STUDIES IN THE INTERACTIONS BETWEEN SPECIES OF VERTICILLIUM

It is shown that five parasitic species of Verticillium , viz. V. alboatrum, V. Dahliae, V. ticorpus, V. nigrescens and V. nubilum , developed together on agar media or wheat grains as saprophytes without causing mutual antagonism but, when these were injected together, as a mixed spore suspension, into antirrhinum and tomato, suppression of V. tricorypus, V. nigescens and V. nubilum by either one or other of the remaining two invariably resulted. The two most virulent pathogens, V. alboatrum and V. Dahliae , were not recoverable from soil after the elapse of 6 months from the time of inoculation; while the other three less virulent pathogens could be isolated after about 12 months in the soil. It is concluded that V. alboatrurn and V. Dahliae may be described as 'root inhabiting' and V. nigrescens and V. nubilurn as 'soil inhabiting', while V. tricorpus may be considered as an intermediate type.     

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.     

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.     

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 .     

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.     

STUDIES IN THE ANTAGONISM BETWEEN BLASTOMYCES LUTEUS AND SPECIES OF VERTICILLIUM

Blastomyces luteus has been shown to be antagonistic in culture to both Verticillium albo-atrum and V. dahliae over a wide range of temperature and irrespective of the pH value of the medium. When B. luteus is grown on Dox's and potato-dextrose solutions it produces an exudate which, added to agar media, has an inhibiting effect upon the growth of these two species of Verticillium. The effectiveness of this inhibiting material is slightly reduced by boiling for 15 min.
When 'spent' Dox's liquid medium, which has supported the growth of B. luteus , is injected into tomato and antirrhinum seedlings inoculated with Verticillium no control of the disease is effected, but when B. luteus mycelium is added to soil infected with Verticillium , marked reduction in the incidence of disease results when the antagonistic organism and the pathogens have co-existed in the soil a sufficient length of time for the exudate of the former to be effective upon the development of the latter.     

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.     

Effect of the chemical and physical condition of the soil on Verticillium wilt of antirrhinum

Summary In vitro study showed thatVerticillium dahliae Kleb. grew well in a wide range of acid and alkaline media (viz. pH 3.5 to 10.5). The best growth of the fungus was observed in pH 5.5. Soil pH 3.5 was toxic for growing antirrhinum seedlings. Development of Verticillium wilt of antirrhinum was affected by soil pH. The severity of the disease was greater in alkaline soil conditions compared with acid conditions. Soil of pH 3.5 gave very good control of the symptom expression by the infected plants. Rhizosphere analysis results showed that fungal population with the exception ofPenicillium spp., was drastically reduced in the rhizosphere of the plants grown in acid soil. Although the overall population of fungi was reduced in theV. dahliae infected antirrhinum rhizosphere in acid soil, the population ofPenicillium spp. markedly increased. The antagonistic activity of thePenicillium spp. in the rhizosphere might also have reduced the disease severity. Since the seedlings did not grow properly in very dry and very wet soil, rhizosphere analysis of these soils was not possible. Disease severity was much less in wet soil compared with plants grown in medium moisture level and dry soil, but the plant growth was very poor. Dedicated to the memory of the late Prof. H. K. Baruah from whom I had the inspiration for research     

Effects of organic amendments to soil on the rhizosphere microflora of antirrhinum infected withVerticillium dahliae Kleb.

Summary Organic (e.g. chitin, green manure, cellulose) amendments to soil induced quantitative and qualitative changes in the rhizosphere microflora of antirrhinum plants infected withVerticillium dahliae Kleb. Whereas reduction in disease severity occurred with chitin and green manure amendments, an increase in disease severity was observed with the application of cellulose. The reduction of the disease severity with chitin and green manure may be correlated with the increased population of actinomycetes in the antirrhinum rhizosphere.     

VERTICILLIUM WILT OF BRUSSELS SPROUT

A wilt disease of Brussels-sprout plants caused by Verticillium dahliae Kleb, is described. Field observations indicate that the disease is more severe in a wet than in a dry season, the various stages of the pathological symptoms appearing earlier and developing more rapidly. This was corroborated by experiment; under dry conditions the onset of wilt symptoms was delayed and the severity of attack diminished. Since nine different strains and/or species of Verticillium wound-inoculated into Brussels sprouts failed to induce wilt, and since the isolate from this host proved to be non-pathogenic to a wide range of plants usually susceptible to attack by Verticillium spp., it is suggested that the V. dahliae from Brussels sprouts is a distinct physiological strain. Variations in the amounts of the different chemical constituents of the soil (calcium, nitrogen from two different sources, phosphate and potassium) have no apparent effect upon the incidence of disease. The pathogen is not seed-borne but it may be spread by the dissemination of infected plant tissues. Some control measures are suggested and farmers are advised to grow in the infected soil runner beans, cauliflower and broccoli which are resistant to attack by this fungus.     

Effects of inorganic amendments (N,P and K) to soil on the rhizosphere microflora of antirrhinum plants infected withVerticillium dahliae Kleb

Summary A study of the inorganic amendments (N, P and K) to soil, and their effect on the rhizosphere microflora, as well as their relation to the control of wilt of antirrhinum plants caused byVerticillium dahliae Kleb. was done. Ammonium sulphate was the only chemical found to be significantly inhibitory toV. dahliae in vitro. Soil amendments (NPK) affected the rhizosphere microorganisms of the antirrhinum plants. Higher concentration of the chemicals were phytotoxic. It was further observed that ammonium sulphate, and the combined chemicals (NPK 25%) in soil delayed the senescence in healthy plants, suggests that chemical fertilisers affected the host plants directly. Addition of ammonium sulphate (0.25%), calcium nitrate (0.25%, 0.5%) combined NPK (0.25%) to soil caused considerable reduction in disease severity. It is assumed that this reduction may be caused by the (1) fungitoxic nature of the chemicali.e. ammonium sulphate, (2) antagonistic environment for the pathogen in the rhizosphere was boostedi.e. where calcium nitrate was added as soil amendments and (3) reduction in disease severity in soil-amended with combined NPK, may be due to the fact that antagonistic actinomycete population was boosted in the rhizosphere.     

Control of Verticillium wilt transmission in strawberry runners with systemic benzimidazole-type fungicides

Pre-planting root dips and soil drenches with benomyl, carbendazim (MBC), thiabendazole and thiophanate-methyl were used to reduce disease severity in mother plants. Transmission of the pathogen Verticillium dahliae through stolons and into young runners was prevented by two applications of benomyl or thiophanate-methyl at o-i% active ingredient applied at stolon appearance and 1 month later. Sufficient concentrations of fungistatic residues were detected in all plant tissues to suppress the internal spread of the pathogen.     

Factors influencing infection of the tomato by Verticillium albo-atrum

Infection of tomato plants by Verticillium albo-atrum was encouraged by application of nitrogenous manures. Application of phosphate had no significant effect on the progress of the disease, but a deficiency of potash tended to encourage it. Steam-sterilized soil inoculated with Verticillium immediately after treatment produced a very high total of infected plants. When inoculation of the steamed soil by Verticillium was delayed for 17 days or longer after steaming, the steamed soil was no more favourable for development of the disease than untreated soil. Spread of Verticillium from the roots of an infected plant to those of neighbouring healthy plants was hastened by killing the infected plant.     

Effect of Paenibacillus alvei, strain K165, on the germination of Verticillium dahliae microsclerotia in planta

Verticillium wilt is a devastating disease of a wide range of herbaceous and woody plant hosts, incited by the soilborne fungus Verticillium dahliae. In the present study, the effect of the potential biocontrol isolate Paenibacillus alvei, strain K165, on the germination of V. dahliae microsclerotia (msc) was investigated. Strain K165 was isolated from tomato root tips and its activity against V. dahliae has been shown in glasshouse and field experiments. In the present study, the application of K165 resulted in the reduction of msc germination of V. dahliae, in the root tips and the zone of elongation, of eggplants by 50% compared to the control treatment; whereas 10 and 12 cm away from root tips and in soil without plants the percentage of msc germination was reduced by 26% and 40%, respectively. However, K165 did not significantly affect the number and length of hyphae per germinated msc. In a split-root system, K165 triggered induced systemic resistance in eggplants against V. dahliae by reducing disease severity and msc germination by 27% and 20%, respectively. In addition, K165 colonised the rhizosphere of eggplants and soil in a population density of 5 and 3 log₁₀ cfu g⁻¹, 7 dpi, respectively. This is the first report of evaluating the direct/indirect effect of a rhizospheric bacterium on msc germination in the rhizosphere of eggplants, indicating that strain K165 reduces msc germination.     

THE INFECTION OF PLANTS BY VIRUSES THROUGH ROOTS

Roots of young tomato plants became infected when inoculated with tomato bushy stunt, tobacco mosaic, and potato X viruses. Root infections also occurred when these viruses were added to soil or culture solutions in which plants were growing.
The viruses were sometimes localized around their initial entry points in roots; sometimes they invaded the root system but not the shoots, and sometimes they produced full systemic infection of roots and shoots. In some experiments, but not all, systemic infections were more frequent when the upper tap root or superficial roots were inoculated than when fibrous roots were inoculated.
In both tomato and potato, virus X spread from diseased to healthy plants sharing the same culture solution, if their roots were in contact, but not otherwise. Infection of the roots of potato plants by inoculation, produced only one plant with virus-infected haulms, although several had infected tubers.     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt disease.     

Arbuscular mycorrhizal fungi and biological control of Verticillium-wilted cotton plants

The interaction of arbuscular mycorrhizal fungi (Glomus etunicatum, Glomus intraradices, and Glomus versiforme) with a wilt-causing soil-borne pathogen, Verticillium dahliae, was studied in cotton. It was found that establishment by arbuscular mycorrhizal fungi reduced disease index. In diseased cotton plants colonised by G. etunicatum, the disease index was less than other diseased mycorrhizal and non-mycorrhizal ones. In diseased cotton plants, chlorophyll content was lower than others. Three Glomus species significantly increased content of sugar and protein in shoot and root. Pathogen-infected plants had higher proline concentration in shoot and root than healthy plants. On the other hand, the increased content of proline as stress sensor showed that Verticillium accelerates senescence and reduces yield. These results suggest that the beneficial effects of mycorrhiza can alleviate the pathogenesis effects of V. dahliae partly, and also there is a competitive interaction between the pathogenic and symbiotic fungi.