Germination of Sclerotinia minor and S. sclerotiorum Sclerotia Under Various Soil Moisture and Temperature Combinations

ABSTRACT Sclerotial germination of three isolates each of Sclerotinia minor and S. sclerotiorum was compared under various soil moisture and temperature combinations in soils from Huron and Salinas, CA. Sclerotia from each isolate in soil disks equilibrated at 0, -0.03, -0.07, -0.1, -0.15, and -0.3 MPa were transferred into petri plates and incubated at 5, 10, 15, 20, 25, and 30 degrees C. Types and levels of germination in the two species were recorded. Petri plates in which apothecia were observed were transferred into a growth chamber at 15 degrees C with a 12-h light-dark regime. All retrievable sclerotia were recovered 3 months later and tested for viability. Soil type did not affect either the type or level of germination of sclerotia. Mycelial germination was the predominant mode in sclerotia of S. minor, and it occurred between -0.03 and -0.3 MPa and 5 and 25 degrees C, with an optimum at -0.1 MPa and 15 degrees C. No germination occurred at 30 degrees C or 0 MPa. Soil temperature, moisture, or soil type did not affect the viability of sclerotia of either species. Carpogenic germination of S. sclerotiorum sclerotia, measured as the number of sclerotia producing stipes and apothecia, was the predominant mode that was affected significantly by soil moisture and temperature. Myceliogenic germination in this species under the experimental conditions was infrequent. The optimum conditions for carpogenic germination were 15 degrees C and -0.03 or -0.07 MPa. To study the effect of sclerotial size on carpogenic germination in both S. minor and S. sclerotiorum, sclerotia of three distinct size classes for each species were placed in soil disks equilibrated at -0.03 MPa and incubated at 15 degrees C. After 6 weeks, number of stipes and apothecia produced by sclerotia were counted. Solitary S. minor sclerotia did not form apothecia, but aggregates of attached sclerotia readily formed apothecia. The number of stipes produced by both S. minor and S. sclerotiorum was highly correlated with sclerotial size. These results suggest there is a threshold of sclerotial size below which apothecia are not produced, and explains, in part, why production of apothecia in S. minor seldom occurs in nature.     

Comparative survival of Sclerotia of Sclerotinia minor and S. sclerotiorum

Survival of sclerotia of Sclerotinia minor and S. sclerotiorum was compared in irrigated fields during the summer in two major lettuce production areas in California. More than 50% sclerotia of S. sclerotiorum compared with 4 and 35% of S. minor remained viable after 24 weeks of burial at 15 and 5 cm depths, respectively, in the San Joaquin Valley while >80% of sclerotia survived in the Salinas Valley for both species. The results explain in part the lower infections from S. minor in the San Joaquin Valley. To identify factors that contribute to the rapid decline in the viability of sclerotia, the effects of soil moisture, temperature, and oxygen levels were studied in laboratory. More than 90% of sclerotia of both species survived for at least 3 months in sterilized dry soils at temperatures between 15 and 40 degrees C. Soil moisture did not affect survival at 15 and 25 degrees C. At 35 degrees C, however, survival rates were significantly lower at high (-0.3 to -0.01 MPa) water potential than at low (<-1.0 MPa) water potential. Incubation under ultralow oxygen concentration (0.01%) significantly reduced survival of sclerotia in nonautoclaved moist soils at 25 degrees C, with less than 2% sclerotia surviving over 4 weeks compared with about 45% sclerotia surviving at the ambient oxygen level (21%). The combination of high temperature, high soil moisture, and reduced oxygen in irrigated fields contribute to the lower survival of both Sclerotinia species and the responses of the two species to these conditions shape their relative geographical distribution.     

链孢粘帚霉寄生核盘菌菌核相关基因的EST生物信息学分析

链孢粘帚霉Gliocladium catenulatum是一种重要的菌寄生真菌,为了明确该菌与寄主核盘菌Sclerotinia sclerotiorum互作中参与菌寄生过程的功能基因,本实验室前期采用抑制消减杂交技术(SSH)构建了消减cDNA文库.本文从该库里随机挑选420个阳性克隆进行测序分析,结果表明克隆片段大小在200～600bp的序列占81.07%.去除载体和小于100bp的序列,获得391条有效序列,其平均长度为409bp.通过序列拼接得到181个单值基因(unigenes),其中包括27个重叠群(contigs)和154个单拷贝的ESTs(singletons).将得到的ESTs与NCBI非冗余蛋白数据库(NR)进行BLASTx分析,结果显示有38个基因的编码蛋白与已知功能的蛋白有较高的相似性,其中包括可能与生物防治功能相关的内切葡聚糖酶、脂滴包被蛋白、MFS转运蛋白、过氧化物酶等;有39条序列在NCBI数据库中未找到相似序列,可能为新基因;其他基因的编码蛋白与数据库中功能未知蛋白相似性高.本研究为明确与重寄生功能相关的基因奠定了基础.     

Effect of Inoculum Rates and Sources of Coniothyrium minitans on Control of Sclerotinia sclerotiorum Disease in Glasshouse Lettuce

Coniothyrium minitans isolate Conio grew on both maizemeal-perlite and ground maizemeal-perlite, producing high numbers (1.6×10⁷ conidiag^–1 inoculum) of germinable conidia. Coniothyrium minitans isolate Conio applied as a preplanting soil incorporation of maizemeal-perlite inoculum at full application rate (0.6lm^–2; 10¹¹ colony forming units (cfu)m^–2) significantly reduced Sclerotinia disease in a sequence of three lettuce crops grown in a glasshouse. No reduction in disease was achieved with any of the reduced rate treatments (10⁸cfum^–2) of a range of C. minitans isolates (Conio ground maizemeal-perlite at reduced rate, Conio and IVT1 spore suspensions derived from maizemeal-perlite, IVT1 spore suspension derived from oats and Contans® WG spore suspension). After harvest of the second and third crops, C. minitans maizemeal-perlite at full rate reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore suspension and reduced rate maizemeal-perlite inocula. Coniothyrium minitans was recovered from the soil throughout the trial, between 10⁵ and 10⁷cfucm^–3 in maizemeal-perlite inoculum full rate treated plots and 10¹–10⁴cfu cm^–3 in all other inoculum treated plots.Pot bioassays were set up corresponding to the inoculum used in the glasshouse, with the addition of Conio ground maizemeal-perlite at a rate corresponding to the full rate maizemeal-perlite. Coniothyrium minitans maizemeal-perlite and ground maizemeal-perlite at full rate significantly decreased carpogenic germination, recovery and viability of sclerotia and increased infection of sclerotia by C. minitans in comparison with spore suspension treatments, reflecting results of the glasshouse trials. Additionally, reduced maizemeal-perlite treatment also decreased apothecial production, recovery and viability of sclerotia compared with the spore suspension treatment, despite being applied at similar rates. Simultaneous infection of sclerotia by several isolates of C. minitans was demonstrated. Inoculum level in terms of colony forming unitscm^–3 of soil appears to be a key factor in both control of Sclerotinia disease and in reducing apothecial production by sclerotia.     

Interactions Between the Mycoparasite Pythium oligandrum and Sclerotia of the Plant Pathogen Sclerotinia sclerotiorum

Pythium oligandrum Drechsler is a mycoparasite which parasitizes hyphae of many fungal species. A detailed study of the interactions between P. oligandrum and the sclerotia of the plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary is presented. Pythium oligandrum was present in Danish soils at concentrations between 4 and 26 cfu g^–1 soil. An increase in the natural population of P. oligandrum by addition of P. oligandrum zoospores to a soil reduced the ability of sclerotia of S. sclerotiorum to germinate myceliogenically and the sclerotia were colonized internally by P. oligandrum. This colonization and reduction of germination of sclerotia were also seen when sclerotia and P. oligandrum were incubated together in water. Small sclerotia were significantly more susceptible to parasitism by P. oligandrum than large sclerotia, and increasing the incubation time caused a further reduction in the germination ability of the sclerotia. P. oligandrum was able to pass through its entire life-cycle from zoospores to oogonia both with sclerotia as sole nutrient-source and in water containing exudates from the sclerotia. The cell wall degrading enzymes N-acetyl--D-glucosaminidase (NAGase), endo-chitinase, protease, -glucanase, -glucosidase and cellobiohydrolase were detected in culture filtrates of P. oligandrum cultivated with S. sclerotiorum. These findings suggest that P. oligandrum has a potential to reduce the survival of S. sclerotiorum sclerotia present naturally in soils, through mycoparasitic activity.     

链孢粘帚霉寄生核盘菌菌核差异表达基因的筛选及分析

为克隆和研究链孢粘帚霉Gliocladium catenulatum寄生核盘菌菌核的相关基因,应用抑制消减杂交技术构建了cDNA消减文库并进行了筛选。通过PCR技术从文库中共筛选到1315个阳性克隆,克隆中插入片段大小主要集中于300～600bp之间。随机挑取120个克隆,经测序和同源性分析,获得60条有效序列,其中部分序列所编码的血红素加氧酶、核糖体蛋白L11、细胞色素P450及热激蛋白等均参与机体对胁迫条件的应答反应。11条序列在NCBI数据库中未找到显著匹配的序列,可能为新基因片段。分别将寄生于核盘菌菌核上的粘帚霉cDNA和粘帚霉与核盘菌纯培养的cDNA混合物经RasⅠ酶切后进行标记作为探针,利用反向Northern杂交技术验证了所选取的25条序列全部为差异表达基因片段。     

Effects of Irrigation and Tillage on Temporal and Spatial Dynamics of Sclerotinia minor Sclerotia and Lettuce Drop Incidence

ABSTRACT The temporal and spatial dynamics of Sclerotinia minor sclerotia and the resulting incidence of lettuce drop were studied under furrow irrigation with conventional tillage and subsurface-drip irrigation with minimum tillage during 1993-95. Lettuce crops were grown each year during the spring and fall seasons. All plants were inoculated immediately after thinning in the spring of 1993. Grids of 24 contiguous quadrats (1 by 1 m(2)) were demarcated in the centers of each 150-m(2) plot. Lettuce drop incidence in each quadrat was evaluated each season prior to harvest. One soil sample (100 cm(3)) was collected from each quadrat at harvest and after tillage prior to planting of the next crop for both spring and fall crops and assayed for S. minor sclerotia using wet sieving. Lloyd's index of patchiness, the beta-binomial distribution, and variance of moving window averages were used to evaluate the spatial patterns of sclerotia and lettuce drop incidence under the two irrigation systems and associated tillage treatments. Disease incidence remained significantly higher under furrow irrigation than under subsurface-drip irrigation throughout the study period, and was significantly higher on fall crops than on spring crops. Under furrow irrigation, the number of sclerotia at the end of a crop season increased significantly over that at the beginning of the season, but no significant changes were detected over years. In contrast, the number of sclerotia within a single season did not increase significantly under subsurface drip irrigation, nor was year-to-year accumulation of sclerotia statistically significant. The degree of aggregation of sclerotia increased significantly during a cropping season under furrow irrigation, but not under subsurface drip irrigation. The conventional tillage after harvest under furrow irrigation decreased the degree of aggregation of sclerotia after each season, but the distribution pattern of sclerotia under subsurface-drip irrigation changed little by the associated minimum tillage. Spatial pattern analyses suggested that the aggregation of S. minor sclerotia occurred at a scale of no more than 1 m, and distribution of diseased lettuce plants was random at a scale larger than 1 m. The combination of fewer sclerotia produced by each crop and its unaltered distribution under subsurface drip irrigation and associated minimum tillage makes it a valuable cultural practice for lettuce drop management.     

Analyses of Lettuce Drop Incidence and Population Structure of Sclerotinia sclerotiorum and S. minor

ABSTRACT To understand the geographical distribution of lettuce drop incidence and the structure of Sclerotinia minor and S. sclerotiorum populations, commercial lettuce fields were surveyed in the Salinas, San Joaquin, and Santa Maria Valleys in California. Lettuce drop incidence, pathogen species, and mycelial compatibility groups (MCGs) were determined and analyzed using geostatistic and geographical information system tools. Lettuce drop incidence was lowest in the San Joaquin Valley, and not significantly different between the other two valleys. Semivariogram analysis revealed that lettuce drop incidence was not spatially correlated between different fields in the Salinas Valley, suggesting negligible field-to-field spread or influence of inoculum in one field on other fields. Lettuce drop incidence was significantly lower in fields with a surface drip system than in fields with furrow or sprinkler irrigation systems, suggesting that the surface drip system can be a potential management measure for reducing lettuce drop. In the San Joaquin Valley, S. sclerotiorum was the prevalent species, causing drop in 63.5% of the fields, whereas S. minor also was identified in 25.4% of the fields. In contrast, in the Salinas Valley, S. minor was the dominant species (76.1%) whereas S sclerotiorum only observed in only 13.6% fields, in which only a few plants were infected by S. sclerotiorum. In the Santa Maria Valley, both species frequently were identified, with S. minor being slightly more common. Although many MCGs were identified in S. minor, most of them consisted of only one or two isolates. In all, approximately 91.4% of the isolates belonged to four MCGs. Among them, MCG-1 was the most prevalent group in all three valleys, accounting for 49.8% of total isolates. It was distributed all over the surveyed areas, whereas other MCGs were distributed more or less locally. Populations of S. sclerotiorum exhibited greater diversity, with 89 isolates collected from the Salinas and San Joaquin Valleys belonging to 37 different MCGs. Among them, the most recurrent MCG-A contained 16 isolates, and 30 MCGs contained only 1 isolate each. Many MCGs occurred within only one or a part of the two valleys. Potential reasons for this abundant diversity are discussed.     

Potential for Integrated Control of Sclerotinia sclerotiorum in Glasshouse Lettuce Using Coniothyrium minitans and Reduced Fungicide Application

ABSTRACT All pesticides used in United Kingdom glasshouse lettuce production (six fungicides, four insecticides, and one herbicide) were evaluated for their effects on Coniothyrium minitans mycelial growth and spore germination in vitro agar plate tests. Only the fungicides had a significant effect with all three strains of C. minitans tested, being highly sensitive to iprodione (50% effective concentration [EC(50)] 7 to 18 mug a.i. ml(-1)), moderately sensitive to thiram (EC(50) 52 to 106 mug a.i. ml(-1)), but less sensitive to the remaining fungicides (EC(50) over 200 mug a.i. ml(-1)). Subsequently, all pesticides were assessed for their effect on the ability of C. minitans applied as a solid substrate inoculum to infect sclerotia of Sclerotinia sclerotiorum in soil tray tests. Despite weekly applications of pesticides at twice their recommended concentrations, C. minitans survived in the soil and infected sclerotia equally in all pesticide-treated and untreated control soil trays. This demonstrated the importance of assessing pesticide compatibility in environmentally relevant tests. Based on these results, solid substrate inoculum of a standard and an iprodione-tolerant strain of C. minitans were applied individually to S. sclerotiorum-infested soil in a glasshouse before planting lettuce crops. The effect of a single spray application of iprodione on disease control in the C. minitans treatments was assessed. Disease caused by S. sclerotiorum was significantly reduced by C. minitans and was enhanced by a single application of iprodione, regardless of whether the biocontrol agent was iprodione-tolerant. In a second experiment, disease control achieved by a combination of C. minitans and a single application of iprodione was shown to be equivalent to that of prophylactic sprays with iprodione every 2 weeks. The fungicide did not affect the ability of C. minitans to spread into plots where only the fungicide was applied and to infect sclerotia. These results indicate that integrated control of S. sclerotiorum with soil applications of C. minitans and reduced foliar iprodione applications was feasible, did not require a fungicide tolerant isolate, and that suppression of Sclerotinia disease by C. minitans under existing chemical control regimes has credence.     

New Populations of Sclerotinia sclerotiorum from Lettuce in California and Peas and Lentils in Washington

ABSTRACT Four populations of Sclerotinia sclerotiorum in North America were inferred previously, based on analyses of both rapidly evolving markers (DNA fingerprint and mycelial compatiblity), and multilocus DNA sequence spanning the range between fast and slow evolution. Each population was defined as an interbreeding unit of conspecific individuals sharing a common recent ancestor and arising in a unique evolutionary event. The present study applies this standard to extend characterization of S. sclerotiorum populations to the Western United States. Isolates of S. sclerotiorum (N = 294) were determined to represent three genetically differentiated populations: California (CA, lettuce), Washington (WA, pea/lentil), and Ontario (ON, lettuce). CA was the most diverse population yet sampled in North America. Clonality was detected in ON and WA. No DNA fingerprints were common among the populations. The index of association (I(A)), based on fingerprint, was closer to zero (0) for CA than it was for the other populations. High diversity and lack of association of markers in California are consistent either with genetic exchange and recombination, or with large population size and high standing genetic variation. Intra- and interlocus conflict among three DNA sequence loci was consistent with recombination. The coalescent IGS genealogy confirmed subdivision and showed CA to be older than WA or ON. The Nearest Neighbor statistic on combined data confirmed subdivision among all present and previously defined populations. All isolates had both MAT1-1 and MAT1-2, consistent with uniform homothallism.     

核盘菌致病性分化研究

为了解核盘菌的致病性分化,本研究用活体定位穿刺接种法,以油菜为供试寄主,对采自四川省10个地区23个县、9种寄主的108个菌株进行了致病性测定,结果表明,所有菌株对供试油菜品种均能致病,但各菌株所致病斑长度差异很大(2.7～82.0 mm),说明核盘菌种群内存在明显的致病性分化,这种分化与地理来源和寄主来源没有明显的关系。     

高温低氧对菌核存活率和土壤微生物组成的影响

核盘菌Sclerotinia sclerotiorum可引起多种重要经济作物的菌核病,造成严重损失。降低土壤中菌核数量是防治该病害的关键。本研究分析了土壤类型、土壤温度、水分含量和氧气水平对核盘菌菌核萌发率的影响,并通过高通量测序分析了相应处理对土壤微生物组成及丰度的影响。研究发现湿润条件下,35℃低氧处理2~4周可导致土壤中核盘菌菌核100%死亡。测序结果表明,处理4周后土壤中微生物的群落结构发生了显著变化。其中15℃正常氧水平条件下,菌核周围木霉菌属Trichoderma的丰度显著增加,35℃正常氧水平芽孢杆菌属Bacillus和篮状菌属Talaromyces相对丰度显著提高,而低氧条件下狭义梭菌属Clostridiumsensu stricto 1、11、12丰度显著提高。这一发现为通过调控土壤微生态防控作物菌核病提供了依据。     

Predicting field diseases caused by Sclerotinia sclerotiorum: A review

Predicting diseases caused by Sclerotinia sclerotiorum in field crops remains difficult, and published literature is largely inconsistent in finding significant relationships with environmental and agronomic factors for various life stages of the fungus. A scoping review was performed to synthesize the current quantitative insights into the role of the environment on the life cycle of S. sclerotiorum and the relationships between various life stages of the fungus and final disease expression under field conditions. For most variables, relationships with stages of the life cycle showed a wide range of responses ranging from closely related (high correlations or r² values) to not related at all. The effects were often moderated by year, location and/or the presence of another variable such as irrigation, soil type, row spacing or cultivar. Studies that based analysis on a more nuanced understanding of pathogen biology rather than looking only at linear relationships tended to find stronger associations between variables. Yield was consistently negatively associated with disease levels, but cultivar, year, location and planting density were all important determinants of yield. Suggestions for improvement to future research in predictive model development of S. sclerotiorum diseases are discussed.     

A method for inducing apothecia from sclerotia of Sclerotinia sclerotiorum

An isolate of Sclerotinia sclerotiorum from oilseed rape was grown on sterilized wheat grain for 3 weeks at 20 C, followed by 4 weeks at 4^oC. Harvested sclerotia were buried 1 cm deep in compost in plastic containers and kept at 10^oC until apothecial stipes appeared (c. 6 weeks). When these dishes were placed under near-UV light (14 h/day) at 22^oC, apothecia matured in 5 days. The method also induced apothecia from sclerotia of 35 other isolates of S. sclerotiorum obtained from 17 different hosts.     

Studies on the germination of sclerotia of Sclerotinia sclerotiorum

Journal of Plant Diseases and Protection - Sclerotial germination of 33 isolates of Sclerotinia sclerotio-rum, which were collected from different regions and crops, was compared at 10†C in a...     

核盘菌对菌核净的抗药性机制初探

经药剂筛选获得对菌核净不同表型的核盘菌(Sclerotinia sclerotiorum ) 抗药性突变体。与敏感亲本菌株相比, 抗药突变体MN 61 (MR ) 和MN 91 (HR) 在含1% 和8% 葡萄糖的PDA 上生长受到抑制,MN 113 (LR) 只对1% 葡萄糖敏感。通过测定抗药突变体MN 61 和野生敏感菌株PN 061 的电导率, 发现抗药突变体能在更短的时间里渗出更多的电解质。抗药突变体苯丙氨酸解氨酶活性比敏感亲本菌株高出1 倍以上, 当用不同浓度菌核净处理或饥饿处理时, 抗药突变体和敏感亲本菌株PAL 活性均上升, 但抗药突变体的酶活始终高于敏感亲本菌株。     

A Glasshouse Cropping Method for Screening Lettuce Lines for Resistance to Sclerotinia Sclerotiorum

A soil-based glasshouse crop procedure was developed to screen lettuce lines for resistance to Sclerotinia sclerotiorum. Six sequential crops of 19 different lettuce lines with a range of cultural morphologies, reported previously to exhibit some form of resistance to S. sclerotiorum, were planted in a glasshouse infested with S. sclerotiorum and natural disease development compared with a standard susceptible commercial butterhead cultivar, Rachel. Concomitantly, the same lettuce lines were planted in pots in a nearby glasshouse, were artificially inoculated with ascospores of S. sclerotiorum, assessed for infection and scored for disease severity. Most of lines exhibited resistance in at least one of the crop or direct inoculation assessments with wild form, PI 251246, and stem lettuce, Taiwan, exhibiting resistance in three of the assessments and wild form, PI 271938 (Lactuca serriola), and Iceberg (crisp) line, 74-1076, exhibiting resistance in all four assessments. Cos line, PI 250427, was less resistant than the standard control in all assessments. The crop based screen with predictable, natural disease development was the most discriminating overall assessment and enabled growth habit to be taken into account during the screening process which was not possible through the direct inoculation procedures. Nevertheless, the novel ascospore inoculation screening process provided information on the type of resistance expressed that could not be identified from the cropping procedure.     

First record of Sclerotinia sclerotiorum on tree tomato in Ecuador

Cyphomandra betacea (tree tomato) plants with stem lesions were observed in an orchard in Tungurahua province, Ecuador. Dissection of the stems showed the presence of black sclerotia. Further laboratory analysis and pathogenicity tests revealed that Sclerotinia sclerotiorum was the causal agent of the disease. Although recorded in New Zealand, this is the first report of the pathogen affecting tree tomato in Ecuador.     

利用栽培措施控制油菜菌核病的综合研究

通过以3个甘蓝型油菜品种作为材料,采用田间自然鉴定方法,研究了不同播期和不同密度等栽培措施对控制油菜茵核病的作用.结果表明,在油菜生育期间,无任何药剂防治的条件下,品种和播期对油菜菌核病的影响均达显著水平,而密度对菌核病无明显影响,要与其他栽培条件,特别是氮肥的用量结合起来,才具有显著作用.因此,选用抗病性的品种和适当的晚播可有效控制或避开油菜菌核病的发生,合理密植可提高产量.     

粉红粘帚霉67-1菌株寄生核盘菌研究

 用诱捕法从海南乐东县菜园土壤中获得一株对核盘菌菌核具有强寄生能力的粉红粘帚霉(Gliocladium roseum)菌株67-1,寄生频率为100%。该菌的PDA平板回接核盘菌菌核,一周后寄生率可达100%。对峙培养发现其对核盘菌有明显的抑菌带。保湿条件下,该菌孢子在24h内成功侵入核盘菌菌核。切片显微观察证明该菌能高效侵染菌核,造成组织溃解。寄生过程中菌核内蛋白组成发生明显变化。这一菌株在22~35℃均能很好生长,菌丝及产孢最适温度为24℃,产孢量大。认为这一菌株具有良好的菌核病生防应用潜力。