Genetic and Molecular Characterization of Verticillium dahliae Isolates from Woody Ornamentals in Belgian Nurseries

Isolates of Verticillium dahliae were collected from affected trees (Acer spp., Tilia spp. and Robinia spp.) and soils in Belgian ornamental nurseries. Nitrate non-utilizing mutants were produced and vegetative compatibility groups (VCGs) were classified based on complementation tests with reference tester strains. Of the 30 isolates analysed, 12 were classified as VCG2B and 18 as VCG4B following the American classification. In order to distinguish VCG2B from VCG4B, specific polymerase chain reaction primers were designed based on the sequence of a VCG2B-associated Direct Amplification of Minisatellite-region DNA (DAMD) band generated with the core sequence of the phage M13 minisatellite DNA. Using this test, amplification products were generated for all the VCG2B isolates characterized in this study. In contrast, no signal was seen on ethidium–bromide agarose gel for VCG4B isolates. Pathogenicity tests were carried out in a glasshouse on maple-rooted cuttings inoculated with conidial suspensions of V. dahliae belonging to both groups (VCG2B/VCG4B). Some strains proved to be highly aggressive, while others did not. However, these different behaviours were not correlated with the VCGs.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.     

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.     

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum^® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.     

Incidence of Verticillium wilt of artichoke in eastern Spain and role of inoculum sources on crop infection

Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield. http://www.phytoparasitica.org posting July 21, 2005.     

Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. I. Taxonomy

The genusVerticillium is divided into four sections (two new) and a residual group. The new sectionNigrescentia comprises the well-known plant-pathogenic and some other saprophytic species with dark resting structures. The new sectionAlbo-erecta is characterized by white (or yellowish) colonies and erect conidiophores and contains mainly fungicolous species. Seven (two new) species and two new varieties are described and keyed out. Three other species have known teleomorphs ofNectriopsis (one new and two new combinations). This genus had been merged withNectria by Samuels, but its retention is justified, as the original and the here described fungicolous (and myxomyceticolous) species are more suitably accomodated inNectriopsis than inHypomyces.The common causal agent of dry bubble inAgaricus bisporus, Verticillium fungicola var.fungicola, is redescribed and defined more narrowly than by Gams (1971) by its maximum growth temperature below 27°C. A similar fungus with a maximum growth temperature near 33°C, causing brown spots inA. bitorquis, is described asV. fungicola var.aleophilum. Isolates from wild agarics with a strongly reduced growth at 24°C and a maximum below 27°C, a yellowish mycelium and inconspicuous sclerotia, are described asV. fungicola var.flavidum. V. biguttatum W. Gams,sp. nov., with cylindrical biguttulate conidia, is a common soil fungus and hyperparasite ofRhizoctonia solani. Gliocladium microspermum (Sacc.) W. Gams,comb. nov., the anamorph ofNectriopsis broomeana (Tul.) W. Gams,comb. nov., which is intermediate betweenGliocladium andVerticillium, is included because of its fungicolous habit.Samenvatting Het geslachtVerticillium wordt onderverdeeld in vier secties (waaronder twee nieuwe) en een restgroep. De nieuwe sectieNigrescentia bevat onder meer de bekende ziekteverwekkende soorten met donkere ruststructuren. De nieuwe sectieAlbo-erecta wordt gekarakteriseerd door witte (of geelachtige) kolonies en rechtopstaande conidioforen en bevat hoofdzakelijk op andere fungi groeiende soorten. Zeven soorten (waaronder twee nieuwe) en twee nieuwe variëteiten worden beschreven en in een sleutel verwerkt. Drie andere soorten bezitten perfecte vormen vanNectriopsis (waaronder één nieuwe en twee nieuwe combinaties). Dit geslacht werd door Samuels metNectria samengevoegd, maar kan gehandhaafd worden en lijkt beter geschikt om de hier beschreven ascomyceten onder te brengen danHypomyces. Verticillium fungicola var.fungicola, de algemene veroorzaker van droge mollen bij de champignon (Agaricus bisporus), wordt beschreven en met zijn temperatuur-maximum beneden 27°C nauwkeuriger gedefiniëerd dan in een vroegere studie (Gams, 1971). Een vergelijkbare schimmel met een temperatuurmaximum van ongeveer 33°C, die bijA. bitorquis bruine vlekken veroorzaakt, wordt beschreven alsV. fungicola var.aleophilum. Isolaten van wilde paddestoelen met een optimum duidelijk beneden 24°C en een maximum beneden 27°C, geelachtig mycelium en onopvallende sclerotiën, worden beschreven alsV. fungicola var.flavidum. V. biguttatum W. Gams, sp. nov., met cilindrische, biguttate conidiën, is een algemene grondschimmel en hyperparasiet vanRhizoctonia solani. Gliocladium microspermum (Sacc.) W. Gams, comb. nov., de imperfecte vorm vanNectriopsis broomeana (Tul.) W. Gams, comb. nov., een tussenvorm tussenGliocladium enVerticillium, wordt opgenomen wegens zijn voorkomen op vruchtlichamen van de dennemoorder.     

Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. II. Pathogenicity

In recent years in the Netherlands a second mushroom species,Agaricus bitorquis, which prefers higher temperatures thanA. bisporus and is less susceptible to certain diseases, is often commercially grown.Verticillium fungicola var.fungicola, the causal agent of dry bubble, is responsible for considerable damage in crops ofA. bisporus. InA. bitorquis, however, dry bubble has hardly been noticed, but brown spots due toV. fungicola var.aleophilum resulted in inferior mushroom quality. The latter variety also caused brown spots ina. bisporus, but to a minor degree. In variety Les Miz 60 ofA. bisporus, however, it also induced fruit-body deformation in a way different from dry bubble. Verticillium psalliotae, isolated fromA. bitorquis in England, induced more confluent brown spots inA. bitorquis. In the netherlands, where moreA. bitorquis is grown than in other countries,V. psalliotae has not yet been encountered in crops ofA. bitorquis. V. psalliotae, which has a high temperature optimum for mycelial growth, likeV. fungicola var.aleophilum andA. bitorquis, did not infectA. bisporus in our trials.Artificial infection ofA. bisporus orA. bitorquis could not be accomplished with the following related and/or fungicolous fungi:Verticillium lamellicola, V. fungicola var.flavidum, V. biguttatum, Nectriopsis tubariicola, Acremonium crotocinigenum andAphanocladium album.Samenvatting Vooral in Nederland wordt sinds een aantal jaren naastAgaricus bisporus ook de warmteminnende champignonsoortAgaricus bitorquis geteeld, die minder vatbaar is voor bepaalde ziekten. TerwijlVerticillium fungicola varfungicola in de teelt vanA. bisporus droge mollen en daardoor veel schade veroorzaakt, komen in de teelt vanA. bitorquis geen droge mollen voor maar wel bruine vlekken, die tot kwaliteitsverlies en dus schade leiden. De vlekken bleken veroorzaakt te worden doorV. fungicola var.aleophilum. Deze schimmel veroorzaakte ook inA. bisporus bruine vlekken, hoewel in minder ernstige mate, maar in het ras Les Miz 60 vanA. bisporus bovendien misvormde champignons, die wel op droge mollen leken, maar daaraan niet gelijk waren.OokV. psalliotae, in Engeland geïsoleerd vanA. bitorquis met vlekken, veroorzaakte wat meer samenvloeiende, bruine vlekken inA. bitorquis. In Nederland, waar meerA. bitorquis geteeld wordt dan in andere landen, isV. psalliotae nog niet aangetroffen in teelten vanA. bitorquis. InA. bisporus kon geen kunstmatige infectie worden verkregen metV. psalliotae, die net alsV. fungicola var.aleophilum enA. bitorquis warmteminnend genoemd zou kunnen worden.Met de volgendeVerticillium-achtige of van paddestoelen geïsoleerde schimmels kon evenmin op kunstmatige wijze een infectie worden opgeroepen inA. bisporus ofA. bitorquis: Verticillium lamellicola, V. fungicola var.flavidum, V. biguttatum, Nectriopsis tubariicola, Acremonium crotocinigenum enAphanocladium album.     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

Regional and varietal differences in the risk of wheat seed infection by fungal species associated with fusarium head blight in Italy

The incidence of seed infection by fungal species pertinent to the fusarium head blight complex was monitored from 1999 to 2002 in two soft and three durum wheat cultivars grown across the northern, central and southern production zones of Italy, in order to characterize the species composition at the seed level. The main species recovered were Fusarium graminearum, F. poae and Microdochium nivale. There was a marked influence of production zone on seed infection incidence for both durum and soft wheat cultivars, with incidence of infection generally decreasing from the northern to the southern zone. Incidence of seed infection by different species of Fusarium was twice to four times higher in durum compared with the soft wheat cultivars in the study. There were no significant differences in terms of seed infection incidence between the two soft wheat cultivars, but the durum cultivars differed in their levels of seed infection for some of the pathogens. The results demonstrated that the durum cultivars were more at risk of seed infection by pathogens associated with fusarium head blight, and that wheat grown in northern Italy is at higher risk of seed infection by these species.     

Vegetative compatibility and pathogenicity of<Emphasis Type="Italic">Verticillium dahliae</Emphasis> isolates from the aegean region of Turkey

A total of 101Verticillium dahliae isolates were recovered from cotton plants at 57 sites in the Aegean region of Turkey between 2003 and 2004. Isolates were tested for vegetative compatibility by observing heterokaryon formation among complementary nitrate-nonutilizing (nit) mutants. Forty-six isolates were assigned to VCG 1, 12 to VCG 2A, 33 to VCG 2B and four to VCG 4B. The remaining six isolates could not be tested for vegetative compatibility because of their inability to yieldnit mutants. All isolates recovered were tested for pathogenicity on cotton cultivars Acala SJ-1 and Deltapine 15-21 by the stem-injection method. The isolates of VCG 2 and 4B, irrespective of their origin, induced weak to severe symptoms on cotton and were similar to the previously described cotton non-defoliating pathotype. In contrast, all cotton isolates of VCG1 caused severe foliar symptoms, stunting, defoliation and often death. This is the first report on VCG 1 ofV. dahliae in Turkey. http://www.phytoparasitica.org posting May 4, 2007.     

<Emphasis Type="Italic">Verticillium</Emphasis> disease of <Emphasis Type="Italic">Agaricus bisporus</Emphasis>: variations in host contribution to total fungal DNA in relation to symptom heterogeneity

The pathogenic fungus Verticillium fungicola, responsible for dry bubble disease of the common mushroom Agaricus bisporus, causes various symptoms on its host, bubbles (undifferentiated spherical masses), bent and/or split stipes (blowout) and spotty caps. Host DNA quantification by real-time PCR was used to observed relationships between the type of symptom and the relative amount of A. bisporus and V. fungicola in diseased mushrooms. Verticillium fungicola is involved in bubble formation but does not appear to regulate its growth. Quantifications in bubbles and stipe-bubbles (morphology between bubble and sporophore with stipe blowout) showed that the pathogen has no effect on the growth of undifferentiated host hyphae but prevents morphological differentiation if not initiated and stops it when initiated hyphae are affected. Mushrooms with stipe blowout exhibiting both mature and abortive lamellae reveal that V. fungicola has a restricted area of action in host tissues. Despite their visual aspect, healthy looking parts of mushrooms showing spots or stipe blowout were actually contaminated. Discolouration and symptom development are two distinct events. The colour of the tissues was correlated to the percentage of A. bisporus DNA, suggesting that discolouration is not an efficient defensive mechanism, and occurs at the time V. fungicola developed enough to induce tissues necrosis.     

Assessment of real-time PCR as a method for determining the presence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in different Solanaceae cultivars

Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.     

Verticillium dahliae Modifies the Concentrations of Proline, Soluble Sugars, Starch, Soluble Protein and Abscisic Acid in Pepper Plants

The objective of this research was to study the levels of some organic solutes, such as total protein, total soluble sugars, starch and proline in leaves, as well as abscisic acid concentration in xylem of pepper plants inoculated with Verticillium dahliae Kleb. Healthy and inoculated plants were always kept well watered. Measurements were made at time intervals after inoculation. Leaf water potential rapidly decreased as a consequence of fungal infection. However, relative water content in leaves only changed significantly from day 20 after inoculation, and such decreases coincided with a sharp build up of proline and total soluble sugars in leaves. Starch and protein levels, as well as abscisic acid concentration in xylem, declined in healthy and inoculated peppers as they became older. However, such decreases were more pronounced in infected plants, especially soon after inoculation. Results suggest that proline and total soluble sugars accumulation could be sensors of the damage caused by the fungal infection.     

Compatible Biological and Chemical Control Systems for Rhizoctonia solani in Potato

A series of chemical and biological control agents were tested for compatibility with the Rhizoctonia-specific biocontrol fungus Verticillium biguttatum aimed at designing novel control strategies for black scurf (Rhizoctonia solani) and other tuber diseases in potato. The efficacy of chemicals, alone and in combination with V. biguttatum was tested in in vitro assays on nutrient agar plates, in bio-assays with minitubers and in the field. Generally, there were both antagonistic, neutral and additive interactions with V. biguttatum among the combinations tested; there were no indications for synergistic interactions. Broad-spectrum fungicides (azoxystrobin, chlorothalonil, thiabendazole) were fungitoxic to V. biguttatum as shown in in vitro assays, and hampered black scurf control by V. biguttatum in bio-assays. Oomycete-specific chemicals (cymoxanil and propamocarb) and various biocontrol strains (Gliocladium spp., Pseudomonas spp. and Trichoderma spp.) did not interfere with the growth of V. biguttatum on agar nutrient plates and did not affect black scurf control by V. biguttatum in co-applied treatments in the minituber bio-assay. Rhizoctonia-specific (pencycuron, flutalonil) fungicides co-applied with V. biguttatum showed additive effects on black scurf control. When combinations of V. biguttatum and cymoxanil or propamocarb were applied to immature potato tubers at green crop lifting, a reduction of both black scurf and Pythium- or Phytophthora-incited tuber rot was observed at harvest. In conclusion, the biocontrol fungus V. biguttatum is compatible with selected chemical control systems and may improve control efficacy in combination with Rhizoctonia-specific fungicides or may extend control spectrum in combination with Oomycete-specific fungicides.     

Survey of Bacterial and Fungal Seedborne Diseases in Imported and Domestic Potato Seed Tubers

Potato seed tubers are imported to Israel from northern Europe and planted in spring; tubers harvested early from the spring crop are used as seed for the autumn crop. Although only seed lots registered as certified are imported, a previous survey (1984–1994) indicated that most imported lots were affected by latent or active infections caused byErwinia carotovora,Streptomyces scabies, Rhizoctonia solani, Fusarium spp. andSpongospora subterranae. The survey was extended until 1998, and included additional pathogens:Ralstonia solanacearum,Helminthosporium solani, Colletotrichum coccodes andVerticillium dahliae. Most of these pathogens were also monitored in domestic seed tubers, and are reported for the first time. Brown rot was not observed in any of the imported lots. Blackleg and soft rot caused byErwinia spp. were detected in most of the imported lots; however, less than 7% of the lots were contaminated at high levels, while approximately 65% were contaminated at moderate levels. Common scab was detected in most of the imported lots; 51% of the imported lots were contaminated at moderate or high levels, whereas only 6.5% of the domestic seed lots were contaminated at these levels. Black scurf was detected in most of the imported lots; on average, 47.3%, 44.2% and 1.4% of the lots were contaminated at low, moderate and high levels, respectively, and only 7.1% were disease-free. In contrast, most of the domestic lots were either disease-free (45.4%) or had a low disease incidence (37.3%). Only 16.7% of the lots were moderately infected and 0.2% were highly contaminated. Silver scurf was observed in most of the imported lots during all years of the survey, with no differences among the producing countries; on average, 22.7%, 66.1% and 7.5% of the lots were contaminated at low, moderate and high levels, respectively, and only 3.7% were disease-free. Most of the domestic lots (76%) were disease-free and only 6.6% were infected at moderate or high levels. Black dot was observed in a considerable portion of the shipments from Holland during all years of the survey, particularly in 1998, when 34% of the lots were infected. The shipments from France and Germany were infected at low levels, except in 1998, when 19% and 11% of the lots, respectively, arrived infected. In shipments from Scotland and Ireland low incidences of the disease were observed in 1994 and 1995. In the domestic lots, black dot incidence was low (<2.4%) except in 1996, when 11% of the lots were infected.V. dahliae was monitored only in domestic seed tubers. The incidence of disease-free lots was 56–64%, whereas in 20–30% of the lots the level of infection was <5%, and in 6–16% of the lots the level was >5%. The survey findings demonstrate transmission of seedborne pathogens; most of these pathogens can become established in the soil and eventually cause severe outbreaks of disease in potatoes grown in Israel. http://www.phytoparasitica.org posting May 16, 1999.     

Pathogenic variation and molecular characterization of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from wilted Welsh onion in Japan

Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.     

Powdered neem seed and cake for management of the banana weevil,cosmopolites sordidus, and parasitic nematodes

Field trials were conducted in Kenya with ‘Nakyetengu’, an East African highland banana cultivar (AAA-EA), highly susceptible to banana pests. Regardless of soil fertility levels, incorporation around the plant base of powdered neem(Azadirachta indica A. Juss.) seed or cake at 60-100 g/mat at 4-month intervals, gave better control of the banana weevil,Cosmopolites sordidus (Germar), and of parasitic nematodes, than that achieved with soil application of Furadan 5G (carbofuran) at 60 g/mat at 6-month intervals. Compared with untreated control, fruit yield in most of the neem treatments was significantly higher, particularly during the second cycle of crop production. Neem application conferred a net economic gain, whereas Furadan application proved uneconomical. Application of powdered neem seed or cake at higher rates (200–400 g/mat) at 6-month intervals caused phytotoxicity, resulting in drying up of banana plants before fruiting, or in ‘chokethroat’,i.e., inflorescence emergence failure.     

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl⁻¹) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.