Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.     

Verticillium albo-atrum as a parasite ofSenecio vulgaris

Many weeds, includingSenecio vulgaris, are virtually symptomless hosts ofVerticillium albo-atrum. Plants ofSenecio vulgaris were inoculated with spore suspensions ofV. albo-atrum by two different methods: root-dipping or injection. Infected plants rarely wilted but were stunted, their lower leaves died prematurely and numbers of lateral shoots were decreased.V. albo-atrum was isolated from all parts of the host including achenes. When placed on agar, infected achenes germinated more rapidly than healthy ones. Only small amounts of mycelium developed in the vessels. Severe wilting was possibly avoided because there is insufficient time for damaging amounts of mycelium to develop inSenecio which has a short period of vegetative growth. On the other hand its tolerance to infection may be attributed to the morphology of its vascular system with its narrow vessels and sparse parenchyma cells.In co-operation withH. van Geest, J. P. Overdulve andC. J. J. Richter.     

Weed hosts of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in cotton fields in Turkey and characterization of <Emphasis Type="Italic">V. dahliae</Emphasis> isolates from weeds

A weed survey conducted in 2004 and 2005 in Aydin province of Turkey showed that Solanum nigrum, Xanthium strumarium, Amaranthus retroflexus, Portulaca oleracea, Sonchus oleraceus and Datura stramonium were the most prevalent weeds in the cotton fields exhibiting Verticillium wilt. Verticillium dahliae Kleb. was recovered from A. retroflexus and X. strumarium in those cotton fields. This is the first report of V. dahliae occurring naturally in A. retroflexus in Turkey. Pathogenicity tests on cotton and weeds showed that the virulence of V. dahliae isolates from weeds was higher on cotton plants than on weeds, with the disease severity ranging from 31.7% to 98.0%. Disease severity of V. dahliae isolates was 54.7–93.9% on eggplant, 23.7–51.6% on cucumber and 11.0–16.4% on tomato, whereas it did not cause any disease symptoms, or only low levels, on pepper and bell pepper. Two vegetative compatibility groups (VCGs) were identified among seven tested weed isolates: VCG2A (two isolates) and VCG2B (three isolates) using international reference strains.     

Recent progress in understanding relationships betweenVerticillium species and subspecific groups

Improved understanding of the genetic diversity within fungi in the genusVerticillium has resulted from recent studies based on vegetative compatibility analysis and several techniques of molecular biology. Although the method used to identify vegetative compatibility groups (VCGs) does affect the results, vegetative compatibility appears to be a stable characteristic among isolates. Fairly low VCG diversity has been detected withinV. dahliae andV. albo-atrum using nitrate non-utilizing mutants. VCGs do not appear to be related to pathogenicity to particular host species, with the exception ofV. albo-atrum on alfalfa. However, there is some correlation with virulence on certain hosts and with the ability ofV. dahliae to interact with root-lesion nematodes. Studies based on DNA analysis indicate thatV. dahliae andV. albo-atrum are closely related but separate species. Restriction fragment length polymorphism (RFLP) studies have identified several subspecific groups withinV. dahliae, including two non-host-adapted groups and two that are host-adapted. They also have confirmed that alfalfa strains ofV. albo-atrum are a distinct subgroup that is probably a separate population of clonal origin. Using polymerase chain reaction (PCR), a second non-host-adapted subgroup withinV. albo-atrum was identified that was previously unknown.     

Weed hosts ofVerticillium dahliae in crete: Susceptibility, symptomatology and significance

A survey of common and uncommon weed species usually showing Verticillium wilt symptoms was carried out during 1992–2000 in Crete, Greece.Verticillium dahliae was isolated in 48 out of 182 sampled fields, in which several weed species were grown, from several locations in Oropedio, Lasithi. Altogether, 124 isolates ofV. dahliae were recovered from the vascular stem-tissue of 19 weed species, belonging to ten botanical families. Pathogenicity trials with 13 out of 19 weed species that have never been reported as hosts of the fungus, using for inoculation isolates which originated from the same weed species, resulted in infection of all of them, showing various disease symptoms. Seven weed species (Anthemis melanolepis, Cardaria draba, Convolvulus arvensis, Erodium sp.,Euphorbia helioscopia, Helminthotheca echioides andSinapis alba) are new hosts worldwide, and six additional species (Euphorbia sp.,Lactuca serriola, Raphanus raphanistrum, Sinapis arvensis, Sonchus oleraceus andTrifolium sp.) are new hosts for Greece. The most susceptible (isolation frequency: 27.9–52.8%, moderate disease severity) species were:Capsella bursa-pastoris, C. draba, Chenopodium album, Senecio vulgaris andSolanum nigrum. Less susceptible (isolation frequency: 4.8–17.8%, slight disease severity) were:Amaranthus sp.,A. melanolepis, C. arvensis, Erodium sp.,Euphorbia sp.,E. helioscopia, H. echioides, L. serriola, Malva sylvestris, R. raphanistrum, S. alba, S. arvensis, S. oleraceus andTrifolium sp. Some species —C. draba, C. album, L. serriola andS. nigrum L. — that usually showed external and vascular wilt symptoms, occasionally exhibited only reduced growth. Visible symptoms under natural field conditions in all 13 weed species that had never been reported as hosts ofV. dahliae were similar to those observed after their artificial inoculation. The fungus was not isolated fromFoeniculum vulgare ssp.piperitum, Oxalis corniculata andStellaria media, among other species. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Cross-pathogenicity of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> between potato and sunflower

This study examined cross-pathogenicity of the soilborne pathogen Verticillium dahliae between potato and sunflower. Four week-old potato and sunflower seedlings were inoculated with ten isolates from each of the two host species. Potato cultivars (Kennebec, susceptible, and Ranger Russet, moderately resistant) and sunflower hybrids (IS8048, susceptible, and 6946, moderately resistant) were assessed for disease severity and percent infection at 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after inoculation (w.a.i), and for vascular discolouration at 6 w.a.i., using visual scales developed for each host species. The experiments were conducted in 2006 and repeated in 2007. Based on percent infection and disease severity, most V. dahliae isolates were highly aggressive on both host species. The tested isolates caused higher disease levels in the susceptible than in the moderately resistant phenotypes. They also caused more vascular discolouration in their original than in the alternative host. However, the isolates originating from sunflower caused less infection and disease severity on both hosts, compared to their potato counterparts. Cluster analysis based on all of the criteria used to assess pathogenicity led to three groups of isolates: (i) most V. dahliae potato isolates, which ranged with the highly aggressive control isolates, (ii) one V. dahliae sunflower isolate, which showed a similar pathogenicity level to the weakly-aggressive V. albo-atrum sub-group II control isolate, with no more symptoms than in the non-inoculated plants, and (iii) most V. dahliae sunflower isolates with mildly- to weakly-aggressive levels. Based on these results, V. dahliae cross-pathogenicity is very effective between potato and sunflower. Therefore, rotations involving these species should be avoided, especially where sunflower follows potato.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Retrotransposon-like elements in the genome of Verticillium dahliae may be used as DNA markers for fungal species and pathotypes

Using differential hybridization, two DNA fragments, VDf35 and VDf90, specific to Verticillium dahliae, were isolated. These fragments contained truncated open reading frames (ORFs) homologous to the gypsy-type retrotransposon. The ORFs of VDf35 and VDf90 were pol and gag homologs, respectively. In addition, VDf90 had a pol homolog without an ORF sequence. The pol homologs in VDf35 and VDf90 were similar to each other, and these two DNA fragments had completely identical sequences. Genomic Southern analysis revealed that numerous copies of these homologs existed in V. dahliae, suggesting that V. dahliae carries a gypsy-like retroelement. Genomic Southern and polymerase chain reaction (PCR) analysis also indicated that a large number of these homologs exist in V. longisporum as well as in V. dahliae, but only a few were present in V. albo-atrum. No homolog was found in either V. nigrescens or V. tricorpus. The uneven distribution of these homologs of the retroposon-like elements among Verticillium species suggested a close genetic kinship between V. dahliae and V. longisporum. PCR primers designed from VDf35 showed species- or pathotype-specific amplification. Therefore, this sequence may be useful as a DNA marker to identify species and pathotypes of V. dahliae. This is the first report on a retrotransposon-like sequence in the genome of phytopathogenic Verticillium species.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB095264 (VDf90) and AB095265 (VDf35)     

Involvement of two different types of Verticillium dahliae in lettuce wilt in Ibaraki Prefecture, Japan

Although wilt diseases of various plants by Verticillium dahliae occur worldwide, Verticillium wilt of lettuce is relatively new and rare. In 2009, this disease appeared suddenly in commercial fields in Ibaraki Prefecture, Japan. To investigate the cause of the disease, we characterized each lettuce isolate for pathogenicity, mating types, and genetic characteristics and found two types of V. dahliae. A population that included at least two types of V. dahliae was the putative infection source of the disease. Effective management systems against V. dahliae isolates of different races and having different host ranges are necessary.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

Control of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> at a strawberry nursery by paddy-upland rotation

Effects of crop rotation between rice paddy fields and strawberry nurseries on the control of Verticillium wilt of strawberry were studied. For detecting Verticillium dahliae, the causal agent of Verticillium wilt, in soil, eggplant was used as an indicator plant. We were thus able to detect as low as 1 microsclerotium/g dry soil. In field surveys of Chiba and Hokkaido from 2000 to 2003, V. dahliae was detected in 9 of 10 upland fields but in none of 21 paddy-upland fields. In Hokkaido during 2000–2007, strawberry mother plants were planted, and plantlets were produced in upland and paddy-upland fields to assess V. dahliae infestation. Verticillium wilt of strawberry had never occurred in 72 tested paddy-upland fields, compared to 13.2–73.9% of plantlets infected with V. dahliae in upland fields. In a pot experiment in a greenhouse, two flooding treatments or two paddy rice cultivations suppressed Verticillium wilt symptoms on eggplant. In field experiments, one paddy rice cultivation in Chiba and two in Hokkaido prevented development of Verticillium wilt symptoms on eggplant. Verticillium wilt of strawberry was controlled completely with one paddy rice cultivation in infested fields in Chiba. In these field experiments, the number of microsclerotia of V. dahliae decreased under the flooding conditions for paddy rice cultivation. Based on the reduction in microsclerotia, a crop rotation system with paddy rice for 3 years (three times), green manure for 1 year, and strawberry nursery for 1 year was designed for Hokkaido.     

Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.     

Verticillium wilt in nursery trees: damage thresholds,spatial and temporal aspects

Verticillium wilt can cause high losses in tree nurseries. To be able to predict disease and unravel disease dynamics over time and space, the relationship between verticillium wilt and soil inoculum densities of Verticillium dahliae and the nematode Pratylenchus fallax was studied in two 4-year field experiments with Acer platanoides and Catalpa bignonioides in the Netherlands. Best-fit regression equations showed that pre-planting inoculum densities of V. dahliae can be used to predict verticillium wilt over a period of at least 4 years. Pratylenchus fallax contributed significantly to disease severity in A. platanoides in some years. Disease can already occur at the detection limit of the pathogens. The 5% infection thresholds for V. dahliae were at 1 (A. platanoides) vs. 3 (C. bignonioides) colony-forming units (CFU) g⁻¹ soil. Analysis of spatial relationships indicated that diseased plants had a higher influence on neighbouring plants at low V. dahliae inoculum densities (<5 CFU g⁻¹ soil) than at high densities (≥5 CFU g⁻¹ soil). Seventy-four percent of the diseased plants recovered during the following year. After that year, recovered plants had a significantly higher probability of becoming diseased again than plants that were healthy during the two previous years, at high inoculum densities of V. dahliae, indicating that inoculum density in the soil, rather than incomplete recovery, was the most important factor for disease development.     

Low Temperature–Short Duration Steaming of Soil Kills Soil-Borne Pathogens, Nematode Pests and Weeds

Agricultural soil samples containing survival structures of the fungal crop pathogens Verticillium dahliae, Sclerotinia sclerotiorum, Sclerotium cepivorum, Pythium ultimum, potato cyst nematodes Globodera rostochiensis and G. pallida and weeds Chenopodium album and Agropyron repens [Elymus repens] were treated in the laboratory with aerated steam at temperatures ranging from 40 to 80°C in a specially constructed apparatus. Steaming at 50 or 60°C for 3min, followed by an 8-min resting period in the steamed soil and immediate removal from the soil thereafter, resulted in 100% kill of all weeds, diseases and nematodes. When steamed at 45°C, there was a small but significant reduction in the survival of V. dahliae microsclerotia but no reduction in survival of S. cepivorum.     

Wilt diseases in Gerbera

In The netherlands, the glasshouse area used for cultivation of gerbera as a cutflower was strongly restricted in the past since dying off of the plants generally occurred and constituted a great risk for growers.In most casesPhytophthora cryptogea appeared to be the cause of the disease and this fungus is probably responsible for the main difficulties in this crop since the introduction. Besides foot rot, caused byP. cryptogea, Verticillium albo-atrum andV. dahlinae cause a wilt disease of gerbera. Newly developed cultural methods prevent heavy losses, resulting in a strong expansion of the culture.Samenvatting Schade door bodemschimmels heeft lange tijd een sterke uitbreiding van de gerberateelt in Nederland verhinderd. Hierin is verandering gekomen doordat een goede bedrijfshygiëne en verbeterde cultuurmaatregelen (grondontsmetting, grondverwarming, drainage) het risico voor de kweker aanzienlijk hebben verminderd.De oorzaak van de belangrijkste ziekte in Nederland, het zogenaamde voetrot, is de schimmelPhytophthora cryptogea. Er zijn verschillende argumenten die er voor pleiten dat deze vanaf de invoering van de teelt in Nederland verantwoordesijk is geweest voor de grootste moeilijkheden op de bedrijven. Daarnaast kunnenVerticillium albo-atrum enV. dahliae optreden als oorzaak van verwelkingsziekte.Stationed at the Proefstation voor de Bloemisterij in Nederland, Aalsmeer.     

Reliable detection of unevenly distributed Verticillium dahliae in diseased olive trees

Verticillium wilt caused by Verticillium dahliae is one of the most threatening diseases of olive worldwide. For pre‐planting and post‐planting control of verticillium wilt in olive trees, availability of a rapid, reliable and non‐destructive method for detection of V. dahliae is essential. For such a method, suitable and easily performed sampling and efficient processing of samples for extraction of DNA are necessary. In this study, the suitability of young twig and leaf samples of olive trees, which are easy to collect and extract DNA from, were assessed for the detection of V. dahliae in routine procedures. The lower (about 50 cm from the tip) and top parts (about 5 cm from the tip) of twigs, as well as leaves from infected olive trees were screened for V. dahliae infection and distribution using real‐time PCR. The biomass of V. dahliae detected in individual twigs was highly variable, but there was no significant difference between mean quantities of V. dahliae DNA detected in top and lower parts of twigs. Furthermore, it was demonstrated that analysis of combined samples containing DNA extracted from five twigs of an infected tree accurately detected the presence of the pathogen. Similarly, testing combined samples of 5–10 leaves enabled reliable detection of the pathogen in an infected tree. The development of this assay enables reliable detection of V. dahliae in infected olive trees that can aid in management decisions for the implementation of integrated disease management.     

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.     

新疆棉花黄萎病的发生现状及其病原菌的分子鉴定与ISSR分析

为掌握新疆主要植棉区棉花黄萎病的发生现状及其病原菌大丽轮枝菌Verticillium dahliae的落叶型菌系分布以及遗传变异情况,于2015年对26个新疆主要植棉区棉花黄萎病的发生情况进行了随机调查,统计新疆大丽轮枝菌的培养性状,利用大丽轮枝菌落叶型特异引物D1/D2、INTD2F/INTD2R与非落叶型特异性引物ND1/ND2、INTNDF/INTNDR对新疆大丽轮枝菌菌系进行互补鉴定,并对部分菌系的遗传变异进行简单序列重复区间(inter simple sequence repeat,ISSR)分析。结果表明:2015年新疆棉花黄萎病发病田比例为54.0%,其中病情指数在10.0以上的发病田与2013年持平,而病情指数在20.0以上的严重发病田比例为10.8%,比2013年增加3.8个百分点;新疆大丽轮枝菌的培养性状以菌核型为主,比例为70.1%,菌丝型与中间型比例分别为13.4%和16.5%;新疆大丽轮枝菌落叶型菌系比例为53.2%,26株菌株的来源地全部检出落叶型菌系;聚类分析结果显示,当遗传相似系数为0.66时,新疆大丽轮枝菌落叶型与非落叶型菌系聚为2个谱系,菌系地理来源、培养性状与大丽轮枝菌的遗传分化无明显相关性。     

Asexual fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> is potentially heterothallic

Verticillium dahliae, a soilborne plant pathogen, causes wilt disease in many important crops. We reported previously that the mating type gene MAT1-2-1 is spread to isolates of this asexual fungus. However, we did not determine whether V. dahliae is homothallic or heterothallic because the opposite mating type gene, MAT1-1-1, had not been identified. In the present study, we identified the MAT1-1-1 gene from an isolate lacking MAT1-2-1 and the mating type idiomorphs of V. dahliae. Each isolate we tested contained either the MAT1-1 or MAT1-2 idiomorph, indicating that the asexual fungus V. dahliae is potentially heterothallic.     

Plant Phenology Influences the Effect of Mycorrhizal Fungi on the Development of Verticillium-induced Wilt in Pepper

Verticillium dahliae alters water status and consequently, growth and production of pepper plants. On the other hand, arbuscular mycorrhizal fungi (AMF) can reduce damage caused by specific soil-borne plant pathogens and improve drought resistance of pepper. Therefore, one objective of this research was to assess if AMF can modify the development of Verticillium-wilt in pepper plants. A second objective was to study the influence of plant phenology at the moment when V. dahliae was inoculated on the possible biocontrol of the disease by AMF. Results suggested that AMF reduce the deleterious effect of V. dahliae on pepper growth and yield. However, bioprotection against Verticillium-wilt was conditioned by plant phenology at the moment of pathogen attack. The highest efficacy of AMF occurred when V. dahliae was inoculated during the vegetative stage of plants. AMF allowed leaf relative water content to be maintained for longer and delayed both the appearance of disease symptoms and the decrease of photosynthesis in Verticillium-inoculated plants. These benefits on plant physiology increased pepper yield.