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     

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.     

Towards a mechanistic model for the Verticillium dahliaepotato system

Development ofVerticillium dahliae in potato was modelled by coupling aVerticillium subroutine to an existing potato crop growth model, using mainly biologically meaningful and measurable parameters. The model, written in FORTRAN77, describes disease development in single plants. Stem base infection is modelled as a stochastic process. The average effect on the crop is calculated by taking the average of model reruns. The parameters of the potato crop growth model were calibrated in advance of those of theVerticillium subroutine. By doing so, corrections can be made for lack of fit in the potato growth model. Calibration of theVerticillium subroutine was performed using experimental field data of two years at two microsclerotial density levels. The results of the calibration procedure of theVerticillium subroutine improved strongly when the results of runs of both microsclerotial densities were expressed in one combined goodness of fit value. Simulation of root infection and incidence of stem colonization byV. dahliae were in line with experimental data. However, deviances from data from the literature at inoculum densities which were not used in the calibration procedure indicate that calibration has to be performed using more field data and also incorporating more parameters like those describing fungal dynamics. Optimal runs were obtained at pathozones of c. 30 μm, which may indicate that infections occur only when a microsclerotium is very close to the root.     

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.     

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.     

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.     

Effect of crop rotation on the incidence of soil-borne fungal diseases of potato

Effects of crop rotation on the incidence of soil-borne pathogens and on the performance of potato were investigated in five field experiments. Rotations differed in cropping frequency of potato and in crop sequence.Incidence of stem canker caused byRhizoctonia solani was strongly influenced by the cropping frequency of potato and not by crops with which the potato was alternated in the rotation. Cropping frequency of potato also affected the occurrence of black scurf, but less pronounced than for stem canker. The antagonistVerticillium bigutatum slightly reducedR. solani (black scurf) in plots on sandy soil continuously cropped with potato. Incidence of stem canker was also strongly affected by granular nematicides applied to the soil, nitrogen level and the cultivar grown.Stem infections byVerticillium dahliae depended on the cropping frequency of potato, by the crop with which the potato was alternated in the rotation and by the density and virulence of endoparasitic nematodes, especiallyMeloidogyne spp.Crop rotation had no effect at all on incidence of common scab on tubers, whereas the effect of cropping frequency of potato on netted scab was highly significant. When cultivars were grown susceptible to both scab types, netted scab supressed common scab.     

Germination of individual microsclerotia ofVerticillium dahliae

Viability of individual microsclerotia (MS) ofVerticillium dahliae of different sizes (<37 μm, 37–74 μm, 75–125 μm) was studied throughout repeated transfers in a moist chamber. Germination rate and number of germinating hyphae were positively correlated with the size of the MS. After 13 transfers, some MS of the above size ranges still germinated; some MS germinated until they lost their viability; others did not germinate for a while and then started germinating again. The length of germination time of MS on a synthetic medium was 8 days, with slight differences between MS originating from soil and those from one-year-old potato stems. Fresh MS from potato stems, however, completed their germination after 36 hours. Fewer MS (size <37 μm) were isolated from soil by the wet-sieving than by the dry-sieving technique. This finding and microscopic observations lend further support to the assumption that MS exist in soil partly in clusters.     

Effects of crop rotation and removal of crop debris on the soil population of two isolates of Verticillium dahliae

Microsclerotia of Verticillium dahliae are produced in large numbers on senescing parts of host plants and remain viable in the soil for many years. Changes in the population density, i.e. density of microsclerotia, in the soil were measured in micro-plots using two isolates of V. dahtiae , specific to either field bean or potato, several crop sequences comprising potato, field beans and barley, and either the removal of aerial debris of the crops or incorporation into soil.
Potato was more susceptible to the potato isolate and field bean more susceptible to the field bean isolate. Removal of debris of potato and field bean reduced numbers of microsclerotia in the soil in the subsequent years, but removal of barley straw had no effect. Initially non-infested control micro-plots became infested, probably by the growth of potato roots into the naturally infested subsoil. The rate of increase of the microsclerotial population in the non-infested control micro-plots was larger than in the initially infested treatments, because more colonized debris was produced. It is concluded that removal of aerial debris of host crops is important to reduce the soil population of V. dahtiae.     

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.     

Effect of crop rotation on the incidence of soil-borne fungal diseases of potato

Effects of crop rotation on the incidence of soil-borne pathogens and on the performance of potato were investigated in five field experiments. Rotations differed in cropping frequency of potato and in crop sequence.Incidence of stem canker caused byRhizoctonia solani was strongly influenced by the cropping frequency of potato and not by crops with which the potato was alternated in the rotation. Cropping frequency of potato also affected the occurrence of black scurf, but less pronounced than for stem canker. The antagonistVerticillium bigutatum slightly reducedR. solani (black scurf) in plots on sandy soil continuously cropped with potato. Incidence of stem canker was also strongly affected by granular nematicides applied to the soil, nitrogen level and the cultivar grown.     

An Efficient Inoculation Method for Colonization of Chinese Cabbage Seedlings by the Root Endophytic Fungus Heteroconium chaetospira

To develop an efficient method to inoculate Chinese cabbage seedlings with the root endophytic fungus Heteroconium chaetospira, an appropriate nursery soil and glucose concentration for the nutrient medium for fungal colonization were determined. A grid-sheet method was established for estimating the degree of colonization of entire roots by the fungus. The fungus colonized at high frequencies when peat moss was used as the rooting medium. Colonization was highest (75%) when peat moss was amended with 0.1% glucose. Under these conditions, fungal hyphae developed intracellularly in root cortical cells. In contrast, under high glucose conditions, fungal colonization was restricted mostly to intercellular regions of epidermal or cortical root tissues. Here, hyphae formed inter- or intracellular microsclerotia. Received 19 August 2002/ Accepted in revised form 5 December 2002     

棉秆生物炭对大丽轮枝菌生长及毒素作用的影响

棉花黄萎病难以防治的根源在于大丽轮枝菌Verticillium dahliae Kleb在土壤中形成的微菌核能抵抗不良环境,并在土壤中长期存活,将棉秆加工成生物炭施入棉田,可克服棉秆直接还田的缺点,从源头上防止黄萎病菌进入土壤。为探索棉秆生物炭还田对棉花黄萎病原菌的影响机理,采用液态摇床培养及平皿固态培养法研究生物炭对大丽轮枝菌菌丝生长及微菌核萌发的影响;通过平皿内棉花和甜瓜种子发芽试验研究生物炭对大丽轮枝菌毒素的减毒作用。结果表明:液态培养条件下,棉秆生物炭对大丽轮枝菌菌丝生长抑制作用不明显,在固态培养下对菌丝体生长有一定促进作用;生物炭能减慢微菌核的萌发速率,但对最终萌发率无影响;液态培养中加入棉秆生物碳对大丽轮枝菌毒素有较强的减毒作用。在摇床培养中期,加入2.0 g·L-1生物炭处理与对照棉种在培养12天时的发芽率分别为53.3%与33.3%(P0.05);前期、中期及后期加入0.5 g·L-1生物炭处理的棉花幼苗总长度分别为对照的4.9、2.1倍及3.2倍;加入棉秆生物碳处理甜瓜种子发芽率、胚根长度、胚轴长度及总苗长较对照均有不同程度增加。表明棉杆生物炭对棉花黄萎病菌大丽轮枝菌菌丝生长、微菌核萌发的抑制作用较弱,对大丽轮枝菌毒素危害有较强的减毒作用。     

Detection of Colletotrichum coccodes and Helminthosporium solani in soils by bioassay

The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.     

Theoretical approach to the dynamics of the inoculum density of Verticillium dahliae in the soil: first test of a simple model

A mathematical equation was developed that describes the inoculum densities of microsclerotia of Verticillium dahliae in the soil over a long time span. The equation was based on measurable parameters and ecologically meaningful principles. In the model, the number of systemic infections of plant roots during crop growth was related to soil inoculum density. In turn, formation of microsclerotia in debris and reduction of the amount of crop growth were related to the number of systemic infections. Finally, a gradual release and mortality of microsclerotia in the soil were included to calculate subsequent inoculum densities in the soil.
Fitting the function to experimental data of potato cvs Element, Ostara, Mirka and Astarte, flax, pea, barley, sugar beet, onion and faba bean gave a very high correlation between observed and predicted soil inoculum densities. The clear differences in inoculum production among potato cultivars and other crops were expressed in quantitative terms. The highest inoculum density after incorporation of the debris of a susceptible crop was estimated to occur at 2.3 thermal time units of 3600 degree days (base 0°C). Ten per cent of the initial input of inoculum was still present after 4.5 thermal time units. The model was used to predict the dynamics of soil inoculum densities for V. dahliae under various cropping frequency schemes and performed satisfactorily.     

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.     

韭菜等几种植物根系分泌物对棉花黄萎病菌的影响

通过水培法收集韭菜、大葱、野薄荷、藿香和薰衣草的根系分泌物,研究其对大丽轮枝菌菌丝生长、孢子萌发的影响,并通过室内盆栽试验研究了植物根系分泌物对大丽轮枝菌微菌核萌发的影响。结果表明,5种植物根系分泌物对大丽轮枝菌菌丝生长和孢子萌发均表现为抑制作用;随着浓度的升高,抑制作用增强,而在同一浓度下,随着处理时间的延长,根系分泌物对孢子萌发的抑制作用逐渐减弱。盆栽试验表明,植物根系分泌物对大丽轮枝菌微菌核的萌发均表现为抑制作用,且微菌核对韭菜根系分泌物最为敏感。5种植物中,韭菜根系分泌物的抑制作用最强,野薄荷次之,藿香最弱。     

Formation of chlamydospores and microsclerotia in Alternaria dauci

Alternaria dauci (Kühn) Groves and Skolko, the cause of leaf blight of carrot, was observed to produce chlamydospores and microsclerotia in vitro. Four different isolates produced chlamydospores on potato dextrose agar, V8 agar and Czapek-Dox agar at 10, 18 and 28°C, from neighbouring cells of hyphae of the submerged mycelium, forming long chains. Formation of chlamydospores was completed in a multiple stage process, including swelling of cells, development of secondary septa, rounding up of cells and cell wall thickening. Mature chlamydospores were released from the mycelium mostly in clusters and in short or long chains, and sometimes in pairs. They were globose to ellipsoid, smooth or verrucose, pale brown in colour, and averaged 13.87?×?11.37 μm, (length × width). Microsclerotia were formed on PDA, by only one isolate, at 28°C first at the edges of the colony, by close contact of hyphae that exhibited extensive branching and intermingling. In their final form microsclerotia were multi-celled, closely attached masses of short and broad dark grey cells, less compact woven at the edges of the microsclerotium. This is the first illustration of chlamydospore and microsclerotia formation in Alternaria dauci.     

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.