Systemic applications of neem in the control ofCameraria ohridella, a pest of horse chestnut (Aesculus hippocastanum)

Systemic injection of neem (azadirachtin) was tested in the control of horse chestnut leafminer,Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae), a pest of horse chestnut trees (Aesculus hippocastanum L.: Hippocastanaceae). Three concentrations, 0.08, 0.15 and 0.25 g of active ingredient (a.i.) per cm of diameter at breast height (dbh), provided long-lasting insecticidal activity. The 0.15 and 0.25 g a.i. concentrations resulted in 100% pupae reduction in all generations; the 0.08 g a.i. concentration was effective against the horse chestnut leafminer for at least 23 weeks and led to 70–80% pupae reduction. Treated trees did not lose their leaves before winter. Neem applicationvia injection is cheap, with no environmental hazards, and thus this treatment is recommended for the control ofC. ohridella in horse chestnut trees. http://www.phytoparasitica.org posting Dec. 19, 2004.     

Analysis of the natural infection of European horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi

Pseudomonas syringae pv. aesculi (Psa) is an emerging bacterial pathogen responsible for a recent epidemic of bleeding canker of European horse chestnut (Aesculus hippocastanum) in northwest Europe. Very little is known about the infection biology of this pathogen, which can cause lethal cankers in the branches and stem of its host. In this study, branches and whole trees of European horse chestnut naturally infected with Psa were subjected to detailed morphological and histological examination to identify the primary infection sites, the time of infection, and the patterns of subsequent lesion expansion within the host. Lesions developed during the host dormant season on the 2003–2009 extension growth increments and were centred mainly on lenticels, leaf scars and nodes. The oldest lesion developed in the 2004/2005 dormant season and the number of new lesions increased in each subsequent year. The lesions developed in the cortex and phloem and extended into the cambium to cause cankers, but there was no evidence of necrosis in the xylem. All lesions on the branches were discrete and apparently contained by a necrophylactic periderm, although there was evidence that Psa could survive within such periderms and subsequently breach them. Examination of two whole 30‐year‐old trees revealed extensive, continuous cankers in the phloem and cambium which had formed within a single growing season. Thus, the success of Psa as a tree pathogen and the causal agent of a large‐scale epidemic may in part reflect an ability to infect the aerial woody parts of its host directly.     

Survival,cold tolerance and seasonality of infection of European horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi

Bleeding canker of European horse chestnut, caused by the pathogenic bacterium Pseudomonas syringae pv. aesculi (Pae), is now an established disease throughout several countries in northwest Europe after first emerging in 2001–2002. Pae infects the woody branches of horse chestnut directly via natural discontinuities in the bark, such as lenticels, leaf scars and leaf traces, and nodes. However, the timing of infection in relation to seasonality of host development, and the dispersal mechanisms of Pae, in particular its ability to survive and spread in soil and water, remains unknown. In this study, infection of freshly cut horse chestnut shoots by Pae was assessed at monthly intervals over a 12 month period. Pae infected the greatest numbers of lenticels and leaf scars of horse chestnut when inoculated onto actively elongating shoots in spring and early summer, whereas lesion extension from artificial wound sites was greatest after inoculation during early dormancy. Soil survival experiments showed that Pae was still detectable and viable after 50 weeks' incubation in sterile soil and 41 weeks' incubation in nonsterile soil in the absence of host debris. Pae also remained viable and pathogenic after 1 year's storage in King's B broth at ?20 and ?80°C, and was not killed by freeze/thaw treatments. Thus Pae is able to survive independently for extended periods in soil and water, and can tolerate lengthy periods of freezing at very low temperatures. Such information may facilitate a greater understanding of the epidemiology and spread of Pae in northern Europe.     

Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real-time PCR

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.     

Aesculus pavia foliar saponins: defensive role against the leafminer Cameraria ohridella

BACKGROUND: Recently, the leafminer Cameraria ohridella Deschka & Dimic has caused heavy damage to the white‐flowering horse chestnut in Europe. Among the Aesculus genus, A. pavia L. HBT genotype, characterised by red flowers, showed an atypical resistance towards this pest. Its leaves, shaken in water, originated a dense foam, indicating the presence of saponins, unlike the common horse chestnut tree. The aim was to isolate and identify these leaf saponins and test their possible defensive role against C. ohridella. RESULTS: Spectroscopic analyses showed that A. pavia HBT genotype leaves contained a mixture of saponins, four of which were based on the same structure as commercial escin saponins, the typical saponin mixture produced by A. hippocastanum and accumulated only within bark and fruit tissues. The mixture showed a repellent effect on C. ohridella moth. The number of mines detected on the leaves of A. hippocastanum plants treated with A. pavia HBT saponins through watering and stem brushing was significantly lower than the control, and in many cases no mines were ever observed. CONCLUSION: The results showed that the exogenous saponins were translocated from roots/stem to the leaf tissues, and their accumulation seemed to ensure an appreciable degree of protection against the leafminer. Copyright © 2010 Society of Chemical Industry     

Bleeding canker of horse chestnut (Aesculus hippocastanum) in Ireland: incidence,severity and characterization using DNA sequences and real‐time PCR

A survey of bleeding canker disease, caused by Pseudomonas syringae pv. aesculi, was undertaken across Ireland. Incidence has become severe and can be considered epidemic, as 61% of the 1587 horse chestnut trees surveyed showed symptoms of the disease. Bacteria were isolated from a sample of trees and characterized using gyrBDNA sequencing. DNA was also extracted directly from wound tissue. The Irish P. syringae pv. aesculi genotype was identical to genotypes previously sequenced with gyrB from the UK and some other locations in Europe. Real‐time PCR, using existing primers and a newly designed, more pathovar‐specific primer set, was assessed for use in disease screening. With molecular screening, a total of 11 trees from a sample of 55 tested positive for P. syringae pv. aesculi in Ireland. It was more efficient to extract DNA directly from wound tissue, especially fresh bark, for disease detection than to undertake bacterial isolation with subsequent molecular analysis. A further set of sequencing primers was developed for the amplification of the gyrB gene from P. syringae pv. aesculi and their specificity was shown using a diverse sample of bacterial isolate DNAs. The study also isolated and identified other bacterial species from diseased material; some of these are known pathogens (Brenneria nigrifluens, P. marginalis and P. syringae) or have previously been identified as potentially beneficial endophytes of host trees (Erwinia billingiae, E. tolentana, P. fluorescens, P. putida and Raoultella).     

Aqueous solubilities,photolysis rates and partition coefficients of benzoylphenylurea insecticides

The aqueous solubilities and octanol–water partition coefficients (K_OW) of the benzoylphenylurea (BPU) insecticides teflubenzuron, chlorfluazuron, flufenoxuron and hexaflumuron were determined in comparison with the more extensively studied diflubenzuron. Both teflubenzuron and hexaflumuron were substantially less water‐soluble (9.4 (± 0.3) µg litre⁻¹ and 16.2 (± 0.5) µg litre⁻¹ in water, respectively) than the value previously reported for diflubenzuron (89 (± 4) µg litre⁻¹ in water). Log K_OW values for diflubenzuron, teflubenzuron, hexaflumuron, flufenoxuron and chlorfluazuron were 3.8, 5.4, 5.4, 6.2 and 6.6, respectively, as determined using reverse‐phase HPLC. Photodegradation of hexaflumuron, teflubenzuron and diflubenzuron in water indicated hexaflumuron to be the most rapidly degraded of the three compounds at pH 7.0 (t_1/2 = 8.6 (± 0.4) h) and pH 9.0 (t_1/2 = 5 (± 1) h); diflubenzuron was the slowest of the three pesticides to degrade in pH 7.0 (17 (± 4) h) and pH 9.0 (8 (± 2) h) buffered water. In a solar simulator using river water buffered to pH 9.0, teflubenzuron, hexaflumuron and diflubenzuron half‐lives were 20 (± 4), 15 (± 2) and 12 (± 1) h, respectively; dark controls showed no loss of parent BPU over similar time periods. © 2000 Society of Chemical Industry     

The effect of diflubenzuron upon larvae of the geometrid Boarmia (Ascotis) selenaria on avocado leaves

Small (70 mg) Boarmia selenaria larvae fed for 4 days on avocado leaves or alfalfa dipped in aqueous diflubenzuron suspensions suffered from severe developmental disturbances. Similar results were obtained with leaves sprayed in an avocado orchard, with which, in addition, the considerable persistence of diflubenzuron under field conditions could be demonstrated. The substance was also active by topical application against large (500–600 mg) B. selenaria larvae, whereas by contact it was only moderately toxic. Some other non-conventional control agents, viz., the antifeedant AC-24055 and several juvenile hormone analogues, were of medium and negligible activity, respectively, against this insect.     

Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β-tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov.     

The inhibition of chitin synthesis in spodoptera littoralis larvae by flufenoxuron,teflubenzuron and diflubenzuron

The chitin precursor [¹⁴C] N-acetylglucosamine injected into the haemolymph of Spodoptera littoralis (Boisduval) larva was incorporated into the chitin exponentially with time. When caterpillars were injected with precursor at the commencement of feeding on acylurea-treated leaf discs, flufenoxuron, teflubenzuron and diflubenzuron were found to be equally effective inhibitors of chitin synthesis, measured after 21 h. The dose response curves by feeding are not parallel, indicating that the relative potency of the compounds will vary across the dose range. When chitin precursor was injected simultaneously with topically applied diflubenzuron, flufenoxuron or teflubenzuron, all three acylureas were found to be equally effective as inhibitors of chitin synthesis when measured after five hours. The I₅₀values (50% inhibition of chitin synthesis) were not significantly different; average 600 ng, compared with LD₅₀values (50% lethal dose) of ～ 13 ng for flufenoxuron and teflubenzuron but 130 ng for diflubenzuron (topical application). Injection of precursor 24 h after topical application of insecticide gave an I₅₀value which had dropped 670- and 150-fold for flufenoxuron and teflubenzuron respectively but only 20-fold for diflubenzuron. It is postulated that the reason for the low increase in diflubenzuron effectiveness with time was due either to less diflubenzuron than flufenoxuron reaching the site of action, or more probably, a faster rate of metabolism and excretion for diflubenzuron. The lower toxicity of diflubenzuron compared with flufenoxuron and teflubenzuron may not be due to any inherent differences in biochemical effectiveness, but rather to different penetration/metabolism properties.     

Entomopathogenic fungi and nematodes against larvae of the chestnut weevil,Curculio elephas (Coleoptera: Curculionidae): a laboratory evaluation

Pest control in chestnut orchards is mainly achieved by chemicals, but there is growing focus on the potential use of biological control agents. The larval susceptibility of the chestnut weevil Curculio elephas L. (Coleoptera: Curculionidae) to different strains/species of entomopathogenic fungi and nematodes was evaluated in laboratory by soil cup bioassays. In the experiments with fungi, a wild strain of Metarhizium anisopliae caused the highest larval mortality (about 90%) compared with a commercial and a wild strain of Beauveria bassiana (80% and 77% respectively). Regarding nematodes, the commercial strain of Heterorhabditis bacteriophora was more effective in the control of chestnut larvae (77% insect mortality) than Steinernema carpocapsae (43%).     

Acute spread in France of an invasive pest, the horse chestnut leafminer Cameraria ohridellaDeschka & Dimic (Lep., Gracillariidae)

The horse chestnut leafminer, Cameraria ohridella Deschka & Dimic (Lepidoptera, Gracillariidae) spread over the last 10 years from central Europe to western Europe and was observed for the first time in 2000 in the east of France and near Paris. This study presents the results of surveys carried out in France in 2001 to investigate the distribution and infestation level of the leafminer. One year after its first discovery, the moth was present in a large part of the country and for the first time has been observed in Paris. The spread of the moth is due mainly to natural dispersal and to passive dispersal by human activities.     

Acute spread in France of an invasive pest, the horse chestnut leafminer Cameraria ohridellaDeschka & Dimic (Lep., Gracillariidae)

The horse chestnut leafminer, Cameraria ohridella Deschka & Dimic (Lepidoptera, Gracillariidae) spread over the last 10 years from central Europe to western Europe and was observed for the first time in 2000 in the east of France and near Paris. This study presents the results of surveys carried out in France in 2001 to investigate the distribution and infestation level of the leafminer. One year after its first discovery, the moth was present in a large part of the country and for the first time has been observed in Paris. The spread of the moth is due mainly to natural dispersal and to passive dispersal by human activities.     

Einsatz der Pheromonverwirrungstechnik zur Regulierung der Rosskastanien-Miniermotte Cameraria ohridella Deschka u. Dimić (Lep.: Gracillariidae)

The horse chestnut leafminer is an introduced invasive pest that every year causes extensive damage to the leaves of the white flowering horse chestnut tree. An environmentally friendly method to control insect pests involves the use of sex pheromones for mating disruption. A large quantity of artificial pheromone is released into the insect plant system confusing males to the extend that they are unable to locate receptive females. In order to test the potential efficacy of the mating disruption technique on the horse chestnut leafminer we set up semi-field trials with closed experimental units and varying leafminer densities. We counted the number of leaf mines on pheromone treated horse chestnut trees and compared them with an untreated control. Mating disruption lead to a significant reduction in damage by more than 90% in the first and second generation of the leafminer, irrespective of the initial leafminer density. Although these results are very encouraging, high population densities of the horse chestnut leafminer in nature and the patchy distribution of horse chestnut trees in the cities will challenge the efficacy of mating disruption in the field. Further experiments are planned with open experimental units and typical horse chestnut trees in various parks and gardens.     

A laboratory evaluation of the effectiveness of diflubenzuron against Dermestes maculatus De Geer and other storage insect pests

The chitin synthesis inhibitor diflubenzuron, applied as a wettable powder spray to woven polypropylene at 100–500 mg m^?2, was effective against Dermestes maculatus De Geer for at least 12 weeks. D. maculatus was unable to develop on ox hide dipped in a suspension of diflubenzuron (125 mg litre^?1), or on fishmeal dusted at 1–10 mg of active ingredient kg^?1. Diflubenzuron prevented the development of infestations of Callosobruchus maculatus (L.) on peas and of Acanthoscelides obtectus (Say) on beans dusted at 1–5 mg kg^?1. The compound was also very effective against early instar larvae of Trogoderma granarium Everts on wheat. The persistence and activity of diflubenzuron at low dosage rates against D. maculatus appear to justify larger scale trials.     

The effect of Cryphonectria parasitica on water relations of American chestnut

Chestnut blight has commonly been regarded as a phloem disease due to conspicuous stem cankers that result from infection by the fungal pathogen Cryphonectria (Endothia) parasitica. Stomatal conductance (g_s), transpiration rate (E) and leaf water potential were measured throughout the day on leaves distal to naturally-occurring virulent (sunken bark with abundant stromata) or hypovirulent (swollen bark lacking stromata) cankers and cankers induced by inoculation with virulent or hypovirulent strains of C. parasitica. Relative to control stems, hydraulic conductivity (K_h), g_s and E were reduced significantly (α = 0·05) for both natural virulent cankers, and cankers that were induced by a virulent strain. These effects were less pronounced for both natural and induced hypovirulent cankers. Isolation experiments revealed that the percentage of xylem tissue chips yielding C. parasitica was greater for virulent than for hypovirulent cankers. The data provide evidence that the localized presence of C. parasitica in cankers of American chestnut results in stomatal closure, possibly as a direct result of xylem dysfunction.     

Effects of buprofezin and diflubenzuron on various developmental stages of Asian citrus psyllid, Diaphorina citri

BACKGROUND: Diaphorina citri populations in Florida are developing resistance to commonly used neurotoxic insecticides. Alternatives to neurotoxins, such as insect growth regulators, are needed to control this season-long subtropical pest to prevent or delay development of insecticide resistance. In the present investigation, two insect growth regulators (IGRs), buprofezin and diflubenzuron, were evaluated against various developmental stages of D. citri. RESULTS: The 0–1-day-old D. citri eggs were more susceptible to buprofezin and diflubenzuron than the 3–4-day-old eggs. Adult emergence was completely suppressed by treating first- or third-instar nymphs with buprofezin or diflubenzuron at 30–240 or 23–184 µg mL⁻¹ rates respectively. Treatment of fifth-instar nymphs with diflubenzuron at a rate of 184 µg mL⁻¹ and with buprofezin at 30–240 µg mL⁻¹ rates resulted in approximately 20 and 15–80% reductions in adult emergence respectively. The mean number of eggs per plant was reduced at 5 days after topical treatment with diflubenzuron. Mean egg hatch per plant was reduced at 5 and 6–15 days after topical treatments with buprofezin and diflubenzuron respectively. CONCLUSION: Buprofezin and diflubenzuron effectively suppressed D. citri adult emergence. D. citri were more susceptible as early (first–third-instar) than late (fifth-instar) nymphs. Both IGRs inhibited egg production and egg hatch. Reduction in the number of subsequent offspring suggests reduced vertical transmission of Candidatus Liberibacter asiaticus, the pathogen thought to cause citrus greening disease. The present results indicate that both IGRs tested here should be effective tools for rotation in insecticide-based D. citri management programs. Copyright © 2012 Society of Chemical Industry     

The effect of diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on soybean [Glycine max (L.) Merr.] photosynthesis,respiration, and leaf ultrastructure

The effect of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on photosynthesis, respiration, and leaf ultrastructure of soybean [Glycine max (L.) Merr., cv. Swift] was examined on plants treated at the second trifoliate leaf stage with 0, 0.067, and 0.269 kg of active ingredien/ha of diflubenzuron. Photosynthesis and respiration were measured with an infrared CO₂ analyzer in an open flow system prior to diflubenzuron application and at 4, 24, 48, and 96 hr after treatment with diflubenzuron. Diflubenzuron had no effect on soybean photosynthesis at any rate examined. Respiration was stimulated by the high rate (0.269 kg/ha) in a transitory manner. Tissue samples removed from both old and new leaves, 9 days after diflubenzuron application, were used for the ultrastructure study with the transmission electron microscope. The lower trifoliate leaves contained more starch grains than the upper ones being formed after treatment, but no aberrations or degradation of leaf ultrastructure due to diflubenzuron treatment were evident.     

Assessing the potential distribution of insect pests: case studies on large pine weevil (Hylobius abietis L) and horse‐chestnut leaf miner (Cameraria ohridella) under present and future climate conditions in European forests

Forest insect pests represent a serious threat to European forests and their negative effects could be exacerbated by climate change. This paper illustrates how species distribution modelling integrated with host tree species distribution data can be used to assess forest vulnerability to this threat. Two case studies are used: large pine weevil (Hylobius abietis L) and horse‐chestnut leaf miner (Cameraria ohridella Deschka & Dimi?) both at pan‐European level. The proposed approach integrates information from different sources. Occurrence data of insect pests were collected from the Global Biodiversity Information Facility (GBIF), climatic variables for present climate and future scenarios were sourced, respectively, from WorldClim and from the Research Program on Climate Change, Agriculture and Food Security (CCAFS), and distributional data of host tree species were obtained from the European Forest Data Centre (EFDAC), within the Forest Information System for Europe (FISE). The potential habitat of the target pests was calculated using the machine learning algorithm of Maxent model. On the one hand, the results highlight the potential of species distribution modelling as a valuable tool for decision makers. On the other hand, they stress how this approach can be limited by poor pest data availability, emphasizing the need to establish a harmonised open European database of geo‐referenced insect pest distribution data.     

Impact of Cameraria ohridella on Aesculus hippocastanum growth and long-term effects of trunk injection with pesticides

Defoliation caused annually by Cameraria ohridella has a considerable impact on the general condition of horse chestnut trees. We investigated the consequences of tree endotherapy against this insect. Wood samples, leaves and insect specimens were collected ten years after the treatment (injection) with pesticides. Insect invasion was manifested in all investigated trees by distinct radial growth depression. Treatment resulted in a growth upturn, while non-treated trees maintained decreased growth. The overall foliage infestation level was lower in treated trees, but pupal mass and potential fecundity of insect females were not affected. Our study revealed a long-lasting, positive effect of endotherapy on trees’ appearance and growth.