Factors affecting the control of Pythium ultimum damping-off of sugar beet by Pythium oligandrum

Application of Pythium oligandrum to a soil-based compost as a mycelial suspension (5 × 10² CFU g⁻¹ of dry compost) and oospore alginate pellets (10⁵ oospores/g of dry compost) controlled pre- and postemergence damping-off of sugar beet caused by Pythium ultimum to a level similar to metalaxyl seed treatment. Oospore seed treatments and aqueous suspensions of oospores applied to compost failed to control disease. Problems in the use of P. oligandrum oospore inocula for the control of damping-off were highlighted. It was shown that treatment of oospores with cellulase (20 g L⁻¹) increased germination approximately three-fold in comparison to untreated spores. Untreated and cellulase pretreated oospores were subsequently evaluated as seed treatments for their ability to control damping-off of sugar beet. The highest rate of pretreated oospores (10⁴ oospores/seed) gave levels of emergence and establishment in infested compost that were not significantly different from the uninfested controls, whereas seed treatment with untreated oospores gave no significant reduction in disease. In a trial carried out in a controlled environment to assess the effect of pH (4.5–8.0), P. oligandrum (10⁴ cellulase pretreated oospores/seed) was shown to control pre- and postemergence damping-off of sugar beet at pH 7.0 and 7.5 only.     

Viability, germination and infection potential of oospores of Phytophthora infestans

Matings between five A1 and five A2 wild-type isolates of Phytophthora infestans from potato and tomato crops in the United Kingdom produced oospores in vitro in all cases examined. Oospores from the majority of crosses germinated, albeit at a low level (max 13-4%), after extraction from agar cultures by high-speed blending and treatment with novoZym 234. Viability of oospores from 20 crosses was tested by three methods. Two methods involved stains, either tetrazolium bromide (MTT) or phloxine B, and the third measured plasmolysis in 2 M NaCl. Both staining methods indicated a high percentage viability but gave false-positive results with heat-killed oospores. The plasmolysis method gave a lower percentage viability but no false positives. Oospores produced in vitro and stored either in sterile H₂O or in soil at temperatures between 0 and 20 C survived for between 5 and 7 months, the length of the experiments. Oospores buried in non-sterile field soil survived for up to 8 months (January-August). Inoculation of potato with zoospores of AI and A2 isolates produced oospores in stems but not in leaf tissue. In some, but not all cases, rapidly growing potato shoots (15-mm long) were successfully infected with oospores produced in vitro.     

Extraction of oospores of Peronospora viciae from soil

A method for the extraction of oospores of Peronospora viciae from soil is described. Approximately 75% of the oospores added to silty clay loam and loamy sand soil samples were recovered. Percentage recovery was independent of oospore density. This extraction method did not affect viability as determined by the tetrazolium bromide test and a germination assay and may therefore be used to study survival of oospores. Numbers of oospores extracted from soil samples taken from seven fields naturally infested with oospores of P. viciae f.sp. pisi ranged from 2 to 21 oospores per g soil. Oospore density was not significantly correlated with disease incidence as determined by a bioassay.     

Biological control of Pythium ultimum–induced damping-off by treating cress seed with the mycoparasite Pythium oligandrum

Incorporation of the aggressive mycoparasite Pythium oligandrum into a carboxymethyl cellulose-based seed coating decreased damping-off of cress seedlings by Pythium ultimum in both naturally infested soil and artificially infested sand. This effect is attributed to the germination of P. oligandrum oospores on the seed surface with subsequent generation of a protectant mycelium around the seedling.
P. oligandrum oospores survived storage periods of 10—20 weeks at zero relative humidity both on glass slides and within seed coatings. The mycoparasite also survived an 18–month burial in natural soil, probably in the form of oospores.
The implications of these observations for biocontrol of seedling diseases by P. oligandrum are discussed.     

Phytophthora infestans Produces Oospores in Fruits and Seeds of Tomato

ABSTRACT Tomato fruits at the mature green stage coinoculated with A1 + A2 sporangia of Phytophthora infestans, the late blight causal fungus, showed abundant oospores in the vascular tissues, pericarp, columella, and placenta. Oospores were also formed on the surface of fruits kept in moisture-saturated atmosphere. Occasionally, oospores were enclosed between the epidermal hairs of the seed coat. In a few seeds, oospores were detected inside the embryo. The data suggest that blighted tomato fruits may carry a large number of oospores, thus making them a threatening source of blight inoculum. Such fruits may also release airborne oosporic inoculum that may introduce recombinant genotypes within a growing season. Although Phytophthora infestans is seedborne in tomato, to our knowledge, this is the first report on the occurrence of oospores in tomato seeds. Whether such tomato seeds produce blighted seedlings remains to be shown.     

棉铃疫菌的越冬存活

 用MTT活体染色法,对棉铃疫苗(Phytophthora boehmeriae Sawada)的存活越冬进行了研究,结果表明:孢子囊和卵孢子均可在棉田土中的病残体(烂钤壳,棉籽)上存活。其中孢子囊可存活3~4个月,且其存活率可高达58.0%,认为孢子囊在棉铃疫病的再侵染过程中起重要作用。但孢子囊在1月份温度达零度时就全部死亡,不能越冬。而卵孢子在历经1990年至1991年冬季零度以下76天(其中最低温度达-2.8℃)后仍可以存活,在存活300天以后其存活率达48.8%,证明卵孢子是毫无疑问的越冬菌态。生物测定,越冬后卵孢子形态观察,卵孢子萌动变化与温度的关系表明,越冬存活的卵孢子可以成为棉铃疫病的初侵染来源。     

Environmental and Genetic Factors Influencing Self-Fertility in Phytophthora infestans

ABSTRACT Phytophthora infestans is generally regarded as heterothallic-requiring physical proximity of two individuals of different mating type (A1 and A2) for oosporogenesis. Recent reports of limited selfing in young cultures of this oomycete stimulated us to investigate factors contributing to the phenomenon. The ability to produce oospores rapidly (within 2 weeks) in pure, single individual cultures (self-fertility) was tested in 116 individual isolates. The 116 isolates were from geographically diverse locations (16 countries) and were genetically diverse. Mating type and growth medium were the most prominent factors in determining if an isolate would be self-fertile. The majority of A2 isolates (45 of 47 tested) produced oospores when grown on a 50:50 mixture of V8 and rye B medium. In contrast, the majority of A1 isolates (65 of 69 tested) did not produce oospores on this medium. None of the 116 isolates produced oospores when grown on rye B medium (with no V8 juice). Further tests on representative A1 and A2 isolates revealed that oatmeal agar, tomato juice agar, and V8-juice agar all induced the A2 mating type isolate to produce oospores but did not induce the A1 mating type isolate to produce oospores. Calcium carbonate and pH did not alter the self-fertile oospore production in either A1 or A2 mating type isolates. For in vivo tests, the application of fungicide to potato or tomato leaf tissue either before or after inoculation did not stimulate any individual isolate (one A2 and one A1 isolate) to produce oospores in infected tissue. However, in all of the controls for all experiments (in vivo and in vitro), many oospores were produced rapidly if both strains grew in physical proximity.     

Survival Dynamics of Aphanomyces cochlioides Oospores Exposed to Heat Stress

ABSTRACT To determine how exposure to heat effects their survival, oospores of Aphanomyces cochlioides isolate C22 were exposed in water to 35, 40, 45, or 50 degrees C for prescribed times and then examined for viability. The Weibull model was modified to represent the effects of temperature on survival of oospores. The final fitted model gave lethal doses for 50% of the oospores of 251, 49.8, 9.8, and 1.9 h at 35, 40, 45, and 50 degrees C, respectively. To determine if alternating high and low temperatures resulted in (i) recovery from heat damage during low temperature periods, (ii) increased susceptibility to heat damage, or (iii) if effects of heat damage were cumulative, oospores were examined after each of four 24-h cycles at 45 degrees C for 4 h and 21 degrees C for 20 h. Survival of oospores exposed to alternating high and low temperatures fit the cumulative effects model. Significant variability in heat tolerance among five isolates was observed (P< 0.001) but model parameters successfully accommodated this variability (R(2) = 0.96, P < 0.001). This research shows that under wet conditions, there are predictable patterns to mortality for A. cochlioides oospores exposed to continuous or fluctuating high temperatures.     

Real-time PCR assay for quantification of Tilletia caries contamination of UK wheat seed

A quantitative real-time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat-seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log₁₀ (number of spores per seed + 1) on mean log₁₀ DNA ( µ g) produced a coefficient of determination ( R ²) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real-time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed.     

Occurrence of the A2 mating type and oospores ofPhytophthora Infestans in potato crops in Israel

Seventeen metalaxyl-sensitive and 21 metalaxyl-resistant isolates ofPhytophthora infestans collected from blighted potato fields during the years 1983–1988 were tested for mating type on rye seed agar medium. All isolates except one (MS3, collected in 1986 at Sufa, in the western Negev) were found to belong to the A2 mating type. A2 isolates produced oospores within 2 weeks when cultured together with isolate 163 (A₁ from the U.S.A.) or with the A₁ isolate MS3 from Israel. When cultured singly, A₂ isolates produced some oospores within 4–8 weeks. Blighted potato tubers harvested from potato crops artificially inoculated with a mixture of A1+A2 sporangia were found to contain some oospores. No oospores were detected in blighted tubers harvested from A₂ + A₂ inoculated crops. It was concluded that the A2 mating type ofP. infestans has occurred in Israel since 1983 or even earlier. The rare occurrence of the A1 mating type was unexpected and indicated that sexual reproduction of the fungus in the country might be limited.     

Factors affecting germination of oospores of Peronospora viciae f.sp. pisi in vitro

The effects of host plant exudates, light and temperature on germination of oospores of Peronospora viciae f.sp. pisi in vitro were investigated. Seed and root exudates did not increase percentage germination, whereas light inhibited germination. The first germ tubes appeared after 4, 7, and 14 days of incubation at 15, 10 and 5 °C, respectively. The eventual level of germination was highest and had similar values at 5 and 10 °C. At 20 °C germination was poor and at 25 °C no germination was observed. Oospores placed on membrane filters were incubated on soil. When oospores were retrieved from the membrane filters after six days and placed in water at 10 °C, they germinated within 2 days. On soil significantly less oospores germinated than in water. Germinability of oospores stored in the dark at 5 or 20 °C at 30 or 76% RH was studied over a two-year period. Germinability generally increased over time, but fluctuations were observed indicating the occurrence of secondary dormancy. Time courses of germinability were generally similar for oospores stored at several temperatures and humidities. No effect of light on time course of germinability was found when oospores were exposed to alternating light-dark periods or stored in continuous dark for 140 days. Percentage germination observed in a germination assay was correlated with percentage infection determined in a bioassay.     

大豆疫霉Phytophthora sojae卵孢子在黑龙江省土壤中的越冬存活率

为探究大豆疫霉Phytophthora sojae卵孢子在黑龙江省土壤中的越冬存活率及其与所处土壤深度和媒介的相关性,以增强型绿色荧光蛋白为报告基因,将培养基及病残体中的大豆疫霉卵孢子分别接种到试验田框栽土壤表层下不同深处,检测其卵孢子的越冬存活率,同时在框栽中定量播种不含任何已知抗疫霉根腐病基因的大豆品种Sloan(rps),苗期调查其发病率。结果表明,大豆疫霉卵孢子在黑龙江省土壤中的适生性较强,可在5~15 cm深度土壤中安全越冬,越冬存活率高达81.67%~96.33%。卵孢子越冬存活率与其所处的越冬媒介关系不大,而与土壤深度有关。在5~15 cm范围内,随着土壤深度的增加,卵孢子越冬存活率增加。处于深层土壤中的卵孢子更容易打破休眠,进入萌发前的萌动状态。各处理间卵孢子越冬存活率的显著性差异并未在发病率上表现出来,说明除了土壤深度外,还有其它因素影响发病率。     

恶疫霉有性杂交后代的生物学研究

 将2个带有不同抗药性标记的恶疫霉菌株配对,培养36 d后诱导其卵孢子萌发,从3760个单卵孢株中,获得50株带有双亲标记的杂交个体。对其中32株杂交个体的生长速率、有性和无性繁殖能力、卵孢子和游动孢子萌发率、致病力及对高温的耐受力等生物学性状进行测定。结果显示,测试的32株杂交个体在LBA培养基上均能较好地生长,有24株的杂交个体的生长速率介于2亲本之间;在LBA和SL培养基上,大多数杂交个体均能产生较大数量的卵孢子,有37%的杂交个体在SL培养基上产卵孢子能力显著大于双亲,仅有1个杂交个体不产生卵孢子;在人工培养条件下,大多数供试杂交个体可以产生较大数量的孢子囊,其中15株杂交个体产孢子囊能力处于双亲之间;被测的杂交个体产生的卵孢子或游动孢子均可以萌发形成有效的单孢株,有22株的杂交个体的卵孢子萌发率大于50%;被测的杂交个体接种在苹果上都有较强的致病力,有16株杂交个体的致病力显著大于双亲。表明在群体水平上恶疫霉有性杂交后代具有较强的生活能力,提示同宗配合恶疫霉不同菌株间的有性重组,对该种的群体遗传多样性可能具重要作用。     

大豆疫霉病菌在中国的发现及其生物学特性的研究

 本文通过对从黑龙江、吉林和北京等地的大豆上分离的7个疫霉菌株的一系列生物学特性的研究,并以美国的Phytophthora megasperma标准菌株为对照,将该7个菌株鉴定为Phytophthoramegasperma f.sp.glycinea。接种试验表明,该菌在不同大豆品种上致病力表现差异明显,而且对三叶草和紫花苜蓿不侵染。用MTT染色法定期检测埋在不同处理的土壤中的卵孢子活性。经一年后,所有处理中90%以上的卵孢子均处于活性状态;而温度对卵孢子的休眠期有较大的影响。     

Sprinkling irrigation enhances production of oospores of phytophthora infestans in field-grown crops of potato

ABSTRACT Two field experiments were conducted to study the effect of overhead sprinkling irrigation on oospore formation by the late blight fungus Phytophthora infestans in potato. Total rain (natural + sprinkling) accumulated in treatments of experiment 1 (winter 1997 to 1998) were 765, 287, and 219 mm and treatments of experiment 2 (winter 1999 to 2000) were 641, 193, and 129 mm. Sporangia from 11 isolates of P. infestans were combined in eight pairs, seven of A1 and A2 and one of A2 and A2 mating type, and were sprayed on field-grown potato crops (42 plants per plot at 7 m(2) each) and examined for their ability to form oospores in the host tissues. In experiment 1, oospores were recorded in a total of 132 of 1,680 leaflets (7.9%), 24 of 105 stems, and 2 of 90 tubers. In experiment 2, oospores were recorded in 40 of 519 leaflets (7.7%), but not in any of the 90 stems or the 45 tubers examined. Both the proportion of leaflets containing oospores and the number of oospores per leaflet increased with time after inoculation and were dependent on the rain regime, the position of leaves on the plant, and the isolate pair combination. In both field trials, increasing the rainfall significantly enhanced oospore production in leaves. Leaf samples collected from the soil surface had significantly more oospores than those collected from the midcanopy. Two pairs in experiment 1 were more fertile than the others, whereas the pair used in experiment 2 was the least fertile. The total number of oospores per leaflet usually ranged from 10 to 100 in experiment 1, but only from 2 to 10 in experiment 2. Maximal oospore counts in the field were 200 and 50 in experiments 1 and 2, respectively, but ranged from approximately 2,000 to 12,000 oospores per leaflet in detached leaves in the laboratory. We concluded that P. infestans can produce oospores in the foliage of field-grown potato crops, especially when kept wet by regular overhead sprinkling irrigation, but production was far below that in the laboratory.     

掘氏疫霉卵孢子萌发研究

 掘氏疫霉(Phytophthora drechsleri)种内菌株直接交配产生的卵孢子分别用0.1% KMnO₄处理20分钟和0.3% H₂O₂处理2分钟在S+L培养基上(26℃)培养7天萌发率>70%。H₂O₂是本研究首次报道的一种刺激掘氏疫霉卵孢子萌发的处理剂,其效果略优于KMnO₄。用KMnO₄和H₂O₂处理均可有效地抑制卵孢子悬浮液中菌丝片段及菌丝膨大体的萌发生长。在一定时期内卵孢子萌发率随卵孢子保存时间的增加而增加,保存30天和45天的卵孢子分别用KMnO₄和H₂O₂处理萌发率最高。卵孢子保存期间有无光照对萌发率无显著影响,但卵孢子荫发时给予光照对萌发有明显的促进作用,保存30天的卵孢子在光照下萌发率为60-70%,在黑暗中仅0-16%。卵孢子萌发过程中的光照条件以黑光灯8小时,日光灯16小时交替连续照射7天效果最好,其次为黑光灯单独连续光照,以日光灯单独照射效果较差。所测定的6种培养基中以S+L培养基对卵孢子萌发的效果最好,其次为V₈+L和WA+L。蜗牛酶、纤维素酶、土壤浸出液和黄瓜果提取液对掘氏疫霉卵孢子萌发无明显刺激作用。     

Effect of metalaxyl on formation and germination of oospores of Phytophthora infestans

Oospores of Phytophthora infestans were produced in potato leaf discs floating on metalaxyl solution (100 μg mL⁻¹ a.i.) and inoculated with all combinations of two metalaxyl-sensitive and two -resistant parental isolates. Numbers of oospores produced varied between different matings, depending on parents, in the absence of the fungicide and when metalaxyl was added 0, 7, 14 and 21 days after inoculation. Oospores were not produced when metalaxyl was added at the time of inoculation (0 days) when either one or both parents were sensitive to metalaxyl. In two of three such matings further oospore formation was arrested when metalaxyl was added either 7 or 14 days after inoculation. Oospores extracted from leaf discs 14, 21 and 28 days after inoculation were assessed for germination on water agar after 21 days. Germination of oospores from water control treatments varied between 6 and 30% depending on the cross. Germination was significantly reduced in oospores of metalaxyl-sensitive parents extracted 28 days after inoculation of leaf discs treated with metalaxyl 0, 7 and 14 days after inoculation compared with the 21-day treatment. Minimal differences in germination were observed for oospores from the mating of resistant parents irrespective of metalaxyl treatment, although germination was generally low, not exceeding 8.5%.     

AnthracnoseTracer: A Spatiotemporal Model for Simulating the Spread of Anthracnose in a Lupin Field

ABSTRACT A spatiotemporal model has been developed to simulate the spread of anthracnose, initiated by infected seed, in a lupin field. The model quantifies the loss of healthy growing points of lupin in all 1-m(2) subunits of a field throughout a growing season. The development of growing points is modeled as a function of temperature using a 1-day time step, and disease-induced compensatory growth is accounted for. Dispersal of spores is simulated explicitly using Monte Carlo techniques. Spread of spores occurs during rainfall events on a 1-h time step. The distance traveled by spores is partially dependent on wind speed and is generated by adding the values selected from half-Cauchy distributions. The direction of travel of the spores is influenced by wind direction. The model has been employed to produce a theoretical assessment of damage from disease in two environments at five levels of seed infection. It was calculated that in a susceptible lupin cultivar with a 0.01% initial seed infection, anthracnose would cause approximately 15% loss of healthy growing points in a high rainfall environment in Western Australia. In a low rainfall environment, similar damage would be unlikely even with a much higher (1%) level of seed infection.     

具防病并提高植物耐旱功能的小链霉菌HS57的筛选

本研究旨在寻找防治黄瓜枯萎病并提高植物抗逆能力的多功能菌株。从连作田健康黄瓜根际土中分离筛选到1株放线菌HS57。平板对峙法测定菌株HS57对尖孢镰刀菌、茄病镰刀菌和立枯丝核菌的抑制作用,平皿对扣共培养法测定其挥发性物质对尖孢镰刀菌的抑制作用,温室盆栽法测定其对黄瓜枯萎病的防病效果及对小麦耐旱能力的影响,结合菌株形态特征和16S rRNA基因序列分析对其进行鉴定。结果表明,菌株HS57可抑制3种病原菌生长,其中对尖孢镰刀菌的抑制率为52.4%;其挥发性物质对尖孢镰刀菌的抑制率为32.1%。浓度为10^7个/mL的HS57孢子悬浮液浸种对黄瓜枯萎病的防效最好,为51.0%;小麦种子经浓度为10^7、10^8个/mL的HS57孢子悬浮液浸种,麦苗经连续7 d缺水处理后再浇水,比对照恢复得更好。初步鉴定HS57菌株为小链霉菌Streptomyces parvus,是一株非常有潜力的多功能菌株。     

Control of damping-off in cress and sugar-beet by commercial seed-coating with Pythium oligandrum

Seeds of cress and sugar-beet were coated with oospores of Pythium oligandrum using commercial seed-pelleting or film-coating procedures. Following either procedure approximately 10⁴ oospores were recovered from both seed types, achieving 75.94% of the targeted dose. Oospore germination (9.19%) was unaffected by the coating treatments. Both types of treatment reduced damping-off of cress caused by P. ultimum in artificially infested sand and potting compost and by Rhizoctonia solani in artificially infested sand. In some cases, the level of control was equivalent to fungicide drenches. In general, pelleting of P. oligandrum on cress gave better control than film-coating treatments. P. oligandrum also reduced damping-off of sugar-beet in soil naturally infested with Aphanomyces cochlioides and Pythium spp. Control was equivalent to that achieved with hymexazol fungicide seed-coating treatments and was related to the inoculum potential of A. cochlioides in the soil; neither standard hymexazol coatings nor P. oligandrum treatments gave control at high inoculum potentials. P. oligandrum was not rhizosphere competent on cress or sugar-beet.