THE SPREAD OF DISEASES CAUSED BY SPECIES OF VERTICILLIUM

An investigation has been made of the spread of Verticillium disease of tomato and antirrhinum by root contact between diseased and healthy plants, and by the growth of the fungi through the soil. V. alboatrum and V. Dahliae spread rapidly by root contact, while V. tricorpus, V. nigrescens and V. nubilum showed no appreciable spread. Where the tomato and antirrhinum hosts wilted and died the causal organism was invariably isolated from the soil near the roots of the dead plants, but where the inoculated host harboured species without showing disease symptoms, as in the case of V. tricorpus and V. nubilum in antirrhinum, the parasite was never obtained from the soil. Moreover, these species in antirrhinum, and V. nigrescens and V. nubilum in tomato would appear to be mild pathogens since when they were already well established V. alboatrum was later actually found to enter and parasitize the respective hosts and bring about its own typical disease symptoms.
Spread through the soil in the absence of hosts was tested by the insertion into it of infected wheat grains followed by later attempts to isolate the fungi at varying distances from the point of infection. V. alboatrum, V. Dahliae and V. tricorpus showed little or no growth through the soil, whereas V. nigrescens and V. nubilum spread saprophytically to some extent, though never attaining sufficient concentration to cause appreciable infection of later planted tomatoes.
The conclusion is reached that spread of disease caused by species of Verticillium takes place mainly by root contact, with a rapidity relatively proportional to that of the death of the host. This finding, together with the lack of spread from antirrhinum plants infected with the non-lethal V. tricorpus and V. nubilum , suggests that the parasites remain in the vascular tracts until the death of the hosts.     

Verticillium wilt of sainfoin

A wilt disease of sainfoin caused by Verticillium Dahliae Kleb. is described, and it is shown that the fungus can penetrate sainfoin seedlings through unwounded roots as well as through ruptures caused by the emergence of lateral rootlets. V. Dahliae was isolated from naturally infected soil only in June, July and August, although another species, V. nigrescens , was obtained throughout the year.
Comparative studies of the longevity of cultures of V. Dahliae, V: albo-atrum and V. nigrescens proved that all are viable for at least 3 years on agar media. On sterilized wheat grains V. Dahliae dies within 8 weeks after inoculation, V. albo-atrum and V. nigrescens within 12 weeks, while the hyaline variants of the first two remain viable for 6 months.
Evidence was obtained that in artificially inoculated soil V. Dahliae persists mainly as microsclerotia. The fungus may also exist in the soil as hyaline mycelium or conidia, but only for a relatively short time.
The incidence of this disease in sainfoin is reduced by an increase in soil-water content, but is unaffected by the application of lime to the soil.     

SOME SOIL FACTORS AFFECTING VERTICILLIUM WILT OF ANTIRRHINUM

Variations in the application rates of chalk and superphosphate and the omission of all the fertilisers had no visible effect on the incidence of wilt caused by any of the five Verticillium species.
he literature dealing with the influence of soil conditions upon the Verticillium wilt of a wide range of host plants is reviewed. It is shown that the species V. alboatrum, V. dahliae, V. nigrescens, V. nubilum and V. tricorpus vary inter se in their pathogenicity to Antirrhinum majus, and that their infectivity may be influenced by soil treatments. Thus, in pot cultures, the incidence of antirrhinum wilt induced by Verticillium dahliae and V. nigrescens was reduced by increasing sulphate of potash or ammonium sulphate; or by decreasing soil moisture. Very wet soil and heavy dressings of hoof-and-horn were the only conditions under which V. nubilunt and V. tricorpus induced wilt symptoms. Z7. albo-atrunt was the most virulent species tested; none of the soil treatments decreased its pathogenicity.     

THE EFFECTS OF NITROGEN SUPPLY UPON THE VERTICILLIUM WILT OF ANTIRRHINUM

Isolates of Verticillium albo-atrum, V. dahliae and V. nigrescens grown on media adequately supplied with sodium nitrate induced wilt rather more rapidly in antirrhinum plants growing in soils with a normal and an excessive amount of nitrogen than in plants in nitrogen-deficient soil, though plants became diseased in all soils. Similarly treated isolates of V. nubilum and V. tricorpus induced a greater incidence of wilt in plants in soil supplied with heavy dressings of organic nitrogenous fertilizer than in plants in soil deficient in nitrogen, although V. tricorpus from a medium containing much sodium nitrate, in contrast to V. nubilum , was pathogenic to plants in such deficient soil. The ability of the nitrogen-starved isolates to penetrate the host plant was significantly diminished, and even when wound-inoculated into stems their effect upon the host was much reduced.     

WILT OF LUCERNE CAUSED BY SPECIES OF VERTICILLIUM

The incidence and symptoms of a disease of lucerne induced by Verticillium spp. are much less severe immediately after the crop has been first cut than later when the crop has matured. Infection becomes more marked as the season advances and the age of the ley increases, and cuts taken in the third harvest year are proving unprofitable.
In cross-inoculation studies the causal organisms of lucerne wilt, V. albo-atrum and V. dahliae prove to be only mild pathogens to hop, potato, tomato and antirrhinum, and are not pathogenic to sainfoin and clover. The last two may therefore be cultivated as fodder crops in those areas most severely affected by lucerne wilt, but the cultivation of potatoes after lucerne should be discouraged, particularly if lucerne is to be sown in future years, since the potatoes might build up a reservoir of inoculum of the lucerne pathogen. In studies of host-parasite relations it is shown that V. albo-atrum and V. dahliae from lucerne, V. albo-atrum from hop, V. tricorpus from tomato and V. nigrescens and V. nubilum from potato vary in their effects upon lucerne seedlings growing on agar media, V. albo-atrum from lucerne being, as in the field, the most virulent and the only one to reach the xylem vessels under the conditions of the experiments.
The dressings Agrosan-GN (containing mercury) and Fernasan (containing thiram) are toxic both to spores of Verticillium on lucerne seed coats and to infected plant material carried with seed, and although they are also toxic in vitro to Rhizobium mellelotus this did not prevent dressed seeds which had been inoculated with the bacterium from developing into healthy seedlings bearing normal root-nodules.     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Denaturing gradient gel electrophoresis analysis with different primers of subgingival bacterial communities under mechanical debridement

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.     

WILT OF LUCERNE CAUSED BY SPECIES OF VERTICILLIUM

A wilt disease of lucerne caused by species of Verticillium is described: at twenty-eight disease areas in England and Wales the pathogen was V. albo-atrum , while at one site it was V. dahliae . Both pathogens form superficial conidia on the basal regions of infected stems. It is shown that V. albo-atrum is introduced into a new area in contaminated plant material harvested with the seed from an infected crop. Rapid secondary spread of the disease follows the dissemination of spores from infected stems, and by contact of these and transported fragments of diseased tissues with the wounded surfaces of recently cut lucerne plants. It is recommended that seed should be collected only from healthy crops and that machinery, footwear, etc. should be disinfected before leaving a site of infection.
Manurial trials showed that the incidence of wilt induced by V. albo-atrum was very severe under all soil conditions tested, whereas V. dahliae is a virulent pathogen only to plants in soil rich in superphosphate. Generally the more vigorous the growth of the lucerne–in soils rich in potash and hoof and horn–the more rapid is both the onset of wilt and the resulting secondary spread of the disease throughout the crop.     

Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.     

Friends and foes: streptomycetes as modulators of plant disease and symbiosis

The ecological role of soil streptomycetes within the plant root environment is currently gaining increased attention. This review describes our recent advances in elucidating the complex interactions between streptomycetes, plants, pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-associated microbial communities. Some act as biocontrol agents, inhibiting plant interactions with pathogenic organisms. Owing to the antagonistic properties of streptomycetes, they exert a selective pressure on soil microbes, which may not always be for plant benefit. Others promote the formation of symbioses between plant roots and microbes, and this is in part due to their direct positive influence on the symbiotic partner, expressed as, e.g., promotion of hyphal elongation of symbiotic fungi. Recently, streptomycetes have been identified as modulators of plant defence. By repressing plant responses to pathogens they facilitate root colonisation with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or enhance plant growth. In conclusion, while streptomycetes have a clear potential of acting as biocontrol agents, care has to be taken to avoid strains that select for virulent pathogens or enhance disease development. We argue towards the use of an integrated screening approach in the search for efficient biocontrol agents, including assays on in vitro antagonism, plant growth, and disease suppression.     

Influence of soil fauna on fungal plant pathogens in agricultural and horticultural systems

An understanding of the ecology of plant pathogens is crucial for the development of agricultural and horticultural growing systems that enhance plant health. One important group of organisms that influences plant pathogen survival and dispersal is the soil fauna. This review deals with known and possible interactions between soil animals and different groups of fungal plant pathogens. It is suggested that facultative saprophytes may be affected considerably by mycelial grazing by soil animals, while obligate pathogens may be more influenced by animals that ingest spores and other types of propagules. Both types of pathogens can also be influenced by indirect interactions with the soil fauna. Manipulating the soil environment for enhancement of beneficial soil animals that consume pathogens seems to be a realistic possibility for sustainable agriculture and horticulture.     

How spatial heterogeneity of cover affects patterns of shrub encroachment into mesic grasslands

We used a multi-method approach to analyze the spatial patterns of shrubs and cover types (plant species, litter or bare soil) in grassland-shrubland ecotones. This approach allows us to assess how fine-scale spatial heterogeneity of cover types affects the patterns of Cytisus balansae shrub encroachment into mesic mountain grasslands (Catalan Pyrenees, Spain). Spatial patterns and the spatial associations between juvenile shrubs and different cover types were assessed in mesic grasslands dominated by species with different palatabilities (palatable grass Festuca nigrescens and unpalatable grass Festuca eskia). A new index, called RISES ("Relative Index of Shrub Encroachment Susceptibility"), was proposed to calculate the chances of shrub encroachment into a given grassland, combining the magnitude of the spatial associations and the surface area for each cover type. Overall, juveniles showed positive associations with palatable F. nigrescens and negative associations with unpalatable F. eskia, although these associations shifted with shrub development stage. In F. eskia grasslands, bare soil showed a low scale of pattern and positive associations with juveniles. Although the highest RISES values were found in F. nigrescens plots, the number of juvenile Cytisus was similar in both types of grasslands. However, F. nigrescens grasslands showed the greatest number of juveniles in early development stage (i.e. height<10 cm) whereas F. eskia grasslands showed the greatest number of juveniles in late development stages (i.e. height>30 cm). We concluded that in F. eskia grasslands, where establishment may be constrained by the dominant cover type, the low scale of pattern on bare soil may result in higher chances of shrub establishment and survival. In contrast, although grasslands dominated by the palatable F. nigrescens may be more susceptible to shrub establishment; current grazing rates may reduce juvenile survival.     

The airborne metagenome in an indoor urban environment

The indoor atmosphere is an ecological unit that impacts on public health. To investigate the composition of organisms in this space, we applied culture-independent approaches to microbes harvested from the air of two densely populated urban buildings, from which we analyzed 80 megabases genomic DNA sequence and 6000 16S rDNA clones. The air microbiota is primarily bacteria, including potential opportunistic pathogens commonly isolated from human-inhabited environments such as hospitals, but none of the data contain matches to virulent pathogens or bioterror agents. Comparison of air samples with each other and nearby environments suggested that the indoor air microbes are not random transients from surrounding outdoor environments, but rather originate from indoor niches. Sequence annotation by gene function revealed specific adaptive capabilities enriched in the air environment, including genes potentially involved in resistance to desiccation and oxidative damage. This baseline index of air microbiota will be valuable for improving designs of surveillance for natural or man-made release of virulent pathogens.     

Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor

Oral malodor is considered to originate primarily from tongue microbiota populations. However, the relationship between oral malodor and tongue microbiota remains unclear. In this study, tongue periodontal pathogens were analyzed via real-time PCR, and the association between oral malodor and tongue periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola, was examined. The subject population consisted of 29 individuals with and 10 healthy persons without oral malodor. Oral malodor was assessed by organoleptic test and volatile sulfur compound (VSC) levels as measured by gas chromatography. Real-time PCR was conducted for anaerobes in tongue biofilm samples employing a LightCycler system; furthermore, bacterial proportion served as a quantitative parameter. Among the five anaerobes, only T. forsythia displayed higher proportions in malodor subjects than corresponding values in healthy controls. Proportions of P. intermedia and P. nigrescens correlated strongly with hydrogen sulfide concentration. Proportions of P. gingivalis and P. nigrescens also exhibited strong correlation with methyl mercaptan concentration. The correlation coefficient between the proportion of the total of the five anaerobes and total VSC level (r = 0.88) was greater than that between bacterial proportion and organoleptic score (r = 0.29). When a linear regression analysis was performed utilizing the proportion of each of the five periodontal pathogens as an independent variable, the explanatory power of these independent variables revealed 81% for total VSC level and 16% for organoleptic score. These results suggest that these five periodontal pathogens on tongue dorsa may contribute greatly to VSC production.     

Susceptibility of Prevotella intermedia/Prevotella nigrescens (and Porphyromonas gingivalis) to propolis (bee glue) and other antimicrobial agents

Black-pigmented gram-negative anaerobes such as Porphyromonas gingivalis and Prevotella intermedia are suspected pathogens in adult periodontitis, whereas Prevotella nigrescens has been associated with health. Antimicrobial resistance among bacteria from this group has been reported in the past decade. This research aimed to evaluate and compare the susceptibility profile of 17 P. intermedia/P. nigrescens isolates recovered from patients with periodontitis and three reference strains to six antimicrobials, prescribed in dentistry in Brazil, and propolis (bee glue). The antimicrobial agents tested were tetracycline, penicillin, clindamycin, erythromycin, metronidazole, meropenem and six ethanolic extracts of propolis (EEPs) from Brazil. The reference strains P. gingivalis ATCC 33277 and P. intermedia ATCC 25611 were used for determination of minimum bactericidal concentration (MBC) and for time-kill assay to the EEPs. All of the strains were susceptible to penicillin, erythromycin, meropenem, metronidazole and 95% of them (n=19) to tetracycline. Thirty six percent (n=7) of the P. intermedia/P. nigrescens strains tested were resistant to clindamycin. As for propolis activity, all strains were susceptible and the minimum inhibitory concentration values ranged from 64 to 256 microg/mL. For the reference strains P. gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611 the MBC was 256 microg/mL and death was observed within 3 h of incubation for P. gingivalis and within 6 h for P. intermedia. The action of propolis (bee glue) against suspected periodontal pathogens suggests that it may be of clinical value.     

Virulence of oomycete pathogens from Phragmites australis‐invaded and noninvaded soils to seedlings of wetland plant species

Soil pathogens affect plant community structure and function through negative plant–soil feedbacks that may contribute to the invasiveness of non‐native plant species. Our understanding of these pathogen‐induced soil feedbacks has relied largely on observations of the collective impact of the soil biota on plant populations, with few observations of accompanying changes in populations of specific soil pathogens and their impacts on invasive and noninvasive species. As a result, the roles of specific soil pathogens in plant invasions remain unknown. In this study, we examine the diversity and virulence of soil oomycete pathogens in freshwater wetland soils invaded by non‐native Phragmites australis (European common reed) to better understand the potential for soil pathogen communities to impact a range of native and non‐native species and influence invasiveness. We isolated oomycetes from four sites over a 2‐year period, collecting nearly 500 isolates belonging to 36 different species. These sites were dominated by species of Pythium, many of which decreased seedling survival of a range of native and invasive plants. Despite any clear host specialization, many of the Pythium species were differentially virulent to the native and non‐native plant species tested. Isolates from invaded and noninvaded soils were equally virulent to given individual plant species, and no apparent differences in susceptibility were observed between the collective groups of native and non‐native plant species.     

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.     

Evolution of immunological memory and the regulation of competition between pathogens

Memory is a central characteristic of immune responses. It is defined as an elevated number of specific immune cells that remain after resolution of infection and can protect the host against reinfection. The evolution of immunological memory is subject to debate. The advantages of memory discussed so far include protection from reinfection, control of chronic infection, and the transfer of immune function to the next generation. Mathematical models are used to identify a new force that can drive the evolution of immunological memory: the duration of memory can regulate the degree of competition between different pathogens. While a long duration of memory provides lasting protection against reinfection, it may also allow an inferior pathogen species to persist. This can be detrimental for the host if the inferior pathogen is more virulent. On the other hand, a shorter duration of memory ensures that an inferior pathogen species is excluded. This can be beneficial for the host if the inferior pathogen is more virulent. Thus, while in the absence of pathogen diversity memory is always expected to evolve to a long duration, under specific circumstances, memory can evolve toward shorter durations in the presence of pathogen diversity.     

Mechanisms of Pathogenesis, Infective Dose and Virulence in Human Parasites

The number of pathogens that are required to infect a host, termed infective dose, varies dramatically across pathogen species. It has recently been predicted that infective dose will depend upon the mode of action of the molecules that pathogens use to facilitate their infection. Specifically, pathogens which use locally acting molecules will require a lower infective dose than pathogens that use distantly acting molecules. Furthermore, it has also been predicted that pathogens with distantly acting immune modulators may be more virulent because they have a large number of cells in the inoculums, which will cause more harm to host cells. We formally test these predictions for the first time using data on 43 different human pathogens from a range of taxonomic groups with diverse life-histories. We found that pathogens using local action do have lower infective doses, but are not less virulent than those using distant action. Instead, we found that virulence was negatively correlated with infective dose, and higher in pathogens infecting wounded skin, compared with those ingested or inhaled. More generally, our results show that broad-scale comparative analyses can explain variation in parasite traits such as infective dose and virulence, whilst highlighting the importance of mechanistic details.     

Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin

Virulence properties of pathogenic bacteria, as well as resistance to antibiotics, are thought to arise through a specialization process favoured by the strong selection pressure imposed in clinical treatments. Nevertheless, in the case of opportunistic pathogens, it is unclear whether strains can be classified into virulent and non-virulent isolates. Clones of the opportunistic pathogen Pseudomonas aeruginosa do not seem to be associated to a particular biovar or pathovar, which suggests that virulence characteristics in opportunistic pathogens may already be present in environmental (non-clinical) isolates. We have explored this possibility, studying environmental isolates (mainly from oil-contaminated soils) and clinical isolates (from bacteraemia and cystic fibrosis patients) of P. aeruginosa . All environmental strains were found to actively efflux quinolones, which are synthetic antibiotics not expected to be present in the environment. These strains contained multidrug resistance determinants, were capable of invading epithelial cells and presented genes from the quorum-sensing and type III secretion systems. Some of them expressed either haemolytic or proteolytic activities or both, characteristics considered to be typical of virulent strains. All the strains tested, of clinical or environmental origin, could use alkanes (oil hydrocarbons) as a carbon source. Our results suggest that clinical and non-clinical P. aeruginosa strains might be functionally equivalent in several traits relevant for their virulence or environmental properties. Selection of clinically relevant traits, such as antibiotic resistance or cellular invasiveness, in opportunistic pathogens present in soil ecosystems is discussed.