EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 41

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Evidence Project Final Report

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Project identification

1. Defra Project code PH0437

2. Project title

Supporting risk management and risk assessment for invertebrate pests of statutory concern

3. Contractor organisation(s)

The University of Warwick

54. Total Defra project costs £ 257,440

(agreed fixed price)

5. Project: start date ................ 1 February 2010

end date ................. 31 July 2012

EVID4 Evidence Project Final Report (Rev. 06/11) Page 2 of 41

6. It is Defra‟s intention to publish this form.

Please confirm your agreement to do so. ........................................................................................................ YES

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Executive Summary

7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Two of the current main research priorities for Plant Health policy and operations are 1) development of management approaches for current or future outbreaks of quarantine or emerging pests and 2) improvement of risk assessments for emerging pests, by filling in biological gaps in Pest Risk Assessments. In particular, management approaches for controlling soil dwelling phases (life-stages) of specific quarantine pest groups or specific species is a key scientific need and operational gap in various cropping systems. In addition there are two specific areas of risk that require further investigation which are 1) a variety of specific emerging pests of solanaceous plants and crops which are of current concern. These include: Tuta absoluta (tomato leaf miner moth), Keiferia lycopersicella (tomato pin worm) which are both moths of concern to glasshouse-grown tomatoes in the UK, Tecia solanivora (potato moth) and Epitrix spp (flea beetles), both of which are of concern to potato crops; Aceria kuko (Gogi gall mite), Tetranychus evansi (a new red spider mite species) and Trialeurodes abutiloneus (banded-winged whitefly), which are currently of lesser concern and 2) bonsai plants and large tree specimens with quarantine soil-inhabiting nematodes and mealy-bugs. These represent an on-going issue since effective treatment approaches (active ingredients and appropriate application methods) are lacking, both prior to export from third countries or on arrival as an alternative to destruction. The overall aim of this project was to better inform risk assessment and risk management approaches for specific emerging pests of solanaceous crops and to develop or strengthen risk management methods for soil phases of invertebrate pest groups or species of statutory concern. The specific objectives were as follows:

1. Collate information on biology and control of pests of solanaceous crops.

2. Devise and undertake a programme of experimental work on moth pests of solanaceous

crops aimed at risk assessment, risk management and control.

3. Identify types of soil pests and soil „environments‟ for which further information about

control measures is required.

4. Review soil treatments for a range of pest groups that include taxa of statutory concern

and make recommendations for experimental work.

5. Devise and undertake a programme of experimental work on treatments to control soil

EVID4 Evidence Project Final Report (Rev. 06/11) Page 3 of 41

inhabiting pests of containerised plants.

6. For the work on solanaceous pests, information was collated on Keiferia lycopersicella,

Tecia solanivora and Tetranychus evansi. Experimental work was proposed and

undertaken on Tuta absoluta and Epitrix species. However, although work on K.

lycopersicella was proposed, it was not possible to source experimental material.

For T. absoluta, experiments were undertaken to evaluate survival of all development stages at low temperatures. In general, batches of insects in each stage of development (egg, larva, pupa, adult) were exposed either to -5

oC for short periods of time (1-5 days) or to 0

oC for longer periods of time (up to one

month). In general, all stages survived short periods of exposure to -5oC and longer periods of exposure

to 0oC and survival declined with duration of exposure. The precise relationship between survival and

temperature varied with development stage. There appeared to be no strong effect of pupal age on survival at low temperatures and there was no major difference in survival ability of pupae from larvae reared either at 20

oC with an18 hour day or reared at a lower temperature/short daylength (15

oC and 12

hour day). Thus although previous studies have shown that development of T. absoluta ceases at temperatures below 6-7

oC, each stage might be able to survive „short‟ periods of exposure to low

temperatures and then resume development if conditions became more favourable. This has implications for how UK tomato growers might manage the period between one crop and the next. Preferences of female T. absoluta for a range of plant species as oviposition sites were determined, making comparisons with tomato. Females laid eggs freely on potato and black nightshade (Solanum nigrum), pepper and Datura were not accepted as oviposition sites and Nicotiana and Petunia were less preferred. Tomato, potato and black nightshade were equally good host plants for supporting full development of T. absoluta. Larvae also mined Nicotiana foliage and were able to develop to adulthood. Black nightshade is a relatively common weed in the UK, but observations in this study indicated that the foliage died back in winter. None of the other host plants tested would be viable in a normal winter in the UK, probably succumbing to the first frost. Samples of two species of Epitrix (E. similaris and E. cucumeris), originating from Portugal, were obtained from the Food and Environment Research Agency (Fera) and placed on potato plants. Numbers of E. similaris increased sufficiently to undertake small scale experiments. Numbers of E. cucumeris remained low. Adult E. similaris were exposed either to -5°C for 1-5 days or to 0°C for 1- 4 weeks. Following exposure to the low temperatures, the beetles were placed at 20°C and survival was assessed. Survival decreased with increasing exposure time and virtually all the beetles were dead after 3 days at -5°C or two weeks at 0°C. Epitrix similaris adults were also used in insecticide tests. There were two sets of treatments, each of which had an untreated control and used deltamethrin as a standard. Potato leaves were dipped in solutions of the insecticides. The leaves were allowed to dry, placed in small containers and then adult beetles were added to the containers. Products either killed, or immobilised, the majority of beetles very rapidly (7) or were ineffective (2). This was also reflected in the numbers of feeding holes in the potato leaves. The work on soil pests focused on nematodes infesting bonsai plants. Bonsai plants were obtained from suppliers in the UK and a series of experiments was undertaken to investigate the between-pot distribution of nematodes, the within-pot distribution of nematodes, overwintering survival of nematodes, effects of root washing treatments to remove nematodes and dispersal of nematodes in the soil. First of all, plants were sampled to determine numbers and type/species of nematode in each pot. The plants were numbered so that they could be identified and re-sampled later. The samples consisted of compost and root material taken from the two ends of each (rectangular) pot. The spaces in pots were back-filled with clean compost. The samples were sent to Fera for analysis and identification. The plants were left to grow in a greenhouse and further samples of soil and root material were taken once the plants had grown a new root system. A range of nematode species were found in the samples and the distribution was far from uniform. There was some indication of differences between plant species and this may be because they originated from different locations before being potted up. Nematode numbers were much lower on the second sampling occasion, with the exception of the microbivorous nematodes. In a second experiment, fresh plants were sampled more intensively by dividing the soil/root ball into 9 sections and placing each section in a separate polythene bag. In general, if one of the 9 samples from a pot contained a significant number of nematodes of a particular group then the others did also. This suggests that it may be appropriate to take sub-samples when assessing plant for infestations, which might for example, consist of two samples taken from diagonally opposite ends of the pot. In most cases the „trunk‟ was in the centre of the pot, but there was no evidence that higher numbers of nematodes were found under the trunk. The aim of a third experiment was to determine how well the nematodes imported with bonsai plants might survive outdoor conditions in the UK during the winter; if for example, the pot was placed outside or discarded on a compost heap. For this experiment the plants were potted into larger

EVID4 Evidence Project Final Report (Rev. 06/11) Page 4 of 41

containers containing sandy loam soil and strawberry plants to provide an alternative host if the bonsai plants died (as might happen if the plants were put outside). There was a control treatment which contained strawberry plants alone. The plants were divided into groups and on 28 January 2011, one batch was placed outdoors, one batch of plants in the greenhouse with the stock plant material and one batch in a poly tunnel which was unheated. All containers were placed in large saucers/trays to ensure no liquid escapes. Loggers were used to record soil temperatures in the pots. The plants were left outside until March 2011 when the soil close to roots of the bonsai and strawberry plants was sampled and the nematodes identified and counted. The pots in the poly tunnel suffered the greatest fluctuation in temperature whilst the pots in the cold frame appeared to be buffered to a certain extent. The majority of plants kept in the poly tunnel or cold frame were dead. There was no evidence that nematode numbers were decreased by exposure to low temperatures. To evaluate root-washing as a method of removing nematode infestations, a sample of stock plant material was divided into two groups (one group to be an untreated control). Sub-samples of soil were taken from each plant for identification and counts of nematodes. The roots of the „treated‟ plants were washed (protocol supplied by Plant Health). The plants were re-potted and placed in the same greenhouse compartment as the untreated control plants. The plants were left to grow for 3 months before sampling the soil for nematode counts. There was good evidence from the data that root washing removed nearly all Helicotylenchus spp. (which are likely to be ectoparasitic or semi-endoparasitic) Information on other species (Pratylenchus spp. and Tylenchus sensu lato) was less conclusive as

numbers were low prior to root washing.

Finally, to examine the potential for dispersal of nematodes, individual bonsai plants were re-planted with all the soil from the original pot at one end of a 1 metre long plastic trough containing sandy loam soil. Strawberry plants were added (1 per trough) at different distances from the Bonsai plants e.g. 5 cm, 10 cm, 20 cm, 40 cm, 80 cm. The strawberry plants were separated from the bonsai plants with coarse mesh to allow the passage of free-living nematodes but prevent the roots of the plants growing together. The troughs were placed in trays in a greenhouse compartment and grown for 6 months. They were watered carefully using drip irrigation. Soil samples were taken from around the strawberry plants, placed in polythene bags and sent to Fera for nematode identification and counts. There are obviously a number of constraints when working with potential new pest species, one of which is that they must be confined to containment facilities, which limits the scope of experimental work, and secondly, it can be difficult to find and subsequently build-up sufficiently large populations from which to take samples for experimental work. However, despite these constraints, the experimental studies indicated that all the species investigated had some potential to survive „cold‟ conditions in the UK, although in some cases that would be for limited periods. The studies on Epitrix and nematode pests of bonsai indicated that relatively effective control methods were available (insecticidal and physical respectively). Parallel studies have indicated a potentially effective integrated control strategy for T. absoluta using a combination of a novel method of insecticide application and predators. Future studies should confirm the efficacy of the control methods for Epitrix species, identify alternative control methods for nematode pests of bonsai and other containerised plants and determine the overwintering potential of Epitrix species.

Project Report to Defra

8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:

the objectives as set out in the contract;

the extent to which the objectives set out in the contract have been met;

details of methods used and the results obtained, including statistical analysis (if appropriate);

a discussion of the results and their reliability;

the main implications of the findings;

possible future work; and

any action resulting from the research (e.g. IP, Knowledge Exchange).

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Introduction

Two of the current main research priorities for Plant Health policy and operations are: 1) Development of management approaches (eradication, containment and pest management methods) for current or future outbreaks of quarantine or emerging pests; 2) Improvement of risk assessments for emerging pests, by filling in biological gaps in Pest Risk Assessments, including knowledge related to pest management, thereby helping the development of policy. In particular, management approaches for controlling soil dwelling phases (life-stages) of specific quarantine pest groups (e.g. dipteran and lepidopteran leaf miners, thrips, Coleoptera, etc.) or specific species is a key scientific need and operational gap in various cropping systems. In addition there are two specific areas of risk that require further investigation. Firstly there are a variety of specific emerging pests of solanaceous plants and crops are of current concern. These include: a) Tuta absoluta (tomato leaf miner moth) and Keiferia lycopersicella (tomato pin worm) which are both moths of concern to glasshouse-grown tomatoes in the UK; b) Tecia solanivora (potato moth) and Epitrix spp (flea beetles), both of which are of concern to potato crops; T. solanivora also presents a considerable potential threat to [unchilled] stored potatoes; c) Aceria kuko (Gogi gall mite), Tetranychus evansi (a new red spider mite species) and Trialeurodes abutiloneus (banded-winged whitefly), which are currently of lesser concern. The second area that requires further investigation is bonsai plants and large tree specimens with quarantine soil-inhabiting nematodes and mealy-bugs. These pests represent an on-going issue since effective treatment approaches (active ingredients and appropriate application methods) are lacking, both prior to export from third countries or on arrival as an alternative to destruction.

The overall aim of the project was to better inform risk assessment and risk management approaches for specific emerging pests of solanaceous crops and to develop or strengthen risk management methods for soil phases of invertebrate pest groups or species of statutory concern. The specific objectives were as follows:

1. Collate information on biology and control of pests of solanaceous crops.

2. Devise and undertake a programme of experimental work on moth pests of solanaceous crops aimed at

risk assessment, risk management and control.

3. Identify types of soil pests and soil „environments‟ for which further information about control measures is

required.

4. Review soil treatments for a range of pest groups that include taxa of statutory concern and make

recommendations for experimental work.

5. Devise and undertake a programme of experimental work on treatments to control soil inhabiting pests of

containerised plants.

A project steering group was formed at the start of the project to provide guidance throughout the project and a

forum for discussion.

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Objective 1 Collate information on biology and control of pests of solanaceous crops. The steering group and project team discussed and prioritised the pests to form the focus of the work. The agreed focus was as follows:

Pest species Focus of work

Keiferia lycopersicella (tomato pin worm)

Focus information collection on this species with the aim of collating information for a Pest Risk Analysis (PRA). Scope out experimental work to fill in gaps once information collated.

Tuta absoluta (tomato leaf miner moth).

Propose work on survival at low temperatures and on alternative hosts.

Tecia solanivora (potato moth) Review newer literature and scope out experimental work based on topics for further work outlined in PRA.

Epitrix species (flea beetles) EPPO working group is developing a PRA. Once produced, produce a work plan (with advice from others) for the steering group to consider, taking account of the uncertainties and research gaps highlighted at the end of the PRA.

Aceria kuko (Gogi gall mite) Not high priority. Pathway now closed.

Tetranychus evansi Undertake review on more recent work. Re-visit gaps and produce an outline plan of work to address any gaps for steering group to consider.

Trialeurodes abutiloneus (banded-winged whitefly)

Lower priority as ornamental pest – hold in reserve.

Keiferia lycopersicella (tomato pin worm) A draft literature review/PRA was prepared. Keiferia persicella prefers a warm climate and has been found in Mexico, California, Texas, Hawaii, Cuba, Haiti, Bahamas and Florida. It is a sporadic pest in Georgia (Poe, 1999; Sparks & Riley, 2008). It has also been found in glasshouses elsewhere in the USA (Delaware, Mississippi, Missouri, Pennsylvania and Virginia) and Canada and it is possible for the pest to spread into nearby fields, although it cannot overwinter in cold climates (Fera, 2010). The first outbreak in Canada occurred in 1946 and it appeared in Ontario in 1991, since when it has been a recurrent pest (Ferguson & Shipp, 2009; Elmhirst, 2006). It is found in glasshouses in USA and Canada (Fera, 2010). Therefore it is possible that it could survive under protected environments in the UK unless careful hygiene practices are carried out between crops. Host plants are tomato, eggplant, potato, solanaceous ornamentals and weeds (Poe, 1999; Drees & Jackman, 1998; Sorenson, 2005). Pepper and tobacco are not thought to support the pest (Ferguson & Shipp, 2009). To date, in Europe, the only outbreak of K. lycopersicella was in Italy in 2008 from where it is thought to have been eradicated (Fera, 2010). Potential questions to address experimentally for this species are:

Survival at low temperatures - investigate survival of different life stages. In terms of temperature requirements, development in the laboratory from oviposition to adult required 456D° at a threshold temperature of 9.5°C and development in a greenhouse required from 444 to 495D° based on the 9.5°C threshold (Weinberg & Lange, 1980). The low temperature threshold was estimated at 11°C by Lin & Trumble (1985).

• Potential for survival outdoors on solanaceous weeds on which no information is available currently.

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• Efficacy of insecticidal control and novel insecticides. Potential control methods from the literature are summarised in Table 1.1

• Biological control approaches – also summarised in Table 1.1

Proposed work on Keifeira lycopersicella A programme of work on K. lycopersicella was proposed. This would consider: 1) potential for survival outdoors on solanaceous weeds; 2) efficacy of insecticidal control and novel insecticides and 3) biological control approaches. Professor John Trumble (University of California, Riverside) was contacted as a possible source of K. lycopersicella. He replied „We no longer have colonies of Keiferia. In fact, we have not seen the tomato pinworm in our crops in California for many years. The mating disruption technique appears to have seriously depressed populations. I will inquire of my colleagues in central California, but I suspect your best bet will be to contact some Mexican scientists. You might consider contacting Dr. Martin Aluja, the General Director of the Instituto de Ecología, A.C. in Xalapa, Veracruz, Mexico’. Dr Aluja was contacted and he sent out a general e-mail request to colleagues in Mexico in early December 2011 but there was no response. Tuta absoluta A literature search on T. absoluta was undertaken and gaps were identified relating to development/survival at low temperatures (as in winter in the UK) and survival on other potential hosts (weeds, potato volunteers). Potential control methods identified in the literature are shown in Table 1.2. In addition to these, the insecticide Spinosad was showing useful activity (R. Jacobson, personal communication). Experimental work was proposed under Objective 2.

Table 1.1 Potential control methods for Keiferia lycopersicella (tomato pin worm) identified in the literature. The pesticides in bold capitals are approved for use on tomato in the UK.

Insecticides

• ABAMECTIN • Cyfluthrin • Emamectin benzoate • Esfenvalerate • Fenpropathrin • INDOXACARB • LAMBDA-CYHALOTHRIN • Permethrin • PYMETROZINE • Rynaxypyr • SPINOSAD • Zeta-cypermethrin

Other methods

• Bt • Mating disruption • Egg parasitoids • Larval parasitoids

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Tecia solanivora (potato moth) The more recent literature on this species was reviewed. Tecia solanivora is a pest of potato tubers and is found in Central and South America. It requires high temperatures for survival and reproduction and this may limit its spread unless there is significant climatic

Table 1.2 Potential control methods for Tuta absoluta identified in the literature. The pesticides in bold capitals are approved for use on tomato in the UK.

Insecticides ABAMECTIN Acephate Azinphos-ethyl Beta cyfluthrin Cartap Cypermethrin DELTAMETHRIN Diflubenzuron Flubendiamide INDOXACARB LAMBDA-CYHALOTHRIN Lufenuron Methamidophos Permethrin Phenthoate Propiofos Teflubenzuron Triflumuron Biological controls Metarhizium BEAUVERIA BASSIANA BACILLUS THURINGIENSIS VAR. KURSTAKI NEMATODES (STEINERNEMA CARPOCAPSAE, STEINERNEMA FELTIAE) Parasitoids:- Trichogramma pretiosum TRICHOGRAMMA BRASSICA TRICHOGRAMMA EVANESCENS Dineulophus phthorimacae Bracon lulensis Bracon tutus Bracon lucileae Conura sp. Apanteles gelechiidivoris Predators:- Metacanthus tennulllus Nesidiocoris tenuris Macrolophus pyghaeus MACROLOPHUS CALIGINOSUS Brachygastra lecheguana Vicosa solution Eucalyptus extract Trichilia pallida extract Neem extract Other Gamma irradiation (eggs)

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warming in areas at present too cold to sustain the pest (Dangles et al., 2008). It was first reported in Ecuador in 1996 and, along with strict phytosanitary measures, the cooler, wetter climatic conditions in 1997 eradicated all signs of the pest. However, a survey in 2000 found that it was still widespread (EPPO, 2004). In 1999, it was reported from the Canary Islands where a severe outbreak occurred in Tenerife since when pheromone traps have been used to record moth numbers. The moths have since been found on Lanzarote, Gran Canaria, La Gomera and La Palma as well as along the entire northern side of Tenerife (EPPO, 2001; Carnero et al., 2008). Under Article of Regulation (EC NO 1857/2006), Spanish farmers were granted compensation by the European Union to cover phytosanitary measures necessary to eradicate T. solanivora found on the Canary Islands (JEU, 2009). The moths are nocturnal and only fly if the temperature is above 11°C. They can produce 10 generations per year at temperatures of 25°C (Bosa Ochoa, 2005; Notz, 2002; NPAG, 2000). Development of T. solanivora was studied at temperatures of 10, 15, 20, 25 and 30 °C and, as well as the rate of development, fertility was affected by temperature, 25°C being most favourable. All larvae were killed at 30°C. For oviposition, 15°C was the optimum (Notz, 2002). Data from Notz (2002) have been used to estimate low temperature thresholds for each stage (Figure 1.1; Table 1.3), which were close to 7°C.

y = 0.1527x - 1.1932

y = 0.8952x - 6.832

y = 0.3953x - 2.7691

y = 1.4873x - 10.989

y = 0.4356x - 3.6151

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35

Temperature°C

Pe

rce

nt

de

velo

pm

en

t p

er

day

egg-adult egg larva pre-pupa pupa

Linear (egg-adult) Linear (egg) Linear (larva) Linear (pre-pupa) Linear (pupa)

Figure 1.1 Linear fits relating development rate of Tecia solanivora to temperature – data from Notz, 2002.

Phytosanitary measures to control the spread of T. solanivora in the Canary Islands included the removal of infested tubers to controlled landfill sites and the selection of only unaffected tubers for storage (EC, 2009). Barragan et al. (2004) stressed the necessity for vigilance and stringent control measures to prevent further spread of the pest through Europe. Crop hygiene is important in control of this pest and storage <8°C will prevent reproduction (NPAG, 2000). Good soil preparation can destroy pupae and early stages of the moth and planting when conditions are cool and wet prevents attack. Burying seed tubers >5-10 cm and deep ridging prevents oviposition. Good irrigation to prevent cracks in the soil can remove access by moths to the tubers. Removal of plant residues from the field and protection of tubers from the moths can prevent carry over infestation as can removal of infested tubers from store. Potato stores should be thoroughly cleaned and disinfected (Palacios & Cisneros, 1996). The larvae can survive in tubers left in the ground or in store so it is important to destroy all unwanted tubers (Wale et al., 2008).

Table 1.3 Extrapolated low temperature thresholds for Tecia solanivora Egg 7.4 Larva 7.0 Pre-pupa 8.3 Pupa 7.6 Egg-adult 7.8

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Potential control methods identified in the literature are summarised in Table 1.4.

Potential questions to address experimentally for this species are:

• Control methods – are there effective treatments? • Overwintering – in field and in stores. Store temperature could be 7°C or lower depending on how long

potatoes are going to be stored • Dispersal – if there was an outbreak, could it be „isolated‟? • Role of solanaceous weeds

Tetranychus evansi A review of more recent work was undertaken. Tetranychus evansi is thought to have originated in South America but, as it is easily confused with other Tetranychus species, this may be incorrect (EPPO, 2007). It is now found in the USA, central America, Hawaii, parts of Africa, India, Spain, Portugal, France, Italy, Greece, Israel, Jordan and Taiwan (EPPO, 2007; Migeon, 2005; Tagore & Putatunda, 2004; Tsagkarakou et al., 2007). It was found on eggplant from Chiayi County, Taiwan in 2001 and on Solanum nigrum in Nantou County in 2002 (Ho, 2006). The distribution and spread is described by Migeon & Dorkeld (2007). A distribution map for T. evansi was produced by CABI in 2006. In Europe, the Canary Islands, France, Portugal and Spain are listed. In 2009, Migeon et al. produced a CLIMEX model to predict global spread of the mite. This shows that the entire Mediterranean region could be at threat from infestation and, as this is the main area for tomato and aubergine production, the threat is serious. However, low temperatures should limit its spread northwards. Boubou et al. (2009) found that mites from the Mediterranean countries (Algeria, France, Greece, Israel, Italy, Spain and Tunisia) fell into two groups using ITS sequencing. All specimens from France and Catalonia, Spain were in one group while the remainder came from a second group. In 2005, T. evansi was found in the UK on imports of aubergines from Kenya (CSL, 2008). T. evansi develops by arrhenotokous parthenogenesis where fertilised eggs produce diploid females and unfertilised eggs produce haploid males. There does not appear to be a diapause for these mites so they can reproduce throughout the year. However, the optimal temperature is 34˚C and the minimum is between 10-14˚C. Development time from egg to adult is 6-13 days at 25˚C and 80-250 eggs can be laid per female depending on temperature. Thus increase of the pest under favourable conditions is exponential. When populations are very high, the mites climb to the upper foliage and are spread by wind (EPPO, 2007). There are particular issues around the identification of this species. Tetranychus evansi varies from T. urticae with the orange-brown colour, absence of two dark dorsal spots, the arrangement of tarsal setae on first leg and the shape of the aedeagus. It has longer legs than many other Tetranychus species (Castagnoli et al., 2006; EPPO, 2007). Tetranychus marianae and T. takafuji are thought to be misidentifications and are actually T.

Table 1.4. Potential control methods for Tecia solanivora (potato moth) identified in the literature. The pesticides in bold capitals are approved for use on potato in the UK.

Insecticides

• RANGE OF PYRETHOIDS • Spinosad • Lufenuron • Indoxacarb • Chlorpyrifos • Beta-cyfluthrin • Baits with insecticides • Lure and kill - pheromones

Other methods

• Bt • Baculovirus • Granulosis virus • Parasitic wasps • Predatory bugs • Repellents • Talc • Cultural methods

EVID4 Evidence Project Final Report (Rev. 06/11) Page 11 of 41

evansi. It is not possible to distinguish the two species using the shape of the male empodium II but DNA sequencing and cross breeding prove that T. takafuji is a junior synonym of T. evansi (Ho, 2006; EPPO, 2007; Gotoh et al., 2009). Work has been done to identify T. evansi and T. urticae Koch using molecular techniques (Knapp et al., 2003) and to identify, and distinguish between, Panonychus citri and other Tetranychus species e.g. T. evansi, T. urticae, T. ludeni and T. turkestani (Abercrombie et al., 2009; Hurtado Ruiz et al., 2006; Tsagkarakou et al., 2007). Hurtado et al. (2008) found that PCR-RFLP analysis of ITS2 gave rapid results for field screening. In Japan, Ikeshima et al. (2009) identified T. takafujii from 11 congeneric species of mites using the gene normally used to distinguish T. evansi from T. urticae. Host plants include tomato, aubergine, potato, tobacco, okra and other vegetables, ornamentals and weeds e.g. Solanum nigrum, S. americanum and S. caroliniense. Damage on beans, Phaseolus vulgaris, has been reported from Africa; on peanuts in Mauritius and Brazil and on water melon in Spain (EPPO, 2007; Tsagkarakou et al., 2007). Ho (2006) reported that T. evansi had been found on Lycopersicon esculentum, S.melongena, S. aculeatissimum, Passifora fortida L. var. hispisa, Dioscorea alata, Pueraria sp., Ricinus communis and Aristolochia sp. Mainly it had been reported on S. nigrum. Migeon & Dorkeld (2007) have produced a list of host plants colonised by T. evansi. Potential control methods are listed in Table 1.5.

Potential questions to address experimentally for this species are:

• Identification – molecular or otherwise • Control methods – chemical or biological

Objective 2 Devise and undertake a programme of experimental work on moth pests of solanaceous crops aimed at risk assessment, risk management and control. Experimental work on Tuta absoluta A protocol was written describing experimental work on Tuta absoluta to determine whether any of the developmental stages will survive periods of prolonged cold at 0

oC and short periods of exposure to temperatures

below 0oC. Further experiments were designed to investigate survival potential on alternative hosts. The

experiments were undertaken in the containment area in Glasshouse C at Warwick University, Wellesbourne Campus. A licence application to cover this work was submitted and approved. The insect material was supplied by Phil Northing at Fera and arrived in April 2011. Food material (tomato and other plants) was grown in a separate greenhouse. The culture was allowed to complete one generation to establish how to manage the insects. The culture was maintained in constant environment room at 20+2

oC. The experiments were done in this room, in incubators and

a freezer. Treatment temperatures were monitored using a logger. The larvae are relatively robust and survive both wet and dry conditions. The culture decreased in numbers in late autumn 2011, mainly because too many insects were used in experiments where they subsequently died as a result of the treatments, but was restored with a further batch of insects from Fera.

Table 1.5 Potential control methods for Tetranychus evansi identified in the literature. Insecticides

• Propargite • Bifenthrin • Abamectin • Other newer acaricides e.g. bifenazate?

Other methods

• Beauveria • Metarhizium • Predatory mites Phytoseiulus fragariae and P. longipes • Phytoseiulus persimilis and Neoseiulus californicus ineffective • Quite a lot of work done on predators etc since 2000 – species available in UK?

EVID4 Evidence Project Final Report (Rev. 06/11) Page 12 of 41

The adult T. absoluta were maintained in sleeved Perspex cages. Eggs were collected by placing a piece of host plant foliage (generally tomato cv Gardeners Delight), in a small conical flask filled with water, within the cage. To prevent the moths drowning, a piece of cotton wool was placed around the plant stem at the top of the flask. Once collected, the pieces of foliage supporting the eggs were placed on a damp sheet of blotting paper or paper towel or latterly a layer of silver sand, inside a clear Perspex box (8 x 13 x 6 cm) and clearly labelled. The boxes were maintained in the constant environment room and once the larvae had hatched and started to feed, fresh plant material was added every 2 days. When the larvae were relatively large, additional sand was added to the culture box and the larvae generally formed pupae in cocoons made from the sand. The pupae were then removed by sieving the sand and were placed in the adult cage to emerge. Development rate under UK glasshouse conditions An HDC-funded study at Fera described development of T. absoluta at temperatures between 7 and 25°C (Cuthbertson, 2011). The optimum temperature range for development was 19-23°C and there was 52% survival from egg to adult at 19°C. The study found that development ceased between 7 and 10°C, only 17% of eggs hatched at 10°C and no larvae developed to adult moths. Development from egg to adult took 58 days at 13°C, 37 days at 19°C and 23 days at 25°C. Adult longevity was greatest at 10°C with adult moths living for 40 days (when supplied with food) and lowest at 19°C when they survived for 16 days. Survival at low temperatures Apart from the study by Cuthbertson (2011), other earlier experimental work on the effect of temperature on development of T. absoluta indicated that development stops between 6 and 9ºC (Barrientos et al., 1998; Bentacourt et al., 1996), depending on the life stage. The aim of the work in the current project was to investigate survival of the different life stages at low temperatures and to use two „types‟ of insects: 1) kept at high temperature/long daylength (20

oC and 18 hour day) and 2) reared at a lower temperature/short daylength (15

oC

and 12 hour day). Survival of eggs Foliage supporting newly-laid eggs was placed in 7 cl plastic containers with snap-on lids and kept in a cooling incubator for 7 days at either 0

oC or 5

oC. The containers were then placed at 20

oC. Control treatments

consisting of eggs laid on the same day were kept at 20oC for the full period of egg development. Once hatching

had occurred, all samples were examined under the microscope to determine the percentage hatch. The results are shown in Table 2.1. Viability was reduced by <10% after exposure to lower temperatures for 7 days. Table 2.1. Tuta absoluta – egg viability at lower temperatures (after 7 days exposure to lower temperatures)

Temperature oC Percentage hatch Standard error (s.e.)

0 87 2.8

5 89 2.8

20 (control) 96 1.1

Experiments were undertaken to determine the impact on egg viability of exposure to 0

oC for a longer period of

time, or of a short period of exposure to -5oC. Preliminary experiments showed that newly-laid eggs are able to

survive 1 or 2 days exposure to -5oC. In both instances (0 and -5° tests), the preliminary experiments showed that

it is necessary to treat the eggs off the plant as the plant material rots too rapidly once replaced at 20 oC and

affects the eggs adversely. In further experiments, eggs were removed from the oviposition site (tomato foliage) with a fine paintbrush and placed in batches of 20 on small pieces of black filter paper. Each batch of eggs was placed in a small plastic pot with a snap-on lid and placed either at -5°C for 1,2,3,4 or 5 days or at 0°C for 7, 14, 21 or 28 days. Following exposure to the low temperature for the required period of time the pots were placed at 20°C until the eggs hatched. The number of eggs that hatched successfully was then recorded using a microscope. Control batches of eggs were kept at 20°C. The results are summarised in Figures 2.1 and 2.2. In both cases, the numbers of eggs hatching decreased with increased duration of exposure to low temperatures. However, a small number of eggs survived 5 days at -5°C and 28 days at 0°C showing that the egg stage would be likely to survive the range of low temperatures that eggs might be exposed to in the UK for a reasonable period of time.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 13 of 41

y = -2.7694x + 17.471

R2 = 0.9449

-5

0

5

10

15

20

25

30

0 1 2 3 4 5 6

Days at -5°C

Nu

mb

er

of

eg

gs

ha

tch

ed

Figure 2.1 Effect of exposing eggs to -5°C for 1-5 days.

y = -0.6116x + 18.377

R2 = 0.7919

-5

0

5

10

15

20

25

0 5 10 15 20 25 30

Days at 0°C

Nu

mb

er

of

eg

gs h

atc

hed

Figure 2.2 Effect of exposing eggs to 0°C for 7-28 days. Survival of larvae In a first series of experiments, larvae of different ages were exposed to a temperature of 0°C for a period of 30 days. These larvae were previously reared at 15

ºC (from oviposition until treatment) and then replaced at 15°C

following treatment. The exposure to 0°C was begun 14, 21, 28 and 35 days after oviposition. Control batches of larvae were maintained continuously at 15°C. Although larvae in the control treatments survived, none of the larvae exposed to 0°C for 30 days survived. Larvae of different ages were exposed to either 0°C for 7, 14, 21 or 28 days or -5°C for a period of 1-4 days and then placed at a higher temperature to record emergence of moths following pupation. These tests showed that some larvae can survive 2 days at -5

oC or 7 days at 0

oC. However, the plant material was generally badly-

affected at these temperatures.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 14 of 41

Survival of pupae Newly-formed pupae, within their sand cocoons, were placed in small plastic containers with snap-on lids (7 cl) and held at temperatures of 0 or 5

oC for periods of 7 days before being placed at 20

oC. Control treatments

(pupae of the same age) were kept continuously at 20oC. The pupal stage lasts about 28 days at 15°C and 19

days at 20°C. The results are shown in Table 2.2. Over a period of 7 days, pupa viability was reduced by 16% and 10% for the 0 and 5

oC treatments respectively.

Table 2.2 Pupal viability at low temperatures (after 7 days exposure to the lower temperatures) – pupae

reared at 20°

Percentage emergence (s.e.)

Temperature Test temperature Control at 20oC

0o 64 (5.6) 80 (5.5)

5o 68 (7.7) 78 (5.8)

In further experiments, pupae reared either at 20

oC with an 18 hour day or at 15

oC with a 12 hour day were

exposed to -5oC for periods of 1-4 days or 0

oC for periods of up to 28 days. The pupae were exposed to low

temperatures either when newly-formed, or after different time periods at the rearing temperature, to determine whether pupa age had an effect on survival at low temperatures. Figure 2.3 shows the effect of exposures of different durations to 0

oC on the percentage of moths that emerged

subsequently. Data are shown both for pupae reared at 20°C that were exposed to the low temperature when newly-formed and for samples of mixed-age pupae (1-3 days old). Both sets of data showed that survival declined with increased duration of exposure to 0

oC and that survival was relatively low following 21 days

exposure. Figure 2.4 shows the effect of rearing conditions on survival of pupae exposed to -5oC for periods of 0-

4 days. All pupae were newly-formed. Again, both sets of data indicated that survival declined with increased duration of exposure to -5

oC and survival was relatively low following 3 days exposure.

Data on the effect of pupal age on survival are presented in two ways. Figure 2.5 shows the relationship between percentage survival and pupa age for pupae reared at 15

oC and exposed to – 5 for 2 days. Table 2.3 shows the

percentage survival of pupae that were reared at 20oC and were 0 or 7 days old and exposed either to 0

oC for 21

days or -5oC for 2 days.

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30

Days at 0oC

Pe

rce

nt

live

Newly-formed Mixed-age

Figure 2.3 Effect of exposing pupae to 0°C for 0-28 days. Data are shown both for pupae reared at 20°C that

were exposed to the low temperature when newly-formed and for samples of mixed-age pupae (1-3 days old).

EVID4 Evidence Project Final Report (Rev. 06/11) Page 15 of 41

0

10

20

30

40

50

60

0 1 2 3 4 5

Days at -5oC

Pe

rce

nt

live

Reared at 15° Reared at 20°

Figure 2.4 Effect of rearing conditions on survival of pupae exposed to -5

oC for periods of 0-4 days. All

pupae were newly-formed. Table 2.3 Percentage survival of pupae that were reared at 20

oC and were 0 or 7 days old and exposed

either to 0oC for 21 days or -5

oC for 2 days.

Age of pupae (days at 20oC) Percentage live (s.e.)

21 days at 0oC 2 days at -5

oC

0 1 (0.05) 14 (1.07)

7 7 (1.28) 19 (2.26)

Survival of adults Newly-emerged adults were confined in small batches (1-5 moths) in plastic containers with snap-on lids (125 ml) and then placed at -5°C for periods of 1-5 days, or at 0

oC for longer periods, before being replaced at 20°C.

Survival was assessed. The results are shown in Figures 2.6 and 2.7. Survival decreased with increasing duration of exposure to low temperatures. Some of the moths exposed to the low temperatures were subsequently provided with tomato foliage as an oviposition site. The moths laid eggs and some of these eggs were viable.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 16 of 41

0

10

20

30

40

50

60

70

80

1 2 3 4 5

Pe

rce

nt

surv

ival

Days at -5oC

8

96

50

37 19

Figure 2. 5 Percentage survival of moths placed at -5°C for 1-5 days (numbers of moths exposed at each

temperature are shown on the figure).

0

10

20

30

40

50

60

70

80

7 10 15 30

Pe

rce

nt

surv

ival

Days at 0oC

4541

53

40

Figure 2.6. Percentage survival of moths placed at 0°C for up to 30 days (numbers of moths exposed at each

temperature are shown on the figure). Alternative host plants The aim of these experiments was to determine 1) whether female T. absoluta would lay eggs on host plants apart from tomato and to obtain an indication of their ovipostion preferences and 2) to evaluate larval survival on alternative host plants on which eggs were laid. Alternative host plants - number of eggs laid in choice tests Adult female moths maintained in the culture cages described above were presented with leaves of potential

EVID4 Evidence Project Final Report (Rev. 06/11) Page 17 of 41

alternative host plants in a choice situation, using tomato foliage as the alternative. The pieces of foliage of the test and control plants were similar in size and were presented in separate conical flasks as describe above. The results are summarised in Table 2.4. Female moths laid relatively large numbers of eggs on potato (cv Marfona) and black nightshade (Solanum nigrum). Pepper (variety not specified) and Datura were not acceptable as oviposition sites. Table 2.4. Number of eggs laid in choice tests (standard error in parenthesis)

Control No. eggs No. eggs Test species

Tomato 155 (25) 135 (18) Potato

Tomato 132 (29) 33 (6) Black nightshade

Tomato 100+ 4 Pepper

Tomato 100+ 0.2 Datura

Emergence of adults from larvae fed on different host plants Insects were reared from eggs to adults on the three „preferred‟ host plants. Pieces of foliage with known numbers of eggs were placed in Perspex culture boxes and fresh foliage was added at least every 2 days. As the larvae reached the third instar, silver sand was added to the culture boxes to allow the insects to form pupal cocoons. Emergence of adult moths was recorded. The results are summarised in Table 2.5 and Figure 2.7. Between 54% (tomato) and 40% (potato) of eggs survived to adulthood, showing that all three host plants supported larval development. Table 2.5. Emergence of adults from larvae fed on different diets

Diet Mean percentage emergence of adults (s.e.)

Tomato 54 (13.3)

Potato 40 (7.7)

Black nightshade 49 (17.6)

Inspection of the data suggested that the initial number of eggs per replicate also affected survival and so the percentage of insects surviving was plotted against initial egg number for each treatment. This confirmed that insects reared at a lower density were more „successful‟.

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50

Number of eggs at start

Perc

en

t em

erg

ing

as a

du

lts

Black nightshade

Potato

Tomato

Figure 2.7. Emergence of adults from larvae fed on different diets – effect of initial number of eggs Further tests were done to determine whether commonly-grown bedding plants – Nicotiana (Nicotiana alata) and

EVID4 Evidence Project Final Report (Rev. 06/11) Page 18 of 41

Petunia (Petunia x hybrida) - were also suitable host plants. Foliage was presented to moths in conical flasks as above, in „choice‟ and „no choice‟ situations – using tomato as the alternative in the „choice‟ tests (Table 2.6). Eggs were laid on both Nicotiana and Petunia but tomato was much preferred in the „choice‟ tests. Table 2.6 Numbers of eggs laid by female T. absoluta on foliage of Nicotiana alata and Petunia x hybrida in

„choice‟ and „no choice‟ situations.

Petunia Tomato Nicotiana Tomato

No choice Mean 10.1 17.9

s.e. 4.4 4.3

Choice Mean 2.0 28.8 5.7 28.5

s.e. 0.6 7.9 2.8 5.5

The pieces of Nicotiana and Petunia foliage with eggs were placed on sand in plastic boxes and observed for egg hatch and subsequent larval development. Larvae mined the Nicotiana foliage and a single moth emerged, but there was no evidence of feeding/development on Petunia. Control of Tuta absoluta on tomato in the UK A range of insecticides are approved for use on tomato crops in the UK. Insecticides that will control Lepidoptera include Deltamethrin, Indoxacarb, Lambda-cyhalothrin and Spinosad. A major concern is that methods of control should be compatible with biological control agents used on the crop. A draft control strategy for Tuta absoluta This research was undertaken by Rob Jacobson and Phil Morley and was funded principally by the HDC. For the purpose of this strategy, the UK growing season is divided into four distinct periods: 1. The first period is from planting in December until early-mid spring which is the key period for establishing

Macrolophus caliginosus. The predators are released immediately after planting at rates varying from 0.5-2.0 per m

2. However, they are slow to establish and usually do not produce useful populations for

biocontrol for at least four months. Other methods are employed throughout this period to slow down T. absoluta population growth. These include exclusion, de-leafing, sticky floor treatments and mass trapping with pheromone and / or UV light traps. Regular treatments with Bacillus thuringiensis may also make a contribution although the cost-benefit has been questioned. It is hoped that mating disruption will soon become an additional option for this period.

2. The second period is from mid to late spring. Despite the measures taken during the first period to delay

T. absoluta population growth, it seems inevitable that a second line of defence (SLoD) treatment will be required before the predatory bugs start to have a significant impact. The aim here is to apply a product that is quick to act and compatible with all the biocontrol agents being used in the greater IPM programme. Chemical options for organic crops are currently limited to spinsosad as a high volume spray but other IPM compatible products are becoming available for conventional crops. The entomopathogenic nematode, Steinernema feltiae, provides a useful alternative and this technique is being further refined to optimise efficacy.

3. The third period is from early summer through to early autumn. The predatory bugs should now be

numerous and should suppress the T. absoluta population growth by feeding on eggs and larvae. However, careful monitoring is required to determine whether it becomes necessary to apply additional SLoD treatments.

4. The fourth period is from early to late autumn when the main objective is to reduce the number of pests

that survive to infest the following crop. For conventional crops, it is possible to broaden the range of chemical pesticides used during this period to include those which are less compatible with biocontrol agents as their role has been completed for the season.

Increasing the available chemistry against Tuta absoluta to provide a second line of defence: In parallel to the above studies, the Tomato Growers Association took the initiative to work with Plant Health, CRD, HDC and insecticide suppliers to increase the range and efficacy of the products available to UK growers. As a consequence, there have been new Extensions of Authorisations for Minor Use for high volume sprays of Rynaxypyr (Coragen) and Spinosad (Conserve). More recently a Plant Health Authorisation has been issued for the use of Conserve through the irrigation system. This method of application should be more compatible with the

EVID4 Evidence Project Final Report (Rev. 06/11) Page 19 of 41

primary biocontrol agents, the predatory bugs. Experimental work on Epitrix spp. As an EPPO working group were developing a Pest Risk Analysis for Epitrix species, no desk-based research was undertaken. However, a decision was made to undertake some experimental studies towards the end of the project that would increase the information available. Samples of two species of Epitrix (E. similaris and E. cucumeris), originating from Portugal, were taken to the containment area in Glasshouse C at Warwick Crop Centre, Wellesbourne on 9

th March 2012. They had been

cultured on aubergine at Fera, following a procedure adopted by INRB, Portugal. As the work at Warwick was to be associated with their status as a pest of potato they were placed on potato plants produced from sprouting tubers. The adults fed freely on the foliage of the plants and numbers of E. similaris had increased sufficiently by mid-May to start small scale experiments. Numbers of E. cucumeris have remained low; they have reproduced but not as well as E. similaris. Further adults were obtained from Fera on 15

th June to try and increase the E.

cucumeris culture. Once numbers of E. similaris increased sufficiently to be able to „spare‟ some insects, attempts were made to develop a small-scale culturing system, in small plastic containers, that could be used undertake experiments on the immature stages. In the first approach, potato foliage as an adult food source and small potato tubers as an „oviposition site‟ and these were part-covered in sand. No adults were produced using this technique. Subsequently a report from INRB suggested that the substrate may also make a difference, so compost was used instead of sand and tests were done with small tubers in peat-based compost and also with sprouting tubers placed in Bugdorm® pint pots. The latter method produced adult beetles. The original priorities for research were:

1. Tuber damage especially comparison of the damage caused by E.similaris with that of E.tuberis 2. Potential alternative hosts (particularly weeds such as Solanum nigrum) 3. Studies on survival at low temperatures 4. Insecticidal control (current and novel insecticides)

Since research in Portugal focused on 1 and 2, the work at Warwick focused on 3 and 4. Low temperature studies In the time available, the aim was to investigate survival of adult and immature stages at low temperatures and with exposure for periods of time that might realistically occur outdoors in the UK. The aim was to investigate survival of different stages after exposure to at -5

oC for short periods (1-4 days) and to 0° for longer periods (7-28

days). Within the current project, because of time constraints and limited experimental material, these studies focused on the adult stage of E. similaris. Previous research in North America indicates that Epitrix species are likely to overwinter as adults in the soil (Kabaluk & Vernon, 2000). Adult E. similaris were collected from the culture cage (at 20°C) in small plastic containers with snap-on lids (125 ml) and exposed either to 0° for 1-5 days or to 0°C for 1- 4 weeks. Following exposure to the low temperatures for the required period of time, the beetles were placed at 20°C and survival was assessed. The results are summarised in Figure 2.8. Survival decreased with increasing exposure time and virtually all the beetles were dead after 3 days at -5°C or two weeks at 0°C. These results suggest that beetles reared under „summer‟ conditions are unable to survive for very long at low temperatures. However, it is possible that beetles that are exposed to „autumn‟ conditions for a period prior to their exposure to low temperatures become more resilient. If time had permitted, this aspect would have been followed up.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 20 of 41

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Duration of exposure to low temperature (days)

Perc

en

t ali

ve

Exposure to -5° Exposure to 0°

Figure 2.8. Percentage survival (with standard error) of adult Epitrix similaris exposed to low temperatures for

different periods. Insecticidal control The aim was to investigate potential products for insecticidal control of both species. Logically the adult is the best stage to target as it is most accessible to insecticide treatments. Soil treatments to control larvae would be more difficult for a number of reasons. There is a range of current and novel insecticides to test as foliar sprays. As E. similaris adults were more abundant they were used in insecticide tests. Potato leaves were dipped in solutions of the insecticides, which were made up to simulate the recommended field rate. The leaves were allowed to dry, placed in small containers and then adult beetles were added to the containers. There were two sets of treatments, each of which had an untreated control and used deltamethrin as a standard (Table 2.7). Each set of treatments was replicated twice on two occasions. Mortality was assessed after 24 and 48 hours and the number of feeding holes was recorded after 48 hours. The results are summarised in Figures 2.9 and 2.10. Products either killed or immobilised the majority of beetles very rapidly (7) or were ineffective (2). This was also reflected in the numbers of feeding holes in the potato leaves. The results were so clear-cut that statistical analysis of the data was unnecessary. Table 2.7 Treatments used to control Epitrix similaris

Set 1 Set 2

Active ingredient Product Active ingredient Product

Coded treatment Acetamiprid Gazelle

Lambda-cyhalothrin Hallmark Flonicamid Teppeki

Spinosad Tracer Pyrethrum

Thiacloprid Biscaya Thiamethoxam Cruiser

Deltamethrin Decis Deltamethrin Decis

Untreated Untreated

EVID4 Evidence Project Final Report (Rev. 06/11) Page 21 of 41

0

10

20

30

40

50

60

70

80

90

100

Coded Lambda-cyhalothrin Spinosad Thiacloprid Deltamethrin Untreated

Perc

en

t

% dead and moribund Day 1 % dead and moribund Day 2 Feeding holes per beetle

Figure 2.9 Percentage of adult Epitrix similaris that were dead and moribund and number of feeding holes

per beetle following exposure to potato leaves treated with insecticides (Treatment Set 1)

0

10

20

30

40

50

60

70

80

90

100

Acetamiprid Flonicamid Pyrethrum Thiamethoxam Deltamethrin Untreated

Pe

rce

nt

% dead and moribund Day 1 % dead and moribund Day 2 Feeding holes per beetle

Figure 2.10 Percentage of adult Epitrix similaris that were dead and moribund and number of feeding holes per beetle following exposure to potato leaves treated with insecticides (Treatment Set 2)

Objective 3 Identify types of soil pests and soil ‘environments’ for which further information about

control measures is required. The key target pests are nematodes and particularly those imported with bonsai trees or other shrubby species.

Objective 4 Review soil treatments for a range of pest groups that include taxa of statutory concern and make recommendations for experimental work.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 22 of 41

A review of potential treatments for soil pests (nematodes and mealy bugs) was undertaken. Chemical controls that are approved in the UK are listed in Table 4.1:

The steering group and project team discussed some of the issues associated with identification of infestations and survival of nematodes in imported bonsai and decided that the first priority was to address some of these issues before undertaking experiments on control strategies. Objective 5 Devise and undertake a programme of experimental work on treatments to control soil

inhabiting pests of containerised plants. A source of imported bonsai was identified and a total of 100 plants (3 species) were obtained. These were collected and kept in a greenhouse at Warwick University, Wellesbourne campus. A series of experiments was planned using these plants as follows: Experiment 5.1 – between-pot distribution of nematodes Experiment 5.2 – within pot distribution of nematodes Experiment 5.3 – overwintering survival of nematodes Experiment 5.4 – effects of root washing Experiment 5.5 – dispersal of nematodes Experiment 5.1 – between-pot distribution of nematodes Twenty plants were sampled to determine numbers and type/species of nematode in each pot. The plants were numbered so that they could be identified and re-sampled later. The samples consisted of compost and root material taken from the two ends of each (rectangular) pot and placed in labelled polythene bags. The spaces in pots were back-filled with clean compost. The samples were boxed and sent to Fera (Sue Hockland) for analysis and identification. The 20 plants were left to grow in a greenhouse and further samples of soil and root material were taken once the plants had grown a new root system. The areas containing fresh compost were sampled as above and again sent to Fera (Sue Hockland) for analysis and identification. Nematodes were identified to species where possible and counted. Compost samples were weighed and the weights were recorded. Nematode counts from the first and second samples are shown in Tables 5.1 and 5.2. Figure 5.1 shows the numbers of Heliocotylenchus sp. per sample for the two sampling occasions. A range of nematode species were found in the samples and the distribution was far from uniform. There was some indication of differences between plant species and this may be because they originated from different locations before being potted up (i.e.Tylenchorhynchus sp. were only found in association with Zelkova plants (variety 2)). Nematode numbers were much lower on the second sampling occasion, with the exception of the microbivorous nematodes.

Table 4.1 Potential control methods for soil pests (nematodes and mealy bugs) identified in the literature.

• Metam sodium - fumigant • Dazomet - fumigant Both pre-planting treatments (raspberry)

• Fosthiazate – non-fumigant • Oxamyl – non-fumigant • Ethoprophos Pre-planting – potato Non-chemical controls include: • Hot water • Heat • Marigold • Various phytochemicals (reviewed by Chitwood, 2002) • Cranberry juice • Thyme oil • Entomopathgenic nematodes • Nematophagous fungi

EVID4 Evidence Project Final Report (Rev. 06/11) Page 23 of 41

It was also recognised that it would be very useful to know more about the within pot distribution of plant parasitic nematodes associated with bonsai plants (particularly in association with the roots). It was agreed that smaller samples could be taken by dividing up the plant to investigate this further (Experiment 2).

Table 5.1 Nematode counts from first sample. Plants 1, 2 and 3 are different species. Species 1 = unidentified; 2 = Zelkova; 3 = Sageretia.

Heliocotylenchus sp. spiral nematodes

Meloidogyne Sp. root knot nematodes

Ditylenchus sp. anguinid nematodes

Pratylenchus sp. root lesion nematodes

Safianema sp. anguinid nematodes

Tylenchorynchus sp. stunt nematode

Microbivorus nematodes

Hel

ioco

tyle

nch

us

sp.

Mel

oid

og

yne

Sp.

Dit

ylen

chu

s sp

.

Pra

tyle

nch

us

sp.

Safi

an

ema

sp

.

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

Me

dia

we

igh

t (g

)

Nu

mb

ers

pe

r 2

00

g m

ed

ia

Hel

ioco

tyle

nch

us

sp.

Mel

oid

og

yne

Sp.

Dit

ylen

chu

s sp

.

Pra

tyle

nch

us

sp.

Safi

an

ema

sp

.

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

1A 34 307 130 1A 52 472

1B 1479 124 1B 2385

1C 173 220 3 1674 140 1C 247 314 4 2391

1D 59 3 3171 107 1D 110 6 5927

1E 36 69 871 126 1E 57 110 1383

1F 61 690 134 1F 91 1030

1G 37 112 1G 66

2A 8 1 283 117 2A 14 2 484

2B 1 1 1 16 770 125 2B 2 2 2 26 1232

2C 277 1 1 2614 116 2C 478 2 2 4507

2D 10 2 735 92 2D 22 4 1598

2E 91 367 116 2E 157 633

2F 5 27 1390 143 2F 7 38 1944

2G 386 1796 147 2G 525 2444

3A 2 127 266 143 3A 3 178 372

3B 252 113 3B 446

3C 1 78 651 124 3C 2 126 1050

3D 3569 670 107 3D 6671 1252

3E 197 122 3E 323

3F 2111 579 129 3F 3273 898

EVID4 Evidence Project Final Report (Rev. 06/11) Page 24 of 41

Table 5.2. Nematode counts from second sample. Plants are as in Table 5.1.

Hel

ioco

tyle

nch

us

sp.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

Me

dia

we

igh

t (g

)

Ro

ot

we

igh

t (g

)

Ro

ot

Re

gro

wth

Nu

mb

ers

pe

r 2

00

g m

ed

ia

Hel

ioco

tyle

nch

us

sp.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

Nu

mb

ers

pe

r 5

g ro

ot

Hel

ioco

tyle

nch

us

sp.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

1A 7 1 691 76 1 Moderate 1A 18 3 1818 1A 35 5 3455

1B 314 64 4 Good 1B 981 1B 393

1C 10 1571 80 0.5 Low 1C 25 3928 1C 100 15710

1D 2 4700 59 0.8 Dead 1D 7 15932 1D 13 29375

1E 2 497 57 5 Good 1E 7 1744 1E 2 497

1F 6 1706 65 3 Moderate 1F 18 5249 1F 10 2843

1G 2 5000 48 0.6 Dead 1G 8 20833 1G 17 41667

2A 7000 65 6 Good 2A 21538 2A 5833

2B 1 10000 74 2 Low 2B 3 27027 2B 3 25000

2C 1 8000 86 3 Moderate 2C 2 18605 2C 2 13333

2D 912 66 2 Moderate 2D 2764 2D 2280

2E 226 10000 87 2 Moderate 2E 520 22989 2E 565 25000

2F 3900 110 1 Low 2F 7091 2F 19500

2G 6 1137 93 1 Good 2G 13 2445 2G 30 5685

3A 506 75 1 Dead 3A 1349 3A 2530

3B 1 5700 105 0.5 Dead 3B 2 10857 3B 10 57000

3C 1369 101 0.2 Dead 3C 2711 3C 34225

3D 40 3700 86 0.5 Dead 3D 93 8605 3D 400 37000

3E 1327 78 1 Dead 3E 3403 3E 6635

3F 28 1793 92 0.3 Dead 3F 61 3898 3F 467 29883

1

10

100

1000

10000

A B C D E F G A B C D E F G A B C D E F

1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3

Nu

mb

er

First sample Second sample

Figure 5.1. Numbers of Heliocotylenchus sp. per sample on the two sampling occasions Experiment 5.2 – within pot distribution of nematodes In the second experiment, 5 fresh plants (Zelkova) were sampled more intensively by dividing the soil/root ball into 9 sections and placing each section (labelled) in a separate polythene bag. The samples were boxed and sent to Fera (Sue Hockland) for analysis and identification. The compost and root material were weighed and the nematodes were identified to group and counted.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 25 of 41

In general, if one of the 9 samples from a pot contained a significant number of nematodes of a particular group then the others did also (Table 5.3; Figures 5.2-5.5). This suggests that it may be appropriate to take sub-samples, which might for example, consist of two samples taken from diagonally opposite ends of the pot. In most cases the „trunk‟ was in the centre of the pot, but there was no evidence that higher numbers of nematodes were found under the trunk. Table 5.3. The numbers of nematodes in each of the 9 sub-samples taken from 5 pots containing bonsai

plants (Zelkova).

Po

t

Sub

-sam

ple

Hel

ioco

tyle

nch

us

sp.

Dit

ylen

chu

s sp

.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

Me

dia

we

igh

t (g

)

Ro

ot

we

igh

t (g

)

Po

t -

nu

mb

ers

in 2

00

g m

ed

ia

Hel

ioco

tyle

nch

us

sp.

Dit

ylen

chu

s sp

.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

Po

t -

nu

mb

er

pe

r 5

g ro

ot

Hel

ioco

tyle

nch

us

sp.

Dit

ylen

chu

s sp

.

Tyle

nch

us

sen

su la

to

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

ne

mat

od

es

21 1 406 64 1 579 35 3 21 2320 366 6 3309 21 677 107 2 965

2 310 33 31 3 551 34 6 1771 194 182 18 3241 258 28 26 3 459

3 134 14 479 21 3 766 133 4562 223 23 798

4 533 44 1 327 39 5 3046 226 5 1677 533 44 1 327

5 370 2 128 12 4 2114 33 2133 463 3 160

6 568 12 73 20 3 3246 120 730 947 20 122

7 294 14 637 18 4 1680 156 7078 368 18 796

8 405 24 671 19 5 2314 253 7063 405 24 671

9 231 2 1163 16 3 1320 25 14538 385 3 1938

22 1 35 2 6713 26 5 22 200 15 51638 22 35 2 6713

2 82 6 47 439 19 7 469 63 495 4621 59 4 34 314

3 134 52 330 26 5 766 400 2538 134 52 330

4 747 2 28 397 44 5 4269 9 127 1805 747 2 28 397

5 1050 3 161 23 3 6000 26 1400 1750 5 268

6 12 1 480 241 37 6 69 5 2595 1303 10 1 400 201

7 169 3 757 21 4 966 29 7210 211 4 946

8 461 7 3 271 25 7 2634 56 24 2168 329 5 2 194

9 966 178 28 6 5520 1271 805 148

23 1 1 1 47 24 3 23 6 8 392 23 2 2 78

2 28 3 121 19 6 295 32 1274 23 3 101

3 2 6 264 26 7 15 46 2031 1 4 189

4 2 2 103 26 6 11 15 792 2 2 86

5 147 18 5 1633 147

6 129 24 3 1075 215

7 37 26 5 285 37

8 35 787 14 3 500 11243 58 1312

9 4 829 24 5 33 6908 4 829

24 1 1 14 1 291 27 6 24 6 104 7 2156 24 1 12 1 243

2 0 28 3 0 0

3 540 31 4 3484 675

4 0 24 8 0 0

5 74 16 2 925 185

6 981 28 4 7007 1226

7 497 26 6 3823 414

8 4520 18 3 50222 7533

9 216 27 4 1600 270

25 1 5 897 30 2 25 33 5980 25 13 2243

2 8 432 21 4 76 4114 10 540

3 629 27 4 4659 786

4 3 453 49 7 12 1849 2 324

5 449 18 4 4989 561

6 3 3 541 32 5 17 19 3381 3 3 541

7 2 12 377 32 5 11 75 2356 2 12 377

8 1 13 210 22 4 6 118 1909 1 16 263

9 1 12 302 36 6 6 67 1678 1 10 252

EVID4 Evidence Project Final Report (Rev. 06/11) Page 26 of 41

Figure 5.2. The distribution of Helicotylenchus between the 9 sub-samples taken from 5 pots containing

bonsai plants (Zelkova). Row 1 Number, Row 2 Nematodes/g media, Row 3 Nematodes/g root

Figure 5.3. The distribution of Microbivorous nematodes between the 9 sub-samples taken from 5 pots

containing bonsai plants (Zelkova). Row 1 Number, Row 2 Nematodes/g media, Row 3 Nematodes/g root

Figure 5.4. The distribution of Ditylenchus between the 9 sub-samples taken from pots containing bonsai plants

(Zelkova). Only pots 21 and 22 are shown as these were the only ones containing significant numbers. Row 1 Number, Row 2 Nematodes/g media, Row 3 Nematodes/g root

0

10

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0.0

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EVID4 Evidence Project Final Report (Rev. 06/11) Page 27 of 41

Figure 5.5. The distribution of Tylenchorynchus between the 9 sub-samples taken from pots containing

bonsai plants (Zelkova). Only pots 21 and 22 are shown as these were the only ones containing significant numbers.

Experiment 5.3 – overwintering survival of nematodes The aim of Experiment 5.3 was to determine how well the nematodes imported with bonsai plants might survive outdoor conditions in the UK during the winter; if for example, the pot was placed outside or discarded on a compost heap. For this experiment the bonsai plants were potted into larger containers containing sandy loam soil and strawberry plants to provide an alternative host if the bonsai plants died (as might happen if the plants were put outside). There was a control treatment (Variety 4) which contained strawberry plants alone. The plants were divided into groups and on 28 January 2011, one batch was placed outdoors, one batch of plants in the greenhouse with the stock plant material and one batch in a poly tunnel which was unheated. All containers were placed in large saucers/trays to ensure no liquid escapes. Delta-T loggers (Model DL2, Delta-T Devices Ltd; Thermistors No. 590-39AA07-103, Fenwall Electronics Inc) were used to record soil temperatures in the pots. The plants were left outside until March 2011 when the soil close to roots of the bonsai and strawberry plants was sampled and the nematodes identified and counted by Fera. Figure 5.6 shows soil temperatures in the pots over the whole experimental period and Figure 5.7 show the temperatures during the coldest weather event. The pots in the poly tunnel suffered the greatest fluctuation in temperature. The pots in the cold frame appeared to be buffered to a certain extent. Table 5.4 and Figure 5.8 show the numbers of nematodes recovered from the pots subsequently. The majority of plants kept in poly tunnel or frame were dead – except Zelkova plants in the cold frame. There was no evidence that nematode numbers were decreased by exposure to low temperatures.

0

0.5

1

1.5

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3.5

1 2 3 4 5 6 7 8 9

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0.1

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0.1

0.1

1 2 3 4 5 6 7 8 9

0.0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6 7 8 9

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100

200

300

400

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600

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6.0

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0.0

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EVID4 Evidence Project Final Report (Rev. 06/11) Page 28 of 41

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Poly tunnel

Cold frame

Glasshouse

Figure 5.6. Temperature records during the whole experimental period

Figure 5.7. Temperature records during coldest „event‟

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Cold frame Poly tunnel Glasshouse

EVID4 Evidence Project Final Report (Rev. 06/11) Page 29 of 41

Table 5.4. Survival of nematodes surrounding bonsai plants kept in a glasshouse, cold frame or poly tunnel between January and March 2011.

Per sample

Per 20g root

Loca

tio

n

vari

ety

Re

plic

ate

Sam

ple

Hel

ioco

tyle

nch

us

sp.

Dit

ylen

chu

s sp

.

Pra

tyle

nch

us

sp.

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

nem

ato

des

Me

dia

we

igh

t (g

)

Ro

ot

we

igh

t (g

)

Hel

ioco

tyle

nch

us

sp.

Dit

ylen

chu

s sp

.

Pra

tyle

nch

us

sp.

Tyle

nch

ory

nch

us

sp.

Mic

rob

ivo

rus

nem

ato

des

Cold frame 1 A 2 1 59 200 1 20 1180

Cold frame 1 A 1 1 4 52 200 7 3 11 149

Cold frame 1 B 1 2 11 200 5 8 44

Cold frame 1 B 2 116 200 37 63

Cold frame 1 C 1 7 68 200 21 7 65

Cold frame 1 C 2 11 108 200 21 10 103

Cold frame 1 D 1 68 200 12 113

Cold frame 1 D 2 11 129 200 28 8 92

Cold frame 2 A 1 227 1482 200 6 757 4940

Cold frame 2 B 2 1 4 371 200 12 2 7 618

Cold frame 2 B 1 19 2 286 200 25 15 2 229

Cold frame 2 B 1 89 200 9 198

Cold frame 2 C 1 113 1 17 1382 200 8 283 3 43 3455

Cold frame 2 C 2 32 7 586 200 13 49 11 902

Cold frame 2 D 1 82 111 200 9 182 247

Cold frame 2 D 2 97 200 7 277

Cold frame 3 A 2 8 53 200 8 20 133

Cold frame 3 A 1 1 4 6 180 200 16 1 5 8 225

Cold frame 3 B 2 88 200 23 77

Cold frame 3 B 1 6 62 200 47 3 26

Cold frame 4 A 2 1 107 200 21 1 102

Cold frame 4 A 1 2 1 1 85 200 10 4 2 2 170

Cold frame 4 B 2 3 1 78 200 16 4 1 98

Cold frame 4 B 1 3 111 200 15 4 148

Cold frame 4 C 2 2 5 89 200 14 3 7 127

Cold frame 4 C 1 106 200 15 0 141

Glasshouse 1 A 1 2 44 200 6 7 147

Glasshouse 1 A 2 1 1 2 283 200 8 3 3 5 708

Glasshouse 1 B 1 9 313 200 11 16 569

Glasshouse 1 B 2 48 200 17 56

Glasshouse 1 C 1 60 8 19 973 200 12 100 13 32 1622

Glasshouse 1 C 2 2 4 502 200 6 7 13 1673

Glasshouse 1 D 2 17 172 200 5 68 688

Glasshouse 1 D 1 62 7 543 200 18 69 8 603

Glasshouse 2 A 2 3 89 200 18 0 3 99

Glasshouse 2 A 1 886 200 34 0 521

Glasshouse 2 B 2 71 3 8 509 200 8 178 8 20 1273

Glasshouse 2 B 1 113 303 200 21 108 289

EVID4 Evidence Project Final Report (Rev. 06/11) Page 30 of 41

Glasshouse 2 C 2 9 16 437 200 30 6 11 291

Glasshouse 2 C 1 12 4 202 200 27 9 3 150

Glasshouse 2 D 1 1211 200 16 1514

Glasshouse 2 D 2 862 200 48 359

Glasshouse 3 A 2 3 227 200 14 4 324

Glasshouse 3 A 1 7 43 200 45 3 19

Glasshouse 3 B 1 71 200 6 237

Glasshouse 3 B 2 117 200 30 78

Glasshouse 4 A 1 3 42 200 1 60 840

Glasshouse 4 A 2 2 2 662 200 15 3 3 883

Glasshouse 4 B 1 40 200 1 800

Glasshouse 4 B 2 48 200 23 42

Glasshouse 4 C 1 139 200 27 103

Glasshouse 4 C 2 119 200 14 170

Poly tunnel 1 A 2 32 2003 200 11 58 3642

Poly tunnel 1 A 1 11 2 1263 200 14 16 3 1804

Poly tunnel 1 B 1 76 467 200 10 152 934

Poly tunnel 1 B 2 47 1 13 2982 200 13 72 2 20 4588

Poly tunnel 1 C 2 537 200 15 716

Poly tunnel 1 C 1 109 200 13 168

Poly tunnel 1 D 2 311 1 8 1826 200 6 1037 3 27 6087

Poly tunnel 1 D 1 21 1981 200 26 16 1524

Poly tunnel 2 A 1 9 567 200 8 23 1418

Poly tunnel 2 A 2 8 8 3643 200 6 27 27 #####

Poly tunnel 2 B 2 227 3826 200 6 757 #####

Poly tunnel 2 B 1 698 200 8 1745

Poly tunnel 2 C 2 4 2 228 200 18 4 2 253

Poly tunnel 2 C 1 77 200 10 154

Poly tunnel 2 D 2 11 3 482 200 11 20 5 876

Poly tunnel 2 D 1 23 1113 200 8 58 2783

Poly tunnel 3 A 2 118 200 11 215

Poly tunnel 3 A 1 106 200 13 163

Poly tunnel 3 B 1 5 3 90 200 13 5 138

Poly tunnel 3 B 2 37 200 13 57

Poly tunnel 4 A 2 21 200 4 105

Poly tunnel 4 A 1 101 200 6 337

Poly tunnel 4 B 2 21 200 10 42

Poly tunnel 4 B 1 19 200 5 76

Poly tunnel 4 C 1 867 200 3 5780

Poly tunnel 4 C 2 3638 200 10 7276

EVID4 Evidence Project Final Report (Rev. 06/11) Page 31 of 41

Figure 5.8. Survival of nematodes surrounding bonsai plants kept in a glasshouse, cold frame or poly tunnel

between January and March 2011. Experiment 5.4 – effects of root washing A sample of stock plant material was divided into two groups (7 plants per group and one group to be untreated control). Sub-samples of soil were taken from each plant and sent to Fera (Sue Hockland) for identification and counts of nematodes (Table 5.5). The roots of the „treated‟ plants were washed (protocol supplied by Ray Cannon) on 25 July 2011 and it took about 2 hours to wash the roots of 7 plants. The plants were re-potted and placed in the same greenhouse compartment as the untreated control plants. The plants were left to grow for 3 months. Samples of soil were sent to Fera (Sue Hockland) for identification and counts of nematodes in each group. These results are also displayed in Table 5.5 and the total numbers of plant-parasitic nematodes pre- and post-washing are compared in Figure 5.9. The data indicate that nearly all nematodes were removed by root-washing but the plants were affected badly by the procedure (Table 5.5d – see root weights and information on plant condition). Table 5.5a. Effects of root washing – number of nematodes in pre-wash samples

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Wash 1 35 6 41 1119 33 8

Wash 2 17 2 19 179 47 19

Wash 3 12 12 279 46 12

Wash 4 71 71 1391 50 12

Wash 5 14 14 1711 42 8

Wash 6 30 2 32 727 39 9

Wash 7 84 84 279 29 13

Control 1 45 45 1121 41 11

Control 2 49 9 2 2 62 631 34 9

Control 3 1 1 97 44 8

Control 4 16 16 129 43 8

Control 5 0 71 41 15

Control 6 2 2 81 34 9

Control 7 0 172 52 5

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Table 5.5b. Effects of root washing – number of nematodes in pre-wash samples. Counts adjusted for media weight of 50g.

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Wash 1 53 9 0 0 0 62 1695

Wash 2 18 2 0 0 0 20 190

Wash 3 13 0 0 0 0 13 303

Wash 4 71 0 0 0 0 71 1391

Wash 5 17 0 0 0 0 17 2037

Wash 6 38 3 0 0 0 41 932

Wash 7 145 0 0 0 0 145 481

Control 1 55 0 0 0 0 55 1367

Control 2 72 13 3 3 0 91 928

Control 3 0 0 1 0 0 1 110

Control 4 19 0 0 0 0 19 150

Control 5 0 0 0 0 0 0 87

Control 6 3 0 0 0 0 3 119

Control 7 0 0 0 0 0 0 165 Table 5.5c. Effects of root washing – number of nematodes in pre-wash samples. Counts adjusted for root weight of 10g.

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Wash 1 44 8 51 1399

Wash 2 9 1 10 94

Wash 3 10 10 233

Wash 4 59 59 1159

Wash 5 18 18 2139

Wash 6 33 2 36 808

Wash 7 65 65 215

Control 1 41 41 1019

Control 2 54 10 2 2 69 701

Control 3 0 1 1 121

Control 4 20 20 161

Control 5 0 47

Control 6 2 2 90

Control 7 0 344

EVID4 Evidence Project Final Report (Rev. 06/11) Page 33 of 41

Table 5.5d Effects of root washing – number of nematodes in post-wash samples.

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Wash 1 0 237 Live Yes 67 1

Wash 2 0 301 live Yes 81 1

Wash 3 0 137 Dead Yes 84 5

Wash 4 1 1 2102 Live Yes 88 1

Wash 5 0 201 Live Yes 114 5

Wash 6 0 101 Live Yes 99 2

Wash 7 0 3000 Dead No 124 2

Control 1 46 2 1 49 1121 Dead No 88 8

Control 2 45 2 2 49 982 Live No 74 7

Control 3 14 1 2 17 137 Live No 96 4

Control 4 67 67 687 Live No 88 10

Control 5 8 8 1163 Dead No 77 11

Control 6 20 20 162 Live No 99 10

Control 7 6 6 113 Live No 89 4 Table 5.5e Effects of root washing – number of nematodes in post-wash samples. Counts adjusted for media weight of 50g.

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Wash 1 0 0 0 0 0 0 177

Wash 2 0 0 0 0 0 0 186

Wash 3 0 0 0 0 0 0 82

Wash 4 1 0 0 0 0 1 1194

Wash 5 0 0 0 0 0 0 88

Wash 6 0 0 0 0 0 0 51

Wash 7 0 0 0 0 0 0 1210

Control 1 26 1 1 0 0 28 637

Control 2 30 1 1 0 0 33 664

Control 3 7 1 1 0 0 9 71

Control 4 38 0 0 0 0 38 390

Control 5 5 0 0 0 0 5 755

Control 6 10 0 0 0 0 10 82

Control 7 3 0 0 0 0 3 63

EVID4 Evidence Project Final Report (Rev. 06/11) Page 34 of 41

Table 5.5f Effects of root washing – number of nematodes in post-wash samples. Counts adjusted for root weight of 10g.

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Wash 1 2370

Wash 2 3010

Wash 3 274

Wash 4 10 10 21020

Wash 5 402

Wash 6 505

Wash 7 15000

Control 1 58 3 1 61 1401

Control 2 64 3 3 70 1403

Control 3 35 3 5 43 343

Control 4 67 67 687

Control 5 7 7 1057

Control 6 20 20 162

Control 7 15 15 283

Figure 5.9. Effects of root washing – number of plant-parasitic nematodes in samples pre-washing and 3

months post-washing Experiment 5.5 – dispersal of nematodes A new batch of Bonsai plants was purchased from a UK supplier. Each plant was re-planted with all the soil from its original pot at one end of a 1 metre long plastic trough containing sandy loam soil. Strawberry plants were planted (1 per trough) at different distances from the Bonsai plants e.g. 5 cm, 10 cm, 20 cm, 40 cm, 80 cm. The strawberry plants were separated from the bonsai plants with coarse mesh to allow the passage of free-living

EVID4 Evidence Project Final Report (Rev. 06/11) Page 35 of 41

nematodes but prevent the roots of the plants growing together. The troughs were placed in trays in a greenhouse compartment and grown for 6 months. They were watered carefully using drip irrigation. Soil samples were taken from around the strawberry plants, placed in polythene bags and sent to Fera for nematode identification and counts. The results are summarised in Figures 5.10-5.12. The numbers of nematodes were lower in this batch of plants than in the previous batch and there were insufficient numbers to draw any firm conclusions as to how far they will travel. The results also illustrate the difficulties associated with sampling these species, particularly when numbers are low. Table 5.6a. Numbers of nematodes in samples before the start of the trial.

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10a 13 101 24 17 10a 27 210 10a 8 59

10b 81 343 29 6 10b 140 591 10b 135 572

10c 52 186 32 21 10c 81 291 10c 25 89

10d 1 62 27 13 10d 2 115 10d 1 48

10e 1327 46 14 10e 1442 10e 948

20a 8 123 24 10 20a 17 256 20a 8 123

20b 127 35 26 20b 181 20b 49

20c 90 44 6 20c 102 20c 150

20d 237 43 18 20d 276 20d 132

20e 2 362 51 29 20e 2 355 20e 1 125

40a 216 41 17 40a 263 40a 127

40b 3 101 27 13 40b 6 187 40b 2 78

40c 61 35 21 40c 87 40c 29

40d 3 39 22 11 40d 7 89 40d 3 35

40e 17 136 34 11 40e 25 200 40e 15 124

80a 62 54 14 80a 57 80a 44

80b 117 39 11 80b 150 80b 106

80c 42 39 10 80c 54 80c 42

80d 22 41 9 80d 27 80d 24

80e 90 33 18 80e 136 80e 50 Table 5.6b. Numbers of nematodes in samples at the end of the trial.

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10a 360 328 1190 200 21 10a 17 16 57

10b 30 580 200 17 10b 2 34

10c 70 190 1440 200 13 10c 5 15 111

10d 590 64 1830 200 4 10d 148 16 458

10e 280 180 1050 200 27 10e 10 7 39

20a 200 128 1090 200 31 20a 6 4 35

20b 10 160 148 670 200 20 20b 1 8 7 34

20c 130 210 700 200 22 20c 6 10 32

20d 86 690 200 84 20d 1 8

20e 12 254 596 1450 200 20 20e 1 13 30 73

40a 158 256 1680 200 36 40a 4 7 47

40b 144 90 1260 200 48 40b 3 2 26

40c 405 380 1640 200 20 40c 20 19 82

40d 138 88 1510 200 31 40d 4 3 49

40e 130 158 1030 200 27 40e 5 6 38

80a 174 54 1270 200 28 80a 6 2 45

80b 160 140 2190 200 9 80b 18 16 243

80c 202 152 2210 200 35 80c 6 4 63

80d 10 26 18 1290 200 11 80d 1 2 2 117

80e 32 264 1680 200 33 80e 1 8 51

Cont 1 20 1690 200 16 Cont 1 1 0 106

Cont 2 10 1020 900 200 11 Cont 2 1 93 82

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Figure 5.10. Numbers of Heliocotylenchus sp. per sample from bonsai before the start of the trial and from

strawberry at the end of the trial. There were no Heliocotylenchus sp. on the control strawberry plants.

0

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Figure 5.11. Numbers of plant parasitic nematodes per sample from strawberry at the end of the trial.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 38 of 41

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Figure 5.12. Numbers of microbivorous nematodes per sample from bonsai before the start of the trial and from strawberry at the end of the trial.

Discussion There are a number of constraints when working with potential new pest species, one of which is that they must be confined to containment facilities, which limits the scope of experimental work, and secondly, it can be difficult to find and subsequently build-up sufficiently large populations from which to take samples for experimental work. This means that sample sizes are sometimes limited, although it can still be possible to get useful presence/absence information. Despite these constraints, this study produced some useful information on pest survival under cold conditions and on potential methods of control. Pests of solanaceous crops Tuta absoluta is now a well-established pest in parts of Europe and outbreaks occur regularly in greenhouses in the UK (http://www.fera.defra.gov.uk/plants/plantHealth/pestsDiseases/tomatoMoth.cfm and R. Jacobson, personal communication). A number of research projects funded by the HDC have addressed other aspects of the biology and life-cycle of the pest and evaluated management strategies (PC 302, 302 a, b; PE 002). It appears that due to new EMUAs for high volume sprays of Rynaxypyr (Coragen) and Spinosad (Conserve) and a recent Plant Health Authorisation for the use of Conserve through the irrigation system, there are currently sufficient tools to manage the pest using an integrated approach that does not compromise the IPM strategies used to control the other pests of protected tomato crops. This study has shown that T. absoluta is relatively „robust‟ and that each stage in the life-cycle will survive, albeit relatively short, periods of exposure to low temperatures that are not unrealistic for the UK, be this „outdoors‟ or in unheated structures. The larval stage is possibly the most vulnerable to cold, because low temperatures also have adverse effects on the preferred host plants, including black nightshade. Epitrix species are a potential threat to the UK potato crop since they have caused crop damage in Portugal and Spain (http://www.fera.defra.gov.uk/plants/plantHealth/pestsDiseases/epitrix.cfm). Two small-scale experiments were undertaken to test adult survival at low temperatures, as previous research in North America indicates that Epitrix species are likely to overwinter as adults in the soil (Kabaluk & Vernon, 2000). However, the adults exposed to low temperatures in this study survived for relatively short periods. These adults were reared under „summer‟ conditions and it is possible that adult beetles become more resilient if they are „pre-conditioned‟ through exposure to „autumn‟ conditions prior to their exposure to low temperatures. If time had permitted, this aspect would have been followed up. Reassuringly, it appears that a number of insecticide products are likely to be effective against this species, although these require evaluation on a field scale.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 39 of 41

This study has confirmed that there is considerable potential for soil pests to enter the UK on the roots of bonsai plants. Sampling consignments to detect such pests is obviously an issue, constrained by the small size of the pests and their uneven distribution between plants. This study has provided information to inform such sampling strategies. It has also confirmed that root washing is an effective way of removing most of a nematode infestation. As with, the other pests investigated experimentally, there appears to be the potential for introduced nematode pests to survive outdoors in the UK, for example if they are discarded on a compost heap. This must be an area for concern, although the likely impact on the environment, particularly on plants and other soil-inhabiting species is not well known. Outputs A presentation on the work on Tuta absoluta was made to the Tomato Growers Association Technical Committee on 5

th September 2012.

The information on T. absoluta will also be part of a presentation made by Rob Jacobson at the Tomato Conference on 27 September 2012 and will be incorporated into an HDC Factsheet due at the end of 2012 and in an article for HDC News in 2012-13 (both authored by Rob Jacobson). A paper on the ability of T.absoluta to survive exposure to low temperatures will be submitted to a peer-reviewed journal.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 40 of 41

References to published material

9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

Abercrombie, L.G., Anderson, C.M., Baldwin, B.G., Bang, I.C., Beldade, R., Bernardi, G., Boubou, A., Branca,

A., Bretagnolle, F., Bruford, M.W., Buonamici, A., Burnett, R.K. Jr., Canal, D., Cardenas, H., Caullet, C., Chen, S.Y., Chun, Y.J., Cossu, C., Crane, C.F., Cros-Arteil, S., Cudney-Bueno, R., Danti, R., Davila, J.A., Rocca, G., della Dobata, S. & Dunkle, L.D. 2009. Permanent genetic resources added to molecular ecology resources database. Molecular Ecology Resources 9(5): 1375-1379.

Bosa Ochoa, C.F. 2005. Pheromone-mediated communication disruption in Guatemalan potato moth, Tecia solanivora. Licentiate Thesis, Swedish University of Agricultural Sciences. (http://diss-epsilon.slu.se:8080/archive/00001013/01/FelipeBosa.pdf ) and Entomologia Experimentalis et Applicata 114(2): 137–142.

Boubou, A., Migeon, A., Lebdi-Grissa, K.& Navajas, M. 2009. Genetic diversity of the invasive tomato spider mite Tetranychus evansi (Acari: Tetranychidae) in the Mediterranean basin, assessed by sequences of the ribosomal internal transcribed spacers (ITS). IOBC/wprs Bulletin 49: 115-119. (http://www.iobc-wprs.org/pub/bulletins/bulletin_2009_49_table_of_contents_abstracts.pdf)

Carnero, A., Padilla, A., Perera, S., Hernández, E. & Trujillo, E. 2008. Pest status of Tecia solanivora (Povolny 1973) (Lepidoptera: Gelechiidae), Guatemalan Potato Moth, in the Canary Islands. IOBC/wprs Bulletin 31. Working Group “Insect Pathogens and Insect Parasitic Nematodes”, Proceedings of the Meeting at Alés (France), 3-7 June, 2007. Eds: Ralf-Udo Ehlers, Jürg Enkerli, Itamar Glazer, Miguel-López-Ferber & Cezary Tkaczuk. (http://www.iobc-wprs.org/pub/bulletins/bulletin_2008_31_table_of_contents_abstracts.pdf )

Castagnoli, M., Nannelli, R. & Simoni, S. 2006. Tetranychus evansi (Baker and Pritchard) (Acari Tetranychidae), a new pest for Italy [Lycopersicon esculentum Mill.; Liguria]. Agris record IT2007600796. (http://agris.fao.org/agris-search/search/display.do;jsessionid=C2463DE3B922A63C0AC4D1884642B125?f=2008/IT/IT0716.xml;IT2007600796)

Chitwood, D.J. 2002. Phytochemical based strategies for nematode control. Annual Review of Phytopathology 40: 221–49.

CSL. 2008. Pest risk analysis for Tetranychus evansi. (http://www.fera.defra.gov.uk/plants/plantHealth/pestsDiseases/documents/tetranychus.pdf)

Cuthberson A. 2011. Development rate of Tuta absoluta under UK glasshouse conditions. HDC Final Report Project.

Dangles, O., Carpio, C., Barragan, A.R., Zeddam, J.-L. & Silvain, J-F. 2008. Temperature as a key driver of ecological sorting among invasive pest species in the tropical Andes. Ecological Applications 18(7): 1795-1809. (http://www.esajournals.org/doi/abs/10.1890/07-1638.1)

Drees, B.M. & Jackman, J.A. 1998. A Field Guide to Common Texas Insects. Gulf Publishing Company. Texas. (http://insects.tamu.edu/fieldguide/cimg248.html)

Elmhirst, J. 2006. Crop Profile for Greenhouse Tomato in Canada. Pest Management Centre, EPPO 2001. Tecia solanivora introduced into Islas Canarias, Spain. EPPO reporting service. No. 7.

(http://archives.eppo.org/EPPOReporting/2001/Rse-0107.pdf) EPPO 2004.History of a disastrous biological invasion Tecia solanivora in Ecuador.

(http://www.invasive.org/library/eppo/Rse-0402.pdf) EPPO 2007. Draft data sheet on quarantine pests 07-13924. Tetranychus evansi. Fera 2010. A new moth pest to look out for on tomato, Keiferia lycopersicella. Plant Clinic News.

(http://www.fera.defra.gov.uk/plants/plantClinic/documents/plantClinicNews/march10Issue.pdf) Ferguson, G. & Shipp, L. 2009. Tomato Pinworm - Biology And Control Strategies For Greenhouse Tomato

Crops. Ontario Ministry of Agriculture. (http://www.omafra.gov.on.ca/english/crops/facts/04-025.htm) Gotoh, T., Araki, R., Boubou, A., Migeon, A., Ferragut, F. & Navajas, M. 2009. Evidence of co-specificity

between Tetranychus evansi and Tetranychus takafuji (Acari: Prostigmata, Tetranychidae): comments on taxonomic and agricultural aspects. International Journal of Acarology 35(6): 485-501. (http://pdfserve.informaworld.com/333203_751308121_917917912.pdf)

Ho, C-C. 2006. Monitoring on two exotic spider mites in Taiwan. (Paper-246339602[1]) Hurtado Ruiz, M., Cros-Arteil, S., Ansaloni, T., Jacas Miret, J.A. & Navajas, M. 2006. Molecular discrimination of

Tetranychidae mite species present in citrus orchards in Eastern Spain. IOBC wprs Bulletin 29(3): 139. Hurtado Ruiz, M., Cros-Arteil, S., Ansaloni, T., Jacas Miret, J.A. & Navajas, M. 2006. Molecular discrimination of

Tetranychidae mite species present in citrus orchards in Eastern Spain. IOBC wprs Bulletin 29(3): 139. Hurtado, M.A., Ansaloni, T., Cros-Arteil, S., Jacas, J.A. & Navajas, M. 2008. Sequence analysis of the ribosomal

internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination. Annals of Applied Biology 153(2): 167-174.

Ikeshima, K., Sakamaki, Y., Miyagi, A., Ohno, S., Fukuda, T. & Tsuda, K. 2009. Occurrence of the spider mite Tetranychus takafujii Ehara & Ohashi in Kyushu and Okinawa. Kyushu Plant Protection Research 55: 136-140.

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JEU. 2009. Official Journal of the European Union. C 190/16. (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:C:2009:190:0016:0021:EN:PDF)

Kabaluk, J.T. & Vernon, R.S. 2003. Effect of crop rotation on populations of Epitrix tuberis (Coleoptera: Chrysomelidae) in potato. Journal of Economic Entomology 93: 315-322.

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