Assessing Eucryptorrhynchus brandti (Coleoptera: Curculionidae) as a potential carrier for Verticillium nonalfalfae (Phyllachorales) from infected Ailanthus altissima

Two potential biological control agents for the invasive tree-of-heaven, Ailanthus altissima (Mill.) Swingle, have been extensively studied: a vascular wilt fungus, Verticillium nonalfalfae Inderb. (formerly Verticillium albo-atrum Reinke and Berthold), and a host-specific weevil from China, Eucryptorrhynchus brandti Harold, which is currently pending quarantine release. In 2002, V. nonalfalfae was observed in Pennsylvania causing significant mortality to A. altissima. Quarantine experiments were conducted to test different forms of transmission of the fungus by E. brandti to A. altissima. In one experiment, 75% of adult E. brandti transmitted V. nonalfalfae to A. altissima seedlings after contact with a culture and feeding on infected plant material. In another study, 14.7±1.3% of adult E. brandti ingested and passed viable V. nonalfalfae propagules into faeces after feeding on infected A. altissima stems for 24, 48 and 72 h, respectively, and the number of propagules increased at 72 h. Eighty-three per cent of surviving weevils overwintering in naturally infested potting mix carried viable V. nonalfalfae propagules externally. All weevil progeny that emerged from infected A. altissima billets appeared to be as healthy as weevils reared from non-infected billets. Our studies have shown that ingestion of V. nonalfalfae after feeding on infected plant material plays an important role in assessing E. brandti as a potential carrier. Results from these laboratory studies indicate E. brandti has the ability to transfer V. nonalfalfae to A. altissima seedlings in a laboratory setting.     

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.     

Genetic diversity of Miscanthus sinensis in US naturalized populations

Miscanthus is increasingly gaining popularity as a bioenergy grass because of its extremely high biomass productivity. Many clones of this grass were introduced into United States over the past century from East Asia where it originated, and planted for ornamental and landscaping purposes. An understanding of the genetic diversity among these naturalized populations may help in the efficient selection of potential parents in the Miscanthus breeding program. Here, we report our study analyzing the genetic diversity of 228 MiscanthusDNA samples selected from seven sites in six states (Ohio, North Carolina, Washington D.C., Kentucky, Pennsylvania, and Virginia) across the eastern United States. Ten transferable DNA markers from other plant species were employed to amplify genomic DNA of Miscanthus because of the paucity of molecular markers in Miscanthus. There were significant genetic variations observed within and among US naturalized populations. The highest genetic diversity (0.3738) was found among the North Carolina genotypes taken from Biltmore Deer Park and Biltmore, Madison County, Cody Rd. The lowest genetic diversity (0.2776) was observed among Virginia genotypes that were diverged from those from other states, suggesting Virginia genotypes might be independently introduced into the United States from the different origin. By the cluster and structure analysis, 228 genotypes were categorized into two major groups that were further divided into six subgroups at the DNA level and the groups were generally consistent with geographic region.     

Clonal growth in Ailanthus altissima on a natural site in West Virginia

Abstract. : The exotic tree Ailanthus altissima is usually confined to open sites. As an exception, Ailanthus established in a densely closed forest in West Virginia, which was analyzed with the aim to elucidate possible pathways of persistence in forest succession of this light-demanding pioneer species. Demographic analysis revealed a seedling mortality of 100 %. Instead, the understory is populated by clonal ramets, ranging from one to more than 19 yr of age, with a mean of 5 yr. Growth averages only 0.11 m/yr, and height is correlated with age. This clonal growth contrasts with the performance of Ailanthus on open sites. The possible ecological benefits of establishing a ramet bank in a resource-poor habitat are considered in terms of space occupation of a pioneer species.     

Einsatz von V. lecanii als biologisches Schädlingsbekämpfungsmittel gegen den Bohnenrostpilz U. appendiculatus var. appendiculatus im Feld und im Gewächshaus

The use of Verticillium lecanii as a biological control agent against the bean rust fungus Uromyces appendiculatus var. appendiculatus in the field and in the glasshouse The deuteromycete V. lecanii parasites uredo- and teliospores of the bean-rust-fungus U. appendiculatus var. appendiculatus. We investigated the conditions for the use of the hyperparasite as biological control agent in the field and in glasshouses. The growth rate of the hyperparasite was 0,3 cm per day at 25 °C. Under suitable conditions in the lab (25 °C, 100 % r. h.) it took about 20 days to invade 100 % of uredospores and 65 % of teliospores. We failed to prevent the spread of bean-rust-fungus spores in the field, but we succeeded in the glasshouse by 68 %, compared to the untreated controls, using the hyperparasite V. lecanii as biological control agent.     

Effects of cotton–maize rotation on soil microbiome structure

Verticillium wilt is a disastrous disease in cotton-growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton–maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long-term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton–maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control.     

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.     

Assessing the Risk of Biological Control Agents on the Indigenous Microbial Communities: <Emphasis Type="Italic">Serratia plymuthica</Emphasis> HRO-C48 and <Emphasis Type="Italic">Streptomyces</Emphasis> sp. HRO-71 as Model Bacteria

The phytopathogenic fungus Verticillium dahliae Kleb. causes high yield losses in strawberry production. As effective chemical control of this fungus is no longer available, biological control based on natural antagonists might provide new control strategies. The aim of this study was to assess the impact of the two biological control agents S. plymuthica HRO-C48 and Streptomyces sp. HRO-71 on the rhizosphere community of the Verticillium host plant strawberry in field trials at two different sites in Germany. Therefore, we determined the abundances of culturable bacteria and investigated the community structure of the total rhizosphere microbiota by PCR-single strand conformation polymorphism analysis of the 16S rRNA and fungal ITS1 region. The abundances of culturable rhizobacteria on R2A medium as well as the proportion of in vitro Verticillium antagonists did not differ significantly. Additionally, no treatment specific differences were obtained in the composition of species of the non-target antagonistic bacteria in the rhizospheres. The culture-independent analysis revealed only transient differences between the bacterial communities not due to the treatments rather than to the plant growth stage. Fungal and bacterial community fingerprints showed the development of a microbiota, specific for a field site. However, no sustainable impact of the bacterial treatments on the indigenous microbial communities was found using culture-dependent and -independent methods.     

Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

Verticillium dahliae (Kleb.) Infecting Pumpkin Seed

This study investigated the natural occurrence of Verticillium dahliae (Kleb.) infection in pumpkin (Cucurbita pepo L.) seed. The mean incidence of infection was found to be 21.0%. Isolates recovered from seeds were pathogenic to pumpkin (cultivar ‘Jamaican squash’). Surface sterilization by immersion in 0.6% sodium hypochlorite for 20 min eradicated V. dahliae from infected pumpkin seeds without affecting germinability. Plating of seed components revealed that the fungus was present in the seed coat but not in the embryo or cotyledons. In a growing‐on test, 25% of 6‐week‐old plants grown from untreated seeds were infected. Germination and production of normal seedlings were unaffected by V. dahliae infection of seeds. Verticillium dahliae in pumpkin seed was found to be external and transmissible to plants. The findings of this study are important in devising disease control strategies.     

CGTase,a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP-10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP-10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis-related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

KARYOTYPES OF FIVE PLANT SPECIES WITH DISJUNCT DISTRIBUTIONS IN VIRGINIA AND THE CAROLINAS

Five species (Actaea pachypoda, Aralia nudicaulis, Cornus alternifolia, Panax quinquefolius, and Symplocarpus foetidus) display a disjunct distribution between the Mountains and Piedmont in North Carolina, South Carolina, and Virginia. The Piedmont populations are small and have apparently been isolated from the Mountain populations for several thousand years. Karyotype differences among populations were found only in A. pachypoda, and even in this species the differences were slight. The absence of population differences in four species, and the moderate differentiation found in A. pachypoda, indicate considerable cytological stability in these species.     

Control of aphids by the fungus, Verticillium lecanii: Effect of spore concentration

In laboratory bioassays, aphid mortality rose while killing time decreased with increasing spore concentrations of Verticillium lecanii. However, in the glasshouse, the ability of the fungus to be spread among aphid populations on chrysanthemums quickly masked the initial effects of spraying different spore concentration. Hundred-fold differences in spore concentrations produced equally good control of the aphid, Myzus persicae, in the glasshouse.

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Controle des pucerons par le champignon: Verticillium lecanii: effet de la concentration en spores

Résumé Dans des essais de laboratoire, la mortalité des pucerons augmente et est plus rapide quand la concentration en spores de Verticillium lecanii s'élève. Cependant, en serre, la possibilité de pulvériser le champignon sur les populations de pucerons sur chrysanthème a masqué rapidement les effets initiaux des différentes concentrations de spores. Ainsi des concentrations de spores différent de cent fois produisent des résultats aussi bons en serre, contre le puceron Myzus persicae.

     

The interactions of potato cyst nematodes and Verticillium dahliae on early and maincrop potato cultivars

The effects of potato cyst nematodes (Globodera rostochiensis and G. pallida) on the growth of early and later maturing potato cultivars, with or without the H, resistance gene, were compared in pot experiments and the effects of introducing Verticillium dahliae into the system were studied. The influence of the nematodes on root function and structural integrity was inferred from their effects on calcium and water uptake. Early maturing cultivars were less tolerant of nematode attack than later maturing ones, and those with the H₁ resistance gene were more tolerant of G. rostochiensis than G. pallida. When V. dahliae was introduced, Verticillium wilt symptoms appeared much earlier on the early than the maincrop cultivars. Also, Maris Anchor (first early) died before Maris Peer (second early) and death of both when infected by nematodes and fungus was much earlier than uninfected control plants or plants infected with G. rostochiensis only. Maris Peer infected by both organisms snowed symptoms much more severe than when infected by either organism alone, whereas Maris Anchor was almost as severely affected by V. dahliae alone as both organisms together. Pentland Javelin (first early) developed Verticillium wilt symptoms several weeks later than other cultivars and the addition of nematodes failed to increase the severity of the symptoms. These differences in cultivar susceptibility to the combination of nematodes and fungus may be due to the nematodes causing different degrees of trauma in the roots of the three cultivars: measuring calcium uptake per unit water uptake showed that nematodes dramatically increase calcium uptake but that the proportional change is least in Pentland Javelin. If this is a reflection of less damage by nematodes to the endodermis of this cultivar, it may explain the greater resistance of its roots to penetration by V. dahliae.     

Observations on the introduction of Verticillium chlamydosporium and other parasitic fungi into soil for control of the cereal cyst-nematode Heterodera avenue

Isolates of Verticillium chlamydosporium and a sterile fungus added to soil on ground oat grain reduced the numbers of Heterodera avenae on wheat by between 26 and 80%. Nematode populations in uninoculated soil increased from 15 eggs/g soil before planting to 218 eggs/g after harvest. V. chlamydosporium was isolated from oat grain that had been air-dried and milled before introduction into soil. Applications of the fungi on attapulgite clay or as suspensions of mycelium and spores in water had no effect on nematode multiplication. The effect of the fungi on numbers of H. avenae eggs was similar in autoclaved and non-sterilised soil. V. chlamydosporium added on attapulgite clay to a calcareous sand and a calcareous silty loam could be re-isolated after at least 6 months. Some isolates colonised the roots of wheat without causing lesions or affecting the dry weights of shoots or roots. These results indicate that V. chlamydosporium may be useful for the biological control of cyst nematode pests.     

Significant mortality of eggs and young larvae of two pine processionary moth species due to the entomopathogenic fungus Metarhizium brunneum

Bioassays were conducted to determine the susceptibility of egg masses and young larvae of two pine processionary moth species, Thaumetopoea pityocampa and Thaumetopoea wilkinsoni, to two strains (ARSEF4556, V275) of the entomopathogenic fungus Metarhizium brunneum. Mortality of treated eggs by both strains ranged from 96% to 99% but not all of this was caused by M. brunneum since control groups also experienced egg mortality due to saprophytic fungi. Still, larvae hatched in the laboratory from eggs treated with M. brunneum were all killed by this fungus, acquiring M. brunneum conidia, whereas larval mortality was 0% in the control groups. Young larvae of both pine processionary moth species were also highly susceptible to ARSEF4556 and V275 with larval mortality ranging between 94% and 100%, 8 days post-inoculation, with the vast majority of larvae being killed within the first 2–4 days. Larval mortality was dose dependent. Results were consistent across the two pine processionary moth species, showing that the pathogenicity of M. brunneum to both eggs and young larvae might be promising for biological control of these insect pests. The study also showed that non-target parasitoids of pine processionary moth eggs were also susceptible to M. brunneum. Further work is required to understand and reduce the M. brunneum effect on non-target insects.     

Chemical Profiling and Antifungal Activity of Ziziphora clinopodioides Leaf Essential Oil against the Pathogen Verticillium dahliae

Essential oils (EOs) are often used as natural antifungal agents to control the growth of phytopathogenic fungi. The aim of this study was to determine the effect of Ziziphora clinopodioides leaf EO against Verticillium dahliae, a pathogenic fungus of cotton. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of 15 compounds of the total of extracted oil, which was consisted of 98.79 % monoterpenes and 0.61 % sesquiterpenes. The major constituents were pulegone (62.17 %), isomenthone (18.42 %), l-menthone (5.55 %) and piperitenone (3.99 %). The mycelial growth of Verticillium dahliae was completely inhibited at 0.24 μL/mL air under vapor phase condition. Considerable morphological variations were also observed in the fungal sclerotia at the contact phase at 3 μL/mL. This study demonstrated for the first time that Z. clinopodioides EO can effectively inhibit the growth of V. dahliae, implying that it has the potential to be explored as an antifungal agent against Verticillium Wilt of cotton.     

Interactions between seedlings of the invasive tree Ailanthus altissima and the native tree Robinia pseudoacacia under low nutrient conditions

The allelopathic effect of A. altissima and the nitrogen fixing ability of R. pseudoacacia make the interaction between these two species important to community dynamics. A replacement series greenhouse experiment was used to investigate the type of interaction between seedlings of A. altissima and R. pseudoacacia at high and low soil nutrition states. Also, seeds of A. altissima from its native (China) and invasive ranges (USA) were used to compare the effect of the different seed sources on the interaction with R. pseudoacacia. Robinia pseudoacacia was the better competitor. The presence of A. altissima significantly inhibited nodulation of R. pseudoacacia roots. In summary, in low nutrient early succession sites, seedlings of R. pseudoacacia and A. altissima will coexist and R. pseudoacacia will be the better competitor. However, A. altissima can increase its competitive ability by inhibiting nitrogen fixation by R. pseudoacacia. Differences between the competitive ability of the Chinese and US seed lots of A. altissima did not support the rapid evolution theory for invasive species success.     

Untersuchungen über die in Lagerhäusern der DDR Trockenfäule an Kartoffeln verursachenden Fusarium-Arten

The virulence of two strains of the entomopathogenic fungus Verticillium lecanii to the aphids Myzus persicae, Aphis gossypii and Brevicoryne brassicae was bioassayed. The strain (V24 and V18) tested were capable of infecting all three aphids species, but their virulence determined by LC50 and LT50 varied. The strain (V24) originally isolated from M. persicae showed the highest virulence against the homologous aphid species, but the whitefly (Trialeurodes vaporariorum) derived strain (V18) showed also the highest virulence against an aphid species. The variability of the bioassay results and the impossibility of defining clearly traits associated with the virulence of a fungus strain toward a specific insect species are discussed. Effect of additives (glycerin, soyflour and rape oil) was studied for strain improvement. Considerable improvement of the strains virulence was not observed with glycerin and soyflour. The results are discussed in conjunction with the non-convincing results published earlier.