Factors influencing infection of the tomato by Verticillium albo-atrum. II

Tomato plants receiving adequate supplies of mineral nutrient acquired great resistance to infection by Verticillium albo-atrum if the leaf-shoot ratio was reduced; this effect is attributed to reduction in carbohydrate content of the host. Wide variation in potash manuring did not affect susceptibility of tomatoes to Verticillium .     

Studies on the nature of resistance in tomato plants to Verticillium albo-atrum

Extracts of healthy resistant and of healthy susceptible plants of tomato had the same effect on growth of Verticillium albo-atrum in vitro. Tracheal saps from resistant and from susceptible plants showed no difference in their effect on spore germination and mycelial growth of V. albo-atrum. Cuttings from resistant plants, inoculated with V. albo-atrum and fed with low concentrations of casamino acids, or glucose, at first wilted more than controls but soon recovered. Continuous treatment with dilute ethanol solutions for 2 weeks induced marked wilting in inoculated cuttings of resistant plants: treatment for shorter periods caused less severe symptoms, from which cuttings recovered slowly. Metabolic inhibitors did not break resistance of cuttings, but the pathogen survived longer in cuttings treated with sodium diethyldithiocarbamate, salicylaldoxime or 8-hydroxyquinoline than in controls. When one end of segments of stems of resistant plants was inoculated with the pathogen, and 48 h later the uninoculated end was placed near a colony of V. albo-atrum on agar, growth of the fungus colony towards the stem segment was sometimes inhibited. There was no such inhibition when segments from susceptible plants were used. Both tracheal sap and diffusates from segments of inoculated resistant plants supported less growth of germ tubes of V. albo-atrum than sap and diffusates from uninoculated plants. These differences were not obtained with the susceptible variety and production of fungitoxic substances in resistant plants after infection is inferred.     

The response of tomato seedling roots to infection by Verticillium albo-atrum or Fusarium oxysporum f. sp. lycopersici

The response of seedling roots of near-isogenic tomato varieties to infection by Verticillium albo-atrum or Fusarium oxysporum f. sp. lycopersici was investigated. Studies of the infection of seedling roots not artificially damaged indicated that there was an extra-vascular expression of resistance towards V. albo-atrum but not to F. oxysporum. Roots of resistant tomato seedlings infected by V. albo-atrum contained the fungus in the epidermis and outer cortex while susceptible roots became heavily colonised. Observations made by transmission electron microscopy showed that the fungus appeared to be abnormal in growth and appearance in the epidermal and cortical cells of resistant seedling roots but normal in susceptible roots. Two preformed antifungal terpenoids were detected in seedling roots in greater amounts in resistant that in susceptible varieties. The possible mechanisms of seedling root resistance to vascular wilts are discussed.     

The reactions of susceptible and resistant tomato cultivars to strains of Verticillium albo-atrum

Three strains of Verticillium albo-atrum causing severe wilt of tomato (T), progressive (Hp ) and fluctuating (HF) wilt of hop, were inoculated through the roots of four tomato cultivars at different inoculum concentrations. Symptoms were assessed visually 42 days after inoculation, and quantitatively on the change in total leaf area compared with controls. Distribution of mycelium and tyloses was determined by sections at 2 cm intervals of root, stem and petiole. Cultivars Loran Blood and Moscow showed resistance to disease expression at all levels of inoculum concentration with the T strain. Bonny Best and Potentate were both susceptible to this strain, but whereas in Potentate, disease severity increased from mild to severe with increase in inoculum concentration, Bonny Best was severely diseased at the lowest level of inoculum. All cultivars showed some susceptibility to the HP and HF strains; the ‘resistance’ of Loran Blood and Moscow was no longer apparent and Bonny Best was most severely affected. The relative susceptibilities to the strains were HF Bonney Best > Loran Blood > Potentate > Moscow, HP Bonny Best > Loran Blood, Moscow > Potentate, T Bonny Best > Potentate > Loran Blood, Moscow. In general, vascular colonization was less in the cultivars Loran Blood and Moscow with all three fungal strains at io⁵propagules/ml level of inoculum, but this was not always correlated with an increase in disease severity. With the exception of the host-pathogen combinations Bonny Best/T, Bonny Best/HF, Potentate/T and Moscow/T, increasing the inoculum concentration to 10⁷propagules/ml increased disease severity but had little or no effect of increasing vascular colonization. In Bonny Best/T, Bonny Best/HF and Potentate/T vascular colonization was reduced with the higher level of inoculum. Moscow showed complete resistance to symptom expression and little vascular colonization with the T strain at 10⁵prop./ml. At 10⁷prop./ml resistance was maintained but there was very extensive growth of mycelium in the vessels. Tylosis resulted from an interaction of host, fungal strain and the level of inoculum and was not always correlated with the degree of vascular colonization. Contrary to previous reports the resistant varieties Loran Blood and Moscow developed acute disease symptoms after inoculation with HP and HF and these were associated with a high level of tylosis rather than mycelial growth. Tylosis and disease severity but not mycelial growth increased with higher levels of inoculum. The results suggested that susceptibility to Verticillium wilt was a complex response depending on host cultivar, fungal strain and the initial inoculum concentration. In some cultivar-pathogen combinations susceptibility was directly proportional to the amount of mycelium present in the vessels, while in others a physiological resistance mechanism independent of the degree of colonization appeared to operate. In a third category, increased disease development rather than resistance was associated with high levels of tylosis.     

Elicitation of Ethylene by Verticillium albo-atrum Phytotoxins in Potato

Petioles from a susceptible cultivar (Désirèe) of Solanum tuberosum treated with a low‐molecular mass toxin, separated from culture fluid of Verticillium albo‐atrum, produced greater quantities of ethylene than did petioles of a tolerant cultivar (Home Guard). Pretreatment of leaflets from cv. Désirèe with silver thiosulphite, which inhibits perception of ethylene, prevented the chlorosis and necrosis normally associated with exposure to the toxin. Similarly, application of aminoethoxyvinylglycine (AVG) an inhibitor of aminocyclopropane‐1‐carboxylic acid (ACC) synthase, to petioles of cv. Désirèe reduced toxin‐induced ethylene synthesis and symptom development. The data indicate that, in part, Verticillium‐toxin acts through induction of ethylene biosynthesis in the host tissues, and different responses of susceptible and tolerant potato cultivars to V. albo‐atrum are the result of differential production of ethylene.     

Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces

Background

Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus.

Methodology/Principal Findings

We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum on a range of wounded plant tissues. Fungal transformants were obtained from co-cultivation on a range of plant tissue types, demonstrating that plant tissue provides sufficient vir gene inducers to allow A. tumefaciens to transform fungi in planta.

Conclusions/Significance

This work raises interesting questions about whether A. tumefaciens may be able to transform organisms other than plants in nature, or indeed should be considered during GM risk assessments, with further investigations required to determine whether this phenomenon has already occurred in nature.     

Verticillium albo－atrum激发子诱导的番茄组织培养细胞的反应性氧迸发

从对Ｖｅｒｔｉｃｉｌｌｉｕｍａｌｂｏ－ａｔｒｕｍ感抗性不同的两株近等位基因番茄获得细胞悬浮培养物Ｓ－ＧＣＲ２６和Ｒ－ＧＣＲ２１８。用．ａｌｂｏ－ａｔｒｕｍ激发子处理后,敏感性的Ｓ－ＧＣＲ２６未表现出明显的反应性氧改变。抗性的Ｒ－ＧＣＲ２１８则响应以反应性氧迸发。反应性氧迸发中的细胞,其反应性氧累积量与激发子浓度的关系呈近Ｓ形。悬浮介质、某些无机离子、培养物的生长状况和培养日龄等因素均影响细胞对激发子的反应性氧迸发响应。     

Properties of wilt-toxins produced by Verticillium albo-atrum Reinke & Berth.

Cultures of Verticillium albo-atrum grown in a liquid, mineral salts/sucrose medium, were harvested at daily intervals and subjected to gel-filtration on Sephadex G-25 columns eluted with demineralized water. Ultraviolet scanning of the eluate indicated a broad division into high and low molecular weight fractions (HMW and LMW). The HMW material was found to be a variable mixture of a protein component (M.W. c. 100,000) possessing mild cellulase activity, and fructosan (M.W. 5,000–10,000). Both components produced wilting in a lucerne detached-leaf bioassay. Heat denaturation resulted only in the loss of the protein component with a corresponding reduction in wilt-toxicity. A number of LMW components were found to produce leaf necrosis, but only in high concentrations and long-duration assays. These were considered to be of doubtful significance in the wilt syndrome. The possible action mechanisms and the basis of resistance are discussed.     

Properties of β-Galactosidase from Verticillium albo-atrum

An intracellular, inducible β-galactosidase [EC 3.2.1.23] was partially purified from Verticillium albo-atrum. The activity was associated with a particle of about one million molecular weight and required polyhydroxyl compounds for stabilization and activation. It was inhibited by various sulfhydryl inhibitors and EDTA. The latter inhibition could be overcome by adding Mn²⁺ to reaction mixtures. The β- galactoside (ONPG) activity toward lactose (apparent K_m= 0.08 M) and o-nitrophenyl-β-D-galactoside (ONPG) (apparent K_m= 2×10^-23M) purified in parallel. Lactose competitively inhibited the degradation of ONPG with a K_i of 0.1 M. When activated by glycerol, the enzyme produced not only glucose and galactose from lactose, but also other unidentified products, perhaps by transglycosylation.