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.

     

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease‐resistance proteins with nucleotide binding site (NBS) and leucine‐rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS‐LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS‐LRR disease‐resistance proteins and triggers the production of trans‐acting (ta)‐siRNAs, most of which target mRNAs of defense‐related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e‐derived ta‐siRNA‐mediated silencing on NBS‐LRR‐disease‐resistance proteins. It is speculated that a miR482‐mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.     

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star‐shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

Survival of the causal agents of 'early-dying disease' (Verticillium wilt) of potatoes

Although the isolation of Verticillium albo-atrum and V. dahliae from soil and dried moribund stems following infection of a potato crop proved extremely difficult, both fungi were equally capable of overwintering in these substrates and of inducing disease in a subsequently planted susceptible crop. In the absence of a susceptible crop some weed species became colonized. The two species, however, appeared to differ in their capacity for survival both beneath a monocotyledonous crop and within the potato tubers. Colonization of the roots of wheat, barley, oats, rye and maize was observed with V. dahliae but not with V. albo-atrum. The latter appeared to be capable of prolonged survival in the tubers, whereas V. dahliae did not remain viable in storage over winter. Consequently only tubers infected with V. albo-atrum produced infected plants. The presence of the fungi within the tubers affected neither dormancy nor the initial development of the sprouts. Some correlation was noted between tuber size, the percentage of tubers infected, the distribution of V. albo-atrum within the tubers and the development of disease in plants subsequently grown from these tubers.     

Effect of vesicular-arbuscular mycorrhizal fungi on verticillium wilt of cotton

The development of vesicular-arbuscular mycorrhizal fungi (VAMF): Glomus mosseae (Nicol and Gerd.) Gerdemann and Trappe, Glomus versiforme (Karsten) Berch, Sclerocystis sinuosa Gerdemann and Bakhi and Verticillium dahliae and the effects of the VAMF on the verticillium wilt of cotton (Gossypium hirsutum L. and Gossypium barbadense L.) were studied with paper pots, black plastic tubes and clay pots under natural growth conditions. All of the tested VAMF were able to infect all the cotton varieties used in the present experiment and typical vesicles and arbuscules were formed in the cortical cells of the cotton roots after inoculation. The cap cells, meristem, differentiating and elongating zones of the root tip were found to be colonized by the VAMF. In the case of most V. dahliae infection, the colonization occurred mostly from the root tip up to 2 cm. VAMF and V. dahliae mutually reduced their percentage of infection when inoculated simultaneously. VAMF inoculation reduced the numbers of germinable microsclerotia in the soil of the mycorrhizosphere, while the quantity of VAM fungal spores in the soil was not influenced by infection of with V. dahliae. The % of arbuscule colonization in roots was negatively correlated with the disease grades, while the numbers of vesicles in roots were not. These results suggest that certain vital competition and antagonistic reactions exist between VAMF and V. dahliae. VAMF reduced the incidence and disease indices of verticillium wilt of cotton during the whole growth phase. It is evident that cotton seedling growth was promoted, flowering was advanced, the numbers of flowers and bolls were increased, and this resulted in an increase in the yield of seed cotton. Among the VAMF species, Glomus versiforme was the most effective, and Sclerocystis sinuosa was inferior. So far as the author is aware, such an effect of VAMF on the increase of cotton wilt tolerance/resistance is reported here far the first time.     

The Relationship Between Peroxidase Activity and the Resistance of Tomatoes (Lycopersicum esculentum) to Verticillium dahliae

The levels of peroxidase activity in roots of non-infected tomato plants resistant to Verticillium dahliae Kleb., were found to be considerably higher than those in the roots of susceptible plants. Similar differences were found in the leaves but were not significant as in the roots. Peroxidase activity increased in leaves, stems and roots of both susceptible and resistant plants after infection with V. dahliae. The ratio of the increase in leaves and roots of susceptible plants was greater than those of resistant plants. A positive correlation was found between the peroxidase activity in the roots of eleven cultivars/breeding lines and presence of Ve gene.     

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.     

Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and V. longisporum

A collection of 24 isolates of Verticillium dahliae, 11 isolates of V. longisporum and one isolate of V. albo‐atrum originating from different host plants and geographical regions was tested for genetic variability by random amplified polymorphic DNA‐polymerase chain reaction (RAPD–PCR). Based on nine primers, the three Verticillium species could be clearly differentiated. Likewise, this analysis provided a distinct separation of vegetative compatibility groups (VCG) 2B, 4A and 4B of V. dahliae by specific DNA banding patterns. Additionally, V. longisporum was found to segregate into two subgroups with only 88% similarity. This molecular‐genetic approach was used for the analysis of randomly selected Verticillium isolates from a field with high intensity of oilseed rape cultivation (33% in crop rotation). RAPD‐PCR analysis revealed that 95 of 100 isolates tested belonged to V. longisporum and 5 to VCG 2B of V. dahliae. This study demonstrates an adaptation of Verticillium soil populations to a specific cropping history.     

Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana

Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.     

Appartenance Raciale et Pathogenie Comparée de Quelques Isolats de Verticillium dabliae (Kleb.) Obtenus a Partir de Tomateś Résistantes au Maroc

Pathogenecity and race classification of some isolates of V. dahliae from resistant tomato in Morocco In Morocco, the cv. H 204, resistant to Verticillium dahliae race 1 is widely used under plastic tunnels. Use of this cultivar controlled the disease for many years but recently a high incidence of Verticillium on this hybird was obsereved. V. dahliae was isolated from 90 % of the wilted plants.The other 10 % were infected by F. oxysporum f. sp. lycopersici alone (7 %) or in association with V dabliae (3 %). The inoculation of the Verticillium susceptible cv. Vemone and of the race 1 resistant cv. H 204 by 120 isolates obtained from the resistant hybird, showed that (69 %) of the isolates are race 1 and 31 % are race 2. There are different virulence levels of V. dahliae race 2 both on the susceptible cv. Vemone and on the race 1 resistant cv. H 204. Race 2 on the average was less virulenton the susceptible than on the resistant cultivar.     

Association between the potato cyst-nematode, Heterodera rostochiensis Woll., and Verticillium dahliae Kleb. in the early-dying disease of potatoes

Investigation of a possible association between Verticillium dahliae and H. rostochiensis (pathotype E, British notation) was based on field observations and an examination of disease development in single-stemmed potato plants grown in pots. An association was found in the distribution of the nematode and the fungus in the field, and the disease was far more severe with a combined infection than with either pathogen alone. Studies on leaf-area development and yield reduction suggest there is synergism between fungus and nematode, the reductions produced by the combined infection exceeding the sum of those produced by either pathogen alone. Fungal mycelium and the extent of host colonization by V. dahliae were greatly increased by the presence of the nematode. The possible benefits to V. dahliae in the fungus-nematode complex are discussed.     

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.     

Soil inoculum density of Verticillium dahliae and Verticillium wilt of olive in Lebanon

In the Mediterranean basin, Verticillium Wilt of Olive (VWO) is diffused throughout its range of cultivation, causing severe yield losses and tree mortality. The disease was reported in almost all the Mediterranean and Middle East countries, and in Lebanon it is of increasing significance also on many valuable crops. The disease has already been reported on potato, peach and almond in the Bekaa valley; however, to date no information is available about the incidence of VWO and the inoculum density of Verticillium dahliae microsclerotia in soil of the main agricultural areas of Lebanon. Results from the present investigations demonstrate a high V. dahliae frequency in soils (75.3%), coupled with a mean soil inoculum density of 17.0 MS g^?1, clearly indicating a great impact on the production of susceptible hosts in Lebanon, mainly in Bekaa region. Molecular method to assess the microsclerotia inoculum density in soil allowed the detection of a higher frequency of infested soils, as compared with the traditional plating, thus confirming its higher sensitivity. The overall Verticillium wilt prevalence in the inspected olive orchards was 46.2%, and the frequency of V. dahliae‐infected trees was 25.7%. The widespread presence of V. dahliae in all olive growing areas of Lebanon enforces the adoption of measures aimed at reducing the soil inoculum density before any new olive plantation, and the use of strong phytosanitary regulations to improve the certification schemes of propagating material.     

Verticillium wilt of cacao in Uganda: symptoms and establishment of pathogenicity

Pathogenicity tests have shown that Verticillium dahliae Kleb. causes a vascular wilt disease of cacao (Theobroma cacao L.) in Uganda. Isolates of V. dahliae from cacao, okra (Abelmoschus esculentus Medik.), and cotton (Gossypium hirsutum L.) induced similiar symptoms on artificially inoculated cacao seedlings. External symptoms of the disease include acute wilting, foliar chlorosis and abscission, stunting of roots and shoots, and production of suckers at lower nodes of severely affected plants. The internal symptoms are vascular discoloration, tylosis, and presence of gum deposits and mycelium of V. dahliae in xylem vessels.     

Isolation of microsatellite markers from an interspecific hybrid isolate of the fungal plant pathogen Verticillium dahliae

Ten microsatellite markers have been isolated from an amphihaploid isolate of Verticillium dahliae using genomic libraries enriched for (AGT), (GAC), (GCC), (TAC) and (TTA) repetitive elements. These were characterized on four amphihaploid isolates and two haploid isolates each of V. dahliae and Verticillium albo‐atrum. Nine were polymorphic, with between two and five alleles, and the tenth was polymorphic when tested on a further 20 haploid V. dahliae isolates (three alleles). Only one primer pair gave the expected double bands from the amphihaploid isolates, supporting the view that V. albo‐atrum is not one of the parents of the interspecific hybrid amphihaploid isolates.     

The symptoms and causal agents of early-dying disease (Verticillium wilt) of potatoes

The only characteristic symptom produced by Verticillium albo-atrum and V. dahliae in infected potato plants is unilateral chlorosis and necrosis: this was not shown until the approach of host maturity, and was distinguishable from symptoms of natural senescence only in its slightly earlier expression. Of six species of Verticillium tested against potato (King Edward), V. albo-atrum, V. dahliae, V. nigrescens and V. nubilum were pathogenic (all produced ‘wilt’ symptoms and relative virulence was in that decreasing order) but V. tricorpus and V. lateritium did not induce disease. Isolates of V. albo-atrum and V. dahliae, obtained from a number of other host plants, were also pathogenic to potato. Possible reasons are given for the fewness of records of ‘Early dying’ disease (Verticillium wilt) of potatoes in the field.     

Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton

Two isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.