Epidemiology of Grey Mould in Annual Waiting-bed Production of Strawberry

The epidemiology of Botrytis cinerea was studied in five annual strawberry crops using waiting-bed transplants, a system widely adopted in the Netherlands. On dead leaves of transplants the incidence of B. cinerea varied from 26.7% to 52.6%, but the leaf area with potential sporulation was low (3.5–15.6%). During each crop cycle, the availability of necrotic leaf substrate for spore production of B. cinerea was generally low and varied between seasons and with the quality of transplants. B. cinerea sporulated on a maximum of 15.5 cm² of leaf area per plant, measured as potential sporulation. The aerial concentration of B. cinerea conidia in untreated plots did not differ from the concentration in plots where all dead leaves had been removed, nor from the concentration at 25–50 m distance from the strawberry plots. B. cinerea incidence on flowers ranged from 5% to 96%, but no correlation was found with the potential spore production on necrotic leaves. Grey mould at harvest varied from 1.4% to 11.3% and was correlated with the average precipitation during the harvesting period but not with B. cinerea incidence on flowers. Post-harvest grey mould ranged from 2.1% to 32.6% and was correlated with petal colonisation by B. cinerea. The results suggest that in the annual cropping system with waiting-bed transplants, necrotic leaves are not a significant source of B. cinerea inoculum, unlike in other strawberry production systems. Therefore, control measures of grey mould in this annual system should focus on protection of flowers and young developing fruits, and not on the reduction of inoculum production on leaf debris.     

Fungicide resistance inBotrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain)

The severity of disease caused byBotrytis cinerea in strawberries is very high and chemical control is common practice; low residue levels of chemical products are required. Thus, it is important to be aware of the development of fungicide resistance in order to choose the best strategies of chemical control. In the present study we evaluated the response of 36B. cinerea isolates against eight different fungicides. The isolates were sampled twice, at the beginning and the end of the season, in 11 commercial strawberry fields located in the area of Huelva (Spain). In addition, two reference isolates, SAS56 and SAS405, were evaluated. The proportion of isolates resistant to benomyl was very high (86%). Resistance to dicarboximides was detected in 44% of the isolates and resistance to pyrimethanil in 25% of the isolates. Different degrees of sensitivity to captan and dichlofluanid were recorded. No resistance was found to diethofencarb plus carbendazim. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.     

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.     

番茄灰霉病菌对腐霉利的抗药性检测及生物学性状研究

采用区分剂量法,检测了来自福建省、上海市、辽宁省和内蒙古自治区不同采样点的番茄灰霉病菌Botrytis cinerea对腐霉利的抗性频率;选取未施药地区的敏感菌株,建立了灰霉病菌对腐霉利的敏感基线;比较了常年施药地区与未施药地区灰霉病菌的抗性频率差异,测定了施药10年以上地区番茄灰霉病菌的抗性指数;研究比较了抗、感菌株的生物学性状。结果表明:腐霉利对127株敏感菌株的平均EC50值为(0.31±0.08) μg/mL;供试4省市灰霉病菌对腐霉利均表现出了很高的抗性频率,其中内蒙古自治区和辽宁省采样点的抗性菌株频率高于90%;在施药历史超过10年的215株田间菌株中,抗性水平最高的菌株来自辽宁省,抗性指数为44.3;施药地区灰霉病菌的抗性频率均高于未施药地区;抗性菌株的抗药性可稳定遗传;供试11株抗性和敏感菌株在产孢量和孢子萌发率、菌丝生长速率、活体致病力等生物学性状方面的差异与其抗性水平之间无明显相关性。     

两种蚕豆赤斑病菌对常用杀菌剂的抗药性检测及比较

对采自浙江、湖北和安徽3省的蚕豆赤斑病样品进行了病原菌的分离和鉴定,采用菌丝生长速率法检测了引起赤斑病的2种病原菌——蚕豆葡萄孢Botrytis fabae和灰葡萄孢B.cinerea 的抗药性发生情况,并在离体条件下通过抗药性诱导试验比较了二者的抗药性风险。结果共分离得到153个菌株,其中蚕豆葡萄孢122株(占79.7%),灰葡萄孢31株(占20.3%)。共检测到37株多菌灵高水平抗药性菌株(其中蚕豆葡萄孢9株)和42株异菌脲低水平抗药性菌株(其中蚕豆葡萄孢17株);嘧霉胺对153个菌株的EC50值在0.01~5.13 μg/mL之间,平均EC50值为0.72±0.15 μg /mL;表明蚕豆赤斑病菌对常见杀菌剂已表现出一定的抗药性,且灰葡萄孢的抗药性问题比蚕豆葡萄孢要严重得多。抗药性诱导试验进一步证实,灰葡萄孢的抗药性风险明显高于蚕豆葡萄孢。     

Biological Control of Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×10³conidia ml⁻¹), but smaller lesions at high concentration (5×10⁴ conidia ml⁻¹ or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + benomyl (50 μg ml⁻¹) and benomyl (100 μg ml⁻¹) in 2003, and between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + vinclozolin (100 μg ml⁻¹) and vinclozolin (500 μg ml⁻¹) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.     

Field evaluation of a supervised control system forBotrytis leaf blight in spring sown onions in the Netherlands

Between 1988 and 1992 two existing weather based advisory models to control leaf blight in onions, were evaluated in the Netherlands. The first model, BOTCAST, can be used to time the initial spray while the second model, SIV, can be used to advise on every subsequent spraying. The evaluation based on field trials showed that application of both BOTCAST and SIV can reduce the number of sprays by 54% compared to a weekly spraying program without any yield loss or a higher disease severity. There was no relation between yield losses of untreated plots and disease severity expressed as lesion counts or leaf dieback. Relative disease growth rate was significantly but not closely related to weather based model characteristics or the observed crop micro-climate using linear regression analysis. The model characteristics did not yield better regressions than the climatological characteristics. Two changes to improve BOTCAST are proposed. Introduction of a supervised control system based on BOTCAST and SIV seems only economically feasible when the system is used as a regional warning system.     

N-(2-三氟甲基-4-氯苯基)-2-吡唑酰氨基环己烷基磺酰胺类化合物的合成与杀菌活性

吡唑酰胺类杀菌剂是近年新农药开发的热点。本研究采用EDCI/HOBt酰胺化法合成了14个结构新颖的N-(2-三氟甲基-4-氯苯基)-2-吡唑酰氨基环己烷基磺酰胺类化合物 ( 3a ~ 3n ),其结构均经1H NMR、13C NMR、质谱和元素分析确认,并用X-射线衍射法确定了化合物 3g 的单晶结构和立体构型。菌丝生长速率法试验结果表明,化合物 3a 、 3e 和 3j 对番茄灰霉病菌Botrytis cinerea KZ-9的EC₅₀值分别为4.28、10.08和11.31 mg/L,抑菌活性不及对照药剂啶酰菌胺和腐霉利;但在孢子萌发试验中,目标化合物表现出与对照药剂相近的抑菌活性,在10 mg/L下有7个化合物对灰霉病菌孢子萌发的抑制率超过了85%;在番茄活体盆栽试验中,化合物 3e 在200 mg/L下对番茄叶片及其花上灰霉病菌的防治效果分别为77.5%和65.2%,高于对照药剂啶酰菌胺 (防效分别为59.8%和30.3%),有进一步研究的价值。     

油菜菌核病抗(耐)病性筛选方法的研究

本文评价了不同接种鉴定方法在油菜菌核病抗(耐)病性筛选上的应用。结果表明:花期离体或活体叶片菌丝体接种和限期接种法能显著区分不同品种(系)的抗感水平;苗期菌丝体悬浮液接种和试管苗接种法适于早期育种世代进行大规模筛选。对83份品种(系)鉴定结果,821、083等呈轻度感病,大部分属中感至高感范围。     

Influence of calcium pretreatment on pectic substance evolution in cucumber fruit (cucumis sativus) duringbotrytis cinerea infection

The role of calcium in protecting plant tissue from infection byBotrytis cinerea was studied in cucumber fruit. A self-defense reaction, consisting of an increase in cell-wall-bound Ca, occurred in the plant tissue after an injury or an infection. However, it was not sufficient to protect the tissue from the spread of the fungus, which is accompanied by pectic substance digestion. The degradation induced mainly a decrease in acid- and chelator-soluble pectin levels, and an increase in water-soluble pectin. Ca treatment of the fruit prior to infection could amplify the cell-wall-bound Ca and therefore lessen pectin digestion by fungal pectinolytic enzymes.     

A new leaf blight disease of <Emphasis Type="Italic">Trifolium dasyurum</Emphasis> caused by <Emphasis Type="Italic">Botrytis fabae</Emphasis>

A new disease was observed on Trifolium dasyurum, with symptoms beginning as a halo spot and developing into a leaf blight. The causal organism was identified by microscopy and DNA sequence studies as Botrytis fabae. This strain of B. fabae was also demonstrated to cause disease on foliage of a range of pulse crops, including Vicia faba, Pisum sativum, and Lens culinaris. This study demonstrates the potential of this strain of B. fabae to not only pose a significant threat to T. dasyurum but also to pulses grown in rotation with T. dasyurum that are susceptible to this strain of B. fabae.     

不同覆盖处理对甘肃中部地区甘蓝型冬油菜越冬率及产量的影响

试验研究了地膜覆盖、土覆盖、农家肥覆盖处理对甘肃中部地区甘蓝型冬油菜的越冬率、生育期、产量的影响。试验结果表明,覆盖处理较露地栽培可缩短总生育期2~11 d,各生育时期也相应缩短。其中地膜覆盖效果最好,可缩短生育期11 d左右,农家肥覆盖与土覆盖处理分别可缩短生育期2 d与5 d。地膜覆盖、土覆盖、农家肥覆盖越冬率分别为95.45%、81.25%、80.77%,露地栽培越冬率为66.67%。覆盖处理较露地栽培的越冬率增加14.1~28.78个百分点。覆盖处理的株高、主花序长度、单株角果数、千粒重、单株产量以及角粒数等性状优于露地栽培。覆盖处理可提高产量,其中地膜覆盖产量达到5 358.3 kg/hm2,土覆盖为3 854.25 kg/hm2,农家肥覆盖产量为3 704.25 kg/hm2,分别较对照增产50.94%、8.57%和4.34%。覆盖对提高土壤温度有良好影响,无论地膜覆盖、土覆盖还是农家肥覆盖,土壤温度均高于露地栽培的处理。根据覆盖栽培越冬保苗效果、增产效果以及农事作业的可操作性,建议甘肃中部地区甘蓝型冬油菜栽培以地膜覆盖和土覆盖为主要覆盖方式。     

Methods for Sampling and Assessment in Relation to the Spatial Pattern of Phoma Stem Canker (Leptosphaeria maculans) in Oilseed Rape

A sound assessment of phoma stem canker symptoms is needed to develop epidemiological, agronomical and physiological studies on the pathosystem. A specific analysis was therefore carried out to: (i) compare four methods of crown canker assessment; (ii) test the among and within assessor repeatability of one of the methods compared; (iii) characterise the spatial pattern of the disease; and (iv) define the sample size required to achieve a given level of disease assessment precision. The methods compared examined the symptoms with different procedures and graded the plants observed into six severity classes. A disease index (DI) summarised the severity distribution observed. Examination of crown cross-sections was the most precise method for assessing crown cankers. The method was repeatable, though an 'assessor effect' was apparent. The disease generally had a random pattern although significant spatial correlations were detected for four out of the fifteen plots studied at the scales examined. A relationship between the coefficient of variation of the DI and the sample size was established, evaluated with experimental field data and exemplified for typical severity distributions.     

Relationships of aphid and mite infestations to control ofBotrytis cinerea byClonostachys rosea in rose(Rosa hybrida) leaves

Infestations of aphids(Macrosiphum rosae L.) and of twospotted spider mites(Tetranychus urticae Koch) were examined in relation to growth and sporulation ofClonostachys rosea andBotrytis cinerea, and to suppression of the pathogen by the agent, in green rose leaves. Leaves were infested artificially with 10 aphids/leaflet for 3 h, or naturally with 15-30 aphids/leaflet for 7-12 days or with undetermined numbers of mites for 10-12 days. Leaves that had or had not been infested were inoculated withC. rosea, withB. cinerea, or withC. rosea plusB. cinerea. Germination incidence and germ tube growth ofC. rosea andB. cinerea on the phylloplane in most instances were much greater in leaves previously infested with aphids or mites compared with noninfested leaves. After combined inoculation,C. rosea suppressed germination ofB. cinerea from 47% to 19% in noninfested leaves, but in leaves that had been infested the agent was ineffective and germination incidence of the pathogen increased to 75-93%. Previous infestation with naturally introduced aphids or mites, but not brief infestations of artificially introduced aphids, markedly increased sporulation ofC. rosea after the leaves died during an initial 7-15 days of incubation on a paraquat agar medium, regardless of whether or notB. cinerea was present. Sporulation ofB. cinerea was similarly increased when inoculated alone. After 15-20 days, however, conidiophores of the agent or pathogen covered most of the leaf surface in these treatments. In leaves inoculated withC. rosea plusB. cinerea, the agent suppressed sporulation of the pathogen almost completely in both previously infested and noninfested leaves. Thus, aphid and mite infestations did not compromise the ability ofC. rosea to suppress inoculum production byB. cinerea in the leaves. Increased nutrient availability on the phylloplane through exudation or as honeydew or frass is proposed as a basis to explain effects of the pest infestations onC. rosea andB. cinerea.     

Identification of Greenhouse Characteristics which Affect the Incidence of Sclerotinia sclerotiorum (Lib.) de Bary in Pepper Crops in a Mediterranean Climate

The effect of the main construction factors and design features of greenhouses on the incidence of white rot disease, caused by Sclerotinia sclerotiorum (Lib.) de Bary, was studied in peppers grown under plastic-roofed greenhouses typical of Almería (SE Spain). At the height of the pepper-growing season in the province, fifty greenhouses were repeatedly sampled. Incidence of the disease was measured and the different construction features of the greenhouses were evaluated: types of roof cover, colour and age of the plastic cover, dimensions (ground plan, surface area, average height), estimated ventilation capacity and the presence of netting on the sides of the greenhouses (laterals). Statistical analyses were undertaken to see if the choice of various design options for each factor had any significant influence on the incidence of the disease. To achieve this single factor ANOVA was undertaken. The incidence of this disease was significantly affected (p<0.05) by the following greenhouse characteristics: ground plan, surface area, mean height, colour of the plastic cover, estimated ventilation and use of lateral netting.     

Reactive oxygen intermediates and oxalic acid in the pathogenesis of the necrotrophic fungus Sclerotinia sclerotiorum

An effective colonization of the host plant tissue by the necrotrophic fungus Sclerotinia sclerotiorum requires the secretion of the non-host specific toxin oxalic acid (OA), which is known to suppress the generation of reactive oxygen intermediates (ROI). A full-length cDNA coding for an oxalate decarboxylase (TOXDC), which converts OA into CO₂ and formate, was isolated from the basidiomycete Trametes versicolor. It was overexpressed in tobacco plants to study the role of ROI and OA in the interaction between tobacco and S. sclerotiorum. The transgenic plants contained less OA and showed a delayed colonization of S. sclerotiorum; furthermore a strong ROI accumulation and nearly no catalase activity compared to the wild type (WT) plants could be detected. In addition, inoculation experiments with transgenic catalase-deficient plants (CAT1AS) and in vitro studies showed that S. sclerotiorum copes with strong ROI stress. Our results indicate that OA supports the infection process caused by S. sclerotiorum and the fungus itself is able to tolerate high ROI concentrations. The nucleotide sequence data is available from the NCBI Genbank nucleotide-sequence database under the number AY370675