Annual Report 2011

Biological control of perennial pepperweed, Lepidium latifolium

Jointly prepared by:

Esther Gerber, Hariet L. Hinz and Danielle Fife (CABI)

Massimo Cristofaro, Francesca Di Cristina, Francesca Lecce and Alessandra Paolini (BBCA, Rome, Italy)

Margarita Dolgovskaya (Zoological Institute, Russian Academy of Sciences, St Petersburg, Russia)

February 2012

CABI Ref: VMO1732 Issued February 2012

Biological control of perennial pepperweed, Lepidium latifolium

Annual Report 2011

Jointly prepared by: Esther Gerber, Hariet L. Hinz and Danielle Fife (CABI)

Massimo Cristofaro, Francesca Di Cristina, Francesca Lecce and Alessandra Paolini (BBCA, Rome, Italy)

Margarita Dolgovskaya (Biological control group, Russian Academy of Sciences, Zoological Institute, St Petersburg, Russia)

CABI Rue des Grillons 1, CH-2800 Delémont, Switzerland

Tel: ++ 41 32 421 4870 Fax: ++ 41 32 421 4871 Email: e.gerber@cabi.org

BBCA onlus Via del Bosco 10, 00060 Sacrofano (Rome), Italy

Tel: ++ 39 06 3048 3480 Fax: ++ 39 06 3048 6044 Email: massimo.cristofaro@casaccia.enea.it

Zoological Institute, Russian Academy of Sciences 199034 St Petersburg, Russia

Tel: ++7 812 714 5167 Fax: ++ 7 812 714 0444 Email: bcongroup@gmail.com

Sponsors: CABI received funding from the Wyoming Biological Control Steering Committee, USDA-APHIS-CPHST and the Bureau of Land Management, Idaho. BBCA received funding from the California Department of Food and Agriculture and the USDA-ARS Western Region Research Center, Reno, Nevada.

This report is the Copyright of CAB International, on behalf of the sponsors of this work where appropriate. It presents unpublished research findings, which should not be used or quoted without written agreement from CAB International and BBCA. Unless specifically agreed otherwise in writing, all information herein should be treated as confidential.

Table of Contents

Summary 1

1. Introduction 3

2. Work Programme for Period under Report 5

3. Field Surveys 5

3.1. Turkey 5

3.2. Russia 7

4. Ceutorhynchus marginellus SCHULTZE (Col., Curculionidae) 7

4.1. Overwintering and rearing 7

4.2. Oogenesis tests 7

4.3. Host-specificity tests 8

4.3.1. No-choice oviposition and larval devlopment tests 8

4.3.2. Multiple-choice cage tests 10

4.4. Conclusions and outlook 11

5. Phyllotreta reitteri (HEIKERTINGER) (Col., Chrysomelidae) 12

5.1. Overwintering and rearing 12

5.2. Host-specificity tests 13

5.2.1. No-choice larval transfer tests 13

5.2.2. Multiple-choice cage test 14

5.3. Conclusions and outlook 15

6. Open Field Test with Metaculus lepidifolii MONFREDA & DE LILLO (Acari, Eriophyidae) and Melanobaris sp. near semistriata (Col., Curculionidae) 16

6.1. Conclusions and outlook 17

7. Work Programme Proposed for 2012 18

8. Conferences 18

9. Acknowledgements 18

10. References 19

Annexes 21

1

Summary

1. Field surveys in 2011 focused on collecting four prioritized biological control agents. Several field trips were conducted in southern Russia by our Russian partners Drs Boris Korotyaev, Sergey Reznik and Alexey Moseyko (Russian Academy of Sciences, Zoological Institute, St Petersburg) and Sergey Grigoriev (Krasnodar). The Biotechnology and Biological Control Agency (BBCA) conducted four field trips to Turkey, one of which CABI joined.

2. Host-specificity tests with the gall-forming weevil Ceutorhynchus marginellus worked well under quarantine conditions at CABI in Switzerland and a considerable number of tests were conducted. Results indicate that perennial pepperweed, Lepidium latifolium, (PPW) is clearly the preferred host plant of C. marginellus. So far, only ten other species (eight Lepidium species, Rorippa sinuata and Stanleya pinnata) have been found to support adult development. Several of these species have already been tested in multiple-choice cage tests in southern Russia. Rorippa sinuata was not attacked under these conditions, but four Lepidium species, the native North Americans L. crenatum, L. huberi and L. latipes and the European L. perfoliatum, were attacked. Two of these species, L. crenatum and L. huberi, were subsequently exposed in an open-field test and were not attacked, indicating that the risk of non-target attack by C. marginellus on these species under natural conditions can be considered low. In order to further assess the risk to L. perfoliatum and L. latipes from C. marginellus, we plan to conduct an open-field test in 2012. In addition, species that supported development to adult under no-choice conditions, but have not been exposed under more natural conditions so far, will be tested in 2012. Work scheduled for 2012 includes further no-choice tests under quarantine conditions and the continuation of a test with L. crenatum and L. huberi to see if weevils reared from these test plants are able to sustain viable populations on them.

3. No-choice larval transfer tests with the stem-mining flea beetle Phyllotreta reitteri under quarantine conditions at CABI were continued to identify which test plant species will need to be tested in the field. In 2011, adults emerged from one additional species, the native North American Lepidium fremontii, resulting in a total of 17 plant species identified so far which can support development of this flea beetle. However, while the larval host range of Ph. reitteri under laboratory test conditions is rather broad, the species turned out to be very specific when tested in the field. Among the 14 test plants exposed in the field so far, only one single replicate of the North American species L. lasiocarpum was attacked, and only three dead larvae and no exit holes were found upon dissection. Based on these results we will continue considering Ph. reitteri as a potential agent. No-choice larval transfer tests in quarantine will continue in 2012. Preparations to carry out additional multiple-choice cage tests and an open-field test in 2012 have already been initiated in collaboration with our Russian partners. As in previous years, test plants will be simultaneously exposed to both C. marginellus and Ph. reitteri.

4. An open-field test was carried out to investigate acceptance of nine critical test plant species by the gall-forming mite Metaculus lepidifolii and the root-mining weevil Melanobaris sp. near semistriata in cooperation with Ortahisar Wine in Ortahisar, central Turkey. Due to a miscommunication, watering of plants in the experiment by our local collaborators was unfortunately stopped too early in the season. Upon harvest, most plants were dead and we could therefore not reliably evaluate whether they were attacked by the potential agents. Field tests are however essential at this

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point to decide if prioritized agents are host specific enough to be further considered. We will therefore continue our collaboration with Ortahisar Wine. Multiple-choice cage tests are planned for M. sp. near semistriata to clarify the acceptance of several test plant species under natural conditions. A detailed work plan has been prepared and an agreement between Ortahisar Wine, CABI and BBCA signed.

5. The main emphasis in 2011 was on host-specificity tests with prioritized potential agents, conducted both in quarantine and in the field in the native range of the insects. Work will continue with the four potential agents and will, as in 2011, focus on host-specificity tests.

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1. Introduction

Perennial pepperweed (PPW), Lepidium latifolium L. (syn.: Cardaria latifolium), (Brassicaceae) is a herbaceous, semi-woody perennial that typically reaches 0.5–1.5 m in height and reproduces vegetatively and by seed (Renz, 2000). Plants regrow early each year from a dense network of creeping, horizontal roots, flower in June/July, and set seeds in July/August. It is a prolific seed producer, capable of producing more than six billion seeds per acre of infestation (Miller et al., 1986).

PPW is native to central Asia and is believed to have been introduced into the USA through California around 1900 as a contaminant of sugar beet seeds (Renz and DiTomaso, 1998). It is now widely distributed in the western USA, coastal New England, Mexico and Canada (Young et al., 1995; Chen et al., 2002). Although recorded in the USA for more than 100 years, the species only started invading a wide range of habitats throughout the western part of the country in the past two decades (Young et al., 1998). It is especially prevalent in Nevada, Oregon, Utah and California, and declared noxious or prohibited in 15 US states (USDA, NRCS, 2010) and one Canadian province (Rice, 2010).

PPW is often associated with mesic habitats, such as river banks, drainage ditches, and subirrigated pastures and hay meadows. However, it can invade a wide range of habitats including pastures, open fields, roadsides and residential areas (Young et al., 1998). Downstream movement of seeds and root fragments along waterways and irrigation systems is the primary mode of spread for this weed. PPW is highly competitive and invasions result in dense monocultures and subsequent loss of biodiversity through the exclusion of native vegetation. When dense stands establish along rivers and streams, the plant interferes with the regeneration of important native willow species and cottonwoods (Young et al., 1995), reducing availability of cover and food for nesting waterfowl (Trumbo, 1994), displacing threatened, endangered and rare species such as the salt marsh harvest mouse (Reithrodontomys raviventris) (Trumbo, 1994) and Suisun marsh aster (Aster lentus) (Skinner and Pavlik, 1994), and eliminating shading for fish and aquatic insects (Blank and Young, 1997). In areas that are not mown annually, semi-woody stems can accumulate over the years, negatively impacting on nesting habitat for birds and hindering the grazing and movement of livestock and wildlife (Renz, 2000). In addition, PPW can alter the habitat it invades by increasing salinity (Blank and Young, 1997), thereby hindering regeneration of native flora. In agricultural settings, the species competes with crop plants and reduces agricultural yields (Eiswerth et al., 2005). PPW contains fewer nutrients for livestock than naturally occurring vegetation and therefore decreases the quality of hay and reduces livestock-carrying capacity in rangelands (Eiswerth et al., 2005). Furthermore, secondary plant compounds produced by PPW are reported to be toxic to livestock (Whitson, 1987).

PPW is difficult to control because of its large, stout root system. Mechanical or cultural control techniques usually provide no permanent reduction of populations (Young et al., 1998; Renz and DiTomaso, 2006). Plants quickly resprout after mowing, and cultivation severs and transports root fragments that rapidly sprout new plants. When feasible, deep, repeated cultivation can suppress PPW. Grazing by sheep or goats may reduce weed density temporarily, but does not provide long-term control (Allen et al., 2001). Application of herbicides containing chlorsulfuron is most effective for suppressing PPW; however, the substance is not registered for use in many invaded habitats, such as in areas adjacent to water (Renz and DiTomaso,

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2006). Phenoxy herbicides, such as 2,4-D and clopyralid, will kill the shoots of PPW, but root crowns will quickly sprout new foliage. Applications may need to be repeated for up to five years to deplete its abundant root reserves.

In 2004 and 2005, CABI in Switzerland had received modest start-up funding from the Idaho State Department of Agriculture through Dr Mark Schwarzlaender at the University of Idaho to conduct preliminary literature and field surveys for insects, mites and fungi associated with PPW in Europe. In addition BBCA (Biotechnology and Biological Control Agency) has been conducting opportunistic surveys in conjunction with other weed biocontrol projects since 2002. Because PPW may prove to be a challenging target for weed for biological control, and to avoid overlap, CABI and BBCA decided to share resources and collaborate closely in their effort towards developing biological control for PPW. Since 2006, field surveys have been conducted in cooperation and results from both research teams are presented jointly in this report.

Based on field collections in various countries within the native range of PPW, 113 phytophagous organisms were sampled or reared, five of which were prioritized as potential biological control agents: the root-mining weevil Melanobaris sp. near semistriata (Coleoptera, Curculionidae), the gall-forming weevil Ceutorhynchus

marginellus Schultze (Coleoptera, Curculionidae), the stem-mining flea beetle Phyllotreta reitteri (Heikertinger) (Coleoptera, Chrysomelidae), the gall-forming eriophyid mite Metaculus lepidifolii Monfreda & de Lillo (Acari, Eriophyidae) and the stem-mining fly Lasiosina deviata Nartschuk (Diptera, Chloropidae). Investigations on these potential agents have focused on their biology, phenology, distribution and host range.

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2. Work Programme for Period under Report

The following work plan was developed by CABI and BBCA for 2011 and proposed in the last annual report (Gerber et al., 2011).

Ceutorhynchus marginellus

• Continue no-choice oviposition and larval development tests in quarantine; • Conduct oogenesis tests with Lepidium huberi and L. crenatum; • Collect weevils at field sites in Russia and import insects into quarantine for

host-specificity tests in 2012; • Conduct a multiple-choice cage test in southern Russia, provided sufficient

insects are available and Rorippa sinuata is available for testing.

Phyllotreta reitteri

• Continue no-choice larval transfer tests; • Collect infested plant material and/or adults in southern Russia and import

insects into quarantine for host-specificity tests in 2012; • Conduct a multiple-choice cage test in southern Russia on test species that

supported adult development in no-choice larval transfer tests.

Melanobaris sp. near semistriata

• Repeat the open-field test in Turkey.

Metaculus lepidifolii

• Repeat the open-field test in Turkey.

Lasiosina deviata

• Potentially include this species in the open-field test with Melanobaris sp. near semistriata and Metaculus lepidifolii in Turkey.

3. Field Surveys

3.1. Turkey

Several trips were made by Dr Massimo Cristofaro and his team: on 18 April, test plants were taken to Ortahisar, central Turkey for an open-field test (section 6). In mid May, these plants were set up in the field and from 19 to 21 May, adults of M. sp. near semistriata were collected in eastern Turkey, taken to Ortahisar and released at the site. On 24 May, mite-infested PPW shoots were collected at a field site near Kayseri and used for infestation in the open-field test. Five weeks later, on 7 July, PPW shoots were removed and the state of plants was recorded. From 23 to 26 August, Massimo Cristofaro, Francesca Di Cristina and Dr Esther Gerber travelled to Ortahisar to wind up the experiment. On the same occasion, a short survey trip was made and an additional PPW site discovered near the town of Avanas. At this site, a PPW patch was discovered where several pupae and fully developed adults of M. sp. near semistriata were found in the roots of plants (Fig. 1). Attacked plants in this patch were noticeably smaller than plants growing in the vicinity that showed no sign of M. sp. near semistriata attack. Another site with high abundance of M. lepidifolii

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was visited in the area of Ürgüp. Various degrees of damage were observed on PPW: on some plants, only single inflorescences were attacked, while on others, all inflorescence was affected; some shoots failed to produce inflorescences altogether (Fig. 2, shoots on the right). We hypothesize that different damage levels reflect different timing of attacks and/or different infestation levels.

Figure 1. Mining (left) and fully developed adult (right) of Melanobaris sp. near semistriata in PPW roots (photos: Massimo Cristofaro).

Figure 2. Different degrees of deformation of PPW inflorescences due to attack by Metaculus lepidifolii compared to an unattacked shoot on the left (photo: Esther Gerber).

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3.2. Russia

In early April 2011, Dr Gena Davidyan (Russian Academy of Sciences, Zoological Institute, St Petersburg) collected 40 adults of the gall-forming weevil Ceutorhynchus marginellus, and more than 150 of the shoot-mining flea beetle Phyllotreta reitteri in the region close to the Sea of Azov in southern Russia, most of which were later used in the multiple-choice cage tests (sections 4.3.2 and 5.2.2). Between 29 and 31 May 2011, Dr Boris Korotyaev (Russian Academy of Sciences, Zoological Institute, St Petersburg) and Sergey Grigoriev collected an additional 281 C. marginellus and shoots containing larvae and pupae of Ph. reitteri in the same area (see sections 4 and 5). Collected material was transferred to the laboratory of Dr Margarita Dolgovskaya and her team, where Ph. reitteri adults were successfully reared. Shipments of 253 adult C. marginellus and 81 adult Ph. reitteri were sent on 2 July from St Petersburg to Switzerland to augment our rearing colonies in quarantine.

4. Ceutorhynchus marginellus SCHULTZE (Col., Curculionidae)

Ceutorhynchus marginellus is a gall-forming weevil native to Asia. The species was found at field sites in Kazakhstan, southern Russia, western China and eastern Turkey. The population used in host-specificity testing originates from southern Russia. Ceutorhynchus marginellus lays eggs in early spring. Galls are formed on leaf midribs, leaf stalks and stems, and adults emerge from mid April onwards. The weevil has one generation per year.

4.1. Overwintering and rearing

All C. marginellus reared from PPW plants in quarantine and collected in the field were kept in cylinders for overwintering in incubators at 2°C and regularly provided with cut PPW leaves and shoots (see Gerber et al., 2011, section 5.1). On 7 March 2011, weevils were taken out of incubators. Of the 104 adults initially set up in cylinders, 87 (84%) survived (i.e. 50 females and 37 males).

On 21 April, ten freshly field-collected C. marginellus (seven females and three males) were sent from St Petersburg and tested for egg laying. All but two females laid eggs, indicating that shipping them to Switzerland did not affect oviposition. Field collection of weevils early in the season can therefore be considered in years with low winter survival of weevils kept in quarantine.

All C. marginellus reared from PPW plants during 2011 and collected in the field (see sections 3.2 and 5.2) were kept in cylinders and regularly provided with cut PPW leaves and shoots until 19 October, at which time the weevils (133 females and 97 males) were transferred to an incubator with an 8/16 h day/night cycle. The temperature was gradually lowered to reach 2°C on 21 November and will be kept at this level until the end of February 2012.

4.2. Population viability tests

Tests are being conducted to check if viable populations can be sustained on non-target hosts. Tests in previous years have shown that C. marginellus can complete its development in two North American species, Lepidium crenatum and L. huberi. Both species were also attacked in multiple-choice cage tests, but not under open-field

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conditions (Gerber et al., 2011, section 5.2.1, 5.2.2). To further elucidate the suitability of these two plant species as potential alternative hosts for C. marginellus, we initiated a test to see if weevils reared on these non-targets could produce fertile eggs. This would indicate whether or not C. marginellus is able to sustain a population on these non-targets in the absence of PPW.

METHODS Between 15 March and 1 April, two female and one male C. marginellus were released upon individually potted, gauze-covered L. crenatum and L. huberi plants. Prior to being used in tests, adults were tested for oviposition. Single pairs were placed into cups provided with a PPW leaf inserted in a moist florist foam block. After 3 days, the plant material was dissected to record eggs; only egg-laying pairs were subsequently used in tests. After 4–6 days, adults were retrieved and the plants re-covered with gauze bags. From 25 May onwards, plants were regularly checked for emerged adults.

RESULTS Unexpectedly, emergence was rather low. For L. crenatum, a total of five adults (two females and three males) emerged from just three out of seven replicates. For L. huberi, six females and ten males emerged from five out of seven replicates. However, most (N = 12) emerged from one L. huberi plant; all other plants produced a single adult only. Retrieved adults were set up in cylinders and provided with cut leaves and shoots of their respective host plant species inserted in a florist foam block. Plant material was regularly changed, and at the end of October, weevils were transferred to an incubator for overwintering (see also section 4.1). Tests will continue in March 2012.

4.3. Host-specificity tests

4.3.1. No-choice oviposition and larval development tests

METHODS From 15 March onwards, two female and one male C. marginellus were released upon individually potted, gauze-covered test or PPW plants. Prior to being used in tests, females were tested for oviposition (see section 4.2). Only females that laid eggs were subsequently used. After 4–7 days, adults were retrieved from the plants and all adults re-tested for oviposition (see section 4.2). Weevils that continued to lay eggs were placed onto a new series of plants. Up to 17 May, 1–5 replicates of 28 test plant species and 31 plants of PPW were exposed in this way. All plants were checked for galls at least two weeks after infestation and all plants with galls were kept for adult emergence. All other test plant species were dissected but as soon as eggs and/or larvae were found in one replicate of a test plant species, the remaining replicates were also kept for adult emergence. All PPW were kept to rear adults for tests in 2012. From 12 May onwards, test and PPW plants were regularly checked for emerged adults. Retrieved adults from PPW were set up in cylinders, and on 19 October the cylinders were transferred to an incubator for overwintering (see also section 4.1).

RESULTS All but two of the PPW plants exposed were attacked (i.e. mines and/or larvae were recorded upon dissection) and adults emerged from 20 plants (Table 1). Of the 28 test plant species and varieties tested, 18 were not attacked at all (Table 1). In three species, Caulanthus crassicaulis, Raphanus sativus and Thelypodium laciniatum, only eggs were found. Larvae were found in six test plant species. Adults emerged from three Lepidium species: the native North American species L. latipes

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and L. papilliferum as well as L. perfoliatum. In addition, one single adult developed in the North American species Stanleya pinnata.

Table 1. Results of no-choice oviposition and development tests with Ceutorhynchus marginellus in 2011.

# replicates # eggs

Plant species valid attackedb with adult

emergence and/or larvae

found # adults

emergedc

Brassicaceae

Lepidium latifolium Europe 19 19 11 25 70 (5.4 ± 0.9) USA 12 10 9 2 78 (8.7 ± 2.7) Alyssum saxatilis 1 0 - 0 Arabidobsis thaliana 3 0 - 0 Arabis aculeolataa 2 0 - 0 Boechera blepharophyllaa 3 0 - 0 Boechera holboelliia 4 2 0 3 Brassica napus 1 0 - 0 Brassica oleracea var. sabauda 1 0 - 0 Brassica oleracea var. italica 2 0 - 0 Brassica rapa 1 0 - 0 Cardamine cordifoliaa 3 0 - 0 Caulanthus crassicaulisa 2 1 - 1 Descurainia nelsonii a 1 0 - 0 Lepidium appelianuma 2 1 0 7 Lepidium fremontiia 5 0 - 0 Lepidium latipesa 2 2 2 4 7 (3.5 ± 1.5) Lepidium papilliferuma 2 2 1 5 4 Lepidium perfoliatum 3 3 3 4 17 (5.7 ± 1.7) Lepidium sordiduma 2 0 - 0 Nasturtium gambeliia 3 0 - 0 Parrya nudicaullisa 2 0 - 0 Raphanus sativus 2 1 - 1 Sisymbrium linifoliuma 4 1 0 1 Stanleya pinnataa 2 1 1 0 1 Streptanthus farnsworthianusa 2 0 - 0 Streptanthus glandulosus ssp. nigera 2 0 - 0 Thelypodium laciniatuma 3 1 0 14 Thlaspi arvense 2 0 - 0

Cleomaceae

Peritoma lutea 2 0 - 0 - Not recordable (plants completely dissected). a Species native to North America. Nomenclature according to Boufford et al. (2010). b Containing mining and/or larvae (living and/or dead). c Values given in brackets are mean ± SE emerged weevils, calculated for replicates with adult

emergence.

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4.3.2. Multiple-choice cage tests

Tests conducted in 2009 revealed that C. marginellus also develops in Rorippa sinuata, a species native to North America (Gerber et al., 2010). We therefore included this species in multiple-choice cage tests, otherwise mainly designed to test acceptance of several test plant species by Ph. reitteri (section 5.2.2).

METHODS In December 2010, seeds of L. lasiocarpum, L. latifolium (PPW) L. latipes, L. oblongum, L. perfoliatum, L. sativum, L. squamatum, Lesquerella fendleri, Nasturtium officinale and R. sinuata were sent to St Petersburg, grown in a laboratory greenhouse and the plants taken to southern Russia in early spring 2011. At the beginning of April, 1–4 replicates of the test plant species as well as 5–6 PPW were randomly arranged in four 1 × 1 × 1 m plots. On 23 April, screen cages were established over each plot (Fig. 3) and 12 Ph. reitteri were released in each cage. In addition, 12 C. marginellus (six female and six male) were released in two of the cages. All insects had been field collected in spring 2011 (see section 3.2). After 15 days, the cages were removed.

On 2 June, i.e. approximately 40 days after releasing the insects, all plants were harvested. Plants were taken to the laboratory in St Petersburg, dissected under a stereo microscope, and the number of galls and/or mines and larvae recorded. In most plants, only empty galls and/or mines were found, suggesting that larvae had already left the plants. These plants were considered as attacked.

Larvae found in each plant were transferred into vials filled with alcohol and sent to CABI, where the head-capsule diameters were measured and compared with head-capsule sizes recorded for C. marginellus. In addition, plant material was dried in laboratory conditions (room humidity c. 50%) and the dry weight of each plant was measured.

Figure 3. Dr Sergey Reznik releasing insects in multiple-choice test cages in the garden of our collaborator Sergey Grigoriev in southern Russia (left) and plants prior to harvest (right) (photos: Sergey Grigoriev).

RESULTS Head-capsule measurements of larvae recorded in PPW and test plant species revealed that all were well within the range recorded for C. marginellus. All

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but three PPW were attacked by C. marginellus, i.e. on average 71.6 ± 11.6% of the plants exposed in each cage (Table 2). None of the R. sinuata plants exposed was attacked. Galls were only found on two Lepidium species, i.e. on the native North American species L. latipes, and on L. perfoliatum, native to Europe. Attack rates were however lower than on PPW, and L. latipes was only attacked in one of the cages (Table 2). Most C. marginellus had already left the plants to pupate in the soil, therefore no attack levels could be calculated; larvae were found in only one PPW and two replicates established with L. perfoliatum.

Table 2. Results of multiple-choice cage test on Brassicaceae species with Ceutorhynchus marginellus in 2011.

Cage 1 Cage 2 Plant species # plantsb % attacked # plantsb % attacked

Lepidium latifolium (PPW) 5 (3) 60 6 (5) 83.3 Lepidium lasiocarpuma 3 (0) 0 2 (0) 0 Lepidium latipesa 2 (1) 50 2 (0) 0 Lepidium oblonguma 3 (0) 0 3 (0) 0 Lepidium perfoliatum 3 (1) 33.3 3 (1) 33.3 Lepidium sativum 2 (0) 0 2 (0) 0 Lepidium squamatum 1 (0) 0 2 (0) 0 Lesquerella fendleria 1 (0) 0 - - Nasturtium officinale 2 (0) 0 2 (0) 0 Rorippa sinuataa 3 (0) 0 3 (0) 0

a Species native to North America. Nomenclature according to Boufford et al. (2010). b Values given in brackets are number of replicates attacked (including plants with mining only).

4.4. Conclusions and outlook

Host-specificity tests under quarantine conditions advanced well in 2011 and included 28 test species, 18 of them native to North America. Adults emerged from four test plant species, i.e. from Lepidium perfoliatum and from three North American species: L. latipes, L. papilliferum and Stanleya pinnata. Since the start of testing in 2008 we have been able to test 52 test species and/or varieties, 27 of them native to North America (Annex 1). While results so far indicate that PPW is clearly the preferred host of Ceutorhynchus marginellus, complete adult development was also recorded in ten other species. Eight of these are in the same tribe as PPW (Lepidieae), one from a closely related tribe (Cardamineae) and only one from a more distantly related tribe (Schizopetaleae) within the family Brassicaceae (Annex 1).

Adults emerged from eight out of 13 Lepidium species tested, including five species native to North America. For two of these, L. crenatum and L. huberi, a test was initiated in 2011 to see if weevils reared from these non-targets would be able to produce fertile eggs in 2012 when overwintered on the plant species they emerged from. Unexpectedly few adults emerged from the test plants, in particular from L. crenatum, indicating that these species are only suboptimal hosts for C. marginellus. Adults emerged also from Rorippa sinuata in the tribe Cardamineae (Gerber et al., 2010, Annex 1). However, when exposed to C. marginellus in multiple-choice cage tests in 2011, the species was not attacked. This result indicates that the risk of attack under natural conditions is low.

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In no-choice tests conducted in 2011, a single adult developed in S. pinnata in the tribe Schizopetaleae. Stanleya pinnata has been found to be attacked by other potential agents investigated for brassica weeds at CABI, e.g. by the gall-forming weevil C. cardariae on hoary cress (Lepidium draba) (Hinz et al., 2004). Naturally growing Stanleya species accumulate selenium, which has been shown to act as a defence against insect herbivores (Hanson et al., 2003; Freeman et al., 2007) and attack by C. marginellus might stem from the fact that our potting soil does not contain selenium. The influence of selenium on attack by the flea beetle Psylliodes isatidis was tested; this species is currently being investigated for the biological control of dyer’s woad (Isatis tinctoria). The tests revealed that selenium-treated plants of the closely related Stanleya viridiflora did not support adult development, while non-treated plants did (Hinz et al., 2012).

Multiple-choice cage tests carried out in 2011 in the native range of C. marginellus revealed that both L. perfoliatum and L. latipes, a species native to North America, were attacked. Combined with data from previous years, four out of ten Lepidium species tested in multiple-choice cage tests were attacked. Two of these species, L. crenatum and L. huberi, were subsequently exposed in an open-field test and were not attacked, indicating that the risk of non-target attack by C. marginellus on these species under natural conditions can be considered low. In order to further assess the risk to L. perfoliatum and L. latipes from C. marginellus, we plan to conduct an open-field test in 2012. In addition, the acceptance of S. pinnata and L. papilliferum, two species that supported development to adult in 2011 (see above), will be tested in multiple-choice cage tests. As in previous years, these tests will be conducted in combination with Ph. reitteri (see below). Finally, we will also continue with no-choice tests in quarantine at CABI.

5. Phyllotreta reitteri (HEIKERTINGER) (Col., Chrysomelidae)

The stem-mining flea beetle Ph. reitteri was first found at field sites in Kazakhstan in May 2006, where larval mining caused the dieback of shoots. The species was subsequently also found in western China, southern Russia and central Turkey. The population used in host-specificity testing originates from southern Russia. In quarantine, eggs are laid in early spring. Larvae mine in petioles and shoots, and adults emerge from mid April onwards.

5.1. Overwintering and rearing

Adults that emerged in 2010 (N = 97) were overwintered in an incubator at 2°C with an 8/16 h day/night cycle (see section 6.1, Gerber et al., 2011). Up to 7 March 2011, 33 (34%) survived. They were placed in cylinders and provided with cut leaves of PPW inserted in blocks of florist foam. Leaf material was regularly changed and the foam blocks checked for eggs. Beetles started to lay eggs on 16 March. Eggs were placed into Petri dishes and hatched larvae transferred onto potted test or PPW plants (see section 5.2).

Adults reared from plant material collected in the field (section 3.2) as well as adults that emerged at CABI in 2011 were kept in cylinders and regularly provided with cut PPW leaves and shoots until 19 October, at which time the beetles (N = 110) were transferred to an incubator with an 8/16 h day/night cycle. The temperature was

13

gradually lowered to reach 2°C on 21 November and will be kept at this level until the end of February 2012.

5.2. Host-specificity tests

5.2.1. No-choice larval transfer tests

METHODS Between 5 April and 24 June, 914 larvae were transferred. Eight to 33 first-instar larvae, depending on plant size, were transferred with a fine paint brush onto 70 plants, i.e. 20 controls (PPW) and 27 test species with one to four replicates of each. At least one week after the last transfers, test plant species were dissected but as soon as larvae were found in one replicate of a test plant species, the remaining replicates were kept for adult emergence. For each PPW plant, only one leaf and/or petiole with larval entrance holes was dissected to confirm attack, while the rest of the plant was kept for adult emergence.

RESULTS Eighteen out of 20 PPW infested were attacked and adults emerged from 11 plants (Table 3). For both PPW populations combined (i.e. Europe and USA), 23.7 ± 5.7% of the larvae transferred developed into adults. Valid replicates were obtained from 26 of 27 test plant species tested. Nine species were not attacked at all (Table 3). In three test plant species, i.e. Descurainia nelsonii, Draba albertina and Lepidium appelianum, only mining was found. Further, three species only contained dead larvae, i.e. Berteroa incana, Iberis umbellata and L. ostleri. Adults emerged from just one species, the North American L. fremontii (Table 3).

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Table 3. Results of no-choice larval transfer tests with Phyllotreta reitteri in 2011.

Total # # of replicates % larvae

developed

Plant species larvae

transferred valid repl. attackedb

with adults emerged (mean±SE)c

Lepidium latifolium USA 222 16 14 8 19.2 ± 4.8 Europe 80 4 4 3 36.1 ± 5.2 Arabidopsis thaliana 24 2 2 0 Arabis aculeolataa 20 2 2 0 Aubrieta deltoidea 29 2 0 0 Berteroa incana 33 2 1 0 Boechera ligniferaa 24 2 2 0 Brassica juncea 20 1 1 0 Brassica rapa 19 2 0 0 Cardamine cordifolia 41 2 1 0 Descurainia nelsoniia 22 2 1 0 Draba albertinaa 30 2 1 0 Iberis umbellata 10 1 1 0 Isatis tinctoria 20 2 0 0 Lepidium appelianum 10 1 1 0 Lepidium barnebyanuma 8 0 0 0 Lepidium didymum 20 2 2 0 Lepidium fremontiia 52 3 3 2 12.3 ± 7.7 Lepidium montanuma 17 2 2 0 Lepidium ostleria 26 2 1 0 Lepidium sordiduma 38 4 0 0 Lepidium virginicuma 14 1 1 0 Noccaea fendleri ssp. siskiyouensea 10 1 0 0 Noccaea fendleri ssp. idahoensea 10 1 0 0 Physaria chambersiia 20 2 0 0 Sisymbrium linifoliuma 45 3 2 0 Streptanthus farnsworthianusa 20 2 0 0 Streptanthus gladulosus ssp. nigera 10 1 0 0 Thlaspi arvense 20 2 1 0

a Species native to North America. Nomenclature according to Boufford et al. (2010). b Containing mining and/or larvae (living and/or dead). c Calculated for replicates with adult emergence.

5.2.2. Multiple-choice cage test

Tests conducted in quarantine in previous years indicated a rather wide physiological host range for Ph. reitteri both under no-choice and single-choice conditions. Multiple-choice cage tests in 2010 in southern Russia did however reveal a high degree of specificity of this beetle; none of the test plant species exposed was attacked (Gerber et al., 2011, section 6.3.1). In 2011 we continued testing plant species that supported development to adult in quarantine under cage conditions. These tests were set up in conjunction with C. marginellus (for methods see section 5.3.2).

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RESULTS Over all four cages, on average 81.6 ± 6.9% of PPW plants were attacked. One test species, L. lasiocarpum, was attacked, although only three dead larvae and no emergence holes were found in this plant (Table 4). As for C. marginellus, most larvae had already left the plants to pupate in the soil. In 12 replicates established with PPW, a few larvae were still present, although most mines were empty.

Table 4. Results of multiple-choice cage test on Brassicaceae species with Phyllotreta reitteri in 2011.

Plant species Total # plantsb % plants with miningc Attackc,d Lepidium latifolium (PPW) 21 (17) 81.6 ± 6.9 1.0 ± 0.7 Lepidium lasiocarpuma 13 (1) 7.7 ± 6.2 0.2 ± 0.4 Lepidium latipesa 10 (0) Lepidium oblonguma 13 (0) Lepidium perfoliatum 12 (0) Lepidium sativum 8 (0) Lepidium squamatumd 4 (0) Nasturtium officinale 8 (0) Physaria fendleria 1 (0) Rorippa sinuataa 10 (0)

a Species native to North America. Nomenclature according to Boufford et al. (2010). b Values given in brackets are number of replicates attacked (including plants with mining only). c Values are means ± SE. d Number larvae in attacked replicates. e Tested in three cages.

5.3. Conclusions and outlook

No-choice larval transfer tests advanced very well in 2011 and included 27 test species, 16 of them native to North America. Adults emerged from only one test plant species, the North American Lepidium fremontii. Summarizing data from 2007 to 2011, no-choice larval transfer tests have been conducted on 60 test plant species and varieties (34 native to North America); 41 showed signs of larval mining and adults emerged from 17 of these (see Annex 2). The larval host range of Phyllotreta reitteri is therefore rather broad. Non-target attack occurred mainly on closely related species; adults emerged from 13 out of 19 Lepidium species tested. In addition, species from three other tribes were attacked (Annex 2). However, these results are from larval transfer tests, and thus do not take into account the oviposition behaviour of females. Test plant species supporting adult development in previous years were subsequently tested under multiple-choice conditions in the field (see below). Lepidium fremontii will be tested in 2012.

As in previous years, multiple-choice cage tests carried out in the native range of Ph. reitteri in southern Russia worked well. On average 81% of PPW plants were attacked. Among test plants, only one single replicate of the North American species L. lasiocarpum was attacked, and only three dead larvae and no exit hole were found upon dissection. Results therefore further confirm the narrow field host range of Ph. reitteri, i.e. so far no other plant species has been consistently attacked.

Based on these results we will continue considering Ph. reitteri as a potential agent. No-choice larval transfer tests in quarantine will continue in 2012 to identify which test

16

plant species will need to be tested in the field. Preparations to carry out tests in southern Russia in 2012 have already been initiated in collaboration with our Russian partners. Both multiple-choice cage tests (to test acceptance of L. fremontii) and open-field tests (to further assess the risk to L. lasiocarpum) will be conducted. As in 2011, test plants will be simultaneously exposed to both C. marginellus (see section 4.3) and Ph. reitteri.

6. Open Field Test with Metaculus lepidifolii MONFREDA & DE

LILLO (Acari, Eriophyidae) and Melanobaris sp. near semistriata (Col., Curculionidae)

To further explore the host range of two additional potential agents, Metaculus lepidifolii and Melanobaris sp. near semistriata, an open-field test was conducted with a selection of critical test plants, i.e. Barbarea orthoceras, Descurainia incana, Lepidium crenatum, L. huberi, L. oblongum, L. campestre, L. draba, Nasturtium officinale and Rorippa sinuata. Metaculus lepidifolii is a newly described eriophyid mite species, sampled on several occasions in Turkey. Particularly heavy mite attack completely inhibits flowering and/or seed production (Fig. 2). Melanobaris sp. near semistriata is root-mining weevil that has been found at several field sites in Turkey (see section 3.1). Females lay eggs in spring; under quarantine conditions, oviposition continued until the beginning of June. Weevils used in host-specificity tests originated from sites near Erzurum, eastern Turkey.

METHODS All test and PPW (control) plants used in the experiment were grown from seeds and were propagated at BBCA. On 18 April, plants were transported to Ortahisar, central Turkey. Between 15 and 19 May, nine test plant species and PPW were arranged in a Latin square design in a 10 × 10 m field plot (Fig. 4). On 24 May, 43 M. sp. near semistriata adults, previously collected in eastern Turkey, were released. The same day, bouquets of cuttings of PPW infested with Metaculus lepidifolii, collected at a field site near Kayseri, were placed next to young leaves in each potted test species or PPW. Five weeks later, on 7 July, PPW cuttings were removed and plant survival recorded. It was agreed with Philipp Gfeller and his local collaborators (Ortahisar Wine) that plants in the experiment should be watered twice a week from the start of the experiment until August, and that the surrounding vegetation should be regularly cut to provide optimal growing conditions for the test plants. On 24 August, all plants were harvested.

RESULTS During a visit on 7 July by Massimo Cristofaro, it was noted that plant survival varied between 10% and 100% according to species. For three species, D. incana, L. crenatum and L. huberi, survival was low (10–30%). For at least one of these species, L. huberi, mortality occurred immediately after plants were taken out into the field (M. Cristofaro, pers. obs.), i.e. this species did not survive transplantation well. All surviving plants looked well, indicating that the watering regime was sufficient. Unfortunately, due to a misunderstanding, watering was stopped at the beginning of August and when we visited the site on 24 August to harvest the plants, most plants were dead. Where still possible, roots were dissected and signs of what have might been mining by Melanobaris sp. near semistriata were recorded on two PPW.

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Figure 4. Open-field test with Metaculus lepidifolii and Melanobaris sp. near semistriata in Turkey (left); PPW plant exposed in the experiment (right) (photos: Massimo Cristofaro).

6.1. Conclusions and outlook

Carrying out the field test in Turkey proved again to be challenging. However, and unlike in previous years, this time the failure was mainly due to a communication problem and not to climatic conditions and/or lack of maintenance. As described above for Ceutorhynchus marginellus and Phyllotreta reitteri, field tests are essential at this point to decide if prioritized agents found in Turkey are specific enough to be further considered as potential agents. We will therefore continue our collaboration with Ortahisar Wine.

Multiple-choice cage tests are planned for Melanobaris sp. near semistriata to clarify the acceptance of several test plant species that have been attacked in previous no-choice tests. In order to improve growing conditions, i.e. to increase soil moisture, plants will be planted in ground depressions. In addition, we will explore if the weevil is able to complete development in potted PPW plants in the field. Complete adult development on PPW in quarantine has been observed; it is however unreliable and results of no-choice development tests are therefore less credible, which makes conclusions drawn from them less reliable. Tests in 2012 will indicate if conducting no-choice tests with some plant species in Turkey is a viable option. In addition, host-range evaluations for Metaculus lepidifolii will be included in the multiple-choice cage tests.

A detailed work plan has been prepared and an agreement between Ortahisar Wine, CABI and BBCA signed.

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7. Work Programme Proposed for 2012

Based on results obtained during the field season 2011, it is proposed that work in 2012 should focus on:

Ceutorhynchus marginellus

• Continue no-choice oviposition and larval development tests in quarantine; • Continue tests with Lepidium huberi and L. crenatum to see if females produce

fertile eggs when continuously reared on these non-targets; • Collect weevils at field sites in Russia and import insects into quarantine for

host-specificity tests in 2013; • Conduct a multiple-choice cage test and open-field tests in southern Russia on

test species that supported adult development in no-choice oviposition and larval development tests.

Phyllotreta reitteri

• Continue no-choice larval transfer tests in quarantine; • Collect infested plant material and/or adults in southern Russia and import

insects into quarantine for host-specificity tests in 2013; • Conduct a multiple-choice cage test in southern Russia on test species that

supported adult development in no-choice larval transfer tests.

Melanobaris sp. near semistriata

• Conduct multiple-choice cage and no-choice tests in Turkey.

Metaculus lepidifolii

• Conduct multiple-choice cage tests in Turkey.

8. Conferences

A poster presentation on ‘Developing biological control for perennial pepperweed in the US: progress so far’ was presented by Esther Gerber, Hariet Hinz, Massimo Cristofaro, Franca Di Cristina, Francesca Lecce, Alessandra Paolini, Margarita Dolgovskaya, Rüstem Hayat and Levent Gültekin at the 13th International Symposium on Biological Control of Weeds, Waikoloa, Hawaii, 11–16 September 2011.

The same poster was also presented at the 2nd research forum ‘Responding to invasive species’ organized by the Invasive Plant Council of British Columbia, Richmond, Canada, 18–19 October 2011.

9. Acknowledgements

We thank Cornelia Closca and Leah Blair for technical assistance in the laboratory, as well as Florence Willemin and Christian Leschenne for plant propagation (all CABI).

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We would like to specifically thank our Russian partners Drs Boris Korotyaev, Sergey Reznik, Gena Davidyan and Alexey Moseyko (Zoological Institute, Russian Academy of Sciences, St Petersburg) and Sergey Grigoriev (Krasnodar) for their invaluable help with carrying out field surveys in southern Russia, insect rearing and shipments, and for carrying out the multiple-choice cage test. Special thanks are also due to Philipp Gfeller, Coni Zieglmaier and Ines Rebentrost for their very much appreciated collaboration and for facilitating our field tests in Turkey.

Financial support for this project was provided to CABI by the Wyoming Biological Control Steering Committee, USDA-APHIS-CPHST (United States Department of Agriculture - Animal and Plant Health Inspection Service – Center for Plant Health Science and Technology), and BLM (Bureau of Land Management), Idaho, and to BBCA by the Californian Department of Food and Agriculture (CDFA), and the USDA-ARS (Agricultural Research Service) Western Region Research Centre, Reno, Nevada. This project would not be possible without the continuing effort and dedication of Nancy Webber (PPW Consortium chair, Wyoming), Dr Mark Schwarzlaender (University of Idaho), Dr Brian Rector (USDA-ARS, Reno, Nevada) and Dr Mike Pitcairn (CDFA).

10. References

Allen, J.R., Holcombe, D.W., Hanks, D.R., Surian, M., McFarland, M., Bruce, L.B., Johnson, W. and Fernandez G. (2001) Effects of sheep grazing or mowing on the control of perennial pepperweed (Lepidium latifolium L.). Journal of Animal Science 79, 129.

Blank, R. and Young, J.A. (1997) Influence of invasion of perennial pepperweed on soil properties. USDA Agricultural Experiment Station, Oregon State University, Special Report 972, pp. 11–13.

Boufford, D.E., Freeman, C.C., Gandhi, K., Hill, M.J., Kiger, R.W., Poole, J.M., Schmidt, H.H., Shultz, L.M., Strother, J.L. and Zarucchi, J.L. (2010) Flora of North America: North of Mexico. Vol. 7: Magnoliophyta: Salicaceae to Brassicaceae. Oxford University Press, New York.

Chen, H., Qualls, R.G. and Miller, G.C. (2002) Adaptive responses of Lepidium latifolium to soil flooding: biomass allocation, adventitious rooting, aerenchyma formation and ethylene production. Environmental and Experimental Botany 48, 119–128.

Eiswerth, M.E., Singletary, L., Zimmermann, J.R. and Johnson, W.S. (2005) Dynamic benefit-cost analyses for controlling perennial pepperweed (Lepidium latifolium): a case study. Weed Technology 19, 237–243.

Freeman, J.L., Lindblom, S.D., Quinn, C.F., Fakra, S., Marcus, M.A. and Pilon-Smits, E.A.H. (2007) Selenium accumulation protects plants from herbivory by Orthoptera via toxicity and deterrence. New Phyologist 175, 490–500.

Gerber, E., Hinz, H.L., Hensel, J., Cristofaro, M., Di Cristina, F., Lecce, F., Paolini, A. and Dolgovskaya, M. (2010) Biological control agents of perennial pepperweed, Lepidium latifolium. Unpublished Annual Report 2009, CABI Europe – Switzerland, Delémont, Switzerland, 25 pp.

Gerber, E., Hinz, H.L., Five, D., Cristofaro, M., Di Cristina, F., Lecce, F., Paolini, A. and Dolgovskaya, M. (2011) Biological control agents of perennial pepperweed,

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Lepidium latifolium. Unpublished Annual Report 2010, CABI Europe – Switzerland, Delémont, Switzerland, 24 pp.

Hanson, B, Garifullina, G.F., Lindblom, S.D., Wangeline, A., Ackley, A., Kramer, K., Norton, A., Lawrence, C. and Pilon-Smits, E.A.H. (2003) Selenium accumulation protects Brassica juncea from invertebrate herbivory and fungal infection. New Phytologist 162, 655–662.

Hinz, H.L., Cripps, M., Renteria, J.L. and Wins-Purdy, A. (2004) Biological control of whitetops, Lepidium draba and L. appelianum. Unpublished Annual Report 2003, CABI Europe – Switzerland, Delémont, Switzerland, 26 pp.

Hinz, H.L., Blair, L., Closca, C. and Gerber, E. (2012) Biological control of dyer’s woad, Isatis tinctoria. Annual Report 2011. Unpublished Report, CABI Europe – Switzerland, Delémont, Switzerland.

Miller, G.K., Young, J.A. and Evans, R.A. (1986) Germination of seeds of perennial pepperweed (Lepidium latifolium). Weed Science 34, 252–255.

Renz, M.J. (2000) Perennial pepperweed. The Nature Conservancy – Elemental Stewardship Abstract. Available at: www.invasive.org/weedcd/html/esas.htm [accessed March 2011]

Renz, M.J. and DiTomaso, J.M. (1998) The effectiveness of mowing and herbicides to control perennial pepperweed in rangeland and roadside habitats. Proceedings of the 1998 California Weed Science Society Conference Vol. 50, p. 178.

Renz, M.J. and DiTomaso, J.M. (2006) Early season mowing improves the effectiveness of chlorsulfuron and glyphosate for the control of perennial pepperweed (Lepidium latifolium). Weed Technology 20, 32–36.

Rice, P.M. (2010) INVADERS Database System. Division of Biological Sciences, University of Montana, Missoula, Montana. Available at: http://invader.dbs.umt.edu [accessed February 2010]

Skinner, M.W. and Pavlik, B.M. (1994) Inventory of Rare & Endangered Plants of California, 5th edn. California Native Plant Society, Sacramento, California, p. 336.

Trumbo, J. (1994) Perennial pepperweed: a threat to wildland areas. CalEPPC Newsletter 2, 4–5.

USDA, NRCS (2010) The PLANTS Database. National Plant Data Center, Baton Rouge, Louisiana. Available at: http://plants.usda.gov [accessed February 2010]

Whitson, T.D. (ed) (1987) Weeds and Poisonous Plants of Wyoming and Utah. Cooperative Extension Service, University of Wyoming, pp. 56–57.

Young, J.A., Turner, C.E. and James, L.F. (1995) Perennial pepperweed. Rangelands 17, 121–123.

Young, J.A., Palmquist, D.E. and Blank, R. (1998) The ecology and control of perennial pepperweed (Lepidium latifolium L.). Weed Technology 12, 402–405.

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Annexes

Annex 1. Summary of no-choice oviposition and development tests with Ceutorhynchus marginellus 2007–2011.

# replicates # eggs

Plant species valid attacked with adult

emergence and/or larvae

# adults emerged

Brassicaceae Lepidieae

Lepidium latifolium 57 54 37 185 301 (7.7 ± 0.9)

Lepidium appelianuma 3 2 0 9 Lepidium campestre 1 1 0 2 Lepidium crenatuma 9 9 4 15d 17

(4.3 ± 2.7) Lepidium densifloruma 2 2 1 15 3 Lepidium draba 3 2 1 14 3 Lepidium fremontiia 5 0 - 0 Lepidium huberia 7 7 5 -e 16

(3.2 ± 2.2) Lepidium lasiocarpuma 2 1 - 1 Lepidium latipesa 2 2 2 4 7 (3.5 ± 1.5) Lepidium papilliferuma 2 2 1 5 4 Lepidium perfoliatum 3 3 3 4 17

(5.7 ± 1.7) Lepidium sordiduma 3 0 - 0 Lepidium squamatum 1 1 1 1 2 Alysseae Alyssum alysoides 2 0 - 0 Alyssum (Aurinia) saxatilis 3 0 - 0 Arabideae Arabis aculeolataa 2 0 - 0 Aubrieta sp. 1 0 - 0 Boechereae Boechera blepharophyllaa 3 0 - 0 Boechera holboelliia 7 4 0 10 Brassiceae Brassica napus 3 0 - 0 Brassica oleracea var. gemmifera 2 0 - 0 Brassica oleracea var. italica 3 0 - 0 Brassica oleracea var. sabauda 4 0 - 0 Brassica rapa 3 0 - 0 Raphanus sativus 4 1 0 1 Sinapis alba 4 1 0 4

22

Annex 1 continued

# replicates # eggs

Plant species valid attacked with adult

emergence and/or larvae

# adults emerged

Camelineae Arabidobsis thaliana 3 0 - 0 Camelina sativa 1 0 - 0 Capsella bursa-pastoris 1 0 - 0 Erysimum cheiri 3 0 - 0 Erysimum inconspicuuma 1 0 - 0 Cardamineae Barbarea orthocerasa 3 0 - 0 Cardamine cordifoliaa 3 0 - 0 Nasturtium gambeliia 3 0 - 0 Nasturtium officinale 1 0 - 0 Rorippa sinuataa 3 3 3 3 4 (1.3 ± 0.3) Anchonieae Parrya nudicaulisa 2 0 - 0 Descurainieae Descurainia incanaa 1 0 - 0 Descurainia nelsoniia 1 0 - 0 Heperideae Hesperis mationalis 1 0 - 0 Physarieae Physaria condensataa 3 1 - 2 Schizopetaleae Caulanthus crassicaulisa 2 1 - 1 Stanleya pinnataa 3 1 1 0 1 Streptanthus farnsworthianusa 2 0 - 0 Streptanthus glandulosus ssp. nigera 2 0 - 0 Thelypodium laciniatuma 3 1 0 14 Sisymbrieae Sisymbrium linifoliuma 4 1 0 1 Thlaspideae Thlaspi arvense 2 0 - 0

Cleomaceae Peritoma lutea 2 0 - 0

- Not recordable (plants completely dissected). a Species native to North America. Nomenclature according to Boufford et al. (2010). b Containing mining and/or larvae (living and/or dead). c Values given in brackets are mean ± SE emerged weevils, calculated for replicates with adult

emergence. d Only one plant dissected. e Plants not dissected.

23

Annex 2. Summary of larval transfer tests with Phyllotreta reitteri 2007–2011.

Total #

# of replicates % larvae

developed

Plant species

larvae transferred

valid repl.

attackedb with living organisms

w. adults emerged

(mean ± SE)c

Lepidieae

Lepidium latifolium 2129 107 97 82 67 17.7 ± 1.7

Lepidium appelianum 10 1 1 0 0 Lepidium campestre 100 5 4 4 4 36.4 ± 14.1 Lepidium crenatuma 110 5 3 3 1 Lepidium densifloruma 105 5 3 3 3 13.6 ± 6.8 Lepidium didymum 60 4 4 3 2 25.0 ± 5.0 Lepidium draba 150 7 6 6 5 15.4 ± 3.8 Lepidium fremontiia 52 3 3 3 2 12.3 ± 7.7 Lepidium huberia 100 5 3 3 3 26.7 ± 16.7 Lepidium lasiocarpuma 60 3 2 2 2 32.5 ± 12.5 Lepidium latipesa 45 3 1 1 1 Lepidium montanuma 17 2 2 2 0 Lepidium oblonguma 45 2 2 1 1 Lepidium ostleria 26 2 1 0 0 Lepidium papilliferuma 80 5 5 5 4 8.6 ± 1.3 Lepidium perfoliatum 65 3 3 3 2 42.5 ± 7.5 Lepidium sativum 110 5 5 5 3 18.3 ± 8.3 Lepidium sordiduma 38 4 0 0 0 Lepidium squamatum 110 6 2 1 0 Lepidium virginicuma 14 1 1 1 0

Alysseae Alyssum allysoidesa 20 1 0 0 0 Berteroa incana 33 2 1 0 0

Arabideae Arabis aculeolataa 20 2 2 1 0 Aubrieta deltoidea 29 2 0 0 0 Draba albertinaa 30 2 1 0 0

Boechereae Boechera holboelliia 20 1 0 0 0 Boechera ligniferaa 24 2 2 2 0

Brassiceae Brassica juncea 20 1 1 1 0 Brassica napus 40 2 0 0 0 Brassica nigra 110 5 4 4 3 11.0 ± 2.1 Brassica oleracea var. gemmifera 40 2 0 0 0 Brassica rapa 19 2 0 0 0 Raphanus sativus 45 3 0 0 0 Sinapis alba 115 5 4 3 0

Camelineae Arabidopsis thaliana 24 2 2 2 0 Capsella bursa-pastoris 65 3 1 0 0 Erysimum asperuma 55 3 1 0 0 Erysimum cheiri 20 1 0 0 0 Erysimum inconspicuuma 40 2 0 0 0

24

Annex 2 continued

Total # # of replicates % larvae

developed

Plant species

larvae transferred

valid repl.

attackedb with living organisms

w. adults emerged

(mean ± SE)c

Cardamineae Armoracia rusticana 40 2 0 0 0 Barbarea orthocerasa 90 5 2 1 0 Cardamine cordifoliaa 41 2 1 1 0 Nasturtium officinale 100 4 3 3 1 Rorippa sinuataa 55 3 2 2 2 12.5 ± 2.5 Rorippa sylvestris 65 3 0 0 0

Descurainieae Descurainia incanaa 65 3 1 1 0 Descurainia nelsoniia 22 2 1 0 0

Iberideae Iberis umbellata 10 1 1 0 0

Isatideae Isatis tinctoria 45 3 0 0 0

Noccaeeae Noccaea fendleri ssp. idahoensisa 10 1 0 0 0 Noccaea fendleri ssp. siskiyouensea 10 1 0 0 0

Physarieae Physaria chambersiia 20 2 0 0 0 Physaria fendleria 45 3 3 2 2 6.7 ± 0.0

Schizopetaleae Stanleya pinnataa 55 3 0 0 0 Stanleya viridifloraa 75 4 1 0 0 Streptanthus farnsworthianusa 80 5 1 0 0 Streptanthus gladulosus ssp. nigera 10 1 0 0 0 Thelypodium laciniatuma 45 3 2 1 0

Sisymbrieae Sisymbrium linifoliuma 45 3 2 1 0

Thlaspideae

Peltaria alliacea 40 2 0 0 0 Thlaspi arvense 80 5 2 0 0 a Species native to North America. Nomenclature according to Boufford et al. (2010). b Containing mining and/or larvae (living and/or dead). c Calculated for replicates with adult emergence.

25

Distribution list

Jennifer Andreas Jerry Marks John L. Baker Brian Marschman Dan Bean Joseph Milan Larry Beneker Sergei Mosyakin Ken Bloem Radmila Petanovic Maurizio Biondi Mike Pitcairn Dave Burch Carol Randall Steve Burningham Brian Rector Craig McClure Brett Richardson Ray Carruthers Lesley Richman Tim Collier Mark Schwarzländer Eric Coombs Bruce Shambaugh Enrico de Lillo Josh Shorb Margarita Dolgovskaya John Simons Liz Galli-Noble Lincoln Smith John Gaskin Jeanne Standley Levent Gültekin Atanaska Stoeva Vili Harizanova Joe DiTomaso Rich Hansen Shaharra Usnick Rüstem Hayat Janet Valle Bruce Helbig Rick VanBebber Jim Hull Nancy Webber Jeff Littlefield USDA ARS EBCL Roman Jashenko CABI library Boris Korotyaev

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