An optimized method for in vitro production of Verticillium dahliae microsclerotia

Verticillium dahliae causes wilt disease of many crops worldwide. Microsclerotia are the main resting structure of V. dahliae in soil and can survive for more than 10 years, serving as an important source of primary inoculum. Mass production of microsclerotia in laboratory is valuable for studying various aspects of V. dahilae, such as its biology, epidemiology and control. We failed to produce a sufficient amount of microsclerotia in vitro for one strain of our interest using several previously published methods. Therefore, we developed a new protocol for mass in vitro production of viable microsclerotia. Verticillium strains were cultured in a modified basal agar medium at pH of 11.5 under 20 °C and incubated for 25 days in dark. When 16 strains were subjected to this condition, large numbers of microsclerotia were produced although varied greatly among strains, including the strain that failed to produce microsclerotia with previously published methods. Microsclerotia from 14 of the 16 strains resulted in wilt development on inoculated cotton seedlings.     

Effect of plant roots on the germination of microsclerotia ofVerticillium dahliae

Induction of germination of microsclerotia by exudates from plant roots may be important for the control ofV. dahliae. Laboratory experiments with root observation boxes were carried out to assess the influence of root tips of seven crop species and cultivars on the germination of microsclerotia ofVerticillium dahliae in soil under controlled conditions. The root density of crops was measured in a field experiment. The results of the laboratory experiments and the field experiment were combined to estimate the total effect of crops on the population of microsclerotia in the field. Germination of microsclerotia was stimulated by all crops compared to a control without a crop. Among crops, roots of potato cvs Element and Astarte had a larger stimulation effect on microsclerotia than that of potato Ostara, pea, flax, sugar beet or onion. The number of hyphae per microsclerotium decreased with distance from the root surface regardless of the crop species or cultivar. Differences in root densities, in the affected root zones and in the stimulation effect on germination of microsclerotia caused large differences among crops in the effect on the population of microsclerotia in the soil. However, growing a rop with the special purpose to reduce the level ofV. dahliae inoculum in the soil is an inefficient control measure, because only a small part of the total soil volume is affected by roots and the number of hyphae per microscleroium affected is too low.Abbreviations MS microsclerotia, microsclerotium     

Inoculum levels ofVerticillium dahliae in the soils of the hot semi-arid negev region of Israel

In the Negev area,Verticillium dahliae Kleb. produces 50 times more microsclerotia (MS) on potato plants harvested in the winter than on those harvested in the summer. Number and distribution of viable fungal units, subsequent to incorporation of infected potato plants and during a sequence of non-susceptible hosts, were estimated in February of the following years by counting fungal units in four sublayers: 0–10, 10–20, 20–30 and 30–40 cm. Inoculum density ofV. dahliae was the highest in the top layer, decreasing to the deepest sublayer. Reduction of population density of viableV. dahliae units was less in the field than in the laboratory. No viable units were detected after 5 years of storage in the laboratory, whereas 4% of the original population remained viable after 7 years of crop rotation. Although crop rotation did not control the fungus completely, it did reduce inoculum density markedly.     

Application to soil of disinfectants through irrigation reduces Verticillium dahliae in the soil and verticillium wilt of olive

The application of disinfectants through drip irrigation could be a feasible practice against verticillium wilt (Verticillium dahliae) of olive. OX-VIRIN (activated peroxide) and OX-AGUA AL25 (quaternary ammonium compounds) are two disinfectants that have shown efficacy against V. dahliae in irrigation water and potential for reducing the disease in young olive plants. In this work, various post-planting application strategies incorporating OX-VIRIN (once a month, or twice a month on alternate or successive weeks) or OX-AGUA AL25 (once a month, or twice a month on alternate weeks) were assessed for their effect on V. dahliae in soil, disease in olive trees, and olive yield, in a 2-year pot-experiment under natural environmental conditions. The disinfectants were injected via metering pumps into a drip irrigation system that irrigated olive trees planted in V. dahliae-inoculated soil. All the application strategies significantly reduced the total inoculum density in soil compared to controls with no disinfectants and noninoculated soil. The microsclerotia density was also significantly reduced in disinfested soils by 73.6–86.8%, depending on the strategy. The symptoms and infection incidence were always lower in treatments subjected to disinfestation. The treatment with OX-AGUA AL25 applied twice a month on alternate weeks most reduced the symptoms (by 53.0%) and colonization index (by 70.8%) with respect to untreated water control. This soil disinfestation also significantly strengthened the symptom remission. Tree growth and production were negatively affected by soil inoculation (reduced by 45.6% and 88.7%, respectively), but not so by disinfectants, which even relieved the reduction in inoculated soils, especially when OX-AGUA AL25 was applied.     

Evaluation of organic amendments from agro‐industry waste for the control of verticillium wilt of olive

Biological control of plant diseases using soil amendments such as animal manure and composted materials can minimize organic waste and has been proposed as an effective strategy in crop protection. In this study, 35 organic amendments (OAs) and 16 compost mixtures were evaluated against Verticillium dahliae by assessing both the antagonistic effect on the mycelial growth of two representative isolates of V. dahliae and the effect on the reduction of microsclerotia viability of the pathogen in naturally infested soil. Eleven OAs and five compost mixtures showed a consistent inhibition effect in in vitro sensitivity tests, with solid olive‐oil waste compost one of the most effective. Therefore, a bioassay with olive plants was conducted to evaluate the suppressive effect against V. dahliae of these selected OAs and compost mixtures. Significant reduction in the severity of the symptoms of V. dahliae indicates the potential use of grape marc compost (100% disease severity reduction) and solid olive‐oil waste, combined with other OAs. Microorganism mixtures and dairy waste OAs had a potential suppressive effect when they were combined with compost, showing a 73% and 63% disease severity reduction, respectively. A mixture of agro‐industrial waste with other biological control agents is a promising strategy against verticillium wilt of olive. To the authors' knowledge, this is the first report on the effectiveness of compost extracts (compost teas) on the inhibition of natural microsclerotia of V. dahliae, and also on verticillium wilt suppression in olive with solid olive‐oil waste.     

Prospects and strategies in controlling verticillium wilt of olive1

Control of verticillium wilt of olive currently depends on preventive measures. Since systemic fungicides are unable to prevent or control the disease, its control should primarily be based on cultural methods, including irrigation systems which restrict dissemination of Verticillium dahliae propagules by irrigation water and avoidance of intercropping with hosts susceptible to V. dahliae. Since leaves from affected olive trees contribute, through formation of microsclerotia, to the inoculum in the soil, pruning should be practised prior to branch defoliation. As for resistant olive rootstocks or cultivars, promising verticillium-wilt resistance has been found in two rootstocks selected in California (US). However, these have to be tested under local conditions before they can be released to Mediterranean growers, while further search for other resistant rootstocks is needed. Soil solarization applied to individual diseased trees in established olive groves could substantially contribute to recovery or long-lasting symptom remission in the treated trees. This effect is mainly attributed to the decrease or eradication of V. dahliae microsclerotia in the treated soil but also to heat-tolerant fungal antagonists of the pathogen. Using herbicides to control weeds, and limiting soil rotovation, can restrict symptom development. Biological control can also be considered as a promising trend in controlling the disease by searching, testing and exploiting potential fungal or bacterial antagonists.     

Sensitivity to chloropicrin of microsclerotia ofVerticillium dahliae as related to their germination on growth media and in soil

Sensitivity of microsclerotia ofVerticillium dahliae to chloropicrin increased considerably during incubation on Czapek’s medium and in moist soil. However, whereas on a growth medium maximum sensitivity was obtained after 48–96 h, a period of 216–288 h was required to obtain a similar degree of sensitivity of microsclerotia incubated in moist soil. At the end of the incubation period, the microsclerotia which had been incubated in moist soil gave rise to an average of four hyphae per microsclerotium as compared with 130 observed on Czapek’s agar. It is concluded that germination does not play a major role in the increase of sensitivity to chloropicrin during the incubation of microsclerotia ofV. dahliae in moist soil.     

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.     

Distribution of Verticillium dahliae through watering systems in widely irrigated olive growing areas in Andalucia (southern Spain)

Verticillium dahliae Kleb. causes Verticillium wilts in many herbaceous and woody species. Many hosts of the pathogen are commonly cultivated in Andalucía (southern Spain), particularly major crops such as cotton, vegetables, almond, peach and, particularly, olive, in which the fungus causes Verticillium wilt of olive. Infective structures of the pathogen (microsclerotia), produced in the late phases of the infection cycle in senescent tissues of the infected plants, can be spread over short or long distances by a number of dispersal methods. Irrigation water is one of the factors implicated in this spread of V. dahliae. Indeed, increasing irrigation dosages in crops or an inadequate irrigation schedule have been identified as cultural practices favouring Verticillium wilt onset and severity in olive and other hosts. Most of the cultivated areas in the Guadalquivir Valley of Andalucía are irrigated by pumping stations using modern infrastructures that supply water to thousands of hectares of farm land, which are usually associated with irrigation communities. This study demonstrates that the pathogen survives in the sediment and particles suspended in water used for irrigation in different facilities of an irrigation community, that were involved in distributing water (main canal and reception tank of a investigated pumping station, irrigation pools and sand from filters). Thus microsclerotia moves from the pumping station to individual plots (olive and cotton cultivated farm) as viable microsclerotia, free or embedded in soil particles and plant debris, suspended in the irrigation water, or deposited in the sludge in piping systems or water storage ponds. We have detected amounts of inoculum in the solid pellet samples in these facilities that ranged from 2.7 to 6.7 microsclerotia per gram. Besides this, water from drippers in cultivated plots released into the soil a variable amount of infective propagules of the pathogen over time that accounted for 3.75 microsclerotia/m³ in some of the recording times. Therefore, irrigation water becomes an important source of inoculum that is very effectively involved in medium and long-distance spread of the pathogen.     

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.     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.     

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.     

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.     

Effect of plant roots on the germination of microsclerotia ofVerticillum dahliae

Root observation boxes were used to study the effects of hosts and non-hosts on the germination of microsclerotia ofV. dahliae. The effects of roots on microsclerotia were examined within a radius of 1 mm around the root tip. Host plants such as potato and field bean induced a higher percentage of germination of the microsclerotia than a non-host such as barley. A susceptible potato cultivar stimulated germination more than a resistant cultivar. The germination percentage and the number of hyphae per microsclerotium decreased with distance from the root surface regardless of the plant species or cultivar.     

Quantification of microsclerotia ofVerticillium dahliae in plant material by image analysis

A procedure for the quantification of microsclerotia ofVerticillium dahliae with an image analysis system was compared with counting by eye. Colonised potato plant material was used from plants grown in pathogen-free soil in a greenhouse and from twelve crops (including four potato cultivars) grown outdoors in pots filled with pathogen-free soil under natural conditions. The values obtained from the potato material from the greenhouse were comparable for both methods. Variation in the results mainly resulted from sampling errors. The numbers of microsclerotia in plants grown outdoors were overestimated by image analysis for most crops. The source of the error was related to the presence of plant and soil particles that did not discolour during boiling of the samples in sodium hydroxide. Image analysis was a suitable and reliable method for assessing the number of microsclerotia only in potato haulm samples from plants grown in pathogen-free soil in the greenhouse.Abbreviations cv coefficient of variation - IA image analysis - MS microsclerotia, microsclerotium     

Disruption of Cerevisin via Agrobacterium tumefaciens‐mediated transformation affects microsclerotia formation and virulence of Verticillium dahliae

Agrobacterium tumefaciens‐mediated transformation (ATMT) was used to obtain a large number of Verticillium dahliae (Vd991) T‐DNA insertion mutants that were randomly integrated. Insertion mutants that produced significantly fewer microsclerotia were chosen for further analysis. Mutant T0065 was identified as having the Cerevisin gene interrupted by T‐DNA, and it was named cerevisin. The cerevisin protein showed a high amino acid sequence similarity with the vacuolar protease B. The mutant strain cerevisin displayed decreased production of microsclerotia and conidia, significantly reduced growth rate and reduction in virulence compared to the wild type. Moreover, the composition of secreted proteins differed between the cerevisin mutant and the wild type. Loss of function of Cerevisin decreased secretion of proteins of low molecular weight (14–25 kDa). Upon treatment with the secreted proteins of the mutant, the degree of leaf wilting decreased, indicating that Cerevisin is implicated in production of these proteins, which are putative pathogenicity factors of V. dahliae. The results suggest that Cerevisin is involved in controlling multiple processes of development and metabolism, plays an important role in vegetative growth and microsclerotia formation and affects virulence of V. dahliae.     

Single-site root inoculations on eggplant with microsclerotia ofVerticillium dahliae

For many soilborne plant pathogens, disease results from multiple root infections. Studying the infection dynamics of single or multiple propagules of these pathogens applied at one site of the root system may be the basis for understanding the development of disease caused by multiple root infections. The effect of single-site inoculations of roots of eggplant seedlings with microsclerotia of the wilt-causing fungusVerticillium dahliae, was studied. Experiments were conducted using specially designed pots which enabled the incorporation and removal of inoculum in the soil. Inoculations were carried out by placing microsclerotia, firmly embedded in small sections of polypropylene screen filter, directly below the growing tip of the main root of young eggplant seedlings. Three to 4 days after inoculation, the root had grown over the screen filter, and the filter was removed. Root platings showed high infection levels at the inoculation site, but also several (discrete) root infections were noted some distance above and below the site of inoculation. Exposure of the root to the lowest number of microsclerotia (26/inoculation site) was sufficient to lead to up to 65% root infections. Number of plants with root infections declined over time, ranging from a maximum of 65–100% 2–4 wk after inoculation, to 10–29% at 6–7 wk after inoculation. Apparently,V. dahliae died in nonsystemic infections after some time.     

Effect of soil solarization on the survival of fungal antagonists of Verticillium dahliae1

Talaromyces flavus, a fungal antagonist of Verticillium dahliae, naturally occurring in clay loam artichoke fields or sandy loam olive groves, is able to survive following application of soil solarization. Survival was almost always linked to an increase in T. flavus populations detected in the rhizosphere of artichoke plants or olive trees with a verticillium wilt history as compared with the untreated control soils. It was evident that soil solarization resulted in the control of the disease in artichoke fields and the recovery of olive trees from V. dahliae infection. It was furthermore proved that solarization had a beneficial long-term effect in controlling V. dahliae for a period of 2 or 3 consecutive years. This could at least partially be attributed to the activity of T. flavus in inhibiting the germination of microsclerotia or causing their death. Aspergillus terreus, another potential V. dahliae antagonist, was also found to survive and occasionally increase following the application of the technique.     

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.     

Association of Verticillium dahliae and Verticillium longisporum with Chinese cabbage yellows and their distribution in the main production areas of Japan

Chinese cabbage yellows caused by Verticillium dahliae and V. longisporum is one of the most economically important diseases in Japan. Verticillium isolates from infected Chinese cabbage in Japan were identified as V. dahliae and V. longisporum based on morphological characteristics and newly developed molecular genotyping methods using group I intron of 18S rDNA, mitochondrial-SSU rDNA and cob gene. Although the two species were equally virulent to Chinese cabbage in the greenhouse, V. longisporum was more virulent in the field compared with V. dahliae. Among the 67 Verticillium isolates from Chinese cabbage, 53 were V. longisporum and considered to be the major pathogen. The isolation frequency of V. longisporum was 98?% in Ibaraki and eastern Gunma where Chinese cabbage was cropped from autumn to winter. In contrast, the frequency was 48?% in Nagano and north to west Gunma where the plant was cropped in cool summer and highland conditions and the rests were V. dahliae. This is the first report to show the different geographical distribution of V. longisporum and V. dahliae in Chinese cabbage production area of Japan.