Disease management in organic apple orchards in Vermont is focused on controlling diseases with sulfur fungicides. The objective of this two year study was to evaluate the target and non-target effects of an organic disease management system containing agricultural biostimulants compared to two sulfur-based systems on foliar and fruit diseases, pest and beneficial arthropods, tree growth, yield and fruit quality on four cultivars, `Ginger Gold', `Honeycrisp' and `Liberty' and `Zestar!'. Trees were arranged in a complete randomized design of five three-tree replications in a certified organic orchard. The two sulfur-based systems differed in the number of applications; in the third system, sulfur was replaced with biostimulants including pure neem oil, liquid fish, an activated microbial inoculant plus equisetum and stinging nettle teas. Each biostimulant application also included kelp meal, unsulfured organic molasses and yucca extract emulsifier. The biostimulant system did not successfully manage apple scab and rust diseases as well as the sulfur-based fungicide systems, and had variable results with other diseases. No differences were observed among the three systems in tree growth parameters; however, the length of the study may not have been sufficient to determine effects. Differences in the incidence of disease among the three systems were reflected in extrapolated figures for gross income per hectare which takes into account fruit yield and quality. In the higher fruit-bearing year of the study, it was estimated that the gross income per hectare of the biostimulant system would be significantly lower than the reduced-sulfur system and the full-sulfur system by at least $5,800 and $12,000, respectively. In that same year, it is estimated that the full-sulfur system would have generated approximately $6,500 more gross income per hectare than the reduced-sulfur system suggesting the number of sulfur sprays can influence fruit quality and income. The use of the agricultural biostimulants had very limited non-target effects and when present, they were beneficial in suppressing insect pest incidence and/or damage on foliage compared to one or both of the sulfur-based fungicide systems. However, many insect pests or their damage were not observed on the foliage or had incidence of less than 1% in any of the systems. The biostimulant system did appear to suppress European red mites in both years compared to both sulfur-based systems when data were averaged across cultivars. On fruit, no differences in non-target impacts on arthropod pests were observed among the three systems except for surface-feeding Lepidoptera and San Jose scale damage. In a separate phytophagous mite study on the cultivar `Zestar!' leaf samples were evaluated for the number of motile phytophagous mites every 14 days from 1 July through 26 August each year. When there were differences, the biostimulant system had less mite incidence per leaf than one or both of the sulfur-based systems in both years. The difference in the number of sulfur sprays did not have a major effect on the mite populations. In summary, the use of the biostimulant system resulted in insufficient disease management which led to lower estimated gross income compared to the sulfur-based systems. These results show more research and further evaluation of new organic disease management tools, including the use of agricultural biostimulants, are necessary before growers consider replacing the use of standard sulfur fungicides for disease management in Vermont orchards.

The diploma thesis is focussed on understanding relationships between soil chemical characteristics, actinobacterial communities of agricultural field soils and occurrence of potato common scab, a disease caused by members of the genus Streptomyces. The aim of monitoring study, on thirty-three sites covering main potato- growing regions in the Czech Republic, was to find relationships suitable for prediction of common scab severity. The second part of the thesis compared actinobacterial communities and incidence of Streptomyces harboring a pathogenic determinant, gene txtA (gene of biosynthetic pathway of phytotoxin thaxtomin A), in soils differing in occurrence of common scab. In the screening study, analysis of terminal restriction fragment length polymorphism (T-RFLP) was employed to compare composition of soil actinobacterial communities. Real-time PCR was used to quantify total actinobacteria and streptomycetes harboring txtA gene in soils differing in scab incidence. The screening study revealed negative correlations between the scab severity and (i) available phosphorus in soil and (ii) diversity of actinobaterial community. The results were used to design a model for scab prediction. A qPCR analysis showed difference in numbers of total actinobacteria and the strains harboring txtA gene in...

Fusarium head blight (FHB) is a serious disease of wheat (Triticum aestivum) and other small grains; disease severity is affected by temperature and rainfall. This research comprised three studies: an artificially warmed experiment during 2016-2017, a morphology study and an FHB resistance screening study in 2015-2016, using approximately 250 wheat cultivars and breeding lines from programs in the eastern US. The location was the University of Kentucky Spindletop Research Farm in Lexington, KY. Higher levels of Fusarium damaged kernels and the toxin deoxynivalenol (DON) were observed in the warmed treatment compared to the control, and plant development was accelerated. In the FHB resistance screen, significant (p < 0.05) genotype differences for all traits were observed. A GWAS identified 16 SNPs associated with resistance and susceptibility, ranging from -2.14 to 4.01%. Three DON-associated SNPs reduced toxin levels by 3.2, 2.1, and 1.5 ppm. In the morphology study, negative correlations were observed among morphological and disease traits. Small effect SNPs were identified for all morphological traits, which might be useful in genomic selection; traits like spike length, spikelet number and inclination could be used in phenotyping. Response to warming indicates that existing resistance sources may be less effective in a warming climate.

In 1992, a research program was initiated to select suitable antagonists against the saprophytic (or winter) phase of the apple scab pathogen, Venturia inaequalis. An improved method for the mass screening of a vast collection of fungi was developed for this purpose. Some of the previously reported criteria such as leaf rheology and overwintering structure production proved unreliable or fastidious and cannot be used for in vitro antagonist selection. The main antagonism selection criterium retained was the in vitro inhibition of ascospore formation. To measure ascospore production, a simplified method of in vitro pseudothecia production was devised. This new method eliminates the need for prior conidia production by using a mycelial suspension and greatly reduces the risks of sterile mating by using a cocktail of Venturia inaequalis strains of different origins. Finally, the production cycle duration was reduced by varying the incubation temperature during the simulated winter. Moreover, a quick and efficient method of ascospore collection was developed. Ascospores were forcibly ejected in a large scale bubbler type apparatus in only one hour. This bubbler can be used for other studies including axenically produced ascospores. This new method compared favourably to the previously reported method and was used to screen a collection of about forty-two fungi. Six proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of saprophytes can now be based on a reliable and simple in vitro screening methodology.

Venturia inaequalis, the causal agent of apple scab, overwinters in apple leaves on the orchard floor. To develop a control strategy based on the prevention of the maturation of overwintering pseudothecia, a sampling of fungi colonizing dead apple leaves was conducted from different orchard floors in Quebec during the spring and fall of 1993. A total of 345 different isolates were obtained, from which fifteen genera have never been previously recorded as colonizers of apple leaves in North America. Small differences were detected in genera richness among orchards but the fungal composition of each orchard was fairly unique. Different tests on growth on amended media and leaf decomposition demonstrated that leaf degradation is not a reliable parameter alone to screen antagonist against V. inaequalis. No significant relation between growth on amended media, leaf rheology and ascospore inhibition was detected. However, 40 fungi reduced significantly ascospore production more than 87% compared to the control (V. inaequalis only). Of these antagonists, 30% decomposed apple leaves, suggesting that competition for the substrate is involved in the mode of action of at least one third of the antagonits detected. Other possible modes of antagonism are discussed.

Fusarium head blight (FHB) or head scab, caused by Fusarium graminearum Schwabe [telomorph: Gibberella zeae Schwein.(Petch)], is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Numerous strategies for scab resistance breeding are in use, including phenotypic selection for low severity and marker-assisted selection for resistance QTL. The most destructive consequences of scab are evidenced through a reduction in grain quality, and the presence of mycotoxins, the most common of which is deoxynivalenol (DON). Thus, there is great interest among breeders in selecting for resistance to both of these traits. To this end, a study was devised as follows. In 2010, 20 bulk F3 SRW wheat populations with scab resistant parents in their pedigrees were harvested by population from unreplicated plots near Lexington, KY. The plots were affected by a naturally occurring mild-moderate scab epidemic. The grain was sorted on a USDA/ARS and National Manufacturing Seed Sorter System with color camera according to a calibration that reflected visual differences between asymptomatic grain and grain showing FHB symptoms. This process was repeated in 2011 using grain from plots that had conidial suspension applied at anthesis. In 2012, an unreplicated plot study of the C0, C1 and C2 cycles of selection, inoculated with grain spawn and conidial suspension, was evaluated for Fusarium damaged kernels (FDK) and DON concentration. An additional cycle of selection was conducted by running the bulk grain through the sorter. In October 2012, 4 selection cycles of the 20 populations were planted in a RCB experiment at Lexington and Princeton, KY. Bulk populations were planted in both scab nursery and plots, and C3 accepted and rejected of all populations and derived lines of 2 populations were planted in the scab nursery in Lexington, KY. Some populations had FDK and DON reduction with selection, and some derived lines had either numerical or significant reduction with selection. Although the accepted fraction had non-significant reduction compared with the rejected fraction over the populations, FDK and DON means were obviously lower in accepted than in rejected fractions.

Apple scab is one of the greatest apple management problems throughout the world Much work has been done on cultivars resistant to Venturia inaequalis (Cke.) Wint., but few have been a commercial success. This frequently leaves fungicides as the only control method used. As Quebec growers select new cultivars for planting, more information is needed on their relative susceptibility for efficient scab control. In this light, 21 cultivars common to central and eastern Canada, were examined for their relative susceptibility using several components of partial resistance; disease severity, incubation period, latent period, lesion size, and conidial production. The cultivars used were Cortland, Early Geneva, Empire, Golden Delicious, Golden Russet, Idared, Jersey Mac, Jonagold, Jonamac, Lobo, Lodi, Summerland McIntosh, Mutsu (Crispin), Northern Spy, Paulared, Red Cortland, Red Delicious, Royal Gala, Spartan, Sunrise, and Vista Bella. A final ranking of the cultivars and selection of partial resistance components was done using the principal components analysis. (Abstract shortened by UMI.)

The use of a biofungicide on the perfect stage of V. inaequalis on leaf litter is one potential way to reduce the number of fungicides used to control apple scab. The previous in vitro screenings of Quebec mycoflora have shown that several isolates are able to significantly reduce the primary inoculum of the pathogen. Among the screened fungi, P176A and P130A, reduced over 98% of the ascospore production and were as effective as Athelia bombacina. However, because in vitro tests are generally poor predictors of in vivo assays a re-evaluation of the antagonists was done under field conditions. Eight fungal isolates, leaf shredding, and two comparative treatments (A. bombacina, 5% urea) were applied to intact scabbed leaves in October 1994 and 1995. After the treatments, the leaves overwintered on the orchard ground until the next spring. In April, samples of treated leaves were randomly selected and placed in spore traps to collect the ejected ascospores during rainfall. Since the primary inoculum was ejected during a four-month period, antagonism was based upon ratings taken throughout the whole ejection season. To evaluate the effect of incubation conditions on the antagonistic performance we incubated separately, in vitro and in vivo, sterile leaf disks which were artificially inoculated with V. inaequalis and fungal isolates. (Abstract shortened by UMI.)

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.