Recipient Organization
CONNECTICUT AGRICULTURAL EXPERIMENT STATION
PO BOX 1106
NEW HAVEN,CT 06504
Performing Department
Plant Pathology & Ecology
Non Technical Summary
First discovered in 2012 in Lake County, Ohio, Beech Leaf Disease (BLD) has since spread to stands of American beech (Fagus grandifolia) across much of northern Ohio, western and northern Pennsylvania, New York, and Ontario, Canada, and has also been found on European beech (Fagus sylvatica) in Ohio. In 2019, BLD was identified for the first time in southern New York State and southwestern Connecticut. The disease, characterized by dark interveinal banding of leaves appearing soon after spring flush, reappears and advances in trees in subsequent years, with a reduction in buds and therefore foliage, increasing canopy thinning, and tree mortality within five to seven years. Also in 2019, researchers in Ohio, Ontario, and at the USDA confirmed that BLD is caused by a foliar nematode, Litylenchus crenatae mccannii (Lcm), closely related to Litylenchus crenatae crenatae, the nematode associated with leaf gall symptoms on Japanese beech (Fagus crenata). The American beech is an important hardwood forest species of eastern North America, occurring from Nova Scotia and southern Ontario to northern Florida and eastern Texas. With no reports to date of BLD in other New England states, Connecticut is now the gateway for the incursion of this disease into New England, where American beech is a foundational tree species, playing an important role in forest ecosystems, its cavities and canopies supporting nesting sites and shelter, and its nuts constituting a hard mast food source essential to the survival of a variety of vertebrates, from birds to black bears. Dense beech foliage also plays an important role in the forest ecosystem, both in the canopy by modulating light levels in the understory, and as leaf litter, contributing to nutrient cycling on the forest floor. Also susceptible to BLD, the European beech is an important tree of urban landscapes, often occupying iconic roles on town greens and other public spaces in New England's historic towns. The overall goal of this project, strongly grounded in both laboratory and field work, is to track the spread of BLD in Connecticut, and to develop and deploy genetic tools essential to improving our understanding of how Lcm moves within sites and from one site to another. An annual distribution survey for presence of BLD in Connecticut's forests will allow us to firmly determine the current extent of BLD in Connecticut, and track the disease as it advances in each subsequent year. In the laboratory, we will use the Lcm genome to identify molecular markers known as microsatellites - the same type of marker used in human forensics, and for many other organisms. These genetic markers will allow us to genotype individual Lcm nematodes, and thereby to track the origins of new incidences of BLD that are identified by surveys, and test hypotheses about how the nematode disperses to begin new infections. There are no reports to date of BLD in other New England states, and as of September of 2019, BLD was found only in the very southwest corner of Connecticut. Therefore, Connecticut can be considered the gateway for the incursion of this disease into New England, where American beech increases in representation and importance. Additionally, the genetic markers developed for this project will continue to serve an important role in studying the dispersal of Lcm as BLD spreads further into New England and into other eastern states. Insights into modes of transmission of Lcm gained through molecular genotyping will also be important to the development of management strategies. This project will involve collaborations with researchers in Ohio, West Virginia, Ontario, Canada, and the USDA, building upon strategic relationships based on complementary and overlapping approaches and areas of expertise.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The goals of this project are to (1) undertake a distribution survey of Beech Leaf Disease in Connecticut in order to establish its current extent, with subsequent annual surveys to chart the spread of the disease; (2) using a whole-genome sequence, develop a minimum of 15 polymorphic microsatellite markers to be used for testing hypotheses on the origins and movement of the nematode in North America; (3) using these microsatellite markers, study the population genetics of Lcm; and (4) conduct public outreach through print (e.g. fact sheets), internet (CAES website), and public presentations.
Project Methods
1. BLD Distribution Survey: This will consist of field work occurring at various locations in Connecticut. To conduct this survey, we will first generate an inventory of forests, parks, and preserves in Connecticut with significant populations (≥20%) of American beech. Municipal parks with significant specimens of European beech will also be inventoried. After leaf-out (~mid-late May), we will visit target sites, systematically radiating outward north and east from 2019 sites with BLD in Fairfield County. Frequency of site visits will depend on staff availability. The data to be collected will include:Site data: slope, aspect, soil type, GPS coordinates;Percentage of beech;Co-occurring tree species;Percentage of symptomatic trees;Dbh range;Percentage of symptomatic foliage.Symptomatic foliage will be collected from trees at the edges of infected sites as well as the center. All sampled trees will be flagged and recorded. Leaves will be collected in Ziploc-type bags and stored at 4°C for downstream nematode isolations, for use in Objective 2.To identify new sites and track the spread of the disease in subsequent years, with each new season the survey will focus on the outermost sites recorded the previous year and radiate outward north and east from those sites.2. Polymorphic microsatellite markers for Litylenchus crenatae mccannii (and possibly L. crenatae crenatae): This laboratory-based work will be conducted in the Marra Lab at CAES, in New Haven, CT. Sequencing and microsatellite fragment analysis will be conducted at Yale University, New Haven, CT.This process will require the extraction of high-quality high-molecular-weight DNA from a large number (approximately 100,000) Lcm nematodes, which will be isolated from freshly collected symptomatic infested leaves.The DNA will then be processed at the Yale University Center for Genome Analysis (alternatively, Ohio State University) using current whole-genome sequencing (WGS) technology (e.g., PacBio, Illumina, or Nanopore); the resulting "reads" will then be assembled by the sequencing facility.The resulting assembled "contigs" will then be used to screen the WGS for microsatellite loci using Genome-wide Microsatellite Analyzing Tool (gMATo) (software freely available at sourceforge.net).Once the microsatellite loci are identified, PCR primers flanking each candidate locus will be designed using Primer3-Plus software (freely available at primer3plus.com). The primers will then be ordered from a primer-synthesis company (e.g., Sigma).Each microsatellite locus will then be queried for length polymorphisms among a screening population consisting of 24-32 isolates, which will be obtained from sites representing the range of current infestations in North America (and, if possible, Japan, in order to query the suitability of these primers for the Lcc subspecies.Candidate microsatellite loci will be tested in this way until a minimum of 15 informative (i.e., polymorphic) loci are obtained, generating a "suite" or microsatellite markers to be used and tested for segregation of polymorphisms from within trees, and within and among sites.3. Population genetics of Litylenchus crenatae mccannii. From each site sampled in CT, the following sampling scheme will be used to collect symptomatic leaves for nematode extraction and microsatellite genotyping.From each site, one tree will be sampled at the center of the site, along with four trees evenly distributed at the perimeter of the site, approximating four ordinal corners. Hierarchical sampling will be conducted as follows: from each tree, symptomatic leaves will be collected from each of the four ordinal corners of the tree, at ground level and at a height that is within reach with a pole pruner, yielding eight leaves per tree. From each leaf, a symptomatic portion will be sampled using a standard hole-punch to obtain 3-4 discs; these will be floated in water in a 60 mm petri dish overnight at room temperature. The following day, 8-12 individual nematodes will be transferred individually to buffer and frozen prior to a standard freeze-boil lysis. Resulting lysates will be used for microsatellite genotyping using markers developed in Procedure 2.4. Public Outreach: The goal of this procedure is to keep stakeholders informed on the occurrence of BLD in CT, and as new information is generated through research. Fact sheets and the CAES website will be updated with new information on the disease, and on locations of new infestations. Presentations suitable for the general public will be developed and used for presentations at various venues; e.g. garden clubs and tree and landscape professionals.