Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against ...which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.
Pythium oligandrum, strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and ...elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum. The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.
Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been ...identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17.
We present here a detailed analysis of Class I retrotransposons and Class II "cut-and-paste" DNA elements detected in the sequenced Verticillium genomes. We report also of their distribution in other Vd and Va isolates from various geographic origins. In VdLs.17, we identified and characterized 56 complete retrotransposons of the Gypsy-, Copia- and LINE-like types, as well as 34 full-length elements of the "cut-and-paste" superfamilies Tc1/mariner, Activator and Mutator. While Copia and Tc1/mariner were present in multiple identical copies, Activator and Mutator sequences were highly divergent. Most elements comprised complete ORFs, had matching ESTs and showed active transcription in response to stress treatment. Noticeably, we found evidences of repeat-induced point mutation (RIP) only in some of the Gypsy retroelements. While Copia-, Gypsy- and Tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other Verticillium spp. strains surveyed. In particular, neither complete nor defective "cut-and-paste" TEs were found in VaMs.102.
Copia-, Gypsy- and Tc1/mariner-like transposons are the most wide-spread TEs in the phytopathogens V. dahliae and V. albo-atrum. In VdLs.17, we identified several retroelements and "cut-and-paste" transposons still potentially active. Some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. Others, such as the ripped Copias, have been potentially acquired by horizontal transfer. The observed biased TE insertion in gene-rich regions within an individual genome (VdLs.17) and the "patchy" distribution among different strains point to the mobile elements as major generators of Verticillium intra- and inter-specific genomic variation.
Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to ...Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.
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.
Since 1992 elm trees have been treated with a biological control product Dutch Trig® to protect them from infection by Ophiostoma novo-ulmi causing Dutch elm disease (DED). The active ingredient of ...the biocontrol product consists of the fungus Verticillium albo-atrum strain WCS850. A conidiospore suspension of this fungus is injected into the vascular system of elm trees at a height of 1.3 m. This biocontrol product prevents healthy elm trees from fungal infection transmitted by elm bark beetles. Dutch Trig®, however, does not protect already infected trees or trees connected with diseased trees via root grafts. Since 2010, only 0.1 % of the injected elms became infected with DED through beetle transmission and an additional 0.4 % of the treated elms were infected through root contact in the Netherlands. Regression analysis considering all injected elm trees in the Netherlands since 1992 indicated that DED infection through beetle transmission had significantly decreased during the 24 years application of Dutch Trig®. In 2015, approximately 28,300 trees in five countries (Netherlands, USA, Germany, Canada and Sweden) were treated with Dutch Trig® to protect valuable susceptible elm trees, mainly in urban environments.
Although new, rapid detection and identification technologies are becoming available more and more for various plant pathogens, pathogen quantification remains one of the main challenges in the ...disease management of many crops. Currently, real-time polymerase chain reaction (PCR) is the most straightforward technique to quantify pathogen presence. This manuscript describes the use of real-time PCR to quantitatively assess the presence of a number of economically important fungal and oomycete tomato pathogens in biological samples. We demonstrate that pathogen DNA can be accurately quantified over at least four orders of magnitude. Additionally, we demonstrate the feasibility of the technique to quantify pathogen biomass in complex biological samples.
The cruciferous fungal pathogen
Verticillium longisporum
represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other
Verticillium
species.
V
.
...longisporum
evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop
Brassica napus
. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents
Verticillium dahliae
. Eleven
V
.
longisporum
isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to
V
.
dahliae
. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the
Verticillium
transcription activator (
VTA
)
2
regulatory isogene pair allow identification of
V
.
longisporum
hybrids by a single polymerase chain reaction and the separation from haploid species as
V
.
dahliae
or
Verticillium albo
-
atrum
. The combination between
VTA2
signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid
Verticillia
and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards
B. napus
.
Verticillium albo-atrum and V. dahliae have both been identified as causing wilt and mortality of the invasive tree species Ailanthus altissima (Chapter I). Verticillium albo-atrum is more virulent ...and aggressive to A. altissima than V. dahliae, under both greenhouse and field conditions. Overwintering (survival) occurs within infected A. altissima trees, on fallen A. altissima leaves, and in soil. Overwintering may also occur within striped maple trees. Inoculation likely takes place in the spring at time of leaf emergence. Wounding of A. altissima seedlings roots increased the rate of symptom development by 4 weeks following inoculation of V. albo-atrum, as compared to non-wounded A. altissima seedling inoculations. Examination of infected plant tissues by means of histology revealed that phenols were deposited in most outer xylem parenchyma within at the least 1 week following inoculation, indicating that the fungus had began to invade the circumference of seedling initially, and then spread both upward and downward until plant mortality occurred. Extensive fungal colonization was not observed until 4 weeks after inoculation, and the seedling had completely wilted. Dissemination potentially involve wind disseminated leaflets, seeds transmission, or ambrosial beetle transmission. Within 1 year of inoculation V. albo-atrum had spread from five initially inoculated trees in two stands of 39 and 95 trees, to 94.9% to 86.3%, respectively, of the A. altissima trees within the two stands. Rate of spread is fairly rapid. Between 200 and 2006, the average spread of mortality caused by V. albo-atrum downwind is approximately 116.2 m/yr and upwind is 64.6 m/yr.
A piece of polychrome and gilded cartonnage mask was excavated by the Ministry of Antiquities in Saqqara region. It belongs to the late period. The mask upon mummy presented a gilded face with heavy ...wig height 30-40 cm was painted with green, black eye brow and red mouth. Cartonnage is a composite material consists of organic support contains many glued layers of linen or papyri that have ground painting layers. It suffers from many deterioration aspects (microbial pigments, and flaking most likely due to inappropriate storage. Examination study was performed to analyze and investigate the ground painting and gilding layer of the cartonnage by optical light microscopy, Scanning electron microscopy (SEM) equipped with energy dispersive X-ray analysis, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The results confirmed that the degradation factors affecting on the painted, gilded cartonnage was attributed to the direct effects of microbial phenomena, which have led to many deterioration forms (macro- and micro cracks, flaking, coloration, scaling and defoliation microbiological spots). Three Gram-positive bacteria (Bacillus cereus, Bacillus licheniformis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and four fungal species (Aspergillus flavus, Botrytes cinerea, Fusarium moniliforme, and Verticillium albo-atrum) were isolated from archeological gilded painted cartonnage. Antibacterial and antifungal activities of three shapes (rods, spheres and prisms) of gold nanoparticles (AuNPs) were tested against the isolated deteriorated microbial species. The rod-shaped gold nanoparticles have high potential antibacterial and antifungal activity reaching to the maximum significant inhibition (8- and 9-cm inhibition zones) against the most sensitive bacterial and fungal species (B. cereus and A. flavus, respectively) accompanied with leakage of protein and nucleic acid, sugars and electrolytes. Disturbance in the blood picture of guinea pigs infected with the most resistant species (P. aeruginosa and F. moniliforme) were showed. The rod-shaped gold nanoparticles were safe and successful in curing the tested inoculated guinea pigs.
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