The black walnut, Juglans nigra L., is native to the United States (USA) and is a valuable timber and nut tree. Just before the beginning of the 21st century, several western states observed a decline in the health and, later, death of black walnut trees. The pathogen-vector complex now known as thousand cankers disease (TCD) was shown to be the cause. The disease, caused by Geosmithia morbida Kolařik, is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis Blackman. Thousands of WTB will swarm and enter a tree vectoring the fungus at each entry point where cankers then develop, quickly expand, coalesce and kill the branch or stem. The disease has been confirmed across the USA and in parts of Europe. The research and development of resistant cultivars is important to maintain native populations and livelihoods. The purpose of this project was to evaluate black walnut and hybrid trees for resistance to TCD through direct inoculation with the pathogen G. morbida. Inoculation of limbs took place in early summer of 2015, 2016 and 2017 at the Cyril Reed Funk Research Farm in Richmond, UT and Dayton, ID. Inoculated limbs were removed from the tree after senescence and canker size measured. An average of 336 trees were inoculated. One tree consistently exhibited resistance to TCD indicated by no canker staining. An additional 14 trees exhibited resistance for two of the three years. The results of this project indicate that breeding for resistance to TCD could be a management option for the disease.

In the early 1900's the American chestnut (Castanea dentata (Marsh.) Borkh.) was nearly destroyed by the introduction of the orange-pigmented, chestnut blight fungus (Cryphonectria parasitica (Murr.) Barr). Chestnut blight is less severe in Europe, where hypovirulent (= reduced virulence) strains of the fungus are found to be associated with healing cankers. These European hypovirulent strains are infected with a dsRNA virus, Cryphonectria hypovirus 1 (CHV1), and have a white phenotype when grown in culture. Transmission of CHV1 in C. parasitica is limited by incompatibility between isolates in different vegetative compatibility (vc) types. In 1982-83, naturally formed blight cankers on American chestnut grafts, derived from large survivors, were inoculated with a mixture of four European (white) hypovirulent strains of C. parasitica. After 14 years the white strains were recovered throughout the inoculated grafts, which had low levels of blight damage. CHV1 had infected at least 45 new vc types, and was present in four different fungal colony morphology groups, including one type that had intermediate or partial pigmentation. However, CHV1 was unable to move throughout a single vc type within a natural canker. The objectives of this study were: 1) to determine the frequency and phenotypic diversity of CHV1-infected C. parasitica isolates recovered from stromata and canker tissue from natural cankers on the grafted American chestnut trees and artificially established cankers on forest American chestnuts; 2) to determine the presence or absence of CHV1 in intermediate-pigmented isolates recovered from the American chestnut research plots; 3) to investigate the roles of colony age, resistance to hypovirus infection, and functional mycelial units in the failure of CHV1 to move throughout a vc type of C. parasitica in vitro, and; 4) to examine the role of low temperatures and a high elevation topographic site on CHV1 survival within C. parasitica colonies in vivo and in vitro. The results indicated that there was no direct correlation between the amount of colony pigmentation and the presence of dsRNA. Within each of the three colony phenotype categories (pigmented, intermediate and white), several C. parasitica isolates tested positive for the presence of CHV1. This presence of CHV1 in intermediate isolates, coupled with the relatively large number of intermediate isolates collected from stromata on cankers, indicates that intermediate isolates may perform an important, and previously overlooked, function in biological control of chestnut blight. In this study, all CHV1 movement trials indicated that the age of the C. parasitica colony limited the movement of CHV1 throughout the colony. The majority of the CHV1 movement through a C. parasitica colony occurred between 0 and 7 days following challenge with an isogenic CHV1-infected strain. Isolation data using a lattice grid did not indicate a consistent pattern of CHV1 movement throughout a C. parasitica colony. Low temperatures associated with high altitude had no effect on hypovirus survival in vivo or in vitro. Additionally, no long-term C. parasitica resistance to CHV1 infection or movement was identified in this study. This research has identified new insights into CHV1 spread and survival that may be important in understanding the role of CHV1 in the biological control of chestnut blight. / Ph. D.

Isolates of Monilinia fructicola (Wint.) Honey obtained from stone fruit orchards in Hawkes Bay, North Island and from Californian fruit exported to New Zealand, were tested for resistance to methyl benzimidazole carbamate (MBC). Resistant isolates from the North Island had EC₅₀ values of >30,000, and most isolates from the imported fruit had of values approximately 1.5 mg a.i./l carbendazim. Sensitive isolates failed to grow on 1 mg a.i./l carbendazim. A detached peach shoot system was used in controlled conditions for estimation of values for incubation period, latent period and rate of spore production on flowers (cv Glohaven). The same variables and the rate of colonisation of host tissue were measured on fruit (cv Fantasia) in controlled conditions. An inoculum density of 1x10⁴ spore/flower or fruit greatly increased fitness in vivo compared to an inoculum density of 1x10² spore/flower (fruit). Isolates varied considerably, but there was no consistent relationship between the degrees of resistance and fitness. This was in contrast to earlier studies with dicarboximide resistant strains of M. fructicola. The survival in the field of 10 isolates resistant or sensitive to MBC or dicarboximide fungicides on twig cankers and mummified fruit was compared. The ability to produce conidia on twig cankers inoculated in late spring 1989 was maintained by all sensitive and MBC resistant isolates for at least 1 year. The production of conidia on mummified fruit inoculated in February 1990 decreased after 2-3 months in the field but some conidia were still produced on all fruit in the following spring. Dicarboximide resistant isolates produced less conidia than either the MBC resistant and the sensitive isolates. The pathogenicity and fitness of all isolates were similar to the original values after survival for 1 year. A technique was developed to produce apothecia reliably from inoculated peach (cv Black Boy) and nectarine (cv Fantasia) fruit in controlled conditions in the laboratory. The fruit were inoculated with resistant or sensitive isolates, or combinations, and were incubated for 8 weeks at 25°C (±1°C) with 12 hours photoperiod of fluorescent light (Sylvania 2x65 W, daylight) to produce mummified fruit. The fruit were then buried in moist autoclaved peat moss for 10 weeks at 25°C (±1°C) in the dark to form stromata. These fruit were then hydrated with running tap-water (total hardness (CaCO₃) = 47 g/m³ and conductivity at 20°C = 12.7 mS/m) for 72 hours. The hydrated mummified fruit were placed in moist peat moss and were incubated for 13-14 weeks at 8°C (±0.5°C) in the dark. At the end of this period, stipe initials were visible. Differentiation of stipe initials into mature apothecia occurred within 15-20 days after transfer to 12°C (±2 °C) with a 12 hour photoperiod of fluorescent and incandescent light. All isolates produced apothecia when treated in this way. A technique for isolation of ascospore sets in linear arrangement was developed for tetrad analysis of the inheritance of resistance. At least 3 hours of fluorescent and incandescent light at 12°C (±2°C) was essential to allow ascospore ejection from individual asci taken from apothecia previously maintained in a 12 hour photoperiod at 12°C (±1°C). A water film on the surface of water agar was necessary to hold a set of ejected ascospores in linear sequence. Single ascospores were obtained in sequence with the aid of a micromanipulator. Genetic analysis of MBC resistant isolates was carried out on ascospores derived from apothecia produced in the laboratory. Analysis of ascospore sets in linear arrangement and ascospore populations indicated that resistance to >30,000 mg a.i./l carbendazim (high-resistant) is governed by a single major gene and is affected by gene conversion mechanisms. Crossing over was frequent, suggesting that recombination of resistance with other characters, such as pathogenicity and fitness, may occur readily. The segregation ratio (1:1) from most resistant isolates revealed that heterokaryons containing both resistant and sensitive alleles were common in resistant populations and that resistance is dominant. Allozyme analysis of ascospore progeny through electrophoresis revealed a narrow genetic base of M. fructicola in New Zealand. The technique for reliable apothecial production in controlled conditions developed in this study provided an important step for the determination of the biology of M. fructicola strains resistant to MBC fungicides, and the complexity of its life cycle. Genetic heterogeneity in field populations can be conserved in one isolate through heterokaryosis, thus providing for adaptability of the pathogen to the changing environmental conditions. Knowledge on genetic variability, overwintering ability, pathogenicity and fitness factors may be useful for future management strategies of stone fruit brown rot. Special emphasis should be made in particular to prevent primary infection on blossoms, which would delay the establishment of recombinant strains of M. fructicola and the onset of brown rot epidemics.