Vegetative Compatibility Groups 4B Research Articles

Complex Molecular Relationship Between Vegetative Compatibility Groups (VCGs) in Verticillium dahliae: VCGs Do Not Always Align with Clonal Lineages

Verticillium wilts caused by the soilborne fungus Verticillium dahliae are among the most challenging diseases to control. Populations of this pathogen have been traditionally studied by means of vegetative compatibility groups (VCGs) under the assumption that VCGs comprise genetically related isolates that correlate with clonal lineages. We aimed to resolve the phylogenetic relationships among VCGs and their subgroups based on sequences of the intergenic spacer region (IGS) of the ribosomal DNA and six anonymous polymorphic sequences containing single-nucleotide polymorphisms (VdSNPs). A collection of 68 V. dahliae isolates representing the main VCGs and subgroups (VCGs 1A, 1B, 2A, 2B, 3, 4A, 4B, and 6) from different geographic origins and hosts was analyzed using the seven DNA regions. Maximum parsimony (MP) phylogenies inferred from IGS and VdSNP sequences showed five and six distinct clades, respectively. Phylogenetic analyses of individual and combined data sets indicated that certain VCG subgroups (e.g., VCGs 1A and 1B) are closely related and share a common ancestor; however, other subgroups (e.g., VCG 4B) are more closely related to members of a different VCG (e.g., VCG 2A) than to subgroups of the same VCG (VCG 4B). Furthermore, MP analyses indicated that VCG 2B is polyphyletic, with isolates placed in at least three distinct phylogenetic lineages based on IGS sequences and two lineages based on VdSNP sequences. Results from our study suggest the existence of main VCG lineages that contain VCGs 1A and 1B; VCGs 2A and 4B; and VCG 4A, for which both phylogenies agree; and the existence of other VCGs or VCG subgroups that seem to be genetically heterogeneous or show discrepancies in their phylogenetic placement: VCG 2B, VCG 3, and VCG 6. These results raise important caveats regarding the interpretation of VCG analyses: genetic homogeneity and close evolutionary relationship between members of a VCG should not be assumed.

Structural and phylogenetic analysis of the rDNA intergenic spacer region ofVerticillium dahliae

The nuclear ribosomal intergenic spacer (IGS) region was structurally analyzed and exploited for molecular discrimination and phylogenetic analysis of vegetative compatibility groups (VCGs) of Verticillium dahliae. A structural study of 201 available IGS sequences of the fungus was performed, and four classes of ubiquitous repetitive elements, organized in higher-order repetitive structures or composite blocks, were detected in a variable IGS subregion. This subregion was amplified from an international collection of 59 V.dahliae isolates covering all VCGs, together with nine representative V.albo-atrum and V.longisporum isolates, and sequenced. Structural and phylogenetic analyses of the sequences of this polymorphic IGS subregion were consistently informative and allowed the identification of two main lineages in V.dahliae, that is, clade I including VCGs 1A, 1B, 2A, 4B, and 3 and clade II containing VCGs 2B, 4A, and 6. Analysis of IGS sequences proved a highly suitable molecular tool for (a) rapid interspecific differentiation, (b) intraspecific discrimination among VCGs of V.dahliae, facilitating high-throughput VCG confirmation and prediction/profiling, and (c) phylogenetic analysis within and among V.dahliae VCGs.

Vegetative compatibility of Verticillium dahliae isolates from potato and sunflower using nitrate non-utilizing (nit) mutants and PCR-based approaches

Vegetative compatibility groups (VCGs) of 12 potato and nine sunflower isolates of Verticillium dahliae were determined using both nitrate non-utilizing (nit) mutants and multiplex-nested-PCR approaches. Based on analysis of nit mutants, most potato isolates showed strong compatibility with VCG 4A and weak compatibility with VCG 4B; one potato isolate was also weakly compatible with VCGs 2A, 2B and 3. Sunflower isolates were more variable and most of them showed weak reactions with VCG 4A and 4B. One sunflower isolate, Vs06-14, was compatible only with VCG3 testers, whereas another isolate, Vs06-20, was compatible with all VCG groups except VCG2A. Vegetative compatibility of four selected isolates, two from potato and two from sunflower, was also investigated after passing them through potato or sunflower for four generations. The VCG of one potato isolate did not change, whereas the other one changed from VCG4B to 4A. One sunflower isolate, initially compatible with both VCG4A and 4B, became incompatible with both. The other sunflower isolate was not compatible with any VCG and remained so. In a second part of this work, multiplex-nested-PCR was carried out to characterize VCGs of the same isolates of V. dahliae. Vegetative compatibility grouping using this approach assigned the V. dahliae isolates to VCG1, 2A, 3 or 4A. Six of the 12 potato isolates and four of the nine sunflower isolates of V. dahliae could not be identified or placed in any VCG group based on PCR results. In the present study, the multiplex-nested-PCR results were confirmed using nit mutants. As such, PCR-based methods will require more specific primers before they can replace the approach based on nit mutants. Current address of A.F. El-Bebany: Department of Plant Pathology, Faculty of Agriculture, Alexandria University, El-Shatby, 21545, Alexandria, Egypt.

Vegetative compatibility groups and pathogenicity of verticillium dahliae ısolates from potato plants in Erzurum province

One hundred eleven isolates of Verticillium dahliae were obtained from potato plants in Erzurum province, Turkey. The pathogen was isolated from 7.6% of the stems collected. All isolates were assigned to vegetative compatibility groups (VCGs) using nitrate-nonutilizing ( nit ) mutants. In total, 240 nit mutants were obtained from V. dahliae isolates, and classified as nit 1 (71%) and nit M (29%). Two VCGs were found and identified as VCG 2B (34 isolates) and VCG 4B (77 isolates) by using tester isolates of known VCGs. Pathogenicity of V. dahliae isolates was tested on potato (cv. Marfona) by the root-dip method. Both VCG 2B and VCG 4B isolates showed similar aggressiveness on potato. This is the first study of VCGs of V. dahliae isolates from potato plants in Turkey.

Verticillium wilt of potato – the pathogen, disease and management†

Verticillium wilt is a major disease of potato caused by either Verticillium dahliae or Verticillium albo-atrum. Both species are soilborne fungi that invade xylem elements, disrupt water transport in plants and cause vascular wilt in a variety of hosts. Despite the broad host range of V. dahliae, a degree of host adaptation occurs with some isolates exhibiting different levels of aggressiveness depending on the host species and even cultivar within a species. In addition, isolates can be separated into vegetative compatibility groups (VCGs) based on the ability to undergo hyphal anastamosis with other isolates. Distinct sub-specific groups have been recognized which contain host-adapted pathotypes that are aggressive and prevalent on particular hosts. Isolates of V. dahliae collected from potato stems, tubers and potato fields in the USA mostly belong to VCG 4A and VCG 4B, with the VCG 4A isolates being highly aggressive on potato compared with isolates of VCG 4B and other VCGs. In contrast, isolates collected from potatoes grown in Israel were predominantly VCG 2A and VCG 4B. Initial inoculum of V. dahliae is primarily soilborne microsclerotia. Verticillium dahliae can be transmitted in certified seed tubers, but tuber-borne inoculum appears to have little effect on verticillium wilt symptoms and potato yields. Vascular infection by V. dahliae of seed tubers of the moderately susceptible potato cultivar ‘Russet Burbank’ resulted in negligible effects on the development of verticillium wilt symptom, did not significantly contribute to aboveground stem infection or the formation of microsclerotia in debris, and did not significantly contribute to progeny tuber infection. An importance of tuber-borne inoculum is the potential to introduce inoculum to soils not previously used to grow potato or where a management practice such as fumigation has been applied to reduce soilborne inoculum. Management strategies should focus on reducing initial inoculum in soil and restricting vascular infection and pathogen development within the host. Tactics that may reduce initial inoculum include crop rotation, soil fumigation, green manures, soil solarization, and bio-fumigants. Cultivar resistance restricts pathogen infection and colonization of plants. Integration of several management tactics and a better understanding of soil health will be needed for lasting and economic management of the disease.

Genetic and pathogenic characterization of Verticillium dahliae isolates from eggplant in Turkey

During 2005 to 2007, eggplant fields in 19 provinces from three different regions (western, southern and southeastern Anatolia regions) of Turkey were surveyed for Verticillium wilt. Sixty-seven isolates of Verticillium dahliae from wilted eggplants were collected and used for vegetative compatibility analysis using nitrate non-utilizing mutants and reference tester strains of vegetative compatibility groups (VCGs) 1A, 2A, 2B, 3, 4A and 4B. Among all isolates, 33 (12 from western, 15 from southern and six from southeastern Anatolia) were assigned to VCG2B, 23 (four from western, eight from southern and 11 from southeastern Anatolia) to VCG2A, six (four from southern, one from western, and one from southeastern Anatolia) to VCG4B and five (one from western, one from southern and three from southeastern Anatolia) to VCG1A, whereas VCG3 and VCG4A were not defined among isolates. In order to test if there is a correlation between VCG and pathogenicity in V. dahliae, pathogenicity of 30 isolates, representing the four multimember VCGs, were tested on Solanum melongena cvs. ‘Kemer’ and ‘Aydin Siyahi’ in an unheated greenhouse. All isolates were found to be pathogenic on both cultivars and there was no difference in susceptibility between the two cultivars. VCG4B isolates collectively led to higher vascular discoloration index (VDI) on both cultivars and higher disease severity index (DSI) on ‘Kemer’ compared with other VCGs. Similarly, VCG1A caused lower VDI on both cultivars and lower DSI on ‘Kemer’. Isolates within each of VCGs 1A, 2A and 4B caused similar VDI on both cultivars. Isolates of VCG2B were found to vary in their VDI values on both cultivars. To the best of our knowledge, the present study is the first report of natural infections of eggplant by VCG1A.

VEGETATIVE COMPATIBILITY GROUPS OF VERTICILLIUM DAHLIAE ISOLATES FROM WEEDS IN POTATO FIELDS

Fifteen species of weedy plants were collected from potato fields between July and September 2006 in Erzurum (Turkey). In total, 21 isolates of Verticillium dahliae were obtained from Cirsium arvense, Convolvulus arvensis, Chenopodium album, Polygonum lapathifolium and Sinapis arvensis. Of these, 15 were isolated from C. arvense, and one or two isolates from the other weed species. This is the first report of V. dahliae isolated from these weed species in Turkey. All isolates from weeds were assigned to vegetative compatibility groups (VCGs) using nitrate-nonutilizing (nit) mutants. Two VCGs were found and identified as VCG 2B (11 isolates) and VCG 4A (10 isolates) by using tester isolates of known VCGs. Pathogenicity of V. dahliae isolates was tested on potato by the root-dip method. Both VCG 2B and VCG 4A isolates from weeds were pathogenic to potato.

Morphology, vegetative compatibility and pathogenicity of Verticillium dahliae isolates from woody ornamentals in Germany

Isolates of Verticillium dahliae were collected from affected woody ornamentals in German tree nurseries. Nitrat non-utilizing mutants were produced and vegetative compatibility groups (VCGs) were identified based on complementation tests. The results of this study indicate that VCG diversity of V. dahliae in German is limited, because only two VCGs were detected. Of the 32 isolates analysed, 19 were classified as VCG D1 and 13 as VCG D2. VCG D1 was compatible with the American VCG 2B, whereas VCG D2 was compatible with VCG 4B. In morphological studies a significant correlation between the conidial length of the V. dahliae isolates and their VCG could be detected. Isolates from VCG D1 consistently formed small conidia with an average length of 3,78:gmm, whereas isolates from VCG D2 produced larger conidia with an average length of 4,36:gmm. Pathogenicity tests were carried out with isolates on Acer platanoides, Acer pseudoplatanus, Catalpa bignonioides, Castanea sativa, Fraxinus excelsior and Tilia cordata. The isolates showed little significant differences in their aggressiveness to certain hosts. No major differences in virulence between VCGs were observed. The results imply that VCG identification does not contribute to verticillium disease prediction for a range of woody hosts.

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

AbstractA 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.

Morphological and Physiological Differentiation among Vegetative Compatibility Groups of Verticillium dahliae in Relation to V. longisporum

AbstractTwenty‐four isolates of Verticillium dahliae from various geographic regions and host origins were assigned to vegetative compatibility groups (VCG) based on complementation between nitrate‐non‐utilizing (nit) mutants. The VCG assignment was associated with clearly distinguishable morphological and physiological characteristics. Most isolates of VCG 2B produced deep black colonies, small conidia (3.62 × 0.02 μ m), and spherical microsclerotia. They exhibited polyphenol oxidase (PPO) activity, high sporulation rate in shake cultures, fluorescence on sanguinarine‐amended PDA (snPDA) and excretion of dark pigment on Czapek Dox agar. The isolates assigned to VCG 4B had significantly longer conidia (4.73 × 0.04 μ m), spherical microsclerotia and white, fluffy colonies due to enhanced production of aerial mycelium. They excreted only traces of dark pigment, exhibited PPO activity, did not fluoresce on snPDA and had limited ability to sporulate in shake cultures. Except for lacking PPO activity the only heterokaryon self‐incompatible isolate (HSI) was similar to VCG 4B. Ten isolates of Verticillium longisporum from cruciferous hosts did not produce nit mutants. They had clearly longer conidia (7.41 × 0.05 μ m), formed elongate microsclerotia, and only poorly sporulated in shake cultures. They did not excrete dark pigment, lacked PPO, and failed to fluoresce on snPDA. The results indicate a clear morphological and physiological differentiation not only between the two species V. longisporum and V. dahliae but also among the VCGs of V. dahliae.

Vegetative Compatibility and Pathogenicity of Verticillium dahliae from Spearmint and Peppermint.

The vegetative compatibility of 128 isolates of Verticillium dahliae from spearmint and peppermint in the western and midwestern United States was determined. Nit mutants were used to assign isolates to vegetative compatibility groups (VCGs). All isolates were assigned to VCG 2B except for one assigned to VCG 2A and two assigned to VCG 4A. VCG 2 isolates were found in all commercial mint growing regions, while the two VCG 4A isolates originated from southern Idaho. Pathogenicity assays on mint were performed using isolates from mint and other hosts, and pathogenicity assays were conducted on the potato cultivar Russet Norkotah using mint and potato isolates. Isolates originating from mint were significantly more aggressive on mint than were other host isolates, indicating that mint isolates were host-adapted. VCG 4A isolates from mint and potatoes were significantly more aggressive on potato than VCG 4B potato isolates and VCG 2B mint isolates. We speculate that the low VCG diversity of mint isolates may be due to the introduction of a single aggressive strain into Washington State mint fields via infected rhizomes.

Molecular Characterization of Vegetative Compatibility Group 4A and 4B Isolates of Verticillium dahliae Associated with Potato Early Dying.

Forty isolates of Verticillium dahliae, collected from potato seed tubers and potato plants from various regions in North America and previously assigned to vegetative compatibility groups (VCGs) 4A or 4B, were characterized using molecular markers. The VCG 4A isolates were previously shown to be a highly virulent pathotype of potato and to interact synergistically with the root-lesion nematode Pratylenchus penetrans to cause potato early dying. All but one of the VCG 4A isolates characterized in this study lacked the subspecies-specific repetitive DNA sequence E18 and could be differentiated from the remaining isolates by restriction fragment length polymorphisms (RFLPs) in the nuclear rDNA and Trp1 loci. The E18 RFLP patterns of several VCG 4B isolates from Maine and New York were highly similar to those of VCG 4B isolates previously collected from potato and tomato fields in Ontario. The data presented here suggest that the molecular markers will be useful for the detection and classification of isolates of V. dahliae associated with potato early dying.