Verticillium Nonalfalfae Research Articles

Effects of phosphite on the in vitro growth of Verticillium nonalfalfae and Verticillium dahliae and on their in vivo ability to infect potato plants

Verticillium spp. are soil-borne fungi containing many pathogenic species that affect a wide range of cultivated plants. In potatoes, Verticillium dahliae, V. nonalfalfae and V. albo-atrum cause Verticillium wilt or, together with other pathogens, are responsible for the occurrence of the potato early dying disease. Verticillium pathogens are notoriously difficult to control, and techniques proved to be efficient, such as chemical soil fumigation, are causing environmental concerns worldwide. Phosphites (Phi) represent a class of chemicals with a good environmental footprint, that are currently used to control oomycete pathogens, such as the potato late blight agent, Phytophtora infestans. To determine the potential inhibitory effects of Phi on Verticillium spp., isolates of V. dahliae and V. nonalfalfae were tested in vitro and the IC50 of Phi was found to vary between 60.9 and 481.9 μg/mL. The effects of Phi were further tested in field trials, carried for two years, using Phi-treated potato plants infected with a V. nonalfalfae isolate. Infection progression was assessed in both years by qPCR and was found to be significantly reduced in Phi-treated plants. The concentration of Phi in these plants, determined by high performance ion chromatography, was found to vary between 25.0 and 86.2 μg/mL in leaves and between 55.7 and 113.8 μg/mL in tubers. Even though the concentrations of Phi required to control Verticillium spp. were found to be relatively high compared to those needed to inhibit the development of oomycete pathogens, the results from this study indicate that Phi can limit the development of Verticillium spp.

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity.

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

Preliminary pathogenicity screening of <i>Verticillium</i> spp. on kiwifruit in New Zealand

Plant-pathogenic Verticillium species have been present in New Zealand for many years, and have been considered minor wilt pathogens of kiwifruit. However, an outbreak of Verticillium nonalfalfae (previously identified as Verticillium alboatrum) causing wilt and death of the kiwifruit cultivar Actinidia chinensis var. chinensis ‘Hort16A’ in Chile has raised questions around the pathogenicity and significance of New Zealand Verticillium species. This study investigated the pathogenicity of New Zealand isolates of Verticillium spp. to ‘Hort16A’. Three isolates of Verticillium dahliae and one of V. alboatrum sensu stricto, previously recovered from kiwifruit in New Zealand, were tested for pathogenicity against ‘Hort16A’ by artificial inoculation of young vines. Disease assessments were carried out monthly. Symptoms observed ranged from minor wilt to vine death. The V. alboatrum isolate appeared the most aggressive. Although there is evidence of some pathogenicity on kiwifruit within this group of isolates from Verticillium species in New Zealand, they appear less aggressive than those recorded in Chile. However, this cannot be confirmed without testing isolates from both countries concurrently under the same conditions.

Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

RNA interference core components identified and characterised in Verticillium nonalfalfae, a vascular wilt pathogenic plant fungi of hops

The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.

Verticillium dahliae and Verticillium nonalfalfae occurrence and abundance in several agricultural fields from Nova Scotia, Canada, assessed by real-time quantitative PCR

Verticillium dahliae and Verticillium nonalfalfae are soil-borne fungal pathogens with a ubiquitous geographical distribution affecting a wide range of plants of economic importance. Though Verticillium wilt represents a major problem for crop production in Nova Scotia, Canada, little is known about the distribution of these pathogens in the major agricultural areas from this province. To address the paucity of available data, a molecular-based survey of these Verticillium pathogens, documenting their distribution, incidence, and in-sample abundance, was carried out for two successive years, in several representative agricultural locations in this province. Soil and plant samples (potato and strawberry) were analyzed using real-time quantitative PCR targeting the Intergenic Spacer ribosomal DNA locus of the Verticillium pathogens. Molecular data revealed that V. dahliae has a wider distribution and a higher incidence than V. nonalfalfae (former V. albo-atrum group 1) in the tested fields while V. albo-atrum sensu stricto (former V. albo-atrum group 2) was not identified in any of the samples collected from this region. Also, V. dahliae was found to have a higher incidence in fields used to grow potatoes as compared to fields used to grow strawberries, irrespective of the rotating crops.

Genome Sequence of a Lethal Vascular Wilt Fungus, Verticillium nonalfalfae, a Biological Control Used Against the Invasive Ailanthus altissima.

Verticillium nonalfalfae, a cosmopolitan soil-borne phytopathogen, causes vascular wilt in agricultural crops and perennial woody plants. Select strains of V. nonalfalfae can cause lethal disease in the invasive tree Ailanthus altissima and several have since been utilized as a biological control (biocontrol) against this widespread invader. Here, we report the genome sequence and annotation of V. nonalfalfae strain VnAa140/NRRL 66861.

Effects of Verticillium nonalfalfae on Ailanthus altissima and associated indigenous and invasive tree species in eastern Austria

The naturally occurring Verticillium nonalfalfae shows promise for biocontrol of the highly invasive Tree of Heaven (Ailanthus altissima), but might also bear a risk for non-target tree species. In this study, we conducted inoculations on potted seedlings of A. altissima as well as on eight indigenous and two invasive tree species associated with Tree of Heaven in Austria. Although vascular discolourations developed in all inoculated tree species, V. nonalfalfae was reisolated from Ailanthus and eight of the ten non-target-species, whereas typical disease symptoms and mortality only occurred on A. altissima. Results confirmed high susceptibility (S) of A. altissima to V. nonalfalfae but indicated tolerance (T) of Acer campestre, Acer pseudoplatanus and Quercus robur, possible resistance (PR) of Fraxinus excelsior, Populus nigra, Tilia cordata, Ulmus laevis and Ulmus minor and resistance (R) of Fraxinus pennsylvanica and Robinia pseudoacacia to this potential biocontrol agent. Results from seedling inoculations were confirmed by cursory field observations in Ailanthus-inoculated forest stands, where admixed A. campestre, A. pseudoplatanus, F. excelsior, Populus alba, R. pseudoacacia and U. laevis canopy trees remained asymptomatic, while mortality was induced in Ailanthus.

Comparative transcriptional analysis of hop responses to infection with Verticillium nonalfalfae

Key MessageDynamic transcriptome profiling revealed excessive, yet ineffective, immune response toV. nonalfalfaeinfection in susceptible hop, global gene downregulation in shoots of resistant hop and only a few infection-associated genes in roots.Hop (Humulus lupulus L.) production is hampered by Verticillium wilt, a disease predominantly caused by the soil-borne fungus Verticillium nonalfalfae. Only a few hop cultivars exhibit resistance towards it and mechanisms of this resistance have not been discovered. In this study, we compared global transcriptional responses in roots and shoots of resistant and susceptible hop plants infected by a lethal strain of V. nonalfalfae. Time-series differential gene expression profiles between infected and mock inoculated plants were determined and subjected to network-based analysis of functional enrichment. In the resistant hop cultivar, a remarkably low number of genes were differentially expressed in roots in response to V. nonalfalfae infection, while the majority of differentially expressed genes were down-regulated in shoots. The most significantly affected genes were related to cutin biosynthesis, cell wall biogenesis, lateral root development and terpenoid biosynthesis. On the other hand, susceptible hop exhibited a strong defence response in shoots and roots, including increased expression of genes associated with plant responses, such as innate immunity, wounding, jasmonic acid pathway and chitinase activity. Strong induction of defence-associated genes in susceptible hop and a low number of infection-responsive genes in the roots of resistant hop are consistent with previous findings, confirming the pattern of excessive response of the susceptible cultivar, which ultimately fails to protect the plant from V. nonalfalfae. This research offers a multifaceted overview of transcriptional responses of susceptible and resistant hop cultivars to V. nonalfalfae infection and represents a valuable resource in the study of this plant-pathogen interaction.

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants.

Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant's xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen's virulence/pathogenicity and its interaction with hop plants. In this protocol, we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.

Xylem Sap Extraction Method from Hop Plants.

Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many different virulence factors. Xylem sap of infected plants is therefore a rich source for investigating the molecules that are involved in molecular interactions of Verticillium - hop plants. This protocol provides instructions on how to infect hop plants with V. nonalfalfae artificially and how to obtain xylem sap from hop plants.

Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae.

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker.

Intraspecific Root Grafts and Clonal Growth Within Ailanthus altissima Stands Influence Verticillium nonalfalfae Transmission.

Verticillium nonalfalfae, causal agent of Verticillium wilt, is being considered as a biocontrol for the highly invasive Ailanthus altissima in Pennsylvania. This soilborne fungus is extremely virulent on Ailanthus and rapidly transmitted from diseased to healthy trees within Ailanthus stands. The rapid transmission of the fungus could be facilitated by root grafts, but neither root graft formation in Ailanthus nor Verticillium transmission by root grafts in trees has been reported. Here, V. nonalfalfae transmission between diseased and healthy Ailanthus trees via intraspecific root grafts and clonal growth is evaluated. Using air-spade excavation, dye translocation, and root graft inoculations, functional root grafts were detected between Ailanthus trees and transmission of V. nonalfalfae across root grafts demonstrated. Inoculation of one Ailanthus parent stem resulted in 187 root sprouts showing Verticillium wilt symptoms 12 months after inoculation. This study revealed that clonal growth and root grafts, normally advantageous growth habits, leave Ailanthus stands vulnerable to widespread V. nonalfalfae infection. This study also broadens the understanding of the Ailanthus-Verticillium pathosystem, growth strategies of invasive Ailanthus, and epidemiology of Verticillium wilt within trees.

Expanded Host Range Testing for Verticillium nonalfalfae: Potential Biocontrol Agent Against the Invasive Ailanthus altissima.

The naturally occurring Verticillium nonalfalfae has been proposed as a biocontrol agent against the highly invasive Ailanthus altissima in the eastern United States. We tested 71 nontarget woody species for susceptibility to the potential biocontrol agent. In the field, only devil's walkingstick (17% incidence) and striped maple (3%) acquired infections through natural spread from infected A. altissima (100%). Staghorn sumac (16% incidence) also exhibited wilt in close proximity to diseased Ailanthus, although V. nonalfalfae was never recovered. Stem inoculations, which are highly artificial in that they bypass root defenses and flood the xylem with millions of conidia, induced varying levels of wilt and mortality in 10 nontarget species from which V. nonalfalfae was reisolated, although recovery and crown rebuilding occurred following initial wilt in several species including sassafras and northern catalpa. Thirty-seven of the 71 inoculated species exhibited vascular discoloration, although 23 of these species exhibited no outward symptoms (wilt, dieback) for up to 6 years postinoculation. However, V. nonalfalfae was reisolated from three of the 23 species, indicating a tolerant host response. Our results suggest that V. nonalfalfae is generally host-adapted to A. altissima with 78 of 78 A. altissima seed sources from 26 states and Canada showing susceptibility, and offers support for adoption and dissemination of V. nonalfalfae to combat the highly invasive A. altissima.

Biocontrol of Ailanthus altissima: inoculation protocol and risk assessment for Verticillium nonalfalfae (Plectosphaerellaceae: Phyllachorales)

Verticillium nonalfalfae has been proposed as a biocontrol for invasive Ailanthus altissima (tree-of-heaven) in Ohio, Pennsylvania and Virginia. However, previous studies evaluating this potential biocontrol utilised a conidial suspension with a short shelf life as inoculum. Anticipating future expanded use of V. nonalfalfae, we evaluated other inoculum formulations, inoculation protocols and sensitivity of non-target (non-Ailanthus) plant species within Pennsylvania. The most effective inoculum formulation, with an extended shelf life, was prepared by mixing water with stored, refrigerated soil containing V. nonalfalfae. Less successful, but positive infections were obtained by simply using infected Ailanthus wood and leaves as inoculum. Monthly inoculation of Ailanthus trees demonstrated that the optimal time for successful inoculations was April to May, but limited infections were achieved during all months, including the winter. The health of Ailanthus and non-target species was evaluated within a decade-old natural Verticillium wilt epicentre, where all mature Ailanthus trees had been killed by V. nonalfalfae. Verticillium wilt was observed on a few small Ailanthus trees, likely newly established seedlings, whereas non-target species were asymptomatic. Our findings reveal that soil formulated and natural inocula are effective biocontrols against Ailanthus, and V. nonalfalfae appears to pose little threat to non-target plants.

Comparative Pathogenicity, Biocontrol Efficacy, and Multilocus Sequence Typing of Verticillium nonalfalfae from the Invasive Ailanthus altissima and Other Hosts

Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and highly efficacious against Ailanthus and, thus, is a strong candidate for use as a biocontrol agent.

Survey of Verticillium nonalfalfae (Phyllachorales) on tree-of-heaven in the southeastern USA

Ailanthus altissima (Mill.) Swingle commonly known as tree-of-heaven is an invasive tree species that has spread through much of the USA. Significant mortality of A. altissima by a highly host-specific strain of the fungus, Verticillium nonalfalfae Inderb. (formerly Verticillium albo-atrum Reinke and Bethold), has been recently documented in Pennsylvania. To detect the regional distribution of this biological control agent in the Southeast, a windshield survey was conducted in Virginia, North Carolina and South Carolina to locate V. nonalfalfae-infected A. altissima stands. The survey covered 26,500 km of interstate highways and primary and secondary roads. There were 90 potential sites located throughout the three states. Of those, six were found to be infested with the fungus, all in the mountain regions of Virginia. Inoculation tests suggest no difference in virulence among the six V. nonalfalfae isolates on A. altissima, which resulted in 100% mortality in 9–11 weeks post-inoculation. Tree ratings revealed differences in health among sites, but all sites had an increase in disease incidence from 2011 to 2012 (mean = 4.7 ± 3.8%), suggesting this fungus is dispersing. This survey provides evidence that biological control using this fungus may be possible in Virginia.

Initial Steps towards Biocontrol in Hops: Successful Colonization and Plant Growth Promotion by Four Bacterial Biocontrol Agents

Verticillium wilt, caused by Verticillium nonalfalfae and V. dahliae, is a devastating disease in hops that can cause considerable economic crop losses. The perennial use of hops combined with the long persistence of the pathogen in soil make it difficult to suppress the disease with conventional measures. Biological control agents (BCA) are the basis of an environmentally friendly plant protection strategy that uses plant promotion and antagonistic effects of microorganisms. We evaluated the effect of four selected beneficial bacterial strains, Burkholderia terricola ZR2-12, Pseudomonas poae RE*1-1-14, Serratia plymuthica 3Re4-18, and Stenotrophomonas rhizophila DSM14405T for their use in hops. All strains were shown to be both rhizosphere and endorhiza competent, and their abundances ranged from log10 3.0 to log10 6.2 CFU g−1 root fresh weight in the endorhiza and from log10 2.9 to log10 4.7 CFU g−1 root fresh weight in the rhizosphere with B. terricola ZR2-12 showing the highest overall cell densities. Microscopic visualization of DsRed-labeled transformants with confocal laser scanning microscopy showed different colonization patterns and confirmed the rhizosphere competence. Growth promoting effects on seedlings treated with bacteria were found for S. plymuthica 3Re4-18 and S. rhizophila DSM14405T. Competent colonization and plant growth promoting effects are the most important prerequisites towards efficient biocontrol.

First Report of Verticillium Wilt Caused by Verticillium nonalfalfae on Tree-of-Heaven (Ailanthus altissima) in Ohio.

Verticillium wilt of the highly invasive tree-of-heaven [Ailanthus altissima (Mill.) Swingle], caused by Verticillium nonalfalfae Interbitzin et al. (1), formerly classified as V. albo-atrum Reinke and Berthold, has been reported in the United States from two states: Pennsylvania (2) and Virginia (3). Infected A. altissima in both states exhibited similar symptoms of wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality. In June 2012, the second author observed dead and dying A. altissima trees in southern Ohio (Pike County) that exhibited symptoms similar to those on diseased A. altissima trees in Pennsylvania and Virginia. Samples were collected from stems of three symptomatic A. altissima trees and sent to Penn State for morphological and molecular identification. Immediately upon arrival, samples were surface-disinfected and plated onto plum extract agar (PEA), a semi-selective medium for Verticillium spp., amended with neomycin and streptomycin (2). The samples yielded six isolates, two from each of the three symptomatic trees, all of which were putatively identified as V. nonalfalfae based on the presence of verticillate conidiophores and formation of melanized hyphae. DNA was extracted from three isolates and molecular analyses performed using known primers (1) coding for elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS). A BLAST search generated sequences that revealed 100% similarity to V. nonalfalfae for all three protein coding genes among the three Ohio isolates and reference sequences from Ailanthus, including isolates VnAaPA140 (GenBank Accession Nos. KC307764, KC307766, and KC307768) and VnAaVA2 (KC307758, KC307759, and KC307760), as well as isolate PD592 from potato (JN188227, JN188163, and JN188035), thereby confirming taxonomic placement of the Ohio Ailanthus isolates among those recovered from Ailanthus in Pennsylvania and Virginia. Aligned sequences from one representative isolate, VnAaOH1, were deposited into GenBank as accessions KC307761 (EF), KC307762 (GPD), and KC307763 (TS). In August 2012, the pathogenicity of all six isolates was confirmed by root-dipping 10 healthy 3-week-old A. altissima seedlings (seeds collected in University Park, PA) into conidial suspensions of 1 × 107 cfu/ml, wherein all inoculated seedlings wilted and died within 4 and 9 weeks, respectively. V. nonalfalfae was reisolated from all inoculated seedlings; control seedlings inoculated with distilled water remained asymptomatic. Ohio is the third state from which V. nonalfalfae has been reported to be pathogenic on A. altissima. If V. nonalfalfae proves to be widespread, it may represent a natural biocontrol for the invasive A. altissima. Also, since USDA APHIS evaluates and regulates new potential biocontrol agents on a state-by-state basis, it is important to document each state in which V. nonalfalfae is killing A. altissima, so that in-state inoculum can be used for biocontrol efforts, simplifying the regulatory process.