BBCA Bi-Annual Report 2003-2004 by blu pubblicità e comunicazione srl

Biotechnology and Biological Control Agency

Bi-annual Report 2003 â&#x20AC;&#x201C; 2004

Edited by Massimo Cristofaro and Carlo Tronci

Table of contents Acknowledgements........................................................................................................................ 3 Introduction .................................................................................................................................... 6 Weed biocontrol ............................................................................................................................. 8 1. Yellow starthistle ...................................................................................................................... 8 1.1. 1.2. 1.3. 1.4. 1.5. 1.6. 1.7.

Ceratapion basicorne .........................................................................................................................8 Psylliodes sp. nr. chalcomerus.........................................................................................................10 Tingis grisea......................................................................................................................................19 Aceria solstitialis De Lillo, Cristofaro and Kashefi on Centaurea solstitialis (YST) in Apulia.............23 Seed head survey.............................................................................................................................24 Larinus filiformis ...............................................................................................................................25 Preliminary field screening of potential biocontrol agents of Centaurea spp. in Slovakia..............25

2. Russian thistle ........................................................................................................................ 26 2.1.

Future plans: work to do in 2005.....................................................................................................27

3. Rush skeleton weed ............................................................................................................... 28 3.1.

Work to do in 2005...........................................................................................................................28

4. Field bindweed ....................................................................................................................... 29 4.1.

Field bindweed stem borer, Melanagromyza albocilia (Diptera: Agromyzidae) .............................29

5. Other target weeds................................................................................................................. 31 6. Work plans for 2005................................................................................................................ 32 7. Exploration trips...................................................................................................................... 33 7.1. 7.2.

2003..................................................................................................................................................33 2004..................................................................................................................................................36

Biological and integrated control of insect pests ......................................................................... 42 8. Agriculture pests..................................................................................................................... 42 8.1. 8.2. 8.3. 8.4. 8.5.

Evaluation of attractants for Ceratitis capitata and Bactrocera oleae in two localities in Southern Italy. ..................................................................................................................................42 Evaluation of bioinsecticides and attractants ..................................................................................45 Integrated Mediterranean fruit fly control in an organic peach orchard in Southern Italy using different lure-based mass-trapping systems and bioinsecticide spraying. ....................................46 Effects of an azadirachtin-based compound on the host-parasitoid interactions between the Mediterranean fruit fly, Ceratitis capitata Wied. (Diptera: Tephritidae) and the braconid wasp Opius concolor Szepl. (Hymenoptera: Braconidae).........................................................................47 Biological control of the citrus flatid planthopper (CFP), Metcalfa pruinosa with the parasitoid wasp Neodrynus typhlocybae (Hymenoptera: Drynidae) in Latium, Italy. ......................................48

9. Human and animal parasitic insects ....................................................................................... 49 9.1. 9.2. 9.3. 9.4.

Effects of NeemAzal速 on the life cycle of Anopheles stephensi (Diptera: Culicidae).....................49 Effects of Spinosad速 on larval instars of three mosquito species of medical importance ............52 Experimental trap monitoring of mosquito populations in the urban area of San Benedetto del Tronto, Italy, and evaluation of the effects of chemical disinfection of manholes and other reservoirs in the urban drainage system. ........................................................................................53 Evaluation of new mass-trapping attractants for noxious mosquito species.................................53

Acknowledgements BBCA is grateful to Chuck Quimby, former Director of USDA ARS European Biological Control Laboratory (until summer 2004), Richard Greene, International Program Leader of USDA ARS, for their help in coordinating the projects; Heather Phelps and Sherri Carroll, USDA ARS Beltsville, for their capability to administrate the USDA grant; Lincoln Smith, USDA ARS Albany, and Mike Pitcairn, CDFA Sacramento, Ca., for their input in carrying out field and lab bioassays on the biological control of yellow starthistle and Russian thistle; Alex Konstantinov, USDA ARS Washington D.C.; Mark Volkovitsh, Margarita Dolgovskaya, Sergey Reznik, Boris Korotyaev, Prof. Vadim Zaitsev, Zoological Institute S. Petersburg, Russia; Alexander Popov, Volgograd, Russia; and Roman Jashenko, Almaty, Kazakhstan, for their help in the research and during foreign explorations; Enzo Colonnelli and Alberto Zilli, Museo Civico di Zoologia, Rome, Italy; Maurizio Biondi, University of l’Aquila, Italy, for their help in insect determination; Tim Widmer, René Sforza, Rouhollah Sobhian, Brian Rector and Marie-Claude Bon, USDA ARS EBCL; Javid Kashefi, USDA ARS Thessaloniki, Greece; Gaetano Campobasso, USDA ARS EBCL Rome, Italy, for the “friendship” feeling in their involvement in field explorations and laboratory work; Thanks also to Doug Luster, USDA ARS Ft. Dietrick, Rick Bennett, Alan Kirk, Dominique Coutinot, Steve Clement, Ray Carruthers, Eric Jang, USDA ARS, Nada Carruthers, USDA APHIS, Dan Bean, U.C. Davis, Hank McNeel, USDI, for their support in several administrative and scientific aspects; Rüstem Hayat, Göksel Tozlu, and Levent Gültekin, Atatürk University, Erzurum, Turkey; Sibel and Nezihi Uygur, Çukurova University of Adana, Turkey, for their help in carrying out field bioassays on biocontrol candidate agent of YST; Ludovit Cagan and Peter Toth, Agricultural University of Nitra, Slovakia, for their help during foreign explorations for natural enemies of field bindweed; Enrico De Lillo, Rosita Monfreda and Margherita Baldari, University of Bari, Italy, for their help in the taxonomy of eriophyid mites; Paolo Audisio, Alessio De Biase and Emiliano Mancini, University of Rome, for their help in genetic analyses; Augusto Vigna Taglianti, University of Rome Antonio De Cristofaro, University of Molise, Campobasso, Italy; Gianfranco Anfora, ISMAA, S. Michele all’Adige, Italy; Marilena Porto, Dow AgroSciences; Prof. Sebastiano Barbagallo and Giuseppe Cocuzza, University of Catania; and Ferdinando Baldacchino, ENEA-Trisaia, Italy; for their help in field and laboratory bioassays on biological control of insect pests; Annette Habluetzel, Fulvio Esposito, Guido Favia and Luciano Pasqualini, University of Camerino, Italy, Roberto Romi and Michele Maroli, Istituto Superiore Sanità, Rome, Italy, Luca and Paolo Serbelloni, Actima, Rome, Italy, for their help in the work on bioinsecticides for medical entomology; Alberto Brizio and Giorgio Andreoli, Agriconsulting, Rome, Italy; Vincenzo Tropiano, AGER, Italy; Marta Ferrara, Spazio Verde, Padova; Luigi Rossi, Lucio Triolo, Anna Moreno, Roberto Balducchi, ENEA-Casaccia, Rome, Italy; Mircea and Florina Popa, ENERGI-CO, Bucharest, Romania, for their technical support and organization in international projects. We also thank, for their scientific and economic support, Enzo Tescari and Marcello Re, DOW AgroSciences, Italy; Rosaria Tabilio, Istituto Sperimentale per la Frutticoltura, Rome, Italy; and Enzo Casagrande, AgriSense, Cardiff, U.K. Special thanks are also due to Fortunata Minelli, Agostino Letardi, Leonardo Lucantoni, Alessandra La Marca, Gloria Antonini, Giorgia Coletti, Antonio Fenio, Francesca Lecce, Franca Di Cristina, Silvia Arnone, Carlo Tronci, Alessandra Paolini, Paula Gambino, Giuseppe Carbone, Michèle Guedj, Sergio Musmeci, Vincenzo Di Ilio, Daniela Arona, Paola Nobili, Marcello Barlattani and Silvia Catarci, for their technical and scientific support at BBCA laboratory facilities in Rome, Italy.

BBCA staff...

…and BBCA cooperators & friends!

BBCA Bi-annual Report 2003 - 2004

Introduction

Introduction By Massimo Cristofaro The forth year of BBCA is gone, and we are back again! This time we are going to present a biannual report (2003-2004), because we missed last year edition. As I was saying two years ago, the editing of BBCA Annual Report is for me crucial 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 growing fast, and we have to re-organize everything. For this reason, and for new Italian legislations, we decided to improve the organization: the new Company IBIS s.r.l. (International Biocontrol Service) will be involved in all of the work aspects connected to a “profit” business (mainly contracts for performing bioassays for large private Companies), while BBCA will remain involved in dealing with grants for International Universities, Foundations, State Departments and Research Institutes. The changes will not only involve the structure, but also the programs! During 2003 we decided to drastically decrease explorations of new areas, in order to advance laboratory bioassays and open field tests for selected biocontrol candidates (Ceratapion basicorne and Psylliodes sp.). But, still keeping only the “most important” travels, during 2003 BBCA made 6 travels in Turkey, 2 in Russia, 2 in the USA, 2 in Austria, 1 in Switzerland, 4 in Montpellier, 1 in Romania, 2 in Greece and 1 in Tunisia! In particular Eastern 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: within 2004 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 weevil is ready to be petitioned for the release in the US. In addition, we will need only one additional year to finish with another YST agent, the stem boring flea beetle Psylliodes sp. nr. chalcomerus: 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 eriophyid mite Aceria solstitialis (Bari University, Italy: life history), and the seed feeder 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 biocontrol, since last year we started to work with another target weed, Russian thistle (Salsola spp.), and during 2004 we re-started with explorations in new areas. BBCA and Cooperators made explorations in Turkey (5 one-week trips), Tunisia (4 1-week trips), Russia (four 10-days trips), Kazakhstan (one-month trip), Egypt (one 10 days trip), Crete, Greece (one week trip) and (several) in Southern Italy. The travels in Tunisia, Turkey and Kazakhstan, led us to make a list of 6 new promising biocontrol candidate agents to be screened (5 weevil spp. from the genera Anthypurinus, Baris, Lixus and Salsolia, according to our taxonomist co-operators Enzo Colonnelli and Boris Korotyaev). Moreover, together with our Russian co-operators, during 2004 we started explorations in Eastern Turkey, Kazakhstan and Southern Volga Basin for a new target weed, rush skeleton weed (Chondrilla juncea); among the 6 biocontrol agents we found related to RSW, two root borer buprestid beetles (Sphenoptera spp.) and the leaf feeder noctuid moth Symira nervosa have been selected for further screening. Compared to previous years, we improved a lot the cooperations, both in Italy and in other Countries. In particular I would like to mention the creation of BBCA France, with 6 people 6

BBCA Bi-annual Report 2003 - 2004

Introduction

working as Research Assistants under the supervision of EBCL. Moreover, we set up 2 new important cooperations with 3 Italian Research Entomologists, Dr. Enrico de Lillo, eriophyid mite taxonomist; and Dr. Paolo Audisio and Dr. Alessio De Biase, for genetic studies on flea beetles on YST. Of course, the performances in our work can not be achieved without the “storical” 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! We also improved the programs: beside to the classic approach, we improved ecological aspects (impact studies), and we started to perform genetic analyses and (last minute news) plant pathology. BBCA improved the programs not only in biocontrol of weeds: special emphasis was put on new programs on the control of insect pests, with new cooperations with ENEA BIOTEC, CNR, universities in Spain and Greece, and IAEA. A new branch on biocontrol of human and animal parasite insects started with a biocontrol program on Anopheles mosquitoes (and malaria) and on sand flies (and Leishmania). Finally we moved from a small production to a large distribution of organic fruits and vegetables. To do all of these activities, we grew up, and BBCA has at the moment about 15 people under payroll of BBCA Italy and 7 for BBCA France (6 technicians and 1 administrator). We know that the near future will be probably difficult and we can have problems related to budget issue concerning the lack of funds. But the special feeling we have among the BBCA staff and the trust and mutual esteem with our partners, will keep me optimistic, as usual! Plans for 2005. Next year will follow the style of 2004, improving BBCA efforts in terms of new disciplines and new projects. Regarding biological control of weeds, in particular, we will enhance our screening improving genetic aspects of work (for both weeds and biocontrol agents) and adding a plant pathologist position in our staff. The target for 2005 will be to help EBCL in the clarification of several taxonomic question marks on some of the new potential biocontrol agents and to improve impact tests evaluating the possible synergism with plant pathogens. For the new projects our goal will be to be involved on Lepidium latifolium and the European target ragweed, Ambrosia artemisiifolia. Regarding the “historic“ projects, our work on YST will be focussed on the petition of the rootboring weevil Ceratapion basicorne, in cooperation with Lincoln Smith and Mike Pitcairn; the last field and laboratory trials on the stem-boring flea beetle Psylliodes chalcomerus; continue the screening with the lace bug Tingis grisea and the leaf galling mite Aceria solstitialis. Special emphasis will be put on the evaluation of the impact of all these candidates on the target at different phenological stages. We will also start the screening of new insects found on Salsola spp. in Turkey, Italy and Kazakhstan: the approach will be the same, doing part of the work in our facilities but at the same time improving the cooperations with foreign scientists in order to carry out as soon as possible long term field observations and open field tests. The work on rush skeleton weed will be mainly carried out by the Russian team, under our supervision and in cooperation with Javid Kashefi for the screening of a root boring buprestid found in central Anatolia. Also in this case, as for Russian thistle, we will follow the same strategy, sharing the work with other partners selected in the areas where the weed and the new candidates have been found. 7

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

Weed biocontrol 1. Yellow starthistle The work on YST was focused on the study of the four selected candidate agents and on explorations and collections mainly in Turkey and Southern Russia. Carried out the second and the third year of field testing in Turkey for the weevil Ceratapion basicorne (CEBA), obtaining completely satisfactory results. Four collection trips and host range laboratory and greenhouse experiments have been completed on the flea beetle Psylliodes sp. nr. chalcomerus (PSYL), in Russia and Italy, together with lifecycle and diapause studies. Performed six collection trips for the YST lace bug Tingis grisea (TIGR), carrying out Picture 1. BU-3 and BU-4 Yellow starthistle several laboratory preliminary biology and host range flower heads; Turkey, June 2004. observations; the work confirmed the difficulties to deal with this species, especially in terms of survival and feeding, however, it was possible to highlight some start point information and to establish a laboratory diapausing colony. Continued the work on the eriophyid mite Aceria solstitialis (ACER) in co-operation with Dr. De Lillo, selected two Italian locations for future studies, and carried out preliminary biological observations. As for the individuation of new potential biocontrol agents, one leaf beetle, one tip galling moth and one flea weevil species have been collected.

1.1. Ceratapion basicorne By M. Cristofaro, F. Di Cristina, F. Lecce, A. Paolini, C. Tronci, BBCA, Rome, Italy R. Hayat, G. Tozlu, Plant Protection Department, Atatürk University, Erzurum, Turkey During 2003 season, two sets of field experiments on host specificity and impact evaluation of the apionid weevil Ceratapion basicorne (CEBA, picture 2) on YST, have been carried out in Turkey in co-operation with Dr. Hayat team at the Atatürk University of Erzurum. The hostrange field test showed a slight attack on safflower test plants in one location. Nevertheless, the specimens emerged from safflower test plants were identified by Dr. B. Korotyaev (RAS-ZIN, St. Petersburg, Russia), as Picture 2. The root/crown boring weevil Ceratapion scalptum, a polyphagous species very similar to CEBA and known to attack the genus Carthamus. Ceratapion basicorne (L. Smith) The impact evaluation test, (picture 3) carried out using systemic insecticide treated YST plants as control did not show significant differences between attacked and un-attacked plants (final height, dry weight, root crown diameter, number of seed heads were measured). Even though in 2002 and 2003 tests, all adults emerged from safflower test plants, were identified as Ceratapion scalptum, in order to clearly and univocally define CEBA host specificity towards safflower, tests have been repeated for the third year.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Picture 3. C. basicorne insecticide exclusion test plants at the Ataturk Univerisity, June 2003.

Picture 4. R. Hayat inspects the Horasan field plot prior harvesting in June 2004; on the right a YST plant edge row.

During 2004, a commercial safflower variety and local wild biotype YST have been tested in 2 high infestation locations, using large numbers of safflower plants densely planted simulating intensive cultivation, surrounded by YST test plants (picture 4). Following previous years procedure, we monitored the development status of CEBA population sampling wild YST at selected locations; test plants were harvested upon the first findings of CEBA pupae. At harvest, 50% of safflower test plants were dissected and all larvae/pupae found preserved for genetic analyses; the other half, together with YST test plants, has been individually labelled and stored in single bag in laboratory, and monitored for insect emergence. Results confirmed previous findings: attacks on safflower were recorded at Askale location only, all emerged insects were identified by the taxonomist B. Korotyaev as C. scalptum or C.orientale (a well known polyphagous species). On the other hand the majority of insects recovered from YST test plants were identified as CEBA and few specimens ad C. orientale (table 1). Table 1. Identifications of weevils emerged from or collected after dissection of YST and safflower test plants from open field choice tests carried out in Eastern Turkey in 2004. Similar results were obtained in 2003. Location Askale Askale Askale Askale Askale Askale Askale Askale Askale Askale Horasan Horasan Horasan

Specimens 5 1 many Many 1 3 1 24 1 1 3 many 1

Stage Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult

Collected from

Emerged from Identified as Safflower in Lab C. scalptum claviceps Safflower in Lab C. orientale YST dissected in lab C. basicorne YST in lab C. basicorne YST in lab C. orientale Safflower in Lab C. scalptum claviceps Safflower in Lab C. orientale YST dissected in lab C. basicorne YST dissected in lab C. orientale Wild YST outside field plot C. basicorne YST in lab C. basicorne YST dissected in lab C. basicorne YST dissected in lab C. orientale

Such results, merged with CEBA specificity evidences collected by L. Smith team at Albany USDA-ARS PWA quarantine laboratory, would allow the submission of release petition for the weevil in next months. Further work has been planned for CEBA in 2005: molecular analyses on adults from field tests will be carried out during the winter at BBCA and/or EBCL facilities in order to confirm the taxonomic identifications by means of genetic distances. 9

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

In addition, in order to evaluate the possible synergism between the root boring weevil and soil born diseases, pilot tests will be carried out in Italy by a (new) BBCA plant pathologist.

1.2. Psylliodes sp. nr. chalcomerus By R. Dolgovskaya, S. Reznik, M. Volkovitsh, Biocontrol Group, RAS-ZIN, St. Petersbourg, Russia M. Cristofaro, F. Di Cristina, F. Lecce, A. Paolini, C. Tronci, BBCA, Rome, Italy The flea beetle P. chalcomerus (PSYL) was included in our list of potential YST biocontrol agents when an apparently specific population was found in Krasnodar territory in 1998 (picture 5). This population was also sympatric with a more oligophagous one, developing mainly on Scotch thistle. Preliminary biology observations, together with first host range no-choice tests, and DNA analyses carried out in 2001 and 2002 confirmed the existence of 2 different biotypes of PSYL: one, more specific, developing on YST, and one with broader host plant spectrum Picture 5. Psylliodes sp. nr. chalcomerus developing on Scotch thistle. In 2003 more detailed tests on both populations have been carried out by Biocontrol Group, RAS-ZIN, St. Petersburg, Russia, in laboratory and field conditions; in addition choice and nochoice tests on Scotch thistle biotype have been conducted by BBCA in Italy, and DNA analyses have been continued by P. Audisio and A. De Biase at the University of Rome “La Sapienza”. 1.2.1. Field estimation of P. chalcomerus (YST biotype) larval density in relation to

host plant size

In occasion of two field collection trips in Krasnodar Territory, Russia, two field samplings were conducted in May and June 2003 in Volna location. For each sampling, a separate YST patch with relatively high density of PSYL was selected. All YST plants from this patch were collected, measured, weighted and then dissected. Results showed that in natural conditions, two opposite tendencies manifest themselves in turn (table 2, figures 1 and 2). In spring sampling, the number of larvae per plant was linearly correlated both with plant height and plant stem diameter; we consider these correlations as result of selective oviposition by females laying eggs in proportion to the host plant size, which is rather common in phytophagous insects and, particularly, in leaf beetles. In June, the distribution of larvae was almost independent on the plant’s height (accordingly, diameter of stem turned to be the most important factor); this marked change could be possibly explained by the negative larval impact on the host plant. i.e., the rate of plant growth decreased with increase in larval density. Table 2. Results of field sampling at Volna location Sampling date

May 19, 2003

June 16, 2003

Number of YST plants inspected

Total number of larvae and empty mines found

178

297

YST plant height (cm) *

34±12 (15 – 63)

64 ± 14 (40 – 120)

YST plant stem base diameter (mm) *

5.4±2.1 (2 – 11)

4.5 ± 0.9 (2.5 – 7)

10.5 ± 6.7 (2 – 38)

2.7 ± 3.5 (0 – 16)

6.2 ± 4.4 (0 – 19)

YST plant weight (g) * Infestation rate (larval mines per plant) * *) mean±SD, range

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

May 19

June 16

Figure 1. Correlation between YST plant height and number of PSYL larvae per plant. at Volna location; left: May 19 (r=0.47, n=65, p<0.001), right: June 16 (r=0.22, n=48, n.s.).

May 19

June 16

Figure 2. Correlation between YST plant diameter and number of PSYL larvae per plant. at Volna location. Left: May 19 (r=0.32, n=65, p<0.01), right: June 16 (r=0.41, n=48, p<0.01).

1.2.2. Field test of P. chalcomerus (YST biotype) host specificity A pilot adult oviposition and larval feeding field test of (YST-form) specificity was conducted on PSYL in field cages with the possibility of choice (picture 6). Two cages were mounted in natural conditions (Krasnodar territory). In each cage, three plant species: YST, Centaurea calcitrapa, and Centaurea melitensis were planted as young plants grown in greenhouse conditions. Adults, collected from YST in natural conditions were released inside each cage for 15 days. In a month all plants were dissected and inspected for larvae or any traces of larval feeding. In both plots, normal larval mines, as well as mature larvae were recorded only in YST plants. According to this first results, we concluded that in natural conditions PSYL females do not lay eggs on the tested non-host plants, and/or larvae fail to develop on them (table 3). Picture 6. Preparing the pilot field choice test in Krasnodar, spring 2003.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

Table 3. Results of pilot host specificity field test with PSYL (YSTform) in field cages with the possibility of choice. YST C. calcitrapa Plants* Larvae** Plants Larvae #1 8 6 0 2.9±2.3 #2 14 8 0 10.0±6.0 *) Total number of survived plants; **) Larvae per one plant, mean±SD. Plot #

C. molitensis Plants Larvae 5 0 5 0

1.2.3. Laboratory host specificity no-choice tests As a first step we evaluated survival, feeding, fecundity and longevity of YST biotype individual females on 15 plant species in no-choice conditions. Ovipositing females were allowed to feed and oviposit on small bunches of test plants in Petri dishes (picture 7); number of eggs laid and feeding status were recorded. On the basis of the results obtained, tested plant species can be divided into 3 groups (table 4): a) a first group where any or little feeding was recorded, no eggs were laid and life duration of females was very short; considering that all females were ovipositing (on YST) at the Picture 7. Petri dishes used for oviposition and feeding specificity beginning of the experiment, it is possible to tests. assume that a behavioural interruption of oviposition was observed. b) a second group, including several Centaurea and Cirsium spp. on which females occasionally oviposited and fed, showing a significantly higher survival rate; this suggests that oogenesis may be inhibited by feeding on the plants of this group (physiological interruption of oviposition). c) a third group, formed by YST only where feeding, oviposition, and oogenesis were recorded in all females. Both mean survival time and mean lifetime fecundity were significantly higher. Table 4. Survival, feeding and oviposition of adult PSYL (YST-form) females in no-choice conditions (Petri dishes). Plant No. of Plant species Survival (days)* group females Calendula officinalis a) 3 3.7±1.5 (2 – 5) Bracteantha bracteata a) 3 4.0±1.0 ( 3 – 5) Chrysantnhemum sp. a) 3 6.0±2.7 (4-9) Callistephus chinensis a) 3 4.6±2.5 (2 – 7) Tagetes patula a) 3 4.0±1.0 (3 – 5) Cucumis sativus a) 3 5.3±1.5 (4-7) Carthamus tinctorius a) 3 7.0±2.0 (5 – 9) Cirsium californicum b) 3 15.0±15.6 (5 – 33) Cirsium occidentale b) 3 14.7±19.4 (2 – 37) Cirsium brevistylum b) 3 6.0±2.6 (4 – 9) Helianthus annuus b) 3 16.7±10.6 (7 – 28) Centaurea sulphurea b) 3 >40.3±2.9 (37 – >42) Centaurea calcitrapa b) 3 16.3±10.7 (7 – 28) Centaurea diffusa b) 3 >25.0±17.5 (7 – >42) Centaurea solstitialis c) 4 >33.8±16.5 (9 – >42) *) Mean SD (variation range). **) Percentage of readings with feeding traces recorded

Feeding** 0 0 0 0 0 30 17 73 67 55 87 92 81 94 100

Oviposition (eggs/female)* 0 0 0 0 0 0 0 0.4±0.6 (0 – 1) 0.7±1.2 (0 – 2) 3.4±5.8 (0 – 10) 4.7±3.5 (1 – 8) 3.7±4.0 (0 – 8) 2.7±3.8 (0 – 7) 10.7±2.5 (8 – 13) >99.3±62.5 (32 – >153)

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Laboratory no-choice tests were repeated on potted plants showing similar results (table 5). Table 5. Host specificity test: survival, feeding and progeny of PSYL (YST-form) individual females in no-choice conditions (potted plants). Plant Plant species group a) Calendula officinalis a) Bracteantha bracteata b) Cirsium californicum b) Cirsium occidentale b) Cirsium buv. b) Helianthus annuus b) Centaurea sulphurea b) Centaurea calcitrapa c) Centaurea solstitialis *) Mean SD (variation range).

No. of females 3 3 2 3 3 3 2 3 3

Survival in 5 days (%)

Feeding(%)

66 0 100 100 33 66 100 33 100

0 0 100 100 33 33 100 66 100

Progeny, larvae/female* 0 0 0 0 0 0 0 0 7.7Âą3.8(5 â&#x20AC;&#x201C; 12)

During 2004 two field collection trips have been carried out in Russia and one in Italy in order to provide living material for specificity studies and genetic analyses; a first preliminary shipment has been sent to USDA-ARS PWA quarantine at Albany, CA. Continued laboratory host-range evaluation of YST-specific biotype on 30 test plant species/varieties; feeding, fecundity and larval survival have been assessed in Petri dishes and on potted plants in no-choice conditions, and in choice conditions on potted plants only. 1.2.4. No-choice conditions on potted plants Females survival, feeding, oviposition and progeny development of P. chalcomerus (YSTform) were tested at the S.Petersburg facilities under no-choice conditions on potted plants. To carry out the tests, ovipositing females were individually placed on potted plants of different species covered with transparent cages. After 5 days cages were opened, recording adult survival and feeding traces. All surviving females were re-placed in Petri dish with YST leaves for two days. Females laying eggs in Petri dish were used again for a new test with other test plant. Each exposed test plant was periodically checked for the detection of larval mines. In a month after the beginning of each test final reading of the results was made. During final reading, all plants being carefully inspected, larval mines (if present) dissected and rate of larval development estimated. Each test was considered as independent observation, data obtained with YST plants were used as controls. Results showed (table 6) that a limited number of larvae were able to develop on Centaurea americana, C. cyanus, C. melitensis, C. montana, and C. sulphurea in no-choice conditions, although complete adult development was not assessed.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Table 6. Results of laboratory host specificity tests with PSYL (YST-form) in no-choice conditions on potted plants No. of Females females survival (%) Acroptilon repens 5 80 Carthamus tinctorius 5 60 Centaurea americana 2 100 Centaurea calcitrapa 4 75 Centaurea cineraria 2 0 Centaurea cyanus 5 80 Centaurea diffusa 5 80 Centaurea maculosa 3 100 Centaurea melitensis 5 100 Centaurea montana 4 50 Centaurea pratense 5 40 Centaurea orientalis 4 25 Centaurea rothrockii 3 67 Centaurea solstitialis (USA) 8 100 *Centaurea solstitialis (USA) 4 100 *Centaurea solstitialis (Italy) 5 100 *Centaurea solstitialis (Turkey) 4 100 *Centaurea solstitialis (Russia) 4 100 Centaurea sulphurea 5 100 Centaurea virgata var. squarrosa 1 100 *Carduus nutans 2 100 Carduus pycnocephalus 5 80 Cirsium brevistylum 5 40 Cirsium californica 4 50 Cirsium hydrophilum 4 75 Cirsium loncholepis 4 100 Cirsium occidentale 5 40 Cirsium vinaceum 4 25 Cynara scolymus (artichoke) 2 100 *Cynara scolymus (artichoke) 5 100 Helianthus annuus (sunflower) 6 83 Lactuca sativa (lettuce) 3 0 Onopordum acanthium 4 50 *) Tests carried at BBCA facilities, Rome, Italy. Plant species (biotype)

Ovipositing Females 2 0 2 0 0 3 3 1 1 1 1 1 2 7 4 4 4 4 0 0 1 1 0 1 2 2 1 0 2 1 2 0 0

Mean Fecundity 19 0 10 0 0 19 8 5 8 18 5 4 15 13 7 20 12 13 0 0 5 2 0 5 5 13 2 0 10 10 8 0 0

Mature larvae 3 0 1 0 0 2 0 0 2 1 0 0 0 16 8 11 15 27 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1.2.5. Choice conditions in large cages in laboratory In these experiments, several females were placed in large cages with a set of potted plants of different species. Four experiments were carried out, using 5 ovipositing females in the first two, and 9 ovipositing females in experiments 3 and 4. After 10 â&#x20AC;&#x201C; 15 days, beetles were removed from the cages and adult feeding traces were recorded on each plant. Then, 30 days later, all plants were checked for larval mines. Results of these tests agree with previous experiments. However, as expected, in choice conditions insects showed stricter host specificity than in starvation tests. Particularly, only five on 22 plant species tested (see list in table 6) were slightly or moderately damaged by adults (A. repens, safflower, C. melitensis, C. sulphurea and C. cyanus), and only one larval mine was detected on C. cyanus, in spite of the relatively high population density (ca 2 females per plant). Interestingly, both adult damage and larval infestation level turned to be markedly different among YST plants used in the same experiments, suggesting high intraspecific selectivity of feeding and oviposition.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

1.2.6. Field cage host specificity choice tests Field test of PSYL (YST-form collected in Volna location) adult oviposition and larval feeding specificity were established in 10 field cages, each covering 1 sq m plot in natural conditions (Krasnodar territory). In each cage, 5 replicates of five plant species were grown. At April 9, six adults, collected from YST in natural conditions, were released inside each cage. Totally, 13 plant species were tested in different combinations (pictures 8 and 9).

Picture 8. Field cage choice test at the beginning of the experiment; Krasnodar, April 2004.

Picture 9. One of the 1 sq m. portions of the field cage test at harvest in May 2004

Five plots were organized as starvation tests (different combinations of non-target plants), while additional 5 plots included also YST plants. At 18 May, all plants were dissected and inspected for living or dead larvae. The results of this test (table 7) clearly indicated that in natural conditions only one of the tested plant species, Centaurea diffusa, could be attacked by the beetle. Interestingly, this result was obtained even in the presence of the target weed. However, not all of C. diffusa plants were attacked, suggesting significant variations in either plants suitability or female host specificity (or both). As for C. orientalis and O. acanthium, in both cases only single small mine was discovered on one of many plants of these species. Besides, the larva was dead or absent.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

Table 7. Results of host specificity field test with PSYL (YST-form) carried out in Krasnodar Territory

1.2.7. Genetic analyses on taxonomic status and mtDNA variation in natural

populations of Psylliodes spp. cfr. chalcomerus (Coleoptera, Chrysomelidae, Alticinae) By A. De Biase, G. Antonini, E. Mancini, P. Audisio, Department of Animal and Human Biology, University of Rome “La Sapienza” Introduction PSYL populations feeding on different host plants and putatively belonging to the Psylliodes spp. cfr. chalcomerus assemblage of forms (species ?) were sampled from four Russian, three Turkish, three Italian and two Tunisian localities. 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 (Konstantinov in verbis). Materials and Methods Genetic analyses were carried out on 48 adults and 10 larvae chosen among the available samples. The Italian populations were sampled in Central Italy while feeding on Carduus nutans (Asteraceae; European Musk Thistle=CAR) and on Onopordum illyricum (Asteraceae; =ONI); the six Russian populations were collected in four localities of the Krasnodar region (Black Sea) feeding on Centaurea solstitialis (Asteraceae; Yellow Star Thistle=YST) and Onopordum acanthium (Asteraceae; Scotch Thistle=ONO). 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. The Turkish populations were sampled on both host plants (YST and ONO). The Tunisian samples were both collected on Centaurea cyanus (=CCY). Finally two populations 16

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

of Psylliodes hyoscyami (Linné), previously recognised as possibly separated species within the same complex, were sampled in Turkey on their known host plant (Hyoscyamus niger, Solanaceae; =HYN). These samples were characterized by means of the same genetic marker – Cytochrome C Oxidase subunit I gene (mtDNA) – and included in all the performed analyses. Results and Discussion The genetic distances computed among all pairs of sequences showed values ranging from 0.000 to 0.046 when including the contrasts among the P. hyoscyami individuals and all others. Anyway these values, are still not able to give us a clear-cut clue on the relationships among PSYL forms, and more insights are likely when looking at figure 3 portraying the results of the unrooted NJ analysis, showing four groups supported by high bootstrap values. The matrix of the pair-wise genetic distances (not showed) is almost cumbersome and of difficult interpretation when looking for patterns or trends among groups of individuals sharing features of our interest. Therefore it seemed interesting to compute the genetic distances among the above cited four groups instead of contrast among a prior groups that cluster individuals sharing the same feature (i.e. the same host-plant were scored at the sampling time, or the individuals come all from the same location etc) that sometimes could be due to convergence phenomena rather than to evolutionary relationships. In Table 8 these groups are labeled as those depicted in Fig. 3. Table 8 - Net Distances (Tamura & Nei) among the four clusters characterized by means of NJ and MP [Standard errors in square brackets] Turkey (HYN)

Italy (CAR)

0.027 [0.007]

Russia (YST)

0.014 [0.005]

0.025 [0.007]

Russia (ONO)

0.024 [0.007]

0.018 [0.005]

Russia (YST)

0.018 [0.006]

Russia (ONO) and Russia (YST) seem to be distinct genetic pools well characterized and disjunct, but incomplete lineage sorting or even hybridization events cannot be excluded due to the presence of the above cited un-clumped individuals that seem to be in intermediate position between these two pools. On the other hand the evidenced relationships of these two forms with P. hyoscyami individuals, along with the structure of the fourth group [Italy (CAR)] clustering individuals from four very distant locations (Russia, Turkey, Tunisia, Italy) and feeding on five distinct host plants (YST, ONO, CCY, CAR, ONI), seem to suggest the existence of a plastic genetic pool that enable this insects to shift among different host plants when "required". The shifted forms could eventually evolve in highly local specialized forms (and, in fact, Russia (YST) and Russia (ONO) are very well characterized from the genetic point of view), but it is still unclear what mechanisms drive those shifts and their stability. Finally it should be noted the inclusion in the Italy (CAR) cluster of several individuals (PS3.5, PS. 3.1, PS18.3, PS18.1, PS10.1) from Russia (Volna and Tuzla) and feeding on YST. It could be hypothesized that these individuals represent a distinct genetic pool inhabiting the same region of Russia (YST) and that probably reached the region from the area of origin (more southern areas? as for instance the Anatolian areas?) in a different time.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Conclusions The available data still suggest the existence of three distinct forms inside the Psylliodes spp. cfr. chalcomerus taxon, that are not distinguishable by morphological traits. These forms seem also reflect a trophic 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. Different genetic markers, more data and maybe a statistical phylogeographic approach (through a Nested Clade Analysis) could shed more light on the whole matter. However it should be once more stressed that the assay of samples of the widespread European Psylliodes dulcamarae (Koch), that is a very closely related species of the taxon under study but showing a distinct morphology, would provide data that could let us calibrate the levels of the scored genetic divergence, and also give a clue on the evolutionary dynamics of the host plant shifts of the studied taxa. Turkey (YST) PS15.1

Russia (YST)

Turkey (HYN)

PS15.2

PS7.3

PS18.2

PH1.2 PH2.1

PS9.1 PS7.2

PS11.1

PS9.2 PS9.3

PS7.1 PS5.1

PH2.2

PS3.7

PS3.6 62

PS3.3 57

PS3.2

PH1.1

PS19.2

PS16.1

PS10.3

PS17.1

PS10.2

Russia (ONO)

PS6.7

Russia (ONO)

PS8.3

Turkey (YST)

PS6.5

PS8.2 PS10.1

PS8.1

PS12.5

PS18.1

PS6.1

PS18.3

PS4.2

PS22.1 69

PS4.70

PS23.1

PS3.1 PS3.5

PS12.2

PS12.4 PS12.1

PS1.1

PS24.3 PS1.2 PS2.1

PS1.3

Russia (YST), Turkey (ONO), Tunisia (CCY), Italy (CAR, ONI)

Figure 3. Unrooted Neighbour Joining Tree on Tamura & Nei Distances. Bootstrap Analysis on 1000 replicates.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

1.2.8. Work planned for 2005 Further host specificity tests will be necessary with a set of non-target plant species, especially the ones that showed to be potential alternative hosts for larval development. The work will be performed, as for 2004, at the St. Petersburg facilities, in Krasnodar territory and at the USDA ARS Albany facilities. No-choice and choice tests will be carried out only in laboratory. No-choice tests will be conducted confining a single female in Petri dish, and transferring hatching eggs on the test plants. Choice tests should be carried out with single female per cage. In both cases the progeny will be monitored, sacrifying the mothers and adults from the progeny for genetic analyses. Moreover, impact studies will be carried out at the Rome facilities testing both adult and larval impact on the host plant. Complete genetic analyses, by including two outgroup species (Psylliodes sp.) and keeping the track of the progeny of each female belonging to the Volna “population”, in order to estimate the actual distance among the already highlighted sub-groups and to identify potential “contaminations” of the “Onopordum” form in the YST group.

1.3. Tingis grisea By M. Cristofaro, F. Di Cristina, F. Lecce, A. Paolini, C. Tronci, BBCA, Rome, Italy 1.3.1. Field collections and preliminary host specificity tests Live adults and nymphs of T. grisea (TIGR) were collected in Central and Eastern Turkey in May, June and July 2003. Despite extensive searches, only few adults recovering from diapause, have been collected in spring (mid-May); a preliminary no-choice test has been set up using 10 plant species / biotypes. Each treatment was presented to insects as a stem / flowerhead bouquet, kept in a capped vial with water, together with a smaller tube providing water through a wick, in 5x5x12 cm transparent Picture 10. The lace bug Tingis grisea plastic containers, covered with a fine mesh nylon net (picture 11). Four replicates of each treatment have been set up, using two insects each; the test has been conducted at room conditions. Unfortunately most of the insects died during the first two days. In addition, few or no activity was observed on tested plants and only few feeding scars were found on YST US biotype only. We concluded that insects needed some kind of substrate (soil or sand) at the bottom of testcontainers. Picture 11. Preliminary no-choice tests with T. grisea, May 2003.

In June numerous live samples were collected; 50% of them as immature stages.

The same experimental conditions described above were used, using sand as a substrate, and using 4 individuals for each replicates (4 each plant species/variety). Since the population collected in June likely belonged to the new generation, we did not expect any oviposition, therefore only feeding and adult/nymph survival observations were made.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

As expected, mortality was lower than in previous preliminary observations; unfortunately, feeding activity on all treatments was scarce, in addition, no significant differences in feeding levels were highlighted between the treatments. The test was continued for four weeks, than the remaining insects were transferred in diapause conditions to establish a lab colony to re-activate during the next season for oviposition tests. Objectives of the work on TIGR in 2004 were to artificially induce diapausing state in overwintering adults by means of controlled photoperiod and temperature and, subsequently, to induce the emergence from diapause in late winter. Second goal was to evaluate insects fecundity and fertility, and at the same time, to assess their host specificity in terms of oviposition and feeding preferences. 1.3.2. Diapausing Newly emerged adults collected in summer 2003 at Horasan, Eastern Turkey, were transferred in laboratory and maintained in glass jars at room temperature, fed with fresh YST bouquets (picture 12). From early August to early September, about 175 adult lace bugs were put in variable controlled environment from 24-15 °C and 14:10 L/D, to 12-8°C and 8:16 L/D; starting from the second week of September the insects have been put in dark conditions at 8°C. Bugs were kept at room temperature (15-20°C) Picture 12. Diapause storage of T. grisea approximately 8h each week and allowed to feed on adults fresh YST. In late January, emergence from diapause was induced transferring the insects at 20°C and then at 25 °C with increasing photoperiod up to 16:8 L/D. About 50% of insects survived the overwintering period (sex ratio approximately 1:1); the majority of deaths were recorded in the first weeks of artificial diapause. 1.3.3. Oviposition substrate Preliminary tests were carried out on confined parts of YST stems and leaves at 25°C and 16:8 L/D. Since we firstly wanted to individuate the more suitable oviposition substrate, the plant was exposed to couples of bugs (male+female), using 3 methods: a) fresh stems or leaves on humid filter paper in Petri dishes; b) living young rosettes transplanted in big Petri dishes; c) portions of stems of potted plants isolated by transparent polypropylene tubes closed with rubber foam at the bottom and a fine mesh net at the top (pictures 13, 14, 15). Plant parts were changed every 3 days and examined at the stereoscope in order to count eggs (opercula).

Pictures. 13, 14 and 15. Ovipositing substrates used; from left to right: cut stem and leaf in Petri dish, living rosette transplanted in petri dish, transparent tubes on potted plants

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Methods a) and b) were rejected because of fast desiccation of cut plant parts and for the difficulty to deal with soil in Petri dishes. 1.3.4. Fecundity and nymph development tests Because of the extreme small size of egg opercula (0,1 mm reported in literature), we were not able to spot eggs on tested plants; inspected stems and leaves were than kept alive in water as long as possible, to indirectly evaluate daily and total fecundity by retrieving newly emerged nymphs. Obviously fertility was not assessed. Results of nymph emergence suggest that at 25Â°C and 16:8 L/D, eggs hatch 7 to 10 days after oviposition and that the metamorphosis between the instars takes place approximately every 7 to 10 days (figure 4) 45

Number of nymphs

1st instar

2nd instar

3rd instar

4th instar

20 15 10 5 0

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 27 28 29

Days from oviposition Figure 4 â&#x20AC;&#x201C; Results of fecundity tests; each area represents the number of individuals of each immature stage retrieved at different time intervals from oviposition.

1.3.5. Nymph development host specificity tests First and second instar nymphs were collected daily and transferred to 8 different plant species/varieties in transparent tubes to evaluate the host preference of immature stages development in no-choice conditions. Survival, development status and weight (for 4th and 5th instars and adults only) were recorded. Results showed that T. grisea 1st and 2nd instar nymphs are able to completely develop to adults only on yellow starthistle and C. cyanus. On both hosts the development period is about 30 days, confirming results of oviposition tests (table 9) Picture 16. Transparent tube used for tests.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

Table 9. Results of nymph development no-choice tests

Plant YST OLE LIN CYA CYN CRU MAC DIF

1st instar nymphs Adults Replicates retrieved 15 8 15 0 15 0 13 8 10 0 14 0 10 0 10 0

2nd instar nymphs Adults Replicates retrieved 7 5 8 0 5 0 4 2 5 0 4 0 0 0 0 0

1.3.6. Oviposition tests The aim of this test was to assess oviposition preference among the above-cited test plants, on overwintering adults collected in Eastern Turkey in March 2004. Sixteen couples of lace bugs were allowed to mate and oviposit in plastic tubes put around portion of stems of potted plants for 3 days. In order to grant the insects the possibility to feed on the host plant, couples were kept on YST for 3 days prior to be put on any other plant species. Due to the impossibility to spot egg opercula, we indirectly evaluated oviposition (successes) by retrieving emerged 1st and 2nd instar nymphs that were left on the plant to assess the ability to complete development on different hosts. We estimated that oviposition as well as adult development took place only on 3 close related Centaurea spp. (YST, C. cyanus and C. maculosa), even if the small number and the high mortality of insects did not allow us to perform a large number of replicates (table 10) Table 10. Summary of results of oviposition and nymph development no-choice test Plant

Replicates

Adults retrieved

OLE

LIN

CYA

CYN

CRU

MAC

DIF

YST

1.3.7. .Work to do •

Carry out field collections of overwintering adults and, later in the season, of new generation individuals

•

Complete nymphal development and oviposition no-choice tests on wider test plant list

•

Develop a technique to easily spot egg opercula on stems and leaves (coloration?)

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

1.4. Aceria solstitialis De Lillo, Cristofaro and Kashefi on Centaurea solstitialis (YST) in Apulia By E. De Lillo, R. Monfreda, G. Rondinone, Department Of Entomology, University of Bari, Italy Aims of study: •

finding for possible overwintering sites for Aceria solstitialis with particular regard on young rosettes or on dead YST skeletons;

•

description of the seasonal occurrence of mites and symptoms on the plants;

•

ascertainment of American YST suitability development and reproduction of the mite evaluation of the most suitable YST plant parts;

for Picture 17. Aceria solstitialis and damage on YST, Turkey (photo J. Kashefi)

detection of A. solstitialis geographical distribution in Apulia.

•

1.4.1. Overwintering location of A. solstitialis This step is going to start in December 2004, collecting 10 YST rosettes and 10 YST skeletons per site. Plant samples are going to be separated in four categories: •

rosette leaves;

•

roots;

•

root adhering soil;

•

meristems.

Samples are going to be pooled into a single unit for each category and processed for mite extraction to ascertain the mite presence and its distribution on the plants. 1.4.2. Seasonal occurrence of A. solstitialis on YST Four sites with large wild populations of YST were selected in February 2004 in the area around Castel del Monte, Apulia. Starting from February 2004, 20 plants per site were sampled every four weeks, avoiding to extinguish the plant populations in the field. When A. solstitialis was found in a site, the sampling period was reduced at two weeks until the drying of the YST plants (August) and mites disappearance. Four categories of samples, when available, were separately collected from each plant and transported in laboratory forming the following groups: •

new rosette leaf (with length ≤ 2 cm);

•

mature rosette leaf (with length ≥10 cm);

•

stem (without flowerhead);

•

flowerhead.

Plant abnormalities were recorded. Mites were extracted from each sample by “washing and sieve method” (De Lillo, 2001), identified, counted and recorded per part of plant for each sampling. Specimens of the same sampling and category were pooled all together to save processing time until mites were found, then they were processed separately. 23

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Yellow Starthistle

Results showed that no eriophyoids were detected in February and March samplings. Aceria solstitialis was identified only in June, July and August samplings from 2 of 4 sites. In these sites, the seasonal occurrence of mites appeared to be selective for the different plant parts considered in the study: in June, mites were found on new rosette leaves and young stems; on stems, the population density was higher and persistent until the end of August; the occurrence on pre-flowering flower buds was limited to mid August, while no mites were recorded on flowering and senescent buds. The mite population was not pure and other species were contemporary present. In particular, Aculodes sp., Epitrimerus sp. and Aceria sp. could strongly be new species. About Aculodes sp., species belonging to this genus are usual on grasses and, therefore, its detection could be related to the neighbouring plants to the YST. 1.4.3. Ascertainment of American YST suitability for development and reproduction

of the mite and evaluation of the most suitable YST plant parts Seedlings of American (50 plants) and Italian (50 plants) strains of YST were prepared by BBCA and transplanted in a net greenhouse in Bari, at May. The greenhouse was built in field using an anti-aphid net. Unfortunately, the mite density in the field was too low in 2004 to collect them and release in the greenhouse. A larger mite collection will be performed in late June 2005 to carry out host specificity tests in greenhouse environment at the peak of population density. 1.4.4. Detection of A. solstitialis in Apulia Surveys were carried out in numerous Apulian localities during spring and summer to look for YST and its possible abnormalities. Samplings were carried out in localities where plant deformations probably caused by eriophyoid mites were detected. Just in one site (Valenzano) Aceria centaureae was found in April. Other species were not found.

1.5. Seed head survey By M. Cristofaro, F. Di Cristina, F. Lecce, A. Paolini, C. Tronci, BBCA, Rome, Italy Flower bud survey has started in 2002 in order to compare the occurrence of natural enemies on Italian and Turkish YST wild populations with the group of introduced biocontrol agents in the US. YST flower heads have been collected three sites in Italy and three in Turkey following the same protocol: 10 flower heads (stage Bu-3 or older) have been collected from each of 20 plants and kept in separate bags (one each plant); once in laboratory, flower heads have been individually separated in 5x1cm vials capped with cotton plugs. Plants have been numbered and flower heads catalogued. Collections have been organised in three times: early, middle and late summer 2002, also according to each area climatic conditions. The vials have been checked for emerging insects two times in the following spring (2003): in march and June. Emerged insects have been pinned and labelled. Results showed low infestation rates in all locations; tephritid flies, in particular Urophora sp. (often more than one specimen for capitula) in Turkey and Chaetorellia sp. in Italy were the most common associated species, while the weevil population was extremely low. As expected high numbers of parasitoids were recovered. 24

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Yellow Starthistle

In 2004 collections have been repeated at the 6 Italian and Turkish locations and flower buds from 2003 collections have been dissected. Insects are currently being prepared to be identified.

1.6. Larinus filiformis By L. Gültekin, Atatürk University, Erzurum, Turkey L. filiformis (picture 18) is a weevil distributed in Central and Eastern Anatolia, apparently highly specific to YST; the feeding activity of the weevil destroys young flower buds; single eggs are laid in larger buds where developing larvae completely destroy the seeds. Purpose of the project is to estimate the potential competition among L. filiformis and E. villosus, already released and established agent, which feeding and ovipositing patterns are very similar.

Picture 18. The weevil L. filiformis on a

During 2004, Dr Gultekin completed field survey and YST flower bud. collections finding for the first time both insects in sympatric conditions in Central Anatolia.

The first part of the project was focussed on the assessment of feeding and ovipositing capitula stage preference (picture 19): first analyses of results show that while E. villosus prefers BU-4 stage capitula for oviposition, L. filiformis seems to prefer BU-2 and BU3.

Picture 19. Field cages used for the tests.

In the second part of the project the daily fecundity for both species was evaluated; during the tests it was possible to highlight different feeding and oviposition holes position and sealing patterns among the two species.

During the third part of the project, concerning the evaluation of feeding, oviposition and larval development competition, none of the eggs that were laid by both species did hatch. This may be caused by the need of mating immediately before oviposition. Therefore, in 2005, we will complete competition tests; in particular, it will be important to repeat the oviposition and larval development test, giving the opportunity to the females to mate ad libitum. Moreover, since it seems clear that L. filiformis is preferring early flower bud instars, it is important to understand if buds infested with its larva are suitable for E. villosus oviposition or if they do show deterrence due to marking pheromones. Second step of the 2005 season will be to complete field surveys in order to define distribution areas for both species

1.7. Preliminary field screening of potential biocontrol agents of Centaurea spp. in Slovakia By L. Čagan, P. Toth, Agricultural University, Nitra, Slovakia In 2003, we extended our research also on plants from the genus Centaurea growing on dry localities of Slovakia. The research involves several collecting explorations to find yellow starthistle (C. solstitialis) distribution and to screen of promising biocontrol agents from family Curculionidae (feeding also on other natural Slovakian Centaurea species). The main aim of the research was to help BBCA to better understand insect populations characterization on the northern border of C. solstitialis distribution. 25

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Russian thistle

Although, there were 12 locations with C. solstitialis occurrence in Slovakian literature, we found (until now) the species only at one location (Bratislava). The population consisted of about 150 individuals. Collected seeds were sent to BBCA for analyses. Even if, there is one old record of Ceratapion basicorne from Slovakia, we did not record the species during the preliminary surveys. Besides, we screened weevil fauna associated with plants from the genus Centaurea growing in dry locations (table 11) Table 11 Host plants and plant association of weevils recorded in Slovakia on Centaurea spp. Species A. austriacum A. onopordi A. penetrans L. jaceae L. sturnus P. cinereus R. ermischi R. horioni R. pratensis

Locations 6 42 20 14 15 5 12 1 20

Host plant C. scabiosa C. calcitrapa, C. scabiosa Centaurea sp. C. scabiosa C. jacea, C. scabiosa Centaurea sp. C. scabiosa Centaurea sp. C. jacea, C. nigra

Plant association* leaves (A) leaves (A) roots (L) leaves (A) leaves (A) leaves (A) leaves (L, A) leaves (A) leaves (L, A)

2. Russian thistle By M. Cristofaro, F. Di Cristina, F. Lecce, A. Paolini, C. Tronci, BBCA, Rome, Italy The work on Salsola sp. started in the summer of 2003, under the supervision of L. Smith, USDA-ARS PWA, Albany, CA. Dedicated surveys were carried out in Turkey and Tunisia, selecting two associated insects: a flea beetle (picture 20) and a weevil (Lixus sp., picture 21).

Picture 20. Unidentified flea beetle associated with Salsola sp. during preliminary specificity assessment.

Picture 21. preliminary laboratory choice tests with Lixusa salsolae.

Preliminary specificity tests have been carried out showing that both species were able to feed and develop on several Chenopodiaceous plants. Both species have been rejected. During 2004 more dedicated explorations have been carried out: three trips in Tunisia, one in Eastern Kazakhstan, two in Turkey, one in Crete (Greece), and several short trips in Southern Sicily. Several new potential biocontrol agents have been selected. Among them the weevil Anthypurinus biimpressus (ANBI), found in E Tunisia, was considered the most promising and preliminary tests have been carried out in laboratory (picture 22). This weevil was found in May and identified by E. Colonnelli as a probable monophagous species. Larvae and/or 26

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Russian thistle

adults feed on young leaves causing severe damage. Oviposition and larval development were obtained in laboratory from adults collected in the field, although larvae could not complete pupation.

Picture 22. The new selected agent for Russian thistle A. biimpressus

Picture 23. Laboratory tests with the lace bug Piesma salsolae.

Laboratory preliminary observations showed that larvae appear to be external feeders; this is quite uncommon for weevils. The insect is likely univoltine: a new trip has been carried out in September in order to collect the overwintering generation; differently from the ones collected in spring, the insects fed and mate on host plant but did not oviposit, thus we started diapause induction in order to perform first laboratory preliminary host and biology tests in late winter. Beside ANBI, preliminary fecundity assessment tests were carried out on the lace bug Piesma salsolae (PISA, picture 23), already screened by R. Sobhian (USDA-ARS EBCL, Montpellier, France); a tentative establishment of a laboratory colony is in progress starting with diapause induction. The wide host range will not allow as to keep this insect as promising candidate. Among a list of several biocontrol agents, other interesting natural enemies have been found and selected particularly in Eastern Kazakhstan: •

The weevil Salsolia morgei

•

New population of the gall midge Desertovellum stackelbergii

•

The weevil Baris przewalskyi

2.1. Future plans: work to do in 2005 •

Complete insect’s diapause and induce emergence in late winter with the weevil Anthypurinus biimpressus;

•

Start laboratory no-choice specificity test in controlled laboratory conditions on both species;

•

Define a priority list of new identified natural enemies. Carry out field collections and preliminary host specificity tests.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Rush Skeletonweed

3. Rush skeleton weed A research project finalized to the selection and study of new biocontrol agents for Chondrilla juncea, was established in summer 2003 under the supervision and in cooperation with G. Markin USDA Forest Service, Bozeman, MT. During 2003 only opportunistic surveys have been carried out in Turkey and Italy. C. juncea plants have been sampled in numerous locations finding only the already known natural enemies such as the rust Puccinia chondrillina, and the mite Eriophyes chondrillae. In 2004 dedicated exploration trips have been conducted in Volgograd Territory, Russia, Eastern Kazakhstan and Turkey; opportunistic surveys in Italy, Tunisia, Crete (Greece). Among the numerous insect species we found, five new natural enemies have been selected for further studies: •

The jewel beetle Sphenoptera foveola

•

The leaf-feeding moth Simyra nervosa

•

The flower-feeding moth Schinia cognate

•

A not yet identified leaf-feeding sawfly

•

The root boring moth Oporopsamma wertheimsteini

3.1. Work to do in 2005 •

Carry out field collections of overwintering larvae of Sphenoptera spp. in Turkey and Kazakhstan and, later in the season, of new generation individuals;

•

Start preliminary field cage and laboratory host range tests and biological observations with both Sphenoptera spp.;

•

Continue opportunistic explorations on new biocontrol agents in Kazakhstan, Volga Region, Central and Eastern Turkey, and Southern Italy.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Field Bindweed

4. Field bindweed 4.1. Field bindweed stem borer, Melanagromyza albocilia (Diptera: Agromyzidae) By L. Čagan, P. Toth, Agricultural University, Nitra, Slovakia Starting in 1998, we cooperated with BBCA/USDA-ARS, working mainly on biological control of field bindweed (Convolvulus arvensis). During the cooperation a key player was Melanagromyza albocilia Hering (Diptera: Agromyzidae), which was selected as a most promising biocontrol candidate agent. Its general distribution, biology and symptoms on host according to literature were described in the report 1998. During the previous years was outlined the life history of the stem borer fly; assessed the damage caused to the target weed (reports 1999 and 2000); determined the seasonal parasite complex and natural parasitization of M. albocilia (report 2000); set up and evaluated laboratory host range tests for Melanagromyza albocilia (report 2001) and set up and evaluate open field host range tests for M. albocilia (report 2002). In 2003, we summarized the results of previous years (seasonal biology and seasonal patterns of field bindweed infestation), wrote down a paper and submitted the paper to Entomologica Fennica (abstract see below). In addition, we recapitulated the data of parasitism rate and seasonal parasite complex of M. albocilia to produce another scientific paper (nowadays in the final stage of preparation). The field research (in 2003), was aimed on hedge bindweed (Calystegia sepium) to confirm M. albocilia host preference to field bindweed under field conditions in Slovakia. 4.1.1. Melanagromyza albocilia: short summary of the paper submitted to scientific

journal (Entomologica Fennica) Melanagromyza albocilia Hendel (Diptera: Agromyzidae), a native European stem borer of field bindweed (Convolvulus arvensis L.), was considered as a very promising candidate for its biological control. To determine the seasonal biology of the stem borer, and its seasonal patterns of field bindweed infestation under field conditions in Slovakia, thirty randomly selected plants were sampled weekly at three farmers' field sites during 1999 and 2000. The insect over-wintered as pupa. The emergence of the first-generation adults started in the beginning of May and continued until the end of May in an outdoor insectary. The second-generation adults emerged during June-August with maximum in August. M. albocilia larvae generally infested field bindweed throughout the season. Infestation was initially low and reached its peak from August through September. Percentage of infested plants varied from 46.7 to 99.2% during the period. Number of infested field bindweed stems also increased generally from June to September, rising from 4.1 to 37.2%. Infested stems contained an average of 0.84±0.40 to 1.34±0.50 stages (larvae, parasitized larvae, pupae) of the stem borer. Sometimes it was found up to 5 stages per stem which did not indicate strong intraspecific competition of the species. The numbers were stable within each locality and year. The population of M. albocilia was the highest in 1999, with mean densities of 1.57±1.01 to 3.21±2.13 stages per infested plant at single localities. Numbers declined in 2000, with an average of 1.40±0.89 to 2.23±1.55 stages per plant. All parasitoids that emerged from larvae were identified as Bracon picticornis Wesmael (Hymenoptera: Braconidae). The larval parasitization was 36% in 1999 and 56% in 2000. In this study, M. albocilia was found as a new host for B. picticornis. In addition to the three weekly sampled localities, M. albocilia was found in another 88 localities throughout the country, confirming that it is a common insect in Slovakia and it is closely related to its host.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Field bindweed

4.1.2. Field survey of hedge bindweed (Calystegia sepium) to confirm M. albocilia

host preference Table 12; Infestation of hedge bindweed by M. albocilia under field conditions of Slovakia in 2003. Location*

Nr of plants Nr of sprouts ** sprouts infested

Kamenica n/Hronom (W) 40

303

Tehla (W)

291

Vráble (W)

267

Nitra (W)

175

Svodín (W)

314

Urmince (W)

200

Hajnáčka (M)

152

Bátka (M)

148

Sliač (M)

197

Turňa n/Bodvou (E)

222

Trebišov (E)

338

Svätuše (E)

277

* W = West Slovakia; M = Middle Slovakia; E = East Slovakia ** Number of sprouts was counted about 1 cm above the roots

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Other Target Weeds

5. Other target weeds Opportunistic observations and collections on other target weeds were carried out in the course of BBCA field surveys during 2003 and 2004. In particular, it is important to mention stem and leaf gall-like damage (Eriophyid mite?) found on Arundo sp. in Northern Tunisia on Sept. 2003 (picture 24.); populations of two root borer weevils were recorded on Lepidium latifolium in Eastern Turkey during July 2003 and identified as Melanobaris sp. pr. semistriata and Lixus myagri (picture 25); an undetermined crown-galling weevil was found in Northern Caucasus on hoary cress, Lepidium (Cardaria) draba in early April 2003 (picture 28); two seed feeder agents, an undetermined tephritid fly (probably Urophora sp. picture 26) and an unidentified moth on Russian knapweed (Acroptilon repens) as well as an eriophiyd mite on Canada thistle, C. arvense(picture 27), during July 2004 in Eastern Kazakhstan; 4 weevils (2 Geranorrhynchus spp., 1 Coniatus sp. and one Corimalia sp.) and 2 defoliator moths (one undetermined and the monophagous Lepidogma tamaricalis) (picture 29) on salt cedar, Tamarix sp. in Egypt during September 2004.

Picture 24. Stem and leaf gall-like damage on Arundo sp. found in Tunisia, 2003.

Picture 25. Adult of Lixus myagri in L. latifolium roots; Turkey, July 2003.

Picture 26. Probable Urophora sp. attack on Russian knapweed (right flower bud); Kazakhstan, 2004

Picture 27. C. arvense plant attacked by eriophyid mites, likely Aceria anthocoptes; Kazakhstan, 2004.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Other Target Weeds

Picture 28. Galls of a still undetermined crown-galling weevil on L. draba found in N Caucasus in spring 2003.

Picture 29. Lepidogma tamaricalis damage on salt cedar and detail of larva in the web system; Egypt, 2004.

6. Work plans for 2005 Several new candidate agents for different target weeds have been selected in 2003 and 2004. Those species will be subject of study at different extents, based on their priority level. In particular, field collections, biology and life history notes will be performed for most of the new species, while preliminary host specificity laboratory and field tests will be carried out on rush skeletonweed selected agents and on A. biimpressus for Russian thistle. Beside the new agents, the experimental work in 2005 will focus on completion of host specificity assessment of the flea beetle PSYL and of the lace bug TIGR. For these species, special emphasis will be placed on the evaluation of the impact on the target weed. Impact studies will be also performed on C. basicorne in order to improve already collected research data with the assessment of its possible role transmitting soil-borne diseases to YST. The work on the eriophiyd mite A. solstitialis will focus on the first host specificity assessment in laboratory conditions. Table 13. Summary of BBCA and cooperators working plans for 2005; in orange, the tasks to be performed, in green the tasks completed during previous years. Field Host Range observations

Weed* Candidate agent

Exploration trips

Biological notes

Host Range Host Range lab field

YST

C. basicorne

Completed

Start

YST

P. chalcomerus

collections

Completed

Complete

Start

YST

A. solstitialis

Complete

Start

YST

T. grisea

collections

Complete

Completed

Continue

A. biimpressus

Continue

Start

Prliminarly selected agents Start

Start

Prliminarly selected agents Start

RSW

Sphenoptera foveola (KZ)

Start

RSW

Sphenoptera sp. (TK)

Start

RSW

Simyra nervosa

Start

RSW

O. wertheimsteini

Start

Impact Studies

Petition In progress

Start

Start Start Start

*) YST=yellow starthistle; RT=Russian Thistle; PP=perennial pepperweed; RSW=rush skeletonweed

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol â&#x20AC;&#x201C; Exploration Trips 2003

7. Exploration trips

Figure 5. Summary of the areas explored during the years 2003 and 2004.

7.1. 2003 The collection and exploration season 2003 was almost entirely dedicated to yellow starthistle with the aim to collect more samples of Psylliodes sp. nr. chalcomerus for laboratory studies and to implement the population dataset for genetic analyses; supervise and help the work of our co-operators at Erzurum, Turkey, on Ceratapion basicorne; to continue YST seedhead survey in Italy and Turkey; set up a lab colony of the lace bug Tingis grisea. Beside this, additional and opportunistic work has been conducted on common teasel hoary cress, rush skeletonweed and Russian thistle. Trips to Turkey have been repeated monthly from April to September; one trip has been done in Northern Caucasus, one in Tunisia, one in Romania and Slovakia and several short trips in Italy (Sicily, Sardinia and Apulia). In addition, we did two trips to the US for business meetings and to attend the W1185 workshop at Monterey, CA; we also attended the XI International Symposium on the Biological Control of Weeds at Canberra in April. 7.1.1. Field trip in Russia (March 30 â&#x20AC;&#x201C; April 9) By M. Cristofaro, C. Tronci, B. Rector, M. Volkovitsh, B. Korotyaev, S. Reznik This trip was focused on the collection of live samples of P. chalcomerus populations for genetic analyses and laboratory testing purposes. The trip took place along the north-eastern coast of the Black Sea, West of Krasnodar, where previous observations and collections were carried out. Two inland and five coastal locations were visited, adult flea beetles were collected feeding and resting on the host plants YST and Scotch thistle, both in sympatric and allopatric conditions (picture 30.). Other purpose of the trip was to help Dr. Rector (USDA- Picture 30. Field collection of adult ARS EBCL, Montpellier, France), to collect samples of PSYL on the Black Sea coast; N. teasel species, Dipsacus sp., for genetic analyses; Caucasus, April 2003. 33

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2003

several locations were visited for this purpose. In addition, weevil root-crown galls were found on Lepidium draba. 7.1.2. Field trip in Turkey (May 1-10) By M. Cristofaro, F. Di Cristina In early May started the exploration and collections of the tingid lace bug to start host specificity, biology and impact evaluation in laboratory. Visited known locations in Erzurum and Cappadocia Regions finding only few adult specimens emerging from winter diapause, probably due to low average temperatures. A second aim of the trip was to supervise and help the start up of the work of our cooperators at the Atatürk University of Erzurum: laboratory and field host specificity tests with Larinus filiformis and competition tests between L. filiformis and Eustenopus villosus performed by L. Gültekin (see chapter 1.7); and field plot specificity and impact evaluation tests of Ceratapion basicorne conducted by Dr. Hayat team (see chapter 1.1). During exploration trip, in Central and Eastern Anatolia, several new and known agents have been recorded and collected in different locations: an unknown damage on YST rosette, as well as a YST rosette tip gall moth larvae; two rusts on L. draba and C. juncea leaves, and few samples of the flea beetle P. chalcomerus on Scotch thistle. 7.1.3. Field trip in Greece (June 1-15) By M. Cristofaro, J. Kashefi Aims of the trip were to help J. Kashefi (EBCL, Thessaloniki Substation) to solve several problems connected with YST candidate biocontrol agent Botanophila turcica rearing and host range testing, and to collect C. basicorne (CEBA) samples for genetic analyses. 7.1.4. Field trip in Turkey (June 14-24) By C. Tronci, B. Rector This trip was mainly focused on the collection of T. grisea live samples for biological notes and host specificity tests, and to make a quick survey on rush skeletonweed potential biocontrol agents. Central and Eastern Anatolian plateau areas have been explored, visiting known tingid bug locations in Cappadocia and Erzurum regions. Large numbers of 1st, 2nd, and 3rd instar nymphs, and only a small ratio of adults have been found, especially at Horasan site (Erzurum). Other important tasks accomplished include the collection of larvae, pupae and adult C. basicorne specimens from dissected YST plants, for DNA analyses. Opportunistic observations and field dissections have been carried out also on common teasel (Dipsacus fullonum), Russian thistle, hoary cress. 7.1.5. Field trip in Turkey (July 9-15) By F. Lecce, F. Di Cristina The trip was targeted to the collection of T. grisea adults for host feeding and diapause/overwintering tests, and to carry out mid season YST seedhead collections in Turkish locations. Secondary objectives were to gather more locations and live specimens of eriophyid mites species associated with yellow starthistle, and to conduct opportunistic surveys on rush skeletonweed. The selected locations for collection were in Central (two for YST lace bug and two for YST seedheads), and Eastern Anatolia (the same location for tingids and seedheads). 34

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2003

7.1.6. Field trip in Romania and Slovakia (August 20-26) By M. Cristofaro The main purpose of this trip was to set up an official co-operation agreement between BBCA and ENERGI-CO, a Romanian company, for the constitution of the Romanian Biological Control (ROBICON) Company, within a newly built Scientific Park in the city of Bucarest. Aim of the Company will be to promote alternative and low environmental pest and weed control strategies, in particular based upon the sterile insect technique (SIT). Only one day was spent at the University of Nitra, Slovakia, to review the agreement with Dr. Čagan finalized to the biocontrol research on field bindweed, and to visit the small YST population recently found in Slovakia (likely the northernmost distribution point), and collect some samples for genetic analyses (carried out by D. Luster, Ft. Detrick, MD). 7.1.7. Field trip in Turkey (August 20-29) By C. Tronci, A. Paolini The objectives of the fourth trip to Turkey were: to complete late season YST seedhead collection, to continue the survey on rush skeletonweed (RSW) associated organisms, and to conduct field observations on the life-cycle status of the lace bug Tingis grisea performing possible collections of (pre-) overwintering adults. In addition, we dissected the harvested YST and safflower plants from the C. basicorne field plot tests and collected insect specimens for taxonomist identification and DNA analyses. 7.1.8. Field trip in Tunisia (August 29 – September 4) By M. Cristofaro Goals of the trip were to perform a preliminary survey on Russian thistle in this area, not well explored for Salsola sp. and other weeds; in addition, carried out YST seed collection for DNA analyses, and collected olive samples attacked by olive fruit fly for population sampling carried out by Kim Hoelmer at EBCL, Montpellier, France. 7.1.9. Field trip in Turkey (October 13-20) By M. Cristofaro, F. Di Cristina The October trip was organized to accomplish the following tasks: carry out a preliminary survey on new potential biocontrol agents of Russian thistle, conclude the plant dissection of C. basicorne impact evaluation field test at Erzurum University, and to complete the YST seedhead and natural enemies survey season. Opportunistic observations, collections and samplings have been carried out also on common teasel and rush skeletonweed. Started from Istanbul and went east through Northern Turkey, reaching Erzurum on October 16. Discussed with Dr. Hayat and Dr. Gültekin about future research co-operations and signed the agreement for 2004; dissected with Dr. Hayat 60 plants from the insecticide exclusion experiment.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

7.2. 2004 7.2.1. Field trip in Turkey (March 16-23) By F. Lecce, C. Tronci Objectives of the trip were to re-define details of the C. basicorne open field tests to carry out in spring with our cooperators at the Ataturk University, Erzurum, and to carry out a life-history survey, in particular on the overwintering status, of the YST candidate agent T. grisea population at Horasan site (picture 31). Even if the YST population at Horasan was mostly at very early rosette stage and minimum temperatures were below 0°C, it was possible to assess that numerous T. grisea overwintering adults, came out from soil debris to feed at Picture 31. Overwintering T. grisea adult; Eastern Turkey, March 2004. warmest time of day. About 50 living individuals were collected to increase laboratory-reared population for testing purposes. 7.2.2. Field trip in Turkey (April 24-May 3) By M. Cristofaro, F. Di Cristina, E. Colonnelli & B. Rector This trip was organized to help Turkish cooperators from Ataturk University, Erzurum, to establish field tests to evaluate CEBA host specificity in two locations (see chapter 1.1), and to carry out collections of live samples of the YST lace bug T. grisea. Additional surveys and collection were focused on Russian thistle and rush skeleton weed. Other purpose of the trip was to help Dr. Rector (USDA-ARS EBCL, Montpellier, France), to collect samples of teasel species, Dipsacus sp., for genetic analyses; several locations were visited for this Picture 32. Collecting teasel seeds for purpose (picture 32). genetic analyses; Central Turkey, May 2004. For YST, found few specimens of Psylliodes, lace bugs and about 5 Araxia n. sp. at Çat; Galeruca larvae (picture 34) and tip galling moths (picture 33) in Cappadocia.

Picture 33. YST tip galling moth; Cappadocia, Turkey, 2004

Picture 34. Galeruca sp. on YST; Cappadocia, Turkey 2004

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

Concerning RSW, two larvae (probably 2 species of Cerambycidae) feeding in the roots were found. 7.2.3. Field trip in Tunisia (March 12 – 18) By M. Cristofaro & F. Di Cristina Aim of the trip was to perform survey on Russian thistle and other weeds in this area. In different localities several specimens of the plant and of putative natural enemies were collected for genetic analyses. The trip covered almost the entire country diverse habitats from northern shores to southern deserts and including mild-climate areas in the Central Ranges. In few locations we recorded several YST spots, collecting Lepidopteran natural enemies. 7.2.4. Field trip in Crete, Greece (September 20 – 29) By F. Lecce The trip was targeted to the exploration and collection of natural enemies of Russian thistle Salsola sp. The explorations covered the whole island perimeter. In different localities several specimens of the plant and of putative enemies were collected for genetic analyses. Opportunistic observations, seed collections, and field dissections have been carried out also on Chondrilla juncea. Salsola sp. was found on the majority of beaches and coastal environments often associated with lace bugs of the Piesma genus and in a couple of sites with large caterpillars. The insects have been collected and sent to taxonomist for identification. 7.2.5. Field trip in Kazakhstan (June 23 – July 15) By M. Cristofaro, M. Volkovitsh, R. Sobhian, C. Tronci Objectives of this long trip were the search for new potential biocontrol agents of Russian thistle (Salsola sp.) and rush skeletonweed (Chondrilla juncea) in SE Kazakhstan. The trip was carried out by 2 groups of researches and included 2 six day trips and a few daily trips: first group (M. Volkovitsh, C. Tronci, and R. Sobhian) travelled from June 26 to July 3 (1897 km, 22 sites), focussing on Russian thistle; second group (M. Volkovitsh, M. Cristofaro) travelled from July 6 to July 13 (2061 km, 19 sites), focussing on rush skeletonweed. The trips were conducted in cooperation with L. Smith (USDA-ARS PWA) for the Salsola sp. part, and with G. Markin (USDA FS) for the rush skeletonweed part. The whole trip was arranged by Dr. Roman Jashenko (Institute of Zoology, Chairman of Tethys Society, Almaty) who also helped to rent a car with driver and cook Sergey Pachin.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

Figure 6. Field exploration trips carried out in Kazakhstan in 2004; a) first trip (Jun 26 – Jul 3); b) second trip (Jul 6 – 13)

First part of trip (figure 6a) covered mainly lowland and foothill (elevation 340-860 m) desert area between western coast of Balqash lake on the north, foothills of Zailiiskii Alatau Range on the south, eastern foothills of Shu-Ili Mts on the west, and eastern coastal band of Ili river on the east. Area includes sandy and hard (clay, salty) soil deserts with typical North Turanian vegetation (sandy desert plants, numerous bush and semi bush Salsolae and other Chenopodiaceae), as well as foothill hard-soil deserts with bush vegetation. Russian thistle was widespread on almost all locations visited; numerous herbarium samples have been collected, which unfortunately did not include any “Type B”, as learned upon plant identification carried out by Fred Hrusa (CDFA). Nevertheless, several promising agents have been found, together with still interesting but likely oligophagous insects (table 14). In addition, new populations of the gall midge Desertovellum stackelbergii, already subject of study by R. Sobhian and L. Smith, have been found. Table 14. Summary of insects associated with Salsola spp. found in SE Kazakhstan (full trip) as determined by taxonomists. Monophagous insects The weevil Salsolia morgei New population of the gall midge Desertovellum stackelbergii The weevil Baris przewalskyi (figure 22) Oligophagous insects Lixus rubicundus Lixus polylineatus Lixus scabricollis Cosmobaris scolopacea Baris (Baris) sulcata Elasmobaris signifera Baris convexicollis Baris mnemnonia Microlarinus rhinocylloides Temnorhinus elongatus

Second part (figure 6b) (elevation 348-1399 m) covered foothills of Zailiiskii Alatau Range westward of Almaty, an extent desert area between Ili river on the west, Balqash lake on the north, Lepsi river on the east, and foothills and low altitudes of Dzhungarskii Alatau Range on the south. This desert area named “Semirechiye” (seven rivers) is crossed by several big rivers (Karatal, Aksu, Lepsi) and minor ones originated in the high altitudes of 38

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

Zailiiskii and Dzhungarskii Alatau Ranges with desert river woods. Chondrilla spp. were quite common and abundant in almost all the habitats but humid foothills and high altitudes, and, particularly, along the roads. Herbarium samples have been collected. Beside several Bradhirroa spp. for which further identifications are in progress, the most promising agent collected was the jewel beetle Sphenoptera foveola, which was found in numerous localities causing interesting damage to the root system (see next paragraph for details on selected agents for RSW in 2004). Rush skeletonweed biocontrol agents surveys (Russia, Turkey, E Kazakhstan) The following exploration trips had the main goal to carry out a search for new potential biocontrol agents against rush skeletonweed, Chondrilla juncea, in its native area in SouthEast Eurasia. Field collections were conducted in Lower Volga Basin (Russia), in Eastern Turkey, and in Kazakhstan. Surveys in Kazakhstan which is believed to be a centre of biodiversity of Chondrilla spp. turned to be the most productive area, although Lower Volga Basin and SE Turkey also seem to be rather promising. Several potential biocontrol agents (i.e., insects associated with Chondrilla spp. which could be rather specific, but still were never used or tested before as Chondrilla biocontrol agents) were collected: •

The buprestid Sphenoptera foveola, collected in Kazakhstan, widespread, rather abundant, root boring larvae and stem-feeding adult. Larval feeding seems to have significant negative impact on the host plant.

•

The moth Schinia cognate with flower and seed-feeding caterpillar, collected in Volgograd province. Taxonomy literature reported its host range restricted on Chondrilla and relative plant genera.

•

The moth Simyra nervosa with leaf-feeding caterpillar collected in Kazakhstan, rather abundant, leaf-feeding larvae may have negative impact on the host plant.

•

The sawfly (still unidentified) with leaf-feeding larvae was collected in Turkey in two locations.

Among them buprestid Sphenoptera foveola seems to be the most interesting for further investigations. 7.2.6. First field trip in Volga Basin, Russia (May 25 – June 04) By M. Cristofaro, M. Volkovitsh & S. Reznik Region surveyed covered Volgograd province and northern part of Astrakhan province along Volga and Don Rivers. The region is characterised by arid climate and steppe to semi desert landscapes with spots of sandy deserts which are most favourable for natural Chondrilla and Russian thistle growing (pictures 35 and 36). This region is also situated next to Kazakhstan which is considered as the centre of diversity of Chondrilla spp.

Picture 35. Inspecting young Salsola sp. plantlets; Volga region, Russia, May 2004.

Picture 36. A pause dissecting RSW samples; Volga region, Russia, May 2004

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

7.2.7. Second field trip in Volga Basin, Russia (July 15 – 16) By A. Popov (Botanist from Volgograd) This second survey was targeted to the collection of Chondrilla spp. flowerbuds attacked by immature stages of flower parts / seed feeding insects to be reared out in laboratory conditions. Unfortunately, no adults were reared out from more than 600 buds. 7.2.8. Third field trip in Volga Basin, Russia (August 16 – 20) By S. Reznik The third survey was carried out to increase rush skeletonweed flowerbud samplings in known distribution locations of selected natural enemies, with the aim to obtain newly emerged adults to utilise for preliminary laboratory assays. Buds and flowers were collected at three different development stages and stored in laboratory; only some adults of the moth Schinia cognate were obtained. 7.2.9. Trip in Turkey (May 28 – June 8) By A. Paolini & C. Tronci One of the primary aim of the trip was to investigate natural enemies of rush skeleton weed, Chondrilla juncea in SE Turkey, in particular we would like to verify the presence of associated buprestid beetles belonging to the genus Sphenoptera, as it was previously but unclearly reported. The trip was planned to start from and end in the city of Adana, following NE direction to Erzurum, than S to the Iranian border, than W along N coast of Van Lake, crossing the Dyiarbakir region towards Adana. Three days have been spent at Erzurum to complete the host specificity field tests on YST candidate biocontrol agent C. basicorne in cooperation with Dr. Hayat Team (Ataturk University). Several natural enemies were found in almost all sites where RSW was present; the most common were moths belonging to the genus Bradhirroa but also other Lepidopteran larvae associated with crown leaves (picture 37) and coleopteran root-boring larvae were found. All specimens (mainly immature stages) were brought to laboratory and emerged adults have been sent to taxonomist for identification. 7.2.10. Field trip in Turkey (June 26 – July 3)

Picture 37. A crown boring lepidopteran larva on RSW; Eastern Turkey, June 2004.

By M. Cristofaro This trip was focused on the survey on natural enemies of rush skeletonweed (Chondrilla juncea). From Istanbul, drove south in direction of Ankara and then south west. Along the road E90 found a huge population of C. juncea (found one sawfly larva). Near Pamukkale found a large site of C. juncea with a rest of an external cocoon of a pyralid moth. Drove South West, between Kale and Mugla found a large population of Chondrilla: some of the plants had stems showing pathogen disease-like symptoms. Along the beach found a good population of Salsola spp., the plants were generally healthy, but with different shape of the leaves; collected 3 samples of different “ecotypes” for herbarium and DNA analyses.

BBCA Bi-annual Report 2003 - 2004

Weed Biocontrol – Exploration Trips 2004

7.2.11. Field trip in Turkey (July 15-22) By M. Cristofaro & F. Di Cristina The trip was targeted to the search of C. juncea natural enemies and to the collection of T. grisea live adults for host feeding and diapause/overwintering tests. Collected 150 adults of T. grisea on YST in Horasan. On Chondrilla found a moth (Pyralidae, adult) and several eggs around the stem. Few plants had also eriophyid mite symptoms. Drove south, reached Van Lake, and followed the North shore. Found some Chondrilla plants found one sawfly larva outside of the roots. Drove West to Tatvan, found Chondrilla with eriophyid, aphids and one sawfly larva. Between Bayran and Kozluk found Chondrilla sp. with roots infested by “sandy” new cocoons (Lepidopteran larva inside). Drove west to Gaziantep direction. Along the road 400 found a small larva outside of the Chondrilla roots. About 1 km after the intersection with the road to Andirin found a huge attack of Lepidopteran larvae (maybe 2 species) on the roots of Chondrilla. Collected some to rear out adults. Before the rail way coming from Develi going to Cappadocia found a large population of Salsola; some plants were attacked by tingid (?). Found also a root boring larva (weevil), an other stem boring larva (not weevil), and one pupa (?) in the stem. 7.2.12. Field trip in Egypt (September 21 – October 1) By M. Cristofaro & F. Di Cristina The goal of this trip was to perform a preliminary survey on Salsola, Chondrilla and Tamarix in this area. Drove East along the Mediterranean coast. Found two Salsola species attacked by Lepidopteran larvae (likely Pyralidae) and one large green hemipteran. In addition, carried out plants collection for DNA analyses and Herbarium. Few km before the town Abu Zenema found Tamarix plants attacked by the gregarious larvae of the defoliator moth Lepidogma tamaricalis, producing peculiar cocoons (found and collected mature larvae and some pupae). Drove East in the direction of Red Sea. Move in direction of Cairo. Found some Tamarix plants heavily attacked by a Lepidopteran in silken cocoon and 1-2 little weevil species (Corimalia and Coniatus like). Ten km South of Assyut found little weevils (green, with the head yellow and Corimalia sp.) and 1 large weevil. Along the road outside Assyut found Tamarix attacked by little weevils (green and yellow) and tingids. 30 km North of El Kharga found Tamarix attacked by 2 species of little weevils (green and Corimalia), tingids, bruchids, stem galling Lepidopteran and silken cocoon Lepidopteran. Close to Dakhla found on Tamarix a population of the silken web lepidopteran and green (only) weevils. Along the road 10 found a very large population of Tamarix attacked by tingids and the small green weevils. About 15 km northern found on Tamarix two large green weevils, the small green, silken web lepidopterans and stem galling moths. Along the same road found on Tamarix an other weevil, 2 smaller Baris like, two other small weevils, several tingids and large silken web and stem galling moths. Collected damaged olives and checked several Tamarix plants in Baharia Oasis. Found the small green weevils, one yellow, one Baris like. Collected stem galls. At the North Eastern edge of the oasis found on Tamarix the green small weevils, 4 Baris-like, and one coleopteran larva.

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

Biological and integrated control of insect pests 8. Agriculture pests 8.1. Evaluation of attractants for Ceratitis capitata and Bactrocera oleae in two localities in Southern Italy. By M. Porto, Department of Agriculture, University of Catania, Italy A. De Cristofaro, SAVA Department, Molise University, Campobasso, Italy Objective of the present work was the evaluation of fruit fly attractants on two species of economic importance in Italy: Ceratitis capitata and Bactrocera oleae, following the experimental protocol agreed at previous FAO/IAEA CRP Research Coordination Meetings. The work has been conducted in two locations of Southern Italy, one for each tephritid target species: C. capitata experiments have been performed at Paternò, Eastern Sicily, about 40 Km W of Catania, in one of the most typical areas for citrus orchards; the work on B. oleae has been conducted in the vicinity of Foggia, Apulia, in the Italian SE plains, one of the widest and productive areas for olives. 8.1.1. Site I – Paternò, Sicily The experiments have been carried out in a 2ha orange orchard cultivated with Navel cv, one of the most susceptible to medfly attacks. The tests have been carried out from September 26 to November 21, 2003; the experimental plot has been divided in 3 sub-plots: replicates A and B, and control. Each replicate was organised using five PMT type traps baited with treatments A, B, C, D, E from standard protocol as follows: A: 250 ml NL B: AA+PT+ TMA+TRI C: AA+ TMA+TRI D: ½ AA+ TMA+TRI E: ½ AA+PT+ TMA+TRI AA=Ammonium TRI=H2O+Triton

acetate,

PT=Putrescine,

TMA=Trimethylamine,

NL=NuLure,

In the control sub-plot five treatment A traps were used. Replicates A and B have been applied 6 days/week, while the control was applied 1 day per week, when replicates A and B were not applied. In replicates A and B and in the Control sub-plot, the ripeness and abundance of fruits was evaluated according to the RCP standard protocol. Climatic data were recorded by a nearby weather station (SOAT Paternò, n.19). Results The graph in figure 7 reports the relative captures of male and female C. capitata for both replicates, including control treatment;

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

3,5

3,0

Mean Captures

2,5

2,0

1,5

1,0

Females Males

0,0 A

Control

Treatment Figure 7 – Mean captures of male and female C. capitata on both replicates and on control.

Among the 5 different baits used, treatments B (AA+PT+ TMA+TRI) showed the highest attractancy towards C. capitata in both replicates, eliciting better results than NuLure control. Treatments C (AA+ TMA+TRI) and E (½ AA+PT+ TMA+TRI) showed good scores if compared with control. All baits showed marked selectivity for females. 8.1.2. Site II – Serracapriola, Apulia The selected site for the B. oleae experiment is a 40ha olive orchard at Serracapriola, Foggia, Apulia. This area is one of the most important and productive olive production locations of the world. The experiment was carried out between August and November 2003. The plot has been divided in 5 sub-plots: replicates 1-4 and control. Each replicate was organised using six PMT type traps baited with treatments A, B, C, D, E, F from standard protocol as follows: A: 250 ml NL B: AA+PT+ TMA+TRI C: AA+ TMA+TRI D: ½ AA+ TMA+TRI E: ½ AA+PT+ TMA+TRI F: 2 AB+PT+TRI AA=Ammonium acetate, PT=Putrescine, TRI=H2O+Triton, AB=Ammonium bicarbonate

TMA=Trimethylamine,

NL=NuLure,

In the control sub-plot six treatment A traps were used. Traps have been hung at 1,5-2m from the ground and at 20 m distance by width and 30m by length from each other; a 15m by width and 24m by length buffer area has been set all around the plot to avoid “border effect”.

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

Replicates 1-4 have been applied 6 days/week, while the control was applied 1 day per week, when replicates 1-4 were not applied. Traps were serviced every three days, treatment A was renewed at every visit, while treatments B-F, once every 4 weeks. Traps were rotated at every visit to avoid â&#x20AC;&#x153;plant effectâ&#x20AC;?. Climatic data were recorded by a nearby weather station. Results Figure 8 shows the relative captures of Males and females 2,5

Mean Captures

2,0

1,5

1,0

,5 Females Males

0,0 A

Ctrl

Treatment Figure 8 - Mean captures of male and female C. capitata on both replicates and on control.

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

8.2. Evaluation of bioinsecticides and attractants 8.2.1. Effects of Spinosad速 treatments on the life-cycle of Ceratitis capitata and

Bactrocera oleae in laboratory and field cages By A. Fenio, M. Cristofaro, BBCA, Rome, Italy

This series of tests were carried out in the framework of our cooperation with Dow Agrosciences focussed on the evaluation of the bioinsecticide Spinosad速. Preliminary tests were carried out in laboratory on both tephritid flies to assess the effectiveness of various application methods on laboratory strains in laboratory cages. Methods included substrate spraying, solid and liquid food treatment, and topical treatment by spraying of resting substrate. For each thesis the persistence of the effects was also assessed. Moreover, choice tests were conducted to estimate possible repellence effects of the product. All treatment methods elicited high effectiveness at 5 and 10 ppm concentrations on both insects, preserving the insecticide effect for relatively long time after treatment; in addition, the Spinosad速 did not show any repellent effect. A second set of tests was carried out in field cages on C. capitata only using a laboratoryreared medfly strain. In particular, the efficacy of different product application strategies on males and females was investigated, including canopy spraying and bait-based distribution. In addition two side experiments were set up in order to estimate the persistence of such application methods and to test new distribution substrates. Spinosad速 showed a better efficacy when deployed by bait; food distribution also evidenced higher persistence maintaining nearly 100% of the effects after 5 days. 8.2.2. Field cage evaluation of new trapping systems for the medfly developed by

Agrisense By A. Fenio, A. Paolini, M. Cristofaro, BBCA, Rome, Italy The work was focussed on the evaluation of the efficacy of two traps produced by Agrisense. The attractants used in the tests were Trimedlure (TML) and Ammonium Bicarbonate (AB). The bioassays have been carried out in large field cages (picture 38), where standard numbers of medflies were confined, sorted for sex (males and females) and physiological stage (neonate-virgin and 5 day old-mated). Traps were positioned 1.8 m above the ground, hanging on lateral branches of a large potted Picture 38. Field cage tests carried out at BBCAcitrus tree, located in the centre of the cage. The ENEA facilities. efficacy was evaluated recording the number of captures (on sticky traps) or the number of dead insects (on trap treated with deltametrin) vs. the number of alive flies in each cage. Results with mated insects showed an attractive response of the males for both attractants TML and AB (in deltametrin traps). Both attractants worked better with mated than unmated females. 45

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

8.3. Integrated Mediterranean fruit fly control in an organic peach orchard in Southern Italy using different lure-based mass-trapping systems and bioinsecticide spraying. By A. Fenio, G. Carbone,C. Tronci, M. Cristofaro, BBCA, Rome, Italy F. Baldacchino, A. Brunori, ENEA, C. R. Trisaia, Italy In the framework of its cooperation with ENEA (Italian National Agency for the New Technologies Energy, and the Environment), since 2004 BBCA is involved in a three-year project (codenamed BRIMET), finalized to the development of new agricultural strategies and technologies in Basilicata Region, Southern Italy. Beside the fulfillment of BRIMET project primary tasks, BBCA is carrying out the evaluation of new fruit fly monitoring and mass-trapping systems within the IAEA Cooperative Research Programme “Development of Improved Attractants and their Integration into Fruit Fly SIT Management Progams”. Objective of BBCA work in 2004 was the evaluation of the efficacy of two Biolure® based mass trapping systems used in synergy with baited spray application of the bioinsecticide Spinosad® in an organic peach orchard where neem and rotenone treatment are regularly applied. Experimental orchard was divided in four zones separated by buffer areas; in each zone different treatments were applied: a) Biolure® bucket traps (mass trapping, picture 40), b) Spinosad® baited spray, c) Biolure® + Spinosad®, d) Control (no treatment). Trimedlure® and Biolure® baited EasyTrap® (picture 39) and bucket traps were also used for monitoring medfly population in all treatments.

Picture 39. Easy trap baited with biolure used for monitoring.

Picture 40. A bucket trap hung at a peach tree

Treatments were applied from late June until harvest in mid September; traps and treatments servicing/renewing as well as capture count and fruit sampling were performed every week. This first year of experimentation produced several interesting outcomes (figure 9): •

all Biolure® monitoring traps showed the same efficacy as TML traps capturing, as expected, mainly females one or two weeks in advance, confirming that “female” attractants allow a more sensible and more reliable monitoring of medfly infestations;

•

the monitoring traps placed in Biolure® mass trapping alone and Biolure® + Spinosad® treatments captured significantly less females when compared to Spinosad® alone or control treatments; such result suggests a good efficacy of Biolure® based mass trapping;

•

Spinosad® confirmed its high insecticidal capacity: the captures of all monitoring traps within Spinosad® treated zones were significantly lower than in other zones. 46

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

Mean Week ly C ap tures / Trap

60 Trimedlure Monitoring

Biolure® Monitoring

40 30 20 10 0 Control

Biolure®

Spinosad®

Biolure® + Spinosad®

Treatm ent

Figure 9. summary of mean weekly captures on monitoring traps; the female portion of medfly population shows significant decrease where Biolure® mass trapping is applied; Spinosad® shows the highest (non sex selective) efficacy

Experiments will be repeated in 2005 season, adding several modifications to the protocol in order to obtain detailed information on differential response of males and females, and to verify the efficacy of the treatments on medfly population emerged after harvest from dropped fruits.

8.4. Effects of an azadirachtin-based compound on the host-parasitoid interactions between the Mediterranean fruit fly, Ceratitis capitata Wied. (Diptera: Tephritidae) and the braconid wasp Opius concolor Szepl. (Hymenoptera: Braconidae) By G. Carbone, M. Cristofaro, V. Di Ilio, BBCA, Rome, Italy F. Minelli, ENEA C.R. Casaccia, Rome, Italy The host-parasitoid system between the medfly Ceratitis capitata and the oligophagous braconid wasp Opius concolor (also parasitizing the olive fruit fly, Bactrocera oleae, picture 41), has been studied to elicit the existence of indirect effects of an azadirachtin-based treatment of the host, on the life-cycle of the parasitoid. As a first step, we evaluated the fertility, fecundity and longevity of a laboratory strain of O. concolor parasitizing medfly larvae developed on azadirachtin treated larval medium. Next, behavioural choice tests were carried out Picture 41. The braconid wasp O. in order to test host preference of O. concolor ovipositing concolor medfly and olive fruit fly parasitoid. females, between treated and untreated medfly larvae. Third, we tested the direct effects of azadirachtin on fertility and longevity of O. concolor adults fed with treated medium. Finally, histological observations were performed on the ovaries of treated O. concolor females, to evidence any damage to the reproductive system caused by azadirachtin. Results showed that the braconid wasp was able to normally complete its life-cycle on treated medfly larvae, and to produce fertile offspring, even when medfly larval medium was treated with high levels of azadirachtin. On the other hand, the fecundity and longevity of O. concolor adults directly treated with azadirachtin through the feeding medium, showed a 47

BBCA Bi-annual Report 2003 - 2004

Biological and integrated control of agriculture pests

significant reduction, highlighting similar effects to those reported for phytophagous insects, such as C. capitata. These results were confirmed by histological observations of the ovaries of O. concolor treated females which showed high levels of degeneration due to azadirachtin. In behavioural tests, O. concolor ovipositing females used significantly less time to find and oviposit into treated medfly larvae, showing that the treatment with azadirachtin does not affect the chemicals involved in host individuation. This work showed that there are no significant indirect effects of azadirachtin treatment in the host-parasitoid system between C. capitata and O. concolor. Remarkable effects of azadirachtin on the braconid life-cycle were evidenced only upon direct treatment of the insects through the feeding medium. In this framework, it is possible to state that field applications of azadirachtin-based pest control strategies appear to be safe for beneficial insects and therefore, to sustain the synergistic use of this bioinsecticide with biological control agents.

8.5. Biological control of the citrus flatid planthopper (CFP), Metcalfa pruinosa with the parasitoid wasp Neodrynus typhlocybae (Hymenoptera: Drynidae) in Latium, Italy. By A. Fenio, BBCA, Rome, Italy P. Pecora, Phytolab Srl, Latina, Italy This two-year project was partially funded by the Latium Regional Government and was carried out in cooperation with Phytolab s.r.l., Latina, Italy. Purpose of the study was to evaluate the efficacy and feasibility of the biological control of CFP using N. typhlocybae in the extensive kiwi growing region in Latina province. During 2003 extensive surveys have been conducted in the area in order to select the experimental plots area. The presence of CFP populations has been then monitored within the selected areas, not only on kiwi plants but also on several wild plant species including Rubus sp., Urtica sp., Ficus sp., etc. Commercial packages of N. typhlocybae have been released in experimental plots in June. The percentage of parasitized CFP adults and nymphs has been evaluated every 15 days showing a relatively high parasitization rate. Nevertheless, since CFP is an univoltine species, the effects on its population have been evaluated during 2004; collected data are still being evaluated and will be available shortly.

BBCA Bi-annual Report 2003 - 2004

Human and animal parasitic insects

9. Human and animal parasitic insects 9.1. Effects of NeemAzal® on the life cycle of Anopheles stephensi (Diptera: Culicidae). By L. Lucantoni, A. Habluetzel, L. Pasqualini, University of Camerino, Italy M. Cristofaro, BBCA, Rome, Italy The experiments currently in progress at the University of Camerino aim at the assessment of the biological effects of a standardized extract of Neem (Azadirachta indica) seeds on the malaria vector Anopheles stephensi. 9.1.1. Materials and Methods Insects A laboratory strain of Anopheles stephensi is being used. Insects are reared in a growth chamber at standardized humidity (100%) and temperature (30° C) conditions, with a photoperiod of 12:12 l:d. Mosquitoes are provisioned with a sucrose 10% solution during all their lifetime. Blood meals are also given to mature females, using an artificial device made with a plastic cup containing a small amount (1 ml) of human blood, closed with a stretched piece Parafilm “M”® as a skin-like membrane. Neem extract The extract used in the experiments is NeemAzal® (NA), a commercial formulation produced by Trifolio-M, Germany. It is obtained from the seeds of A indica by an extraction procedure using solvents. NeemAzal® is very rich in polar limonoids (57,6%), azadirachtin A in particular (34%). Adult feeding on treated sucrose solutions Adult An. Stephensi of both sexes were fed starting from emergence with sucrose solutions 10% containing the following concentrations of NA: 10, 100, 1000 ppm. Two more solutions were prepared: solvent (containing the same concentration of ethanol and tween as in the 1000 ppm solution) and a simple sucrose 10% solution as the control. Two untreated blood meals were offered to the mosquitoes at days 4 and 9 from the trial start. The experimental cages contained ~50 females and ~ 70 males. Three replications were made. Mortality was registered by daily count of the dead individuals. Blood intake was measured by differential weighing of the artificial devices. Fecundity was assessed counting the laid eggs. Feeding on treated blood meals Two treated blood meals were offered to 6-7 days old An. Stephensi females. The first at the start of the trial and the second at day 6. The following groups were prepared: 10, 100, 1000 ppm, solvent and control, as explained in the previous experiment. An untreated sucrose 10% solution was given to the mosquitoes during all the duration of the assay. The trial cages contained ~50 females. Three replications were made. Mortality data were collected counting the dead individuals daily. Blood intake was measured by differential weighing of the artificial devices. Fecundity was assessed counting the laid eggs. A sample of females from each group was dissected under a stereomicroscope to observe the developing stage reached by the ovaries and the status of them.

BBCA Bi-annual Report 2003 - 2004

Human and animal parasitic insects

9.1.2. Results and Discussion Adult feeding on treated sucrose solutions â&#x20AC;&#x201C; males The statistical analysis of the data is shown in table 15. The presence of NA in the feeding solution determines a highly significant increase in the mortality. Table 15 - Adult feeding on treated sucrose solutions, male mortality: univariate analysis of variance and LSD test. Group

Mean % mortality

Std. Deviation

LSD test

Control

39,65

3,73

Solvent

40,55

3,63

10 ppm

49,77

4,23

100 ppm

42,06

5,03

1000 ppm

62,32

7,70

d.f. = 347

F = 93,26

p < 0,001

Adult feeding on treated sucrose solutions â&#x20AC;&#x201C; females As seen in the case of the males, NA raises the mortality in a concentration-dependent manner. Results from the statistical analysis (table 16) show a high significance of the differences amongst means. Ovipositional and blood intake preliminary data elicited a certain reduction both in the amount of blood ingested by the females and in the number of eggs laid. Only the number of eggs laid by the females belonging to the 1000 ppm group seems to be lower than the other treatment groups, compared to the amount of blood ingested by each neem treated group, which seems to be almost the same. Table 16 - Adult feeding on treated sucrose solutions, female mortality: univariate analysis of variance and LSD test. Group

Mean % mortality

Std. Deviation

LSD test

Control

34,35

2,70

Solvent

41,14

3,00

10 ppm

44,77

3,61

100 ppm

52,89

3,85

1000 ppm

71,06

5,92

d.f. = 397

F = 194,24

p < 0,001

BBCA Bi-annual Report 2003 - 2004

Human and animal parasitic insects

Feeding on treated blood meals NeemAzal® has an effect on female mortality when ingested with blood. Analogously as in the treated sucrose solution assay, the neem extract lowers the lifetime of the mosquitoes on a concentration-dependent way (table 17). Table 17 – Feeding on treated blood meals, female mortality: univariate analysis of variance and LSD test. Group

Mean % mortality

Std. Deviation

LSD test

Control

44,46

2,74

Solvent

40,95

2,98

10 ppm

53,86

3,01

100 ppm

53,90

3,02

1000 ppm

78,82

2,51

d.f. = 361

F = 356,47

p < 0,001

Differently from the previous experiment, a strong antifeedant effect by the solvent can be seen during the blood meal. Consequently, females from the solvent group produced a reduced number of eggs. A reduction in the eggs number is clearly visible in the treatment groups, as well, demonstrating an antifeedant effect by NA. This effect seems to be cumulative with that of the solvent. The number of eggs per µl of ingested blood per female showed that, with the same amount of blood, females from the solvent group laid the same number of eggs as the control females, while females from the treatment groups laid less eggs than control and solvent groups, depending on the concentration, with a minimum of no eggs laid by the 1000 ppm group females. This result allows to distinguish between the antifeedant effect of solvent and NA, and the toxic effect due to NA only. Picture 42 represents a dissected control ovary 24 hours after the blood meal. The eggs appear sub-circular and filled for more than 2/3 with yolk. Picture 43 shows a NA 100 ppm treated ovary at the same time. Less than half of the eggs are similar to those of the control. The development of the most of them, however, is clearly altered: eggs are small, contain few yolk granules and tend to completely degenerate into disorganized masses of vesicles. A raise in the concentration of NA causes in the exposed ovaries an increase in the proportion between damaged and normal eggs, with a maximum effect in the 1000 ppm group, where all the eggs failed to develop correctly (picture 44). Normal eggs in damaged ovaries always developed completely, though often with some delay in comparison with the control.

Pictures 42, 43, 44. Ovaries dissected 24 hours after the blood meal; from left to right: control, 100 ppm and 1000 ppm treated ovaries

BBCA Bi-annual Report 2003 - 2004

Human and animal parasitic insects

Next laboratory activity will include more replications of the trials to allow a statistical analysis of the blood meal and oviposition data. Further experimentations will be also designed to clarify the contribution of the solvent in the antifeedant effect relative to 10 ppm and 100 ppm groups.

9.2. Effects of Spinosad® on larval instars of three mosquito species of medical importance By R. Romi, S. Proietti, Italian National Health institute, Rome, Italy M. Cristofaro, BBCA, Rome, Italy The indiscriminate use of some products causes environmental contamination, adverse effects on non-target organisms and, in time, led to a build up of resistance in pest populations. In this scenario, the use of plant derived pesticides should assure a selective activity against target pests and a shorter persistence in ecosystems. Among these, the bioinsecticide Spinosad® shows a promising activity on mosquito larvae. Spinosad is a naturally-occurring mixture of two spinosyns (21-carbon tetracyclic lactones), A and D, produced by the fermentation of the bacteria Saccharopolyspora spinosa. Spinosad is effective against several insect orders such as Lepidoptera, Coleoptera, Diptera, Thysanoptera, Orthoptera, Hymenoptera, Homoptera, Isoptera, and mites. Spinosad is highly effective on a broad range of agriculturally-important insect pests, with an excellent environmental and mammalian toxicological profile. In this study we evaluated the efficacy of Spinosad in laboratory bioassays against mosquito strains belonging to 3 genera which include the most important species of medical importance ( Aedes aegypti, Anopheles stephensi and Culex pipiens). The results reported in table.18. confirmed the high efficacy of Spinosad® on mosquito larvae belonging to the 3 genera. The range of efficacy on our 3 reference strains is the same of the most common Bacillus thuringiensis israelensis-based formulates (data not showed). Table 18. Susceptibility of Aedes aegypti, Anopheles stephensi and Culex pipiens III-IV instar larvae to Spinosad. Lethal concentrations 50, 90 and 99 (in mg/l). where calculated at 24 and 48 h post treatment. Anopheles stephensi

Aedes aegypti

Culex pipiens

24h

48h

24h

48h

24h

48h

LC50

0.039

0.024

0.0096

0.0070

0.0064

0.0032

LC90

0.101

0.042

0.015

0.0096

0.018

0.013

LC99

0.22

0.068

0.024

0.012

0.043

0.042

χ2

2.55

1.85

2.34

0.56

2.79

6.62

Slope

2.11

1.57

1.47

0.58

2.25

3.03

d.f.

BBCA Bi-annual Report 2003 - 2004

Human and animal parasitic insects

9.3. Experimental trap monitoring of mosquito populations in the urban area of San Benedetto del Tronto, Italy, and evaluation of the effects of chemical disinfection of manholes and other reservoirs in the urban drainage system. By L. Lucantoni, A. Habluetzel, L. Pasqualini, University of Camerino, Italy M. Cristofaro, BBCA, Rome, Italy P. and L. Serbelloni, ACTIMA Srl, Rome, Italy This short-term project aimed to evaluate the efficacy of a chemical control of mosquito population, based on low toxicity compounds such as pyrethroids, and based on preventive monitoring of population by means of an egg-trappingsystem developed by Dr. Romi at the Italian National Health Institute. The work has been carried out between August and November 2004 in cooperation with The university of Camerino, Italy, and ACTIMA srl, Rome, Italy, a company specialized in mosquito disinfection. As a first step, a preliminary survey was conducted in S. Benedetto del Tronto urban area to locate suitable zones for ematophagous insects development (larval reservoirs, adult resting and mating environments, etc.). Several kinds of traps have then been distributed in such zones: mosquito egg-traps, CDC light traps, sticky traps for culicids; GPS coordinates of trap locations were also recorded. Traps were serviced every week, captured insects / eggs / larvae were identified and counted; at the same time the city drainagesystem manholes and other potential larval reservoirs have been periodically treated with larvicides. In addition a survey on the actual noxious effect to humans has been carried out. Results The following species / genera / families have been identified : •

Aedes albopictus (tiger mosquito)

•

Culex spp. (common mosquito)

•

Phlebotominae (sandflies)

Between them the tiger mosquito showed to be the most abundant and widespread, while the other groups showed more marked preference for particular environments such as hills for sandflies. Tiger mosquito showed a weekly average of 150-250 eggs / trap between August and September, decreasing to 10-50 eggs / trap in October. The harm to humans due to A. albopictus has been quantified in 2-4 bites / 30’ in AugustSeptember and 1 bite / 30’ in October, considering one person in private garden or public park during daytime. The experimental treatment of the urban draining system did not cause any decrease in tiger mosquito adult population, as shown by the number of eggs trapped in treated areas. Nevertheless it was not possible to assess the effect of treatment on larval reservoirs due to heavy rains that occurred at the end of September.

9.4. Evaluation of new mass-trapping attractants for noxious mosquito species. During 2004 BBCA started a formal cooperation with the US company American Biophysics and with the Italian company BME Srl, Biella, Italy, involved in the production and distribution of mass-trapping systems for mosquitoes. The formal cooperative agreement will be finalized to the evaluation of new attractants to be used in mosquito trapping systems. The evaluation will be performed in 2005 by means of laboratory and field-cage bioassays on laboratory strains of Anopheles stephensi, Culex pipiens and Aedes albopictus. 53

Contacts Biotechnology and Biological Control Agency onlus Via del Bosco, 10 00060, Sacrofano (RM) Italy Tel: +39 06 3048 3480 Fax: +39 06 3048 6044 Email: info@bbca.it This document is also available in PDF format at the following address: http://www.bbca.it/docs/BBCA0304.pdf