The Nordic Baltic forum for plant health diagnostic laboratories and its use of ‘on‐site’ proficiency testing to improve laboratory testing1

During an Estonia‐UK‐Denmark twinning project, a new forum for Plant Health Diagnostic Laboratories in the Nordic‐Baltic region emerged. At the first meeting held in Estonia in July 2006, a new approach to proficiency testing was evaluated. Proficiency tests are key elements assessing the ability of a laboratory to perform a certain test in comparison with other laboratories. ‘On‐site’ proficiency testing means that experts are meeting to look at exactly the same samples under the same conditions on the same day. This is especially relevant for tests based on morphological interpretation as this minimizes the inherent differences in samples due to natural variation within a given species. The benefits of ‘on‐site’ proficiency testing outweigh the additional costs of organising a regional meeting which might include discussion of related scientific topics. Diagnostic capacity is an essential part of European Plant Health policies but staff may be spread out in different laboratories and countries. ‘On‐site’ proficiency testing helps create a stronger group identity for diagnosticians as well as maintaining quality control. In the plant health field there are also several difficulties in organising conventional proficiency tests. This is further discussed in the paper.     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.     

A laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris

BACKGROUND: This study was undertaken to identify any potential adverse side effects of the use of seven microbiological control agents (MCAs) on the bumblebee, Bombus terrestris L., in the context of combined use in integrated pest management (IPM). AQ10^® (Ampelomyces quisqualis), Binab‐T‐vector^® (Hypocrea parapilulifera + T. atroviride; 1/1), Prestop‐Mix^® (Gliocladium catenulatum J1446), Serenade^® (Bacillus subtilis QST713), Trianum‐P^® (Trichoderma harzianum T22), Botanigard^® (Beauveria bassiana GHA) and Granupom^® (Cydia pomonella granulovirus), comprising five biofungicides and two bioinsecticides, were investigated. Bumblebee workers were exposed under laboratory conditions to each MCA at its maximum field recommended concentration (MFRC) via three different routes of exposure: dermal contact and orally via either treated sugar water or pollen. RESULTS: The tested MCAs were found to be safe for workers of B. terrestris, with the exception of Botanigard^® and Serenade^®. Exposure to Botanigard^® via contact at its MFRC caused 92% mortality after 11 weeks, while the 1/10 MFRC killed 46% of exposed workers. For Serenade^®, topical contact and oral delivery via sugar water resulted in 88 and 100% worker mortality respectively. With lower concentrations (1/2, 1/5 and 1/10 MFRC) the toxicity decreased, but the effect depended on the route of exposure. In addition to lethal effects, nests were also evaluated for sublethal effects after treatment with the seven MCAs at their respective MFRCs over 11 weeks. In these bioassays, only Botanigard^® and Serenade^® gave rise to a significant (P < 0.05) decrease in drone production. Sublethal effects on foraging behaviour were also evaluated, and only Botanigard at its MFRC delivered via treated sugar water induced negative effects. CONCLUSION: The results demonstrated that most of the MCAs tested can be considered safe for use in combination with B. terrestris, based on the International Organisation for Biological Control of Noxious Animals and Plants (IOBC) classification. However, some can be harmful, such as the biofungicide Serenade^® and the bioinsecticide Botanigard^®. Therefore, it is recommended that all should be tested before use in combination with pollinators. In this context, it is also advisable that these MCAs should be evaluated in more realistic field situations for the assessment of potentially deleterious effects on foraging behaviour. Copyright © 2009 Society of Chemical Industry     

Objective evidence for competence1

In the area of plant pest diagnostics, increasing numbers of laboratories apply for accreditation according to the standard ISO 17025 General requirements for the competence of testing and calibration laboratories, which guarantees the compliance with standard operating procedures and the technical competence of the staff involved in the tests. In this standard, each accredited laboratory is required to supervise their staff's skills. This article explains how different French accredited diagnostic laboratories interpret and apply this accreditation requirement.     

Diagnostic sensitivity and specificity of different methods used by two laboratories for the detection of Phytophthora ramorum on multiple natural hosts

Five detection methods were comparatively tested on putative Phytophthora ramorum field samples from 41 wild plant species. The tested methods included two culture‐based assays, a DAS‐ELISA‐based polyclonal assay, a nested PCR‐based assay, and a TaqMan real‐time PCR assay. Diagnostic values including sensitivity, specificity, positive predictive value and negative predictive value were calculated for each method. The effects of host species, seasonality and host location were analysed and compared between two laboratories. Significant effects of season, host species and laboratory were detected. It is concluded that a combination of either culturing and molecular diagnosis or of two molecular assays is the most promising approach to diagnose this pathogen. Based on the results of this and other studies, diagnosis should occur as much as possible during wet and warm periods favourable to the pathogen, and proficiency tests should be performed to compare results obtained with molecular approaches in different laboratories. Furthermore, length of time lapsed between sample collection and processing strongly affected the diagnostic sensitivity of culture‐based methods, and therefore needs to be taken into account when comparing results from different laboratories.     

Effects of juvenile hormone analog formulations on development and reproduction in the bed bug Cimex lectularius (Hemiptera: Cimicidae)

BACKGROUND: Bed bugs (Cimex lectularius L.) have become a common insect pest in urban areas and are often difficult to manage. Eradication is made more problematic by widespread insecticide resistance, raising interest in alternative control products. Juvenile hormone analogs (JHAs) such as methoprene and hydroprene are relatively harmless to non‐arthropods and have proved to be effective against other urban insect pests. Two JHA products (Gentrol^® and Precor^®, Central Life Sciences, Schaumburg, IL) were tested for efficacy against various bed bug stages as direct spray and as dry residue using three bed bug strains. RESULTS: At 1× and 2× the label rate, Precor^® [active ingredient (S)‐methoprene] had no significant effect on the development or fecundity of bed bugs. At 2× the label rate, confinement to residues of Gentrol^® [active ingredient (S)‐hydroprene] had no significant effect, but residues at 3× and 10× the label rate caused a reduction in fecundity and impaired development. Field strains were more susceptible to the reproductive effects of (S)‐hydroprene than a long‐maintained laboratory strain. CONCLUSIONS: While JHAs are attractive alternatives for pest management because of their inherent safety and distinct mode of action, these JHA formulations would have little impact on bed bug populations without relabeling to allow for higher application rates. Copyright © 2012 Society of Chemical Industry     

Suppression of the clubroot pathogen Plasmodiophora brassicae by plant growth promoting formulations in roots of two Brassica species

In this study, the ability of two organic plant growth stimulants, mainly based on algal extracts, amino acids and phosphonate, to reduce clubroot formation in the model plant Arabidopsis thaliana and two economically important Brassica species, Brassia rapa (Chinese cabbage) and Brassica napus (oilseed rape) was investigated. A commercial liquid (Frutogard^®) and a granulate (PlasmaSoil^®) formulation were used to find optimum conditions for both control of the pathogen and plant growth. Both formulations were able to significantly reduce gall formation after Plasmodiophora brassicae infection on Chinese cabbage, but PlasmaSoil^® gave better effects, possibly as a result of the continuous supply of the components to the soil. Individual components did not have the same effect. Clubroots on oilseed rape could also be reduced. In contrast, club formation was not reduced in the model plant Arabidopsis thaliana. This points to interesting differences in the induction of resistance in the different species. Cross‐sections of infected and treated roots stained for different macromolecules (callose, lignin, suberin) indicated differences in anatomy as a result of the two formulations. The results indicate an application for the granulate formulation PlasmaSoil^® in clubroot control.     

A new fire ant (Hymenoptera: Formicidae) bait base carrier for moist conditions

BACKGROUND: A new water‐resistant fire ant bait (T‐bait; cypermethrin 0.128%) consisting of dried distillers grains with solubles (DDGS) as a carrier was developed and evaluated against a standard commercial bait (Advion^®; indoxacarb 0.045%) under both laboratory and field conditions. RESULTS: When applying the normal T‐bait or Advion^® in the laboratory, 100% of Solenopsis invicta Buren worker ants were killed within 4 days. However, when the T‐bait and Advion^® were wetted, 70.6 and 39.7% of the ants were killed respectively. Under field conditions, dry T‐bait and dry Advion^® had almost the same efficacy against ant colonies. However, when T‐bait and Advion^® came in contact with water, the former's ability to kill S. invicta colonies in the field was only marginally reduced, while Advion^® lost virtually all of its activity. In addition, DDGS was also shown to be compatible with a number of other insecticides, such as d‐allethrin, permethrin and pyrethrin. CONCLUSION: Based on its properties of remaining attractive to the fire ants when wetted, combined with its ant‐killing abilities both in the laboratory and in the field, T‐bait is an efficient fire ant bait, especially under moist conditions. Copyright © 2010 Society of Chemical Industry     

Control of postharvest green mould of citrus by haliscosamine isolated from Haliclona viscosa sponge

The aim of this study was to evaluate the antifungal activity of haliscosamine isolated from Haliclona viscosa sea sponge from the Moroccan Atlantic coast against Penicillium digitatum, the causal agent of citrus green mould. The in vitro antifungal activity of haliscosamine, determined using the agar disc‐diffusion method, showed that haliscosamine and DESOGERME SP VEGETAUX^® (reference product) have fungicidal activity with inhibition diameters of 19 and 18 mm, respectively. Haliscosamine and a positive control (DESOGERME SP VEGETAUX^®) were tested in vivo in comparison with a negative control on orange fruits in normal storage conditions. This test showed that after four weeks of storage, the rate of decay of citrus fruits treated with haliscosamine or DESOGERME does not exceed 14%, whereas in the negative control the decay rate is 77%. The results of in vitro and in vivo tests indicate that haliscosamine and DESOGERME SP VEGETAUX^® have an important antifungal potential against P. digitatum and that haliscosamine (a natural product) may represent an alternative to the use of chemical fungicides.     

Evaluation of organically acceptable insecticides as stand-alone treatments and in rotation for managing yellowmargined leaf beetle, Microtheca ochroloma (Coleoptera: Chrysomelidae), in organic crucifer production

BACKGROUND: Yellowmargined leaf beetle, Microtheca ochroloma, is the most damaging pest of organic crucifer crops in the southern United States. Experiments were conducted over four growing seasons (2007–2010) in Alabama to evaluate some botanical and microbial insecticides approved by the Organic Materials Review Institute (OMRI) against M. ochroloma in organically grown crucifer crops. Insecticides evaluated included PyGanic^® (pyrethrum), Aza‐Direct^® (azadiractin), Entrust^® (spinosad), Mycotrol O^® (Beauveria bassiana strain GHA) and NOFLY^® [Isaria ( = Paecilomyces) fumosoroseus strain FE 9901]. Two experimental organic formulations, Tick‐Ex (Metarhizium anisopliae strain F52) and MBI‐203 (Chromobacterium subtsugae), and one non‐OMRI‐listed formulation, Novodor^® (Bacillus thuringiensis subspecies tenebrionis), were also evaluated. The insecticides were applied as stand‐alone treatments at recommended field rates on a weekly schedule. In 2010, some of the treatments were also evaluated in rotation/alternation with Entrust^®. Insecticide efficacy was determined by comparing densities of M. ochroloma larvae and adults and crop damage ratings in treated versus untreated turnip plots. RESULTS: Entrust^® consistently performed well in suppressing M. ochroloma adults, larvae and crop damage. PyGanic^® was the second best treatment. PyGanic^® or NOFLY^? can be applied in rotation with Entrust^® for effective management of M. ochroloma. CONCLUSION: Entrust^® applied weekly or in alternation with PyGanic^® or NOFLY^? provided acceptable control of M. ochroloma in organic crucifer production. Copyright © 2012 Society of Chemical Industry     

Comparison of different PCR tests to identify Monilinia fructicola

Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.     

Comparison of pheromone lures used in mass trapping to control the tomato leafminer Tuta absoluta (Meyrick, 1917) in industrial tomato crops in Kairouan (Tunisia)

An experimental trial of integrated pest management against Tuta absoluta (Meyrick) based on mass trapping with a density of 32 traps per hectare was conducted in a 5 ha open‐field tomato crop. During this trial, the efficiency of three brands of sex pheromone dispenser commercialized in Tunisia was tested. The ‘superdosed’ pheromone lure type TUA‐Optima^® (Russell IPM, Deeside, Flintshire, CH5 2LA, United Kingdom) loaded with 0.8 mg of synthetic pheromone was more attractive to males of T. absoluta, and these lures should be used with high populations. Standard lures Pherodis^® and TUA‐500^® containing 0.5 mg of pheromone were less attractive, and can be used in open‐field tomato crops with lower populations of T. absoluta. An assessment of the susceptibility of crop borders to T. absoluta, based on data collected from traps, revealed that levels of adult T. absoluta were similar between borders with either tomato or cereal crops. Particular attention should thus be paid to crop borders even when not adjacent to other tomato crops.     

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop‐mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (as developed by Kogov?ek and colleagues), with proven applicability for rapid laboratory or on‐site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

Results and experiences from the first EU proficiency test for the detection of Phytophthora ramorum1

The Crop Protection unit of the Institute for Agricultural and Fisheries Research offers plant disease diagnostic services through its ‘Diagnostic Centre for Plants’ (ILVO‐DCP). ILVO‐DCP has requested accreditation (ISO 17025) for a number of diagnostic detection methods in bacteriology, mycology, entomology and nematology. Accreditation forms an essential part of quality control programs such as ISO17025, which can in part be realized by proving the laboratory's competence in inter‐laboratory proficiency or ring tests. In 2006, ILVO‐DCP organized such a proficiency test for the detection of Phytophthora ramorum. The protocol was developed using the standard ILAC‐G13:2000, and defined rules for participation, sample preparation and transport, communication, fraud prevention and reporting. Eight European diagnostic centres participated in the proficiency test including ILVO‐DCP. Each participant received one set of 10 coded samples, each sample consisting of either leaves or stems that were artificially inoculated with either the target or an alternative organism. Participants could use one or more methods listed in the EPPO diagnostic protocol PM 7/66. They had to report within a specific timeframe and received a detailed report of their performance. The success rate of the proficiency test was 100%. This paper lists some of the experiences gained from organising this type of proficiency test.     

The toxicity of cyromazine to Chironomus zealandicus (chironomidae) and Deleatidium sp. (leptophlebiidae)

The toxicity of cyromazine and a commercial formulation, ‘Vetrazin’®, to Chironomus zealandicus (thummi) Hudson and Deleatidium sp. was investigated. Under acute test conditions, the LC₅₀ values for each species were quite comparable. For C. zealandicus, the value varied according to instar, 100–400 mg litre^?1 for second- and third-instar to 1000–10000 mg litre^?1 for older fourth-instars. For the one size class of Deleatidium tested (c.10 mm long), the value was 300–400 mg litre^?1. High control mortalities of C. zealandicus limit that species' usefulness as an acute bioassay candidate. Under chronic test conditions, cyromazine showed a high toxicity to eggs or early-instar larvae of C. zealandicus. The maximum acceptable toxicant concentration for cyromazine against C. zealandicus was approximately 17.5 μg litre^?1. The possibility of water contamination at this level is discussed. Whole-of-life chronic tests with C. zealandicus indicated that the most susceptible stage was in the egg or soon after larval emergence. These results highlight the dangers of using short-term acute toxicity results to formulate environmental exposure limits for modern pesticides that do not have dysfunction of the nervous system as their mode of action.     

Biological control of botrytis bunch rot in organic wine grapes with the yeast antagonist Candida sake CPA‐1

The aim of this research was to confirm the efficacy of the yeast antagonist Candida sake CPA‐1 in suppressing botrytis bunch rot development, in an organic vineyard under Mediterranean conditions for two seasons, and compare its performance with that of two biologically based products currently registered for botrytis bunch rot control in New Zealand. In 2009, treatments applied were: commercial formulations of Ulocladium oudemansii (BOTRY‐Zen^®) and chitosan (ARMOUR‐Zen^®), C. sake CPA‐1 combined with the fatty acid‐based additive Fungicover^® and combinations of these products. All treatments were applied six times between early flowering and harvest and compared with an unsprayed control. In 2010, the treatments focused on C. sake and Fungicover and the number of applications was reduced from six to four. The population dynamics of U. oudemansii and C. sake were measured and wine quality tests were carried out in both seasons. Disease control achieved by C. sake treatments in 2009 were comparable to those achieved by BOTRY‐Zen and ARMOUR‐Zen. Applications of C. sake plus Fungicover between flowering and harvest significantly (P < 0·05) reduced botrytis bunch rot incidence and severity by 64% and 90%, respectively, compared with the untreated control in 2009, and by 67% and 89%, respectively, in 2010. Treatments did not adversely affect wine quality parameters after treated grapes were processed. Candida sake consistently provided effective control of botrytis bunch rot in grapes under different meteorological and disease pressure conditions, thereby improving its potential for future commercial applications.     

粉锈宁和羟锈宁拌种防治玉米丝黑穗病几种影响药效和药害的因子研究

粉锈宁和羟锈宁拌种防治玉米丝黑穗病的田间试验表明,墒情影响药效,墒情好,药效高。正常低土温和正常高墒情相结合,可以达到既压低发病基数,又获得高防效的双重目的。粉锈宁和羟锈宁的防效是近似的。墒情对羟锈宁的影响似更敏感,其防效的稳定性较粉锈宁差,但羟锈宁对幼苗的抑制轻,在逆境下造成的药害小。两种药剂的胶悬剂并不比可湿性粉剂优越。乳剂防效稍高,但药害较重。与促进生长的激素浸种结合应用有降低药效的趋势,不宜采用。室内试验证明,过饱和墒、过低土温和厚复土三者结合是田间按常用量拌种有时造成药害的原因。药害主要发生在干种至种子开始萌动阶段。玉米自交系的抗药性比杂交种差,应用药剂拌种时尤需慎重。     

Effects of some conventional insecticides and insect growth regulators on different phenological stages of the white wax scale,Ceroplastes destructor Newstead (Hemiptera: Coccidae), and its primary parasitoid,Aprostocetus ceroplastae (Girault) (Hymenoptera: Eulophidae)

The toxicity of two juvenile hormone analogues, pyriproxyfen (Nemesis^®) and fenoxycarb (Insegar^®), and two contact insecticides, methomyl (Lannate) and methidathion (Ultracide), was evaluated against immature stages (LI, LII, LIII) of Ceroplastes destructor Newstead in the field. The effects of these chemicals and one moulting inhibitor, triflumuron (Alsystin^®), and three insecticides: methyl-parathion (Penncap-M), profenofos (Selecron) and prothiofos (Tokuthion), on Aprostocetus (= Tetrastichus) ceroplastae (Girault) were assessed in the laboratory. Development of the first and second instar nymphs of C. destructor was completely arrested by the chemicals. Less than 1% of scales sprayed with pyriproxyfen at LII stage survived to adult female. Survival to the adult stage varied significantly between chemical treatments, and between chemicals and untreated controls for scales sprayed at the LIII stage. Female fecundity, fertility and body sizes of survivors of treatments applied at the LIII stage were not significantly affected by any of the chemicals. All the chemicals exhibited high toxicity to A. ceroplastae. Only triflumuron was slightly harmful, while methomyl was the most toxic (harmful), causing 100% mortality in the first 30 min after treatment. Although all the chemicals evaluated had effectively arrested the first and second instars of C. destructor, none of them exhibited sufficient selectivity to A. ceroplastae to warrant recommendation for integrated management of C. destructor in citrus orchards in South Africa, where A. ceroplastae plays an important role.