Butternut Canker Disease: Inoculation Methodology and Host-Pathogen Interactions

James J Jacobs, Purdue University

Abstract

Author: Jacobs, James, J. PhD Institution: Purdue University Degree Received: August 2017 Title: Butternut Canker Disease: Inoculation Methodology and Host-Parasite Interactions Committee Chair: Dr. Keith Woeste Butternut (Juglans cinerea L.) is a relatively short-lived, shade intolerant tree species that has been devastated by butternut canker disease, forestry practices not conducive to seedling establishment, and hybridization with Japanese walnut (J. ailantifolia). Within a relatively short period of time, the introduced fungus Ophiognomonia clavigignenti-juglandacearum (Oc-j), which causes butternut canker disease was able to move throughout the range of butternut in North America. Over the past 50 years, as populations have precipitously declined, many have observed apparently healthy butternut trees surrounded by diseased individuals. Progeny and grafted clones of these trees have been inoculated under controlled conditions and they have been found to be susceptible to Oc-j. To date, complete resistance has not been identified in J. cinerea. One possible explanation for this apparent discrepancy is that our current methodology is a significant deviation from the processes associated with natural infection and therefore we are bypassing resistance mechanisms that protect these naturally canker-free trees. Additionally, current screening methodology for putative resistant germplasm can take many years. It would be beneficial to develop an alternative screening method to better utilize limited financial resources. To that end, we have developed a rapid, detached leaflet inoculation assay using young seedlings that allows researchers to predict the relative size of stem reaction a given half sib seed family would produce if they were stem inoculated with Oc-j (Chapter 2). Stem and leaflet inoculations of hybrid, butternut, and Japanese walnut trees were carried out to determine if differences in the colonization of leaflet and stem tissues existed between the three hosts, as well as determine if the expression of the disease in host leaflets and stems were related to one another. We report a significant positive relationship in two independent experiments carried out with different seedling families in different years between mean necrotic lesion size produced on leaflets 12 days post inoculation (dpi) with Oc-j and mean canker length 31 weeks post inoculation (wpi) with Oc-j in experiment one, and mean necrotic lesion size in leaflets (12 dpi) and mean canker length (32 wpi) in experiment two. These results are promising and support the incorporation of the methodology into the current screening regime, not only to refine the protocol for an operational program but also to test the protocol over longer time frames than 31 wpi and 32 wpi. Current stem inoculation methodology relies on young saplings. Overall, successful canker initiation via wound inoculation in many field inoculation studies has been inconsistent, and putative resistant selections from heavily infected woodlands developed cankers when challenged with the fungus. While compounds present in the phloem of trees in the Juglandaceae have been previously shown to negatively affect the growth of Oc-j, endophytic organisms may be present within phloem and bark tissues that further inhibit disease progression and may explain the inconsistent success of artificial inoculations (Chapter 3). Endophytic and epiphytic fungi were isolated from stems of hybrid, butternut, and Japanese walnut trees previously grafted and planted in Rosemount, MN and West Lafayette, IN. Individual fungal species’ isolation frequencies varied between the two sites and indicated that the fungal populations associated with these three species may be locally derived. Some isolates collected had the ability to inhibit growth of Oc-j in vitro in co-plate and antibiotic assays, indicating the potential for interaction between these fungi and Oc-j under natural conditions. Our knowledge of host-pathogen interactions in the butternut canker pathosystem is incomplete. Recent evidence that Oc-j can cause necrotic lesions on leaflets raises important epidemiological questions as well as an etiological question, e.g., how does Oc-j initiate infection in leaflets? In Chapter 4 we demonstrate that Oc-j likely infects leaflets solely by penetrating stomata in the absence of wounding. Additionally, it has been suggested that wound inoculating trees by removing all tissues exterior to the cambium may circumvent the tissue best able to limit fungal colonization. Characterizing this interaction histologically (Chapter 4) will inform future breeding strategies as well as provide evidence of the presence of resistance mechanisms within these tissues. We report the presence of a distinct barrier zone consisting of suberin and other phenolic compounds in the phloem of wounded as well as wound-inoculated trees. This reaction was more likely to be present in hybrids and Japanese walnut than in butternut trees. Results of this project indicated that some hybrid and Japanese walnut trees may possess a superior wound response when compared to butternut that may translate to greater resistance to colonization by Oc-j.

Degree

Ph.D.

Advisors

Woeste, Purdue University.

Subject Area

Forestry|Plant Pathology

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