Kendra Baumgartner

Contrasting patterns of genetic diversity and population structure of Armillaria mellea sensu stricto in the eastern and western United States.

ABSTRACT Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (theta(ST) = -0.002, P = 0.5 ) or western subpopulations (theta(ST) = 0.004, P = 0.3 ) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens

BackgroundTrunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.ResultsThe integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.ConclusionsThis study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

Ecology of Armillaria spp. in Mixed-Hardwood Forests of California

Incidence of Armillaria root disease and the population structure of associated Armillaria spp. was studied in California mixed-hardwood forests. Four 3,000-m2 plots, consisting of Quercus kelloggii, Q. agrifolia, Lithocarpus densiflorus, Arbutus menziesii, Umbellularia californica, and Pseudotsuga menziesii, were intensively sampled. Root collars were examined for mycelial fans, decayed wood, and rhizomorphs. From 404 trees (333 living, 71 dead), Armillaria mellea and A. gallica were recovered and identified from 148 isolates. The most abundant tree species in each plot was the tree species most frequently infected by A. mellea or A. gallica. The majority of A. mellea isolates were recovered from mycelial fans on living trees. Recent mortality due to A. mellea was found only in one plot. A. gallica was mainly identified from epiphytic rhizomorphs. Only one to three somatic incompatibility groups (SIGs) for each species were found in each plot. Estimated sizes of SIGs varied from the extent of a single root system to the entire width of the plot. Based on our results, both A. mellea and A. gallica are common in mixedhardwood forests, yet A. mellea appears to be a more aggressive pathogen. A. gallica is capable of attacking live hosts, but occurs on living roots more frequently as epiphytic rhizomorphs.

Distribution of Armillaria species in California

To determine the identity and distribution of Armillaria species in California, we collected 589 isolates from managed areas (urban landscapes, or- chards, and vineyards) and 20 natural forest types. From our collection of isolates, we identified A. mel- lea, A. gallica, A. nabsnona, and North American bi- ological species X (NABS X). State-wide ranges of each species were estimated based on distributions of forest types from which they were collected. Only A. mellea and A. gallica are widely-distributed in Califor- nia; A. nabsnona and NABS X appear to have restrict- ed ranges. Armillaria mellea is common in most re- gions of the state dominated by development, such as the Central Valley, the San Francisco Bay area, and Los Angeles. It was the only species, with the excep- tion of a single isolate of NABS X, isolated from symptomatic hosts, including both forest trees and planted hosts. Armillaria mellea does cause mortality in California forests, but large disease centers are rare. The distribution of A. gallica in California in- cludes the greatest variety of forest types of all Ar- millaria species identified from our collection. Al- though it shares many forest types in common with A. mellea, A. gallica is extremely rare in developed areas. The range of A. nabsnona in California ap- pears to be restricted to northern redwood forests. NABS X was only identified in high elevation red and white fir forests of the southern Cascades.

Persistence of Xylella fastidiosa in Riparian Hosts Near Northern California Vineyards

The spread of Pierces disease (PD) from riparian hosts to grapevines in Californias northcoastal grape-growing region is a function of the proportion of Graphocephala atropunctata (blue-green sharpshooters [BGSSs]) that acquire Xylella fastidiosa from infected plant tissue. Riparian hosts that do not maintain sufficient X. fastidiosa populations for acquisition may not be significant inoculum reservoirs. We examined X. fastidiosa populations in systemically infected riparian hosts (California blackberry, California grapevine, elderberry, Himalayan blackberry, periwinkle) at two coastal locations (Mendocino and Napa) with two methods of quantitation (culturing and real-time polymerase chain reaction) from 2003 to 2004. In summer and autumn, X. fastidiosa populations were above the threshold for BGSS acquisition in periwinkle, Himalayan blackberry, and California grapevine at both locations. The only X. fastidiosa-positive plants detected in spring at both locations were periwinkle and Himalayan blackberry, suggesting that these species may contribute to long-term survival of X. fastidiosa. California blackberry and elderberry may not be important reservoirs of X. fastidiosa, given that very few plants of either species maintained infections. Higher X. fastidiosa populations in California grapevine, Himalayan blackberry, and periwinkle in Napa, relative to plants in Mendocino, may partially explain the higher PD incidence in Napa vineyards.

Inferring dispersal patterns of the generalist root fungus Armillaria mellea

Investigating the dispersal of the root-pathogenic fungus Armillaria mellea is necessary to understand its population biology. Such an investigation is complicated by both its subterranean habit and the persistence of genotypes over successive host generations. As such, host colonization by resident mycelia is thought to outcompete spore infections. We evaluated the contributions of mycelium and spores to host colonization by examining a site in which hosts pre-date A. mellea. Golden Gate Park (San Francisco, CA, USA) was established in 1872 primarily on sand dunes that supported no resident mycelia. Genotypes were identified by microsatellite markers and somatic incompatibility pairings. Spatial autocorrelation analyses of kinship coefficients were used to infer spore dispersal distance. The largest genotypes measured 322 and 343 m in length, and 61 of the 90 total genotypes were recovered from only one tree. The absence of multilocus linkage disequilibrium and the high proportion of unique genotypes suggest that spore dispersal is an important part of the ecology and establishment of A. mellea in this ornamental landscape. Spatial autocorrelations indicated a significant spatial population structure consistent with limited spore dispersal. This isolation-by-distance pattern suggests that most spores disperse over a few meters, which is consistent with recent, direct estimates based on spore trapping data.

Cadophora species associated with wood-decay of grapevine in North America

Cadophora species are reported from grapevine (Vitis vinifera L.) in California, South Africa, Spain, Uruguay, and Canada. Frequent isolation from vines co-infected with the Esca pathogens (Togninia minima and Phaeomoniella chlamydospora), and confirmation of its ability to cause wood lesions/discoloration in pathogenicity tests, suggest that C. luteo-olivacea is part of the trunk pathogen complex. In North America, little is known regarding the diversity, geographic distribution, and roles of Cadophora species as trunk pathogens. Accordingly, we characterized 37 Cadophora isolates from ten US states and two Canadian provinces, based on molecular and morphological comparisons, and pathogenicity. Phylogenetic analysis of three loci (ITS, translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (BT)) distinguished two known species (C. luteo-olivacea and Cadophora melinii) and three newly-described species (Cadophora orientoamericana, Cadophora novi-eboraci, and Cadophora spadicis). C. orientoamericana, C. novi-eboraci, and C. spadicis were restricted to the northeastern US, whereas C. luteo-olivacea was only recovered from California. C. melinii was present in California and Ontario, Canada. Morphological characterization was less informative, due to significant overlap in dimensions of conidia, hyphae, conidiophores, and conidiogenous cells. Pathogenicity tests confirmed the presence of wood lesions after 24 m, suggesting that Cadophora species may have a role as grapevine trunk pathogens.

Genes Expressed in Grapevine Leaves Reveal Latent Wood Infection by the Fungal Pathogen Neofusicoccum parvum

Some pathogenic species of the Botryosphaeriaceae have a latent phase, colonizing woody tissues while perennial hosts show no apparent symptoms until conditions for disease development become favorable. Detection of these pathogens is often limited to the later pathogenic phase. The latent phase is poorly characterized, despite the need for non-destructive detection tools and effective quarantine strategies, which would benefit from identification of host-based markers in leaves. Neofusicoccum parvum infects the wood of grapevines and other horticultural crops, killing the fruit-bearing shoots. We used light microscopy and high-resolution computed tomography (HRCT) to examine the spatio-temporal relationship between pathogen colonization and anatomical changes in stem sections. To identify differentially-expressed grape genes, leaves from inoculated and non-inoculated plants were examined using RNA-Seq. The latent phase occurred between 0 and 1.5 months post-inoculation (MPI), during which time the pathogen did not spread significantly beyond the inoculation site nor were there differences in lesion lengths between inoculated and non-inoculated plants. The pathogenic phase occurred between 1.5 and 2 MPI, when recovery beyond the inoculation site increased and lesion lengths of inoculated plants tripled. By 2 MPI, inoculated plants also had decreased starch content in xylem fibers and rays, and increased levels of gel-occluded xylem vessels, the latter of which HRCT revealed at a higher frequency than microscopy. RNA-Seq and screening of 21 grape expression datasets identified 20 candidate genes that were transcriptionally-activated by infection during the latent phase, and confirmed that the four best candidates (galactinol synthase, abscisic acid-induced wheat plasma membrane polypeptide-19 ortholog, embryonic cell protein 63, BURP domain-containing protein) were not affected by a range of common foliar and wood pathogens or abiotic stresses. Assuming such host responses are consistent among cultivars, and do not cross react with other trunk/foliar pathogens, these grape genes may serve as host-based markers of the latent phase of N. parvum infection.

Root Collar Excavation for Postinfection Control of Armillaria Root Disease of Grapevine

Root collar excavation for control of Armillaria root disease of grapevine was investigated in two California vineyards (vineyard K1 and vineyard N1) from 2002 to 2004. The hypothesis tested was that root collar excavation, when timed in early stages of root collar infection, may cause mycelial fans of the pathogen Armillaria mellea to recede from the root collar before severe disease results from vascular tissue decay. In vineyard N1, excavation significantly increased yield and cluster weight of symptomatic grapevines; symptomatic-excavated grapevines had the same high mean cluster weight as healthy grapevines, and there were no significant effects of excavation on yield or pruning weight of healthy grapevines. In vineyard K1, where excavated root collars frequently refilled with soil, excavation had no significant effects on yield or pruning weight of symptomatic grapevines, and significantly reduced pruning weight and shoot weight of healthy grapevines. Reexamination in March 2004 revealed that mycelial fans had receded from root collars of symptomatic-excavated grapevines, but remained on root collars of symptomatic-nonexcavated grapevines. Root collar excavation appears to be a promising cultural approach for control of Armillaria root disease, as long as excavated root collars are kept clear of soil.

Diversity of Diaporthe species associated with wood cankers of fruit and nut crops in northern California

Diaporthe ampelina, causal agent of Phomopsis cane and leaf spot of grapevine (Vitis vinifera L.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthe species, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthe community in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaporthe isolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthe species from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricana and D. novem in North America. Our findings also expand the host ranges of D. ambigua to apricot and willow, D. australafricana to almond and willow, D. chamaeropis to grapevine and willow, D. foeniculina to willow and D. novem to almond. The generalists D. ambigua and D. eres were the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelina was the most pathogenic species to grapevine wood.