) Compare the genetic structure of the population of AYP isolates collected from reservoir hosts and within affected carrot, and II) determine if AYP variability relates to either disease prevalence or infectivity (virulence) of the pathogen.

Accomplishments/Milestones

Throughout this reporting period we have continued to address objectives 1 and 2 in the proposal although greater focus has been on the development of molecular diagnostic tools to ensure aster yellows phytoplasma (AYp) detection is accurate and reflects the underlying biology of the aster yellows disease system (Objective 2). Complimentary to objective 1, we have continued to examine the phenology of the aster leafhopper (ALH), at multiple spatial and temporal scales to better understand which habitats surrounding carrot fields might have the greatest epidemiological importance as pest or inoculum sources in the agricultural landscape.

To study AYp replication and examine the variability of AYp titer in individual ALHs, we developed a quantitative real-time PCR (qPCR) assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the ALH from 0 to 9 days following a 48 hour acquisition access period (AAP) on an AYp-infected plant. Average AYp titer was measured in 107 ALHs and, expressed as Log10 (copies/insect), ranged from 3.53 (±0.07) to 6.26 (± 0.11) occurring at 1 and 7 days after the AAP. AYp titers per insect and relative to an ALH chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle values obtained for ALHs not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the qPCR assay. This method will improve our ability to study biological factors governing AYp replication in the ALH and determine if AYp titer is associated with frequency of transmission.

The relationship between AYp titer and the ALH’s ability to transmit remains unclear and from our limited data and we are continuing to evaluate the limitations of our assay. We are currently setting up experiments that use a combination of qPCR and transmission bioassays to explore AYp titer in ALH individuals as it relates to an insect’s ability to transmit. The use of qPCR will allow us to measure differences in AYp acquisition, growth and transmission of AYp by ALH among AYp pathotypes that may be present in the environment. The improvements to AYp detection will be directly applicable to the AYp management by providing more accurate estimates of ALH infectivity and, subsequently, more accurate calculations of the Aster Yellows Index.

To examine aster leafhopper phenology, historical pest scouting data (spanning 8 years) in commercial carrot fields were used to determine how the critical components of the aster yellows index, ALH abundance and infectivity, vary in time. We also examined these data to detect and exploit general trends that might help refine our current management tactics. As carrot is produced in Wisconsin, the period of time when the plant is above ground (or when an infection court could be established) occurs from May 25 to September 1, approximately 100 calendar days (infection after September 1 will likely not result in severe symptom development prior to harvest). By examining the overlap between periods of above average ALH abundance and infectivity in the historical data sets, we found that the period of time associated with above average AY risk is approximately 40 days, from early June through Mid-July. Similarly, we visualized the impact of plant host resistance to AY on AY-risk and quantified the risk with respect to the long-term predicted AYI (from the historical data). The period of time protection may be necessary to mitigate yield loss can be reduced by 50% or more by planting AY resistant carrot varieties.

Because the historical aster leafhopper data derived from the AY pathosystem in a managed state and it is difficult to determine how a management change might affect the insect’s phenology. Additionally, while it may be feasible to identify average AY risk periods in long term data sets, the variability within a season may continue to limit our ability to manage those, on-average, high risk periods. Continued in-field surveys and data collection of ALH abundance and infectivity will be helpful for research purposes in the future. We have continued to pursue this avenue of research further and have proposed that repetitive applications of synthetic pyrethroid compounds may be unnecessary to achieve adequate disease control. Ongoing (2011) and future work is being conducted to determine if reduced risk and less broad spectrum insecticides can be used to achieve aster yellows control. These insecticide treatments will be deployed at the high AY risk periods identified above, occurring from early-June through mid-July.

Impacts and Contributions/Outcomes

Our work has contributed to the growing body of research of phytoplasma replication in their insect host by describing the AYp growth pattern and titer variation among individual ALH. A primary contribution of our work was to demonstrate qPCR as a reliable and accurate method for measuring AYp titer in ALHs and detecting differences in AYp titers among insect individuals which is consistent with objective 2 of the original grant proposal. A manuscript detailing this new method has been written and submitted to the Journal of Economic Entomology representing a tangible outcome produced from this proposal.
The examination aster leafhopper phenonlogy in Wisconsin has produced new information about the occurrence of seasonal trends in factors associated with elevated AY risk. We are currently using these data as a benchmark to further refine current pest control strategies, reduce the number of pesticide applications and promote the use of reduced risk, less broad spectrum insecticides for the control of aster yellows. Manuscripts detailing this work are currently in preparation.

Project outcomes to date have been consistent with project goals of developing a multidisciplinary approach for the management of AYP in the carrot producing regions of Wisconsin and are complimentary to a core goal of the NCR-SARE program of enhancing environmental quality through a reduction of insecticide inputs.

Additional outputs from this work include:
Abstracts/Presentations:
Frost, K.E., Willis, D.K. and R. L. Groves. 2010. Variation in aster yellows phytoplasma (Candidatus Phytoplasma asteris) titer in its insect vector, Macrosteles quadrilineatus. Entomological Society of America Annual Meeting. Dec. 9-15, 2010, San Diego, CA.

Extension Proceedings (non-refereed)
Frost, K.E., Van Haren, R. and R. Groves. Historical variation in leafhopper abundance and aster yellows phytoplasma infectivity: assessing periods of elevated risk. [proceedings] Wisconsin Potato and Vegetable Growers Association, Inc. Annual Grower Meeting & Convention. February 1-3, 2011, Stevens Point, WI.

Frost, K.E. and R. Groves. Overwintering non-crop sources of aster yellows phytoplasma. [proceedings] Wisconsin Potato and Vegetable Growers Association, Inc. Annual Grower Meeting & Convention. February 2-4, 2010, Stevens Point, WI.

Trade Journal Publications
Groves R. and Frost, K.E. Towards an Improved Understanding of the Aster Yellows Phytoplasma. In The Badger Common’tater. Wisconsin Potato and Vegetable Growers Association, Inc. July 2010.

Szendrei, Z., Frost, K., and R. Groves. 2010. Aster leafhopper index: what is it and how do you use it? In Carrot Country. Columbia Publishing & Design. Summer 2010.

Collaborators: