The Pennsylvania State University The Graduate School College of Agricultural Sciences BIOLOGICAL CONTROL OF AILANTHUS ALTISSIMA: TRANSMISSION, FORMULATION, AND RISK ASSESSMENT OF VERTICILLIUM NONALFALFAE A Thesis in Plant Pathology by Eric S. O’Neal © 2014 Eric S. O’Neal Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2014 ii The thesis of Eric S. O’Neal was reviewed and approved* by the following: Donald D. Davis Professor of Plant Pathology and Environmental Microbiology Thesis Advisor David M. Geiser Professor of Plant Pathology and Environmental Microbiology John A. Pecchia Assistant Professor of Plant Pathology and Environmental Microbiology Dennis R. Decoteau Professor of Horticulture and Plant Ecosystem Health Frederick E. Gildow Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School iii ABSTRACT Tree-of-heaven (Ailanthus altissima), first introduced to the United States into Philadelphia in 1784, is a very aggressive and invasive weed in both forest and urban environments, especially within the Mid-Atlantic region. In 2002 to 2003, in south- central Pennsylvania, Verticillium nonalfalfae, a soilborne, vascular wilt pathogen, was found naturally killing thousands of Ailanthus trees. To date, V. nonalfalfae has been isolated from dying Ailanthus in Pennsylvania, Virginia, and Ohio. Verticillium nonalfalfae causes typical Verticillium wilt symptoms on Ailanthus, including wilt, yellow vascular discoloration, defoliation, and mortality. The fungus naturally spreads rapidly through Ailanthus stands from diseased to healthy Ailanthus trees. Due to its efficacy and relatively small host range, V. nonalfalfae isolate VnAa140 has been proposed as a biological control for the invasive Ailanthus in Pennsylvania. In this thesis there were two main objectives. The first objective was to determine if intraspecific root grafts and clonal root systems in Ailanthus stands play roles in rapid local V. nonalfalfae transmission. The phenomenon of root grafting has been reported in >100 tree species and can aid in the transmission of pathogens from diseased to healthy trees, for example, those causing oak wilt and Dutch elm disease. Ailanthus altissima has not been reported to form root grafts, nor has it been reported that Verticillium spp. can be transmitted by root grafts in trees. Understanding the transmission of this potential biological control is important for optimized deployment and assessing risk. Also, investigating root grafting in Ailanthus altissima will allow for greater understanding of its invasion strategy and biology. The second objective of this thesis was to develop a simple formulation and delivery system for V. nonalfalfae isolate VnAa140 and explore the use of natural V. iv nonalfalfae inoculum sources, while further investigating this potential biocontrol agent’s safety. The level of control achieved by V. nonalfalfae surpasses herbicide treatments currently in use; however it is not formulated for anticipated expanded use by land managers. The development of an optimized formulation and delivery system for V. nonalfalfae will allow for more effective and efficient control of Ailanthus than conventional herbicide control treatments. In addition, developing simple inoculation methods utilizing natural inoculum sources will allow land managers to use V. nonalfalfae without complete dependence on laboratory-prepared inoculum. Lastly, the non-target plant species composition and health was reassessed within the Verticillium wilt epicenter where V. nonalfalfae isolate VnAa140 was originally isolated. In this area of Tuscarora State Forest, V. nonalfalfae has persisted and killed mature canopy Ailanthus trees for over a decade. This assessment will help to further investigate this biological control agent’s risk, efficacy, and host-adaptedness to Ailanthus. v TABLE OF CONTENTS List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Acknowledgements ..............................................................................................................x Chapter I. LITERATURE REVIEW: VERTICILLIUM WILT AND BIOLOGICAL CONTROL OF AILANTHUS ALTISSIMA .....................................................1 Introduction to Ailanthus altissima .................................................................1 Introduction to Verticillium nonalfalfae .........................................................4 Verticillium wilt of Ailanthus altissima ....................................................6 Transmission of Verticillium nonalfalfae .................................................7 Insect vectors ......................................................................................8 Infected leaves, seeds, and wood ........................................................9 Intraspecific root grafts and root contact ..........................................10 Biological Control of Weeds Using Plant Pathogens ...................................13 The use of mycoherbicides in forest management ..................................14 Formulation and delivery of mycoherbicides .........................................16 Formulation of Verticillium nonalfalfae ...........................................18 Literature Cited .............................................................................................21 Figures...........................................................................................................34 Chapter II. ROOT GRAFTING AND CLONAL GROWTH IN AILANTHUS ALTISSIMA AND THEIR ROLE IN VERTICILLIUM TRANSMISSION ..37 Introduction ...................................................................................................38 Materials and Methods ..................................................................................42 Study sites ...............................................................................................42 Detection of root grafts using dye translocation .....................................42 Mapping of dye translocation and estimation of tree connectivity .........43 Air-spade excavation of Ailanthus root systems .....................................44 Culture maintenance, inoculum preparation, and isolation ....................45 DNA extraction and molecular identification of isolates .......................47 Intraspecific root graft inoculations ........................................................47 Ailanthus clone inoculation.....................................................................49 Results ...........................................................................................................50 Detection of root grafts using dye translocation .....................................50 Mapping of dye translocation and estimation of tree connectivity .........52 Air-spade excavation of Ailanthus root systems .....................................52 Intraspecific root graft inoculations ........................................................53 Ailanthus clone inoculation.....................................................................53 Discussion .....................................................................................................54 Acknowledgements .......................................................................................66 Literature Cited .............................................................................................67 Tables and Figures ........................................................................................73 vi Chapter III. BIOCONTROL OF AILANTHUS ALTISSIMA: FORMULATION AND RISK ASSESSMENT OF VERTICILLIUM NONALFALFAE ....................91 Introduction ...................................................................................................92 Materials and Methods ..................................................................................96 Study sites ...............................................................................................96 Culture maintenance, inoculum preparation, and isolation ....................97 DNA extraction and molecular identification of isolates .......................99 Preliminary V. nonalfalfae formulation field testing ............................100 Verticillium nonalfalfae isolate VnAa140 formulation and delivery ...100 Field testing of soil-formulated V. nonalfalfae inoculum .....................102 2012-2013 monthly canopy Ailanthus tree inoculations ......................103 Control of Ailanthus using natural V. nonalfalfae inoculum ................103 Infested soil as inoculum ................................................................104 Infected wood chips as inoculum ....................................................104 Infected wood debris as inoculum ..................................................105 Infected leaves as inoculum ............................................................106 Forest composition and health after a decade of Ailanthus mortality ...107 Statistical analyses ................................................................................107 Results .........................................................................................................108 Preliminary V. nonalfalfae formulation field testing ............................108