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BBCA Biotechnology and Biological Control Agency Bi-annual Report 2007 – 2008

in co-operation with USDA ARS European Biological Control Laboratory - Montpellier, France

BB A Biotechnology and Biological Control Agency

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This report has been completed and edited on February 18, 2009. It is copyright of BBCA-onlus, on behalf of the sponsors and official co-operators of this work where appropriate. It presents unpublished research findings, which should not be used or quoted without written agreement from BBCA-onlus. Unless specifically agreed otherwise in writing, all information herein should be treated as confidential.

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Table of contents

Aknowledgements

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Introduction

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1. Yellow starthistle, Centaurea solstitialis 1.1. Ceratapion basicorne (Coleoptera: Curculionidae) 1.2. Psylliodes sp. nr. chalcomerus (Coleoptera: Chrysomelidae) 1.3. Aceria solstitialis (Acari: Eriophyoidea)

2. Russian thistle, Salsola tragus 2.1. 2.2. 2.3. 2.4.

Aceria salsolae (Acari: Eriophyoidea) Lixus incanescens (Coleoptera: Curculionidae) Lixus rosenschoeldi (Coleoptera: Curculionidae) Cosmobaris scolopacea (Coleoptera: Curculionidae)

3. Rush skeletonweed, Chondrilla juncea 3.1. Sphenoptera foveola 3.2. Schinia cognata (Lepidoptera: Noctuidae) 3.3. Simyra nervosa (Lepidoptera: Noctuidae)

4. Scotch thistle, Onopordum acanthium 4.1. Trichosirocalus briesei (Coleoptera: Curculionidae)

5. Perennial pepperweed, Lepidium latifolium 5.1. Summary of 2007 activities 5.2. Multiple Choice tests with Lasiosina deviata in Turkey 5.3. Metaculus lepidifolii (Acari: Eriophyoidea)

12 12 13 19

25 25 27 27 28

29 29 38 40

46 46

48 48 51 55

6. Russian olive, Elaeagnus angustifolium

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7. Other weeds

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7.1. Saltcedar, Tamarix spp. 7.2. Russian knapweed, Acroptilon repens

8. Exploration trips

59 60

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8.1. Exploration trips 2007 8.2. Exploration trips 2008

62 64

List of publications

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Distribution List

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BBCA Staff ...

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... and Cooperators!

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Aknowledgements BBCA is grateful to Walker Jones, Director of USDA ARS European Biological Control Laboratory, and Dan Strickman, International Program Leader of USDA ARS, for their help in co-ordinating the projects; Bernadette E. Gregor and Deadra Perry, USDA ARS Beltsville, MD, and Xavier Leprieur, EBCL, for their capability to administrate the Grant; Lincoln Smith, USDA ARS Albany, CA, for his great input in the organization and in the development of field and lab bioassays on the biological control of yellow starthistle, Russian thistle and Scotch thistle; Alex Konstantinov, USDA ARS Washington D.C.; Livy Williams and Kristina Schierenbeck, USDA ARS Exotic and Invasive Research Unit, Reno, NV; Mark Volkovitsh, Rita Dolgovskaya, Sergey Reznik, Boris Korotyaev, Dr. Vadim Zaitsev, Russian Academy of Sciences, Zoological Institute, St. Petersburg, Russia; Enzo Colonnelli, Alessandro Biscaccianti and Alberto Zilli, Museo Civico di Zoologia, Rome, Italy; Maurizio Biondi, University of l’Aquila, Italy; René Sforza, Brian Rector, Marie-Claude Bon and Dominique Coutinot, USDA ARS EBCL Montpellier, France; Javid Kashefi, USDA ARS EBCL Thessaloniki, Greece; Gaetano Campobasso, USDA ARS EBCL Rome, Italy; thanks also to Doug Luster, Bill Bruckart and Dana Berner, USDA ARS Ft. Dietrick,MD; Rick Bennett, Steve Clement, Ray Carruthers, USDA ARS; Nada Carruthers, USDA APHIS; Dan Bean, U.C. Ft. Collins, CO; John Simons, BLM, Billings, MT; and Mike Pitcairn, CDFA Sacramento, CA; for their support in several administrative and scientific aspects; Rüstem Hayat and Levent Gültekin, Atatürk University, Erzurum, Turkey, for their help in carrying out field bioassays on biocontrol candidate agents of Lepidium latifolium; George Markin, Justin Runyon and Lynn Kinter, USDA Forest Service; and Jeff Littlefield, Montana State University, Bozeman MT; Hariet Hinz, Esther Gerber, Urs Schaffner and Matthew Cock, CABI-Europe, Délemont, Switzerland; Mark Schwarzlaender, University of Moscow, ID; John Gaskin, USDA ARS, Sidney, MT; David Briese, CSIRO, Australia; Rouhollah Sobhian, former USDA ARS EBCL Research Entomologist; and Maurizio Vurro, CNR ISPA, Bari, Italy; for their support in different research projects and explorations; Reza Ghorbani and Ali Asadi Ghorbanali, Ferdowsi University, Mashhad, Iran; Ivanka Lecheva, Vili Harizanova, Atanaska Stoeva and Anna Karova, Agricultural University of Plovdiv, Bulgaria; Wafaa Mahrous Amer, University of Cairo, Egypt; and Fu Weidong, Chinese Academy of Agricultural Sciences, Beijing, China; for their support in field explorations and research projects in their Countries;

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Peter Toth and Ludovit Cagan, Agricultural University of Nitra, Slovakia, for their help during foreign explorations for natural enemies of Rush skeletonweed and Russian thistle in Slovakia; Enrico De Lillo, Rosita Monfreda and Margherita Baldari, University of Bari, Italy, for their help in the taxonomy of eriophyoid mites and their support during the field tests; Alessio De Biase, Paolo Audisio, Gloria Antonini (BBCA until July 2007), Silvia Belvedere, Emiliano Mancini and Simona Primerano, University of Rome, for their help in genetic analyses; Carlo Tronci (BBCA until June 2007), Silvia Arnone, Marcello Barlattani, Luigi Rossi, Lucio Triolo, Magda Schimberni, Paola Nobili, Fortunata Minelli, Domenico Chiaretti and Agostino Letardi, ENEA-Casaccia, Rome, Italy. We also thank, for their scientific and economic support Rosaria Tabilio, Istituto Sperimentale per la Frutticoltura, Rome, Italy; Augusto Vigna Taglianti, University of Rome “La Sapienza”, Italy; and Valerio Sbordoni, University of Rome “Tor Vergata”, Italy. Special thanks are also due to Alberto Bevilacqua, Blu pubblicità, for his help in editing BBCA Annual Report and the web site.

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Introduction By Massimo Cristofaro The eighth year of BBCA is already gone, and we are back again! This time we are going to present a biannual report (2007-2008), because we missed last year edition. The editing of BBCA Annual Report is for me very important because it’s the end of a cycle: in order to try to keep in track the work in all the fields for all the projects, I need short-term targets. But this time it is more than only a short term cycle: BBCA is still alive despite of the economic problems! During 2007-2008 we decided to increase the explorations of new areas (China, Iran, Spain, Bulgaria, Slovakia and Greece) and the list of the target weeds to work with: overall we made 5 travels in Turkey, 1 in Russia, 2 in the USA, 4 in Tunisia, 3 in Iran, 3 in Switzerland, 4 in Montpellier, 5 in Bulgaria, 1 in China, 1 in Greece,1 in Slovakia and several in southern Italy! In particular Turkey has been explored every 3 weeks from May until September, with a huge amount of data on the ecology of the target weeds and their natural enemies! The strategy was very successful: we finished the screening of Ceratapion basicorne (host range by BBCA and Ataturk University, Erzurum, Turkey; morphological taxonomy by Boris Korotyaev; genetic analyses by BBCA and EBCL) and the root boring weevil was petioned for the release in the US. In addition, within next year we will finish the screening of an other yst agent, the stem boring flea beetle Psylliodes sp. nr. chalcomera: the work of the last two years was a great combination of host range (Russian Academy of Sciences and BBCA), genetic analyses (Rome University, Italy), and biological observations (Russian Academy of Sciences). But, beside to these two most promising candidate agents, during the past two years we screened the lace bug Tingis grisea (BBCA, host range and life history), the eriophyd mite Aceria solstitialis (Bari University, Italy: life history), and the seedfeeder weevil Larinus filiformis (Ataturk University, Erzurum, Turkey: life history and competition tests). Moreover, we found in Eastern Turkey at least 3 new additional insects closely related to YST: two new species of weevils (Araxia sp. and Pseudorchestes sp.) and an undetermined tip galling moth. With the speed-up we gave on the YST biological control, we had the chance to improve the explorations for Russian thistle and, since last year, we started to work with another target weed, Scotch thistle (Onopordum acanthium). BBCA and co-operators made explorations in Turkey (5 one-week-trips), Tunisia (3 one-week trips), Bulgaria (4 five-days trips), China (ten-days trip), Iran(3 ten-days trips) and Slovakia (2 five-days trips). The travels in Iran, Turkey and China, led us to make a list of 6 new promising biocontrol candidate agents to be screened (3 weevil spp. from the genera Elasmobaris and Lixus) and 3 mites species of the genus Aceria, according to our taxonomist co-operators Enzo Colonnelli, Boris Korotyaev, Levent Gultekin and Enrico de Lillo). Moreover, together with our CABI co-operators, since 2006 we started explorations in Eastern Turkey, Kazakhstan and China for a new target weed, perennial pepperweed (Lepidium latifolium); among the 5 biocontrol agents we found together related to PPW, two root borer weevils (Melanobaris spp.), the stem galling weevil Ceutorhynchus marginellus, the stem boring cloropid fly Lasiosina deviata and the leaf galling eriophyoid mite Metaculus lepidofoli have been selected for further screening.

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Compared to previous years, we improved a lot the cooperations, both in Italy and in other Countries. Moreover, we set up 2 new important cooperations with the University of Plovdiv, Bulgaria and the Ferdowsi University of Mashhad, Iran. Of course, the performances in our work can not be achieved without the “historical” cooperators, such as the Russian Academy of Sciences, St. Petersburg, the Ataturk University of Erzurum, Turkey, and the Agriculture University of Nitra, Slovakia. This is our secret: with this great “Task Force”, we have been able to improve a lot the performance of the group. In each Country we have a small team, very professional and well trained, and each contribution is crucial to achieve the performance we can provide! Despite the economic problems of the three last years BBCA is working at the moment with 3 research entomologists, one research assistant, 2 technicians, and 1 administrator to carry on all of these activities. We know that our future is still instable but the enthusiasm for our work and the special feeling we have among the BBCA staff and the mutual trust in our partners, will keep me optimistic, as usual! Plans for 2009 Next year will follow the footsteps of last years, improving BBCA efforts in terms of new disciplines and new projects. Regarding new disciplines, in particular, we will enhance our screening improving genetic aspects of work. The target for 2009 will be to help EBCL in the clarification of several taxonomic question marks on some of the new potential biocontrol agents, having 4 weevil species and one eriophyoid mite in our pipeline. For the classic approach, our goal will be to be involved on 4 different projects for EBCL (Russian thistle, Scotch thistle, saltcedar and YST), one together with EBCL and CABI (perennial pepperweed), and two under the supervision of CABI (Russian olive and Russian knapweed). Regarding the “historical“ projects, our work on YST will be focussed on the final data for the screening and the petition of the stem-boring flea beetle Psylliodes chalcomerus, in cooperation with Lincoln Smith; in addition, we are planning the second year of open field host range test with the eriophyoid mite Aceria solstitialis in Plovdiv, Bulgaria, in cooperation with Dr. Vili Harizanova and Dr. Atanaska Stoeva. Laboratory bioassays with 3 species/biotypes of Lixus spp. nr. incanescens found on Russian thistle in Turkey, Italy and Iran will be carried out in our facilities to evaluate the oviposition and the larval host range in no-choice conditions: the approach includes not only host range observations in or lab, but also molecular analysis with the progeny. An open field test with a population from Kapadokya is planned for the late spring/early summer in Central Turkey. We will start the second year for Scotch thistle, continuing the screening of the root boring weevil Trichosirocalus briesei, carrying out host range bioassays as well genetic analysis. In addition we will work together with Lincoln Smith on two populations of the flea beetle Psylliodes sp. near chalcomerus, associated with Onopordum acanthium in Italy and Turkey. In cooperation with Maurizio Biondi (University of

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L’Aquila, Italy) and Alessio De Biase (Università of Rome, Italy) and under the supervision of Lincoln Smith, we will provide to Albany, CA adults of the seed feeder weevil Larinus latus, collected in central Turkey. Perennial pepperweed project will be focussed on the final steps for the host range screening of the stem boring fly Lasiosina deviata, together with a preliminary screening of an eriophyoid mite in Central Turkey. BBCA will continue to work on collections and preliminary rearing observations on the root galling weevil Liocleonus clathratus on saltcedar, in cooperation with Nada Carruthers, USDA APHIS PPQ, Albany, CA. Field open field host range multiple choice tests will be carried out with the noctuid moths Schinia cognata and Simyra nervosa on Rush skeleton weed in Bulgaria, in co-operation with the University of Plovdiv and Jeff Littlefield, Montana State University, Bozeman, MT. Additional explorations will be performed in Slovakia, Turkey and Iran in order to detect additional potential biocontrol agents. Two open field tests will be carried out on the eriophyoid mites Aceria acroptiloni and A. angustifoliae, on Russian knapweed and Russian olive respectively in Iran and Turkey, evaluating the host specificity and the impact on seed production and germination rate.

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Milestones 2009-2011 for Prioritized Candidate Biocontrol Agents

BCA or Weed

2009

Psylliodes chalcomerus TW: Yellow starthistle

complete DNA analyses submit petition

Tingis grisea TW: Yellow starthistle

complete HR tests in lab and field start impact tests

complete impact tests submit petition

Aceria solstitialis TW: yellow starthistle

complete HR tests in open field condition

submit petition

Psylliodes chalcomerus TW: Scotch thistle

start HR tests in field and lab start impact and crossing tests continue DNA analyses and tassonomic studies

complete HR tests in field and lab complete impact and crossing tests continue DNA analyses and tassonomic studies

Trichosirocalus briesei TW: Scotch thistle

continue HR tests in lab

complete HR tests in lab start quarantine studies

complete quarantine studies submit petition

Eublemma sp. TW: Scotch thistle

foreign explorations start biology studies start HR tests

continue biology studies continue HR tests

complete HR tests complete biology studies

Anthypurinus biimpressus TW: Russian thistle

foreign explorations continue biology studies continue HR tests

complete HR tests start impact tests complete biology studies

complete impact tests start quarantine studies

Gymnancyla sp. TW: Russian thistle

start HR tests

continue HR tests

complete HR tests

Lixus sp. TW: Russian thistle

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

Cosmobaris sp. TW: Russian thistle

2010

2011

complete DNA analyses and tassonomic studies

foreign explorations and collections foreign explorations and collections start genetic analyses continue genetic analyses

Sphenoptera foveola TW: Rush skeletonweed Simyra nervosa TW: Rush skeletonweed

foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

start quarantine studies

Schinia cognata TW: Rush skeletonweed

foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

start quarantine studies

Oporopsamma sp. TW: Rush skeletonweed

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

Lasiosina deviata TW: Perennial pepperweed

foreign explorations continue HR tests

complete HR tests - start quarantine studies

complete quarantine studies submit petition

Psylliodes sp. TW: Perennial pepperweed

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

Oporopsamma sp. TW: Rush skeletonweed

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

Aceria angustifolia TW: Russian olive

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

Scolytis sp. TW: Russian olive

foreign explorations start biology studies start HR tests

continue foreign explorations continue biology studies continue HR tests

complete biology studies complete HR tests

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1. Yellow starthistle, Centaurea solstitialis In cooperation with Lincoln Smith, USDA ARS, Albany, CA, USA. Yellow starthistle is an annual noxious weed that currently infests millions of acres of pasturelands, noncultivated and natural areas in the Western USA. This species is a serious problem in these areas as it displaces native plant communities, reduces plant diversity and drastically reduce forage production for livestock and wildlife. Moreover, it is poisonous to horses, causing a nervous lethal disorder called “chewing disease�. During 2000 BBCA started a biocontrol program for YST in cooperation with USDA-ARS EBCL and PWA with the specific objective to select biocontrol agents attacking the weed at the early developmental stages. Between 2001 and 2005, five new agents were selected; among them the root/crown boring weevil Ceratapion basicorne, distributed in Turkey, Italy, S. France, Greece and S. Russia. The host specificity screening is completed and a release petition was accepted in 2006.

1.1. Ceratapion basicorne (Coleoptera: Curculionidae) During 2007 season, laboratory experiments on host specificity and impact evaluation of the crown boring weevil Ceratapion basicorne on yst, have been carried out at the BBCA facilities. The host-range laboratory test showed a slight more specific behavior of the Italian population compared with Turkish population. The genetic analysis did not show any differences between the Turkish and the Italian populations, confirming the solid stability of this species. Climatic matches computed by the software Climex confirmed that the Italian population has a better chance of establishment because the climatic conditions are very similar of the releasing area. In addition, in cooperation with Brian Rector, USDA ARS EBCL, Montpellier, France, we sequenced more than 50 samples between weevil larvae and adults of an open field host range test on C. basicorne, carried out 3 years ago in Montpellier area, by Brian Rector and Rouhollah Sobhian. Results clearly demonstrate that, except for 5 larvae recorded in the root of Centaurea cyanus and one larva from Carduus pycnocephalus, all the others C. basicorne were associated with the target weed (YST).

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1.2. Psylliodes sp. nr. chalcomerus (Coleoptera: Chrysomelidae) The stem-boring flea beetle P. cfr. chalcomerus is distributed in Italy, Russia and Turkey; the probable existence of two or more sibling species within this group, having different levels of host suitability, has been demonstrated in field surveys, laboratory and field host range tests, and confirmed by means of genetic analyses. In 2007, biological notes and impact evaluation tests have been carried out in BBCA facilities. Experiments were carried out with adults of P. cfr. chalcomerus collected by Russian cooperators during the end of March 2007 near Volna (Krasnodar Province). Once in laboratory, the insects were placed in a 3 L glass beaker with laboratory paper tissue and fresh yellow starthistle leaves at room temperature (18-25°C) and with a 16:8 L/D cycle for a week, in order to allow the flea beetles to feed and complete reactivation after diapause. In addition DNA analyses have been continued by G. Antonini, P. Audisio and A. De Biase at the University of Rome “La Sapienza”.

1.2.1. Biological notes With the aim of selecting ovipositing individuals, females were placed in Petri dishes at room temperature (18-25°C) and with a 16:8 L/D cycle with leaves of fresh yellow starthistle replaced every day. Laid eggs were collected daily, counted, and placed in Petri dishes over wet filter paper to allow hatching. Preliminary data analysis showed that each ovipositing female laid a mean of 233,7 eggs during an oviposition period of 39 days on average. We obtained larvae from 60% of eggs laid.

1.2.2. Impact evaluation experiments Impact evaluation tests have been performed by BBCA in laboratory and greenhouse conditions, in order to evaluate gregarious feeding behaviour of adult males and females and to assess larval damage and impact on biomass and seed production/germination of YST plants using larval transfer techniques. Larval transfer experiment The larval transfer test was carried out at the end of April 2007 on early bolting yellow starthistle plants. Before starting the experiment, plant height and root-crown diameter were recorded for each plant. One treatment and one negative control were set up transferring respectively 10 and 0 larvae per plant, 20 and 10 replicates each. First instar larvae, emerged from eggs stored in Petri dishes were transferred with a fine brush on leaf axils. This first phase of the test was carried out in laboratory at 18-26°C and 16:8 L/D cycle. After two weeks, each plant was enclosed in a nylon cage, and moved to a shade-house outside the laboratory. Forty days after transfer of the last larva, we started to inspect the cages looking for emerged adults. Such inspections were repeated every day until the 60th day after the transfers, when cages were removed and the plants harvested. Because of the bad condition of the plants due to an unknown disease, we were not able

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to record the stage of flower buds, the root-crown diameter the plant height, neither to obtain the seeds. The material from each plant was artificially dried at 65°C for 72h in a ventilated stove and then weighted. Seventeen per cent of transferred larvae became adults. Adult feeding experiment The aim of this experiment was to assess the feeding impact of variable numbers of P. cfr. chalcomerus adults on fresh cut YST leaves by measuring the leaf area eaten per day for ten consecutive days. In addition, we wanted to investigate if the presence of one other individual of the same or opposite sex on the same substrate, would determine any significant change of the amount of leaf tissue consumed by a given adult insect. The following combinations of males (M) and females (F) were tested: 1F, 2F, 1M, 2M, 1F+1M. Each treatment was replicated eleven (1F, 1M), nine (1F+1M), seven (2F) and six (2M) times. The trials were carried out in glass Petri dishes at room temperature (18-25°C) and with a 16:8 L/D cycle. The insects were allowed to feed on one single freshly cut YST leaf, laid on a moist disk of filter paper for 24 hours. The Petri dishes also contained a moist sterile cotton plug in order to allow females to oviposit. Prior to each trial, leaves were scanned into a TIF format digital image using a desktop flatbed scanner. At the end of each 24 h testing session, the leaves were again acquired into digital images. Each insect was tested for ten consecutive days. Eggs were collected daily, counted and placed in Petri dishes over wet filter paper to allow hatching. Each digital image was analyzed in order to measure the eaten areas. Feeding scars were selected using Photoshop LE v.5.0 (Adobe Systems, San Jose, CA, USA) and the amount of area eaten was evaluated utilizing Image v.4.0.3.2 beta for Windows (National Institute of Health, Bethesda, MD, USA). Preliminary results analysis confirmed that females feed more than males: in fact mean surface eaten by one female in 24 h was ten times the surface consumed by one male. Feeding rate analysis per individual didn’t show any correlation with the number of adults per trial.

1.2.3. Genetic analyses on taxonomic status and mtDNA variation in natural populations of Psylliodes spp. cfr. chalcomerus By A. De Biase, G. Antonini, P. Audisio, S. Belvedere, E. Mancini, S. Primerano, M. Trizzino, Dept. of Animal and Human Biology, University of Rome “La Sapienza”, Italy. More than 10 populations feeding on different host plants and putatively belonging to the Psylliodes spp. cfr. chalcomerus assemblage of forms (species ?) were sampled. Populations ranged from Spain to Russia. More than 160 specimens were analyzed and sequenced; they were sampled on the field or were reared in laboratories for testing their feeding specificity in choice or no-choice tests. The studied populations seem to belong to three distinct genetic groups of unclear taxonomic status showing a clear cut different trophic ecology, although they are morphologically undistinguishable. During the last year (Autumn 2007–Autumn 2008) genetic analyses were focused on testing more individuals sampled in the field and, more important, many individuals coming from laboratory specificity tests supplied by L. Smith (VB and VC tagged samples).

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Another very important target was the development of a laboratory protocol aimed at gathering data from biological samples other than adult individuals, in order to build a non-invasive procedure for genetic analyses, to be adopted for field release of the candidate control agent. Standard procedures for extracting DNA can be in fact very highly destructive, making the assayed sample useless for any further analysis. Therefore, we tried to get the genetic profile of samples by analyzing DNA extracted from their feces or from early stages (both embryos and larvae) of their offspring. The feces analyses gave unsatisfactory results owing to the unpredictable results that prevented from establishing a standard protocol for routine analyses (see below). More interesting results came from the experiments carried out on early development stages (embryos and larvae). We tried many different conditions for extracting DNA from samples, both chemical and physical. We were successful in extracting DNA from all stages, although the success rate varied from embryos to larvae. Namely, embryos were very difficult to be tested and results were not satisfactory for setting up a routine protocol. On the other side larvae were successfully amplified and sequenced with a success rate of 75-100% depending of the development stage of the larva with the 100% of success for mature larvae. We can, therefore, set up a standard protocol for routine pre-release analyses aimed at testing larval stages of the offspring of adult individuals reared in laboratory (non-invasive genetic analyses). All sequences gathered from embryos and larvae were included in the current dataset. As final remark, during the last year we began to critically review experimental data that were scored as containing “technical artifact�. We are working on those populations/individuals showing several regions with double nucleotide signals. This situation can be scored as PCR/sequencing errors/artifact or as due to the coexistence of distinct mitochondrial genomes in the same individual. One of the cause of such a situation could be hybridization events occurred in the past or even in the present time. It is clear that if our candidate agents can undergo such events much care and attention must be paid before planning any release program. We have just started to cope with the issue and we hope to have as soon as possible some preliminary results.

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Materials and Methods Genetic analyses were carried out on 110 adults and 45 larvae chosen among the available samples. Six Italian populations were sampled in Central Italy while feeding on Carduus nutans (Asteraceae; European Musk Thistle=CAR), on Onopordum illyricum (Asteraceae; =ONI) and on Onopordum acanthium (Asteraceae; =ONO); six Russian populations were collected in six localities of the Krasnodar region (Black Sea) feeding on Centaurea solstitialis (Asteraceae; Yellow Star Thistle=YST), on Onopordum acanthium (Asteraceae; Scotch Thistle=ONO) and on Centaurea diffusa (Asteraceae; Diffuse knapweed=CDI). Samples from Red October and from Anapa were of great interest due to the presence of both plants (YST and ONO) hosting individuals of Psylliodes spp. cfr. chalcomerus feeding on them. Four Turkish populations were sampled on both host plants (YST and ONO). The analyzed dataset is strongly biased towards Russian samples, obviously due to the strong interest that we have for these populations as candidate control agents. Further, the entire covered area is very large and the sampling scheme was not covering it evenly: we have mainly samples from Italy, Turkey and Russia making the dataset surely mirroring a certain amount of genetic variation due to the spatial factor. We noticed that this variation blur and confound the interpretation of the relationships among the scored cryptic forms; we then decided to split our dataset into distinct subdatasets in order to elucidate our findings more clearly. Hereafter we will discuss results relevant to the IT+RU dataset that is more interesting for the goals of the project. The sequencing work was still focused on the mtDNA fragment (700 bp) encompassing a region of the NADH dehydrogenase subunit 2 gene, the tRNA-Trp, tRNA-Cys and tRNA-Tyr genes and a region of the Cytochrome C Oxidase subunit I gene. All sequences were edited and aligned, and a Neighbor Joining (NJ) analysis using the Tamura & Nei model (1993) was performed without constraining any outgroup (i.e. Unrooted). A bootstrap analyses was carried out with 1000 replicates. Figure 1 shows the topology of the relationships among the tested individuals as unrooted tree. Average distances among largest scored groups were computed by means of Mega 4.

Results and Discussion The IT+RU dataset includes individuals from Italy and Russia associated to YST, ONO, ONI, CAR, CDI and many other individuals whose feeding behavior is not exactly known to us. Therefore, the assayed specimens were tagged and assigned to trophic groups on the base of the information that we have available from the other persons of the research team. We failed to tag several specimens due to unavailable “ecological� data (Fig. 1). Genetic distances computed among all pairs of sequences showed values ranging from 0.00 to 0.03 (meaning roughly a percentage of nucleotide differences between 0% and 3%). We computed also average genetic distances among the scored groups and overall values ranged from 0.01 to 0.02 but they dropped when corrected for intra-group variation (net genetic distances; see Report 2004 for computation formula). It means that only groups #1 and #4 are more distantly related with an average genetic distance equal to 0.02. These values, as stated in previous reports, are not able to give us a clearcut clue on the relationships among the forms of the assemblage of the P. cfr. chalcomerus. More insights are possible when looking at the Fig. 1 portraying results of the NJ analysis for the IT+RU dataset. The tree shows four groups supported by medium-high bootstrap values: NJ tree: 86 (# 1 Russia YST); 88 (#2 Russia ONO); 81 (#3 Russia ONO); 93 (#4 Russia, Italy YST CAR ONO etc).

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All groups are well characterized both geographically and ecologically but overall we scored two discrepancies. The group #1 includes individuals PS.6.7 and PS.18.L2 reported as feeding on ONO and CDI respectively; the group # 4 [including Russia (YST, CDI) and Italy (CAR, ONO, ONI)] is highly heterogeneous clustering several individuals sampled in very distant areas and showing marked differences in their feeding needs. Other few individuals are of uncertain position and suggest caution in the interpretation of the ecological (i.e. trophic) specialization of the populations under investigation. Apart from these observations, our results still evidence a very well characterized group of individuals all from Volna (Krasnodar, Russia) that seem strongly associated to YST. As previously stated (see Technical report 2003-04), we suggest caution in evaluating the degree of differentiation of the Russia (ONO) and Russia (YST) groups and in general the degree of differentiation of all sampled populations. Data could suggest, or even support, different hypotheses regarding the relationships of these genetic pools. Russia (ONO) and Russia (YST) seem to be distinct genetic pools well characterized and distinct, but incomplete lineage sorting or even hybridization events cannot be excluded. Again, the structure of the group #4 seem to suggest the existence of a plastic genetic pool (the true widespread and more generalist P. chalcomerus) that enable this insects to shift among different host plants when “required�, with shifted forms that eventually evolve in highly local specialized forms, as seem to be the YST associated form from Russia.

Conclusions The available data still suggest the existence of three distinct forms inside the Psylliodes cfr. chalcomerus taxon, that are not distinguishable by morphological traits. These forms seem also reflect a feeding specialization at least at local level with two of them feeding on a single host plant. Anyway some data suggest caution in the interpretation of the taxonomic and genetic value to be assigned to the above cited forms, also considering that putative hybridization events, incomplete lineage sorting, or host plant shifts events, seem still suggest only partial genetic differentiation between them, combined with a phenotypic plasticity in their feeding needs. Alternatively, with a more conservative approach and taking into account the invariant morphological traits scored for all the studied populations, it could be hypothesized that the evidenced forms belong to a unique widespread taxon represented by several populations, locally specialized in their feeding needs, but still keeping a cohesive genetic pool through adequate levels of gene flow. We think that more data, and maybe a statistical phylogeographic approach, could help in shedding more light on the whole matter. Next steps could be focused towards few more main goals as for instance: i.To set up protocols for screening individuals to be released in the field by means of cost effective procedures (e.g. DSCP analyses that are less expensive than sequencing reactions). ii.To try to use nuclear genetic markers to cope with question arising from the current data, concerning past or present hybridization events among the scored forms of P. cfr. chalcomerus. iii.To try to develop microsatellite markers in order to plan mode accurate genetic studies enabling to monitor also post-release dynamics. iv.To plan a wider sampling aimed at evaluating the ecological and genetic variation of the assemblage of forms under the taxon P. cfr. chalcomerus. 17


1.2.4. Non-invasive methods for the discrimination among different populations of Psylliodes spp. cfr. chalcomerus (Coleoptera, Chrysomelidae, Alticinae). By G. Antonini, Dept. of Animal and Human Biology, University of Rome “La Sapienza”, Italy. Objective of the project is develop a technique to amplify a COI gene region from faecal secretions of living adults of P. chalcomerus. Trials of DNA extraction and/or amplification were carried out on one Italian population (Vejano, Viterbo) of P. chalcomerus collected on Onopordum sp. and Carduus nutans provided by BBCA. The insects were maintained at BBCA facilities and, for a limited period also at our laboratory at the Sapienza University of Rome (Department of Human and Animal Biology, Zoological Institute). DNA amplification was attempted using two different strategies: 1.

Direct PCR of a suspension of faecal matter

The first strategy involved the direct-PCR of diluted faecal matter without preliminary DNA extraction; two samples of dried faeces from each individual were used: each sample was suspended in 5 uL of pure water, an aliquot of 2 uL of such suspension was used as template for DNA amplification. Such protocol, as is, did not permit successful DNA amplification. In our opinion this was due to the physiological and/or dietary features of the insect (plant secondary metabolites, DNA embedded in the epithelial cells partially degraded). Moreover, Asteraceae leaves are extremely leathery so that faeces don’t melt in water. In order to optimize this approach we are now selecting an appropriate solvent to properly dissolve the faeces and then try to perform a direct-PCR. 2.

DNA extraction and amplification

Faeces were preserved in pure ethanol or ACS grade acetone. DNA extraction trials were carried out using the NaCl extraction protocol (Aljanabi & Martinez, 1997) coupled with the use of a good quality Taq DNA polymerase (Q-BioTaq DNA Polymerase, MP Biomedicals).This latter method gave positive results: we successfully amplified the selected COI fragment of about 300 bp from 3 faecal specimens and from one adult used as positive control. During the experiments, the contamination was monitored by means of a negative PCR control. Thermal cycle parameters were as follows: 94°C for 4 minutes, followed by 37 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 1 min. The last elongation step was extended to 7 min. Reactions were carried out in a 25 µl volume containing incubation Buffer 1x: 67 mM Tris HCl (pH 8.8 at 25°C); 16.6 mM (NH4)2 SO4; 0.01% Tween 20; MgCl2 3.5 mM, 1 mM of each deoxynucleotide, 0.8 pmol of each primer, 1.25 units of Taq DNA polymerase, 1-2 uL template DNA. The PCR products were sent to an external sequencing service. The authenticity of the sequences was confirmed by Blast in GenBank and by comparing with previous sequences obtained from adults of P. chalcomerus. At the moment, our efforts are focussed on the repeatability of the experiments and the optimization of the performance of DNA extraction/amplification method, in order to establish an easily standardized routine protocol for the non-invasive, molecular identification of P. chalcomerus and for other insects/ biocontrol agents too.

18


1.3. Aceria solstitialis (Acari: Eriophyoidea) By A. Stoeva and V. Harizanova, Agricultural University of Plovdiv, Bulgaria. Aims of our studies were the search of new populations of the mite; mite transfer in the laboratory facilities in order to carry out preliminary biological notes; and host range evaluation. During 2007 and 2008, two open field tests were set up near Plovdiv, Bulgaria with some key test plants to better understand the mite dispersal behavior and its host range in more natural conditions.

1.3.1. 2007 field experiment Materials and methods In 2007 a field test on host specificity of Aceria solstitialis was initiated. The test included YST (infested and un-infested with mites) and the plants species on which the mite had developed at the laboratory experiment: Centaurea diffusa (Cd), C. cyanus (Cc), Cynara scolymus (C), and Carthamus tinctorius (Ct). 12 plants from each species (a total of 72) were transplanted outside on 13 August in a Latin square scheme (according to the method, suggested by M. Cristofaro) (see below). The distance between rows was 50 cm, between plants in the row – 1 m. The plants were infested with mites prior the transplantation (except the negative control – YST not infested). Each plant was infested with at least 30 mites (placing on the plant leaf cutting bearing approximately 30 mites, counted under stereoscope). In August the temperatures were extremely high and it was necessary to ensure the survival and normal migration of the mites. The plants were sampled on 26 September. Under stereoscope the plants were checked for presence of mites. Afterwards an extraction was made according De Lillo (2001).

Results and discussion Symptoms of mite feeding Plants of YST with “mite” symptoms were found in the region of Plovdiv, and the villages Klokotnitsa, Bogdanitsa and Spahievo (photos 17-20). The plants were with stunted development, “witches broom”, with less spines. In contrast to the not infested plants which were senescing at the end of summer months, the infested plants remained green. Host specificity testing As a result of feeding by slugs and snails, despite the regular use of pesticides, most of the plants were eaten. At the end of the experiment there were only 39 plants left out of 72 (see the table). In addition on 30 August, 5 and 7 September there was a pouring rainfall of more than 30 mm for each day which probably had influenced the population density of the mite. After extraction mites were found in the samples only from the plants of the positive control (table 1).

19


Tab. 1. Eriophyoid mites (number/plant), extracted from the tested plants

Plant id (row/column)

Plant species

Number of mites/ plant

Plant species (plant id)

YST infested

Plant id

Number of mites/ plant

Cynara scolymus

Plant species (plant id)

Plant id

Number of mites/ plant

Carthamus tinctorius

C/10

5

B/2

0

A/1

0

C/11

5

B/3

0

A/9

0

D/6

7

C/4

0

B/10

0

D/7

11

C/12

0

C/7

0

E/5

0

D/1

0

D/5

0

F/2

2

D/9

0

E/3

0

F/3

14

E/10

0

F/4

0

E/11

0

F/6

0

F/7

0

YST not infested

Centaurea cyanus

Centaurea diffusa

A/6

0

D/2

0

C/1

0

C/2

0

E/4

0

D/11

0

C/3

0

E/6

0

D/4

0

E/7

0

D/12

0

F/5

0

E/1

0

F/10

0

F/11

0

1.3.2. 2008 field experiment Materials and methods The experiment was conducted on a plot of 100 m2 at the experimental field of Agricultural University – Plovdiv. Five species were included in the experiment: YST (C. solstitialis) – infested and non-infested; C. diffusa; C. cyanus; Carthamus tinctorius and Cynara scolymus. The plants were grown from seed starting from March and were transplanted outside at the experimental field at the end of May. The plants were arranged in a pseudo Latin square design (12 replicates - 6 rows, 12 columns), 1 m apart. The plants were infested twice – on 20 and 27 June. For the infestation a laboratory population of the mite was maintained on YST. The initial lab population of the mite originates from the region of Plovdiv. Leaves from the infested YST plants were examined under stereomicroscope to evaluate the population density of the mite. Leaf cuttings with at least 20 mites were used for the infestation. The cuttings were placed on each test plant (except YST which were not to be infested) at the field in the early evening after 7:00 p.m.

20


Table 2. Scheme of the test plantsʼ position

12 11 10 9 8 7 6 5 4 3 2 1

YST+ Cc Cc Ct C YSTYSTC YST+ Cd Cd Ct F

Cd Ct Ct YST+ YSTCc Cc YSTCd C C YST+ E

C YST+ YST+ Cd Cc Ct Ct Cc C YSTYSTCd D

YSTCd Cd C Ct YST+ YST+ Ct YSTCc Cc C C

Cc C C YSTYST+ Cd Cd YST+ Cc Ct Ct YSTB

Ct YSTYSTCc Cd C C Cd Ct YST+ YST+ Cc A

Legend: YST+ (C. solstitialis) – infested; YST- (C. solstitialis) non-infested; Cd - C. diffusa; Cc - C. cyanus; Ct -Carthamus tinctorius and C -Cynara scolymus

Mite sampling The first sampling was done on 29 July. Three 10-cm long apical branches (considered as a sample) were collected from each C. cyanus and C. tinctorius plant, which were already flowering. The rest of the test plants were still at rosette stage. From each YST and C. diffusa plant ten fully developed leaves (a sample) were collected, from C. scolymus – 10 leaf cuttings (3x3 cm) (a sample). The samples were examined under stereomicroscope. Plant height, diameter and number of capitula (for C. cyanus and C. tinctorius) were recorded. The second sampling was done on 28 August. The same sampling procedure, described above, was followed for the different plant species except C. tinctorius which was already senescing. The samples from C. cyanus were examined under stereomicroscope and 50 mites from each sample were stored for further identification by Dr. E. De Lillo. The samples from YST, C. diffusa and C. scolymus were extracted and counted (Monfreda et al., 2007). A number of mites from each sample (depending on the population density) was preserved for identification. The height, diameter and number of capitula were recorded for C. cyanus plants. For the rest of the test plants the diameter of the rosette was recorded. Until the end of the experiment C. diffusa, YST and C. scolymus remained at rosette stage.

Results and discussion First sampling July 29th C. solstitialis - The samples were examined only for presence of mites to evaluate the success of the infestation procedure. Mites were found only in the samples from the intentionally infested plants. The number of mites was not recorded.

21


C. diffusa, C. tinctorius and C. scolymus – no mites were found in any sample. C. cyanus – Mites were found in 4 out of 11 samples. The number of the mites in a sample varied from 45 (B12) to 247 (A1).

A1

E7

Fig. 1. View of the C. cyanus plants on 27 July: to the left – plant A1, with highest mite number per sample; to the right – plant E7, without mites in the sample.

22


Second sampling August 28th Table 3. Results from second sampling of YST on 28 August.

Row

Position

Plant species

Measurements of the rosettes

Number of mites per sample

Diameter (cm) A

2

YST+

39

3

A

3

YST+

A

10

YST-

42

0

A

11

YST-

50

0

B

1

YST-

32

0

B

5

YST+

34

8

B

8

YST+

24

4

B

9

YST-

28

0

C

4

YST-

29

0

C

6

YST+

40

3

C

7

YST+

C

12

YST-

40

0

D

2

YST-

D

3

YST-

40

0

D

10

YST+

47

2

D

11

YST+

E

1

YST+

26

30

E

5

YST-

30

0

E

8

YST-

48

0

E

9

YST+

43

2

F

4

YST+

22

20

F

6

YST-

43

0

F

7

YST-

31

0

F

12

YST+

40

4

23


C. solstitialis - Mites were found only on intentionally infested plants. The number of mites varied from 2 (D10, E9) to 30 (E1). Mites were not found on any of the initially non infested plants, indicating that the mite was not dispersing naturally throughout the field. C.diffusa - Only on a single plant (out of 10) 2 mites were found in 1 sample (B6). C. scolymus – No mites were found. C. cyanus - Mites were found on 8 out of 9 plants. The number of mites varied from 30 (F11) to 915 (A1). The height of the plants with the highest mite population density (up to 915 mites per sample) was around 38-40 cm, the diameter – around 20 cm, and the number of capitula – around 49-130. For the plants with lower population density (up to 300 per sample) the height was around 45-61 cm, the diameter – 19-37 cm, and the number of capitula – 59-444.

Conclusions The results from the field experiment in 2008 show that: •On the target plant species, Centaurea solstitialis, eriophyoid mites (Aceria solstitialis) were found only on intentionally infested plants; •Mites were not found on the initially non infested C. solstitialis plants; •On the nontarget plant species mites were found on Centaurea cyanus (almost on each plant in very high population density) and Centaurea diffusa (only on one plant in very low density); •The mite was not found on any plant of Carthamus tinctorius and Cynara scolymus.

24


2. Russian thistle, Salsola tragus In cooperation with Lincoln Smith, USDA ARS, Albany, CA, USA. Salsola tragus represents one of the most troublesome weed in the drier regions of western North America. It infests range and semi-arid pasture lands as well as cropland, agricultural, residential and industrial areas. As a crop weed it can cause yield losses of greater than 50% in spring wheat. It is also a host for several crop pests and the tumbling old plant can fill irrigation canals and pile against fences. A project for the selection of new biocontrol agents for Russian thistle was started in 2003 in co-operation with USDA-ARS EBCL and PWA. At the present time, 3 potential biocontrol candidates have been selected (fig. 4-7). Preliminary studies on the biology and host specificity of selected organisms are ongoing.

Fig. 4. Anthypurinus biimpressus. stem-boring weevil, found in Tunisia.

Fig. 5. Baris przewalskyi. Root-boring weevil, found in Kazakhstan.

Fig. 6. Eriophyoid mite galls in Eastern Turkey.

Fig. 7. Stem boring larva of Lixus sp. in Central Turkey.

2.1. Aceria salsolae (Acari: Eriophyoidea) In co-operation with L.Smith (USDA ARS, Albany, CA, USA); E.De Lillo (University of Bari, Italy); and J. Kashefi (EBCL, Thessaloniki, Greece). The host range of the mite Aceria salsolae was assessed by means of a field garden experiment carried out from June to October 2007 in BBCA facilities. The aim of the test was to determine mite occurrence on selected non target plants in field conditions. In fact, previous laboratory trials indicated host shifting of the mite, as it survived and multiplied on some non target species that became at risk in the field. Potted plant grown in greenhouse were transferred in June to a plot field outside the laboratory. Pots were placed in rows in randomize positions, with 1 m of distance between each plant. A landscape cloth was used to prevent weeds and a drip irrigation system was installed (Fig. 1).

25


Infested Salsola tragus plants, collected in Greece by J. Kashefi, were used to release mites on the test plants. In June we infested all the nontarget species (Suaeda calceoliformis, Kochia scoparia, Bassia hyssopifolia, Salsola kali) plus 12 specimens of Salsola tragus type A. In addition, twelve specimens of Salsola tragus type A not infested were included to the field trial as a negative control.

Fig. 1. • A. salsolae field garden experiment

Infested plant cuttings from S. tragus collected in Greece, previously checked under microscope, were placed in water vials at the base of the test plants in very close contact to allow the mites to crawl on the

test plant. Cuttings were tied to the test plant to prevent blowing away and were maintained alive, adding water in vials for a week, to allow all the mites to infest the target plant. Plants were sampled twice (mid July and mid September) to extract mites if present. Plant height and approximate diameter (of stems and foliage) were recorded and five cuttings of 10 cm were sampled for each plant. In July, after the cuttings sampling, all the plants (except for S. tragus negative control) were reinoculated with infested S. tragus collected in Greece in mid July by L. Smith, M. Cristofaro and J. Kashefi. Mites extraction was carried out following E. De Lillo protocol. Mites obtained from the extraction were delivered to De Lillo for identification. At the end of extraction activities, all the plants were harvested from the field: all the non target species were destroyed while wet and dry weights of S. tragus infested and not infested were recorded. Results showed that, in July, mite densities were highest on infested S. tragus, followed by S. kali and not infested S. tragus. From July to September, number of mites increased on infested S. tragus and on S. kali but remained extremely low on the non target species (S. calceoliformis, K. scoparia, B. hyssopifolia). At least 98,8% of mites on S.tragus were identified as A. salsolae, whereas mite species different from A. salsolae were recorded on the non target plants. Aceria salsolae caused a considerable damage to infested S. tragus, as showed by the small volume of plants with more than 100 mites per 10 cm cutting (fig. 2). On the contrary, there was no significant damage to the non-target plants, indicating that, under field conditions, these species are not attacked and damaged Fig. 2. Relationship of size of S. tragus plants to total number of eriophyoid mites per sample.

26

by the mite. A paper, showing all the results of this field garden experiment, is currently in press by Biological Control.


2.2. Lixus incanescens (Coleoptera: Curculionidae) In cooperation with L. Smith, USDA ARS Albany, CA, USA; L. Gultekin, Ataturk University, Erzurum, Turkey; and B. Korotyaev, Russian Academy of Science, St. Petersburg, Russia. 30 specimens of Lixus sp. were found on Salsola tragus during a field survey in Kapadokya, Central Turkey, on June 2008. Once in the lab, they were placed singularly in Petri dishes in a climatic cabinet at 21-26°C and with a 14:10 L/D cycle, and allowed to feed on stems of Salsola tragus (type A), in order to find ovipositing females.

2.2.1. Host range experiments Preliminary host range trials were carried out with the selected females in no choice and choice conditions. Choice experiments were carried out in Petri dishes in a climatic cabinet at 21-26°C and with a 14:10 L/D cycle, with one female and one stem of Salsola tragus type A (SATR) plus one stem of one of the following plant species: Salsola soda (SASO); Kochia scoparia (KOSC); Suaeda californica (SUCA); Bassia hyssopifolia (BAHY). Results are summarized in the following table:

PLANT SPECIES

N° REP

SATR+SUCA SATR+BAHY SATR+KOSC SATR+SASO

9 8 10 11

N° REP eggs on SATR 3 1 1 1

N° eggs on SATR 7 1 1 1

N° REP eggs on test plant 0 1 0 1

N° eggs on test plant 0 1 0 1

2.3. Lixus rosenschoeldi (Coleoptera: Curculionidae) In cooperation with L. Smith, USDA ARS Albany, CA, USA; L. Gultekin, Ataturk University, Erzurum, Turkey; and B. Korotyaev, Russian Academy of Sciences, St. Petersburg, Russia. 18 specimens of Lixus rosenschoeldi were found on Salsola kali during a field survey in Sicily, Italy, on July 2008. Insects were collected in the localities of Eraclea Minoa, Caucana and Vaccarizzo (South East Sicily). Once in the lab, they were placed singularly in Petri dishes in a climatic cabinet at 21-27°C and with a 12:12 L/D cycle, in presence of a stem of Salsola tragus (type A) with the aim of selecting ovipositing females. Unfortunately, we were no able to obtain any egg, and adults have been stored in climatic chamber for overwintering diapause.

27


2.4. Cosmobaris scolopacea (Coleoptera: Curculionidae) In cooperation with L. Smith, USDA ARS Albany, CA, USA; M.C. Bon, USDA ARS EBCL, Montpellier, France; Jens Prena, USDA ARS Washington DC; and Enzo Colonnelli, Rome, Italy. The work on the stem boring weevil Cosmobaris scolopacea was started some years ago by Gaetano Campobasso, USDA ARS EBCL Rome Substation. On March 02, 2008 Gaetano suddenly passed away; few months later, together with L. Smith and M.C. Bon, we decided to continue the work started by Gaetano, approaching the project from both genetic and behavioral points of view.

2.4.1. Genetic approach Samples of adults and larvae of the stem boring weevil C. scolopacea were collected in 4 sites in Italy (2 in Sicily and 2 near Rome), in Central Turkey, in Northern Iran, in Spain and in the United States on both Russian thistle (Salsola spp.) and Chenopodium album. The material was kept in separate labeled vials in ETOH 95% and send to Marie Claude Bon for genetic analysis. Collections started in the spring 2008 and will continue through the 2009 field season.

2.4.2. Host range experiments 41 specimens of Cosmobaris scolopacea were collected during a field survey in Sicily, Italy, on July 2008. Insects were found on Salsola kali in the localities of Kamarina, Caucana, Vaccarizzo and Eraclea Minoa (South East Sicily). Once in the lab, they were placed singularly in Petri dishes in a climatic cabinet at 2126°C and with a 14:10 L/D cycle with a stem of Salsola tragus (type A) to obtain eggs. Preliminary host range trials were carried out with the selected females in choice conditions. Experiments were carried out in Petri dishes in a climatic cabinet at 21-26°C and with a 14:10 L/D cycle, with one female and one stem of Salsola tragus type A (SATR) plus one stem of one of the following plant species: Salsola soda (SASO); Salsola kali (SAKA); Kochia scoparia (KOSC); Suaeda californica (SUCA); Bassia hyssopifolia (BAHY). Results are summarized in the following table:

PLANT SPECIES

N° REP

SATR+SUCA SATR+BAHY SATR+KOSC SATR+SASO SATR+SAKA

9 10 9 12 13

28

N° REP eggs on SATR 2 3 0 1 2

N° eggs on SATR 2 4 0 3 4

N° REP eggs on test plant 2 3 1 1 3

N° eggs on test plant 2 3 1 6 5


3. Rush skeletonweed, Chondrilla juncea In cooperation with George Markin and Justin Runyon, USDA Forest Service, USA.

3.1. Sphenoptera foveola By Biological Control Group Zoological Institute, St.Petersburg, Russia. Field and laboratory studies on biology and host specificity of Sphenoptera foveola (Gebler) (Coleoptera, Buprestidae), potential agent for biological control of the rush skeleton weed,Chondrilla juncea L, conducted by Biocontrol Group (Zoological Institute, St.Petersburg, Russia) in 2007.

Summary During 2007, the following work was conducted in Kazakhstan, in Russia, and in Armenia: Reading of the results of the earlier (in 2005) started field tests on host specificity, collecting adults for new field and laboratory studies, and conducting selective field sampling in natural habitats of S. foveola in Almaty region of Kazakhstan. Establishing and reading of the results of field tests on host specificity in Rostov prov. of Russia. Establishing and reading of the results of field tests on host specificity in Armenia. Laboratory experiments on host specificity conducted in laboratory facilities of Biocontrol Group (St. Petersburg, Russia). During these studies, new important data were obtained. In combination, these data suggest that S. foveola host specificity is very strict. It is definitely limited by species of the genus Chondrilla and, moreover, it seems that S. foveola could successfully develop only on a certain group of Chondrilla species. Some data obtained in field and in laboratory conditions suggest that the target weed, Chondrilla juncea, in certain cases can be also suitable for adult and larval feeding. Particularly, S.foveola adult feeding on Ch. juncea was first recorded in test plants in natural conditions in Russia and S. foveola larva feeding on Ch. juncea was first recorded in test plant in natural conditions in Armenia. In addition, larval feeding on Ch. graminea, which is closely related to Ch. juncea was recorded in laboratory. On the other hand, it seems that both in field and in laboratory conditions, only few of the tested individuals were able to develop when feed on Ch. juncea roots which could be possibly considered as a result of intraspecific variability. Thus, the further studies are necessary to finally investigate the host specificity of S. foveola oviposition, larval feeding, and development and to estimate the potential of this buprestid for biological control of the rush skeleton weed.

3.1.1. Field collection of adults for laboratory tests About 140 S. foveola adults were collected from or under Ch. ambigua and Ch. incanescens plants during 19-24 June 2007 in their natural habitats (Fig. 1) in Qumbasy sandy desert, 69 km NNW of Qapshaghay, Almaty Province, Kazakhstan (ca 44°25’34” N and 76°47’42” E) and were later used for field and laboratory tests.

29


Fig. 1. Places where Sphenoptera foveola adults were collected (Kazakhstan, June, 2007).

3.1.2. Field observations on host specificity With this aim, roots of plants from several species of Asteraceae family, naturally growing in close vicinity to Chondrilla plants infested by S. foveola were drawn out and inspected for S. foveola larvae or any traces of their feeding, i.e. root galls (more exactly, latex cases). This sampling was conducted in the same habitats, where adults for filed and laboratory tests were collected. Several sites with various latex containing and not containing xerophilous plant species were inspected, plants for sampling were selected randomly by squares or along transects. The results are summarized in table 1. From our data, it is seen that (at least in the studied population and habitats) S. foveola larvae could be found only on Chondrilla species. As for the difference between Ch. ambigua (sect. Brachyrhynchus) and Ch. incanescens (sect. Euchondrilla), the two species seems to be more or less equally populated, the difference being insignificant, at least at the given sample sizes.

3.1.3. Field tests on host specificity Field tests in Kazakhstan In June 2007, the results of no-choice field tests established in Kazakhstan in 2005 were finally recorded. This work was aimed to evaluate host specificity (oviposition and larval development) of S. foveola in nochoice tests under natural conditions.

30


Table 1.

Number of plants with traces of S. foveola Plant species

Number inspected

empty root galls

root galls with larvae

total root galls

Total roots galls, percentage

Lactuca sp. ?*

10

0

0

0

0%

Scorzonera sp. ?

16

0

0

0

0%

Sonchus sp. ?

31

0

0

0

0%

Echinops sp.

20

0

0

0

0%

Jurinea sp.

12

0

0

0

0%

Cousinia sp.

23

0

0

0

0%

Centaurea diffusa Lam.

4

0

0

0

0%

Chondrilla incanescens Kar. & Kir.*

52

22

15

37

71%

Chondrilla ambigua Fisch.*

133

89

18

107

80%

Plant species containing latex In each test, one naturally growing plant was used. The test plant was covered by cage made of cotton and gauze (Fig. 2).

Fig. 2. Mounting cages for S. foveola no-choice host range tests in natural conditions (Kazakhstan, 2005).

In most of cages, one S. foveola male and one female were placed, but only females were used in a few cases. Inside the cage, the soil was covered with sand to offer a substrate suitable for oviposition. The base of the cage was fixed in soil. Position of each test plant was recorded by GPS with the most possible accuracy, which made possible to locate almost each test plant 2 years later. Most of test plants turned to be died and completely destroyed, but in some other cases the reading of the results was possible. In a few cases, the dead beetles were found inside the cages.

31


From table 2 it is seen that most of test plants died more or less independently of the plant species which was possibly caused by too late removing of cages (when this experiment was started, it was planned to remove the cages in 2005). It seems that the cage itself physically depressed the plant when it was covered during more than 2 years or may be the cage promoted development of some insect pests as e.g. aphids. Anyway, basing on the data obtained (table 2), it is clear that remnants of root galls were found only on plants of Chondrilla species, and in none of other tested plants. However, all of the galls found on test plants were empty: obviously, larvaehave finished feeding and have pupated during two years passed since the beginning of the test. Note that in similar galls on control plants of the same Chondrilla species, S. foveola larvae were found (fig. 3).

Fig. 3. Reading of the results of S. foveola no-choice host range tests in natural conditions in Kazakhstan: opening the cage, digging the root, opening the galls, dissected gall with mature larva from control plant (Kazakhstan, 2007).

Thus, we conclude that in their natural habitats (sandy deserts of Kazakhstan) Sphenoptera foveola larvae develop only on plants of the genus Chondrilla (particularly, C. ambigua and C. canesces), the difference between the last two species being insignificant, al least based on the studied samples. Note that this conclusion was made both from “observations” (selective sampling and inspection of different plant species, table 1) and from “experiments” (no-choice tests of oviposition and larval feeding specificity, table 2).

32


Table 2. Sphenoptera foveola oviposition and larval development no-choice tests in natural conditions (Kazakhstan).

Cage ID

Coordinates

Host Plant

Results

S-01

44.21.16,3 N 76.57.52,3 E

Scorzonera sp. ?

Destroyed plant, no galls

S-02

44.21.16,3 N 76.57.52,4 E

Scorzonera sp. ?

Destroyed plant, no galls

S-03

44.21.17,1 N 76.57.55,8 E

Scorzonera sp. ?

Not found

S-04

44.21.15,8 N 76.57.56,6 E

Scorzonera sp. ?

Destroyed cage

S-05

44.21.15,2 N 76.57.56,2 E

Scorzonera sp. ?

Destroyed cage

S-06

44.21.15,2 N 76.57.56,6 E

Cousinia sp.

Not found

S-07

44.21.14,8 N 76.57.56,8 E

Cousinia sp.

Not found

S-08

44.21.13,8 N 76.57.58,5 E

Cousinia sp.

Not found

S-09

44.21.13,1 N 76.57.58,8 E

Cousinia sp.

Destroyed plant, no galls

S-10

44.21.11,9 N 76.57.59,8 E

Cousinia sp.

Remnants of beetles, destroyed plant, no galls

S-11

44.21.11,4 N 76.57.58,0 E

Taraxacum sp.

Not found

S-12

44.21.11,2 N 76.57.56,8 E

Taraxacum sp.

Destroyed plant

S-13

44.21.10,5 N 76.57.56,6 E

Taraxacum sp.

Destroyed plant

S-14

44.21.09,7 N 76.57.56,2 E

Taraxacum sp.

Destroyed plant

S-15

44.21.09,0 N 76.57.55,2 E

Taraxacum sp.

Destroyed cage

S-16

44.21.20,1 N 76.58.01,1 E

Chondrilla incanescens

Destroyed plant

S-17

44.21.19,9 N 76.58.00,9 E

Chondrilla incanescens

Not found

S-18

44.21.20,5 N 76.58.02,0 E

Chondrilla incanescens

Not found

S-19

44.21.22,0 N 76.58.01,7 E

Chondrilla incanescens

Destroyed plant

S-20

44.21.21,4 N 76.58.06,4 E

Chondrilla incanescens

Not found

S-21

44.25.48,5 N 76.47.35,9 E

Chondrilla incanescens

Remnants of root galls

S-22

44.25.48,1 N 76.47.37,0 E

Chondrilla incanescens

Destroyed plant

S-23

44.25.47,7 N 76.47.36,4 E

Chondrilla incanescens

Destroyed plant

S-24

44.25.48,5 N 76.47.36,8 E

Chondrilla incanescens

Young plant, remnants of old root galls

S-25

44.25.48,0 N 76.47.36,8 E

Chondrilla incanescens

Dead beetles, remnants of root galls

S-26

44.25.45,9 N 76.47.21,0 E

Chondrilla ambigua

Destroyed plant

S-27

44.25.46,5 N 76.47.16,8 E

Chondrilla ambigua

Destroyed plant

S-28

44.25.43,3 N 76.47.13,0 E

Chondrilla ambigua

Remnants of root galls

S-29

44.25.43,1 N 76.47.11,9 E

Chondrilla ambigua

Destroyed plant

S-30

44.25.42,6 N 76.47.04,3 E

Chondrilla ambigua

Not found

33


II. Field tests in Russia Field tests in Southern Russia (Kalitvenskaya village, environs of Kamensk, Rostov province), were established at 5.07.2007 in the population with ca 100 Chondrilla juncea plants naturally growing in sandy clearing of mixed forest. The cages were mounted by the same method which was used in Kazakhstan (see fig. 2) and S. foveola adults collected in Kazakhstan (Qumbasy location) were used. In these tests only native Chondrilla juncea were used as a host plant. In ca 20 days (31.07.2007), the cages were opened and removed from test plants. At the same time, test plants were checked for traces of adult feeding (Fig. 4). Three months later, 15.10.2007, roots of all test plants were drawn out and inspected for any traces of larval feeding. The results of this experiment are presented in table 3. It is seen that the feeding traces were recorded in more than a half of the cages suggesting that Ch. juncea is suitable for S. foveola adult feeding. However, in none of the tested plants any traces of larval feeding were observed.

Fig. 4. S. foveola no-choice host range tests in natural conditions in Russia: cage on the plant, removed cage near the plant, traces of adult feeding (Kamensk, 31.07.2007).

34


Table 3. Sphenoptera foveola oviposition and larval development no-choice tests in natural conditions (Russia).

Cage ID

Coordinates

Host Plant

Adult feeding (traces)

Larvae feeding

R01

48.15.02 N 40.28.44 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R02

48.15.02 N 40.28.44 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R03

48.15.02 N 40.28.44 E

Ch. juncea

R04

48.15.02 N 40.28.44 E

Ch. juncea

No

Healthy plant, no traces of larval feeding

R05

48.15.02 N 40.28.42 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R06

48.15.02 N 40.28.42 E

Ch. juncea

R07

48.15.02 N 40.28.42 E

Ch. juncea

No

Healthy plant, no traces of larval feeding

R08

48.15.02 N 40.28.42 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R09

48.15.02 N 40.28.42 E

Ch. juncea

R10

48.15.02 N 40.28.40 E

Ch. juncea

No

Healthy plant, no traces of larval feeding

R11

48.15.02 N 40.28.40 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R12

48.15.02 N 40.28.40 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R13

48.15.02 N 40.28.40 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

R14

48.15.02 N 40.28.40 E

Ch. juncea

Yes

Healthy plant, no traces of larval feeding

Destroyed plant

Destroyed plant

Destroyed plant

35


Field tests in Armenia Similar no-choice field tests were conducted in 2007 in two locations in Armenia: Uranots near Surenavan (ca 39.44 N, 44.49 E) and Goravan (Vedi environs, ca 39.55 N, 44.46 E ). Same as in Russia, only naturally grown Chondrilla juncea plants were used. In two sites, 20 cages were established by the method described above also using S. foveola adults from Kazakhstan (Qumbasy location. The difference between sites was in the type of soil. In the first site (Surenavan), it was rather stony, while in the second site (Goravan), it was almost pure sand (Fig. 5). In three months after the beginning of the test, roots of all tested plants were inspected. In addition, other 10 “control� naturally growing plants were studied. In none of these control plants any buprestid larvae was recorded, while in one of tested plants growing in sandy site Sphenoptera larva was found (Fig. 5).

-

Fig. 5. No-choice specificity tests in Armenia: stony (Surenavan) and sandy (Gorava) deserts, roots prepared for dissection and Sphenoptera larva found in Goravan location in one of tested Ch. juncea plants.

Laboratory tests For laboratory tests, eggs laid by females collected in Kazakhstan during their transportation to St.Petersburg were used. In addition, the rest of adults which were not used for the field tests were kept in laboratory cage fed with Ch. juncea plants (Fig. 6, 7). Eggs found were collected and placed in Petri dishes filled with slightly moist sand or covered with slightly moist filter paper. S. foveola eggs successfully hatched under both conditions, and neonate larvae were used for no-choice host specificity tests with potted plants. All plants used in these tests were grown from seeds in laboratory conditions. For each test, 3-5 larvae were gently transferred by soft brush into the sandy soil close to the stem base of the tested potted plant. In approximately a month, root of each tested plant was dissected and inspected for S. foveola larvae or any traces of their feeding (table 4). Larvae or traces of larval feeding were found only on species of the genus Chondrilla (Fig.8).

36


Fig. 6. The cage with Sphenoptera foveola females in laboratory.

Fig. 7. Sphenoptera foveola female feeding Chondrilla juncea in laboratory conditions.

Fig. 8. Sphenoptera foveola larvae feeding on Chondrilla ambigua.

Table 4. Sphenoptera foveola larvae host specificity no-choice tests in laboratory conditions.

Number of positive results (at least one larva or traces of larlar val feedeing found)

Number of tested Host plant species

Percentage of larvae survived

plants

larvae

plants

larvae

total root galls

Taraxacum sp.

13

49

0

0

0

0%

Sonchus sp. ?

8

25

0

0

0

0%

Chondrilla graminea Bieb. *

7

21

2

0

2

0%

Chondrilla juncea1

7

20

0

0

0

0%

Chondrilla juncea2

2

6

0

0

0

0%

Chondrilla juncea3

1

3

0

0

0

0%

Chondrilla juncea3

1

3

0

0

0

0%

Chondrilla ambigua Fisch.**

5

25

4

7

0

28%

* grown from seeds originated from Volgograd province, Russia 1) – grown from seeds originated from Roggins (Idaho, USA) 2) – grown from seeds originated from Bantis (Idaho, USA) 3) – grown from seeds originated from Lime bay, Anderson Reservoir (Idaho, USA) ** grown from seeds originated from Kazakhstan

Conclusions Summarizing the results, we conclude that S. foveola host specificity is very strict. It is definitely limited by species of the genus Chondrilla and, moreover, it is obvious from field observations in Kazakhstan that 37


S. foveola could successfully develop only on a certain group of Chondrilla species from Brachyrhynchus (Ch. ambigua) and Euchondrilla (Ch. incanescens) sections. Some data obtained in field and in laboratory conditions suggest that the target weed, Chondrilla juncea (sect. Euchondrilla) can be also suitable for adult and larval feeding. Particularly, S. foveola adult feeding on Ch. juncea was first recorded in natural conditions in Russia (fig. 4), and presumably S. foveola larvae feeding on Ch. juncea was first recorded in natural conditions in Armenia (fig. 5). In addition, larval feeding on Ch. graminea, which is closely related to Ch. juncea was recorded in laboratory (table 4). On the other hand, it seems that both in field and in laboratory conditions, only few of the tested individuals were able to develop when feed on Ch. juncea roots. Note that this could not be explained by low survival, as e.g. in laboratory tests almost all of plants belonged to Chondrilla species native to Kazakhstan gave positive results (table 4). Possibly, this could be a result of intraspecific variability. Thus, the further studies are necessary to finally investigate the host specificity of S. foveola oviposition, larval feeding, and development and to estimate the potential of this buprestid for biological control of the rush skeleton weed.

Biological control of Chondrilla juncea in Bulgaria, 2007 By I. Lecheva, A. Karova, A. Petkova, C. Mincheva, Agricultural University of Plovdiv, Bulgaria.

3.2. Schinia cognata (Lepidoptera: Noctuidae) Methods of observation Observations concerning dynamics of population density of Schinia cognata were carried out in an uncultivated area consisting of 6 ha located between town of Plovdiv and village of Trud, picture 1. Investigations were performed during the active vegetation of the host plant Chondrilla juncea, from April till October. The method “sweeping by entomological net� and visual method (searching leaves, branches, flower buds and flowers of host plant individually) were accomplished mostly. Observations were repeated every 10-14 days. Examination of Sch. cognata’s larvae for verification of restricted feeding specialization of the species was conducted under laboratory conditions using folio isolators. The test was realized in the Biological Control Laboratory of Department of Entomology, Faculty of Plant Protection and Agroecology, University of Agriculture in Plovdiv, picture 2.

Pict. 1. Uncultivated area between Plovdiv and Trud where we carried out field observations.

38

Pict. 2. Biological control laboratory, University of Agriculture, Plovdiv where we set the lab test.


Pict. 3. Plants of Chondrilla juncea raised under laboratory conditions.

Pict. 4. Plants of Cichorium intybus raised under laboratory conditions.

The test was conducted in 2 versions, 10 replications each (first version – with 10 plants of Chondrilla juncea, and second one – with 10 plants of Cichorium intybus). In July 2007 some plants of Ch. juncea and C. intybus were picked up from natural population, planted in pots and raised under laboratory conditions, picture 3 and 4. One folio isolator was placed on each plant and one 3rd instar into each isolator respectively. Feeding and behavior of the larvae were observed daily.

Results and discussion Population density In 2007 the first larvae of Sch. cognata (first generation) emerged at the beginning of July – on July 3rd 5 larvae on 100 sweepings by entomological net were encountered, figure 1. Maximum number of larvae from first generation was reported on July 10th – 20 larvae on 100 sweepings by entomological net. All larvae of first generation pupated normally despite of the extremely high temperatures that took place in July 2007. The first larvae of the second generation appeared on August 3rd. September turned out to be unusually favorable for the development of Sch. cognata larvae. From the beginning of the month till September, 20th, really high density of the caterpillars was recorded. Maximum number of larvae from second generation was reported on September, 4th, when 42 caterpillars were encountered. Larval density decreased after September, 20th, fig. 1.

Fig. 1. Dinamics of population density of larvae of Schinia cognata in Chondrilla juncea population in the region of Plovdiv, 2007 45 35 30 25 20 15 10 5

20-Sep-07

13-Sep-07

6-Sep-07

30-Aug-07

23-Aug-07

16-Aug-07

9-Aug-07

2-Aug-07

26-Jul-07

19-Jul-07

12-Jul-07

5-Jul-07

0

28-Jun-07

Number of larvae

40

Dates of sampling

39


Experiment for verification of restricted feeding specialization In the version with Ch. juncea in all 10 replications each larva was feeding as usual and developed normally into a pupa and then reared to an adult. In the no choice test with C. intybus in all 10 replications not even one larva did feed on the plant. All larvae were wandering about permanently for the first 2 days but after that they began to die. Seven days after the experiment was set all larvae were dead because they refused to feed however they were provided enough food.

3.3. Simyra nervosa (Lepidoptera: Noctuidae) As a result of our observations it was found that the noctuid moth Simyra nervosa develops two generations per year. For the conditions of Plovdiv in 2007 the first generation began its development during the period of the end of June and beginning of July. In 2007 the fist detected larvae were 3rd instar, on July, 3rd. They were feeding openly on the flower heads of Chondrilla juncea. At the next observation at July, 10th, caterpillars’ density was considerably higher – 14 caterpillars, 10 of which were 5th instar and 4 of them - 3rd. Larvae pupated at the end of July.

Fig. 2. Dinamics of population density of larvae of Symira nervosa in Chondrilla juncea population in the region of Plovdiv, 2007 25

Number of larvae

20 15 10 5

19-Sep-07

12-Sep-07

5-Sep-07

29-Aug-07

22-Aug-07

15-Aug-07

8-Aug-07

1-Aug-07

25-Jul-07

18-Jul-07

4-Jul-07

11-Jul-07

27-Jun-07

20-Jun-07

0

Dates of sampling

The second generation began its development on August, 14th. Then first larvae of the second generation were found. At the next observations uninterrupted increasing of the number of caterpillars was encountered and on August, 27th 22 larvae were reported, figure. 2. Simyra nervosa ended its life cycle in the beginning of September.

Conclusion However the summer season of 2007 had extremely hot temperatures and no rains fell down larvae of both noctuid moths Schinia cognata and Simyra nervosa developed normally and were observed at really high density, figure 3.

40


Fig.3. Dinamics of population density of larvae of noctuid moths Schinia cognata and Symira nervosa in Chondrilla juncea population in the region of Plovdiv, 2007 45 40 Number of larvae

35 30

Sch. cognata

25 20 15 10

S. nervosa

5

20-Sep-2007

13-Sep-2007

6-Sep-2007

30-Aug-2007

23-Aug-2007

16-Aug-2007

9-Aug-2007

2-Aug-2007

26-Jul-2007

19-Jul-2007

12-Jul-2007

5-Jul-2007

28-Jun-2007

0

Dates of sampling

In conclusion we would say that the experiment for verification of the feeding specialization of larvae of Sch. cognata once again proves categorically that the species demonstrates restricted feeding specialization and feeds only on Chondrilla juncea. If the host plant is not available larvae die in some days.

41


Field season 2008 Introduction During the summer season 2008, BBCA was involved in the search of Chondrilla juncea natural enemies, focusing the work mainly in 3 aspects: i) conducting collections of the noctuid moth Schinia cognata; ii) performing host range tests with this moth; and iii) carrying out explorations for new natural enemies. In addition, we provide funds to our Russian cooperators and we perform collections and shipments of S. cognata for Jeff Littlefield, Montana State University, Bozeman, MT. LOCATIONS Bulgaria Turkey Bulgaria Bulgaria Slovakia

PERIOD July 08-12 July 19-24 July 25-29 Aug 11-14 Aug 15-18

SITE Plovdiv Cappadocia Plovdiv Plovdiv Nitra

COOPERATORS Mc Ivie & Mayer

Peter Toth

Except for Turkey and Slovakia, all the other travels were focused on the collection of Schinia cognata, and to perform field biological observations. The travel in Turkey was focused in the search of populations of the root boring buprestid Sphenoptera sp., while the work in Slovakia was mainly explorations for natural enemies. The work done by the Academy of Russia will be presented in a different report. As usual, BBCA received a great support from cooperators from Slovakia (Dr. Peter Toth, Entomologist of the Agricultural University of Nitra, Slovakia); Dr. Toth was deeply involved in the exploration and the search of new potential agents in Slovakia, and made several travels in the area before our visit.

Host-range tests in laboratory A total of 26 larvae of Schinia cognata, at different larval instars, was collected during the first two visits in Bulgaria (July). Due to the little number, were tested in no-choice conditions at the BBCA facilities in Rome. Last year skeletonweed (RSW) plants from Bulgaria have been used as control, together with 6 test plant species:

Lactuca sativa

(Lettuce- LAC)

Chicorium intybus

(Chicory- CHI)

Carthamus tictorius

(Safflower-oleic - SOL)

Carthamus tictorius

(Safflower-linoleic - SLI)

Sonchus asper

(SON)

Taraxacum vulgare

(TAR)

We used potted plants, and we confined the larvae on the plant using small transparent cages. Due to the low number of larvae, the test was carried out following “rotation patterns�. Schinia cognata larvae (2-3 per replication, at different instars) were confined with the control for 2 days, then transferred to a test plant. Feeding and larval behavior was recorded for 2 days, then the larvae were moved back on skeleton weed; after two days, the larvae were transferred to a test plant different from the previous. The aim of

42


the protocol was to offer to each larva how many of the 6 test plants was possible (plus the control). Of course some of them died (6) and 5 pupated without feeding on the first test plant.

Results Results are summarized in the following table: PLANT SPECIES N°REP (pots) RSW 2 LAC 2 CHI 3 SOL 2 SLI 2 SON 2 TAR 2 FEEDING 0 = no feeding 1 = faeces on the cage bottom 2 = nibbling 3 = active feeding

N°of OBSERV. 5 5 5 4 4 3 3

FEEDING SCORES 2/3 1 1 0 0 0 1

MORTALITY 1 1 2 0 1 1 0

The results clearly indicate that real feeding was observed only on the target weed; feeding score “1”, recorded on lettuce and chicory (faeces on the bottom without any clear feeding), were probably due to the digestion of previous feeding on the control. The experiment, of course, was far to be perfect: beside to the limited number of larvae, they had at different instars, and some did not feed on the plant because very close to the pupation. In addition, the synchronization of the test plants with the life cycle of the moth was not very easy: the young instars need fresh flower buds, and it is complicate to have all the plants at the same phenological stage in the middle of July. This is the reason why, looking at the number of potted plants, we reported only small numbers (including the target weed). We also brought back a great number (34) of adults, collected only on rush skeleton weed flowers (see pict. 1). We tried to get oviposition using 3 different methods: i) confining the adults in a transparent glass cage, without the target weed but with water and honey supply, and a piece of organdy as oviposition substrate; ii) confining the adult in a plastic cage (30x30x30 cm., with water, honey, and fresh bouquets of RSW; and iii) confining the adults on the upper part of potted Rush skeletonweed bolting plants, confined by a organdy sleeve. All the 3 methods failed and we did not recorded any oviposition; adults dies within 3 days. We recorded (and collect) a S. cognata parasitoid wasp, not yet determined (see pict. 2). The second collection was carried out in mid August, and more than 130 larvae have been collected in just 2 days. We agreed to send about 100 of these larvae to Dr. Jeff Littlefield, to improve his laboratory colony. We kept some of them, trying to get adults in order to replicate the oviposition trial. About 40 % of the larvae died, and the rest pupated and went in diapause. 43


Field explorations From June 19-28 a travel was carried out in Central Turkey together with Dr. Mike Ivie, University of Montana, USA, and his student Crystal Mayer, with the specific goal to detect and collect populations of the stem boring beetle Sphenoptera spp. During the seven days of work, more than 20 sites (BBCA records, for sure more in their report) were recorded, but only one single adult of Sphenoptera sp. was collected (resting on a different pant species) near Nevhseir (see pict. 3).A sample ranging from 10 to 20 plants was dissected (roots and stems) per site, but we recorded only the very common Bradyrrhoa gilveolella (see pict. 4).

Pict. 1. Schinia cognata on RSW flowers.

Pict. 2. Parasitoid of S. cognata in Bulgaria.

Pict. 3. Buprestid site in Turkey (Nevsehir).

Pict. 4. Root feeding moth Bradyrrhoa gilveolella.

A first record of a stem-boring buprestid larva was recorded near Nitra, central Slovakia (see pict. 5), in the middle of August. We found only two larvae of the beetle, while 2 stem-boring tephritid fly species (according to a first determination Ensina sonchi and Campiglossa producta) were relatively more common (two sites), with up to 4 larvae/pupae per infested plant (see pict. 6). In another site of the same region, recorded a massive infection of powder mildew like fungus (see pict. 7). Few mature larvae of S. cognata have been reported in 3 sites.

44


Five samples samples for DNA has been collected respectively in 2 sites in northern Iran and 3 sites in Slovakia.

Pict. 5. Buprestid larvae in RSW stems in Slovakia.

Pict. 6. Stem boring tephritid larva in RSW in Slovakia.

Pict. 7. Chondrilla juncea showing powder mildew like symptoms in Slovakia.

Considerations for the future According to our experience, we recommend to perform open field tests with Schinia cognata, either in the area where the moth occurs and/or at the BBCA facilities in Italy. For this purpose we already started a field plot where 50 Chondrilla juncea plants have been sown, in order to have the plants in perfect phenological stage during next summer. Some of the plants have been sown also in a way to make as well up to 6 large field cages (2x2x2 m), if we would like to consider the option to confine the moths. Scientific reliable laboratory tests can be carried out only if it will be possible to develop a rearing system that will allow moth female oviposition in captivity, possibly on artificial substrate. Of course, a field test at BBCA facilities requires a better survival rate of the adults. Sphenoptera spp., the biological control agent showing the most impressive impact on the target weed (together with the mite), has shown so far a very scattered distribution and is very uncommon. BBCA would like to continue the explorations for this biological control agent as well for the root boring moth Oporopsamma wertheimsteini, but any biological control screening will be subordinated to the finding of consistent and stable insect populations, similar to S. foveola that occurs in Kazakhstan.

45


4. Scotch thistle, Onopordum acanthium In cooperation with Lincoln Smith, USDA ARS, Albany, CA, USA. Scotch thistle is a biennial weed with Eurasian and Central Asian origin that is aggressively invading natural habitats and pastures in North America. This weed is found in most western states and occupies sites characterized by high soil moisture. It competes with desirable foraging species and reduces their growth, with remarkable effects on livestock and wildlife grazing. BBCA started opportunistic field surveys for the selection of new biocontrol agents in 2007. Numerous associated organisms were found; three of them were preliminarily selected (fig. 1-3).

Fig. 1. The root-boring weevil Trichosirocalus briesei found in Spain.

Fig. 2. The root-boring moth Eublemma amoeana in Turkey.

Fig. 3. Seed-feeder weevil Larinus latus in Turkey.

Preliminary host range tests have been carried out with the weevil Trichosirocalus briesei. One hundred specimens of Larinus latus adult weevils were collected in Central Turkey and shipped to the USDA ARS quarantine facility in Albany, Ca (USA) for first host range tests. In 2008 carried out collections of the flea beetle Psylliodes chalcomerus from populations associated to scotch thistle, successively tested in a 3-way choice test at the USDA ARS quarantine facility in Albany, CA (USA).

4.1. Trichosirocalus briesei (Coleoptera: Curculionidae) Trichosirocalus briesei specimens were collected on Onopordum sp. plants during two field surveys in Northern Spain carried out on January and on March 2008. Insects were placed singularly in Petri dishes in a climatic cabinet at 10-14째C in presence of one leaf of Onopordum acanthium, in order to obtain oviposition and select females.

4.1.1 Host range experiments Host range trials were carried out with the selected females in no choice and choice conditions. No choice tests were performed in Petri dishes in a climatic cabinet at 10-14째C, with one female and one leaf of the following plant species: Onopordum acanthium (ONO); Cynara scolymus (CYSC); Sylibum marianum (SYMA); Carduus nutans (CANU); Cirsium hydrophilum (CIHY).

46


Insects collected on January showed a strong preference for O. acanthium, with only a small percentage of insects laying eggs on S. marianum. Plant species ONO CYSC SYMA CANU CIHY

N°REP 52 11 8 9 7

N°REP with eggs 44 0 2 0 0

%REP 84 0 25 0 0

On the other hand, insects collected on March laid eggs preferably on O. acanthium but in small percentage also on all the test plants: Plant species ONO CYSC SYMA CANU CIHY

N°REP 201 40 31 31 29

N°REP with eggs 158 1 1 7 4

%REP 78,6 2,5 3,2 22,5 13,8

Preliminary choice experiments were carried out with insects collected in the field on January. We made 3 sets of trials: one with a pair of insects (1 male + 1 female) and two with four specimens each. Every group of insects was placed in a Petri dish in a climatic cabinet at 10-14°C in presence of one leaf of O. acanthium and one leaf of three of the following test plants:Cynara scolymus (CYSC); Sylibum marianum (SYMA); Cirsium hydrophilum (CIHY); Centaurea cineraria (CECI); Centaurea solstitialis (YST). Results showed a clear preference for O. acanthium with a limited occurrence of oviposition on other test plants, but further investigations are needed.

4.1.2. Plans for 2009 During 2009 experiments will be carried out with scotch thistle associated populations of P. chalcomerus using an ecological, genetic and morphometrical approach. Moreover, a new collection and host range trials with the weevil T. briesei will be performed, in addition to several exploration in order to find new biocontrol agents.

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5. Perennial pepperweed, Lepidium latifolium In cooperation with H. Hinz and E. Gerber, CABI, Delemont, Switzerland; R. Hayat and L. Gultekin, Ataturk Univ. of Erzurum, Turkey; R. Ghorbani and A. Ghorbanali, Ferdowsi Univ., Mashhad, Iran; and Livy Williams, USDA ARS, Reno, NV, USA. Lepidium latifolium is a herbaceous, semi-woody perennial that typically reaches 0.5–1.5 m in height and reproduces vegetatively and by seed. 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. This weed is native to central Asia and is believed to have been introduced into the United States through California around 1900 as a contaminant of sugar beet seeds. Now, it is widely distributed in western United States, costal New England, Mexico and Canada. Perennial pepperweed 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. It is highly competitive and invasions result in dense monocultures and subsequent loss of biodiversity through the exclusion of native vegetation. In agricultural settings, the species competes with crop plants and reduces agricultural yields. In rangelands it decreases the quality of hay and reduces livestock-carrying capacity. In addition, it produces secondary plant compounds that are reported to be toxic to livestock. This weed is difficult to control because of its large, stout root system. Mechanical or cultural control techniques usually provide no permanent reduction of populations. In 2006 CABI E-CH and BBCA decided to share resources and collaborate closely in their effort developing biological control for Lepidium latifolium. Since then, field surveys have been conducted in cooperation and some of the results of both research teams are presented in the following paragraphs.

5.1. Summary of 2007 activities Field surveys in part of the likely area of origin of perennial pepperweed were continued in 2007. Six field trips were conducted, two to central and north-eastern Turkey and one each to southern Russia, western China, southern Ukraine and Iran. The field trips to Turkey, Russia and China focused on collecting potential biological control agents already prioritized in 2006, while surveys in the other countries aimed to find new potential agents. As in 2006, surveys in Turkey were conducted in cooperation with Prof. Rüstem Hayat and Dr Levent Gültekin (both Atatürk University, Erzurum). Special emphasis was given in 2007 to studying the biology of Melanobaris sp. n. pr. semistriata in the field and to collecting adults of this root-mining weevil to conduct host-specificity tests in quarantine at CABI Europe – Switzerland (CABI E-CH). Further, the field

48


phenology and field host range of another potential biological control agent, the stem-mining fly Lasiosina deviata, were investigated. The survey in Russia was concentrated near the shores of the Sea of Azov, Krasnodar Territory, and was carried out together with Dr Boris Korotyaev and Dr Sergey Reznik (both Russian Academy of Sciences, Zoological Institute, St Petersburg, Russia). Lepidium latifolium plants at field sites showed heavy insect and pathogen damage and the region seems very interesting in terms of potential biological control agents. The main objective of the trip was to find the gall-inducing weevil Ceutorhynchus marginellus, identified as a potential biological control agent in 2006. Nearly 100 adults of this species were collected and successfully brought back to CABI E-CH for host-specificity tests in 2008. Larvae of a stem-mining flea beetle were collected and emerged adults resemble Phyllotreta reitteri, another potential biocontrol agent. The survey in China was carried out in the north-west of Xinjiang Province, close to the Kazakhstan border. In contrast to eastern Kazakhstan (which was surveyed in 2006), this part of China is very densely populated and intensively cultivated. We mostly found perennial pepperweed along the border of crop fields, often associated with irrigation ditches. Apart from C. marginellus, several other weevils, flea beetles, a flower gall midge and the fungal pathogen Septoria cf lepidii were found. Preliminary feeding trials with one of the flea beetle species (Psylliodes sp.) revealed a clear preference for Lepidium latifolium and it might therefore be an additional potential agent. Final identifications are still being awaited. The field trip to southern Ukraine (Crimea Peninsula) was made by our colleague Dr Patrick H채fliger. However, few insects and evidence of fungal pathogens were found, indicating that this area is of no particular interest in terms of natural enemies of perennial pepperweed. A field trip to Iran in September 2007 was conducted mostly in connection with two other weed biocontrol projects, i.e. Russian olive and Russian knapweed, and to establish contacts with local collaborators. While perennial pepperweed is recorded to be relatively common in the area visited, few plants were found at that time of the year. More extensive surveys are planned for 2008. In 2007, we started work with six potential biological control agents. Preliminary host-specificity tests conducted with the root-mining weevil Melanobaris sp. n. pr. semistriata in quarantine at CABI E-CH look promising and will be continued in 2008. In addition, specimens were collected from different Lepidium species for molecular analyses to clarify the taxonomy of M. sp. n. pr. semistriata. We successfully reared the gall-forming weevil Ceutorhynchus marginellus in quarantine. Field collected and reared adults are currently being overwintered at CABI E-CH and we will start host-specificity tests in 2008. We were also able to rear the shoot-mining flea beetle Phyllotreta reitteri and started to conduct nochoice larval transfer tests. Apart from perennial pepperweed, adult beetles also emerged from four test plant species. We are planning to conduct single-choice tests in 2008 and depending on results we will decide whether to continue work with this species.

49


Lasiosina deviata is a recently described chloropid stem-mining fly whose congeners are all recorded on monocotyledons. However, field host-range data collected in 2007 indicate that L. deviata does not attack monocot plants. Flies did lay eggs under semi-artificial conditions, but no larvae developed. Emphasis in 2008 will therefore be on developing methods for rearing and host- specificity tests. A new species of eriophyoid mite, Metaculus lepidifolii was described from perennial pepperweed plants collected in Turkey in 2006. Provided sufficient funding is available, studies of this species will continue and preliminary host-range studies will be conducted in 2008. A fungal pathogen isolated from material collected in Kazahkstan in 2006 and in China in 2007 was preliminary identified as Septoria cf lepidii. We were able to inoculate plants under quarantine conditions and first host-specificity tests were carried out. Test revealed that S. cf lepidii also attacked a native North American species and is therefore not specific enough to be further considered as a potential biological control agent.

50


5.2. Multiple Choice tests with Lasiosina deviata in Turkey by R. Hayat, and L. Gultekin, Ataturk University, Erzurum, Turkey.

5.2.1. Field cage test, Erzurum, Turkey, summer 2007 Biology Ten perennial pepperweed shoots showing signs of attack by L. deviata and six grass species (n=10 shoots/ species) that were growing intermixed with the target weed were collected in May from two localities in Central Anatolia. Adult flies observed on perennial pepperweed were sampled and sent to a taxonomist for identification; so far no identification is available. Upon dissection of the perennial pepperweed shoots at Atat端rk University, both eggs and larvae were found. Level of attack was in general quite high (Tab. 1) with a maximum of 16 eggs and 11 larvae being found in the same shoot. Females lay eggs, usually two at the same location, on perennial pepperweed shoots and occasionally also on the leaf base (Fig. 1A). Hatched larvae enter into shoots and mine downwards. Mines are generally situated in the top parts of shoots and no mines were observed lower than 15 cm above shoot bases. Mines reached up to 25 cm in length, and frequently several larvae were found in the same mine. Up to four mines were found in a single shoot. Lasiosina deviata goes through three larval instars prior to pupation in the shoots (Fig. 1B).

A

B

C

D

Fig. 1. Lasiosina deviata eggs laid onto a PPW shoot (A), different larval instars (B), pupa (C) and adult L. deviata (D) (Pictures: R. Hayat).

At the beginning of June, L. deviata were found mostly as pupae, with few larvae still present at this time of the season (Tab. 1). In general, L. deviata pupate at the bottom of mines in groups of up to eleven pupae (Fig. 1C). About 400 pupae were obtained from dissection of infested shoots and transferred into plastic containers.

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Flies emerged from 24 June onwards. By 2 July, emergence ceased by which time 205 adults had been obtained in total. Flies were used to develop rearing techniques and for preliminary host-specificity tests. In addition, 45 parasitoids (Hymenoptera, Chalcidoidea) were reared from L. deviata pupae and will be sent to taxonomists for identification. During a survey to central Turkey conducted in August, our partner Levent Gültekin found pupae in perennial pepperweed shoots, indicating that L. deviata has a bi- or multivoltine life cycle.

Field host-range data and host-specificity tests No attack was recorded in any of the six grass species that grew intermixed with the target weed at the same field sites, while between 90% and 100% of perennial pepperweed shoots were attacked (Tab. 1). Shoot-base diameters of grass species were on average smaller than those of perennial pepperweed. However, the last larval instar of L. deviata has a length of about 5 mm and a maximum diameter of 1 mm. We therefore assume that shoot diameters of some of the grasses were theoretically sufficient for development of the fly. Table 1. Lasiosina deviata attack (eggs, larvae and pupae) in PPW shoots and in six intermixed growing grass species collected at two field sites in Turkey in 2007. Data are means ± SE. SBD: shoot base diameter.

Site /Plant species

# shoots Height (cm) SBD (mm)

# shoots attacked

PPW Bromus tectorum Elymus hispidus Hordeum violaceum

10 10 10 10

Turkey 5 (10 May) 32.4 ± 2.0 4.4 ± 0.1 26.9 ± 1.7 1.1 ± 0.1 48.2 ± 3.6 1.8 ± 0.1 17.8 ± 0.8 1.2± 0.1 Turkey 5 (11 June)

10 0 0 0

PPW

10

72.5 ± 1.1

10

PPW Eleocharis acicularis Phragmites australis Grass2 undetermined

10 10 10 10

Turkey 11 (9 May) 43.4 ± 2.8 5.5 ± 0.3 40.8 ± 2.0 1.7 ± 0.1 80.7 ± 2.9 3.3 ± 0.1 44.3 ± 2.3 1.6 ± 0.1

5.7 ± 0.2

9 0 0 0

Number of eggs

larvae

7.1 ± 1.4 6.0 ± 1.1 0 0 0 0 0 0 0

1.5 ± 0.4

3.6 ± 0.7 4.6 ± 1.5 0 0 0 0 0 0

pupae 0 0 0 0 11.25 ± 2.7 0 0 0 0

Seeds of maize (DKC-4604), barley (Tokak-157/37) and wheat (Dogu-88) were grown at Atatürk University for preliminary host-range tests. Two different methods were tried out. (A) A single potted perennial pepperweed or test plant was placed in a plastic cage (30 × 30 × 35 cm, n=4) in the laboratory and on 1 July, ten adults (7–8 females and 2–3 males) were released into each cage. A mixture of sugar, powdered yeast and water was provided as food. On 11 August, all plants were dissected. Two entry holes and two small mines/galleries were found in the perennial pepperweed plant, but no larvae. Test plants were not attacked. (B) A field cage (1 × 1 × 1 m) was set up in the institute’s garden at Atatürk University and filled with a layer of sand (5 cm). On 15 June, five potted plants of perennial pepperweed, barley, maize and wheat were embedded in the cage and between 24 June and 2 July, 72 females and 29 males of L. deviata were

52


released. On 9 September, all plants were harvested, measured and dissected. As with tests conducted in the laboratory, entry holes and small mines were found in all perennial pepperweed plants, but no larvae or pupae. The test plants were not attacked.

5.2.2. Open field tests in Central Turkey, summer 2008. by R. Hayat, Ataturk University, Erzurum, Turkey. Field and experimental host-range studies carried out in 2007 gave no support that L. deviata (Fig. 2) attacks monocot plants. In 2008 preliminary host-specificity tests were conducted in Turkey with Brassicaceae.

A

B

Fig. 2. Lasiosina deviata adult (A) and entrance hole of larvae in perennial pepperweed shoot (B), (Photos: R. Hayat).

Materials and Methods On 26 April, field collected perennial pepperweed plants were transferred into pots (17 cm diameter x 16 cm height), filled with commercial garden soil. These plants as well as two other test species grown from seeds, Camelina sativa and Berteroa incana, and two commercially available cabbage varieties, Brassica oleracea var. rubra and B. oleracea var. botrytis (optained from the Department of Horticulture, Atatürk University, Erzurum), were kept in a greenhouse at Atatürk University in Erzurum. On 11 June, perennial pepperweed plants were collected at a field site at Yesilhisar (Kayseri Territory, Turkey) and recovered 70 L. deviata pupae. The pupae were transferred individually into plastic containers, enabeling an easy collection of emerging adults. On 21 June, all plants were transferred to the campus of Kayseri Şeker Fabrikası, a sugar factory at Kayseri in central Turkey. Plants were arranged in a common garden in 10 rows, each containing five replicates of perennial pepperweed or one of the test species (Fig. 3). Adjacent to the experimental plot were garden beds with flowering ornamentals, providing food for the adults of L. deviata (Fig. 3). Between 21 and 30 June, 21 L. deviata emerging from rearing were released in the plot (In addition, 11 parasitoid emerged from pupae). On 19 July, all plants were harvested, transferred at the Erzurum University facilities, and subsequently measured and dissected under a stereomicroscope.

Results While all perennial pepperweed plants showed signes of attack by L. deviata (i.e. mining, eggs, larvae and/or pupae), none of the test plants was attacked (Tab. 2). With the exception of Berteroa incana, test 53


species were similar to perennial pepperweed in shoot base diameter and height (Tab. 2). Larval development of L. deviata in regard of the spatial niche provided therefore appears possible in these three species. Table 2. Results of open field multiple-choice test with Lasiosina deviata in 2008.

# replicates

Mean

Mean shoot

exposed

attacked

attacka

height (mm)

base diameter (mm)

perennial pepperweed

10

10

2.0 ± 0.7

285.0 ± 34.1

4.0 ± 0.2

Brassica oleracea var. botrytis

10

0

234.7 ± 11.5

5.5 ± 0.3

Berteroa incana

10

0

81.1 ± 8.6

2.8 ± 0.2

Brassica oleracea var. rubra

10

0

173.6 ± 11.4

4.4 ± 0.2

Camelina sativa

10

0

279.7 ± 18.9

4.0 ± 0.4

a: egg, larvae and/or pupae

A

B

Fig. 3. Prof Rüstem Hayat at a perennial pepperweed site in central Turkey (A); set up of test plants in the multiple-choice test conducted with Lasiosina deviata in 2008 (B).

5.2.3. Conclusions and outlook First host-specificity tests indicate that L. deviata does not attack any other Brassicaceae simultaneously exposed and therefore, the species can be further considered as a potential candidate agent. However, observation form the field so far indicate a rather weak impact of this species on attacked perennial pepperweed. Given that other potential agents are currently at hand whose impact on growth and seed production seems to be higher, work with L. deviata will not continue at this point. If other insects failed to be specific enough, in particular the shoot-mining P. reitteri, host-specificity testing with L. deviata might revive.

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5.3. Metaculus lepidifolii (Acari: Eriophyoidea) In late June 2008, a huge population of perennial pepperweed has been recorded near the town of Kayseri. Several plants were heavily attacked by the eriophyoid mite Metaculus lepidifolii. The impact of the mite was very high, and plants were suitable to the gallina mites at different phenological stages (Fig. 4).

A

B

Fig. 4. Attack by the eriophyoid mite Metaculus lepidifolii preventing flowering (A) and seed production (B).

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6. Russian olive, Elaeagnus angustifolium By U. Schaffner and, H. Hinz (CABI); M. Cristofaro and F. Di Cristina; and R. Ghorbani and A. Ghorbanali (Ferdowsi Univ. of Mashhad, Iran).

Introduction Russian olive is a small tree or multi-stemmed shrub native to south-eastern Europe and Asia. In Central Asia, it is a character species of the tugai forest that is primarily found in riparian habitats, where it often occurs sympatrically with tamarisk, Tamarix spp. Within its native range, Russian olive has been cultivated for many centuries. The garden form, which is characterized by significantly larger fruit (Plate 2), is grown around houses or in orchards. The large and tasty fruits of the garden form are either eaten in the fresh or dry form, or processed to jam, fruit compote or alcoholic beverages. The wild form of Russian olive was intentionally introduced to North America in the late

19th

century as a horticultural plant and was used for hedgerows and

as a shade tree. Moreover, it has been promoted and planted as a nectar source for honey bees, for erosion control, and for reclamation purposes. In the past 50 years, Russian olive has started to spread from its original plantings and is now widely established in at least 17 U.S. states and five Canadian provinces.

Plates 1 -2. Massimo Cristofaro sampling Russian olive in Iran (left); large curculionid mining the trunk of Russian olive in Uzbekistan.

Nevertheless, as recently as in the 1980s and 1990s, many state and federal agencies were subsidizing the distribution of E. angustifolia seedlings in the western U.S. and Canada. At the same time, Russian olive has become a declared noxious weed in four U.S. states (Colorado, Connecticut, New Mexico, Wyoming), and this number is likely to increase in the near future. Developing a classical biological control program for Russian olive could therefore give rise to a conflict of interests. One possible way to solve this potential conflict lies in concentrating on biological control agents that specifically attack the flower buds, flowers, seeds or seedlings. This would allow to slow down the spread of Russian olive without harming established trees. Yet, Russian olive has been spreading to such an extent in recent decades that the eco-

56


nomic and environmental damage caused may soon outweigh its horticultural benefits nationally. Thus, in the longer term, it may be acceptable as a biological control target using all suitable host-specific biological control agents. In 2007, CABI E-CH was provided with start-up funding for a 2-year period to conduct literature and field surveys for potential biological control agents against Russian olive in Asia. To be able to visit different parts of the native range of Russian olive, CABI E-CH and the Biotechnology and Biological Control Agency, BBCA, Rome, Italy agreed to cooperate on this target weed. This report summarizes the work conducted by CABI and BBCA in the first two years of the Russian olive project.

Figure 1. Distribution of Russian olive, Elaeagnus angustifolia, in North America. Source: http://plants.usda.gov.

Work programme for the initial 2-year phase Following work programme was proposed for the initial 2-year phase of the project (2007/08): •conduct a thorough literature survey of herbivores and pathogens associated with Russian olive in the native range; •carry out a quantitative field survey in Uzbekistan of the herbivore community on Russian olive in different regions and at different times of the growing season; •carry out qualitative field surveys on the herbivore community on Russian olive in Iran, China and Turkey during short-term visits; •send samples of natural enemies to experts for identification; •draft the test plant list; •start growing long-lived test plant species (some of which only start flowering after several years only) and Russian olive in the institute’s garden; •write annual Progress Reports, and •participate in a meeting at the end of the two years to discuss the results.

57


Research activities During 2007 and 2008 we accomplished the following goals: The first results of a literature search indicate that the herbivore assemblage associated with Russian olive in its native range is quite species-rich. So far, the search revealed some 55 herbivorous species that feed on Elaeagnus spp., several of which are reported to have a narrow host-range.

Fig 2. Plant attacked by the eriophyoid mite Aceria angustifolia in Central Turkey.

Fig 3. Plant attacked by the stem boring scolytid beetle Scolytis sp, Mashhad, Iran.

During 2007-08, field surveys for insects and mites were conducted in different parts of the native range of Russian olive, i.e. in Uzbekistan, Iran, Turkey and China. In Uzbekistan, the herbivore assemblages of Russian olive were extensively sampled in three climatically distinct regions. Surveys in the other countries were made while conducting field surveys for other weed projects. A total of 25 species have been found during these surveys. Three of these species merit further investigation, i.e. the eriophyoid mite Aceria angustifoliae, the gelechiid moth Ananarsia eleagnella, and a geometrid moth that has yet to be determined. i. A draft version of the test plant list has been compiled. It includes the only four native North American plant species belonging to the Elaeagnaceae family, one cultivated but exotic species of the same family, as well as some 40 additional plant species belonging to closely related families within the Rosales order. ii. Based on the results from the first year of this new project, four species with potential as biological control agents were prioritized. For 2009, it is planned to continue with field surveys in the native range to find new potential biological control agents, and to start preliminary investigations on the biology, the impact and the host-specificity of the eriophyoid mite A. angustifoliae. iii. Of the arthropods found during field surveys or recorded in the literature, at least five appear to have a narrow host-range: the shoot-tip mining weevil Temnocerus elaeagni (Coleoptera, Rhynchitidae) and the stem-boring beetle Scolytis sp., the flower-bud attacking mite Aceria angustifoliae (Acari, Eriophyoidea), a fruit-feeding moth (probably Carpocapsa splendana), an unkown fruit-mining fly, as well as a defoliating geometrid moth (Lepidoptera, Geometridae) found in Turkey. The biology of these species will be studied in 2008.

58


7. Other weeds 7.1. Saltcedar, Tamarix spp. In cooperation with Nada Carruthers, USDA APHIS, Albany, CA, USA. Genus Tamarix consists of 90 different species and 8 of them have been introduced into the United States in the 1800's. Among them, only 2 species are considered a real threat to the natural ecosystems of the Southwest: Tamarix parviflora and T. ramosissima. Once established, saltcedar can out-compete stressed native plants and cover large areas of formerly native habitat, resulting in a less productive and less diverse environment. Very promising results were achieved in the biological control domain by the release of the gregarious leaf beetle Diorhabda elongata.

7.1.1. Liocleonus clatratus (Coleoptera: Curculionidae) 102 specimens of Liocleonus clatratus were found on Tamarix sp. during a field survey in South Turkey in the locality of Kilis on June 2008. Once in the lab, some of them were placed in pair on Tamarix sp. potted plants in the greenhouse in order to find ovipositing females. A nylon cage was fixed on each pot and laboratory paper was added as a shelter. Insects showed a heavy feeding and only few eggs were laid on the paper (fig. 1).At the same time, four pair of the insects were placed in Petri dishes in a climatic cabinet at 21-26째C and with a 14:10 L/D cycle and allowed to feed on stems of Tamarix sp. No oviposition was recorded (fig. 2).

Fig.1. Liocleonus clathratus rearing on saltcedar potted plants.

Fig. 2. Couple of Liocleonus clathratus reared on a bouquet of saltcedar in Petri dish.

Before trials and during both the experiments,insects showed a very high rate of mortality probably due to a pathogen.

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7.2. Russian knapweed, Acroptilon repens In cooperation with U. Schaffner (CABI), R. Ghorbani and A. Ghorbanali (Ferdowsi Univ. of Mashhad, Iran); and E. De Lillo and R. Monfreda, University of Bari, Italy. During the two travel in North Eastern Iran in 2008, CABI asked BBCA to be involved in the supervision of the field activities for the search, selection and preliminary studies of potential biocontrol candidate agents associated with Russian knapweed in its native range. In addition, first field studies were carried out to assess the host–range and the biology of the eriophyoid mite Aceria acroptiloni.

7.2.1. Carry out extensive field surveys A thorough field survey have been carried out in the Chorasan Region, an area dense colonized by A. repens. Care was taken to survey habitats of the selected region with different environmental conditions (temperature, precipitation, altitude etc.). In conclusion, five sites have been selected and visited 3 times during the field season 2008. At each site, samples of the target weed were collected and dissected, recording the occurrence of potential biological control candidate agents, and their density. From each site, additional information have been recorded in a field data sheet (altitude, name of locality, date, weather conditions, GPS coordinates or precise description of Russian knapweed population, etc.) The information and the details of the selected biocontrol candidates will be presented by Dr. Urs Schaffner (CABI, Delemont, CH), Research Leader of the Russian knapweed project, in the RKW 2008 Annual Report.

7.2.2. Carry out preliminary studies on the host-range of the flower mite Aceria acroptiloni. Mite experiment. The field experiment was set up in early May near Shirvan (200 km. NE of Meshhad), Iran. A field plot at the field station of Shirvan (Ferdowsi University) was organized using a blockrandomized design (see Table 1 and Fig 2). Seeds of 2 varieties of safflower (oleic and linoleic), and of Centaurea americana were sown in jiffy pots; 3 weeks later they have been transplanted in 22 cm diameter plastic pots.

Fig. 1. Russian knapweed flowerheads deformed by A. acroptiloni.

Wild Russian knapweed plants were transplanted from the field into plastic pots (22 cm diameter), and kept in a shade area for about a month before being transferred in the field plot together with the other tests plants.

60


Tab. 1. Open field test with A. acroptiloni.

RKW Control

Safflower 88-OL

RKW Mite

Cent. americana

Safflower 1221

Safflower 88-OL

RKW Mite

Cent. americana

Safflower 1221

RKW Control

RKW Mite

Cent. americana

Safflower 1221

RKW Control

Safflower 88-OL

Cent. americana

Safflower 1221

RKW Control

Safflower 88-OL

RKW Mite

Safflower 1221

RKW Control

Safflower 88-OL

RKW Mite

Cent. americana

RKW Control

Safflower 88-OL

RKW Mite

Cent. americana

Safflower 1221

Safflower 88-OL

RKW Mite

Cent. americana

Safflower 1221

RKW Control

RKW Mite

Cent. americana

Safflower 1221

RKW Control

Safflower 88-OL

Cent. americana

Safflower 1221

RKW Control

Safflower 88-OL

RKW Mite

Safflower 1221

RKW Control

Safflower 88-OL

RKW Mite

Cent. americana

At the beginning of June 2008, all the potted plants were transferred in the field plot, inoculated by RKW infested cuttings. During the first week of the experiment the potted plants have been watered twice a day, and then one every two days. At the end of June all the potted plants were harvested and kept singularly in dry condition (as herbarium specimens) in labeled newspaper folders (FIG. 3). During the last visit in Iran mid September 2008), all the folders with the dry plants from the experiment were transferred at the BBCA facilities in Rome, where the mites were extracted. Preliminary data confirm the narrow host range of A. acroptiloni, since the eriophyoid mite has been recorded in big numbers only on the target weed. The results and details of the open field test will be presented by Dr. Urs Schaffner (CABI, Delemont, CH), Research Leader of the Russian knapweed project, in the RKW 2008 Annual Report.

Fig 2. Shirvan, Iran: setting of the open field test for A. acroptiloni, May 2008.

Fig 3. Dry specimens of the test plants exposed to the eriophyoid mite experiment; Shirvan, Iran; September 2008.

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8. Exploration trips 8.1. Exploration trips 2007 8.1.1. Field trip in Turkey (March 6-13, 2007) By M. Cristofaro and F. Di Cristina Aim of the trip was the collection of live specimens of the lace bug Tingis grisea on yellow starthistle (YST) in the vicinity of Horasan, East Turkey. Unfortunately, temperature was very cold and YST population was at an early rosette stage so no insects were found. Stem boring larvae, perhaps Mordellidae, were collected on old YST stem. In the region of Kapadokya (Central Anatolia), found several specimens of Psylliodes sp. on Onopordum sp.

8.1.2. Field trip in Tunisia (May 2-8, 2007) By F. Lecce Main goal of 2007 explorations in Tunisia was the collection of the weevil Anthypurinus biimpressus on Salsola sp. The insect was not found in spite of the good conditions of the Russian thistle population. Carried out further exploration in the area looking for new location with the presence of our target weed. The trip was conducted in co-operation with J. Kashefi (EBCL, Thessaloniki, Greece).

8.1.3. Field trip in Bulgaria (May 9-11, 2007) By M. Cristofaro and F. Di Cristina The aim of this first trip in Bulgaria was the organization of the work on Chondrilla juncea for the next season with the Bulgarian cooperators of Plovdiv Agricultural University. Visited several location with the presence of Schinia cognata and Simyra nervosa and collected some larvae.

8.1.4. Field trip in China (May 21-June 2, 2007) By M. Cristofaro This trip was conducted in cooperation with H. L. Hintz (CABI Bioscience, Switzerland) and with the collaboration of scientists of CAAS (Chinese Academy of Sciences). China was selected as a destination for the collection of Lepidium latifolium and Salsola tragus instead of Kazakhstan. Found a good population of two species of genera Salsola (different from S. Kali or S. tragus) attacked by two weevils, collected to carry out host range experiments in laboratory. Collected plant samples for herbarium. Found also three flea beetle species on L. latifolium.

8.1.5. Field trip in Tunisia (May 25-31, 2007) By F. Di Cristina and M. Barlattani The second survey of the year in the locations where Anthypurinus biimpressus was previously collected didn’t give any result: no specimens of the insect were recorded on Salsola sp. plants. The weevil Broconius biscrensis was collected in the locality of Degache, near Tozeur, on a Chenopodiaceae species. Found few specimens of Elasmobaris sp., about 10 km south of Gabes on Russian thistle. 62


8.1.6. Field trip in Bulgaria (July 6-13, 2007) By A. Paolini, F. Di Cristina and A. Anastasio Target of this field trip was the collection of larvae of Schinia cognata and Simyra nervosa on the weed Chondrilla juncea, in co-operation with scientists of Plovdiv Agricultural University. Visited several localities with Centaurea solstitialis and recorded the presence of the mite Aceria solstitialis.

8.1.7. Field trip in Turkey (July 7-13, 2007) By M. Cristofaro and E. Colonnelli The trip was focused on the exploration of West and Central Anatolia to check out the presence of our target weeds. Found large populations of Salsola sp. Collected adults and pupae of Lixus sp. and recorded the presence of Piesma sp., Conorynchus nigrivictis (cleonine weevil) and Cosmobaris scolopacea as mature larvae in root and soil and as adult near the crown.

8.1.8. Field trip in Greece (July 18-21, 2007) By M. Cristofaro The main purpose of this trip, conducted together with L. Smith (USDA, Albany, CA), was the collection of plants of Salsola tragus infested by the mite Aceria salsolae to be used in the field garden experiment in BBCA facilities in Rome. The collection was carried out in the locality of Kozani. Found also a broad nose cleonine weevil (Conorynchus sp.) on S. tragus.

8.1.9. Field trip in Tunisia (July 18-23, 2007) By F. Di Cristina and F. Lecce Carried out exploration of old and new sites of Russian thistle in order to record the presence of potential biocontrol agents. Found few larvae of the weevil Anthypurinus biimpressus in the locality of Aouinet,north of Gabes. Larvae were placed on artificial diet but didn’t reach the adult stage. Found again few specimens of Elasmobaris sp. near Gabes and of Broconius biscrensis in the site of Degache.

8.1.10. Field trip in Bulgaria (August 26-29, 2007) By M. Cristofaro and F. Lecce This brief field survey was targeted again to the collection of the noctuids Schinia cognata and Simyra nervosa in different localities. Visited also the field garden experiment with the mite Aceria solstitialis performed by bulgarian scientists at Plovdiv Agricultural University.

8.1.11. Field trip in Iran (September 13-22, 2007) By M. Cristofaro Objective of this first trip in Iran was to set up new contacts between BBCA and scientists of the Iranian University and to carry out explorations searching for new potential biocontrol agents. Several plants of Salsola sp. infested by stem boring larvae were found in many sites visited. Collected plant specimens for herbarium and samples to dissect in laboratory. Collected several living adults of Lixus sp. to be tested in laboratory.

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8.2. Exploration trips 2008 8.2.1. Field trip in Spain (January 24-28, 2008) By F. Lecce and F. Di Cristina The main goal of the first trip of the year was to start the collections of the root boring weevil Trichosirocalus briesei on Scotch thistle in central Spain (100 km northern of Madrid). Collected the weevil as adult, egg and larval stages. The insects were used at BBCA facilities to carry out host range tests in laboratory conditions.

8.2.2 Field trip in Spain (March 05-09, 2008) By F. Lecce and A. Paolini Replicated the collection of the root boring weevil Trichosirocalus briesei on Scotch thistle in central Spain (100 km northern of Madrid). Collected the weevil as adult, egg and larval stages. The insects were used at BBCA facilities to carry out host range tests in laboratory conditions. Recorded the presence of a huge population of Russian thistle (Salsola tragus) in the vineyards of the area.

8.2.3. Field trip in Turkey (March 06-14, 2008) By M. Cristofaro and F. Di Cristina The main goal of the trip was the survey and collection of sites of Scotch thistle (Onopordum acanthium) in Central and Eastern Turkey. Found 3 populations of the flea beetle Psylliodes sp. nr. chalcomerus on Scotch thistle and 2 sites in Cappadocia with the root boring moth Eublemma sp. In Cappadocia also found a phytoplasma like damage on Russian olive. Finally recorded 2 new sites for the root galling weevil Liocleonus clathratus on Tamarix.

8.2.4. Field trip in Iran (May 07-17, 2008) By M. Cristofaro and F. Di Cristina This exploration trip was conducted in co-operation with CABI Bioscience, Switzerland, investigating for 6 different target weeds: Russian knapweed, Russian thistle, Russian olive, perennial pepperweed, Scotch thistle and rush skeletonweed. Collected several specimens of Lixus myagri and Ceutorhynchus sp., two flea beetles species and on Lepidium latifolium. Found also a pathogen on the leaves. The Salsola sp. populations were too young. Collected on Russian olive a scolytid beetle, 2 long-horn stem boring beetles and the eriophyoid mite. On Russian knapweed found an eriophyoid mite, a stem boring fly, a stem galling wasp and a root-boring moth. Recorded on Scotch thistle a Larinus sp. (prob. darsi) and a tephritid seed feeder fly.

8.2.5. Field trip in Turkey (May 27-June 05, 2008) By F. Di Cristina and M. Barlattani The main goal of the trip was to collect mite galls on Lepidium draba, survey Central Turkey for Russian olive and collect the root galling weevil Liocleonus clathratus on Tamarix in 2 sites. Found also a root galling weevil larva on Salsola.

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8.2.6. Field trip in Tunisia (June 03-07, 2008) By F. Lecce and M. Paradisi Aim of this trip was the collection of Anthypurinus biimpressus, potential biocontrol agent of Salsola tragus. The weevil wasn’t found but we collected 65 specimens of the weevil Elasmobaris sp. Some of the weevils were sent to Lincoln Smith, while a large portion was tested at the BBCA facilities, in laboratory conditions.

8.2.7. Field trip in Turkey (June 19-28, 2008) By M. Cristofaro The trip was carried out in co-operation with M. McIvie (Montana State University, Bozeman, MT) and U. Schaffner (CABI Bioscience, Switzerland). The trip was mainly targeted to set up a field test in Kayseri with the chloropid fly Lasiosina deviata, candidate agent for perennial pepperweed. Found a large population of perennial pepperweed heavily attacked by a rust, the weevil Lixus myagri and the long horn beetle Phytoecia caerulea. Collected a leaf-rolling moth on Russian olive, a “specific” population of Lixus sp. nr. incanescens on Salsola tragus and a large population of Larinus latus on Scotch thistle in Cappadocia. Collected other specimens of L. clathratus on saltcedar.

8.2.8. Field trip in Sicily (Italy) (July 06-08, 2008) By F. Lecce and F. Di Cristina The main purpose of the trip was the collection of candidate agents on Salsola kali. Collected in 3 sites the root-boring weevil Cosmobaris sp. nr. scolopacea, and in another site the same and the stem boring weevil Lixus rosenschoeldi.

8.2.9. Field trip in Bulgaria (July 25-29, 2008) By M. Cristofaro and F. Di Cristina The trip was carried out for collecting the flower feeder moth Schinia cognata and the leaf feeder moth Simyra nervosa on Chondrilla juncea in the area near Plovdiv. Checked as well the open field test with the mite Aceria solstitialis on yellow starthisle and 5 other test plants.

8.2.10. Field trip in Bulgaria (Aug 13-15, 2008) By F. Lecce and F. Di Cristina The trip was carried out for collecting the flower feeder moth Schinia cognata and the leaf feeder moth Simyra nervosa on Chondrilla juncea in the area near Plovdiv. The larvae were delivered to Jeff Littlefield, Montana State university (Bozeman, MT).

8.2.11. Field trip in Slovakia (Aug 15-18, 2008) By M. Cristofaro and F. Lecce The trip was carried out together with the Slovakian cooperator Peter Toth for collecting potential candidate agents on Chondrilla juncea and Salsola tragus in center/southern Slovakia. Found a stem boring Bu-

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prestid and a stem boring larva (prob. Mordellidae) on skeletonweed and a stem boring moth on Russian thistle.

8.2.12. Field trip in Sicily (Aug 29-31, 2008) By M. Cristofaro and F. Di Cristina The trip was carried out with the aim to collect the stem boring weevil Lixus rosenschoeldi and the root boring weevil Cosmobaris sp. on Salsola kali. Collected as well few specimens of a yellowish flea beetle (prob. Longitarsus sp.) and infested stems with the seed feeder moth Gymnancyla sp.

8.2.13. Field trip in Iran (Sept 19-28, 2008) By M. Cristofaro and F. Di Cristina The trip was targeted to the exploration and collection of natural enemies of Russian thistle, Scotch thistle, Russian olive and Russian knapweed. Totally we visited 18 sites in 5 days of field work, and in all the sites more than one target weed was present. Recorded and collected -both for rearing and for genetic analysis- at least 5 different agents on Russian thistle (2 weevils, one mordellid/cerambycid stem boring, the eriophyoid mite and a small black flea beetle). Two populations of the stem boring weevil Lixus sp. nr. incanescens, have been collected near Shirvan and near Gonabad, 200 km Western and 200 km Southern of Mashhad respectively. Brought back at BBCA facilities dry plant material from the open field host range test with the eriophyoid mite Aceria acroptiloni, harvested in July 2008 by Asadi Ghorbanali.

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List of publications Gültekin, L., Cristofaro, M., Tronci, C. and Smith, L. – 2008. Natural history studies for the preliminary evaluation of Larinus filiformis (Coleoptera: Curculionidae), as a prospective biological control agent of yellow starthistle. Environ. Entomol. 37 (5): 1185-1199. Gültekin L., Borovec R., Cristofaro M., and Smith L. – 2008. Broad-nosed weevils feeding on Centaurea solstitialis L. in Turkey, with a description of the new species Araxia cristofaroi sp. n. (Coleoptera: Curculionidae: Entiminae). Annals of the Entomological Society of America, Volume 101, Number 1, 7-12(6) Smith L., Cristofaro M., De Lillo E., Monfreda R. and Paolini A. 2009. Field assessment of host plant specificity and potential effectiveness of a prospective biological control agent, Aceria salsolae, of Russian thistle, Salsola tragus. Biological Control. 48: 237-243

Contributions to National and International Meetings/Symposia Antonini G., Audisio, P., De Biase, A., Mancini, E., Rector, B. G., Cristofaro, M., Smith, L., Biondi, M., Bon, M.C., Korotyaev, B. and Konstantinov, A. – 2008. The importance of molecular tools in classical biological control of weeds: two case studies with yellow starthistle candidate biocontrol agents. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Cortat, G., Hinz H.L., Gerber, E., Cristofaro, M., Tronci, C., Korotyaev, B.A. and Gültekin, L. – 2008. Giving dyer’s woad the blues: encouraging first results for biological control. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Coutinot, D., Cristofaro, M., Kiss L. and Ehret, P. – 2008. Feasibility of Biological Control of common ragweed (Ambrosia artemisiifolia) in Europe. In proceedings of the XII International Symposium on Biological Control of Weeds (eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Cristofaro, M., Di Cristina, F., Colonnelli, E., Zilli, A. and Amer, W. M.– 2008. Field exploration for saltcedar natural enemies in Egypt. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Gültekin, L., Cristofaro, M., Tronci, C. and Smith, L. – 2008. Bionomics and seasonal occurrence of Larinus filiformis Petri, 1907 (Coleoptera: Curculionidae), potential biological control agent for Centaurea solstitialis L. in eastern Turkey. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK.

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Hinz, H. L., Gerber, E., Cristofaro, M., Tronci, C., Seier, M. K., Korotyaev, B. A., Gültekin, L., Williams, L. and Schwarzlaender, M. – 2008. All against one: first results of a newly formed foreign exploration consortium for the biological control of perennial pepperweed. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Lecce F., Paolini A., Tronci C., Gultekin L., Di Cristina F., Korotoyaev B., Colonnelli E., Cristofaro M. and Smith L. – 2008. Explorations in Central Asia and Mediterranean basin to select biological control agents for Salsola tragus. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Monfreda, R., de Lillo, E. and Cristofaro, M. – 2008. Eriophyoid mites on Centaurea solstitialis L. in the Mediterranean area. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Paolini A., Lecce F., Tronci C., Hayat R., Di Cristina F., Cristofaro M. and Smith L. – 2008. A lace bug as biological control agent of yellow starthistle, Centaurea solstitialis L. (Asteraceae): an unusual choice. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Sforza R., Cristofaro, M. and Jones, W. A. – 2008. Alien poisonous weeds: a challenge for a biological control of weeds program in Europe. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Smith, L., Cristofaro, M., Tronci, C. and Hayat, R. – 2008. Refining methods to improve pre-release. In proceedings of the XII International Symposium on Biological Control of Weeds (eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Tronci C., Lecce F., Paolini A., Di Cristina F., Cristofaro M. and Smith L. – 2008. Impact of larval and adult feeding of Psylliodes chalcomerus (Coleoptera: Chrysomelidae), on yellow starthistle. In proceedings of the XII International Symposium on Biological Control of Weeds (eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK. Volkovitsh, M. G., Dolgovskaya, M. Y., Reznik, S. Y., Zaitsev, V. F., Cristofaro, M., Tronci, C. and Markin, G. – 2008. Sphenoptera foveola (Col.: Buprestidae) as a potential agent for biocontrol of skeleton weed, Chondrilla juncea. In proceedings of the XII International Symposium on Biological Control of Weeds(eds Julien, M.H., Sforza, R., Bon, M.C., Evans, H.C., Hatcher, P.E., Hinz, H.L. & Rector, B.G.), CAB International Wallingford, UK.

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Vurro, M., Cristofaro, M., Casella, F., Boari, A. and Zonno, M. C.-2008. Lotta biologica alle piante infestanti. Workshop “Innovazioni nella difesa delle colture con mezzi a basso impatto ambientale�, Accademia dei Georgofili, Florence, November 27th, 2008

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Distribution List Lloyd ANDRES

Lynn KINTER

Paolo AUDISIO

Lynn LEBECK

Alessandro BISCACCIANTI Dan BEAN Rick BENNETT Maurizio BIONDI Ludovit CAGAN Nada CARRUTHERS Ray CARRUTHERS

Ivanka LECHEVA Jeff LITTLEFIELD Doug LUSTER George MARKIN Boris KOROTYAEV Joseph MILAN Radmilla PETANOVIC

Steve CLEMENT

Mike PITCAIRN

Enzo COLONNELLI

Sergey REZNIK

Eric COOMBS

Justin RUNYON

Alessio DE BIASE

Urs SCHAFFNER

Enrico DE LILLO

Kristina SCHIERENBECK

Margarita DOLGOVSKAYA EBCL (5 copies) John GASKIN André GASSMAN Esther GERBER Asadi GHORBANALI Reza GHORBANI Levent GULTEKIN Kevin HACKETT Vili HARIZANOVA

Magda SCHIMBERNI Mark SCHWARZLAENDER John SIMONS Lincoln SMITH Rouhollah SOBHIAN Atanaska STOEVA Dan STRICKMAN Peter TOTH Mark VOLKOVITSH Maurizio VURRO

Rustem HAYAT

Amer WAFAA

Hariet HINZ

Fu WEIDONG

Eric JANG Roman JASHENKO Javid KASHEFI Alex KONSTANTINOV

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Livy WILLIAMS Dale WOOD Alberto ZILLI


Contacts Biotechnology and Biological Control Agency Via del Bosco, 10 00060 - Sacrofano Rome, ITALY Tel. + 39 06 3048 3480 Fax: + 39 06 3048 6044 email: weeds@bbcaonlus.org www.bbcaonlus.org

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