Spatial Patterns of Microsclerotia of Verticillium dahliae in Soil and Verticillium Wilt of Cauliflower

ABSTRACT The spatial patterns of microsclerotia of Verticillium dahliae in soil and wilt symptoms on cauliflower were determined at three sites in each of two fields in 1994 and 1995. Each site was an 8 x 8 grid divided into 64 contiguous quadrats (2 by 2 m each). Soil samples were collected to a depth of 15 cm with a probe (2.5 cm in diameter), and samples from four sites in each quadrat were bulked. Plants in each quadrat were cut transversely, and the number of plants with vascular discoloration and the number without discoloration were recorded. The soil was assayed for microsclerotia by the modified Anderson sampler technique. Lloyd's index of patchiness (LIP) was used as an indicator to evaluate the aggregation of microsclerotia in the field. Spatial autocorrelation and geostatistical analyses were also used to assess the autocorrelation of microsclerotia among quadrats. The LIP for microsclerotia was greater than 1, indicating aggregation of propagules; however, the degree of aggregation at most sites was not high. Significant autocorrelation within or across rows was detected in some spatial autocorrelograms of propagules, and anisotropic patterns were also detected in some oriented semivariograms from geostatistical analyses for microsclerotia, indicating the influence of bed preparation in the fields on pathogen distribution. The parameter estimates p and theta in the beta-binomial distribution and the index of dispersion (D) associated with the distribution were used to assess the aggregation of diseased plants at each site. A random pattern of wilt incidence was detected at 7 of 12 sites, and an aggregated pattern was detected at 5 of 12 sites. The degree of aggregation was not high. A regular pattern of wilt severity was detected at all sites. The high disease incidence (77 to 98%) observed at 11 of the 12 sites could be explained by high inoculum density.     

Effects of Irrigation and Verticillium dahliae on Cauliflower Root and Shoot Growth Dynamics

ABSTRACT Cauliflower root and plant growth and Verticillium wilt development were evaluated under different moisture regimes in the presence or absence of V. dahliae. Treatments included two main plots (V. dahliae-infested and fumigated), two subplots (furrow and subsurface drip irrigation), and three sub-subplots (deficit, moderate, and excessive regimes) that were arranged in a split-split-plot design in the field. Soil cores with roots were periodically sampled at 5 and 25 cm distance from plants. Total roots in each soil core were extracted with a hydropneumatic root elutriator, and root length from each sample was determined with a digital image analysis system. Incidence and severity of Verticillium wilt, plant height, number of leaves, and dry weights of leaves and roots were determined on 10 plants sampled at 7- to 10-day intervals 1 month after cauliflower transplanting and continued until harvest. To evaluate the effects of Verticillium wilt-induced stress on cauliflower plants, stomatal resistance was measured in upper healthy and lower (or diseased) leaves. Root length density at 5 and 25 cm from plant was significantly (P < 0.05) higher in subsurface drip than in furrow irrigation. Root length density was significantly higher in excessive irrigation regime than in the other regimes. Concomitantly, there was higher wilt incidence and severity in excessive and moderate regimes than deficit regime regardless of the irrigation method. Plant height was affected by irrigation methods and deficit regime. Neither the method of irrigation nor the quantity of water affected the other variables. Stomatal resistance in lower diseased leaves was significantly higher in infested than in fumigated plots but it was not in the upper healthy leaves. In this study, cauliflower yield was not affected by V. dahliae and irrigation method, but the deficit irrigation regime resulted in reduced yield even though it suppressed wilt in cauliflower. Thus, higher moisture levels resulted in higher root length density in V. dahliae-infested plots that in turn lead to greater incidence of Verticillium wilt and severity. The pathogen also affected physiological processes such as hydraulic conductance of cauliflower leaves, but not shoot growth or yield under these experimental conditions.     

Quality of Methods to Quantify Microsclerotia of Verticillium dahliae in Soil

Existing methods used to quantify microsclerotia of Verticillium dahliae in soil are reviewed. Most quantification methods are soil-type dependent, but are useful for disease prediction within certain soils. The major factor determining the accuracy of dry plating methods is the amount of soil plated per Petri dish. Wet plating methods are less sensitive to higher amounts of soil, especially when the fraction smaller than 20 um is removed by wet sieving. Despite general assumptions, wet plating methods do not have lower detection limits than dry plating methods. Dry plating methods are less variable at higher inoculum levels, but more variable at low inoculum levels. Bioassays are helpful tools in answering specific research questions, but are not convenient for large scale use. Molecular quantification techniques are promising, because they are not hampered by antagonistic effects, but data on their disease predictive abilities are still largely lacking. Suggestions are given for a better comparison of techniques, and some original results are presented to illustrate certain arguments.     

Relationships Between Verticillium dahliae Inoculum Density and Wilt Incidence, Severity, and Growth of Cauliflower

ABSTRACT Microplot and field experiments were conducted to evaluate the effects of inoculum density on Verticillium wilt and cauliflower growth. Soil containing Verticillium dahliae microsclerotia was mixed with various proportions of fumigated soil to establish different inoculum densities (fumigated soil was used as the noninfested control). Seven inoculum density treatments replicated four times were established, and the treatments were arranged in a randomized complete block design. Soil was collected from each microplot immediately after soil infestation for V. dahliae assay by plating onto sodium polypectate agar (NP-10) selective medium using the Anderson sampler technique. Five-week-old cauliflower was transplanted into two beds within each 1.2- by 1.2-m microplot. At the same time, several extra plants were also transplanted at the edge of each bed for destructive sampling to examine the disease onset (vascular discoloration) after planting. Cauliflower plants were monitored for Verticillium wilt development. Stomatal resistance in two visually healthy upper and two lower, diseased leaves in each microplot was measured three times at weekly intervals after initial wilt symptoms occurred. At maturity, all plants were uprooted, washed free of soil, and wilt incidence and severity, plant height, number of leaves, and dry weights of leaves and roots were determined. The higher the inoculum density, the earlier was disease onset. A density of 4 microsclerotia per g of dry soil caused 16% wilt incidence, but about 10 microsclerotia per g of soil caused 50% wilt incidence. Both wilt incidence and severity increased with increasing inoculum density up to about 20 microsclerotia per g of soil, and additional inoculum did not result in significantly higher disease incidence and severity. A negative exponential model described the disease relationships to inoculum levels under both microplot and field conditions. Stomatal resistance of diseased leaves was significantly higher at higher inoculum densities; in healthy leaves, however, no treatment differences occurred. The height, number of leaves, and dry weights of leaves and roots of plants in the fumigated control were significantly higher than in infested treatments, but the effects of inoculum density treatments were variable between years. Timing of cauliflower infection, crop physiological processes related to hydraulic conductance, and wilt intensity (incidence and severity) were thus affected by the inoculum density. Verticillium wilt management methods used in cauliflower should reduce inoculum density to less than four micro-sclerotia per g of soil to produce crops with the fewest number of infected plants.     

Ammonia and Nitrous Acid from Nitrogenous Amendments Kill the Microsclerotia of Verticillium dahliae

ABSTRACT This study examined the mechanisms by which nitrogenous amendments such as meat and bone meal kill the soilborne plant pathogen Verticillium dahliae. The effect of nitrogen products from the amendments on the survival of microsclerotia of V. dahliae was examined by solution bioassay and soil microcosm experiments. Ammonia and nitrous acid but not their ionized counterparts, ammonium and nitrite, were toxic to microsclerotia in bioassays. In microcosms, addition of meat and bone meal (2.5%) to an acidic loamy sand resulted in the accumulation of ammonia and death of microsclerotia within 2 weeks. At lower concentrations (0.5 and 1%), microsclerotia were killed after 2 weeks when nitrous acid accumulated (>0.03 mM). In an alkaline loam soil, microsclerotia survived at 3% meat and bone meal and neither ammonia nor nitrous acid accumulated. The toxicity of ammonia to the pathogen was verified by increasing the concentration of meat and bone meal to 4% or addition of urea (1,600 mg of N per kg) to the loam soil resulting in the accumulation of ammonia (>35 mM) and death of microsclerotia. The toxicity of nitrous acid was verified by adding ammonium sulfate fertilizer to an acidic sand soil. Inhibiting nitrification with dicyandiamide revealed that nitrous acid was generated as a result of the accumulation of nitrite and an acidic pH. Thus, levels to which the toxins accumulated and the effective concentration of amendment were dependent upon the soil examined. Of the two mechanisms identified, accumulation of nitrous acid is the more promising strategy to control plant diseases in acidic soil because it is more toxic than ammonia and is formed at lower concentrations of amendments.     

Volatile Fatty Acids in Liquid Swine Manure Can Kill Microsclerotia of Verticillium dahliae

ABSTRACT Liquid swine manure added to acidic soils killed microsclerotia of the wilt fungus Verticillium dahliae. We investigated whether volatile fatty acids (VFAs) in the manure were responsible for this toxicity. The survival of microsclerotia was determined after exposure to various dilutions of manure or its VFA components. Acetic, propionic, and isobutyric acids constituted the major VFAs in the manure, while n-butyric, n-valeric, iso-valeric, and n-caproic acids were present in lesser amounts. Formic acid was not detected. The individual VFAs were more toxic to microsclerotia as the solution pH was decreased, indicating that the protonated forms of the VFAs were toxic (e.g., acetic acid and not acetate). The effective concentration reducing germination of microsclerotia by 95% (EC(95)) for formic and n-caproic acids was approximately 4 mM, the most toxic of the acids tested; for n-valeric, the EC(95) was 9.2 mM, isovaleric was 16.1 mM acids, and acetic, propionic, n-butyric, and isobutyric acids were approximately 30 mM. The toxicity of acetic acid, and likely all the others, was directly related to the duration of exposure. Inhibition of microsclerotia germination followed identical trends in solutions of the manure or in a mixture of VFAs with equivalent concentrations of the individual acids found in the manure. Similarly, germination declined to the same extent in the atmosphere above the manure or the VFA mixture, confirming the toxicity of VFAs to microsclerotia. Thus, under acid conditions, VFAs in liquid swine manure can kill microsclerotia of V. dahliae.     

棉花黄萎病菌提取物对棉花的抗病效果

近年来棉花黄萎病发生严重 ,重病田病株率高达 95 %以上。目前生产上缺少抗病品种 ,防治方法不健全 ,田间防治效果不理想 ,生产上急需新的防治措施。随着环境和生态问题的日益突出 ,诱导抗病性研究日益被人们重视。本研究从诱导抗病性原理出发 ,旨在探索能够有效激发、诱导棉花抗黄萎病的因子 ,并取得一些结果 ,现报道如下。1　材料与方法1 .1　供试材料　棉种为中棉 1 6 ;棉花黄萎病菌 (Verticillium dahliae Kleb)由本室分离鉴定。1 .2　激发子的制备　将棉花黄萎病菌在马铃薯葡萄糖液体培养基中 ,5℃下恒温振荡培养两周后 ,滤纸过滤 ,滤…     

Liquid Swine Manure Can Kill Verticillium dahliae Microsclerotia in Soil by Volatile Fatty Acid, Nitrous Acid, and Ammonia Toxicity

ABSTRACT In previous studies, liquid swine manure (LSM) was sometimes shown to reduce Verticillium wilt of potato caused by Verticillium dahliae. We also observed that microsclerotia of this fungus died within 1 day, or between 3 and 6 weeks, after addition of LSM to some acid soils and within 1 week in some alkaline soils. In this study, we demonstrated that a volatile fatty acid (VFA) mixture with an identical concentration of VFAs as that found in an effective LSM reduced germination in an acid soil (pH 5.1) to the same extent as the LSM after 1 day of exposure. Germination was reduced by 45, 75, and 90% in the 10, 20, and 40% ([wt/wt] soil moisture) treatments, respectively, with the latter being equivalent to an application of 80 hl/ha. Addition to this acid soil of 19 LSMs (30% [wt/wt] soil moisture) collected from different producers resulted in complete kill of microsclerotia with 12 manures. Effective manures had a total concentration of nonionized forms of VFAs in soil solution of 2.7 mM or higher. In some acid soils (pH 5.8), addition of LSM (40% [wt/wt] soil moisture) did not kill microsclerotia until 3 to 6 weeks later. Here, a reduction in viability of microsclerotia was attributed to the accumulation of 0.06 mM nitrous acid in the soil solution at 4 weeks. When an LSM was added (40% [wt/wt] soil moisture) to an alkaline soil (pH 7.9) where VFAs are not toxic, microsclerotia germination was reduced by 80% after 1 week. Here the pH increased to 8.9 and the concentration of ammonia reached 30 mM in the soil solution. An ammonium chloride solution having an equivalent concentration of ammonium as the manure was shown to have the same spectrum of toxicity as the manure in assays ranging from pH 7 to 9, both in solutions and above the solutions. At pH 9, the concentration of ammonia reached 18 mM and 100% mortality of microsclerotia occurred. Thus, in acid soils, LSM can kill microsclerotia of V. dahliae by VFA and/or nitrous acid toxicity and in alkaline soils by ammonia toxicity. In order to take advantage of these mechanisms for disease reduction, the manure chemical composition, rate of addition, and soil characteristics need to be determined for each instance of use.     

Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt

ABSTRACT Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.     

化学农药对棉花黄萎病菌和生防菌的选择毒性

研究了11种化学农药对棉花黄萎病菌和生防菌的选择毒性。对棉花黄萎病菌CV-1的毒力测定结果表明，浓度100μg／g具明显抑制效果的有多菌灵、苯菌灵、甲基硫菌灵和粉锈宁；5000μg／g下具抑制作用的有菌核净和五氯硝基苯。苯并咪唑类杀菌剂对绿色木霉LTR．2的毒性最高，多菌灵、苯菌灵和甲基硫菌灵的EC50值依次为0．7395、1．6784和0．7370μg／g。多菌灵、苯菌灵和甲基硫菌灵对黄萎病菌的最低抑制浓度分别为0．7、2．3、2．6μg／g；对LTR-2最低抑制浓度分别为1．2、3．4、6．4μg／g。多菌灵对几种生防真菌的毒性选择性比值（TSR）介于1～10之间的有LTR-2、2-②、Tc、Tk7a、GLR，分别为4．6、3．1、4．7、1．2和7．3，为正向调控作用；介于10～100之间的有Sxb，其TSR为27．5，为中度选择性；大于1000的有菌株T9，其TSR值为9145．0，为强烈正向调控作用。苯菌灵的TSR值介于1～10之间的有Sxb、2-②、Tc、Tk7a、GLR，依次为2．8、3．9、5．6、4．5和4．7；介于10～100之间的有LTR-2和1、9，其TSR为13．7和66．5；没有大于100的生防菌菌株。     

Crop Rotation,Monoculture and Soil Ecology1

Crop rotation and monocultural systems are discussed in relation to disease management and control. The ecological basis of both systems is examined. In crop rotation the leading principle is that in the absence of host-plants parasite populations invariably decline below threshold levels, mostly within a few years. Knowledge of the behaviour of parasite populations in the soil is becoming vast, especially in the case of plant parasitic nematodes. Analysis of soil samples for nematodes and fungi often helps the farmer to plan rotation schemes. From the ecological point of view, crop rotation is interpreted as a system of interrupted monocultures. Monocultural practices cannot be applied so universally as crop rotation, but in a number of cases monoculture is feasible, e.g. with cereals. In the equilibrium situation between host-plant (e.g. cereals), parasite (Gaeumannomyces graminis [Sacc.] v. Arx & Olivier) and the antagonists of the parasite, growth of the crop is satisfactory. These cases illustrate the role of antagonists, and it is proposed to investigate more systematically monocultures in relation to antagonisms. This may lead to more opportunities for biological control of soil-borne diseases.     

Long-Term Crop Rotation Effects on Organic Carbon,Nitrogen, and Phosphorus in Haplustoll Soil Fractions

Soil organic matter (SOM) or carbon (SOC) consists of a number of fractions (which can be separated by granulometric wet sieving) having different properties among them. Information on fraction nutrient distribution and long-term crop rotations is lacking for semiarid environments. The objective of this research was to study the agronomic effects on soil OC, N, and P fractions. The humified OC was the largest and least variable fraction of the SOC. Soil under continuous mixed pasture had higher OC contents than under annually tilled treatments. Similarly, soil total nitrogen under the cropped treatments decreased from 1.7 g N kg-¹ in noncultivated soils (reference plots) to 1.0, 0.7 an 0.7 g N kg-¹ under mixed pasture, pasture-crop, and wheat-crop respectively, in the fine soil fraction. The reference plots also showed significantly lower levels of organic phosphorus (P o ) in comparison to the other treatment (from 67.1 w g P o g-¹ to greater than 100 w g P o g-¹ in the fine fraction of the treatments and years). The noncultivated soil showed larger values of P o and inorganic P in the large-size granulometric fraction (0.1-2 mm) than in the soil fine fraction (0-01 mm). However, the rotation treatments had greater concentrations of P in the fine fraction. The P o from the coarse fraction appears to be the most labile and sensitive fraction to tillage and environmental conditions, and may be closely related to P availability.     

Crop Rotation and Soil Pathogens — Trends in Germany

The need to cut costs has led to a simplification of crop rotation in several fields of plant cultivation. The results of Thormann, who has grown wheat continuously on parts of his farmland for 10 years, are discussed. Also, the preliminary results of our own experiments with increasing proportions of wheat and barley suggest that more wheat and barley may be cultivated in crop rotation than is allowed according to the conventional limits determined by the increase of foot rot diseases and cereal root eelworm. Even in successive crops of maize the problem of soil-borne pathogens can be tackled: the spread of blister smut is stopped by the control of the frit fly and by selection of tolerant varieties; root and stem rots of maize are overcome by the selection of appropriate varieties, too. On the contrary, the potato root eelworm is a serious pest in all sites of intensive potato production. Only an integrated control including the use of resistant varieties and the application of nematicides gives success. Much research work has to be done mainly to get more information on the biotical defence mechanisms of the soil.     

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.     

棉花黄萎病防治策略

黄萎病是棉花生产中的最主要病害之一,广泛分布于世界各产棉国.由于该病是土壤传播的维管束病害,防治难度较大,至今尚无特效的防治药剂,只能依靠种植抗病品种为主的综合防治措施.但是,我国目前棉花品种的抗病性只能达到抗至耐病水平,致使该病在环境条件适宜时连续流行为害.控制该病的猖獗为害,已成为棉花生产可持续发展的主要问题之一.     

茄子黄萎病菌毒素对茄子愈伤组织几种酶活性的影响

 病原物侵染可引起一些酚类代谢相关酶的活性增强,其中,最常见的是苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO),这些酶活性的变化与植物的抗病机制有密切关系。     

棉花黄萎病菌致病力测定及评价方法研究

 棉花黄萎病菌致病力测定及评价是抗病育种及病害综合防治的基础。本文以我国三大棉区的167个黄萎菌菌株为对象,研究致病力测定及评价中不同批次之间病情指数的校准及致病力类型划分标准等关键技术环节。结果表明,以中等致病力类型菌株Vd076为校正菌株,以感病性稳定的冀棉11为校正鉴别寄主,以校正菌株在校正鉴别寄主上病情指数达到50.0±5.0时的调查结果进行各菌株的致病力评价,可获得较好的校正效果,使不同批次试验数据具有可比性;依据聚类分析结果,制定了致病力类型划分标准,强、中、弱3种致病力类型的平均校正病指分别为﹥40.0、20.1~40.0和20.0;通过对不同鉴别寄主组合的分析,推荐陆地棉中棉所41号、豫棉21、鲁棉研28、中棉所35号、中棉所8号、冀棉11作为鉴别寄主。该研究为棉花黄萎病菌致病力变异等相关研究工作提供了技术支撑。