Verticillium Nonalfalfae Research Articles

The bioherbicide Verticllium nonalfalfae effectively removes Ailanthus altissima but leaves many other nonnative plants

Abstract Ailanthus altissima (Mill.) Swingle readily exploits disturbances, grows quickly into dense monocultures, and suppresses native plant species. The vascular wilt pathogen, Verticillium nonalfalfae Inderb., native to the eastern U.S., has been proposed as a biocontrol agent for the invasive A. altissima. Studies consistently demonstrate the safety and efficacy of the bioherbicide, but they also note that the selective nature of the fungus does not preclude other invasive plants that commonly co-occur with A. altissima from occupying the site. We quantified the standing plant community and seedbank at several sites across Virginia five years after inoculation with V. nonalfalfae to understand which species are present or being naturally recruited. A. altissima remained dominant in untreated areas but was nearly eradicated from the treatment plots. Other nonnative species made up a large portion of the plant community and seedbank across all study areas, with no differences in their respective cover and count between treatments. While variability in plant community composition is high and site-specific context is important for establishing effective management strategies, planting native species and mitigating other invasives will be crucial to ensuring native species successfully establish in bioherbicide-treated areas.

Investigating physiological effects due to artificial infections of grapevine with <i>Verticillium nonalfalfae</i>

Ailanthus altissima is among the most invasive woody species worldwide, outcompeting native trees. The fungus Verticillium nonalfalfae (VN) is promising for A. altissima biocontrol, and its effects on the host have been studied via visual assessment in a range of host species. However, little research was performed to address fungal effects on the physiological processes of non-target woody plants. We investigated the occurrence of visual and non-visually recognisable perturbations of VN infection on potted vines to evaluate the potential risks of the biocontrol pathogen on viticulture. Eighteen four-years-old Vitis vinifera (cultivar Grüner Veltliner grafted on Kober 5BB) potted plants were inoculated with VN conidial suspension of the fungus (F), while nine plants were treated with sterile water (C, control). Disease symptoms and physiological parameters were monitored throughout the experiment (seven evaluation dates), while leaf water potential, leaf mass per area (LMA) and biomass were measured at the end of the study when plant tissue was sampled for re-isolation of the fungus. In our trial, inoculations with VN induced characteristic wilting symptoms only in Ailanthus (used as side control of the inoculum), while vines remained asymptomatic, thus indicating a high degree of host specificity of VN. Limited or no impact was detected on the physiology of the non-target V. vinifera. Furthermore, the LMA and biomass measured in the two experimental groups were not different. Although fungal colonisation induced vascular discolouration in both species, the fungus could only be re-isolated from dying Ailanthus but not from vine tissue. Results suggest that V. vinifera cv Grüner Veltliner is resistant to the applied VN isolate. However, the susceptibility and physiology of additional grapevine cultivars, as well as other native woody species to VN, should be studied before promoting large-scale use of the biocontrol agent.

A protein elicitor PeVn1 from Verticillium nonalfalfae HW recognized as a MAMP triggers plant immunity response.

Protein elicitors can induce plant systemic resistance to pathogens. The recognition of a potential elicitor activates intracellular signaling events, leading to plant resistance against pathogens. In this study, a novel protein elicitor was isolated from the culture filtrate of Verticillium nonalfalfae and named PeVn1, which can induce cell death in several plant species. The PeVn1 gene was then cloned and expressed in Escherichia coli. The recombinant protein PeVn1 triggers cell death in Nicotiana benthamiana in NbBAK1 and NbSOBIR1 dependent manner. Through bioassay analysis showed that the recombinant PeVn1 induced early defense induction events, such as reactive oxygen species burst, callose deposition and the activation of defense hormone signaling pathways and defense enzyme activities. Moreover, PeVn1 significantly enhanced resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum, Botrytis cinerea and N. benthamiana mosaic virus and tomato to Pseudomonas syringae pv. Tomato DC3000. In conclusion, our study reveals that PeVn1 protein as a microbe-associated molecular pattern can induce plant immune responses, which provides a theoretical basis for the development of novel protein-induced disease resistance agents.

Changes in the Microbial Composition of the Rhizosphere of Hop Plants Affected by Verticillium Wilt Caused by Verticillium nonalfalfae.

Verticillium wilt is a devastating disease affecting many crops, including hops. This study aims to describe fungal and bacterial populations associated with bulk and rhizosphere soils in a hop field cultivated in Slovenia with the Celeia variety, which is highly susceptible to Verticillium nonalfalfae. As both healthy and diseased plants coexist in the same field, we focused this study on the detection of putative differences in the microbial communities associated with the two types of plants. Bacterial communities were characterized by sequencing the V4 region of the 16S rRNA gene, whereas sequencing of the ITS2 region was performed for fungal communities. The bacterial community was dominated by phyla Proteobacteria, Acidobacteriota, Bacteroidota, Actinobacteriota, Planctomycetota, Chloroflexi, Gemmatimonadota, and Verrucomicrobiota, which are typically found in crop soils throughout the world. At a fungal level, Fusarium sp. was the dominant taxon in both bulk and rhizosphere soils. Verticillium sp. levels were very low in all samples analyzed and could only be detected by qPCR in the rhizosphere of diseased plants. The rhizosphere of diseased plants underwent important changes with respect to the rhizosphere of healthy plants where significant increases in potentially beneficial fungi such as the basidiomycetes Ceratobasidium sp. and Mycena sp., the zygomycete Mortierella sp., and a member of Glomeralles were observed. However, the rhizosphere of diseased plants experienced a decrease in pathogenic basidiomycetes that can affect the root system, such as Thanatephorus cucumeris (the teleomorph of Rhizoctonia solani) and Calyptella sp.

A droplet digital PCR assay for detection and quantification of Verticillium nonalfalfae and V. albo-atrum.

Verticillium nonalfalfae and V. albo-atrum are notorious pathogenic fungi that cause a destructive vascular disease called Verticillium wilt worldwide. Thus, timely and quantitative monitoring of fungal progression is highly desirable for early diagnosis and risk assessment. In this study, we developed a droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify V. nonalfalfae and V. albo-atrum. The performance of this assay was validated in comparison with that of a quantitative real-time polymerase chain reaction (qPCR) assay. The standard curve analysis of the ddPCR assay showed good linearity. The ddPCR assay indicated similar detection sensitivity to that of qPCR on pure genomic DNA, while it enhanced the positive rate for low-abundance fungi, especially in alfalfa stems. Receiver operating characteristic analysis revealed that ddPCR provided superior diagnostic performance on field tissues compared to qPCR, and the area under curve values were 0.94 and 0.90 for alfalfa roots and stems, respectively. Additionally, the quantitative results of the two methods were highly concordant (roots: R2 = 0.91; stems: R2 = 0.76); however, the concentrations determined by ddPCR were generally higher than those determined by qPCR. This discrepancy was potentially caused by differing amplification efficiencies for qPCR between cultured and field samples. Furthermore, the ddPCR assays appreciably improved quantitative precision, as reflected by lower coefficients of variation. Overall, the ddPCR method enables sensitive detection and accurate quantification of V. nonalfalfae and V. albo-atrum, providing a valuable tool for evaluating disease progression and enacting effective disease control.

Further pathogenicity testing of Verticillium nonalfalfae, a biocontrol agent against the invasive Tree of Heaven (Ailanthus altissima), on non-target tree species in Europe

Verticillium nonalfalfae is an effective biological control agent against the highly invasive Tree of Heaven (Ailanthus altissima) in Europe, and previous research on ten economically and ecologically important tree species occurring in Austria have so far not revealed undesired non-target effects. In this study, another nine tree species including five native, two non-native as well as two invasive alien tree species were tested for susceptibility to the particular strain of V. nonalfalfae (Vert56) used for biological control of A. altissima. Stem inoculations on potted seedlings revealed that this strain of V. nonalfalfae is generally host-adapted to A. altissima. It induced chlorosis, necrosis and wilting already within two weeks post inoculation on A. altissima and resulted in almost completely defoliated or dead seedlings at the end of the vegetation period. Apart from two species (Quercus rubra and Sorbus aucuparia), that suffered from other abiotic/biotic agents, no mortality was observed on all other tree species tested; however, symptoms caused by other abiotic factors were also found on Prunus avium and Ulmus glabra. All tested tree species exhibited vascular discolorations and the fungus could be re-isolated at varying frequencies (6—100%) from inoculated seedlings of all non-target tree species, although five of these species exhibited no external symptoms. Results confirmed high susceptibility (S) of A. altissima to V. nonalfalfae, whereas Acer platanoides, Castanea sativa, Q. rubra, S. aucuparia and U. glabra were considered as tolerant (T), and A. negundo, P. avium, P. serotina and Q. petraea were rated as possible resistant (PR) due to the low rates of re-isolation.

Non-target effects of Verticillium nonalfalfae isolate Vert56 used for biological control of Ailanthus altissima on agricultural crops known to be generally susceptible to Verticillium spp.

Tree of Heaven (Ailanthus altissima), a tree species native to China, was introduced into Europe as an ornamental plant about 250 years ago. It is known for its invasive spread with negative impacts on natural ecosystems as well as human infrastructure. Its control is inevitable in some areas, e.g. transportation corridors, for safety reasons. Furthermore, management actions are mandatory according to EU legislation, but common strategies are time-consuming, costly, only moderately effective and, in case of herbicide treatments, controversially discussed due to environmental issues. Isolate Vert56 of the vascular wilt fungus Verticillium nonalfalfae has been proven in previous studies to be highly adapted and highly virulent to Ailanthus, while inoculation of potentially susceptible tree species with this isolate showed no evidence of negative non-target effects.This study focused on non-target effects of isolate Vert56 on agricultural crops known to be generally susceptible to Verticillium spp. In a greenhouse experiment, seedlings of seven different species (including Ailanthus as a positive control) were artificially inoculated via root dipping or, in the case of seedlings of two potato varieties, by direct addition of the inoculum into the pot.All plants were subsequently assessed for symptoms during the following two months. After assessment, plants were harvested to determine dry biomass. Likewise, tissue samples were used for re-isolation of the pathogen. Besides morphological identification, 25% of the obtained fungal cultures were analyzed with species-specific primers. The pathogen was consistently detected in all species. In addition to Ailanthus, three species (Cucumis sativus, Petunia × hybrid, Spinacia oleracea) developed severe wilting symptoms and showed a decrease in biomass compared to control plants. One species, Solanum lycopersicum, displayed a decrease in biomass although only slight wilting symptoms were observed within a short period after inoculation. Humulus lupulus, Medicago sativa and Solanum tuberosum were not negatively affected by V.nonalfalfae isolate Vert56. These results indicate that safety precautions must be taken, or at least considered, when growing susceptible plants in the vicinity of Vert56-treated Ailanthus, but they also show that this biological control agent is suitable for the treatment of Ailanthus on/next to agricultural land.

Controlling an Invasive Tree with a Native Fungus: InoculatingAilanthus altissima(Tree-of-Heaven) withVerticillium nonalfalfaein Highly Disturbed Appalachian Forests of Ohio

AbstractHighly disturbed forests are commonplace throughout the eastern United States and their residing composition and structure is reflective of their past land use. Management and restoration efforts are complicated by diverse and abundant nonnative invasive plants, including Ailanthus altissima. Verticillium nonalfalfae has been identified as a potential native mycoherbicide option for Ailanthus. To test the efficacy of Verticillium on Ailanthus we designed a study in highly disturbed forests of southern Ohio. At each of five sites, we monitored symptomology, mortality, and rate of spread of stem-inoculated Verticillium on Ailanthus in four inoculated plots and compared it to a control plot. We also monitored native plants for Verticillium symptomology and community responses to Ailanthus control. Our results suggest that Verticillium is an effective tool for controlling Ailanthus with no observed effect on native flora. Further, Verticillium naturally spreads through stands and mortality is slow enough that other resident nonnative invasive plants do not rapidly increase.

Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

First report of Verticillium wilt and mortality of Ailanthus altissima caused by Verticillium dahliae and V. albo-atrum sensu lato in Spain.

Ailanthus altissima (Mill.) swingle is a highly invasive tree that has become established worldwide, especially in the Mediterranean Basin because of its good drought resistance. Ailanthus altissima is included in the list of Invasive Alien Species of the EU, so measures for eradication and management are required. Assessment for potential biological control agents is of great interest to manage this invasive tree in natural ecosystems. Verticillium dahliae Kleb. and Verticillium nonalfalfae Inderb. et al. (formerly V. albo-atrum Reinke & Berthold) have been reported as the causal agents of Verticillium wilt and mortality of ailanthus (Shall and Davis 2009; Rebbeck et al., 2013; Snyder et al., 2013; Brooks et al. 2020). Ailanthus trees with Verticillium wilt symptoms (wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality) were detected for the first time in 2007 in Celrà (42.040466N, 2.864436E) (Catalonia, Northeastern Spain), then spread to neighboring ailanthus populations. In 2018, ailanthus trees in a 570 km2 area in Catalonia were surveyed for disease symptoms. The incidence of wilt disease in ailanthus trees in forest ecosystems ranged from 50 to 90%, and the severity, 60 to 92%. One hundred and fifty branch samples showing wilt symptoms were collected and disinfected by immersion in 1% sodium hypochlorite for 2 min, then cut into 5mm pieces. These were placed onto PDA plates and incubated at 22.5 °C and 12 h light photoperiod for 7-10 days. Eighty-four tentative Verticillium sp. isolates were recovered and subcultured on modified water agar (WA-p) and PDA for identification (Inderbitzin et al. 2011, 2013). The majority of isolates (77 %) were identified as V. dahliae based on morphology; production of brown-pigmented microsclerotia and conidia features and dimensions (5.7 ± 0.9 μm long). Sequencing of mycelial DNA using primer pair ITS1-F and ITS4, resulted in consensus sequences of 503 bp. BLASTn analysis of ITS sequence of native isolate VdGi688 gave 100% identity to the ITS sequences of V. dahliae type strain PD322 (92% coverage) and Vd16_9 (100% coverage). In addition, 23% isolates morphologically corresponded to V. albo-atrum or V. nonalfalfae; melanized resting mycelia and round to oval-shaped conidia (5.2 ± 0.9 μm × 2.2 ± 0.5 μm). The ITS consensus sequence (544 bp) of native isolate VaaGi02 gave 99% identity (90-100 % coverage) to V. albo-atrum isolates CBS 127169, PSU 140, Vaa_TN1 and to V. nonalfalfae type PD592, CBS5451.88 and Vert 18. Sequences from isolates VdGi688 and VaaGi02 were deposited in GenBank as MW624723 and MW624724, respectively. Koch's postulates for seven V. dahliae isolates and eight V. albo-atrum isolates were fulfilled by injection of 1 mL of 1 x 107 conidia/mL suspension into the stem of A. altissima seedlings under greenhouse conditions. Six plants were inoculated per isolate in two independent experiments. Control plants were inoculated with sterile distilled water. All isolates caused leaf chlorosis, defoliation, and apical stem death, as well as internal necrosis and vascular discoloration. Control plants remained asymptomatic. The pathogens were re-isolated from internal symptomatic tissues of inoculated plants. To our knowledge, this is the first report of V. dahliae and V. albo-atrum sensu lato causing Verticillium wilt on A. altissima in Spain. The study suggests the potential of native isolates of Verticillum spp. in the biological control of ailanthus in the Mediterranean Basin. This work was funded by the Diputació de Girona (Spain) (2017/8719, 2019/3091, 2020/7565, and 2021/1468).

Transmission of Verticillium nonalfalfae via root contact from inoculated Ailanthus altissima in close‐to‐nature conditions

AbstractAilanthus altissima is an invasive alien species in Europe. Biological control of this tree species by Verticillium nonalfalfae is a potential alternative control approach. This study investigates host specificity, pathogenicity and transmission of V. nonalfalfae to neighbouring plants with root contact in mini‐ecosystems. V. nonalfalfae led to dieback of all inoculated Ailanthus trees. Furthermore, V. nonalfalfae was transmitted to neighbouring Ailanthus trees, causing wilt and dieback, and, in one case, to Quercus petraea, which did not display any disease symptoms. Lastly, V. nonalfalfae could not be detected in the soil, which suggests transmission via root contact.

Identification and Characterization of Verticillium nonalfalfae-Responsive MicroRNAs in the Roots of Resistant and Susceptible Hop Cultivars.

MicroRNAs are 21- to 24-nucleotide-long, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They can modulate various biological processes, including plant response and resistance to fungal pathogens. Hops are grown for use in the brewing industry and, recently, also for the pharmaceutical industry. Severe Verticillium wilt caused by the phytopathogenic fungus Verticillium nonalfalfae, is the main factor in yield loss in many crops, including hops (Humulus lupulus L.). In our study, we identified 56 known and 43 novel miRNAs and their expression patterns in the roots of susceptible and resistant hop cultivars after inoculation with V. nonalfalfae. In response to inoculation with V. nonalfalfae, we found five known and two novel miRNAs that are differentially expressed in the susceptible cultivar and six known miRNAs in the resistant cultivar. Differentially expressed miRNAs target 49 transcripts involved in protein localization and pigment synthesis in the susceptible cultivar, whereas they are involved in transcription factor regulation and hormone signalling in the resistant cultivar. The results of our study suggest that the susceptible and resistant hop cultivars respond differently to V. nonalfalfae inoculation at the miRNA level and that miRNAs may contribute to the successful defence of the resistant cultivar.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

PM 7/78 (2) Verticillium nonalfalfae and V. dahliae

Specific scopeThis Standard describes a diagnostic protocol for Verticillium nonalfalfae and V. dahliae on hop (Humulus lupulus).This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2007–09. Revised in 2020–04.

The natural persistence and distribution of the proposed biological control agent Verticillium nonalfalfae on Ailanthus altissima in Virginia, USA

AbstractReports of Ailanthus altissima stand declines in south‐central Pennsylvania resulted in the identification of the causal agent, a vascular wilt fungus, Verticillium nonalfalfae. Additional surveys throughout North America and Europe found that this disease outbreak was not an isolated event. One of these surveys identified and monitored six naturally diseased A. altissima stands in Virginia, USA, between 2009 and 2012. To help predict the long‐term outcome of infested stands, these naturally infested sites were monitored again from 2017 to 2018. While A. altissima were still present at all six sites, a reduction in the number of living A. altissima per hectare (from 604 to 66 overall) and A. altissima basal area per hectare (from 16.1 to 2.9 m2/ha overall) was consistently seen. At all but one of these sites, V. nonalfalfae was still causing disease on the remaining A. altissima. This reduction in A. altissima and persistence of V. nonalfalfae highlight the effectiveness of the pathogen at controlling this tree over long periods of time. A walking survey looking closely at all A. altissima stands along roads within 1 km of these sites found additional V. nonalfalfae infections around four sites. Since no correlation existed between tree infection status and distance from the known site, the patchy disease occurrence may indicate this pathogen is able to spread over longer distances or is more abundant than the previous survey suggested. Soil collected at these sites did not infect any A. altissima seedlings in a greenhouse study, suggesting that soil inoculum may not play an important role in V. nonalfalfae persistence. This research further supports the use of V. nonalfalfae as an effective biopesticide that can reduce A. altissima numbers consistently over many years.

Hop Polyphenols in Relation to Verticillium Wilt Resistance and Their Antifungal Activity.

(1) Background: Verticillium wilt (VW) of hop is a devastating disease caused by the soil-borne fungi Verticillium nonalfalfae and Verticillium dahliae. As suggested by quantitative trait locus (QTL) mapping and RNA-Seq analyses, the underlying molecular mechanisms of resistance in hop are complex, consisting of preformed and induced defense responses, including the synthesis of various phenolic compounds. (2) Methods: We determined the total polyphenolic content at two phenological stages in roots and stems of 14 hop varieties differing in VW resistance, examined the changes in the total polyphenols of VW resistant variety Wye Target (WT) and susceptible Celeia (CE) on infection with V. nonalfalfae, and assessed the antifungal activity of six commercial phenolic compounds and total polyphenolic extracts from roots and stems of VW resistant WT and susceptible CE on the growth of two different V. nonalfalfae hop pathotypes. (3) Results: Generally, total polyphenols were higher in roots than stems and increased with maturation of the hop. Before flowering, the majority of VW resistant varieties had a significantly higher content of total polyphenols in stems than susceptible varieties. At the symptomatic stage of VW disease, total polyphenols decreased in VW resistant WT and susceptible CE plants in both roots and stems. The antifungal activity of total polyphenolic extracts against V. nonalfalfae was higher in hop extracts from stems than those from roots. Among the tested phenolic compounds, only p-coumaric acid and tyrosol markedly restricted fungal growth. (4) Conclusions: Although the correlation between VW resistance and total polyphenols content is not straightforward, higher levels of total polyphenols in the stems of the majority of VW resistant hop varieties at early phenological stages probably contribute to fast and efficient activation of signaling pathways, leading to successful defense against V. nonalfalfae infection.

The Inability of Spotted Lanternfly (Lycorma delicatula) to Vector a Plant Pathogen between its Preferred Host, Ailanthus altissima, in a Laboratory Setting.

Simple SummaryThe invasive and accidently introduced insect, the spotted lanternfly, is spreading rapidly and becoming abundant in the mid-Atlantic region of the USA. Though this insect prefers to feed on the also invasive tree-of-heaven, its ability to feed on other native and crop plant species is concerning, and therefore eradication and control efforts are underway. These efforts include targeting the difficult to control tree-of-heaven for removal. Recently, researchers have found that a naturally occurring fungus effectively kills the tree-of-heaven and work towards making this fungus publically available is ongoing. Therefore, we tested whether the spotted lanternfly is capable of spreading the pathogen between symptomatic fungus-inoculated tree-of-heaven seedlings or plant material to healthy tree-of-heaven seedlings in a controlled laboratory setting. In these conditions, we found no evidence that this transmission occurred. This included monitoring the seedlings for symptoms and sampling the seedlings and the insects for the fungus. This lack of transmission may indicate that the spotted lanternfly cannot help spread this fungus to other tree-of-heaven.With the recent introduction of the non-native spotted lanternfly (Lycorma delicatula) to the USA, research and concern regarding this insect is increasing. Though L. delicatula is able to feed on many different plant species, its preference for the invasive tree-of-heaven (Ailanthus altissima) is apparent, especially during its later life stage. Therefore, management focused on A. altissima control to help limit L. delicatula establishment and population growth has become popular. Unfortunately, the control of A. altissima is difficult. Verticillium nonalfalfae, a naturally occurring vascular-wilt pathogen, has recently received attention as a potential biological control agent. Therefore, we studied if L. delicatula fourth instars or adults could vector V. nonalfalfae from infected A. altissima material to healthy A. altissima seedlings in a laboratory setting. We were unable to re-isolate V. nonalfalfae from the 45 A. altissima seedlings or from the 225 L. delicatula utilized in this experiment. We therefore, found no support that L. delicatula could effectively vector this pathogen between A. altissima in laboratory conditions. Since L. delicatula’s ability to vector V. nonalfalfae has implications for the dissemination of both this beneficial biological control and other similar unwanted plant pathogens, future research is needed to confirm these findings in a field setting.

Changes in the Phenolic Compounds of Hop (Humulus lupulus L.) Induced by Infection with Verticillium nonalfalfae, the Causal Agent of Hop Verticillium Wilt.

Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne vascular pathogen Verticillium nonalfalfae causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (Humulus lupulus L.). In this study, we investigated the differential accumulation of phenolics in the susceptible “Celeia” and resistant “Wye Target” hop cultivars during the pathogenesis of Verticillium wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar.

Field-inoculated Ailanthus altissima stands reveal the biological control potential of Verticillium nonalfalfae in the mid-Atlantic region of the United States

Ailanthus altissima, perhaps the best-known example of an entrenched invasive weed tree in North America, was introduced to the Eastern U.S. roughly 240 years ago. The biological control of A. altissima has been a topic of interest since the discovery of a destructive naturally occurring Verticillium wilt disease of A. altissima in 2002. After nearly 20 years of research, an augmentative commercial release of this disease agent, Verticillium nonalfalfae, could be initiated in the near future. However, a few questions still remain: i) does the interaction of V. nonalfalfae with the less virulent V. dahliae inhibit the biological control effectiveness of V. nonalfalfae, and ii) do climate and A. altissima stand variables affect this biological control’s efficacy? To help answer these questions, a three-year field inoculation study including 3,245 A. altissima trees in 13 sites across four hardiness zones of Pennsylvania and Virginia, U.S. was implemented. Disease progressed and spread at similar rates in A. altissima trees co-inoculated with V. nonalfalfae and V. dahliae as those inoculated with V. nonalfalfae alone, with no indication of disease progression changing in co-inoculated trees. Verticillium dahliae alone resulted in lower levels of disease, and no disease spread. Similar results were seen in a supplemental greenhouse inoculation study. Despite slight regional variation of disease progression and spread correlated to climate or stand variables, V. nonalfalfae always caused severe disease and spread rapidly to other A. altissima trees through the forested plots. Our results support the use of V. nonalfalfae as a biological control agent throughout the mid-Atlantic region of the U.S. regardless of stand and climate variables, and including sites where trees are already infected with V. dahliae.

Draft Genome Sequences of Three Strains of Verticillium nonalfalfae Exhibiting Different Levels of Aggressiveness on Ailanthus altissima.

Verticillium nonalfalfae, a soilborne vascular fungus, shows promise for biocontrol of highly invasive Ailanthus altissima strains. This announcement provides draft genome sequences of the aggressive isolate G1/5 (wild-type strain), the highly aggressive isolate Vert56 (improved strain), and the mildly aggressive isolate I3/2, all obtained from symptomatic A. altissima trees in Austria.