Genetic differentiation and spatial structure of Geosmithia morbida, the causal agent of thousand cankers disease in black walnut (Juglans nigra).

The main objectives of this study were to evaluate genetic composition of Geosmithia morbida populations in the native range of black walnut and provide a better understanding regarding demography of the pathogen. The fungus G. morbida, and the walnut twig beetle, Pityophthorus juglandis, have been associated with a disease complex of black walnut (Juglans nigra) known as thousand cankers disease (TCD). The disease is manifested as branch dieback and canopy loss, eventually resulting in tree death. In 2010, the disease was detected in black walnut in Tennessee, and subsequently in Virginia and Pennsylvania in 2011 and North Carolina in 2012. These were the first incidences of TCD east of Colorado, where the disease has been established for more than a decade on indigenous walnut species. A genetic diversity and population structure study of 62 G. morbida isolates from Tennessee, Pennsylvania, North Carolina and Oregon was completed using 15 polymorphic microsatellite loci. The results revealed high haploid genetic diversity among seven G. morbida populations with evidence of gene flow, and significant differentiation among two identified genetic clusters. There was a significant correlation between geographic and genetic distance. Understanding the genetic composition and demography of G. morbida can provide valuable insight into recognizing factors affecting the persistence and spread of an invasive pathogen, disease progression, and future infestation predictions. Overall, these data support the hypotheses of two separate, highly diverse pathogen introductions into the native range of black walnut.

Two-year field analysis of reduced recalcitrance transgenic switchgrass.

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.

Field Resistance to Rose Rosette Disease as Determined by Multi-Year Evaluations in Tennessee and Delaware.

Rose rosette disease (RRD) caused by the rose rosette emaravirus (RRV) and transmitted by the eriophyid mite Phyllocoptes fructiphilus (Pf), both native to North America, has caused significant damage to roses over the last several decades. As cultural and chemical control of this disease is difficult and expensive, a field trial was established to systematically screen rose germplasm for potential sources of resistance. One hundred and eight rose accessions representing the diversity of rose germplasm were planted in Tennessee and Delaware, managed to encourage disease development, and evaluated for symptom development and viral presence for three years. All major commercial rose cultivars were susceptible to this viral disease to varying levels. The rose accessions with no or few symptoms were species accessions from the sections Cinnamomeae, Carolinae, Bracteatae, and Systylae or hybrids with these. Among these, some were asymptomatic; they displayed no symptoms but were infected by the virus. Their potential depends on their ability to serve as a source of viruses. The next step is to understand the mechanism of resistance and genetic control of the various sources of resistance identified.

Rose Rosette Disease Resistance Loci Detected in Two Interconnected Tetraploid Garden Rose Populations.

Rose rosette disease (RRD), caused by the Rose rosette emaravirus (RRV), is a major threat to the garden rose industry in the United States. There has been limited work on the genetics of host plant resistance to RRV. Two interconnected tetraploid garden rose F1 biparental mapping populations were created to develop high-quality tetraploid rose linkage maps that allowed the discovery of RRD resistance quantitative trait loci (QTLs) on linkage groups (LGs) 5, 6, and 7. These QTLs individually accounted for around 18-40% of the phenotypic variance. The locus with the greatest effect on partial resistance was found in LG 5. Most individuals with the LG 5 QTL were in the simplex configuration; however, two individuals were duplex (likely due to double reduction). Identification of resistant individuals and regions of interest can help the development of diagnostic markers for marker-assisted selection in a breeding program.

Identification of QTLs for Reduced Susceptibility to Rose Rosette Disease in Diploid Roses.

Resistance to rose rosette disease (RRD), a fatal disease of roses (Rosa spp.), is a high priority for rose breeding. As RRD resistance is time-consuming to phenotype, the identification of genetic markers for resistance could expedite breeding efforts. However, little is known about the genetics of RRD resistance. Therefore, we performed a quantitative trait locus (QTL) analysis on a set of inter-related diploid rose populations phenotyped for RRD resistance and identified four QTLs. Two QTLs were found in multiple years. The most consistent QTL is qRRV_TX2WSE_ch5, which explains approximately 20% and 40% of the phenotypic variation in virus quantity and severity of RRD symptoms, respectively. The second, a QTL on chromosome 1, qRRD_TX2WSE_ch1, accounts for approximately 16% of the phenotypic variation for severity. Finally, a third QTL on chromosome 3 was identified only in the multiyear analysis, and a fourth on chromosome 6 was identified in data from one year only. In addition, haplotypes associated with significant changes in virus quantity and severity were identified for qRRV_TX2WSE_ch5 and qRRD_TX2WSE_ch1. This research represents the first report of genetic determinants of resistance to RRD. In addition, marker trait associations discovered here will enable better parental selection when breeding for RRD resistance and pave the way for marker-assisted selection for RRD resistance.

Bright-Field and Fluorescence Microscopic Study of Development of Erysiphe polygoni in Susceptible and Resistant Bigleaf Hydrangea.

Temporal development of Erysiphe polygoni and responses of bigleaf hydrangeas (Hydrangea macrophylla) to the fungal attack were investigated using bright-field and fluorescence microscopy. Conidia germinated 2 h after inoculation (HAI) and formed primary appressoria at the tip of the primary germ tubes within 4 HAI. Secondary germ tubes were initiated from primary appressoria or other parts of conidia 12 HAI. Hyphae developed through elongation of secondary germ tubes, and paired lateral appressoria were formed along hyphae within 2 days after inoculation (DAI). Conidiophores and conidia were formed 5 DAI. In the susceptible cultivar Nikko Blue and the resistant cultivar Veitchii, the fungus established a parasitic relationship, which was indicated by the formation of haustoria under primary appressoria and development of secondary germ tubes at 1 DAI. A hypersensitive response (HR) and accumulation of callose were detected in both resistant and susceptible cultivars at 3 DAI. Resistance to powdery mildew in Veitchii was evident by manifestation of early accumulation of callose, relatively high percentage of necrotic infected cells, and restricted colony development compared to the susceptible cultivar Nikko Blue. Restricting hyphal growth and sporulation by early response of callose accumulation and HR are important resistance mechanisms that could be used in screening hydrangeas for resistance to powdery mildew.

Assessment of Alternative Candidate Subcortical Insect Vectors From Walnut Crowns in Habitats Quarantined for Thousand Cankers Disease.

Thousand cankers disease (TCD) results from the combined activity of the fungal pathogen, Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat and its principle vector, Pityophthorus juglandis (Blackman) (Coleoptera: Curculionidae: Scolytinae) in Juglans L. spp. and Pterocarya Kunth spp. host plants. TCD has been reported from the eastern and western United States. To evaluate potential for other beetle species to vector the fungus in east Tennessee, specimens were collected using ethanol-baited traps that were suspended beneath crowns of TCD-symptomatic trees. Associations of G. morbida with insect species collected in traps were assessed in an unsuccessful, preliminary culture-based fungal assay, and then with a molecular-based detection method. For culture-based assays, rinsate from washed, individual insects was plated on nutrient media and growing colonies were subcultured to obtain axenic G. morbida cultures for identification. For the molecular-based method, G. morbida presence was detected by amplifying the previously developed, species-specific microsatellite locus GS004. Capillary electrophoresis was used to detect the amplified amplicons and representative reactions were validated using Sanger sequencing. Eleven beetle species were found to carry G. morbida, including Cnestus mutilatus (Blandford), Dryoxylon onoharaensum (Murayama), Hylocurus rudis (LeConte), Monarthrum fasciatum (Say), Monarthrum mali (Fitch), Xyleborinus saxesenii (Ratzeburg), Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford) (all Coleoptera: Curculionidae: Scolytinae), Stenomimus pallidus (Boheman) (Coleoptera: Curculionidae: Cossoninae), Oxoplatypus quadridentatus (Olivier) (Coleoptera: Curculionidae: Platypodinae), and Xylops basilaris (Say) (Coleoptera: Bostrichidae). These findings raise concerns that alternative subcortical insect species that already occur within quarantined habitats can sustain incidence of introduced G. morbida and contribute to spread within the native range of black walnut, Juglans nigra L., in the eastern United States.

Confirmation of independent introductions of an exotic plant pathogen of Cornus species, Discula destructiva, on the east and west coasts of North America.

Cornus florida (flowering dogwood) and C. nuttallii (Pacific dogwood) are North American native tree species that belong to the big-bracted group of dogwoods. Cornus species are highly valued for their ornamental characteristics, and have fruits that contain high fat content for animals. Also, they are an important understory tree in natural forests. Dogwood anthracnose, caused by Discula destructiva, was observed in the late 1970s on the east and west coasts of the United States and by 1991 had quickly spread throughout most of the native ranges of C. florida and C. nuttalli. We investigated the genetic diversity and population structure of 93 D. destructiva isolates using 47 microsatellite loci developed from the sequenced genome of the type strain of D. destructiva. Clone-corrected data indicated low genetic diversity and the presence of four genetic clusters that corresponded to two major geographic areas, the eastern United States and the Pacific Northwest, and to the two collection time periods when the isolates were collected (pre- and post-1993). Linkage disequilibrium was present in five out of six subpopulations, suggesting that the fungus only reproduced asexually. Evidence of population bottlenecks was indicated across four identified genetic clusters, and was probably the result of the limited number of founding individuals on both coasts. These results support the hypothesis that D. destructiva is an exotic pathogen with independent introductions on the east and west coasts of North America. We also tested the cross-amplification of these microsatellite primers to other Discula species. Genomic DNA from 17 isolates of four other Discula species and two isolates of Juglanconis species (formerly Melanconis species) were amplified by 17 of 47 primer pairs. These primers may be useful for investigating the genetic diversity and population structure of these Discula species.

Natural Occurrence of Microsphaera pulchra and Phyllactinia guttata on Two Cornus Species.

Powdery mildew has become a common foliar disease of Cornus florida and other dogwood species in the eastern United States during the last several years. This study was conducted to determine the identity of powdery mildew fungi on C. florida and C. amomum. Ascocarps of Microsphaera pulchra and Phyllactinia guttata occurred singly and together on both C. florida and C. amomum leaves. M. pulchra ascocarps occurred at a higher density than P. guttata ascocarps on C. florida leaves, whereas P. guttata ascocarps occurred more frequently than M. pulchra ascocarps on C. amomum leaves. Histological studies, however, did not provide supplementary data of infection by the powdery mildew species that occurred less frequently on the leaves of each dogwood species. M. pulchra did not penetrate the cells of C. amomum, and likewise P. guttata did not enter through stomata of C. florida leaves. The presence of ascocarps of both species was not the result of infection of the dogwoods by both pathogens. The ascocarps of M. pulchra probably became airborne and then settled on the C. amomum leaves. Similarly, the ascocarps of P. guttata landed on C. florida leaves. These results emphasize the importance of correct pathogen identification using several criteria such as ascocarp morphology, host-pathogen relationships, distribution of the pathogen, conidial morphology, and histology.

Survival of Conidia of Discula destructiva in Frass of the Convergent Lady Beetle.

The survival of conidia of Discula destructiva in frass of convergent lady beetles (Hippodamia convergens) was investigated. D. destructiva was isolated from frass pellets of 76% of adult convergent lady beetles exposed to D. destructiva for 1 h. Of the beetles from which D. destructiva was initially isolated, more than 80, 32, 19, 13, and 12.5% retained viable conidia of D. destructiva internally for at least 12, 24, 48, 72, and 96 h, respectively. Numbers of conidia, estimated with a hemacytometer, ranged from 0 to 3.2 × 106 conidia per frass pellet.