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.     

Development and application of new molecular markers for analysis of genetic diversity in Verticillium dahliae populations

The aim of this study was to develop new polymorphic markers for analysis of genetic diversity in the fungal soilborne plant pathogen Verticillium dahliae. Twelve polymorphic markers (five microsatellites and seven polymorphic sequences) were developed from a genomic library enriched for microsatellites. Screening of polymorphic loci was done using a collection of 25 V. dahliae isolates of diverse geographic origins, host sources and vegetative compatibility groups (VCGs). Three methods were used to score alleles: polyacrylamide gel electrophoresis (PAGE), sequencing of PCR‐amplified loci, and capillary electrophoresis. The new markers were used to assess genetic differentiation between isolates associated with different host plants. Two collections of isolates were analysed, obtained from artichoke (30 isolates) and potato (20 isolates) from crops grown in rotation located in the same area in eastern‐central Spain. The resolution of genetic differentiation between these two collections using the new markers was compared to that provided by other often‐used markers (SCARs and VCGs). Sequence analysis of the alleles proved to be the most unambiguous technique for scoring microsatellite data. The relatively high genetic differentiation observed between isolates from different crops (genetic differentiation coefficient, G_ST = 0·24) and their high genotypic diversity suggest a divergence between V. dahliae from artichoke and potato. It is hypothesized that evolution of V. dahliae from the local resident population in association with the two host crops has occurred. The new markers are useful for resolving population structure within V. dahliae and may contribute to a better understanding of the population biology of this fungus.     

Effect of crop rotation on Globodera rostochiensis and on potato yield1

A crop rotation field study was conducted in 1981–1989 to assess the effect of six crop rotation sequences on a soil population of potato cyst nematode (PCN), Globodera rostochiensis, and on potato yield. In the plots with potato monoculture, the tuber yield decreased from 35 to 4.6 t ha^?1. All other cropping systems maintained the yield at the original or an even higher level. In monoculture, the population density of the nematode was raised during the first 3 years from 0.1 to 265 larvae g^?1 soil, and the density fluctuated afterwards between 30 and 136 larvae g^?1 soil. Three of the cropping systems, (1) susceptible potato once in 5 years, (2) susceptible potato once in 4 years, and (3) potato once in 3 years using resistant (H1) and susceptible cultivars alternately, decreased the nematode density under the economic threshold or even under the level of detection. The nematicide treatments (oxamyl) did not control nematode multiplication but prevented yield losses. No nematicide is currently registered nor needed for the control of PCN in Finland. The nematode can be controlled effectively with crop rotation, resistant cultivars and early harvesting.     

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.     

Proteomics-based analysis reveals that Verticillium dahliae toxin induces cell death by modifying the synthesis of host proteins

Verticillium dahliae is one of the most destructive soil-borne fungal pathogens that cause vascular wilt diseases in a wide range of important crop plants, including cotton. However, the mechanisms used by this pathogen to infect cotton have not been fully elucidated. In the present study, we first investigated changes in protein abundance during the initial interaction between cotton roots and V. dahliae. Among the proteins that were upregulated upon infection, some were related to reactive oxygen species (ROS); among those downregulated upon infection were proteins involved in normal metabolism or cell structure. Further experiments confirmed that a sudden release of ROS and cell death accompany V. dahliae infection in the cotton vasculature. Further analysis indicated that a culture supernatant of V. dahliae induced lesion formation in tobacco leaves; de novo protein synthesis not active gene expression was required for this induction. Lesion formation was dependent on the age of leaves, but neither the known ROS burst nor the ubiquitin/26S proteasome system are prerequisites.     

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.     

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     

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.     

Sulphur supply impairs spread of Verticillium dahliae in tomato

Vascular wilt caused by the soil-borne fungus Verticillium dahliae is a major yield and quality-limiting disease across a broad spectrum of crop plants worldwide. Sulphur-enhanced plant defence mechanisms provide an opportunity to effectively and environmentally safely constrain the wilt disease levels in planta. To evaluate the influence of sulphur nutrition on the protective potential of these mechanisms, two near-isogenic tomato genotypes differing in fungal susceptibility, were treated with low or supra-optimal sulphur supply. Microscopic analysis revealed a significant sulphur-induced decrease in the amount of infected vascular cells in both genotypes. However, plant shoot and severely pathogen-affected root growth did not display this distinct alleviating influence of sulphur nutrition. Rates of leaf photosynthesis were impeded by Verticillium dahliae infection in both genotypes especially under low sulphur nutrition. However, assimilate transport rates in the phloem sap were enhanced by fungal infection more in the resistant genotype and under high sulphur nutrition suggesting a stronger sink for assimilates in infected plant tissues possibly involved in sugar-induced defence. A SYBR Green-based absolute quantitative Real-Time assay using a species-specific primer was developed which sensitively reflected sulphur nutrition-dependent changes in fungal colonization patterns. High sulphur nutrition significantly reduced fungal spread in the stem in both tomato genotypes. Concentrations of selected sulphur-containing metabolites revealed an increase of the major anti-oxidative redox buffer glutathione under high sulphur nutrition in response to fungal colonization. Our study demonstrates the existence of sulphur nutrition-enhanced resistance of tomato against Verticillium dahliae mediated by sulphur-containing defence compounds.     

Modification of fatty acids in eggplant affects its resistance to Verticilliumdahliae

Expression of the yeast Δ-9 desaturase gene in eggplant increased its levels of 16:1, 18:1, and 16:3 fatty acids and enhanced its resistance to Verticillium dahliae. Eggplants responded to V. dahliae inoculation with transitory upsurges in phospholipase A₂and lipoxygenase activities, as well as levels of free fatty acids and lipid peroxides. The upsurges of certain fatty acids, in particular the 16:1 and 16:3 fatty acids, were more pronounced in the transgenic plants. cis -Δ9 16:1 was found to directly inhibit the growth of V. dahliae. The results suggest that increasing the production of 16:1 in plants could be an approach to enhance their resistance to fungal diseases     

Interaction of spatially separated Pratylenchus penetrans and Verticillium dahliae on potato measured by impaired photosynthesis

Experiments were conducted under growth-chamber conditions to determine if Pratylenchus penetrans systemically alters light use efficiency (LUE) of Russet Burbank potato infected by Verticillium dahliae. Pathogen separation was achieved by inoculating potato roots with the nematode prior to injecting fungal conidia into the stem vasculature. Treatments were P. penetrans alone, V. dahliae alone, nematode and fungus together, and a no-pathogen control. Gas exchange was repeatedly and nondestructively measured on the fifth-youngest leaf with a Li-Cor LI-6200 portable photosynthesis system. By 16 and 20 days after stem injection with the fungus, LUE was synergistically impaired in jointly infected plants. Transpiration in plants infected with both pathogens was significantly reduced. However, the combined effect of nematode and fungus was synergistic in one experiment and additive in the other. Stems were destructively harvested when LUE was synergistically impaired. Coinfected potato plants contained more colony-forming units (CFU) of V. dahliae in stem sap than those infected by the fungus alone in one experiment. Evidence is provided that infection of Russet Burbank roots by P. penetrans systemically affects disease physiology associated with stem vascular infection by V. dahliae . The findings indicate that the role of the nematode in the fungus/host interaction is more than simply to facilitate extravascular and/or vascular entry of the fungus into potato roots.     

Optimization of the metham-sodium dose in controllingVerticillium dahliae in potato

In fields naturally infested withVerticillium dahliae, metham-sodium at 400, 600 and 800 1/ha was applied to reduce the fungal population. Metham-sodium delayed and reduced disease incidence at all doses tested compared with the control; this effect was maximal at 800 1/ha. However, an optimal yield (maximum kg potato/liter metham-sodium) was obtained at 600 1/ha.     

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

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

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.     

灰黄青霉CF3对马铃薯土传病原真菌的拮抗性及其促生作用

为探索灰黄青霉Penicillium griseofulvum CF3对马铃薯土传病害病原真菌立枯丝核菌Rhizoctonia solani、茄病镰刀菌Fusariumsolani、硫色镰刀菌F.sulphureum及大丽轮枝菌Verticillium dahliae的拮抗性及对马铃薯的促生作用,采用菌丝生长速率法和甜瓜种子发芽法分别研究了CF3发酵液对病原菌菌丝的抑制作用、对立枯丝核菌微菌核形成的影响及其发酵液的促生作用,并采用盆栽试验法研究了CF3孢子粉对马铃薯植株的促生作用及对抗逆性的影响.CF3发酵滤液对4株病原真菌菌丝生长的抑菌率达53％～72.1％,对立枯丝核菌微菌核的抑制率达36.8％～100％,并显著促进甜瓜种子胚根、胚轴生长.灰黄青霉孢子粉拌土和包衣接种均能促进马铃薯植株生长并增强植株的抗逆性.其中,拌土接种使马铃薯地上植株鲜重和多酚氧化酶活性较对照分别增加38.3％和9％,丙二醛含量降低28.8％.研究表明灰黄青霉CF3对连作马铃薯常见土传真菌病害有较强的生防潜力,对马铃薯具有良好的促生作用.     

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

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

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

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.     

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.     

Occurrence of Polyscytalum pustulans, Phoma foveata and Fusarium solani var. coemleum in field soils in Scotland

The effectiveness of various methods for detecting three fungal potato pathogens was compared with artificially infested soil, naturally infested tuber-borne soil and field soil. In the spring of 1985 and 1986 field soils from 30 farms in north-east Scotland were sampled just before planting a seed potato crop and 6 months after harvesting such a crop. The minimum statutory gap between crops is 5 years. Polyscytalum pustulans was recovered from 32 out of 60 field soil samples taken 6 months after harvest while from fields sampled in the spring before a potato crop was planted the fungus was isolated from 10 out of 30 soils in 1985 and five out of 30 in 1986. Phoma foveata was isolated from only one out of 60 pre-planting soil samples but Fusarium solani var. coeruleum was recovered from eight of these soils.
Microplant bait plants were grown over 3 years at an experimental farm near Edinburgh in various fields at different intervals after a previous potato crop. Contamination by P . pustulans was not related to interval after potatoes between 1 - 7 years. No contamination was recorded in fields where potatoes had not been grown for more than 30 years.